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Sample records for circulating neutrophil numbers

  1. Functional heterogeneity and differential priming of circulating neutrophils in human experimental endotoxemia.

    PubMed

    Pillay, Janesh; Ramakers, Bart P; Kamp, Vera M; Loi, Adele Lo Tam; Lam, Siu W; Hietbrink, Falco; Leenen, Luke P; Tool, Anton T; Pickkers, Peter; Koenderman, Leo

    2010-07-01

    Neutrophils play an important role in host defense. However, deregulation of neutrophils contributes to tissue damage in severe systemic inflammation. In contrast to complications mediated by an overactive neutrophil compartment, severe systemic inflammation is a risk factor for development of immune suppression and as a result, infectious complications. The role of neutrophils in this clinical paradox is poorly understood, and in this study, we tested whether this paradox could be explained by distinct neutrophil subsets and their functionality. We studied the circulating neutrophil compartment immediately after induction of systemic inflammation by administering 2 ng/kg Escherichia coli LPS i.v. to healthy volunteers. Neutrophils were phenotyped by expression of membrane receptors visualized by flow cytometry, capacity to interact with fluorescently labeled microbes, and activation of the NADPH-oxidase by oxidation of Amplex Red and dihydrorhodamine. After induction of systemic inflammation, expression of membrane receptors on neutrophils, such as CXCR1 and -2 (IL-8Rs), C5aR, FcgammaRII, and TLR4, was decreased. Neutrophils were also refractory to fMLF-induced up-regulation of membrane receptors, and suppression of antimicrobial function was shown by decreased interaction with Staphylococcus epidermis. Simultaneously, activation of circulating neutrophils was demonstrated by a threefold increase in release of ROS. The paradoxical phenotype can be explained by the selective priming of the respiratory burst. In contrast, newly released, CD16(dim) banded neutrophils display decreased antimicrobial function. We conclude that systemic inflammation leads to a functionally heterogeneous neutrophil compartment, in which newly released refractory neutrophils can cause susceptibility to infections, and activated, differentiated neutrophils can mediate tissue damage. PMID:20400675

  2. Extracorporeal immune therapy with immobilized agonistic anti-Fas antibodies leads to transient reduction of circulating neutrophil numbers and limits tissue damage after hemorrhagic shock/resuscitation in a porcine model

    PubMed Central

    2010-01-01

    Background Hemorrhagic shock/resuscitation is associated with aberrant neutrophil activation and organ failure. This experimental porcine study was done to evaluate the effects of Fas-directed extracorporeal immune therapy with a leukocyte inhibition module (LIM) on hemodynamics, neutrophil tissue infiltration, and tissue damage after hemorrhagic shock/resuscitation. Methods In a prospective controlled double-armed animal trial 24 Munich Mini Pigs (30.3 ± 3.3 kg) were rapidly haemorrhaged to reach a mean arterial pressure (MAP) of 35 ± 5 mmHg, maintained hypotensive for 45 minutes, and then were resuscitated with Ringer' solution to baseline MAP. With beginning of resuscitation 12 pigs underwent extracorporeal immune therapy for 3 hours (LIM group) and 12 pigs were resuscitated according to standard medical care (SMC). Haemodynamics, haematologic, metabolic, and organ specific damage parameters were monitored. Neutrophil infiltration was analyzed histologically after 48 and 72 hours. Lipid peroxidation and apoptosis were specifically determined in lung, bowel, and liver. Results In the LIM group, neutrophil counts were reduced versus SMC during extracorporeal immune therapy. After 72 hours, the haemodynamic parameters MAP and cardiac output (CO) were significantly better in the LIM group. Histological analyses showed reduction of shock-related neutrophil tissue infiltration in the LIM group, especially in the lungs. Lower amounts of apoptotic cells and lipid peroxidation were found in organs after LIM treatment. Conclusions Transient Fas-directed extracorporeal immune therapy may protect from posthemorrhagic neutrophil tissue infiltration and tissue damage. PMID:20406470

  3. [Does temperature in extracorporeal circulation affect neutrophil-endothelium interactions?].

    PubMed

    Menasché, P; Peynet, J; Le Deist, F; Carreno, M P; Haeffner-Cavaillon, N; Dillisse, V; Larivière, J; Piwnica, A; Bloch, G; Tedgui, A

    1995-10-01

    The increasing interest in "warm" aerobic cardioplegia requires a critical reevaluation of the systemic effects of the associated normothermic cardiopulmonary bypass (CPB). As activated neutrophils seem to be essential mediators of the inflammatory response to CPB via the cytotoxicity of the products that are released during their adhesion to endothelial cells, the authors undertook a study of the influence of temperature on the interaction between the neutrophils and the endothelium in 95 patients undergoing warm (31-33.5 degrees C, n = 49) and cold (26-27 degrees C, n = 46) CPB surgery. Blood sampling was performed before, during and after CPB. The following markers of neutrophil-endocardium interaction were analysed: complement activation (C3a), cytokine production (tumor necrosis factor alpha, interleukines 1, 6 and 8, and interleukin-1 receptor antagonist); endothelial expression of cytokine-dependent [intercellular adhesion molecule (ICAM)] and cytokine-independent (P-selectin) adhesion molecules (P-selectin); expression of cytokine molecules on the surface of polynuclear neutrophils (CD11a, CD11b, CD11c); and finally, endothelial adhesion and transendothelial migration of neutrophils (interleukin 8 and elastase). The results showed that, irrespective of temperature, CPB was associated with changes strongly suggestive of phenomena of transendothelial adhesion and migration. Moreover, normothermia increased the intensity of the inflammatory response as shown by increased cytokine production, earlier expression of neutrophil adhesion molecules and increased elastase production.

  4. The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils.

    PubMed

    Tole, Soumitra; Mukovozov, Ilya M; Huang, Yi-Wei; Magalhaes, Marco A O; Yan, Ming; Crow, Min Rui; Liu, Guang Ying; Sun, Chun Xiang; Durocher, Yves; Glogauer, Michael; Robinson, Lisa A

    2009-12-01

    In inflammatory diseases, circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not prevent neutrophil recruitment effectively. The Slit family of secreted proteins and their transmembrane receptor, Robo, repel axonal migration during CNS development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types toward a variety of chemotactic factors in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration but not random movement of neutrophils toward fMLP. Slit2 also inhibited neutrophil migration toward other chemoattractants, namely C5a and IL-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2 but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and MIP-2. In all instances, Slit2 reduced neutrophil recruitment effectively (P<0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis but not random motion of circulating neutrophils and point to Slit2 as a potential new therapeutic for preventing localized inflammation.

  5. Granulocyte colony-stimulating factor administration to healthy volunteers: analysis of the immediate activating effects on circulating neutrophils.

    PubMed

    de Haas, M; Kerst, J M; van der Schoot, C E; Calafat, J; Hack, C E; Nuijens, J H; Roos, D; van Oers, R H; von dem Borne, A E

    1994-12-01

    In four healthy volunteers, we analyzed in detail the immediate in vivo effects on circulating neutrophils of subcutaneous administration of 300 micrograms of granulocyte colony-stimulating factor (G-CSF). Neutrophil activation was assessed by measurement of degranulation. Mobilization of secretory vesicles was shown by a decrease in leukocyte alkaline phosphatase content of the circulating neutrophils. Furthermore, shortly postinjection, Fc gamma RIII was found to be upregulated from an intracellular pool that we identified by immunoelectron microscopy as secretory vesicles. Intravascular release of specific granules was shown by increased plasma levels of lactoferrin and by upregulation of the expression of CD66b and CD11b on circulating neutrophils. Moreover, measurement of fourfold elevated plasma levels of elastase, bound to its physiologic inhibitor alpha 1-antitrypsin, indicated mobilization of azurophil granules. However, no expression of CD63, a marker of azurophil granules, was observed on circulating neutrophils. G-CSF--induced mobilization of secretory vesicles and specific granules could be mimicked in whole blood cultures in vitro, in contrast to release of azurophil granules. Therefore, we postulate that the most activated neutrophils leave the circulation, as observed shortly postinjection, and undergo subsequent stimulation in the endothelial microenvironment, resulting in mobilization of azurophil granules. Our data demonstrate that G-CSF should be regarded as a potent immediate activator of neutrophils in vivo.

  6. Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation.

    PubMed

    Wachtfogel, Y T; Hack, C E; Nuijens, J H; Kettner, C; Reilly, T M; Knabb, R M; Bischoff, R; Tschesche, H; Wenzel, H; Kucich, U

    1995-03-01

    Cardiopulmonary bypass causes hemorrhagic complications and initiates a biochemical and cellular "whole body inflammatory response." This study investigates whether a variety of selective inhibitors of the contact pathway of intrinsic coagulation modulate complement and neutrophil activation during simulated extracorporeal circulation. After 60 min of recirculation in the presence of the slow tight-binding boronic acid inhibitor, Bz-Pro-Phe-boroArg-OH (10.7 microM), complete inhibition of kallikrein-C1-inhibitor complex formation and marked inhibition of C1-C1-inhibitor complex formation and the release of human neutrophil elastase were observed. Arg15-aprotinin (3.1 microM), Ala357,Arg358 alpha 1-antitrypsin (2.6 microM), and soybean trypsin inhibitor (48.0 microM) either completely or partially inhibited the generation of kallikrein-C1-inhibitor complexes but were less effective inhibitors of human neutrophil elastase release. The second-order rate constants for the inhibition of kallikrein in purified systems are consistent with the order of effectiveness of the inhibitors in blocking human neutrophil elastase release in heparinized blood. Our results suggest that low-molecular-weight selective inhibitors of kallikrein may be effective agents in the attenuation of the contact-mediated inflammatory response in cardiopulmonary bypass.

  7. Thrombin and human plasma kallikrein inhibition during simulated extracorporeal circulation block platelet and neutrophil activation.

    PubMed

    Wachtfogel, Y T; Kettner, C; Hack, C E; Nuijens, J H; Reilly, T M; Knabb, R M; Kucich, U; Niewiarowski, S; Edmunds, L H; Colman, R W

    1998-10-01

    Cardiopulmonary bypass causes hemorrhagic complications, and initiates a chemical and cellular inflammatory response. Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, complement, and contact systems. Despite the fact that cardiopulmonary bypass is carried out in the presence of high doses of heparin, there is significant activation of both platelets and neutrophils. Thrombin is protected on cell and fibrin surfaces from antithrombin, even in the presence of high doses of heparin (approximately 5 U/ml). We therefore studied the effect of a small (Mr = 497), highly effective (Ki = 41 pM), reversible tripeptide inhibitor of thrombin, DUP 714 (1 microM), in a well characterized model of simulated extracorporeal circulation. In the absence of DUP 714, platelet counts decreased by 75% 5 min after the start of extracorporeal bypass and increased to 48% at 120 min of recirculation. DUP 714 significantly preserved platelet counts, decreased plasma levels of platelet beta-thromboglobulin levels, but did not prevent a decrease in sensitivity of platelets to adenosine diphosphate. Kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively from 0.32 U/ml to 0.67 U/ml and from 4.45 U/ml to 7.25 U/ml, respectively, during 120 min of recirculation without DUP 714. Addition of DUP 714 significantly inhibited kallikrein-C1-inhibitor complex formation but did not affect C1-C1-inhibitor complexes. In the absence of DUP 714, human neutrophil elastase levels rose from a baseline of 0.01 +/- 0.00 microg/ml to 1.18 +/- 0.21 microg/ml during 120 min of recirculation. Human neutrophil elastase release at 120 min was significantly inhibited in the presence of DUP 714 to 37% of the value with heparin alone. These results indicated that addition of this novel thrombin (and kallikrein) inhibitor to heparin preserved platelet counts, decreased platelet secretion, and provided the additional benefit of

  8. Apoptotic neutrophils in the circulation of patients with glycogen storage disease type 1b (GSD1b).

    PubMed

    Kuijpers, Taco W; Maianski, Nikolai A; Tool, Anton T J; Smit, G Peter A; Rake, Jan Peter; Roos, Dirk; Visser, Gepke

    2003-06-15

    Glycogen storage disease type 1b (GSD1b) is a rare autosomal recessive disorder characterized by hypoglycemia, hepatomegaly, and growth retardation, and associated-for unknown reasons- with neutropenia and neutrophil dysfunction. In 5 GSD1b patients in whom nicotin-amide adenine dinucleotide phosphate-oxidase activity and chemotaxis were defective, we found that the majority of circulating granulocytes bound Annexin-V. The neutrophils showed signs of apoptosis with increased caspase activity, condensed nuclei, and perinuclear clustering of mitochondria to which the proapoptotic Bcl-2 member Bax had translocated already. Granulocyte colony-stimulating factor (G-CSF) addition to in vitro cultures did not rescue the GSD1b neutrophils from apoptosis as occurs with G-CSF-treated control neutrophils. Moreover, the 2 GSD1b patients on G-CSF treatment did not show significantly lower levels of apoptotic neutrophils in the bloodstream. Current understanding of neutrophil apoptosis and the accompanying functional demise suggests that GSD1b granulocytes are dysfunctional because they are apoptotic. PMID:12576310

  9. Prognostic value of pretreatment circulating neutrophils, monocytes, and lymphocytes on outcomes in lung stereotactic body radiotherapy

    PubMed Central

    Giuliani, M.; Sampson, L.R.; Wong, O.; Gay, J.; Le, L.W.; Cho, B.C.J.; Brade, A.; Sun, A.; Bezjak, A.; Hope, A.J.

    2016-01-01

    Purpose In the present study, we determined the association of pretreatment circulating neutrophils, monocytes, and lymphocytes with clinical outcomes after lung stereotactic body radiotherapy (sbrt). Methods All patients with primary lung cancer and with a complete blood count within 3 months of lung sbrt from 2005 to 2012 were included. Overall survival (os) was calculated using the Kaplan–Meier method. Factors associated with os were investigated using univariable and multivariable Cox proportional hazards regression. Fine–Gray competing risk regression was performed to test the association of the neutrophil:lymphocyte (nlr) and monocyte:lymphocyte (mlr) ratios with two types of failure: disease-related failure and death, and death unrelated to disease. Results Of the 299 sbrt patients identified, 122 were eligible for analysis. The median and range of the nlr and mlr were 3.0 (0.3–22.0) and 0.4 (0.1–1.9) respectively. On multivariable analysis, sex (p = 0.02), T stage (p = 0.04), and nlr (p < 0.01) were associated with os. On multivariable analysis, T stage (p < 0.01) and mlr (p < 0.01) were associated with disease-related failure; mlr (p = 0.03), nlr (p < 0.01), and sbrt dose of 48 Gy in 4 fractions (p = 0.03) and 54 Gy or 60 Gy in 3 fractions (p = 0.02) were associated with disease-unrelated death. Median survival was 4.3 years in the nlr≤3 group (95% confidence interval: 3.5 to not reached) and 2.5 years in the nlr>3 group (95% confidence interval: 1.7 to 4.8; p < 0.01). Conclusions In lung sbrt patients, nlr and mlr are independently associated with os and disease-unrelated death. If validated, nlr and mlr could help to identify patients who would benefit most from sbrt. PMID:27536185

  10. Beneficial and Adverse Effects of an LXR Agonist on Human Lipid and Lipoprotein Metabolism and Circulating Neutrophils.

    PubMed

    Kirchgessner, Todd G; Sleph, Paul; Ostrowski, Jacek; Lupisella, John; Ryan, Carol S; Liu, Xiaoqin; Fernando, Gayani; Grimm, Denise; Shipkova, Petia; Zhang, Rongan; Garcia, Ricardo; Zhu, Jun; He, Aiqing; Malone, Harold; Martin, Richard; Behnia, Kamelia; Wang, Zhaoqing; Barrett, Yu Chen; Garmise, Robert J; Yuan, Long; Zhang, Jane; Gandhi, Mohit D; Wastall, Philip; Li, Tong; Du, Shuyan; Salvador, Lisa; Mohan, Raju; Cantor, Glenn H; Kick, Ellen; Lee, John; Frost, Robert J A

    2016-08-01

    The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area. PMID:27508871

  11. Beneficial and Adverse Effects of an LXR Agonist on Human Lipid and Lipoprotein Metabolism and Circulating Neutrophils.

    PubMed

    Kirchgessner, Todd G; Sleph, Paul; Ostrowski, Jacek; Lupisella, John; Ryan, Carol S; Liu, Xiaoqin; Fernando, Gayani; Grimm, Denise; Shipkova, Petia; Zhang, Rongan; Garcia, Ricardo; Zhu, Jun; He, Aiqing; Malone, Harold; Martin, Richard; Behnia, Kamelia; Wang, Zhaoqing; Barrett, Yu Chen; Garmise, Robert J; Yuan, Long; Zhang, Jane; Gandhi, Mohit D; Wastall, Philip; Li, Tong; Du, Shuyan; Salvador, Lisa; Mohan, Raju; Cantor, Glenn H; Kick, Ellen; Lee, John; Frost, Robert J A

    2016-08-01

    The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.

  12. [APOPTOSIS AND NECROSIS OF CIRCULATING NEUTROPHILS IN PATIENTS WHILE HIGH RISK OF POSTOPERAIVE PERITONITIS OCCURRENCE].

    PubMed

    Sheyko, V D; Sytnik, D A; Shkurupiy, O O

    2015-11-01

    Processes of apoptosis and necrosis of peripheral neutrophils were investigated in 43 patients, operated on for an acute abdominal organs diseases on the first and fourth postoperative days. Changes of apoptosis and necrosis processes in peripheral neutrophils in dynamics were established. Unfavorable course of early postoperative period in patients with initial high and average risk of postoperative peritonitis occurrence was accompanied by shift in necrosis/apoptosis ratio towards necrosis of peripheral neutrophils.

  13. CIRCULATING CD11B EXPRESSION CORRELATES WITH THE NEUTROPHIL RESPONSE AND AIRWAY MCD-14 EXPRESSION IS ENHANCED FOLLOWING OZONE EXPOSURE IN HUMANS

    EPA Science Inventory

    We recently reported that baseline expression of circulating CD11b is associated with the magnitude of the neutrophil response following inhaled endotoxin. In this study, we examined whether circulating CD11b plays a similar role in the inflammatory response following inhaled ozo...

  14. Neutrophil kinetics in the dog.

    PubMed Central

    Deubelbeiss, K A; Dancey, J T; Harker, L A; Finch, C A

    1975-01-01

    The production of neutrophils in dogs has been estimated from the number of postmitotic neutrophils in the marrow and the transit time of a [3H]-thymidine pulse. The number of postmitotic neutrophils was derived from the erythron iron turnover measurement of erythroid number and the neutrophil-erythroid ratio in bone marrow sections. The mean value for marrow postmitotic neutrophils in dogs was 5.61 plus or minus 0.56 times 10-9 cells/kg. The mean transit time of these neutrophils was calculated to be 82.1 h. A marrow production of 1.65 times 10-9 neutrophils/kg/day was calculated from these data. The turnover of circulating neutrophils was measured by [3H]thymidine and [32P]diisopropylphospho-fluoridate (DF32P) labeling of blood neutrophils. [3H]-Thymidine labeling gave a calculated recovery of 65 per cent, a t1/2 disappearance time of 6.7 h, and a calculated turnover of 1.66 times 10-9 cells/kg/day. Corresponding results with DF32P tagging were 51 per cent, 5.4 h, and 2.89 times 10-9 cells/kg/day. The discrepancy between these two tags persisted in doubly tagged cells and was considered to be due to elution of DF32P. PMID:1120785

  15. Host-protective effect of circulating pentraxin 3 (PTX3) and complex formation with neutrophil extracellular traps

    PubMed Central

    Daigo, Kenji; Hamakubo, Takao

    2012-01-01

    Pentraxin 3 (PTX3) is a soluble pattern recognition receptor which is classified as a long-pentraxin in the pentraxin family. It is known to play an important role in innate immunity, inflammatory regulation, and female fertility. PTX3 is synthesized by specific cells, primarily in response to inflammatory signals. Among these various cells, neutrophils have a unique PTX3 production system. Neutrophils store PTX3 in neutrophil-specific granules and then the stored PTX3 is released and localizes in neutrophil extracellular traps (NETs). Although certain NET components have been identified, such as histones and anti-microbial proteins, the detailed mechanisms by which NETs localize, as well as capture and kill microbes, have not been fully elucidated. PTX3 is a candidate diagnostic marker of infection and vascular damage. In severe infectious diseases such as sepsis, the circulating PTX3 concentration increases greatly (up to 100 ng/mL, i.e., up to 100-fold of the normal level). Even though it is clearly implied that PTX3 plays a protective role in sepsis and certain other disorders, the detailed mechanisms by which it does so remain unclear. A proteomic study of PTX3 ligands in septic patients revealed that PTX3 forms a complex with certain NET component proteins. This suggests a role for PTX3 in which it facilitates the efficiency of anti-microbial protein pathogen clearance by interacting with both pathogens and anti-microbial proteins. We discuss the possible relationships between PTX3 and NET component proteins in the host protection afforded by the innate immune response. The PTX3 complex has the potential to be a highly useful diagnostic marker of sepsis and other inflammatory diseases. PMID:23248627

  16. Parturition in dairy cows temporarily alters the expression of genes in circulating neutrophils.

    PubMed

    Crookenden, M A; Heiser, A; Murray, A; Dukkipati, V S R; Kay, J K; Loor, J J; Meier, S; Mitchell, M D; Moyes, K M; Walker, C G; Roche, J R

    2016-08-01

    Extensive metabolic and physiologic changes occur during the peripartum, concurrent with a high incidence of infectious disease. Immune dysfunction is a likely contributor to the increased risk of disease at this time. Studies using high-yielding, total mixed ration-fed cows have indicated that neutrophil function is perturbed over the transition period; however, this reported dysfunction has yet to be investigated in moderate-yielding, grazing dairy cows. Therefore, we investigated changes in the expression of genes involved in neutrophil function. Blood was collected from cows at 5 time points over the transition period: precalving (-1wk; n=46), day of calving (d 0; n=46), and postcalving at wk 1 (n=46), wk 2 (n=45), and wk 4 (n=43). Neutrophils were isolated by differential centrifugation and gene expression was investigated. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 Dynamic Array, San Francisco, CA) were used to investigate the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using a repeated measures ANOVA. Genes that were differentially expressed over the transition period included those involved in neutrophil adhesion (SELL, ITGB2, and ITGBX), mediation of the immune response (TLR4, HLA-DRA, and CXCR2), maturation, cell cycle progression, apoptosis (MCL1, BCL2, FASLG, and RIPK1), and control of gene expression (PPARG, PPARD, and STAT3). We noted reduced gene expression of proinflammatory cytokines (IFNG, TNF, IL12, and CCL2) on the day of calving, whereas anti-inflammatory cytokine gene expression (IL10) was upregulated. Increased gene expression of antimicrobial peptides (BNBD4, DEFB10, and DEFB1) occurred on the day of calving. Collectively, transcription profiles are indicative of

  17. Parturition in dairy cows temporarily alters the expression of genes in circulating neutrophils.

    PubMed

    Crookenden, M A; Heiser, A; Murray, A; Dukkipati, V S R; Kay, J K; Loor, J J; Meier, S; Mitchell, M D; Moyes, K M; Walker, C G; Roche, J R

    2016-08-01

    Extensive metabolic and physiologic changes occur during the peripartum, concurrent with a high incidence of infectious disease. Immune dysfunction is a likely contributor to the increased risk of disease at this time. Studies using high-yielding, total mixed ration-fed cows have indicated that neutrophil function is perturbed over the transition period; however, this reported dysfunction has yet to be investigated in moderate-yielding, grazing dairy cows. Therefore, we investigated changes in the expression of genes involved in neutrophil function. Blood was collected from cows at 5 time points over the transition period: precalving (-1wk; n=46), day of calving (d 0; n=46), and postcalving at wk 1 (n=46), wk 2 (n=45), and wk 4 (n=43). Neutrophils were isolated by differential centrifugation and gene expression was investigated. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 Dynamic Array, San Francisco, CA) were used to investigate the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using a repeated measures ANOVA. Genes that were differentially expressed over the transition period included those involved in neutrophil adhesion (SELL, ITGB2, and ITGBX), mediation of the immune response (TLR4, HLA-DRA, and CXCR2), maturation, cell cycle progression, apoptosis (MCL1, BCL2, FASLG, and RIPK1), and control of gene expression (PPARG, PPARD, and STAT3). We noted reduced gene expression of proinflammatory cytokines (IFNG, TNF, IL12, and CCL2) on the day of calving, whereas anti-inflammatory cytokine gene expression (IL10) was upregulated. Increased gene expression of antimicrobial peptides (BNBD4, DEFB10, and DEFB1) occurred on the day of calving. Collectively, transcription profiles are indicative of

  18. Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum

    PubMed Central

    Prata, Rhana Berto da Silva; Barbosa, Mayara Garcia de Mattos; Mendes, Mayara Abud; Brandão, Sheila Santos; Amadeu, Thaís Porto; Rodrigues, Luciana Silva; Ferreira, Helen; Costa, Fabrício da Mota Ramalho; dos Santos, Jessica Brandão; Pacheco, Fabiana dos Santos; Machado, Alice de Miranda; Nery, José Augusto da Costa; Hacker, Mariana de Andrea; Sales, Anna Maria; Pinheiro, Roberta Olmo; Sarno, Euzenir Nunes

    2016-01-01

    Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates—a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably

  19. Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

    PubMed

    Schmitz, Veronica; Prata, Rhana Berto da Silva; Barbosa, Mayara Garcia de Mattos; Mendes, Mayara Abud; Brandão, Sheila Santos; Amadeu, Thaís Porto; Rodrigues, Luciana Silva; Ferreira, Helen; Costa, Fabrício da Mota Ramalho; Dos Santos, Jessica Brandão; Pacheco, Fabiana Dos Santos; Machado, Alice de Miranda; Nery, José Augusto da Costa; Hacker, Mariana de Andrea; Sales, Anna Maria; Pinheiro, Roberta Olmo; Sarno, Euzenir Nunes

    2016-08-01

    Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates-a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably

  20. Neutrophil ageing is regulated by the microbiome

    PubMed Central

    Zhang, Dachuan; Chen, Grace; Manwani, Deepa; Mortha, Arthur; Xu, Chunliang; Faith, Jeremiah J.; Burk, Robert D.; Kunisaki, Yuya; Jang, Jung-Eun; Scheiermann, Christoph; Merad, Miriam; Frenette, Paul S.

    2015-01-01

    Blood polymorphonuclear neutrophils provide immune protection against pathogens but also may promote tissue injury in inflammatory diseases1,2. Although neutrophils are generally considered as a relatively homogeneous population, evidence for heterogeneity is emerging3,4. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and the replenishment by newly released neutrophils from the bone marrow5. Aged neutrophils up-regulate CXCR4, a receptor allowing their clearance in the bone marrow6,7, with feedback inhibition of neutrophil production via the IL17/G-CSF axis8, and rhythmic modulation of the haematopoietic stem cell niche5. The aged subset also expresses low levels of L-selectin (CD62L)5,9. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties6,10. Here, we show using in vivo ageing analyses that the neutrophil pro-inflammatory activity correlates positively with their ageing in the circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMβ2 integrin (Mac-1) activation and neutrophil extracellular trap (NET) formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptors (TLRs)- and myeloid differentiation factor 88 (Myd88)-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle cell disease or endotoxin-induced septic shock. These results thus identify an unprecedented role for the microbiota in regulating a disease-promoting neutrophil subset. PMID:26374999

  1. Alpha 1-antitrypsin Pittsburgh (Met358-->Arg) inhibits the contact pathway of intrinsic coagulation and alters the release of human neutrophil elastase during simulated extracorporeal circulation.

    PubMed

    Wachtfogel, Y T; Bischoff, R; Bauer, R; Hack, C E; Nuijens, J H; Kucich, U; Niewiarowski, S; Edmunds, L H; Colman, R W

    1994-12-01

    Cardiopulmonary bypass prolongs bleeding time, increases postoperative blood loss, and triggers activation of plasma proteolytic enzyme systems and blood cells referred to as the "whole body inflammatory response". Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, contact and complement systems. Contact and complement pathway proteins both induce neutrophil activation. alpha 1-antitrypsin Pittsburgh (Met358-->Arg), a mutant of alpha 1-antitrypsin, is a potent inhibitor of plasma kallikrein and thrombin. We investigated whether this recombinant mutant protein inhibited platelet activation, as well as contact and/or complement-induced neutrophil activation during simulated extracorporeal circulation. Arg358 alpha 1-antitrypsin did not prevent the 34% drop in platelet count at 5 min of recirculation, did not block the 50% decrease in ADP-induced platelet aggregation at 120 min of recirculation, nor inhibit the release of 6.06 +/- 1.07 micrograms/ml beta-thromboglobulin at 120 min of recirculation suggesting that the inhibitor had little effect on platelet activation. However, Arg358 alpha 1-antitrypsin totally blocked kallikrein-C1-inhibitor complex formation but not C1-C1-inhibitor complex formation. Most importantly, Arg358 alpha 1-antitrypsin decreased the release of 1.11 +/- 0.16 micrograms/ml human neutrophil elastase by 43%. The attenuation of neutrophil activation in the absence of an effect on complement activation via the classical pathway, supports the concept that kallikrein is a major mediator of neutrophil degranulation during cardiopulmonary bypass.

  2. Neutrophil kinetics of recombinant human granulocyte colony-stimulating factor-induced neutropenia in rats

    SciTech Connect

    Okada, Yuji; Kawagishi, Mayumi; Kusaka, Masaru )

    1990-01-01

    Single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) immediately induced a decrease in the number of circulating neutrophils in rats. This neutropenia occurred 10 minutes after the injection but disappeared 40 minutes after injection. This transient neutropenia was dose-dependently induced by rhG-CSF and also induced by repeated injections. We studied the kinetics of circulating neutrophils in transient neutropenia. rhG-CSF markedly decreased the number of {sup 3}H-diisopropylfluorophosphate ({sup 3}H-DFP) labeled neutrophils in the circulation 10 minutes after injection but the labeled neutrophils recovered to near the control level 40 minutes after the injection. These results indicate that the neutrophil margination accounts for the neutrophenia and the marginated neutrophils return to the circulation.

  3. The influence of Prandtl number on Langmuir circulations

    NASA Astrophysics Data System (ADS)

    Phillips, W. R. C.

    1998-11-01

    Langmuir circulations (LC's) are thought to arise owing to the nonlinear interaction of surface gravity waves with a weak wind-induced shear layer. A model of such interactions is given by the CL-equations which depict two instability mechanisms to LC's, known as CL1 and CL2. The preferred mechanism is CL2, which depends upon three parameters, the Langmuir La, Richardson Ri and Prandtl Pr numbers. Previous work has focussed on the role of La and Ri with Pr fixed at its molecular value in water, say 6.7, but in the ocean, Pr can vary between 1 and 10. Here we vary Pr from zero to infinity in the model problem of LC's in deep water proposed by Leibovich & Paolucci (1981). The model assumes knowledge of the mean current and Stokes drift profiles, and both generic and Smith's (1992) ocean measured profiles were used. The calculations indicate that for non zero Ri, the critical La increases noticeably as Pr decreases. There thus exists a range of La within which the flow can either be destabilizing to, or stabilizing to, LC's, in accord with Smith observations in the ocean. Furthermore, while multiple non-overlapping eigensolutions for zero growth rate are common in such stability problems, here the two lead eigensolutions overlap. At some La, therefore, small variations in Pr cause a jump from one maximum growth curve to the other, causing a dramatic change in LC spacing.

  4. Cross-linking of IgGs bound on circulating neutrophils leads to an activation of endothelial cells: possible role of rheumatoid factors in rheumatoid arthritis-associated vascular dysfunction

    PubMed Central

    2013-01-01

    Background Rheumatoid arthritis is characterized by the presence of circulating auto-antibodies, including rheumatoid factors, which recognize the Fc portion of IgGs. The neutrophil is the most abundant circulating leukocyte and it expresses high levels of FcγRs on its surface. The aim of the present study was to examine the capacity of circulating human neutrophils to be activated by rheumatoid factors and the consequences of these events on endothelium. Methods Neutrophil-bound IgGs were cross-linked with anti-human IgGs to mimick the presence of circulating rheumatoid factors and FcγRs-dependent signalling events and functions were examined. The IgG and IgM composition of rheumatoid factors isolated from the serum of RA patients was characterized. Adhesion of neutrophils to endothelial cells was quantified in response to the addition of rheumatoid factors. Results Cross-linking of IgGs bound on neutrophils leads to FcγRs-dependent tyrosine phosphorylation, mobilisation of intracellular calcium and the extracellular release of superoxide anions and lysozyme. Incubation of endothelial cells with the supernatant of activated neutrophils increases ICAM-1 expression and IL-8 production by endothelial cells. Finally, rheumatoid factors enhance neutrophil adhesion to endothelial cells. Conclusions Our results show that activation of neutrophils’ FcγRs by rheumatoid factors could participate in rheumatoid arthritis-associated vascular damage. PMID:23902799

  5. Decreased numbers of chemotactic factor receptors in chronic neutropenia with defective chemotaxis: spontaneous recovery from the neutrophil abnormalities during early childhood

    SciTech Connect

    Yasui, K.; Yamazaki, M.; Miyagawa, Y.; Komiyama, A.; Akabane, T.

    1987-05-01

    Childhood chronic neutropenia with decreased numbers of chemotactic factor receptors as well as defective chemotaxis was first demonstrated in an 8-month-old girl. Chemotactic factor receptors on neutrophils were assayed using tritiated N-formyl-methionyl-leucyl-phenylalanine (/sup 3/H-FMLP). The patient's neutrophils had decreased numbers of the receptors: numbers of the receptors were 20,000 (less than 3 SD) as compared with those of control cells of 52,000 +/- 6000 (mean +/- SD) (n = 10). The neutropenia disappeared spontaneously by 28 months of age parallel with the improvement of chemotaxis and increase in numbers of chemotactic factor receptors. These results demonstrate a transient decrease of neutrophil chemotactic factor receptors as one of the pathophysiological bases of a transient defect of neutrophil chemotaxis in this disorder.

  6. Circulating Cytokines and Nitric Oxide are Involved in the Inhibition of Neutrophil Migration in Patients with Uterine Cervical Neoplasia

    PubMed Central

    Micheli, Douglas Côbo; Fernandes, Paulo Cesar; Cruvinel, João Celso Garcia; Nomelini, Isabela Destro; Murta, Eddie Fernando Candido; Tavares-Murta, Beatriz Martins

    2012-01-01

    Aim To verify if patients with cervical neoplasia produce mediators that reduce leukocyte function. Methods Control neutrophils incubated with normal serum or serum from pre-invasive or invasive neoplasia patients were assayed for chemotaxis. Mediators were assayed in serum and in leukocyte supernatants. Experiments were also performed in random patients after surgery. Results Neutrophils incubated with patient sera, but not normal sera, failed to migrate towards the chemoattractants. In invasive neoplasia compared to controls, IL-6 and IL-8, and IL-10 and TNF-α were elevated in serum and in neutrophil supernatants, respectively. Nitrite levels were elevated in mononuclear cell supernatants from patients than controls. After surgery, serum cytokine levels were reduced, mainly in pre-invasive patients. Neutrophils treated with serum from pre-invasive patients undergone surgery had restored migration. Conclusion Patients with cervical neoplasia produce mediators, predominantly induced by tumor cells, able to impair the inflammatory response at very early stages of disease. PMID:22693424

  7. Prediction of cloud droplet number in a general circulation model

    SciTech Connect

    Ghan, S.J.; Leung, L.R.

    1996-04-01

    We have applied the Colorado State University Regional Atmospheric Modeling System (RAMS) bulk cloud microphysics parameterization to the treatment of stratiform clouds in the National Center for Atmospheric Research Community Climate Model (CCM2). The RAMS predicts mass concentrations of cloud water, cloud ice, rain and snow, and number concnetration of ice. We have introduced the droplet number conservation equation to predict droplet number and it`s dependence on aerosols.

  8. Nafamostat mesilate, a broad spectrum protease inhibitor, modulates platelet, neutrophil and contact activation in simulated extracorporeal circulation.

    PubMed

    Sundaram, S; Gikakis, N; Hack, C E; Niewiarowski, S; Edmunds, L H; Koneti Rao, A; Sun, L; Cooper, S L; Colman, R W

    1996-01-01

    Activation of humoral and cellular participants in inflammation enhances the risk of postoperative bleeding and multiple organ damage in cardiopulmonary bypass (CPB). We now compare the effects of heparin alone in combination with nafamostat mesilate (NM), a protease inhibitor with specificity of trypsin-like enzymes, in an extracorporeal circuit which simulates CPB. NM significantly inhibits the release of platelet beta-thromboglobulin (beta TG) at 60 and 120 min. Platelet counts do not differ. ADP-induced aggregation decreases in circuits with NM, which is due to a direct effect of NM on platelet function. NM prevents any significant release of neutrophil elastase; at 120 min, plasma elastase-alpha 1-antitrypsin complex is 0.16 micrograms/ml in the NM group and 1.24 micrograms/ml in the control group. NM completely inhibits formation of complexes of C1 inhibitor with kallikrein and FXIIa. NM does not alter markers of complement activation (C1-C1-inhibitor complex and C5b-9), or indicators of thrombin formation (F1.2). However, at 120 min, thrombin activity as measured by release of fibrinopeptide A is significantly decreased. The data indicate that complement activation during CPB correlates poorly with neutrophil activation and that either kallikrein or FXIIa or both may be more important agonists. The ability of NM to inhibit two important contact system proteins and platelet and neutrophil release raises the possibility of suppressing the inflammatory response during clinical CPB.

  9. [Anti-Pseudomonas aeruginosa antibodies, circulating immune complexes, and anticytoplasm antibodies of neutrophils in patients with cystic fibrosis with and without Pseudomonas aeruginosa colonization].

    PubMed

    Chiappini, E; Taccetti, G; Campana, S; Turchini, S; Marianelli, L

    2001-01-01

    Chronic lung infections, mainly due to Pseudomonas aeruginosa (Pa), account for the most of the morbidity and mortality in CF patients. The pathogenic factors predisposing to airway colonization are still nuclear. Host's immune response is not only poorly protective but can also act as a damaging factor in the development of the disease. Moreover, clinical manifestations of an overactive immune response, including vasculitis and arthropathy, have been recently described in the CF population. To deepen factors involved in the pathogenesis of lung injury, we evaluated the presence of anti-Pa precipitating antibodies, circulating immune complexes (CIC) and antineutrophil cytoplasmic antibodies (ANCA) in sera from 50 CF patients colonized and not by Pa. Number of anti-Pa precipitins was significantly different in chronically and in not colonized patients (p < 0.001; t = 7.75). Anti-Pa antibodies were positively correlated to age (p = 0.002, r = 0.42) and inversely correlated to lung function parameters (p = 0.031 r = -0.35 with respect to FVC). Mean C3-CIC levels in the sera were statistically higher in chronically colonized patients (p = 0.013; t = 2.57); while there was not a significant difference with respect to C1q-CIC values. Four patients, all chronically colonized by Pa, were ANCA-positive at indirect immunofluorescence, showing a cytoplasmic pattern. All 50 patients were found to be negative when tested for anti-mpo and anti-pr3 antibodies with ELISA. In conclusion, our data demonstrate that persistence of Pa provides a stimulus for chronic inflammation and the immune response in CF patients, leading to anti-Pa antibodies and CIC production. Even in the face of further research, we speculate that c-ANCA production is secondary to neutrophil activation with a consequent release of cytoplasmic enzymes that perpetuate and increase the inflammatory process. Since anti-mpo and anti-pr3 antibodies rarely occur in CF patients we suggest that other antigenic

  10. Parasite and the Circulating Pool- Characterisation of Leukocyte Number and Morphology in Malaria

    PubMed Central

    Chandrashekhar, Jayaprakash

    2016-01-01

    Introduction Haematological changes are the most common complications encountered in malaria. There is significant correlation between several of the haematological parameters and the clinical profile, prognosis and mortality in malaria. White cell counts and differentials are among the most basic and primary investigations done in a patient presenting with fever of short duration. Aim This study analyzes the numerical and morphological changes in White Blood Cells (WBCs) in peripheral blood in patients with acute malaria in endemic region in an effort to get a picture of specific changes that could be identified by basic investigations. Materials and Methods This study was conducted in tertiary care hospital in a region endemic for malaria. EDTA anticoagulated venous blood samples from 600 patients diagnosed with vivax and falciparum malaria was analysed in Coulter counter LH 500 for the white cell count and differentials. Morphological changes were looked for in Leishman stained peripheral blood smear. Comparison with age matched healthy controls was done by ANOVA with Bonferroni test wherever applicable. Results Patients with malaria showed significant leucopenia, neutrophilia, lymphocytopenia, monocytosis and eosinopenia. Lymphocytopenia was more severe in the falciparum group as compared to the vivax group. A higher White Cell Count (WCC) was seen in patients with higher haemoglobin levels in vivax group. The total leukocyte count showed a negative correlation with neutrophil count in falciparum malaria and a strong positive correlation with neutrophil count in vivax malaria. Band neutrophils were seen in 10% of the patients with falciparum and 1.1% of patients with vivax malaria. Atypical plasmacytoid lymphocytes were the only notable morphological finding. Conclusion Changes in leukocyte number and morphology in the peripheral blood are common. A combination of monocytosis and eosinopenia in a patient presenting with fever should alert the observer to the

  11. High-Reynolds Number Circulation Control Testing in the National Transonic Facility

    NASA Technical Reports Server (NTRS)

    Milholen, William E., II; Jones, Gregory S.; Chan, David T.; Goodliff, Scott L.

    2012-01-01

    A new capability to test active flow control concepts and propulsion simulations at high Reynolds numbers in the National Transonic Facility at the NASA Langley Research Center is being developed. The first active flow control experiment was completed using the new FAST-MAC semi-span model to study Reynolds number scaling effects for several circulation control concepts. Testing was conducted over a wide range of Mach numbers, up to chord Reynolds numbers of 30 million. The model was equipped with four onboard flow control valves allowing independent control of the circulation control plenums, which were directed over a 15% chord simple-hinged flap. Preliminary analysis of the uncorrected lift data showed that the circulation control increased the low-speed maximum lift coefficient by 33%. At transonic speeds, the circulation control was capable of positively altering the shockwave pattern on the upper wing surface and reducing flow separation. Furthermore, application of the technique to only the outboard portion of the wing demonstrated the feasibility of a pneumatic based roll control capability.

  12. ISOLATION OF MOUSE NEUTROPHILS

    PubMed Central

    Swamydas, Muthulekha; Luo, Yi; Dorf, Martin E.; Lionakis, Michail S.

    2015-01-01

    Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT 19.6. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments. PMID:26237011

  13. Neutrophils in the tumor microenvironment: trying to heal the wound that cannot heal.

    PubMed

    Singel, Kelly L; Segal, Brahm H

    2016-09-01

    Neutrophils are the first responders to infection and injury and are critical for antimicrobial host defense. Through the generation of reactive oxidants, activation of granular constituents and neutrophil extracellular traps, neutrophils target microbes and prevent their dissemination. While these pathways are beneficial in the context of trauma and infection, their off-target effects in the context of tumor are variable. Tumor-derived factors have been shown to reprogram the marrow, skewing toward the expansion of myelopoiesis. This can result in stimulation of both neutrophilic leukocytosis and the release of immature granulocytic populations that accumulate in circulation and in the tumor microenvironment. While activated neutrophils have been shown to kill tumor cells, there is growing evidence for neutrophil activation driving tumor progression and metastasis through a number of pathways, including stimulation of thrombosis and angiogenesis, stromal remodeling, and impairment of T cell-dependent anti-tumor immunity. There is also growing appreciation of neutrophil heterogeneity in cancer, with distinct neutrophil populations promoting cancer control or progression. In addition to the effects of tumor on neutrophil responses, anti-neoplastic treatment, including surgery, chemotherapy, and growth factors, can influence neutrophil responses. Future directions for research are expected to result in more mechanistic knowledge of neutrophil biology in the tumor microenvironment that may be exploited as prognostic biomarkers and therapeutic targets. PMID:27558344

  14. Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice.

    PubMed

    Bucher, Kirsten; Schmitt, Fee; Autenrieth, Stella E; Dillmann, Inken; Nürnberg, Bernd; Schenke-Layland, Katja; Beer-Hammer, Sandra

    2015-09-01

    Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC(+) cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE(+) cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC(+) neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G.

  15. Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice.

    PubMed

    Bucher, Kirsten; Schmitt, Fee; Autenrieth, Stella E; Dillmann, Inken; Nürnberg, Bernd; Schenke-Layland, Katja; Beer-Hammer, Sandra

    2015-09-01

    Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC(+) cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE(+) cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC(+) neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G. PMID:26019296

  16. The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment.

    PubMed

    Schloss, Maximilian J; Horckmans, Michael; Nitz, Katrin; Duchene, Johan; Drechsler, Maik; Bidzhekov, Kiril; Scheiermann, Christoph; Weber, Christian; Soehnlein, Oliver; Steffens, Sabine

    2016-01-01

    Myocardial infarction (MI) is the leading cause of death in Western countries. Epidemiological studies show acute MI to be more prevalent in the morning and to be associated with a poorer outcome in terms of mortality and recovery. The mechanisms behind this association are not fully understood. Here, we report that circadian oscillations of neutrophil recruitment to the heart determine infarct size, healing, and cardiac function after MI Preferential cardiac neutrophil recruitment during the active phase (Zeitgeber time, ZT13) was paralleled by enhanced myeloid progenitor production, increased circulating numbers of CXCR2(hi) neutrophils as well as upregulated cardiac adhesion molecule and chemokine expression. MI at ZT13 resulted in significantly higher cardiac neutrophil infiltration compared to ZT5, which was inhibited by CXCR2 antagonism or neutrophil-specific CXCR2 knockout. Limiting exaggerated neutrophilic inflammation at this time point significantly reduced the infarct size and improved cardiac function. PMID:27226028

  17. [The influence of consecutive application of radioprotector B-190 and interleukin-1beta on changes of number of peripheral blood leucocytes and functional status of neutrophils of irradiated mice].

    PubMed

    Grebeniuk, A N; Aksenova, N V; Zatsepin, V V; Nazarov, V B; Vlasenko, T N

    2013-01-01

    The estimation of efficiency of consecutive application of radioprotector B-190 and interleukin-1beta administered at early times after acute irradiation by studying the changes of the number of peripheral blood leukocytes, neutrophils and lymphocytes, as well as cytochemical research into the contents of glycogen, myeloperoxidase and alkaline phosphatase in neutrophils of the irradiated mice was carried out. It is established that administration of radioprotector B-190 at a doze of 50 mg/kg for 15 minutes before irradiation and interleukin-1beta at a doze of 50 mkg/kg for 15 minutes after irradiation reduces the expressiveness of post-irradiation leukopenia, accelerates restoration of the number of neutrophils and lymphocytes in peripheral blood, normalizes the functional status of neutrophils of irradiated mice to a greater degree than their isolated introduction. PMID:24450210

  18. Decreased Number of Circulating Endothelial Progenitor Cells (CD133+/KDR+) in Patients with Psoriatic Arthritis.

    PubMed

    Batycka-Baran, Aleksandra; Paprocka, Maria; Baran, Wojciech; Szepietowski, Jacek C

    2016-08-23

    Cardiovascular diseases are a major cause of mortality in patients with psoriatic arthritis (PsA), but the precise mechanism of increased cardiovascular risk is unknown. Endothelial dysfunction plays a crucial role in the development of atherosclerosis. Circulating endothelial progenitor cells (CEPCs) contribute to endothelial regeneration and their level may be affected by chronic inflammation. The aim of this study was to evaluate the number of CEPCs in patients with PsA (n = 24) compared with controls (n = 26). CEPCs were identified as CD133+/ KDR+ cells in peripheral blood, using flow cytometry. A significantly decreased number of CEPCs was observed in patients with PsA (p < 0.0001). The number of these cells was significantly, inversely correlated with the severity of skin and joint involvement (Psoriasis Area and Severity Index (PASI), DAS28) and the level of C-reactive protein. We hypothesize that the reduced number of CEPCs may indicate and contribute to the increased cardiovascular risk in patients with PsA.

  19. NASA High-Reynolds Number Circulation Control Research - Overview of CFD and Planned Experiments

    NASA Technical Reports Server (NTRS)

    Milholen, W. E., II; Jones, Greg S.; Cagle, Christopher M.

    2010-01-01

    A new capability to test active flow control concepts and propulsion simulations at high Reynolds numbers in the National Transonic Facility at the NASA Langley Research Center is being developed. This technique is focused on the use of semi-span models due to their increased model size and relative ease of routing high-pressure air to the model. A new dual flow-path high-pressure air delivery station has been designed, along with a new high performance transonic sem -si pan wing model. The modular wind tunnel model is designed for testing circulation control concepts at both transonic cruise and low-speed high-lift conditions. The ability of the model to test other active flow control techniques will be highlighted. In addition, a new higher capacity semi-span force and moment wind tunnel balance has been completed and calibrated to enable testing at transonic conditions.

  20. On an instability to Langmuir circulations and the role of Prandtl and Richardson numbers

    NASA Astrophysics Data System (ADS)

    Phillips, W. R. C.

    2001-09-01

    The instability of a weakly sheared density-stratified two-dimensional wavy flow to longitudinal vortices is considered. The instability mechanism is Craik Leibovich type 2, or CL2, and the problem is posited in the context of Langmuir circulations beneath irrotational wind-driven surface waves. Of interest is the influence to the instability of Prandtl Pr and Richardson Ri numbers according to linear theory. The basis for the study is an initial value problem posed by Leibovich & Paolucci (1981) in which the liquid substrate is of semi-infinite extent and the wind-driven current is permitted to grow in the presence of neutral waves. In the present work Pr is varied from zero to infinity, and both stabilizing and destabilizing Ri are considered; so too are monochromatic and measured wave fields, and laminar and turbulent velocity profiles. Only the Ri = 0 results recover those of Leibovich & Paolucci. For stabilizing Ri, it is found in general that diminishing Pr are destabilizing to Langmuir circulations (LCs), and thus that LCs can be present or absent at the same Langmuir number La provided Ri [not equal] 0. It is further found that two branches of neutral curves occur for some combinations of Pr and Ri, and that minor changes in either parameter permit the preferred spacing to switch from one branch to the other. In consequence the preferred spanwise spacing may change from smaller than the wavelength of the dominant waves to larger than it. Furthermore, although LCs will not form at inverse La below a global lower bound given by an energy stability analysis, the actual value of La at onset is found to depend greatly upon local details of the wave and shear fields. Interestingly although this global lower bound is independent of Ri and Pr for Ri [gt-or-equal, slanted] 0, that is not the case for Ri < 0, where it approaches zero as Ri [rightward arrow] [minus sign][infty infinity], indicating that the CL2 instability is viable even at low Reynolds numbers.

  1. Reverse-migrated neutrophils regulated by JAM-C are involved in acute pancreatitis-associated lung injury

    PubMed Central

    Wu, Deqing; Zeng, Yue; Fan, Yuting; Wu, Jianghong; Mulatibieke, Tunike; Ni, Jianbo; Yu, Ge; Wan, Rong; Wang, Xingpeng; Hu, Guoyong

    2016-01-01

    Junctional adhesion molecule-C (JAM-C) plays a key role in the promotion of the reverse transendothelial migration (rTEM) of neutrophils, which contributes to the dissemination of systemic inflammation and to secondary organ damage. During acute pancreatitis (AP), systemic inflammatory responses lead to distant organ damage and typically result in acute lung injury (ALI). Here, we investigated the role of rTEM neutrophils in AP-associated ALI and the molecular mechanisms by which JAM-C regulates neutrophil rTEM in this disorder. In this study, rTEM neutrophils were identified in the peripheral blood both in murine model of AP and human patients with AP, which elevated with increased severity of lung injury. Pancreatic JAM-C was downregulated during murine experimental pancreatitis, whose expression levels were inversely correlated with both increased neutrophil rTEM and severity of lung injury. Knockout of JAM-C resulted in more severe lung injury and systemic inflammation. Significantly greater numbers of rTEM neutrophils were present both in the circulation and pulmonary vascular washout in JAM-C knockout mice with AP. This study demonstrates that during AP, neutrophils that are recruited to the pancreas may migrate back into the circulation and then contribute to ALI. JAM-C downregulation may contribute to AP-associated ALI via promoting neutrophil rTEM. PMID:26841848

  2. Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients.

    PubMed

    Ni, Xiaohui; Zhuo, Minglei; Su, Zhe; Duan, Jianchun; Gao, Yan; Wang, Zhijie; Zong, Chenghang; Bai, Hua; Chapman, Alec R; Zhao, Jun; Xu, Liya; An, Tongtong; Ma, Qi; Wang, Yuyan; Wu, Meina; Sun, Yu; Wang, Shuhang; Li, Zhenxiang; Yang, Xiaodan; Yong, Jun; Su, Xiao-Dong; Lu, Youyong; Bai, Fan; Xie, X Sunney; Wang, Jie

    2013-12-24

    Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.

  3. Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

    PubMed Central

    Ni, Xiaohui; Zhuo, Minglei; Su, Zhe; Duan, Jianchun; Gao, Yan; Wang, Zhijie; Zong, Chenghang; Bai, Hua; Chapman, Alec R.; Zhao, Jun; Xu, Liya; An, Tongtong; Ma, Qi; Wang, Yuyan; Wu, Meina; Sun, Yu; Wang, Shuhang; Li, Zhenxiang; Yang, Xiaodan; Yong, Jun; Su, Xiao-Dong; Lu, Youyong; Bai, Fan; Xie, X. Sunney; Wang, Jie

    2013-01-01

    Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics. PMID:24324171

  4. Neutrophil Depletion Attenuates Placental Ischemia-Induced Hypertension in the Rat

    PubMed Central

    Regal, Jean F.; Lillegard, Kathryn E.; Bauer, Ashley J.; Elmquist, Barbara J.; Loeks-Johnson, Alex C.; Gilbert, Jeffrey S.

    2015-01-01

    Preeclampsia is characterized by reduced placental perfusion with placental ischemia and hypertension during pregnancy. Preeclamptic women also exhibit a heightened inflammatory state and greater number of neutrophils in the vasculature compared to normal pregnancy. Since neutrophils are associated with tissue injury and inflammation, we hypothesized that neutrophils are critical to placental ischemia-induced hypertension and fetal demise. Using the reduced uteroplacental perfusion pressure (RUPP) model of placental ischemia-induced hypertension in the rat, we determined the effect of neutrophil depletion on blood pressure and fetal resorptions. Neutrophils were depleted with repeated injections of polyclonal rabbit anti-rat polymorphonuclear leukocyte (PMN) antibody (antiPMN). Rats received either antiPMN or normal rabbit serum (Control) on 13.5, 15.5, 17.5, and 18.5 days post conception (dpc). On 14.5 dpc, rats underwent either Sham surgery or clip placement on ovarian arteries and abdominal aorta to reduce uterine perfusion pressure (RUPP). On 18.5 dpc, carotid arterial catheters were placed and mean arterial pressure (MAP) was measured on 19.5 dpc. Neutrophil-depleted rats had reduced circulating neutrophils from 14.5 to 19.5 dpc compared to Control, as well as decreased neutrophils in lung and placenta on 19.5 dpc. MAP increased in RUPP Control vs Sham Control rats, and neutrophil depletion attenuated this increase in MAP in RUPP rats without any effect on Sham rats. The RUPP-induced increase in fetal resorptions and complement activation product C3a were not affected by neutrophil depletion. Thus, these data are the first to indicate that neutrophils play an important role in RUPP hypertension and that cells of the innate immune system may significantly contribute to pregnancy-induced hypertension. PMID:26135305

  5. [THE NEW APPROACH TO EVALUATION OF ENDOTHELIUM DYSFUNCTION: DETECTION OF NUMBER OF CIRCULATING ENDOTHELIUM CELLS USING FLOW CYTOMETRY TECHNIQUE].

    PubMed

    Feoktistova, V S; Vavilkova, T V; Sirotkina, O V; Boldueva, S A; Gaikovaia, L B; Leonova, I A; Laskovets, A B; Ermakov, A I

    2015-04-01

    The endothelium dysfunction takes leading place in pathogenesis of development of cardiovascular diseases. The circulating endothelium cells of peripheral blood can act as a direct cell marker of damage and remodeling of endothelium. The study was carried out to develop a new approach to diagnose of endothelium dysfunction by force of determination of number of circulating endothelium cells using flow cytometry technique and to apply determination of circulating endothelium cells for evaluation of risk of development of ischemic heart disease in women of young and middle age. The study embraced 62 female patients with angiography confirmed ischemic heart disease, exertional angina pectoris at the level of functional class I-II (mean age 51 ± 6 years) and 49 women without anamnesis of ischemic heart disease (mean age 52 ± 9 years). The occurrence of more than three circulating endothelium cells by 3 x 105 leukocytes in peripheral blood increases relative risk of development of ischemic heart disease up to 4 times in women of young and middle age and risk of development of acute myocardial infarction up to 8 times in women with ischemic heart disease. The study demonstrated possibility to apply flow cytometry technique to quantitatively specify circulating endothelium cells in peripheral blood and forecast risk of development of ischemic heart disease in women of young and middle age depending on level of circulating endothelium cells.

  6. Marked alterations of neutrophil functions during sepsis-induced immunosuppression.

    PubMed

    Demaret, Julie; Venet, Fabienne; Friggeri, Arnaud; Cazalis, Marie-Angélique; Plassais, Jonathan; Jallades, Laurent; Malcus, Christophe; Poitevin-Later, Françoise; Textoris, Julien; Lepape, Alain; Monneret, Guillaume

    2015-12-01

    Severe septic syndromes deeply impair innate and adaptive immunity and are responsible for sepsis-induced immunosuppression. Although neutrophils represent the first line of defense against infection, little is known about their phenotype and functions a few days after sepsis, when the immunosuppressive phase is maximal (i.e., between d 3 and 8). The objective of the present study was to perform, for the first time, a global evaluation of neutrophil alterations in immunosuppressed septic patients (at d 3-4 and d 6-8) using phenotypic and functional studies. In addition, the potential association of these parameters and deleterious outcomes was assessed. Peripheral blood was collected from 43 septic shock patients and compared with that of 23 healthy controls. In the septic patients, our results highlight a markedly altered neutrophil chemotaxis (functional and chemokine receptor expressions), oxidative burst, and lactoferrin content and an increased number of circulating immature granulocytes (i.e., CD10(dim)CD16(dim)). These aspects were associated with an increased risk of death after septic shock. In contrast, phagocytosis and activation capacities were conserved. To conclude, circulating neutrophils present with phenotypic, functional, and morphologic alterations a few days after sepsis onset. These dysfunctions might participate in the deleterious role of sepsis-induced immunosuppression. The present results open new perspectives in the mechanisms favoring nosocomial infections after septic shock. They deserve to be further investigated in a larger clinical study and in animal models recapitulating these alterations. PMID:26224052

  7. A short-term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA-axis signaling in mice.

    PubMed

    de Kleijn, Stan; Ferwerda, Gerben; Wiese, Michelle; Trentelman, Jos; Cuppen, Jan; Kozicz, Tamas; de Jager, Linda; Hermans, Peter W M; Verburg-van Kemenade, B M Lidy

    2016-10-01

    There is still uncertainty whether extremely low frequency electromagnetic fields (ELF-EMF) can induce health effects like immunomodulation. Despite evidence obtained in vitro, an unambiguous association has not yet been established in vivo. Here, mice were exposed to ELF-EMF for 1, 4, and 24 h/day in a short-term (1 week) and long-term (15 weeks) set-up to investigate whole body effects on the level of stress regulation and immune response. ELF-EMF signal contained multiple frequencies (20-5000 Hz) and a magnetic flux density of 10 μT. After exposure, blood was analyzed for leukocyte numbers (short-term and long-term) and adrenocorticotropic hormone concentration (short-term only). Furthermore, in the short-term experiment, stress-related parameters, corticotropin-releasing hormone, proopiomelanocortin (POMC) and CYP11A1 gene-expression, respectively, were determined in the hypothalamic paraventricular nucleus, pituitary, and adrenal glands. In the short-term but not long-term experiment, leukocyte counts were significantly higher in the 24 h-exposed group compared with controls, mainly represented by increased neutrophils and CD4 ± lymphocytes. POMC expression and plasma adrenocorticotropic hormone were significantly lower compared with unexposed control mice. In conclusion, short-term ELF-EMF exposure may affect hypothalamic-pituitary-adrenal axis activation in mice. Changes in stress hormone release may explain changes in circulating leukocyte numbers and composition. Bioelectromagnetics. 37:433-443, 2016. © 2016 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc. PMID:27553635

  8. Effect of weak rotation on the large-scale circulation in turbulent convection with a Prandtl number Pr = 12 . 3

    NASA Astrophysics Data System (ADS)

    Wei, Ping; Ahlers, Guenter

    2015-11-01

    We report measurements of large-scale circulation properties for high-Rayleigh-number convection in a rotating cylindrical sample with aspect ratio Γ = D / L = 1 . 00 (D is the diameter and L the height). The Prandtl number was Pr = 12 . 3 . The measurements covered the Rayleigh-number range 2 ×1010 <= Ra <= 4 ×1011 and the inverse Rossby-number range 0 <= 1 / Ro <= 1 / Roc = 0 . 28 where the LSC was present. The azimuthal orientation θ0 of the LSC circulation plane remained fixed in the frame of the rotating sample for Ra < Ra0 ~= 5 ×1010 . The sloshing motion of the LSC showed oscillations with a short time period τpl of several tens of seconds. The temperature amplitude < δ > of the LSC increased as 1 / Ro approached 1 / Roc , and decreased rapidly beyond it. For Ra > Ra0 , the circulation plane underwent retrograde rotation and hence caused time-periodic temperature oscillations near the side wall with a large period τac of hundreds of seconds. Remarkably, τac persisted without a discontinuity even for 1 / Ro > 1 / Roc where the LSC ceased to exist, indicating that vortex structures in that regime undergo the same retrograde rotation as the LSC. Supported by NSF Grant DMR11-58514.

  9. Expanded numbers of circulating myeloid dendritic cells in patent human filarial infection reflect lower CCR1 expression.

    PubMed

    Semnani, Roshanak Tolouei; Mahapatra, Lily; Dembele, Benoit; Konate, Siaka; Metenou, Simon; Dolo, Housseini; Coulibaly, Michel E; Soumaoro, Lamine; Coulibaly, Siaka Y; Sanogo, Dramane; Seriba Doumbia, Salif; Diallo, Abdallah A; Traoré, Sekou F; Klion, Amy; Nutman, Thomas B; Mahanty, Siddhartha

    2010-11-15

    APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infection. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c(-)CD123(lo)) in peripheral blood of infected microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c(+)CD123(lo)) in filaria-infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs, monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals had a lower percentage of circulating CCR1(+) mDCs and a higher percentage of circulating CCR5(+) mDCs and pDCs. Finally, live microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations have major implications for regulation of immune responses to these long-lived parasites. PMID:20956349

  10. Heterogeneity of the Mac-1 expression on peripheral blood neutrophils in patients with different types of epithelial ovarian cancer.

    PubMed

    Bednarska, Katarzyna; Klink, Magdalena; Wilczyński, Jacek R; Szyłło, Krzysztof; Malinowski, Andrzej; Sułowska, Zofia; Nowak, Marek

    2016-02-01

    The expression level of Mac-1 on the surface of neutrophils is an important indicator of neutrophil activation. Under pathological conditions, Mac-1 is believed a key adhesion molecule that facilitates cancer progression and mediates the adhesion of tumour cells to the endothelium of blood vessels. Our previous findings indicated that circulating peripheral blood neutrophils in patients with advanced epithelial ovarian cancer (EOC) expressed enhanced levels of Mac-1, which was functionally associated with an increased adhesive function of neutrophils. The objective of the current study was to analyse whether the value of individual components of the differential white cell count, including the neutrophil and lymphocyte ratios, which are markers of blood neutrophil activation, might be associated with certain types of ovarian cancer. We showed the increase in Mac-1 expression along with a parallel decrease of L-selectin and PSGL-1 on peripheral blood neutrophils of patients with EOC of early and advanced FIGO stages, which indicates an activated state of neutrophils in comparison to neutrophils of individuals without cancer. Despite a significant difference between Mac-1 expression in patients with and without cancer, a dramatic increase in Mac-1 expression was observed in the blood of patients with undifferentiated carcinomas compared with patients with other histological types of EOC. Moreover, the expression level of Mac-1 correlated with the number of neutrophils in patients with serous, endometrioid and undifferentiated EOC. The results of an ROC analysis demonstrated that the patients with the undifferentiated type of EOC form a distinct group with regard to Mac-1 expression on blood neutrophils. The results suggested a diverse biological cadre of immune cells in patients with undifferentiated ovarian carcinomas compared with patients with other histological types of EOC. PMID:26563750

  11. Heterogeneity of the Mac-1 expression on peripheral blood neutrophils in patients with different types of epithelial ovarian cancer.

    PubMed

    Bednarska, Katarzyna; Klink, Magdalena; Wilczyński, Jacek R; Szyłło, Krzysztof; Malinowski, Andrzej; Sułowska, Zofia; Nowak, Marek

    2016-02-01

    The expression level of Mac-1 on the surface of neutrophils is an important indicator of neutrophil activation. Under pathological conditions, Mac-1 is believed a key adhesion molecule that facilitates cancer progression and mediates the adhesion of tumour cells to the endothelium of blood vessels. Our previous findings indicated that circulating peripheral blood neutrophils in patients with advanced epithelial ovarian cancer (EOC) expressed enhanced levels of Mac-1, which was functionally associated with an increased adhesive function of neutrophils. The objective of the current study was to analyse whether the value of individual components of the differential white cell count, including the neutrophil and lymphocyte ratios, which are markers of blood neutrophil activation, might be associated with certain types of ovarian cancer. We showed the increase in Mac-1 expression along with a parallel decrease of L-selectin and PSGL-1 on peripheral blood neutrophils of patients with EOC of early and advanced FIGO stages, which indicates an activated state of neutrophils in comparison to neutrophils of individuals without cancer. Despite a significant difference between Mac-1 expression in patients with and without cancer, a dramatic increase in Mac-1 expression was observed in the blood of patients with undifferentiated carcinomas compared with patients with other histological types of EOC. Moreover, the expression level of Mac-1 correlated with the number of neutrophils in patients with serous, endometrioid and undifferentiated EOC. The results of an ROC analysis demonstrated that the patients with the undifferentiated type of EOC form a distinct group with regard to Mac-1 expression on blood neutrophils. The results suggested a diverse biological cadre of immune cells in patients with undifferentiated ovarian carcinomas compared with patients with other histological types of EOC.

  12. Neutrophil Functions in Periodontal Homeostasis

    PubMed Central

    Cortés-Vieyra, Ricarda; Rosales, Carlos

    2016-01-01

    Oral tissues are constantly exposed to damage from the mechanical effort of eating and to microorganisms, mostly bacteria. In healthy gingiva tissue remodeling and a balance between bacteria and innate immune cells are maintained. However, excess of bacteria biofilm (plaque) creates an inflammation state that recruits more immune cells, mainly neutrophils to the gingiva. Neutrophils create a barrier for bacteria to reach inside tissues. When neutrophils are insufficient, bacteria thrive causing more inflammation that has been associated with systemic effects on other conditions such as atherosclerosis, diabetes, and cancer. But paradoxically when neutrophils persist, they can also promote a chronic inflammatory state that leads to periodontitis, a condition that leads to damage of the bone-supporting tissues. In periodontitis, bone loss is a serious complication. How a neutrophil balance is needed for maintaining healthy oral tissues is the focus of this review. We present recent evidence on how alterations in neutrophil number and function can lead to inflammatory bone loss, and how some oral bacteria signal neutrophils to block their antimicrobial functions and promote an inflammatory state. Also, based on this new information, novel therapeutic approaches are discussed. PMID:27019855

  13. The effect of cigarette smoking on neutrophil kinetics in human lungs (see comments

    SciTech Connect

    MacNee, W.; Wiggs, B.; Belzberg, A.S.; Hogg, J.C. )

    1989-10-05

    Neutrophils may play a part in the pathogenesis of the centrilobular emphysema associated with cigarette smoking. The capillary bed of the lungs concentrates neutrophils approximately 100-fold with respect to erythrocytes, producing a large pool of marginated cells. We examined the effect of cigarette smoking on the kinetics of this pool of cells, using 99mTc-labeled erythrocytes to measure regional blood velocity and 111In-labeled neutrophils to measure the removal of neutrophils during the first passage through the pulmonary circulation, their subsequent washout from the lungs, and the effect of local blood velocity on the number of neutrophils retained in each lung region. We observed no difference in these measurements between subjects who had never smoked (n = 6) and smokers who did not smoke during the study (n = 12). However, subjects who did smoke during the study (n = 12) had a significantly slower rate of washout of radiolabeled neutrophils from the lung (0.08 +/- 0.04 of the total per minute, as compared with 0.13 +/- 0.06 in smokers who did not smoke during the experiment and 0.14 +/- 0.08 in non-smokers) (P = 0.02). We also observed an increase in the regional retention of labeled neutrophils with respect to blood velocity in 5 of the 12 subjects who smoked during the study, but in none of the other subjects. We conclude that the presence of cigarette smoke in the lungs of some subjects increases the local concentration of neutrophils, and suggest that the lesions that characterize emphysema may be a result of the destruction of lung tissue by neutrophils that remain within pulmonary microvessels.

  14. Elevation in Peripheral Blood Circulating Tumor Cell Number Correlates with Macroscopic Progression in UICC Stage IV Colorectal Cancer Patients

    PubMed Central

    Molnar, Bela; Floro, Lajos; Sipos, Ferenc; Toth, Bernadett; Sreter, Lydia; Tulassay, Zsolt

    2008-01-01

    Aims: Cytokeratin(CK) based real-time RT-PCR assays (QRT-PCR) are now available for peripheral blood circulating tumor cell (CTC) evaluations in colorectal cancer(CRC) patients. Results are non-existent for the application of these techniques to the determination of progression and therapy response in Dukes stage D CRC patients. Patients and methods: Each month 30 ml peripheral blood of 30 Dukes D patients (17 with progression) were drawn. CEA, CA19-9, CA72-A and TPA-M determinations were made. CK20, thymidilate synthase(TS) QRT-PCR was performed, as well. Buffy coat was used for immunmagnetic cancer cell isolation and CTC counting. Correlation between elevated CTC and macroscopic progression within 3 months was determined by Chi2 test. Results: Microscopic CTC single cell, doublet, cluster number were found in correlation with CK20 QRT-PCR results (p < 0.01). There was a significant increase in microscopic CTC number, CK20 and decrease in TS QRT-PCR levels (p < 0.05) in the peripheral blood of the non-responder as compared to responder patients. Elevation of the CTC was in significant correlation with macroscopic progression of the disease in 3 months (p < 0.01). Conclusions: CTC number reflects the chemotherapeutic sensitivity of CRC patients. Elevation of circulating tumor cell number in peripheral blood is in correlation with macroscopic progression. PMID:18334735

  15. Neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps.

    PubMed

    Jenne, Craig N; Wong, Connie H Y; Zemp, Franz J; McDonald, Braedon; Rahman, Masmudur M; Forsyth, Peter A; McFadden, Grant; Kubes, Paul

    2013-02-13

    Neutrophils mediate bacterial clearance through various mechanisms, including the release of mesh-like DNA structures or neutrophil extracellular traps (NETs) that capture bacteria. Although neutrophils are also recruited to sites of viral infection, their role in antiviral innate immunity is less clear. We show that systemic administration of virus analogs or poxvirus infection induces neutrophil recruitment to the liver microvasculature and the release of NETs that protect host cells from virus infection. After systemic intravenous poxvirus challenge, mice exhibit thrombocytopenia and the recruitment of both neutrophils and platelets to the liver vasculature. Circulating platelets interact with, roll along, and adhere to the surface of adherent neutrophils, forming large, dynamic aggregates. These interactions facilitate the release of NETs within the liver vasculature that are able to protect host cells from poxvirus infection. These findings highlight the role of NETs and early tissue-wide responses in preventing viral infection.

  16. Isolation and Characterization of Low- vs. High-Density Neutrophils in Cancer.

    PubMed

    Sagiv, Jitka Y; Voels, Sandra; Granot, Zvi

    2016-01-01

    Neutrophils are the most abundant of all white blood cells in the human circulation and serve as the first line of defense against microbial infections. Traditionally, neutrophils were viewed as a homogeneous population of myeloid cells. However, in recent years accumulating evidence has suggested that neutrophils are heterogeneous and that distinct neutrophil subsets may play very different roles. Here, we describe the methodology for isolation of high- and low-density neutrophils from the murine and human circulation using a density gradient and antibody based enrichment. We further describe the methodology for functional characterization of these different neutrophil subsets in the context of cancer. PMID:27581022

  17. Influence of minor thermal injury on expression of complement receptor CR3 on human neutrophils.

    PubMed Central

    Nelson, R. D.; Hasslen, S. R.; Ahrenholz, D. H.; Haus, E.; Solem, L. D.

    1986-01-01

    Thermal injury is well known to inhibit functions of the circulating neutrophil related to its role in host defense against infection, but the mechanism(s) of this phenomenon are not fully understood. To gain further clues to these mechanisms, the authors have studied patients with thermal injury in terms of altered expression of neutrophil cell membrane receptors for the opsonic complement-derived ligand C3bi--complement receptor Type 3, or CR3. CR3 expression was selected for study because an increase in the number of receptors on the cell surface can be stimulated by products of complement activation known to accumulate after thermal injury and because of the role of CR3 in phagocytic and adherence functions of the neutrophil. Expression of CR3 was monitored semiquantitatively by flow cytometry with the use of a murine monoclonal antibody (OKM1) specific for an antigen (CD11) associated with this receptor. Patients evaluated were limited in this study to those with minor degrees of thermal injury (second-degree burn involving less than 20% of total body surface area) so that possible confounding effects of major injury and its complications could be eliminated. It was observed that patient neutrophil CR3 becomes significantly up-regulated during the first week, as early as 1 day after injury. The maximum level of expression of CR3 averaged greater than 150% (range, 70-314%) of the respective minimum level observed for each patient. The minimum levels of expression of CR3 on patient neutrophils, reached 11-37 days after injury for 7 of 8 patients, were comparable to the level of expression of CR3 on unstimulated control neutrophils. Such temporal up-regulation of patient neutrophil CR3 suggests the early generation of stimuli of CR3 mobilization in response to thermal injury. Increased numbers of CR3 on patient neutrophils may augment microbicidal function and enhance or inhibit delivery of cells to the burn site. PMID:3541642

  18. [A Large Number of Circulating Tumor Cells(CTCs)Can Be Isolated from Samples Obtained by Using Leukapheresis Procedures].

    PubMed

    Soya, Ryoko; Taguchi, Jyunichi; Nagakawa, Yuichi; Takahashi, Osamu; Sandoh, Norimasa; Hosokawa, Yuichi; Kasuya, Kazuhiko; Umeda, Naoki; Okamoto, Masato; Tsujitani, Shunichi; Tsuchida, Akihiko

    2015-09-01

    We hypothesized that a large number of circulating tumor cells(CTCs)may be isolated from samples obtained by using the leukapheresis procedures that are utilized to collect peripheral blood mononuclear cells for dendritic cell vaccine therapy. We utilized the CellSearch System to determine the number of CTCs in samples obtained by using leukapheresis in 7 patients with colorectal cancer, 5 patients with breast cancer, and 3 patients with gastric cancer. In all patients, a large number of CTCs were isolated. The mean number of CTCs per tumor was 17.1(range 10-34)in colorectal cancer, 10.0(range 2-27)in breast cancer, and 24.0(range 2-42)in gastric cancer. We succeeded in culturing the isolated CTCs from 7 patients with colorectal cancer, 5 patients with breast cancer, and 3 patients with gastric cancer. In conclusion, compared to conventional methods, a large number of CTCs can be obtained by using leukapheresis procedures. The molecular analyses of the CTCs isolated by using this method should be promising in the development of personalized cancer treatments.

  19. Netting neutrophils in autoimmune small-vessel vasculitis.

    PubMed

    Kessenbrock, Kai; Krumbholz, Markus; Schönermarck, Ulf; Back, Walter; Gross, Wolfgang L; Werb, Zena; Gröne, Hermann-Josef; Brinkmann, Volker; Jenne, Dieter E

    2009-06-01

    Small-vessel vasculitis (SVV) is a chronic autoinflammatory condition linked to antineutrophil cytoplasm autoantibodies (ANCAs). Here we show that chromatin fibers, so-called neutrophil extracellular traps (NETs), are released by ANCA-stimulated neutrophils and contain the targeted autoantigens proteinase-3 (PR3) and myeloperoxidase (MPO). Deposition of NETs in inflamed kidneys and circulating MPO-DNA complexes suggest that NET formation triggers vasculitis and promotes the autoimmune response against neutrophil components in individuals with SVV.

  20. Achyrocline satureioides (Lam.) D.C. Hydroalcoholic Extract Inhibits Neutrophil Functions Related to Innate Host Defense

    PubMed Central

    Barioni, Eric Diego; Machado, Isabel Daufenback; Rodrigues, Stephen Fernandes de Paula; Ferraz-de-Paula, Viviane; Wagner, Theodoro Marcel; Cogliati, Bruno; Corrêa dos Santos, Matheus; Machado, Marina da Silva; de Andrade, Sérgio Faloni; Niero, Rivaldo; Farsky, Sandra Helena Poliselli

    2013-01-01

    Achyrocline satureioides (Lam.) D.C. is a herb native to South America, and its inflorescences are popularly employed to treat inflammatory diseases. Here, the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences of A. satureioides on neutrophil trafficking into inflamed tissue were investigated. Male Wistar rats were orally treated with A. satureioides extract, and inflammation was induced one hour later by lipopolysaccharide injection into the subcutaneous tissue. The number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate, and adhesion molecule and toll-like receptor 4 (TLR-4) expressions and phorbol-myristate-acetate- (PMA-) stimulated oxidative burst were quantified in circulating neutrophils. Leukocyte-endothelial interactions were quantified in the mesentery tissue. Enzymes and tissue morphology of the liver and kidney were evaluated. Treatment with A. satureioides extract reduced neutrophil influx and secretion of leukotriene B4 and CINC-1 in the exudates, the number of rolling and adhered leukocytes in the mesentery postcapillary venules, neutrophil L-selectin, β2-integrin and TLR-4 expression, and oxidative burst, but did not cause an alteration in the morphology and activities of liver and kidney. Together, the data show that A. satureioides extract inhibits neutrophil functions related to the innate response and does not cause systemic toxicity. PMID:23476704

  1. Achyrocline satureioides (Lam.) D.C. Hydroalcoholic Extract Inhibits Neutrophil Functions Related to Innate Host Defense.

    PubMed

    Barioni, Eric Diego; Santin, José Roberto; Machado, Isabel Daufenback; Rodrigues, Stephen Fernandes de Paula; Ferraz-de-Paula, Viviane; Wagner, Theodoro Marcel; Cogliati, Bruno; Corrêa Dos Santos, Matheus; Machado, Marina da Silva; de Andrade, Sérgio Faloni; Niero, Rivaldo; Farsky, Sandra Helena Poliselli

    2013-01-01

    Achyrocline satureioides (Lam.) D.C. is a herb native to South America, and its inflorescences are popularly employed to treat inflammatory diseases. Here, the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences of A. satureioides on neutrophil trafficking into inflamed tissue were investigated. Male Wistar rats were orally treated with A. satureioides extract, and inflammation was induced one hour later by lipopolysaccharide injection into the subcutaneous tissue. The number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate, and adhesion molecule and toll-like receptor 4 (TLR-4) expressions and phorbol-myristate-acetate- (PMA-) stimulated oxidative burst were quantified in circulating neutrophils. Leukocyte-endothelial interactions were quantified in the mesentery tissue. Enzymes and tissue morphology of the liver and kidney were evaluated. Treatment with A. satureioides extract reduced neutrophil influx and secretion of leukotriene B4 and CINC-1 in the exudates, the number of rolling and adhered leukocytes in the mesentery postcapillary venules, neutrophil L-selectin, β 2-integrin and TLR-4 expression, and oxidative burst, but did not cause an alteration in the morphology and activities of liver and kidney. Together, the data show that A. satureioides extract inhibits neutrophil functions related to the innate response and does not cause systemic toxicity.

  2. Isolation and Characterization of Neutrophils with Anti-Tumor Properties.

    PubMed

    Sionov, Ronit Vogt; Assi, Simaan; Gershkovitz, Maya; Sagiv, Jitka Y; Polyansky, Lola; Mishalian, Inbal; Fridlender, Zvi G; Granot, Zvi

    2015-01-01

    Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or naïve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a > 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function. PMID:26132785

  3. Pulmonary vascular sequestration of neutrophils in endotoxemia is initiated by an effect of endotoxin on the neutrophil in the rabbit

    SciTech Connect

    Haslett, C.; Worthen, G.S.; Giclas, P.C.; Morrison, D.C.; Henson, J.E.; Henson, P.M.

    1987-07-01

    Endotoxemia causes neutrophil sequestration in the pulmonary vascular bed. Such sequestration may be a critical initiating event in the generation of microvascular injury, although the mechanisms that lead to this localization are not understood. To investigate these phenomena, the following study employed intravenous pulses of /sup 111/Indium-tropolonate-labeled neutrophils (/sup 111/In-neutrophils), which circulated in the rabbit with normal kinetics and responded in a manner indistinguishable from unlabeled, circulating neutrophils in response to an intravenous injection of purified endotoxic lipopolysaccharide (LPS) or epinephrine. Pulmonary sequestration of /sup 111/In-neutrophils was assessed by quantitative external gamma camera scintigraphy of a lung suprahilar region of interest. Noninvasive assessment of radioactivity by this method accurately reflected total lung radioactivity, which was shown by autoradiography to be confined to the injected /sup 111/In-neutrophils. Intravenously administered LPS caused a marked, dose-dependent sequestration of /sup 111/In-neutrophils in the pulmonary vasculature, and exhaustive ultrastructural autoradiography showed discretely radiolabeled neutrophils located within pulmonary capillaries. A distinct effect was seen with an intravenous injection of as little as 100 ng per rabbit (i.e., 500 pg/ml blood). A 5-min ex vivo pretreatment of /sup 111/In-neutrophils with 10 ng to 10 micrograms/ml LPS in heat-inactivated plasma also caused dose-dependent pulmonary sequestration of the pretreated /sup 111/In-neutrophils but did not cause generalized neutropenia in recipient rabbits.

  4. Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: a prospective study.

    PubMed

    Ito, Hiroaki; Inoue, Haruhiro; Kimura, Satoshi; Ohmori, Tohru; Ishikawa, Fumihiro; Gohda, Keigo; Sato, Jun

    2014-07-01

    The identification of circulating tumor cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression and measure treatment effects in several types of malignancies. We have previously used OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. GFP-positive cells (GFP+ cells) were counted under a fluorescence microscope. Our results showed that the number of at least 7.735 µm in diameter GFP+ cells (L-GFP+ cells) in the peripheral blood was a significant marker of prognosis in gastric cancer patients. However, tumor cells undergoing epithelial-mesenchymal transition (EMT) have been reported to be smaller in size than cells without EMT features; thus, CTCs undergoing EMT may escape detection with this technique. Therefore, in this study, we analyzed the relationship between patient outcome and the number of GFP+ cells of any size. We obtained peripheral blood samples from 65 patients with gastric cancer. After infection of OBP-401, GFP+ cells were counted and measured. The relationship between the number of GFP+ cells and surgical outcome was analyzed. The median follow-up period of the surviving patients was 36 months. A significant difference in overall survival was found between patients with 0-5 and patients with ≥6 L-GFP+ cells. No clear relationship was established between the number of small-sized GFP+ cells and patient prognosis. The number of L-GFP+ cells was significantly related to overall survival in patients with gastric cancer. The detection of L-GFP+ cells using OBP-401 may be a useful prognostic marker in gastric cancer.

  5. Two neutrophilic dermatoses captured simultaneously on histology

    PubMed Central

    Wlodek, Christina; Bhatt, Nidhi; Kennedy, Cameron

    2016-01-01

    A number of neutrophilic dermatoses are associated with malignancies and their treatment. These rarely occur together in the same patient. A Caucasian 72-year-old male was treated for acute myeloid leukemia (AML) with chemotherapy including daunorubicin and cytarabine. Within 48 hours of commencing treatment, he developed pyrexia and, two days later, disseminated non-tender pink plaques on the limbs and trunk. A skin biopsy showed a dermal interstitial infiltrate of lymphocytes, histiocytoid cells and predominantly neutrophils. This extended into the subcutis, where a neutrophilic lobular panniculitis was seen. These findings are consistent with Sweet’s syndrome. In addition, a neutrophilic and lymphocytic infiltrate was also present around eccrine coils and lower ducts. The eccrine epithelium showed squamous metaplasia with dyskeratosis and sloughing into the lumen. These latter findings are consistent with neutrophilic eccrine hidradenitis (NEH). These two histologically distinct entities form part of the neutrophilic dermatoses that have been described in oncology patients with reports of concurrent or sequential occurrence of various neutrophilic dermatoses in the same patient. Ours, however, is only the second reported case of simultaneously captured Sweet’s and NEH in the setting of AML. The most likely explanation is that of an epiphenomenon, whereby the neutrophilic infiltrate extended around the sweat glands in the context of the neutrophilic dermatosis. PMID:27648385

  6. Two neutrophilic dermatoses captured simultaneously on histology.

    PubMed

    Wlodek, Christina; Bhatt, Nidhi; Kennedy, Cameron

    2016-07-01

    A number of neutrophilic dermatoses are associated with malignancies and their treatment. These rarely occur together in the same patient. A Caucasian 72-year-old male was treated for acute myeloid leukemia (AML) with chemotherapy including daunorubicin and cytarabine. Within 48 hours of commencing treatment, he developed pyrexia and, two days later, disseminated non-tender pink plaques on the limbs and trunk. A skin biopsy showed a dermal interstitial infiltrate of lymphocytes, histiocytoid cells and predominantly neutrophils. This extended into the subcutis, where a neutrophilic lobular panniculitis was seen. These findings are consistent with Sweet's syndrome. In addition, a neutrophilic and lymphocytic infiltrate was also present around eccrine coils and lower ducts. The eccrine epithelium showed squamous metaplasia with dyskeratosis and sloughing into the lumen. These latter findings are consistent with neutrophilic eccrine hidradenitis (NEH). These two histologically distinct entities form part of the neutrophilic dermatoses that have been described in oncology patients with reports of concurrent or sequential occurrence of various neutrophilic dermatoses in the same patient. Ours, however, is only the second reported case of simultaneously captured Sweet's and NEH in the setting of AML. The most likely explanation is that of an epiphenomenon, whereby the neutrophilic infiltrate extended around the sweat glands in the context of the neutrophilic dermatosis. PMID:27648385

  7. Torsional oscillation of the large-scale circulation in turbulent Rayleigh-Bénard convection at large Rayleigh numbers

    NASA Astrophysics Data System (ADS)

    van Gils, Dennis P. M.; He, Xiaozhou; Ahlers, Guenter; Bodenschatz, Eberhard

    2013-11-01

    We present temperature measurements in turbulent Rayleigh--Bénard convection (RBC) over the Rayleigh number range 3 . 0 ×1013 <= Ra <= 1 . 3 ×1014 and at constant Prandtl number ⪻ ~ 0 . 8 . The RBC sample, known as the High-Pressure Convection Facility (HPCF) of Göttingen, is an upright cylinder of aspect ratio Γ = 1 . 00 . Using three horizontal rows of thermistors at different heights in the sample, we determined the orientation angle of the large-scale circulation (LSC) plane, similar to. Results identify a well established single-roll LSC with a periodic ``torsional'' mode with a frequency fC . The values of fC are consistent with the frequencies fL obtained from power spectra P (f) of temperature time series taken at mid-height of the sample. The non-dimensionalized frequencies f~C are well described by a power law: f~C ~Raζf with ζf = 0 . 427 +/- 0 . 001 . Supported by the Max Planck Society, the Volkswagen Stiftung, the DFD Sonderforschungsbereich SFB963, and NSF grant DMR11-58514.

  8. Distinct Functions of Neutrophil in Cancer and Its Regulation

    PubMed Central

    Granot, Zvi; Jablonska, Jadwiga

    2015-01-01

    Neutrophils are the most abundant of all white blood cells in the human circulation and are usually associated with inflammation and with fighting infections. In recent years the role immune cells play in cancer has been a matter of increasing interest. In this context the function of neutrophils is controversial as neutrophils were shown to possess both tumor promoting and tumor limiting properties. Here we provide an up-to-date review of the pro- and antitumor properties neutrophils possess as well as the environmental cues that regulate these distinct functions. PMID:26648665

  9. Association of microparticles and neutrophil activation with decompression sickness.

    PubMed

    Thom, Stephen R; Bennett, Michael; Banham, Neil D; Chin, Walter; Blake, Denise F; Rosen, Anders; Pollock, Neal W; Madden, Dennis; Barak, Otto; Marroni, Alessandro; Balestra, Costantino; Germonpre, Peter; Pieri, Massimo; Cialoni, Danilo; Le, Phi-Nga Jeannie; Logue, Christopher; Lambert, David; Hardy, Kevin R; Sward, Douglas; Yang, Ming; Bhopale, Veena B; Dujic, Zeljko

    2015-09-01

    Decompression sickness (DCS) is a systemic disorder, assumed due to gas bubbles, but additional factors are likely to play a role. Circulating microparticles (MPs)--vesicular structures with diameters of 0.1-1.0 μm--have been implicated, but data in human divers have been lacking. We hypothesized that the number of blood-borne, Annexin V-positive MPs and neutrophil activation, assessed as surface MPO staining, would differ between self-contained underwater breathing-apparatus divers suffering from DCS vs. asymptomatic divers. Blood was analyzed from 280 divers who had been exposed to maximum depths from 7 to 105 meters; 185 were control/asymptomatic divers, and 90 were diagnosed with DCS. Elevations of MPs and neutrophil activation occurred in all divers but normalized within 24 h in those who were asymptomatic. MPs, bearing the following proteins: CD66b, CD41, CD31, CD142, CD235, and von Willebrand factor, were between 2.4- and 11.7-fold higher in blood from divers with DCS vs. asymptomatic divers, matched for time of sample acquisition, maximum diving depth, and breathing gas. Multiple logistic regression analysis documented significant associations (P < 0.001) between DCS and MPs and for neutrophil MPO staining. Effect estimates were not altered by gender, body mass index, use of nonsteroidal anti-inflammatory agents, or emergency oxygen treatment and were modestly influenced by divers' age, choice of breathing gas during diving, maximum diving depth, and whether repetitive diving had been performed. There were no significant associations between DCS and number of MPs without surface proteins listed above. We conclude that MP production and neutrophil activation exhibit strong associations with DCS.

  10. How neutrophils kill fungi.

    PubMed

    Gazendam, Roel P; van de Geer, Annemarie; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2016-09-01

    Neutrophils play a critical role in the prevention of invasive fungal infections. Whereas mouse studies have demonstrated the role of various neutrophil pathogen recognition receptors (PRRs), signal transduction pathways, and cytotoxicity in the murine antifungal immune response, much less is known about the killing of fungi by human neutrophils. Recently, novel primary immunodeficiencies have been identified in patients with a susceptibility to fungal infections. These human 'knock-out' neutrophils expand our knowledge to understand the role of PRRs and signaling in human fungal killing. From the studies with these patients it is becoming clear that neutrophils employ fundamentally distinct mechanisms to kill Candida albicans or Aspergillus fumigatus. PMID:27558342

  11. How Neutrophils Shape Adaptive Immune Responses

    PubMed Central

    Leliefeld, Pieter H. C.; Koenderman, Leo; Pillay, Janesh

    2015-01-01

    Neutrophils are classically considered as cells pivotal for the first line of defense against invading pathogens. In recent years, evidence has accumulated that they are also important in the orchestration of adaptive immunity. Neutrophils rapidly migrate in high numbers to sites of inflammation (e.g., infection, tissue damage, and cancer) and are subsequently able to migrate to draining lymph nodes (LNs). Both at the site of inflammation as well as in the LNs, neutrophils can engage with lymphocytes and antigen-presenting cells. This crosstalk occurs either directly via cell–cell contact or via mediators, such as proteases, cytokines, and radical oxygen species. In this review, we will discuss the current knowledge regarding locations and mechanisms of interaction between neutrophils and lymphocytes in the context of homeostasis and various pathological conditions. In addition, we will highlight the complexity of the microenvironment that is involved in the generation of suppressive or stimulatory neutrophil phenotypes. PMID:26441976

  12. How Neutrophils Shape Adaptive Immune Responses.

    PubMed

    Leliefeld, Pieter H C; Koenderman, Leo; Pillay, Janesh

    2015-01-01

    Neutrophils are classically considered as cells pivotal for the first line of defense against invading pathogens. In recent years, evidence has accumulated that they are also important in the orchestration of adaptive immunity. Neutrophils rapidly migrate in high numbers to sites of inflammation (e.g., infection, tissue damage, and cancer) and are subsequently able to migrate to draining lymph nodes (LNs). Both at the site of inflammation as well as in the LNs, neutrophils can engage with lymphocytes and antigen-presenting cells. This crosstalk occurs either directly via cell-cell contact or via mediators, such as proteases, cytokines, and radical oxygen species. In this review, we will discuss the current knowledge regarding locations and mechanisms of interaction between neutrophils and lymphocytes in the context of homeostasis and various pathological conditions. In addition, we will highlight the complexity of the microenvironment that is involved in the generation of suppressive or stimulatory neutrophil phenotypes. PMID:26441976

  13. Human neutrophils in auto-immunity.

    PubMed

    Thieblemont, Nathalie; Wright, Helen L; Edwards, Steven W; Witko-Sarsat, Véronique

    2016-04-01

    Human neutrophils have great capacity to cause tissue damage in inflammatory diseases via their inappropriate activation to release reactive oxygen species (ROS), proteases and other tissue-damaging molecules. Furthermore, activated neutrophils can release a wide variety of cytokines and chemokines that can regulate almost every element of the immune system. In addition to these important immuno-regulatory processes, activated neutrophils can also release, expose or generate neoepitopes that have the potential to break immune tolerance and result in the generation of autoantibodies, that characterise a number of human auto-immune diseases. For example, in vasculitis, anti-neutrophil cytoplasmic antibodies (ANCA) that are directed against proteinase 3 or myeloperoxidase are neutrophil-derived autoantigens and activated neutrophils are the main effector cells of vascular damage. In other auto-immune diseases, these neutrophil-derived neoepitopes may arise from a number of processes that include release of granule enzymes and ROS, changes in the properties of components of their plasma membrane as a result of activation or apoptosis, and via the release of Neutrophil Extracellular Traps (NETs). NETs are extracellular structures that contain chromatin that is decorated with granule enzymes (including citrullinated proteins) that can act as neo-epitopes to generate auto-immunity. This review therefore describes the processes that can result in neutrophil-mediated auto-immunity, and the role of neutrophils in the molecular pathologies of auto-immune diseases such as vasculitis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We discuss the potential role of NETs in these processes and some of the debate in the literature regarding the role of this phenomenon in microbial killing, cell death and auto-immunity. PMID:27036091

  14. Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils

    NASA Astrophysics Data System (ADS)

    Wang, Zhenjia; Li, Jing; Cho, Jaehyung; Malik, Asrar B.

    2014-03-01

    Inflammatory diseases such as acute lung injury and ischaemic tissue injury are caused by the adhesion of a type of white blood cell--polymorphonuclear neutrophils--to the lining of the circulatory system or vascular endothelium and unchecked neutrophil transmigration. Nanoparticle-mediated targeting of activated neutrophils on vascular endothelial cells at the site of injury may be a useful means of directly inactivating neutrophil transmigration and hence mitigating vascular inflammation. Here, we report a method employing drug-loaded albumin nanoparticles, which efficiently deliver drugs into neutrophils adherent to the surface of the inflamed endothelium. Using intravital microscopy of tumour necrosis factor-α-challenged mouse cremaster post-capillary venules, we demonstrate that fluorescently tagged albumin nanoparticles are largely internalized by neutrophils adherent to the activated endothelium via cell surface Fcɣ receptors. Administration of albumin nanoparticles loaded with the spleen tyrosine kinase inhibitor, piceatannol, which blocks `outside-in' β2 integrin signalling in leukocytes, detached the adherent neutrophils and elicited their release into the circulation. Thus, internalization of drug-loaded albumin nanoparticles into neutrophils inactivates the pro-inflammatory function of activated neutrophils, thereby offering a promising approach for treating inflammatory diseases resulting from inappropriate neutrophil sequestration and activation.

  15. Anoxia and glucose supplementation preserve neutrophil viability and function.

    PubMed

    Monceaux, Valérie; Chiche-Lapierre, Clarisse; Chaput, Catherine; Witko-Sarsat, Véronique; Prevost, Marie-Christine; Taylor, Cormac T; Ungeheuer, Marie-Noelle; Sansonetti, Philippe J; Marteyn, Benoit S

    2016-08-18

    Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 μg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use. PMID:27402974

  16. Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils.

    PubMed

    Wang, Zhenjia; Li, Jing; Cho, Jaehyung; Malik, Asrar B

    2014-03-01

    Inflammatory diseases such as acute lung injury and ischaemic tissue injury are caused by the adhesion of a type of white blood cell--polymorphonuclear neutrophils--to the lining of the circulatory system or vascular endothelium and unchecked neutrophil transmigration. Nanoparticle-mediated targeting of activated neutrophils on vascular endothelial cells at the site of injury may be a useful means of directly inactivating neutrophil transmigration and hence mitigating vascular inflammation. Here, we report a method employing drug-loaded albumin nanoparticles, which efficiently deliver drugs into neutrophils adherent to the surface of the inflamed endothelium. Using intravital microscopy of tumour necrosis factor-α-challenged mouse cremaster post-capillary venules, we demonstrate that fluorescently tagged albumin nanoparticles are largely internalized by neutrophils adherent to the activated endothelium via cell surface Fcɣ receptors. Administration of albumin nanoparticles loaded with the spleen tyrosine kinase inhibitor, piceatannol, which blocks 'outside-in' β2 integrin signalling in leukocytes, detached the adherent neutrophils and elicited their release into the circulation. Thus, internalization of drug-loaded albumin nanoparticles into neutrophils inactivates the pro-inflammatory function of activated neutrophils, thereby offering a promising approach for treating inflammatory diseases resulting from inappropriate neutrophil sequestration and activation.

  17. Dimethylfumarate Impairs Neutrophil Functions.

    PubMed

    Müller, Susen; Behnen, Martina; Bieber, Katja; Möller, Sonja; Hellberg, Lars; Witte, Mareike; Hänsel, Martin; Zillikens, Detlef; Solbach, Werner; Laskay, Tamás; Ludwig, Ralf J

    2016-01-01

    Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates-in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases. PMID:26763431

  18. [A quantitative analysis of the ultrastructures of the blood polymorphonuclear neutrophils in patients with ischemic heart disease after a session of intravenous laser therapy].

    PubMed

    Khomeriki, S G; Morozov, I A

    1998-01-01

    Circulating neutrophilic granulocytes before and after laser therapy were studied in 10 patients with ischemic heart disease and 5 healthy persons. The patients had severe cytoplasm vacuolization, specific granules number increase, a decrease in thickness of the submembranous actin layer and decrease of surface = volume ratio. Neutrophils indices in patients with ischemic heart disease become closer to those in donor cells after blood irradiation with a helium-neon laser. The results indicate a normalizing effect of helium-neon laser irradiation on the mechanisms of non-specific reactivity in some forms of ischemic heart disease. PMID:9949900

  19. Neutrophils in cancer.

    PubMed

    Treffers, Louise W; Hiemstra, Ida H; Kuijpers, Taco W; van den Berg, Timo K; Matlung, Hanke L

    2016-09-01

    Neutrophils play an important role in cancer. This does not only relate to the well-established prognostic value of the presence of neutrophils, either in the blood or in tumor tissue, in the context of cancer progression or for the monitoring of therapy, but also to their active role in the progression of cancer. In the current review, we describe what is known in general about the role of neutrophils in cancer. What is emerging is a complex, rather heterogeneous picture with both pro- and anti-tumorigenic roles, which apparently differs with cancer type and disease stage. Furthermore, we will discuss the well-known role of neutrophils as myeloid-derived suppressor cells (MDSC), and also on the role of neutrophils as important effector cells during antibody therapy in cancer. It is clear that neutrophils contribute substantially to cancer progression in multiple ways, and this includes both direct effects on the cancer cells and indirect effect on the tumor microenvironment. While in many cases neutrophils have been shown to promote tumor progression, for instance by acting as MDSC, there are also protective effects, particularly when antibody immunotherapy is performed. A better understanding of the role of neutrophils is likely to provide opportunities for immunomodulation and for improving the treatment of cancer patients. PMID:27558343

  20. Myeloperoxidase Stimulates Neutrophil Degranulation.

    PubMed

    Grigorieva, D V; Gorudko, I V; Sokolov, A V; Kostevich, V A; Vasilyev, V B; Cherenkevich, S N; Panasenko, O M

    2016-08-01

    Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation. PMID:27597056

  1. Effects of pentoxifylline on equine neutrophil function and flow properties.

    PubMed Central

    Weiss, D J; Geor, R J; Burris, S M; Smith, C M

    1992-01-01

    Pentoxifylline has been reported to improve peripheral vascular circulation by altering the flow properties of blood. To determine if the hemorrheological effects of pentoxifylline were mediated by alterations in neutrophil function and/or flow properties, we evaluated the drug's effects on equine neutrophils in vitro. Pentoxifylline, at a concentration of 1 x 10(-1) M, but not at concentrations of 1 x 10(-6) M to 1 x 10(-2) M, markedly suppressed neutrophil superoxide production, zymosan phagocytosis and adherence to nylon wool. Pentoxifylline failed to improve neutrophil filterability through 3 mu polycarbonate filters at any concentration tested. We conclude that equine neutrophil function and flow properties are unlikely to be affected by pentoxifylline concentrations achievable in vivo. PMID:1335832

  2. Clearance of apoptotic neutrophils and resolution of inflammation.

    PubMed

    Greenlee-Wacker, Mallary C

    2016-09-01

    The engulfment of apoptotic cells by phagocytes, a process referred to as efferocytosis, is essential for maintenance of normal tissue homeostasis and a prerequisite for the resolution of inflammation. Neutrophils are the predominant circulating white blood cell in humans, and contain an arsenal of toxic substances that kill and degrade microbes. Neutrophils are short-lived and spontaneously die by apoptosis. This review will highlight how the engulfment of apoptotic neutrophils by human phagocytes occurs, how heterogeneity of phagocyte populations influences efferocytosis signaling, and downstream consequences of efferocytosis. The efferocytosis of apoptotic neutrophils by macrophages promotes anti-inflammatory signaling, prevents neutrophil lysis, and dampens immune responses. Given the immunomodulatory properties of efferocytosis, understanding pathways that regulate and enhance efferocytosis could be harnessed to combat infection and chronic inflammatory conditions. PMID:27558346

  3. Major neutrophil functions subverted by Porphyromonas gingivalis.

    PubMed

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil-P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  4. Psychological stress promotes neutrophil infiltration in colon tissue through adrenergic signaling in DSS-induced colitis model.

    PubMed

    Deng, Que; Chen, Hongyu; Liu, Yanjun; Xiao, Fengjun; Guo, Liang; Liu, Dan; Cheng, Xiang; Zhao, Min; Wang, Xiaomeng; Xie, Shuai; Qi, Siyong; Yin, Zhaoyang; Gao, Jiangping; Chen, Xintian; Wang, Jiangong; Guo, Ning; Ma, Yuanfang; Shi, Ming

    2016-10-01

    Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition. Psychological stress has been postulated to affect the clinical symptoms and recurrence of IBD. The exact molecular mechanisms are not fully understood. In the present study, we demonstrate that psychological stress promotes neutrophil infiltration into colon tissues in dextran sulfate sodium (DSS)-induced colitis model. The psychological stress resulted in abnormal expression of the proinflammatory cytokines (IL-1β, IL-6, IL-17A, and IL-22) and neutrophil chemokines (CXCL1 and CXCL2) and overactivation of the STAT3 inflammatory signaling pathway. Under chronic unpredictable stress, the adrenergic nervous system was markedly activated, as the expression of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in bone marrow and colonic epithelium was enhanced, especially in the myenteric ganglia. The β-AR agonist isoproterenol mimicked the effects of psychological stress on neutrophilia, neutrophil infiltration, and colonic damage in DSS-induced colitis. The β1-AR/β2-AR inhibitor propranolol reduced the numbers of the neutrophils in the circulation, suppressed neutrophil infiltration into colonic tissues, and attenuated the colonic tissue damage promoted by chronic stress. Propranolol also abolished stress-induced upregulation of proinflammatory cytokines and neutrophil chemokines. Our data reveal a close linkage between the β1-AR/β2-AR activation and neutrophil trafficking and also suggest the critical roles of adrenergic nervous system in exacerbation of inflammation and damage of colonic tissues in experimental colitis. The current study provides a new insight into the mechanisms underlying the association of psychological stress with excessive inflammatory response and pathophysiological consequences in IBD. The findings also suggest a potential application of neuroprotective agents to prevent relapsing immune activation in the treatment of IBD. PMID

  5. Psychological stress promotes neutrophil infiltration in colon tissue through adrenergic signaling in DSS-induced colitis model.

    PubMed

    Deng, Que; Chen, Hongyu; Liu, Yanjun; Xiao, Fengjun; Guo, Liang; Liu, Dan; Cheng, Xiang; Zhao, Min; Wang, Xiaomeng; Xie, Shuai; Qi, Siyong; Yin, Zhaoyang; Gao, Jiangping; Chen, Xintian; Wang, Jiangong; Guo, Ning; Ma, Yuanfang; Shi, Ming

    2016-10-01

    Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition. Psychological stress has been postulated to affect the clinical symptoms and recurrence of IBD. The exact molecular mechanisms are not fully understood. In the present study, we demonstrate that psychological stress promotes neutrophil infiltration into colon tissues in dextran sulfate sodium (DSS)-induced colitis model. The psychological stress resulted in abnormal expression of the proinflammatory cytokines (IL-1β, IL-6, IL-17A, and IL-22) and neutrophil chemokines (CXCL1 and CXCL2) and overactivation of the STAT3 inflammatory signaling pathway. Under chronic unpredictable stress, the adrenergic nervous system was markedly activated, as the expression of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in bone marrow and colonic epithelium was enhanced, especially in the myenteric ganglia. The β-AR agonist isoproterenol mimicked the effects of psychological stress on neutrophilia, neutrophil infiltration, and colonic damage in DSS-induced colitis. The β1-AR/β2-AR inhibitor propranolol reduced the numbers of the neutrophils in the circulation, suppressed neutrophil infiltration into colonic tissues, and attenuated the colonic tissue damage promoted by chronic stress. Propranolol also abolished stress-induced upregulation of proinflammatory cytokines and neutrophil chemokines. Our data reveal a close linkage between the β1-AR/β2-AR activation and neutrophil trafficking and also suggest the critical roles of adrenergic nervous system in exacerbation of inflammation and damage of colonic tissues in experimental colitis. The current study provides a new insight into the mechanisms underlying the association of psychological stress with excessive inflammatory response and pathophysiological consequences in IBD. The findings also suggest a potential application of neuroprotective agents to prevent relapsing immune activation in the treatment of IBD.

  6. Sitagliptin, a dipeptidyl peptidase-4 inhibitor, increases the number of circulating CD34⁺CXCR4⁺ cells in patients with type 2 diabetes.

    PubMed

    Aso, Yoshimasa; Jojima, T; Iijima, T; Suzuki, K; Terasawa, T; Fukushima, M; Momobayashi, A; Hara, K; Takebayashi, K; Kasai, K; Inukai, T

    2015-12-01

    We investigated the effects of sitagliptin, a dipeptidyl peptidase (DPP)-4 inhibitor, on the number of circulating CD34(+)CXCR4(+)cells, a candidate for endothelial progenitor cells (EPCs), plasma levels of stromal cell-derived factor (SDF)-1α, a ligand for CXCR4 receptor and a substrate for DPP-4, and plasma levels of interferon-inducible protein (IP)-10, for a substrate for DPP-4, in patients with type 2 diabetes. We studied 30 consecutive patients with type 2 diabetes who had poor glycemic control despite treatment with metformin and/or sulfonylurea. Thirty diabetic patients were randomized in a 2:1 ratio into a sitagliptin (50 mg/day) treatment group or an active placebo group (glimepiride 1 mg/day) for 12 weeks. Both groups showed similar improvements in glycemic control. The number of circulating CD34(+)CXCR4(+) cells was increased from 30.5 (20.0, 47.0)/10(6) cells at baseline to 55.5 (31.5, 80.5)/10(6) cells at 12 weeks of treatment with 50 mg/day sitagliptin (P = 0.0014), while showing no significant changes in patients treated with glimepiride. Plasma levels of SDF-1α and IP-10, both physiological substrates of endogenous DPP-4 and chemokines, were significantly decreased at 12 weeks of sitagliptin treatment. In conclusion, treatment with sitagliptin increased the number of circulating CD34(+)CXCR4(+) cells by approximately 2-fold in patients with type 2 diabetes.

  7. Sitagliptin, a dipeptidyl peptidase-4 inhibitor, increases the number of circulating CD34⁺CXCR4⁺ cells in patients with type 2 diabetes.

    PubMed

    Aso, Yoshimasa; Jojima, T; Iijima, T; Suzuki, K; Terasawa, T; Fukushima, M; Momobayashi, A; Hara, K; Takebayashi, K; Kasai, K; Inukai, T

    2015-12-01

    We investigated the effects of sitagliptin, a dipeptidyl peptidase (DPP)-4 inhibitor, on the number of circulating CD34(+)CXCR4(+)cells, a candidate for endothelial progenitor cells (EPCs), plasma levels of stromal cell-derived factor (SDF)-1α, a ligand for CXCR4 receptor and a substrate for DPP-4, and plasma levels of interferon-inducible protein (IP)-10, for a substrate for DPP-4, in patients with type 2 diabetes. We studied 30 consecutive patients with type 2 diabetes who had poor glycemic control despite treatment with metformin and/or sulfonylurea. Thirty diabetic patients were randomized in a 2:1 ratio into a sitagliptin (50 mg/day) treatment group or an active placebo group (glimepiride 1 mg/day) for 12 weeks. Both groups showed similar improvements in glycemic control. The number of circulating CD34(+)CXCR4(+) cells was increased from 30.5 (20.0, 47.0)/10(6) cells at baseline to 55.5 (31.5, 80.5)/10(6) cells at 12 weeks of treatment with 50 mg/day sitagliptin (P = 0.0014), while showing no significant changes in patients treated with glimepiride. Plasma levels of SDF-1α and IP-10, both physiological substrates of endogenous DPP-4 and chemokines, were significantly decreased at 12 weeks of sitagliptin treatment. In conclusion, treatment with sitagliptin increased the number of circulating CD34(+)CXCR4(+) cells by approximately 2-fold in patients with type 2 diabetes. PMID:26209038

  8. Enhancements to the FAST-MAC Circulation Control Model and Recent High-Reynolds Number Testing in the National Transonic Facility

    NASA Technical Reports Server (NTRS)

    Milholen, William E., II; Jones, Gregory S.; Chan, David T.; Goodliff, Scott L.; Anders, Scott G.; Melton, Latunia P.; Carter, Melissa B.; Allan, Brian G.; Capone, Francis J.

    2013-01-01

    A second wind tunnel test of the FAST-MAC circulation control model was recently completed in the National Transonic Facility at the NASA Langley Research Center. The model was equipped with four onboard flow control valves allowing independent control of the circulation control plenums, which were directed over a 15% chord simple-hinged flap. The model was configured for low-speed high-lift testing with flap deflections of 30 and 60 degrees, along with the transonic cruise configuration with zero degree flap deflection. Testing was again conducted over a wide range of Mach numbers up to 0.88, and Reynolds numbers up to 30 million based on the mean chord. The first wind tunnel test had poor transonic force and moment data repeatability at mild cryogenic conditions due to inadequate thermal conditioning of the balance. The second test demonstrated that an improvement to the balance heating system significantly improved the transonic data repeatability, but also indicated further improvements are still needed. The low-speed highlift performance of the model was improved by testing various blowing slot heights, and the circulation control was again demonstrated to be effective in re-attaching the flow over the wing at off-design transonic conditions. A new tailored spanwise blowing technique was also demonstrated to be effective at transonic conditions with the benefit of reduced mass flow requirements.

  9. The lymph node neutrophil.

    PubMed

    Hampton, Henry R; Chtanova, Tatyana

    2016-04-01

    Secondary lymphoid organs provide a specialized microenvironment tailored to foster communication between cells of the innate and adaptive immune systems. These interactions allow immune cells to coordinate multilayered defense against pathogens. Until recently dendritic cells and macrophages were thought to comprise the main innate immune cell subsets responsible for delivering signals that drive the adaptive immune response, while the function of neutrophils was largely confined to the innate immune system. However, the discovery of neutrophils in lymph nodes has raised the question of whether neutrophils might play a more extensive role not only in innate immunity per se, but also in coordinating the interactions between innate and adaptive immune responses. In this review we discuss the mechanisms and consequences of neutrophil recruitment to lymph nodes and how this recruitment influences subsequent immune responses both in situ and at distant sites. PMID:27025975

  10. Circulating plasmacytoid dendritic cells in acutely infected patients with hepatitis C virus genotype 4 are normal in number and phenotype.

    PubMed

    Mansour, Hala; Laird, Melissa E; Saleh, Rasha; Casrouge, Armanda; Eldin, Noha Sharaf; El Kafrawy, Sherif; Hamdy, Maha; Decalf, Jérémie; Rosenberg, Brad R; Fontanet, Arnaud; Abdel-Hamid, Mohammed; Mohamed, Mostafa K; Albert, Matthew L; Rafik, Mona

    2010-12-01

    The incidence of hepatitis C virus (HCV) genotype 4 infection in Egypt provides a unique opportunity to study the innate immune response to symptomatic acute HCV infection. We investigated whether plasmacytoid dendritic cells (pDCs) are activated as a result of HCV infection. We demonstrate that, even during symptomatic acute infection, circulating pDCs maintained a similar precursor frequency and resting phenotype, compared with pDCs in healthy individuals. Moreover, stimulation with a Toll-like receptor 9 agonist resulted in an intact inflammatory response. These data support the growing consensus that pDCs are not directly activated by HCV and therefore are viable targets for immunotherapy throughout HCV infection.

  11. Sexual dimorphism in neuronal number of the posterodorsal medial amygdala is independent of circulating androgens and regional volume in adult rats.

    PubMed

    Morris, John A; Jordan, Cynthia L; Breedlove, S Marc

    2008-02-10

    The posterodorsal medial amygdala (MePD) in rodents integrates olfactory and pheromonal information, which, coupled with the appropriate hormonal signals, may facilitate or repress reproductive behavior in adulthood. MePD volume and neuronal soma size are greater in male rats than in females, and these sexual dimorphisms are maintained by adult circulating hormone levels. Castration of adult males causes these measures to shrink to the size seen in females 4 weeks later, whereas testosterone treatment of adult females for 4 weeks enlarges these measures to the size of males. We used stereological methods to count the number of cells in the MePD and found that, in addition to the sex difference in regional volume and soma size, males also have more MePD neurons than do females, yet these numbers are unaffected by the presence or absence of androgen in adults of either sex. Males also have more glial cells than do females, but, in contrast to the effects on neuronal number, the number of glial cells is affected by androgen in the right MePD of both sexes and, therefore, may contribute to regional volume changes in adulthood in that hemisphere. Thus, regional volume, neuronal size, and glial numbers vary in the MePD of adult rats in response to circulating androgens, but neuronal number does not. These results suggest that the sex difference in neuronal number in the rat MePD may be "organized" by androgens prior to adulthood, whereas regional volume, neuronal size, and glial numbers can be altered by androgens in adulthood. PMID:18076082

  12. Neutrophil depletion delays wound repair in aged mice

    PubMed Central

    Nishio, Naomi; Okawa, Yayoi; Sakurai, Hidetoshi

    2008-01-01

    One of the most important clinical problems in caring for elderly patients is treatment of pressure ulcers. One component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages and lymphocytes. Neutrophils migrate early in the wound healing process. In aged C57BL/6 mice, wound healing is relatively inefficient. We examined the effects of neutrophil numbers on wound healing in both young and aged mice. We found that the depletion of neutrophils by anti-Gr-1 antibody dramatically delayed wound healing in aged mice. The depletion of neutrophils in young mice had less effect on the kinetics of wound healing. Intravenous G-CSF injection increased the migration of neutrophils to the wound site. While the rate of wound repair did not change significantly in young mice following G-CSF injection, it increased significantly in old mice. PMID:19424869

  13. Priming of the neutrophil respiratory burst: role in host defense and inflammation.

    PubMed

    El-Benna, Jamel; Hurtado-Nedelec, Margarita; Marzaioli, Viviana; Marie, Jean-Claude; Gougerot-Pocidalo, Marie-Anne; Dang, Pham My-Chan

    2016-09-01

    Neutrophils are the major circulating white blood cells in humans. They play an essential role in host defense against pathogens. In healthy individuals, circulating neutrophils are in a dormant state with very low efficiency of capture and arrest on the quiescent endothelium. Upon infection and subsequent release of pro-inflammatory mediators, the vascular endothelium signals to circulating neutrophils to roll, adhere, and cross the endothelial barrier. Neutrophils migrate toward the infection site along a gradient of chemo-attractants, then recognize and engulf the pathogen. To kill this pathogen entrapped inside the vacuole, neutrophils produce and release high quantities of antibacterial peptides, proteases, and reactive oxygen species (ROS). The robust ROS production is also called 'the respiratory burst', and the NADPH oxidase or NOX2 is the enzyme responsible for the production of superoxide anion, leading to other ROS. In vitro, several soluble and particulate agonists induce neutrophil ROS production. This process can be enhanced by prior neutrophil treatment with 'priming' agents, which alone do not induce a respiratory burst. In this review, we will describe the priming process and discuss the beneficial role of controlled neutrophil priming in host defense and the detrimental effect of excessive neutrophil priming in inflammatory diseases. PMID:27558335

  14. Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira

    PubMed Central

    Scharrig, Emilia; Carestia, Agostina; Ferrer, María F.; Cédola, Maia; Pretre, Gabriela; Drut, Ricardo; Picardeau, Mathieu; Schattner, Mirta; Gómez, Ricardo M.

    2015-01-01

    NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden. PMID:26161745

  15. P-selectin promotes neutrophil extracellular trap formation in mice.

    PubMed

    Etulain, Julia; Martinod, Kimberly; Wong, Siu Ling; Cifuni, Stephen M; Schattner, Mirta; Wagner, Denisa D

    2015-07-01

    Neutrophil extracellular traps (NETs) can be released in the vasculature. In addition to trapping microbes, they promote inflammatory and thrombotic diseases. Considering that P-selectin induces prothrombotic and proinflammatory signaling, we studied the role of this selectin in NET formation. NET formation (NETosis) was induced by thrombin-activated platelets rosetting with neutrophils and was inhibited by anti-P-selectin aptamer or anti-P-selectin glycoprotein ligand-1 (PSGL-1) inhibitory antibody but was not induced by platelets from P-selectin(-/-) mice. Moreover, NETosis was also promoted by P-selectin-immunoglobulin fusion protein but not by control immunoglobulin. We isolated neutrophils from mice engineered to overproduce soluble P-selectin (P-selectin(ΔCT/ΔCT) mice). Although the levels of circulating DNA and nucleosomes (indicative of spontaneous NETosis) were normal in these mice, basal neutrophil histone citrullination and presence of P-selectin on circulating neutrophils were elevated. NET formation after stimulation with platelet activating factor, ionomycin, or phorbol 12-myristate 13-acetate was significantly enhanced, indicating that the P-selectin(ΔCT/ΔCT) neutrophils were primed for NETosis. In summary, P-selectin, cellular or soluble, through binding to PSGL-1, promotes NETosis, suggesting that this pathway is a potential therapeutic target for NET-related diseases.

  16. Neutrophil swarming: an essential process of the neutrophil tissue response.

    PubMed

    Kienle, Korbinian; Lämmermann, Tim

    2016-09-01

    Neutrophil infiltration into inflamed and infected tissues is a fundamental process of the innate immune response. While neutrophil interactions with the blood vessel wall have been intensely studied over the last decades, neutrophil dynamics beyond the vasculature have for a long time remained poorly investigated. Recent intravital microscopy studies of neutrophil populations directly at the site of tissue damage or microbial invasion have changed our perspective on neutrophil responses within tissues. Swarm-like migration patterns of neutrophils, referred to as 'neutrophil swarming', have been detected in diverse tissues under conditions of sterile inflammation and infection with various pathogens, including bacteria, fungi, and parasites. Current work has begun to unravel the molecular pathways choreographing the sequential phases of highly coordinated chemotaxis followed by neutrophil accumulation and the formation of substantial neutrophil clusters. It is now clear that intercellular communication among neutrophils amplifies their recruitment in a feed-forward manner, which provides them with a level of self-organization during neutrophil swarming. This review will summarize recent developments and current concepts on neutrophil swarming, an important process of the neutrophil tissue response with a critical role in maintaining the balance between host protection and inflammation-driven tissue destruction. PMID:27558329

  17. The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice.

    PubMed

    Deshmukh, Hitesh S; Liu, Yuhong; Menkiti, Ogechukwu R; Mei, Junjie; Dai, Ning; O'Leary, Claire E; Oliver, Paula M; Kolls, Jay K; Weiser, Jeffrey N; Worthen, G Scott

    2014-05-01

    Neonatal colonization by microbes, which begins immediately after birth, is influenced by gestational age and the mother's microbiota and is modified by exposure to antibiotics. In neonates, prolonged duration of antibiotic therapy is associated with increased risk of late-onset sepsis (LOS), a disorder controlled by neutrophils. A role for the microbiota in regulating neutrophil development and susceptibility to sepsis in the neonate remains unclear. We exposed pregnant mouse dams to antibiotics in drinking water to limit transfer of maternal microbes to the neonates. Antibiotic exposure of dams decreased the total number and composition of microbes in the intestine of the neonates. This was associated with decreased numbers of circulating and bone marrow neutrophils and granulocyte/macrophage-restricted progenitor cells in the bone marrow of antibiotic-treated and germ-free neonates. Antibiotic exposure of dams reduced the number of interleukin-17 (IL-17)-producing cells in the intestine and production of granulocyte colony-stimulating factor (G-CSF). Granulocytopenia was associated with impaired host defense and increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in antibiotic-treated neonates, which could be partially reversed by administration of G-CSF. Transfer of a normal microbiota into antibiotic-treated neonates induced IL-17 production by group 3 innate lymphoid cells (ILCs) in the intestine, increasing plasma G-CSF levels and neutrophil numbers in a Toll-like receptor 4 (TLR4)- and myeloid differentiation factor 88 (MyD88)-dependent manner and restored IL-17-dependent resistance to sepsis. Specific depletion of ILCs prevented IL-17- and G-CSF-dependent granulocytosis and resistance to sepsis. These data support a role for the intestinal microbiota in regulation of granulocytosis, neutrophil homeostasis and host resistance to sepsis in neonates.

  18. Neutrophils scan for activated platelets to initiate inflammation

    PubMed Central

    Sreeramkumar, Vinatha; Adrover, José M.; Ballesteros, Ivan; Cuartero, Maria Isabel; Rossaint, Jan; Bilbao, Izaskun; Nácher, Maria; Pitaval, Christophe; Radovanovic, Irena; Fukui, Yoshinori; McEver, Rodger P.; Filippi, Marie-Dominique; Lizasoain, Ignacio; Ruiz-Cabello, Jesús; Zarbock, Alexander; Moro, María A.; Hidalgo, Andrés

    2014-01-01

    Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thrombo-inflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and suggest that their bipolarity allows integration of signals present at both the endothelium and the circulation before inflammation proceeds. PMID:25477463

  19. Fatty acids as modulators of neutrophil recruitment, function and survival.

    PubMed

    Rodrigues, Hosana G; Takeo Sato, Fabio; Curi, Rui; Vinolo, Marco A R

    2016-08-15

    Neutrophils are well-known to act in the destruction of invading microorganisms. They have also been implicated in the activation of other immune cells including B- and T-lymphocytes and in the resolution of inflammation and tissue regeneration. Neutrophils are produced in the bone marrow and released into the circulation from where they migrate to tissues to perform their effector functions. Neutrophils are in constant contact with fatty acids that can modulate their function, activation and fate (survival or cell death) through different mechanisms. In this review, the effects of fatty acids pertaining to five classes, namely, long-chain saturated fatty acids (LCSFAs), short-chain fatty acids (SCFAs), and omega-3 (n-3), omega-6 (n-6) and omega-9 (n-9) unsaturated fatty acids, on neutrophils and the relevance of these effects for disease development are discussed.

  20. Lower numbers of circulating Natural Killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV-1) associated neurological disease.

    PubMed

    Ndhlovu, L C; Snyder-Cappione, J E; Carvalho, K I; Leal, F E; Loo, C P; Bruno, F R; Jha, A R; Devita, D; Hasenkrug, A M; Barbosa, H M R; Segurado, A C; Nixon, D F; Murphy, E L; Kallas, E G

    2009-12-01

    Human T lymphotropic virus type 1 (HTLV-1) infects 10-20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV-1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: São Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4(+) and fewer CD8(+) cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4(+) NK T subset are associated with HTLV-1 disease progression.

  1. LES of High-Reynolds-Number Coanda Flow Separating from a Rounded Trailing Edge of a Circulation Control Airfoil

    NASA Technical Reports Server (NTRS)

    Nichino, Takafumi; Hahn, Seonghyeon; Shariff, Karim

    2010-01-01

    This slide presentation reviews the Large Eddy Simulation of a high reynolds number Coanda flow that is separated from a round trailing edge of a ciruclation control airfoil. The objectives of the study are: (1) To investigate detailed physics (flow structures and statistics) of the fully turbulent Coanda jet applied to a CC airfoil, by using LES (2) To compare LES and RANS results to figure out how to improve the performance of existing RANS models for this type of flow.

  2. Neutrophil secondary necrosis is induced by LL-37 derived from cathelicidin.

    PubMed

    Zhang, Zhifang; Cherryholmes, Gregory; Shively, John E

    2008-09-01

    Neutrophils represent the most common granulocyte subtype present in blood. The short half-life of circulating neutrophils is regulated by spontaneous apoptosis, and tissue infiltrating neutrophils die by apoptosis and secondary necrosis. The mechanism of neutrophil apoptosis has been the subject of many studies; however, the mechanism of neutrophil secondary necrosis is less clear. Human cathelicidin cationic peptide 18, proteolytically processed to its active form, LL-37, is secreted by neutrophils and epithelial cells and shown to have effects in addition to bacterial lysis. We demonstrate here that LL-37 affects neutrophil lifespan by the pathway of secondary necrosis, rapidly converting annexin V-positive (AV(+)), propidium iodide-negative (PI(-); apoptotic) cells into PI(+) (necrotic) cells with the release of IL-8, IL-1R antagonist, ATP, and intact granules. The effects of LL-37 on apoptotic neutrophils are neither energy-dependent nor affected by pretreatment with G-CSF, GM-CSF, TNF-alpha, and LPS and are partially inhibited by human serum. Moreover, LL-37 decreases CXCR2 expression of AV(-)PI(-) (live) neutrophils, suggesting an effect on the neutrophil response to its chemotactic factors, including IL-8. Thus, the lifespan and inflammatory functions of neutrophils are directly affected by LL-37.

  3. Platelets enhance neutrophil transendothelial migration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Platelets are increasingly recognized as important mediators of inflammation in addition to thrombosis. While platelets have been shown to promote neutrophil (PMN) adhesion to endothelium in various inflammatory models, it is unclear whether platelets enhance neutrophil transmigration across inflame...

  4. Major neutrophil functions subverted by Porphyromonas gingivalis

    PubMed Central

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil–P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  5. The Neutrophil Nucleus and Its Role in Neutrophilic Function.

    PubMed

    Carvalho, Leonardo Olivieri; Aquino, Elaine Nascimento; Neves, Anne Caroline Dias; Fontes, Wagner

    2015-09-01

    The cell nucleus plays a key role in differentiation processes in eukaryotic cells. It is not the nucleus in particular, but the organization of the genes and their remodeling that provides the data for the adjustments to be made according to the medium. The neutrophil nucleus has a different morphology. It is a multi-lobed nucleus where some researchers argue no longer function. However, studies indicate that it is very probable the occurrence of chromatin remodeling during activation steps. It may be that the human neutrophil nucleus also contributes to the mobility of neutrophils through thin tissue spaces. Questions like these will be discussed in this small review. The topics include morphology of human neutrophil nucleus, maturation process and modifications of the neutrophil nucleus, neutrophil activation and chromatin modifications, causes and consequences of multi-lobulated segmented morphology, and importance of the nucleus in the formation of neutrophil extracellular traps (NETs).

  6. Retrograde rotation of the large-scale circulation in turbulent rotating Rayleigh-Benard convection at large Rossby numbers up to 200

    NASA Astrophysics Data System (ADS)

    Li, Hui-Min; Zhong, Jin-Qiang

    2014-11-01

    We examine the azimuthal rotation of the large-scale circulation (LSC) for turbulent Rayleigh-Benard convection in the present of week rotations about a vertical axis at angular velocities 1 . 0 ×10-3 <= Ω <= 0 . 1 (rad/s). Over the entire Rossby-number range 1 <= Ro <= 200 studied, linear retrograde rotations of the LSC circulating plane are observed. With increasing Ro (~ 1 / Ω) the retrograde rotating velocity < - θ˙ > decreases monotonically, but the ratio γ = < - θ˙ > / Ω experiences a transition at Ro* ~ 80 above which γ increases sharply. We discuss the Ro -dependence of γ for Ro >Ro* and show that a maximum ratio γmax = 0 . 36 is observed at Ro = 200 , more than twice larger than other results reported before in a lower-Ro regime. The experimental findings may shed new light to interpret the low precession rate under weak Coriolis force within the framework of the LSC models. Supported by NSFC Grant 11202151.

  7. Exogenous melatonin inhibits neutrophil migration through suppression of ERK activation.

    PubMed

    Ren, Da-Long; Sun, Ai-Ai; Li, Ya-Juan; Chen, Min; Ge, Shu-Chao; Hu, Bing

    2015-10-01

    Neutrophil migration to inflammatory sites is the fundamental process of innate immunity among organisms against pathogen invasion. As a major sleep adjusting hormone, melatonin has also been proved to be involved in various inflammatory events. This study aimed to evaluate the impact of exogenous melatonin on neutrophil migration to the injury site in live zebrafish and further investigate whether ERK signaling is involved in this process. Using the tail fin transection model, the fluorescently labeled neutrophil was in vivo visualized in transgenic Tg(lyz:EGFP), Tg(lyz:DsRed) zebrafish. We found that exogenous melatonin administration dramatically inhibited the injury-induced neutrophil migration in a dose-dependent and time-dependent manner. The inhibited effect of melatonin on neutrophil migration could be attenuated by melatonin receptor 1, 2, and 3 antagonists. The ERK phosphorylation level was significantly decreased post injury when treated with melatonin. The blocking of ERK activation with inhibitor PD0325901 suppressed the number of migrated neutrophils in response to injury. However, the activation of ERK with the epidermal growth factor could impair the inhibited effect of melatonin on neutrophil migration. We also detected that PD0325901 significantly suppressed the in vivo neutrophils transmigrating over the vessel endothelial cell using the transgenic Tg(flk:EGFP);(lyz:DsRed) line labeled as both vessel and neutrophil. Taking all of these data together, the results indicated that exogenous melatonin had an anti-migratory effect on neutrophils by blocking the ERK phosphorylation signal, and it led to the subsequent adhesion molecule expression. Thus, the crossing of the vessel endothelial cells of neutrophils became difficult.

  8. Lack of anti-tumour reactivity despite enhanced numbers of circulating natural killer T cells in two patients with metastatic renal cell carcinoma.

    PubMed

    Vyth-Dreese, F A; Sein, J; van de Kasteele, W; Dellemijn, T A M; van den Bogaard, C; Nooijen, W J; de Gast, G C; Haanen, J B A G; Bex, A

    2010-12-01

    Natural killer T (NK T) cells play a central role as intermediates between innate and adaptive immune responses important to induce anti-tumour reactivity in cancer patients. In two of 14 renal cell carcinoma (RCC) patients, treated with interferon (IFN)-α, we detected significantly enhanced numbers of circulating NK T cells which were typed phenotypically and analysed for anti-tumour reactivity. These NK T cells were T cell receptor (TCR) Vα24/Vβ11(+), 6B11(+) and bound CD1d tetramers. No correlation was observed between NK T frequencies and regulatory T cells (T(regs)), which were also enhanced. NK T cells expressed CD56, CD161, CD45RO and CD69 and were predominantly CD8(+), in contrast to the circulating T cell pool that contained both CD4(+) and CD8(+) T cells, as is found in healthy individuals. It is unlikely that IFN-α triggered the high NK T frequency, as all other patients expressed low to normal NK T numbers. A parallel was observed in IFN-α-related increase in activation of NK T cells with that in conventional T and non-T cells. Normal interleukin (IL)-7, IL-12 and IL-15 plasma levels were found. In one of the patients sporadic NK T cells were detected at the tumour site. α-Galactosylceramide (αGalCer) stimulation of peripheral blood mononuclear cells or isolated NK T cell lines from both patients induced IFN-γ, but no IL-4 and no response towards autologous tumour cells or lysates. The clinical course of disease in both patients was not exceptional with regard to histological subtype and extent of metastatic disease. Therefore, despite a constitutive high peripheral frequency and in vitroαGalCer responsiveness, the NK T cells in the two RCC patients did not show anti-tumour responsiveness.

  9. Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

    PubMed

    Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

    2015-05-01

    Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production.

  10. Reduced CD5+CD24hiCD38hi and interleukin-10+ regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies

    PubMed Central

    Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

    2015-01-01

    Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs were evaluated in addition to their CD5+ subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine–phosphate–guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5+CD24hiCD38hi B cells and IL-10+ B cells compared to patients in remission and healthy controls (HCs). As IL-10+ and CD5+CD24hiCD38hi B cells normalized in remission within an individual, ANCA titres decreased. The CD5+ subset of CD24hiCD38hi B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5+ B cells are enriched in the ability to produce IL-10 compared to CD5neg B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs. The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production. PMID:25376552

  11. Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

    PubMed

    Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

    2015-05-01

    Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production. PMID:25376552

  12. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

    PubMed Central

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil. PMID:27034964

  13. In vivo induction of neutrophilia, lymphopenia, and diminution of neutrophil adhesion by stable analogs of prostaglandins E1, E2, and F2 alpha.

    PubMed Central

    Ulich, T. R.; Dakay, E. B.; Williams, J. H.; Ni, R. X.

    1986-01-01

    Stable analogs of prostaglandins E1, E2, and F2 alpha (M-PGE1, DM-PGE2, and M-PGF2 alpha) were found to induce marked changes in circulating white blood cell subsets in Brown-Norway rats after subcutaneous injection. Dose-response studies demonstrated that 1000 micrograms/kg of each prostaglandin induced a maximum neutrophilia in the range of 40-70% of the total white blood cell count (normal, 5-20%) and that as little as 5 micrograms/kg of M-PGE1 induced a significant neutrophilia (P less than 0.05). Kinetic studies demonstrated that the maximum neutrophilia occurred 4-6 hours after injection of each prostaglandin and was not accompanied by the release of morphologically immature neutrophil forms from the bone marrow. Splenectomy slightly diminished the average neutrophilia at 2 hours but not at 4-6 hours after injection, which suggests that release of neutrophils from the spleen partially contributed to the early neutrophilia. Adherence experiments employing whole heparinized blood from rats given prostaglandins 6 hours prior to sacrifice demonstrated that neutrophils exposed to prostaglandins in vivo have diminished adherence to nylon wool columns, which suggests that diminished adherence of the marginated neutrophil pool may contribute to the neutrophilia. The prostaglandin-induced neutrophilia was accompanied not by a significant change in total numbers of circulating white blood cells, but, rather, by a significant decrease in circulating mononuclear white blood cells, including T-helper, T-suppressor, and B cells. The combination of neutrophilia with lymphopenia has classically been attributed to the release of adrenal hormones and suggests 1) that prostaglandins may directly or indirectly cause the release of adrenal hormones, or 2) that adrenal hormones may mediate their effects on circulating white blood cell subsets via prostaglandins, or 3) that prostaglandins activate intracellular messenger systems that are also activated by adrenal hormones. PMID

  14. Characterization of Neutrophil Function in Human Cutaneous Leishmaniasis Caused by Leishmania braziliensis.

    PubMed

    Conceição, Jacilara; Davis, Richard; Carneiro, Pedro Paulo; Giudice, Angela; Muniz, Aline C; Wilson, Mary E; Carvalho, Edgar M; Bacellar, Olívia

    2016-05-01

    Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects.

  15. Characterization of Neutrophil Function in Human Cutaneous Leishmaniasis Caused by Leishmania braziliensis

    PubMed Central

    Conceição, Jacilara; Davis, Richard; Carneiro, Pedro Paulo; Giudice, Angela; Muniz, Aline C.; Wilson, Mary E.; Carvalho, Edgar M.; Bacellar, Olívia

    2016-01-01

    Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects. PMID:27167379

  16. Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus.

    PubMed

    Finsterbusch, Michaela; Hall, Pam; Li, Anqi; Devi, Sapna; Westhorpe, Clare L V; Kitching, A Richard; Hickey, Michael J

    2016-08-30

    Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclear. Here, we examined the contributions of monocytes to antibody- and neutrophil-dependent inflammation in a model of in situ immune complex-mediated glomerulonephritis. Multiphoton and spinning disk confocal intravital microscopy revealed that monocytes patrol both uninflamed and inflamed glomeruli using β2 and α4 integrins and CX3CR1. Monocyte depletion reduced glomerular injury, demonstrating that these cells promote inappropriate inflammation in this setting. Monocyte depletion also resulted in reductions in neutrophil recruitment and dwell time in glomerular capillaries and in reactive oxygen species (ROS) generation by neutrophils, suggesting a role for cross-talk between monocytes and neutrophils in induction of glomerulonephritis. Consistent with this hypothesis, patrolling monocytes and neutrophils underwent prolonged interactions in glomerular capillaries, with the duration of these interactions increasing during inflammation. Moreover, neutrophils that interacted with monocytes showed increased retention and a greater propensity for ROS generation in the glomerulus. Also, renal patrolling monocytes, but not neutrophils, produced TNF during inflammation, and TNF inhibition reduced neutrophil dwell time and ROS production, as well as renal injury. These findings show that monocytes and neutrophils undergo interactions within the glomerular microvasculature. Moreover, evidence indicates that, in response to an inflammatory stimulus, these interactions allow monocytes to promote neutrophil recruitment and activation within the glomerular microvasculature, leading to neutrophil

  17. Changes in Neutrophil Functions in Astronauts

    NASA Technical Reports Server (NTRS)

    Kaur, Indreshpal; Simons, Elizabeth R.; Castro, Victoria; Pierson, Duane L.

    2002-01-01

    Neutrophil functions (phagocytosis, oxidative burst, degranulation) and expression of surface markers involved in these functions were studied in 25 astronauts before and after 4 space shuttle missions. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch (preflight or L-10), immediately after landing (landing or R+0), and again at 3 days after landing (postflight or R+3). Blood samples were also collected from 9 healthy low-stressed subjects at 3 time points simulating a 10-day shuttle mission. The number of neutrophils increased at landing by 85 percent when compared to the preflight numbers. Neutrophil functions were studied in whole blood using flow cytometric methods. Phagocytosis of E.coli-FITC and oxidative burst capacity of the neutrophils following the 9 to 11 day missions were lower at all three sampling points than the mean values for control subjects. Phagocytosis and oxidative burst capacity of the astronauts was decreased even 10-days before space flight. Mission duration appears to be a factor in phagocytic and oxidative functions. In contrast, following the short-duration (5-days) mission, these functions were unchanged from control values. No consistent changes in degranulation were observed following either short or medium length space missions. The expression of CD16, CD32, CD11a, CD11b, CD11c, L-selectin and CD36 was measured and found to be variable. Specifically, CD16 and CD32 did not correlate with the changes in oxidative burst and phagocytosis. We can conclude from this study that the stresses associated with space flight can alter the important functions of neutrophils.

  18. Sequential morphological and quantitative changes in blood and bone marrow neutrophils in dogs with acute inflammation.

    PubMed Central

    Gossett, K A; MacWilliams, P S; Cleghorn, B

    1985-01-01

    Blood and bone marrow morphology were studied sequentially in dogs during experimental inflammation induced by intramuscular injection of turpentine. Depletion of the bone marrow storage pool of mature neutrophils and an increase in mitotic activity and number of early granulocyte precursors were evident within 24 hours. During the next three days, intense granulocytic hyperplasia resulted in replenishment of the bone marrow storage pool. Neutrophils with foamy vacuolation and increased basophilia of the cytoplasm (toxic neutrophils) were present in the blood by eight hours postinjection. The number of toxic neutrophils paralleled the intensity of clinical signs and changes in rectal temperature but not the number of band neutrophils. This indicates that changes in number of toxic neutrophils in sequential leukograms can be a prognostic indicator in dogs with severe inflammation. Images Fig. 3. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:4041973

  19. Increase in filtration coefficient from actions of melittin on neutrophils in isolated rabbit lungs.

    PubMed

    Littner, M R; Lott, F D

    1994-04-01

    Activation of neutrophils may contribute to lung injury in the adult respiratory distress syndrome. We added rabbit neutrophils to the pulmonary circulation of salt-perfused and ventilated isolated rabbit lungs. These neutrophils were activated by adding synthetically pure melittin to the perfusate. This led to lung injury as measured by filtration coefficient under no-flow conditions. We also activated neutrophils in vitro before addition to the pulmonary circulation. These preactivated neutrophils also produced lung injury, indicating a primary action of melittin on neutrophils rather than on lung. The injury was prevented by aristolochic acid, which is an inhibitor of phospholipase A2 (PLA2), and independently by catalase, which is scavenger of hydrogen peroxide (H2O2). Aristolochic acid also appeared to act primarily on neutrophils since addition to neutrophils in vitro prevented injury from in vitro activation by melittin. Aristolochic acid did not appear to act as a free radical scavenger since it did not prevent injury from neutrophils activated by phorbol myristate acetate (PMA). PMA is a direct activator of protein kinase C in neutrophils and leads to formation of H2O2 with consequent lung injury. We conclude that activation of neutrophils by melittin leads to oxidant lung injury possibly from activation of PLA2. Since PLA2 does not directly produce a second messenger, such as diacylglycerol or inositol triphosphate, it is likely that other actions of PLA2 produce an intermediary mediator. We previously showed that an inhibitor of eicosanoid synthesis prevents lung injury from exogenous PLA2. This suggests that the formation of leukotriene B4 (LTB4), a 5-lipoxygenase product of arachidonic acid, may contribute to the oxidant lung injury from melittin.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. [MORPHOLOGICAL FEATURES OF NEUTROPHILS AND EOSINOPHILS GRANULES IN SAPPHIRE MINKS].

    PubMed

    Uzenbaeva, L B; Kizhina, A G; Ilyukha, V A

    2015-01-01

    It has been established that sapphire minks have abnormality of subcellular structure of blood and bone marrow neutrophils and eosinophils. The abnormality consists in forming of abnormal "giant" granules. The si- ze and the number of abnormal granules significantly change during maturation of leucocytes in bone marrow. We have found differences between abnormal granules forming in neutrophils and eosinophils that depend on the maturing stage and the cells life cycle duration as well as morphofunctional features of these granulocytes. PMID:26863773

  1. Pneumolysin activates neutrophil extracellular trap formation.

    PubMed

    G Nel, J; Theron, A J; Durandt, C; Tintinger, G R; Pool, R; Mitchell, T J; Feldman, C; Anderson, R

    2016-06-01

    The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (5-20 ng ml(-1) ) for 30-90 min at 37°C and NET formation measured using the following procedures to detect extracellular DNA: (i) flow cytometry using Vybrant® DyeCycle™ Ruby; (ii) spectrofluorimetry using the fluorophore, Sytox(®) Orange (5 μM); and (iii) NanoDrop(®) technology. These procedures were complemented by fluorescence microscopy using 4', 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min), statistically significant (P < 0·05) dose- and time-related increases in the release of cellular DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis appeared to be restricted to a subpopulation of neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems (10 and 20 ng ml(-1) ) were 4·3 (4·2), 14.3 (9·9) and 16·5 (7·5), respectively (n = 4, P < 0·0001 for comparison of the control with both Ply-treated systems). Ply-induced NETosis occurred in the setting of retention of cell viability, and apparent lack of involvement of reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces vital NETosis in human neutrophils, a process which may either contribute to host defence or worsen disease severity, depending on the intensity of the inflammatory response during pneumococcal infection. PMID:26749379

  2. Oral Neutrophil Transcriptome Changes Result in a Pro-Survival Phenotype in Periodontal Diseases

    PubMed Central

    Lakschevitz, Flavia S.; Aboodi, Guy M.; Glogauer, Michael

    2013-01-01

    Background Periodontal diseases are inflammatory processes that occur following the influx of neutrophils into the periodontal tissues in response to the subgingival bacterial biofilm. Current literature suggests that while neutrophils are protective and prevent bacterial infections, they also appear to contribute to damage of the periodontal tissues. In the present study we compare the gene expression profile changes in neutrophils as they migrate from the circulation into the oral tissues in patients with chronic periodontits and matched healthy subjects. We hypothesized that oral neutrophils in periodontal disease patients will display a disease specific transcriptome that differs from the oral neutrophil of healthy subjects. Methods Venous blood and oral rinse samples were obtained from healthy subjects and chronic periodontitis patients for neutrophil isolation. mRNA was isolated from the neutrophils, and gene expression microarray analysis was completed. Results were confirmed for specific genes of interest by qRT-PCR and Western Blot analysis. Results and Discussion Chronic periodontitis patients presented with increased recruitment of neutrophils to the oral cavity. Gene expression analysis revealed differences in the expression levels of genes from several biological pathways. Using hierarchical clustering analysis, we found that the apoptosis network was significantly altered in patients with chronic inflammation in the oral cavity, with up-regulation of pro-survival members of the Bcl-2 family and down-regulation of pro-apoptosis members in the same compartment. Additional functional analysis confirmed that the percentages of viable neutrophils are significantly increased in the oral cavity of chronic periodontitis patients. Conclusions Oral neutrophils from patients with periodontal disease displayed an altered transcriptome following migration into the oral tissues. This resulted in a pro-survival neutrophil phenotype in chronic periodontitis patients

  3. Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties*

    PubMed Central

    Dalli, Jesmond; Montero-Melendez, Trinidad; Norling, Lucy V; Yin, Xiaoke; Hinds, Charles; Haskard, Dorian; Mayr, Manuel; Perretti, Mauro

    2013-01-01

    Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions. PMID:23660474

  4. Leukocyte subsets and neutrophil function after short-term spaceflight

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Sams, C. F.; Mehta, S. K.; Kaur, I.; Jones, M. L.; Feeback, D. L.; Pierson, D. L.

    1999-01-01

    Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short-term spaceflight (8-15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5-fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10-fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC-1 was significantly decreased while L-selectin was significantly increased. These functional alterations may be of clinical significance on long-duration space missions.

  5. The role of neutrophils in immune dysfunction during severe inflammation.

    PubMed

    Leliefeld, Pieter H C; Wessels, Catharina M; Leenen, Luke P H; Koenderman, Leo; Pillay, Janesh

    2016-01-01

    Critically ill post-surgical, post-trauma and/or septic patients are characterised by severe inflammation. This immune response consists of both a pro- and an anti-inflammatory component. The pro-inflammatory component contributes to (multiple) organ failure whereas occurrence of immune paralysis predisposes to infections. Strikingly, infectious complications arise in these patients despite the presence of a clear neutrophilia. We propose that dysfunction of neutrophils potentially increases the susceptibility to infections or can result in the inability to clear existing infections. Under homeostatic conditions these effector cells of the innate immune system circulate in a quiescent state and serve as the first line of defence against invading pathogens. In severe inflammation, however, neutrophils are rapidly activated, which affects their functional capacities, such as chemotaxis, phagocytosis, intra-cellular killing, NETosis, and their capacity to modulate adaptive immunity. This review provides an overview of the current understanding of neutrophil dysfunction in severe inflammation. We will discuss the possible mechanisms of downregulation of anti-microbial function, suppression of adaptive immunity by neutrophils and the contribution of neutrophil subsets to immune paralysis. PMID:27005275

  6. B–helper neutrophils stimulate immunoglobulin diversification and production in the marginal zone of the spleen

    PubMed Central

    Puga, Irene; Cols, Montserrat; Barra, Carolina M.; He, Bing; Cassis, Linda; Gentile, Maurizio; Comerma, Laura; Chorny, Alejo; Shan, Meimei; Xu, Weifeng; Magri, Giuliana; Knowles, Daniel M.; Tam, Wayne; Chiu, April; Bussel, James B; Serrano, Sergi; Lorente, José Antonio; Bellosillo, Beatriz; Lloreta, Josep; Juanpere, Nuria; Alameda, Francesc; Baró, Teresa; de Heredia, Cristina Díaz; Torán, Núria; Català, Albert; Torrebadell, Montserrat; Fortuny, Claudia; Cusi, Victoria; Carreras, Carmen; Diaz, George A.; Blander, J. Magarian; Farber, Claire-Michèle; Silvestri, Guido; Cunningham-Rundles, Charlotte; Calvillo, Michaela; Dufour, Carlo; Notarangelo, Lucia Dora; Lougaris, Vassilios; Plebani, Alessandro; Casanova, Jean-Laurent; Ganal, Stephanie C.; Diefenbach, Andreas; Aróstegui, Juan Ignacio; Juan, Manel; Yagüe, Jordi; Mahlaoui, Nizar; Donadieu, Jean; Chen, Kang; Cerutti, Andrea

    2011-01-01

    Neutrophils utilize immunoglobulins (Igs) to clear antigen, but their role in Ig production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T-independent Ig responses to circulating antigen. Neutrophils colonized peri-MZ areas after post-natal mucosal colonization by microbes and enhanced their B-helper function upon receiving reprogramming signals from splenic sinusoidal endothelial cells, including interleukin 10 (IL-10). Splenic neutrophils induced Ig class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism involving the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and less preimmune Igs to T-independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial Ig defense by interacting with MZ B cells. PMID:22197976

  7. Reduced iC3b-mediated phagocytotic capacity of pulmonary neutrophils in cystic fibrosis

    PubMed Central

    Morris, MR; Doull, IJM; Dewitt, S; Hallett, MB

    2005-01-01

    Cystic fibrosis (CF) is characterized by a neutrophil-dominated chronic inflammation of the airways with persistent infections. In order to investigate whether neutrophils contribute to an inadequacy in the pulmonary defence mechanism, the phagocytic activity of pulmonary and peripheral blood neutrophils from CF and non-CF respiratory patients were compared. Neutrophils were isolated from both the blood and bronchoalveolar lavage fluid of 21 patients with CF (12 male, 9 female; mean age 7·5 years, range 0·25–16·4 years) and 17 non-CF subjects (9 male, 8 female; mean age 5·4 years, range 0·2–13·1 years). The ex vivo phagocytic rate of normal pulmonary neutrophils to internalize zymosan particles opsonized with iC3b was faster than that of circulating neutrophils (P < 0·05), but the maximum capacity (9 particles/cell) was similar. In contrast, pulmonary neutrophils from patients with CF had a lower phagocytic capacity than circulating neutrophils either from the same patients or from normal subjects. This deficiency could not be attributed to (i) the cell surface density of CR3 (CD18/CD11b) receptors, which were not significantly different between the other groups (ii) the signalling ability of the CR3 receptors, using cytosolic free Ca2+ signalling as the receptor activity read-out or (iii) a decrease in cellular ATP concentration. As CFTR was not detectable on neutrophils from any source by either histochemistry or Western blotting, it was concluded that the reduced phagocytic capacity was not the direct result of a CFTR mutation, but was attributed to a failure of neutrophil phagocytic priming during translocation into the CF lung. PMID:16178858

  8. Cytokine-induced neutrophil-derived interleukin-8.

    PubMed Central

    Strieter, R. M.; Kasahara, K.; Allen, R. M.; Standiford, T. J.; Rolfe, M. W.; Becker, F. S.; Chensue, S. W.; Kunkel, S. L.

    1992-01-01

    During acute inflammation, the first line of cellular response for host defense is the neutrophil. In addition to the historic role of the neutrophil as a phagocyte, recent studies have identified this cell as an important source of a number of cytokines. In this study, we provide evidence that the neutrophil is a significant source of interleukin-8 (IL-8). Neutrophils freshly isolated from whole blood were not found to constitutively express IL-8 mRNA. In contrast, when these leukocytes were cultured on plastic they were activated, leading to the significant expression of de novo steady-state levels of IL-8 mRNA. In addition, when neutrophils were treated with cycloheximide, there was evidence for "superinduction" of steady-state levels of IL-8 mRNA and inhibition of antigenic IL-8 production. Neutrophils were subsequently stimulated with lipopolysaccharide (LPS), tumor necrosis factor-alpha, or interleukin-1-beta and were found to express IL-8 mRNA and antigen in both a time- and dose-dependent manner. Furthermore, neutrophils stimulated with traditional chemotactic/activating factors, such as the split product of the fifth component of complement (C5a), formylmethionyleucylphenylalanine (fMLP), and leukotriene B4 (LTB4) in a dose-dependent manner did not produce significant antigenic IL-8, as compared with unstimulated controls. In contrast, when neutrophils were exposed to either of these neutrophil agonists in the presence of LPS, the production of antigenic IL-8 was significantly elevated, as compared with either of the stimuli alone, suggesting a synergistic response. These data would suggest that the neutrophil can no longer be viewed as only a phagocyte or warehouse for proteolytic enzymes, but is a pivotal effector cell that is able to respond to mediators in its environment and generate cytokines. This latter neutrophil response may be important for either the elicitation of additional neutrophils or to orchestrate the conventional immune response at

  9. Granzyme B-expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques

    PubMed Central

    Mattila, Joshua T.; Maiello, Pauline; Sun, Tao; Via, Laura E.; Flynn, JoAnne L.

    2015-01-01

    Summary The role of neutrophils in tuberculosis (TB), and whether neutrophils express granzyme B (grzB), a pro-apoptotic enzyme associated with cytotoxic T cells, is controversial. We examined neutrophils in peripheral blood (PB) and lung granulomas of Mycobacterium tuberculosis-infected cynomolgus macaques and humans to determine whether mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. We found large numbers of grzB-expressing neutrophils in macaque and human granulomas and these cells contained more grzB+ granules than T cells. Higher neutrophil, but not T cell, grzB expression correlated with increased bacterial load. Although unstimulated PB neutrophils lacked grzB expression, grzB expression increased upon exposure to M. tuberculosis bacilli, M. tuberculosis culture filtrate protein or lipopolysaccharide from Escherichia coli. Perforin is required for granzyme-mediated cytotoxicity by T cells, but was not observed in PB or granuloma neutrophils. Nonetheless, stimulated PB neutrophils secreted grzB as determined by enzyme-linked immunospot assays. Purified grzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration through extracellular matrix and regulating apoptosis or activation in other cell types. These data indicate mycobacterial products and the pro-inflammatory environment of granulomas up-regulates neutrophil grzB expression and suggests a previously unappreciated aspect of neutrophil biology in TB. PMID:25653138

  10. [The expression level of adhesion molecules on neutrophils depending at segmentation of their nuclei].

    PubMed

    Kashutin, S L; Danilov, S I; Vereshchagina, E N; Kluchareva, S V

    2013-11-01

    The article deals with results of detection of expression level of adhesion molecules on neutrophils and segmentation of their nuclei. It is established that in conditions of absence of antigen stimulation neutrophils of circulating pool express molecules of L-selectin in 53.34%, LFA-1 molecules in 65.64%, ICAM-1 in 40.51%, LE4-3 in 58.72% and PECAM-1 in 59.74%. The full readiness to realization of phase of sliding, strong adhesion and immediately transmigration itselfis detected in neutrophils with five segments in nucleus.

  11. Neutrophil Extracellular Traps in ANCA-Associated Vasculitis.

    PubMed

    Söderberg, Daniel; Segelmark, Mårten

    2016-01-01

    A group of pauci-immune vasculitides, characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of antineutrophil cytoplasmic antibodies (ANCAs), is referred to as ANCA-associated vasculitis (AAV). ANCAs against proteinase 3 (PR3) (PR3-ANCA) or myeloperoxidase (MPO) (MPO-ANCA) are found in over 90% of patients with active disease, and these ANCAs are implicated in the pathogenesis of AAV. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally, it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. AAV patients also possess elevated levels of NETs in the circulation. ANCAs are capable of inducing NETosis in neutrophils, and their potential to do so has been shown to be affinity dependent and to correlate with disease activity. Neutrophils from AAV patients are also more prone to release NETs spontaneously than neutrophils from healthy blood donors. NETs contain proinflammatory proteins and are thought to contribute to vessel inflammation directly by damaging endothelial cells and by activating the complement system and indirectly by acting as a link between the innate and adaptive immune system through the generation of PR3- and MPO-ANCA. Injection of NET-loaded myeloid dendritic cells into mice results in circulating PR3- and MPO-ANCA and the development of AAV-like disease. NETs have also been shown to be essential in a rodent model of drug-induced vasculitis. NETs induced by propylthiouracil could not be degraded by DNaseI, implying that disordered NETs might be important for the generation of ANCAs. NET degradation was also highlighted in another study showing that AAV patients have reduced DNaseI activity resulting in less NET degradation. With this in mind, it might be that prolonged exposure to proteins in the NETs due to the overproduction of NETs and/or reduced

  12. Neutrophil Extracellular Traps in ANCA-Associated Vasculitis

    PubMed Central

    Söderberg, Daniel; Segelmark, Mårten

    2016-01-01

    A group of pauci-immune vasculitides, characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of antineutrophil cytoplasmic antibodies (ANCAs), is referred to as ANCA-associated vasculitis (AAV). ANCAs against proteinase 3 (PR3) (PR3-ANCA) or myeloperoxidase (MPO) (MPO-ANCA) are found in over 90% of patients with active disease, and these ANCAs are implicated in the pathogenesis of AAV. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally, it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. AAV patients also possess elevated levels of NETs in the circulation. ANCAs are capable of inducing NETosis in neutrophils, and their potential to do so has been shown to be affinity dependent and to correlate with disease activity. Neutrophils from AAV patients are also more prone to release NETs spontaneously than neutrophils from healthy blood donors. NETs contain proinflammatory proteins and are thought to contribute to vessel inflammation directly by damaging endothelial cells and by activating the complement system and indirectly by acting as a link between the innate and adaptive immune system through the generation of PR3- and MPO-ANCA. Injection of NET-loaded myeloid dendritic cells into mice results in circulating PR3- and MPO-ANCA and the development of AAV-like disease. NETs have also been shown to be essential in a rodent model of drug-induced vasculitis. NETs induced by propylthiouracil could not be degraded by DNaseI, implying that disordered NETs might be important for the generation of ANCAs. NET degradation was also highlighted in another study showing that AAV patients have reduced DNaseI activity resulting in less NET degradation. With this in mind, it might be that prolonged exposure to proteins in the NETs due to the overproduction of NETs and/or reduced

  13. Role of osteopontin in hepatic neutrophil infiltration during alcoholic steatohepatitis

    SciTech Connect

    Apte, Udayan M.; Banerjee, Atrayee; McRee, Rachel; Wellberg, Elizabeth; Ramaiah, Shashi K. . E-mail: sramaiah@cvm.tamu.edu

    2005-08-22

    Alcoholic liver disease (ALD) is a major complication of heavy alcohol (EtOH) drinking and is characterized by three progressive stages of pathology: steatosis, steatohepatitis, and fibrosis/cirrhosis. Alcoholic steatosis (AS) is the initial stage of ALD and consists of fat accumulation in the liver accompanied by minimal liver injury. AS is known to render the hepatocytes increasingly sensitive to toxicants such as bacterial endotoxin (LPS). Alcoholic steatohepatitis (ASH), the second and rate-limiting step in the progression of ALD, is characterized by hepatic fat accumulation, neutrophil infiltration, and neutrophil-mediated parenchymal injury. However, the pathogenesis of ASH is poorly defined. It has been theorized that the pathogenesis of ASH involves interaction of increased circulating levels of LPS with hepatocytes being rendered highly sensitive to LPS due to heavy EtOH consumption. We hypothesize that osteopontin (OPN), a matricellular protein (MCP), plays an important role in the hepatic neutrophil recruitment due to its enhanced expression during the early phase of ALD (AS and ASH). To study the role of OPN in the pathogenesis of ASH, we induced AS in male Sprague-Dawley rats by feeding EtOH-containing Lieber-DeCarli liquid diet for 6 weeks. AS rats experienced extensive fat accumulation and minimal liver injury. Moderate induction in OPN was observed in AS group. ASH was induced by feeding male Sprague-Dawley rats EtOH-containing Lieber-DeCarli liquid diet for 6 weeks followed by LPS injection. The ASH rats had substantial neutrophil infiltration, coagulative oncotic necrosis, and developed higher liver injury. Significant increases in the hepatic and circulating levels of OPN was observed in the ASH rats. Higher levels of the active, thrombin-cleaved form of OPN in the liver in ASH group correlated remarkably with hepatic neutrophil infiltration. Finally, correlative studies between OPN and hepatic neutrophil infiltration was corroborated in a simple

  14. The Aminopeptidase CD13 Induces Homotypic Aggregation in Neutrophils and Impairs Collagen Invasion

    PubMed Central

    Fiddler, Christine A.; Parfrey, Helen; Cowburn, Andrew S.; Luo, Ding; Nash, Gerard B.; Murphy, Gillian; Chilvers, Edwin R.

    2016-01-01

    Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation in vivo. PMID:27467268

  15. Dipeptidyl Peptidase IV Is a Human and Murine Neutrophil Chemorepellent

    PubMed Central

    Herlihy, Sarah E.; Pilling, Darrell; Maharjan, Anu S.; Gomer, Richard H.

    2013-01-01

    In Dictyostelium discoideum, AprA is a secreted protein that inhibits proliferation and causes chemorepulsion of Dictyostelium cells, yet AprA has little sequence similarity to any human proteins. We found that a predicted structure of AprA has similarity to human dipeptidyl peptidase IV (DPPIV). DPPIV is a serine protease present in extracellular fluids that cleaves peptides with a proline or alanine in the second position. In Insall chambers, DPPIV gradients below, similar to, and above the human serum DPPIV concentration cause movement of human neutrophils away from the higher concentration of DPPIV. A 1% DPPIV concentration difference between the front and back of the cell is sufficient to cause chemorepulsion. Neutrophil speed and viability are unaffected by DPPIV. DPPIV inhibitors block DPPIV-mediated chemorepulsion. In a murine model of acute respiratory distress syndrome, aspirated bleomycin induces a significant increase in the number of neutrophils in the lungs after 3 d. Oropharyngeal aspiration of DPPIV inhibits the bleomycin-induced accumulation of mouse neutrophils. These results indicate that DPPIV functions as a chemorepellent of human and mouse neutrophils, and they suggest new mechanisms to inhibit neutrophil accumulation in acute respiratory distress syndrome. PMID:23677473

  16. A Comparative Examination of Demand-Adjusted Shelf Availability Parameters Using Last Circulation Date, Acquisition Date and Imprint Date. Research Report Number 2.

    ERIC Educational Resources Information Center

    Schwarz, Philip

    A theoretical model proposed by Paul Kantor for determining shelf availability for library materials by checking a small sample of items drawn from the checklist against the stacks and circulation records is applied to a working environment, and the following considerations are examined: (1) time required to apply the model to a working library…

  17. Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors.

    PubMed

    Mizuno, Rei; Kamioka, Yuji; Sakai, Yoshiharu; Matsuda, Michiyuki

    2016-01-01

    A number of chemical mediators regulate neutrophil recruitment to inflammatory sites either positively or negatively. Although the actions of each chemical mediator on the intracellular signaling networks controlling cell migration have been studied with neutrophils cultured in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. To understand the mechanisms regulating neutrophil recruitment to the inflamed intestine in vivo, we recently generated transgenic mice expressing biosensors based on FRET (Förster resonance energy transfer) and set up two-photon excitation microscopy to observe the gastrointestinal tract in living mice. By measuring FRET in neutrophils, we showed activity changes of protein kinases in the neutrophils recruited to inflamed intestines. In this chapter, we describe the protocol used to visualize the protein kinase activities in neutrophils of the inflamed intestine of transgenic mice expressing the FRET biosensors. PMID:27246030

  18. Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors.

    PubMed

    Mizuno, Rei; Kamioka, Yuji; Sakai, Yoshiharu; Matsuda, Michiyuki

    2016-01-01

    A number of chemical mediators regulate neutrophil recruitment to inflammatory sites either positively or negatively. Although the actions of each chemical mediator on the intracellular signaling networks controlling cell migration have been studied with neutrophils cultured in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. To understand the mechanisms regulating neutrophil recruitment to the inflamed intestine in vivo, we recently generated transgenic mice expressing biosensors based on FRET (Förster resonance energy transfer) and set up two-photon excitation microscopy to observe the gastrointestinal tract in living mice. By measuring FRET in neutrophils, we showed activity changes of protein kinases in the neutrophils recruited to inflamed intestines. In this chapter, we describe the protocol used to visualize the protein kinase activities in neutrophils of the inflamed intestine of transgenic mice expressing the FRET biosensors.

  19. Neutrophil Elastase Inhibitors

    PubMed Central

    Groutas, William C.; Dou, Dengfeng; Alliston, Kevin R.

    2011-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) constitutes a worldwide health problem. There is currently an urgent and unmet need for the development of small molecule therapeutics capable of blocking and/or reversing the progression of the disorder. Recent studies have greatly illuminated our understanding of the multiple pathogenic processes associated with COPD. Of paramount importance is the key role played by proteases, oxidative stress, apoptosis, and inflammation. Insights gained from these studies have made possible the exploration of new therapeutic approaches. Areas covered An overview of major developments in COPD research with emphasis on low molecular weight neutrophil elastase inhibitors is described in this review. Expert opinion Great strides have been made toward our understanding of the biochemical and cellular events associated with COPD. However, our knowledge regarding the inter-relationships among the multiple pathogenic mechanisms and their mediators involved is till limited. The problem is further compounded by the unavailability of suitable validated biomarkers for assessing the efficacy of potential therapeutic interventions. The complexity of COPD suggests that effective therapeutic interventions may require the administration of more than one agent such as, for instance, an HNE or MMP-12 inhibitor with an anti-inflammatory agent such as a phosphodiesterase-4 inhibitor, or a dual function agent capable of disrupting the cycle of proteolysis, apoptosis, inflammation and oxidative stress PMID:21235378

  20. AUTOINFLAMMATORY PUSTULAR NEUTROPHILIC DISEASES

    PubMed Central

    Naik, Haley B.; Cowen, Edward W.

    2013-01-01

    SYNOPSIS The inflammatory pustular dermatoses constitute a spectrum of non-infectious conditions ranging from localized involvement to generalized disease with associated acute systemic inflammation and multi-organ involvement. Despite the variability in extent and severity of cutaneous presentation, each of these diseases is characterized by non-infectious neutrophilic intra-epidermal microabscesses. Many share systemic findings including fever, elevated inflammatory markers, inflammatory bowel disease and/or osteoarticular involvement, suggesting potential common pathogenic links (Figure 1). The recent discoveries of several genes responsible for heritable pustular diseases have revealed a distinct link between pustular skin disease and regulation of innate immunity. These genetic advances have led to a deeper exploration of common pathways in pustular skin disease and offer the potential for a new era of biologic therapy which targets these shared pathways. This chapter provides a new categorization of inflammatory pustular dermatoses in the context of recent genetic and biologic insights. We will discuss recently-described monogenic diseases with pustular phenotypes, including deficiency of IL-1 receptor antagonist (DIRA), deficiency of the IL-36 receptor antagonist (DITRA), CARD14-associated pustular psoriasis (CAMPS), and pyogenic arthritis, pyoderma gangrenosum, acne (PAPA). We will then discuss how these new genetic advancements may inform how we view previously described pustular diseases, including pustular psoriasis and its clinical variants, with a focus on historical classification by clinical phenotype. PMID:23827244

  1. Identification and characterization of VEGF-A–responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans

    PubMed Central

    Massena, Sara; Christoffersson, Gustaf; Vågesjö, Evelina; Seignez, Cédric; Gustafsson, Karin; Binet, François; Herrera Hidalgo, Carmen; Giraud, Antoine; Lomei, Jalal; Weström, Simone; Shibuya, Masabumi; Claesson-Welsh, Lena; Gerwins, Pär; Welsh, Michael; Kreuger, Johan

    2015-01-01

    Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d+VEGFR1highCXCR4high was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk−/−, tsad−/−), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d+ neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d– neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens. PMID:26286848

  2. The Role of Neutrophils in Alpha-1 Antitrypsin Deficiency.

    PubMed

    McCarthy, Cormac; Reeves, Emer P; McElvaney, Noel G

    2016-08-01

    Alpha-1 antitrypsin deficiency (AATD) is characterized by low levels of circulating alpha-1 antitrypsin and an increased risk for emphysema, liver disease, and panniculitis. The reduced levels of alpha-1 antitrypsin in AATD predispose the lung to unopposed proteolytic activity, predominantly from neutrophil-derived proteases, chiefly neutrophil elastase. This leads to emphysema. The mechanisms subtending the liver disease are less well understood, but are probably due to a "gain-of function" inflammatory process in the liver, stoked by intracellular retention of aberrantly folded alpha-1 antitrypsin. The panniculitis associated with AATD is most likely due to unopposed proteolytic activity in the skin. Although AATD has been traditionally viewed as a condition arising from a protease-antiprotease imbalance in the lung, it is increasingly recognized that AATD is an inflammatory disorder, both in the lung and in the extrapulmonary manifestations associated with the condition. This inflammation is predominantly neutrophil driven, and there are several alpha-1 antitrypsin-related mechanisms involved in potentiating this neutrophilic response. The rationale for AAT augmentation therapy in AATD is classically based on restoring the antiprotease balance in the lung, but its beneficial effects may also be exerted systemically, further exposing the pathogenesis of AATD-related disease and indicating a potential usage for alpha-1 antitrypsin in other inflammatory conditions. PMID:27564664

  3. Neutrophil functional disorder in childhood.

    PubMed

    Mironska, K

    2015-01-01

    Neutrophil functional disorders thought to be uncommon, yet important as a cause of morbidity and mortality in infants and children. During the first years of life, when the immune system is still not completely mature, when the viral infections are frequent and antibiotic overuse can damage and alter the immune response, the inadequate nutrition followed with iron deficient anemia and malnutrition can lead the child`s organism in state of immunodeficiency. Sometimes is difficult to distinguish at the beginning weather the cause of patient suffering from frequent infections is existing of primary immunodeficiency disorder or the cause of the immunodeficiency state is just from exogenous factors. Fortunately, primary immune deficiencies are rare diseases and only 6-7% of all of them, due to the neutrophilic functional disorders. Unfortunately, many exogenous and environmental factors have influence to the immune system, and the percentage of secondary caused neutrophilic functional disorders is much higher and should be considered when children are investigated for immunodeficiency. So, when to suspect neutrophil functional disorder? The hallmarks for diseases related to the neutrophilic functional disorders are discussed in this article.

  4. Neutrophil Extracellular Traps Go Viral

    PubMed Central

    Schönrich, Günther; Raftery, Martin J.

    2016-01-01

    Neutrophils are the most numerous immune cells. Their importance as the first line of defense against bacterial and fungal pathogens is well described. In contrast, the role of neutrophils in controlling viral infections is less clear. Bacterial and fungal pathogens can stimulate neutrophils extracellular traps (NETs) in a process called NETosis. Although NETosis has previously been described as a special form of programmed cell death, there are forms of NET production that do not end with the demise of neutrophils. As an end result of NETosis, genomic DNA complexed with microbicidal proteins is expelled from neutrophils. These structures can kill pathogens or at least prevent their local spread within host tissue. On the other hand, disproportionate NET formation can cause local or systemic damage. Only recently, it was recognized that viruses can also induce NETosis. In this review, we discuss the mechanisms by which NETs are produced in the context of viral infection and how this may contribute to both antiviral immunity and immunopathology. Finally, we shed light on viral immune evasion mechanisms targeting NETs. PMID:27698656

  5. Neutrophils in type 1 diabetes.

    PubMed

    Huang, Juan; Xiao, Yang; Xu, Aimin; Zhou, Zhiguang

    2016-09-01

    Type 1 diabetes is an autoimmune disease that afflicts millions of people worldwide. It occurs as the consequence of destruction of insulin-producing pancreatic β-cells triggered by genetic and environmental factors. The initiation and progression of the disease involves a complicated interaction between β-cells and immune cells of both innate and adaptive systems. Immune cells, such as T cells, macrophages and dendritic cells, have been well documented to play crucial roles in type 1 diabetes pathogenesis. However, the particular actions of neutrophils, which are the most plentiful immune cell type and the first immune cells responding to inflammation, in the etiology of this disease might indeed be unfairly ignored. Progress over the past decades shows that neutrophils might have essential effects on the onset and perpetuation of type 1 diabetes. Neutrophil-derived cytotoxic substances, including degranulation products, cytokines, reactive oxygen species and extracellular traps that are released during the process of neutrophil maturation or activation, could cause destruction to islet cells. In addition, these cells can initiate diabetogenic T cell response and promote type 1 diabetes development through cell-cell interactions with other immune and non-immune cells. Furthermore, relevant antineutrophil therapies have been shown to delay and dampen the progression of insulitis and autoimmune diabetes. Here, we discuss the relationship between neutrophils and autoimmune type 1 diabetes from the aforementioned aspects to better understand the roles of these cells in the initiation and development of type 1 diabetes. PMID:27181374

  6. Neutrophil Extracellular Traps Go Viral

    PubMed Central

    Schönrich, Günther; Raftery, Martin J.

    2016-01-01

    Neutrophils are the most numerous immune cells. Their importance as the first line of defense against bacterial and fungal pathogens is well described. In contrast, the role of neutrophils in controlling viral infections is less clear. Bacterial and fungal pathogens can stimulate neutrophils extracellular traps (NETs) in a process called NETosis. Although NETosis has previously been described as a special form of programmed cell death, there are forms of NET production that do not end with the demise of neutrophils. As an end result of NETosis, genomic DNA complexed with microbicidal proteins is expelled from neutrophils. These structures can kill pathogens or at least prevent their local spread within host tissue. On the other hand, disproportionate NET formation can cause local or systemic damage. Only recently, it was recognized that viruses can also induce NETosis. In this review, we discuss the mechanisms by which NETs are produced in the context of viral infection and how this may contribute to both antiviral immunity and immunopathology. Finally, we shed light on viral immune evasion mechanisms targeting NETs.

  7. Passage of CD18- and CD18+ bovine neutrophils into pulmonary alveoli during acute Pasteurella haemolytica pneumonia.

    PubMed

    Ackermann, M R; Kehrli, M E; Brogden, K A

    1996-11-01

    CD18 is a subunit for three beta 2 integrin molecules (Mac-1, p150, 95, LFA-1), which are expressed on the plasma membrane of neutrophils. These molecules mediate passage of neutrophils into sites of infection. In children and animals that lack CD18 expression, neutrophil infiltration is impaired in most tissues. However, in lung, CD18- neutrophils have been identified in the airway spaces during spontaneous episodes of pneumonia. To determine whether CD18 is vital for passage through the pulmonary alveolar wall, lung lobes of cattle with neutrophils that were deficient in CD18 expression (CD18-) and cattle with normal CD18 expression (CD18+) were inoculated with Pasteurella haemolytica by fiberoptic bronchoscopy; control lobes were inoculated with pyrogen-free saline (PFS). Neutrophil passage into alveolar lumina at 4 and 6 hours postinoculation was measured by computerized image analysis. Blood levels of neutrophils for CD18- cattle ranged from 12- to 26-fold higher than for CD18+ cattle prior to inoculation, and counts in both groups rose slightly postinoculation. In P. haemolytica-inoculated lobes, total numbers of neutrophils in alveolar lumina of the two groups were similar. An increase in the number of neutrophils in the alveolar wall was fourfold greater in CD18- cattle than in CD18+ cattle. In PFS-inoculated lobes, the number of neutrophils in the alveolar wall was sixfold higher in CD18 cattle than in CD18+ cattle. This work shows that by 4 and 6 hours, CD18- neutrophils enter the alveolar lumen at a rate similar to that in CD18+ cattle. Higher numbers of CD18- neutrophils are present in the alveolar wall of control (PFS) and bacteria-inoculated lobes. Thus, the CD18- cells are increased in the walls of alveoli and numbers of neutrophils that enter the alveolar lumen are similar in CD18+ and CD18- cattle. PMID:8952022

  8. Infectious Progeny of 2009 A (H1N1) Influenza Virus Replicated in and Released from Human Neutrophils.

    PubMed

    Zhang, Zhang; Huang, Tao; Yu, Feiyuan; Liu, Xingmu; Zhao, Conghui; Chen, Xueling; Kelvin, David J; Gu, Jiang

    2015-01-01

    Various reports have indicated that a number of viruses could infect neutrophils, but the multiplication of viruses in neutrophils was abortive. Based on our previous finding that avian influenza viral RNA and proteins were present in the nucleus of infected human neutrophils in vivo, we investigated the possibility of 2009 A (H1N1) influenza viral synthesis in infected neutrophils and possible release of infectious progeny from host cells. In this study we found that human neutrophils in vitro without detectable level of sialic acid expression could be infected by this virus strain. We also show that the infected neutrophils can not only synthesize 2009 A (H1N1) viral mRNA and proteins, but also produce infectious progeny. These findings suggest that infectious progeny of 2009 A (H1N1) influenza virus could be replicated in and released from human neutrophils with possible clinical implications. PMID:26639836

  9. Infectious Progeny of 2009 A (H1N1) Influenza Virus Replicated in and Released from Human Neutrophils.

    PubMed

    Zhang, Zhang; Huang, Tao; Yu, Feiyuan; Liu, Xingmu; Zhao, Conghui; Chen, Xueling; Kelvin, David J; Gu, Jiang

    2015-12-07

    Various reports have indicated that a number of viruses could infect neutrophils, but the multiplication of viruses in neutrophils was abortive. Based on our previous finding that avian influenza viral RNA and proteins were present in the nucleus of infected human neutrophils in vivo, we investigated the possibility of 2009 A (H1N1) influenza viral synthesis in infected neutrophils and possible release of infectious progeny from host cells. In this study we found that human neutrophils in vitro without detectable level of sialic acid expression could be infected by this virus strain. We also show that the infected neutrophils can not only synthesize 2009 A (H1N1) viral mRNA and proteins, but also produce infectious progeny. These findings suggest that infectious progeny of 2009 A (H1N1) influenza virus could be replicated in and released from human neutrophils with possible clinical implications.

  10. Infectious Progeny of 2009 A (H1N1) Influenza Virus Replicated in and Released from Human Neutrophils

    PubMed Central

    Zhang, Zhang; Huang, Tao; Yu, Feiyuan; Liu, Xingmu; Zhao, Conghui; Chen, Xueling; Kelvin, David J.; Gu, Jiang

    2015-01-01

    Various reports have indicated that a number of viruses could infect neutrophils, but the multiplication of viruses in neutrophils was abortive. Based on our previous finding that avian influenza viral RNA and proteins were present in the nucleus of infected human neutrophils in vivo, we investigated the possibility of 2009 A (H1N1) influenza viral synthesis in infected neutrophils and possible release of infectious progeny from host cells. In this study we found that human neutrophils in vitro without detectable level of sialic acid expression could be infected by this virus strain. We also show that the infected neutrophils can not only synthesize 2009 A (H1N1) viral mRNA and proteins, but also produce infectious progeny. These findings suggest that infectious progeny of 2009 A (H1N1) influenza virus could be replicated in and released from human neutrophils with possible clinical implications. PMID:26639836

  11. The effect of glutamine supplementation and physical exercise on neutrophil function.

    PubMed

    Lagranha, C J; Levada-Pires, A C; Sellitti, D F; Procopio, J; Curi, R; Pithon-Curi, T C

    2008-04-01

    Glutamine is the most abundant free amino acid in the body. Its primary source is skeletal muscle, from where it is released into the bloodstream and transported to a variety of tissues. Several studies have shown that glutamine is important for rat and human neutrophil function and that these cells utilize glutamine at high rates. Physical exercise has also been shown to induce considerable changes in neutrophil metabolism and function. As neutrophils represent 50-60% of the total circulating leukocyte pool and play a key role in inflammation, both physical exercise and glutamine might be expected to regulate the inflammatory process. In this review, the changes in neutrophil function induced by physical exercise and glutamine supplementation are compared. PMID:17928941

  12. shRNA-Induced Gene Knockdown In Vivo to Investigate Neutrophil Function.

    PubMed

    Basit, Abdul; Tang, Wenwen; Wu, Dianqing

    2016-01-01

    To silence genes in neutrophils efficiently, we exploited the RNA interference and developed an shRNA-based gene knockdown technique. This method involves transfection of mouse bone marrow-derived hematopoietic stem cells with retroviral vector carrying shRNA directed at a specific gene. Transfected stem cells are then transplanted into irradiated wild-type mice. After engraftment of stem cells, the transplanted mice have two sets of circulating neutrophils. One set has a gene of interest knocked down while the other set has full complement of expressed genes. This efficient technique provides a unique way to directly compare the response of neutrophils with a knocked-down gene to that of neutrophils with the full complement of expressed genes in the same environment. PMID:27271902

  13. Clostridium perfringens α-Toxin Impairs Innate Immunity via Inhibition of Neutrophil Differentiation

    PubMed Central

    Takehara, Masaya; Takagishi, Teruhisa; Seike, Soshi; Ohtani, Kaori; Kobayashi, Keiko; Miyamoto, Kazuaki; Shimizu, Tohru; Nagahama, Masahiro

    2016-01-01

    Although granulopoiesis is accelerated to suppress bacteria during infection, some bacteria can still cause life-threatening infections, but the mechanism behind this remains unclear. In this study, we found that mature neutrophils in bone marrow cells (BMCs) were decreased in C. perfringens-infected mice and also after injection of virulence factor α-toxin. C. perfringens infection interfered with the replenishment of mature neutrophils in the peripheral circulation and the accumulation of neutrophils at C. perfringens-infected sites in an α-toxin-dependent manner. Measurements of bacterial colony-forming units in C. perfringens-infected muscle revealed that α-toxin inhibited a reduction in the load of C. perfringens. In vitro treatment of isolated BMCs with α-toxin (phospholipase C) revealed that α-toxin directly decreased mature neutrophils. α-Toxin did not influence the viability of isolated mature neutrophils, while simultaneous treatment of BMCs with granulocyte colony-stimulating factor attenuated the reduction of mature neutrophils by α-toxin. Together, our results illustrate that impairment of the innate immune system by the inhibition of neutrophil differentiation is crucial for the pathogenesis of C. perfringens to promote disease to a life-threatening infection, which provides new insight to understand how pathogenic bacteria evade the host immune system. PMID:27306065

  14. In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation

    PubMed Central

    Kreisel, Daniel; Nava, Ruben G.; Li, Wenjun; Zinselmeyer, Bernd H.; Wang, Baomei; Lai, Jiaming; Pless, Robert; Gelman, Andrew E.; Krupnick, Alexander S.; Miller, Mark J.

    2010-01-01

    Immune-mediated pulmonary diseases are a significant public health concern. Analysis of leukocyte behavior in the lung is essential for understanding cellular mechanisms that contribute to normal and diseased states. Here, we used two-photon imaging to study neutrophil extravasation from pulmonary vessels and subsequent interstitial migration. We found that the lungs contained a significant pool of tissue-resident neutrophils in the steady state. In response to inflammation produced by bacterial challenge or transplant-mediated, ischemia-reperfusion injury, neutrophils were rapidly recruited from the circulation and patrolled the interstitium and airspaces of the lung. Motile neutrophils often aggregated in dynamic clusters that formed and dispersed over tens of minutes. These clusters were associated with CD115+ F4/80+ Ly6C+ cells that had recently entered the lung. The depletion of blood monocytes with clodronate liposomes reduced neutrophil clustering in the lung, but acted by inhibiting neutrophil transendothelial migration upstream of interstitial migration. Our results suggest that a subset of monocytes serve as key regulators of neutrophil extravasation in the lung and may be an attractive target for the treatment of inflammatory pulmonary diseases. PMID:20923880

  15. Lutzomyia longipalpis saliva drives apoptosis and enhances parasite burden in neutrophils.

    PubMed

    Prates, Deboraci Brito; Araújo-Santos, Théo; Luz, Nívea Farias; Andrade, Bruno B; França-Costa, Jaqueline; Afonso, Lilian; Clarêncio, Jorge; Miranda, José Carlos; Bozza, Patrícia T; Dosreis, George A; Brodskyn, Cláudia; Barral-Netto, Manoel; Borges, Valéria Matos; Borges, Valéria de Matos; Barral, Aldina

    2011-09-01

    Neutrophils are considered the host's first line of defense against infections and have been implicated in the immunopathogenesis of Leishmaniasis. Leishmania parasites are inoculated alongside vectors' saliva, which is a rich source of pharmacologically active substances that interfere with host immune response. In the present study, we tested the hypothesis that salivary components from Lutzomyia longipalpis, an important vector of visceral Leishmaniasis, enhance neutrophil apoptosis. Murine inflammatory peritoneal neutrophils cultured in the presence of SGS presented increased surface expression of FasL and underwent caspase-dependent and FasL-mediated apoptosis. This proapoptosis effect of SGS on neutrophils was abrogated by pretreatment with protease as well as preincubation with antisaliva antibodies. Furthermore, in the presence of Leishmania chagasi, SGS also increased apoptosis on neutrophils and increased PGE(2) release and decreased ROS production by neutrophils, while enhancing parasite viability inside these cells. The increased parasite burden was abrogated by treatment with z-VAD, a pan caspase inhibitor, and NS-398, a COX-2 inhibitor. In the presence of SGS, Leishmania-infected neutrophils produced higher levels of MCP-1 and attracted a high number of macrophages by chemotaxis in vitro assays. Both of these events were abrogated by pretreatment of neutrophils with bindarit, an inhibitor of CCL2/MCP-1 expression. Taken together, our data support the hypothesis that vector salivary proteins trigger caspase-dependent and FasL-mediated apoptosis, thereby favoring Leishmania survival inside neutrophils, which may represent an important mechanism for the establishment of Leishmania infection.

  16. Accelerated Apoptosis of Neutrophils in Familial Mediterranean Fever

    PubMed Central

    Manukyan, Gayane; Aminov, Rustam; Hakobyan, Gagik; Davtyan, Tigran

    2015-01-01

    The causative mutations for familial Mediterranean fever (FMF) are located in the MEFV gene, which encodes pyrin. Pyrin modulates the susceptibility to apoptosis via its PYD domain, but how the mutated versions of pyrin affect apoptotic processes are poorly understood. Spontaneous and induced rates of systemic neutrophil apoptosis as well as the levels of proteins involved in apoptosis were investigated ex vivo in patients with FMF using flow cytometry and RT-qPCR. The freshly collected neutrophils from the patients in FMF remission displayed a significantly larger number of cells spontaneously entering apoptosis compared to control (6.27 ± 2.14 vs. 1.69 ± 0.18%). This elevated ratio was retained after 24 h incubation of neutrophils in the growth medium (32.4 ± 7.41 vs. 7.65 ± 1.32%). Correspondingly, the mRNA level for caspase-3 was also significantly increased under these conditions. In response to the inducing agents, the neutrophils from FMF patients also displayed significantly elevated apoptotic rates compared to control. The elevated rates, however, can be largely explained by the higher basal ratio of apoptotic cells in the former group. Monitoring of several proteins involved in apoptosis has not revealed any conventional mechanisms contributing to the enhanced apoptotic rate of neutrophils in FMF. Although the exact molecular mechanisms of accelerated neutrophil apoptosis in FMF remain unknown, it may provide a protection against excessive inflammation and tissue damage due to a massive infiltration of neutrophils in the acute period of the disease. PMID:26042122

  17. Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy.

    PubMed

    Zilio, Serena; Serafini, Paolo

    2016-01-01

    Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer.

  18. Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy

    PubMed Central

    Zilio, Serena; Serafini, Paolo

    2016-01-01

    Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer. PMID:27618112

  19. Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy.

    PubMed

    Zilio, Serena; Serafini, Paolo

    2016-01-01

    Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer. PMID:27618112

  20. Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice

    PubMed Central

    Heestermans, Marco; Salloum-Asfar, Salam; Salvatori, Daniela; Laghmani, El Houari; Luken, Brenda M.; Zeerleder, Sacha S.; Spronk, Henri M. H.; Korporaal, Suzanne J.; Wagenaar, Gerry T. M.; Reitsma, Pieter H.

    2016-01-01

    Recently, platelets, neutrophils, and factor XII (FXII) have been implicated as important players in the pathophysiology of venous thrombosis. Their role became evident in mouse models in which surgical handling was used to provoke thrombosis. Inhibiting anticoagulation in mice by using small interfering RNA (siRNA) targeting Serpinc1 and Proc also results in a thrombotic phenotype, which is spontaneous (no additional triggers) and reproducibly results in clots in the large veins of the head and fibrin deposition in the liver. This thrombotic phenotype is fatal but can be fully rescued by thrombin inhibition. The mouse model was used in this study to investigate the role of platelets, neutrophils, and FXII. After administration of siRNAs targeting Serpinc1 and Proc, antibody-mediated depletion of platelets fully abrogated the clinical features as well as microscopic aspects in the head. This was corroborated by strongly reduced fibrin deposition in the liver. Whereas neutrophils were abundant in siRNA-triggered thrombotic lesions, antibody-mediated depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, or thrombus morphology. In addition, absence of circulating neutrophils did not affect quantitative liver fibrin deposition. Remarkably, siRNA-mediated depletion of plasma FXII accelerated the onset of the clinical phenotype; mice were affected with more severe thrombotic lesions. To summarize, in this study, onset and severity of the thrombotic phenotype are dependent on the presence of platelets but not circulating neutrophils. Unexpectedly, FXII has a protective effect. This study challenges the proposed roles of neutrophils and FXII in venous thrombosis pathophysiology. PMID:26932804

  1. Induction of CD18-mediated passage of neutrophils by Pasteurella haemolytica in pulmonary bronchi and bronchioles.

    PubMed

    Ackermann, M R; Brogden, K A; Florance, A F; Kehrli, M E

    1999-02-01

    Pasteurella haemolytica is an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released by P. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs during P. haemolytica pneumonia are poorly characterized. To determine whether the CD18 family (beta2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected with P. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated with P. haemolytica A1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P < 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study

  2. Site-Specific Neutrophil Migration and CXCL2 Expression in Periodontal Tissue.

    PubMed

    Greer, A; Irie, K; Hashim, A; Leroux, B G; Chang, A M; Curtis, M A; Darveau, R P

    2016-07-01

    The oral microbial community is the best-characterized bacterial ecosystem in the human host. It has been shown in the mouse that oral commensal bacteria significantly contribute to clinically healthy periodontal homeostasis by influencing the number of neutrophils that migrate from the vasculature to the junctional epithelium. Furthermore, in clinically healthy tissue, the neutrophil response to oral commensal bacteria is associated with the select expression of the neutrophil chemokine CXCL2 but not CXCL1. This preliminary study examined the contribution of commensal bacteria on neutrophil location across the tooth/gingival interface. Tissue sections from the root associated mesial (anterior) of the second molar to the root associated distal (posterior) of the second molar were examined for neutrophils and the expression of the neutrophil chemokine ligands CXCL1 and CXCL2. It was found that both the number of neutrophils as well as the expression of CXCL2 but not CXCL1 was significantly increased in tissue sections close to the interdental region, consistent with the notion of select tissue expression patterns for neutrophil chemokine expression and subsequent neutrophil location. Furthermore, mice gavaged with either oral Streptococcus or Lactobacillus sp. bacteria induced a location pattern of neutrophils and CXCL2 expression similar to the normal oral flora. These data indicate for the first time select neutrophil location and chemokine expression patterns associated with clinically healthy tissue. The results reveal an increased inflammatory load upon approaching the interproximal region, which is consistent with the observation that the interproximal region often reveals early clinical signs of periodontal disease. PMID:27013641

  3. [Defects of neutrophil function in chronic gastroduodenitis in children].

    PubMed

    Agafonova, E V; Malanicheva, T G; Denisova, S N

    2013-01-01

    At present, chronic gastroduodenitis (CGD) occupies one ofthe leading places in the structure of diseases of the gastrointestinal tract in children. In the etiology of CGD, along with the leading pathogenic Helicobacterpylori (HP), the role of the fungal flora increased. The aim of the work was to evaluate the functional activity of neutrophils in children with the CGD, associated with HP and Candida albicans. Among 110 children in the age from 7 to 17 years with chronic gastroduodenitis, associated with Helicobacter pylory(HP), as well as the association of HP with Candida albicans and the markers of secondary immune insufficiency, a study of the phagocytic activity and immune phenotype of neutrophils by flow cytofluorimetry was conducted. Differentiated peculiarities of the phagocytic activity of neutrophils in association with bacterial pathogens (HP) and fungal flora were identified. The transformation of the immune phenotype was combined with a significant depression of the phagocytic and microbicidal functions, more pronounced with the association of HP and Candida albicans. Circulating mannano protein antigen of Candida albicans influenced on the surface of phenotype of neutrophils, increasing the expression of protopathic and HLADR-receptors, and decreasing the expression of adhesion receptors and cytolysis. Thus, in case of chronic gastroduodenitis in children, there was a considerable transformation of the phenotype of neutrophil with differentiated characteristics at the association with bacterial (HP) pathogens and fungal flora. The obtained data should be taken into account when carrying out medical activities, and the doctors should include in the composition of complex therapy of CGD, associated with Candida albicans, drugs, aimed at immunocorrection of the identified violations PMID:23951901

  4. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

    SciTech Connect

    Williams, C. David; Bajt, Mary Lynn; Sharpe, Matthew R.; McGill, Mitchell R.; Farhood, Anwar; Jaeschke, Hartmut

    2014-03-01

    Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91{sup phox}−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. - Highlights: • Neutrophil (PMN) function increases during liver repair after acetaminophen overdose. • Liver repair after acetaminophen (APAP)-overdose is not dependent on NADPH oxidase. • Human PMNs do not appear

  5. Neutral serine proteases of neutrophils.

    PubMed

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  6. Lung Circulation.

    PubMed

    Suresh, Karthik; Shimoda, Larissa A

    2016-04-01

    The circulation of the lung is unique both in volume and function. For example, it is the only organ with two circulations: the pulmonary circulation, the main function of which is gas exchange, and the bronchial circulation, a systemic vascular supply that provides oxygenated blood to the walls of the conducting airways, pulmonary arteries and veins. The pulmonary circulation accommodates the entire cardiac output, maintaining high blood flow at low intravascular arterial pressure. As compared with the systemic circulation, pulmonary arteries have thinner walls with much less vascular smooth muscle and a relative lack of basal tone. Factors controlling pulmonary blood flow include vascular structure, gravity, mechanical effects of breathing, and the influence of neural and humoral factors. Pulmonary vascular tone is also altered by hypoxia, which causes pulmonary vasoconstriction. If the hypoxic stimulus persists for a prolonged period, contraction is accompanied by remodeling of the vasculature, resulting in pulmonary hypertension. In addition, genetic and environmental factors can also confer susceptibility to development of pulmonary hypertension. Under normal conditions, the endothelium forms a tight barrier, actively regulating interstitial fluid homeostasis. Infection and inflammation compromise normal barrier homeostasis, resulting in increased permeability and edema formation. This article focuses on reviewing the basics of the lung circulation (pulmonary and bronchial), normal development and transition at birth and vasoregulation. Mechanisms contributing to pathological conditions in the pulmonary circulation, in particular when barrier function is disrupted and during development of pulmonary hypertension, will also be discussed. PMID:27065170

  7. Analysis of the functional characteristics of L-selectin and its expression on normal and CD18-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Higuchi, H; Yamashiki, N; Yamaguchi, M

    2000-06-01

    In vivo responsiveness to epinephrine, expression of L-selectin on neutrophils, changes in intracellular calcium ([Ca2+]i), sulfatide-induced superoxide production and tyrosine phosphorylation in neutrophils were evaluated to elucidate the role of L-selectin-associated functions of normal and CD18-deficient bovine neutrophils. The number of neutrophils in peripheral blood was significantly increased (P < 0.05) in four normal calves at 5-20 min after in vivo administration of epinephrine; however, no significant increase of neutrophils was found in three calves with bovine leucocyte adhesion deficiency (BLAD). Expression of L-selectin on neutrophils from three calves with BLAD was 61-77% of that of normal calves. Pretreatment of neutrophils with phorbol myristate acetate caused a marked decrease in the expression of L-selectin on neutrophils from both normal and BLAD calves. The sulfatide-induced sustained phase of [Ca2+]i concentration in neutrophils from calves with BLAD was significantly (P < 0.05) decreased. Following stimulation with aggregated IgG, the transient phase of [Ca2+]i in neutrophils from normal and BLAD calves was increased; however, the sustained phase of [Ca2+]i in BLAD neutrophils was significantly lower (P < 0.05) than that of controls. Sulfatide-induced O2- production and chemiluminescent response in neutrophils from calves with BLAD were 48-51% of those of normal calves and were inhibited by genistein and wortmannin, respectively, in a dose-dependent manner. The amount of tyrosine phosphorylated 100 kDa protein in neutrophils from BLAD calves stimulated with sulfatides was 57% of that of controls. The degree of L-selectin expression on neutrophils was correlated with the intracellular signalling events and the related superoxide production. PMID:10849115

  8. Type I interferon transcriptional signature in neutrophils and high frequency of low-density granulocytes are associated with tissue damage in malaria

    PubMed Central

    Rocha, Bruno Coelho; Marques, Pedro Elias; Leoratti, Fabiana Maria de Souza; Junqueira, Caroline; Pereira, Dhelio Batista; Antonelli, Lis Ribeiro do Valle; Menezes, Gustavo Batista

    2015-01-01

    SUMMARY Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. Yet, the role of these polymorphonuclear cells in mediating either resistance or pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, the enhanced release of reactive oxygen species and myeloperoxidase, as well as the high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed an intense recruitment of neutrophils to liver sinusoids. Neutrophil migration, IL-1β and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggests that type I interferon signaling have a central role in neutrophil activation and malaria pathogenesis. PMID:26711347

  9. Peeking into the secret life of neutrophils.

    PubMed

    Li, Jackson LiangYao; Ng, Lai Guan

    2012-09-01

    The migration of neutrophils between tissue compartments is an important aspect of innate immune surveillance. This process is regulated by a cascade of cellular and molecular signals to avoid unnecessary crowding of neutrophils at the periphery, to allow rapid mobilization of neutrophils in response to inflammatory stimuli, and to return to a state of homeostasis after the response. Intravital microscopy approaches have been fundamental in unraveling many aspects of neutrophil behavior, providing important mechanistic information on the processes involved in basal and disease states. Here, we provide a broad overview of the current state of research on neutrophil biology, describing the processes in the typical life cycle of neutrophils, from their first appearance in the bone marrow until their eventual destruction. We will focus on novel aspects of neutrophil behavior, which had previously been elusive until their recent elucidation by advanced intravital microscopy techniques. PMID:22407577

  10. Neutrophil extracellular traps - the dark side of neutrophils.

    PubMed

    Sørensen, Ole E; Borregaard, Niels

    2016-05-01

    Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA in complex with histones and granule proteins, which were expelled from dying neutrophils to ensnare and kill microbes. NETs are formed during infection in vivo by mechanisms different from those originally described in vitro. Citrullination of histones by peptidyl arginine deiminase 4 (PAD4) is central for NET formation in vivo. NETs may spur formation of autoantibodies and may also serve as scaffolds for thrombosis, thereby providing a link among infection, autoimmunity, and thrombosis. In this review, we present the mechanisms by which NETs are formed and discuss the physiological and pathophysiological consequences of NET formation. We conclude that NETs may be of more importance in autoimmunity and thrombosis than in innate immune defense.

  11. Microbe-specific unconventional T-cells induce human neutrophil differentiation into antigen cross-presenting cells

    PubMed Central

    Liuzzi, Anna Rita; Tyler, Christopher J.; Khan, Mohd Wajid A.; Szakmany, Tamas; Hall, Judith E.; Moser, Bernhard; Eberl, Matthias

    2014-01-01

    The early immune response to microbes is dominated by the recruitment of neutrophils whose primary function is to clear invading pathogens. However, there is emerging evidence that neutrophils play additional effector and regulatory roles. The present study demonstrates that human neutrophils assume antigen cross-presenting functions, and suggests a plausible scenario for the local generation of APC-like neutrophils through the mobilization of unconventional T-cells in response to microbial metabolites. Vγ9/Vδ2 T-cells and MAIT cells are abundant in blood, inflamed tissues and mucosal barriers. Here, both human cell types responded rapidly to neutrophils after phagocytosis of Gram-positive and Gram-negative bacteria producing the corresponding ligands, and in turn mediated the differentiation of neutrophils into APCs for both CD4+ and CD8+ T-cells through secretion of GM-CSF, IFN-γ and TNF-α. In patients with acute sepsis, circulating neutrophils displayed a similar APC-like phenotype and readily processed soluble proteins for cross-presentation of antigenic peptides to CD8+ T-cells, at a time when peripheral Vγ9/Vδ2 T-cells were highly activated. Our findings indicate that unconventional T-cells represent key controllers of neutrophil-driven innate and adaptive responses to a broad range of pathogens. PMID:25165152

  12. Neutrophils rapidly transit inflamed lymphatic vessel endothelium via integrin-dependent proteolysis and lipoxin-induced junctional retraction.

    PubMed

    Rigby, David A; Ferguson, David J P; Johnson, Louise A; Jackson, David G

    2015-12-01

    Neutrophils are the first leukocyte population to be recruited from the circulation following tissue injury or infection, where they play key roles in host defense. However, recent evidence indicates recruited neutrophils can also enter lymph and shape adaptive immune responses downstream in draining lymph nodes. At present, the cellular mechanisms regulating neutrophil entry to lymphatic vessels and migration to lymph nodes are largely unknown. Here, we have investigated these events in an in vivo mouse Mycobacterium bovis bacillus Calmette-Guérin vaccination model, ex vivo mouse dermal explants, and in vitro Transwell system comprising monolayers of primary human dermal lymphatic endothelial cells. We demonstrate that neutrophils are reliant on endothelial activation for adhesion, initially via E-selectin and subsequently, by integrin-mediated binding to ICAM-1 and VCAM-1, combined with CXCL8-dependent chemotaxis. Moreover, we reveal that integrin-mediated neutrophil adhesion plays a pivotal role in subsequent transmigration by focusing the action of matrix metalloproteinases and the 15-lipoxygenase-1-derived chemorepellent 12(S)-hydroxyeicosatetraenoic acid at neutrophil:endothelial contact sites to induce transient endothelial junctional retraction and rapid, selective neutrophil trafficking. These findings reveal an unexpectedly intimate collaboration between neutrophils and the lymphatic vessel endothelium, in which these phagocytic leukocytes act as pathfinders for their own transit during inflammation.

  13. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

    SciTech Connect

    Galkina, Svetlana I. . E-mail: galkina@genebee.msu.su; Sud'ina, Galina F.; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na{sup +} and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl{sup -} efflux through chloride channels and Na{sup +} influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  14. Cyclic oscillation of blood neutrophils in a patient with multiple myeloma

    SciTech Connect

    Chikkappa, G.; Chanana, A.D.; Chandra, P.; Cronkite, E.P.; Thompson, K.H.

    1980-01-01

    A patient with multiple myeloma developed periodic blood neutropenia (periodicity of 15 to 25 days) after 3 yr of intermittent treatment with cytotoxic agents. Peaks of serum colony-stimulating activity (CSA) level coincided with valleys of blood neutrophils. Fraction of marrow neutrophils in the multiplicative pool was high during blood neutrophil valleys and low during neutrophil peaks. In contrast, the maturation storage pool exhibited the reverse pattern. An increased fraction of marrow neutrophilic cells in the multiplicative pool was in active proliferation during a blood neutrophil valley and a decreased fraction during a blood neutrophil peak. These findings suggest that the marrow granulopoiesis was regulated through CSA. The defect causing the periodicity was probably related to the reduced number of neutrophils in the marrow maturation storge pool, which in turn may be related to a reduced and/or defective granulocytic stem cell pool size consiquent to the long-term administration of cytotoxic drugs and/or infiltration of the marrow by myeloma cells.

  15. Persisting and Increasing Neutrophil Infiltration Associates with Gastric Carcinogenesis and E-cadherin Downregulation

    PubMed Central

    Fu, Hualin; Ma, Yue; Yang, Meng; Zhang, Chunlei; Huang, Hai; Xia, Ying; Lu, Lungen; Jin, Weilin; Cui, Daxiang

    2016-01-01

    H. pylori-induced chronic inflammation is considered the most important cause of gastric cancer. The actual process how chronic inflammation triggers gastric carcinogenesis is still not clear. In this study, neutrophils and relative markers in gastric cancer development were examined with immunohistochemistry and fluorescence RNA in situ hybridization methods. On average, 24 times more neutrophils were found in gastric cancer tissues and about 9 times more neutrophils were found in gastric intestinal metaplasia tissues comparing to normal gastric tissue controls. CagA+ H. pylori infection in cancer adjacent tissues or EBV infection in cancer tissues did not increase neutrophil infiltration into gastric cancer tissues significantly. Neutrophil density was positively correlated with cell proliferation while negatively correlated with E-cadherin intensity. E-cadherin is also transcriptionally downregulated in gastric cancer tissues comparing to adjacent tissue controls. The increased neutrophils in the gastric cancer tissues appear to be related to increased chemoattractant IL-8 levels. In gastric cancers, neutrophil numbers were higher comparing to cancer adjacent tissues and not associated with patient ages, tumor invasion depth, tumor staging, metastasis or cancer types. The conclusion is that persisting and increasing neutrophil infiltration is associated with E-cadherin downregulation, cell proliferation and gastric carcinogenesis. PMID:27412620

  16. Nitric Oxide Regulates Neutrophil Migration through Microparticle Formation

    PubMed Central

    Nolan, Sarah; Dixon, Rachel; Norman, Keith; Hellewell, Paul; Ridger, Victoria

    2008-01-01

    The role of nitric oxide (NO) in regulating neutrophil migration has been investigated. Human neutrophil migration to interleukin (IL)-8 (1 nmol/L) was measured after a 1-hour incubation using a 96-well chemotaxis plate assay. The NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) significantly (P < 0.001) enhanced IL-8-induced migration by up to 45%. Anti-CD18 significantly (P < 0.001) inhibited both IL-8-induced and L-NAME enhanced migration. Antibodies to L-selectin or PSGL-1 had no effect on IL-8-induced migration but prevented the increased migration to IL-8 induced by L-NAME. L-NAME induced generation of neutrophil-derived microparticles that was significantly (P < 0.01) greater than untreated neutrophils or D-NAME. This microparticle formation was dependent on calpain activity and superoxide production. Only microparticles from L-NAME and not untreated or D-NAME-treated neutrophils induced a significant (P < 0.01) increase in IL-8-induced migration and transendothelial migration. Pretreatment of microparticles with antibodies to L-selectin (DREG-200) or PSGL-1 (PL-1) significantly (P < 0.001) inhibited this effect. The ability of L-NAME-induced microparticles to enhance migration was found to be dependent on the number of microparticles produced and not an increase in microparticle surface L-selectin or PSGL-1 expression. These data show that NO can modulate neutrophil migration by regulating microparticle formation. PMID:18079439

  17. Effects of immediate postexercise carbohydrate ingestion with and without protein on neutrophil degranulation.

    PubMed

    Costa R, J S; Walters, Robert; Bilzon J, L J; Walsh, Neil P

    2011-06-01

    The purpose of the study was to determine the effects of carbohydrate (CHO) intake, with and without protein (PRO), immediately after prolonged strenuous exercise on circulating bacterially stimulated neutrophil degranulation. Twelve male runners completed 3 feeding interventions, 1 week apart, in randomized order after 2 hr of running at 75% VO2max. The feeding interventions included a placebo solution, a CHO solution equal to 1.2 g CHO/kg body mass (BM), and a CHO-PRO solution equal to 1.2 g CHO/kg BM and 0.4 g PRO/kg BM (CHO+PRO) immediately postexercise. All solutions were flavor and water-volume equivalent (12 ml/kg BM). Circulating leukocyte counts, bacterially stimulated neutrophil degranulation, plasma insulin, and cortisol were determined from blood samples collected preexercise, immediately postexercise, and every 30 min until 180 min postexercise. The immediate postexercise circulating leukocytosis, neutrophilia, and lymphocytosis (p < .01 vs. preexercise) and the delayed lymphopenia (90 min postexercise, p < .05 vs. preexercise) were similar on all trials. Bacterially stimulated neutrophil degranulation decreased during recovery in control (23% at 180 min, p < .01 vs. preexercise) but remained above preexercise levels with CHO and CHO+PRO. In conclusion, CHO ingestion, with or without PRO, immediately after prolonged strenuous exercise prevented the decrease in bacterially stimulated neutrophil degranulation during recovery. PMID:21719901

  18. Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos.

    PubMed

    Paris, Daniel H; Stephan, Femke; Bulder, Ingrid; Wouters, Diana; van der Poll, Tom; Newton, Paul N; Day, Nicholas P J; Zeerleder, Sacha

    2015-01-01

    Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and

  19. Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos

    PubMed Central

    Paris, Daniel H.; Stephan, Femke; Bulder, Ingrid; Wouters, Diana; van der Poll, Tom; Newton, Paul N.; Day, Nicholas P. J.; Zeerleder, Sacha

    2015-01-01

    Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and

  20. IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium.

    PubMed

    Liu, Rebecca; Lauridsen, Holly M; Amezquita, Robert A; Pierce, Richard W; Jane-Wit, Dan; Fang, Caodi; Pellowe, Amanda S; Kirkiles-Smith, Nancy C; Gonzalez, Anjelica L; Pober, Jordan S

    2016-09-15

    A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell-cell interactions of circulating leukocytes with IL-1- or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17-activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space. PMID:27534549

  1. Altered Innate Immune Responses in Neutrophils from Patients with Well- and Suboptimally Controlled Asthma

    PubMed Central

    Tang, Francesca S. M.; Foxley, Gloria J.; Gibson, Peter G.; Burgess, Janette K.; Baines, Katherine J.; Oliver, Brian G.

    2015-01-01

    Background. Respiratory infections are a major cause of asthma exacerbations where neutrophilic inflammation dominates and is associated with steroid refractory asthma. Structural airway cells in asthma differ from nonasthmatics; however it is unknown if neutrophils differ. We investigated neutrophil immune responses in patients who have good (AGood) and suboptimal (ASubopt) asthma symptom control. Methods. Peripheral blood neutrophils from AGood (ACQ < 0.75, n = 11), ASubopt (ACQ > 0.75, n = 7), and healthy controls (HC) (n = 9) were stimulated with bacterial (LPS (1 μg/mL), fMLF (100 nM)), and viral (imiquimod (3 μg/mL), R848 (1.5 μg/mL), and poly I:C (10 μg/mL)) surrogates or live rhinovirus (RV) 16 (MOI1). Cell-free supernatant was collected after 1 h for neutrophil elastase (NE) and matrix metalloproteinase- (MMP-) 9 measurements or after 24 h for CXCL8 release. Results. Constitutive NE was enhanced in AGood neutrophils compared to HC. fMLF stimulated neutrophils from ASubopt but not AGood produced 50% of HC levels. fMLF induced MMP-9 was impaired in ASubopt and AGood compared to HC. fMLF stimulated CXCL8 but not MMP-9 was positively correlated with FEV1 and FEV1/FVC. ASubopt and AGood responded similarly to other stimuli. Conclusions. Circulating neutrophils are different in asthma; however, this is likely to be related to airflow limitation rather than asthma control. PMID:26663987

  2. Gcsf-Chr19 promotes neutrophil migration to damaged tissue through blood vessels in zebrafish.

    PubMed

    Galdames, Jorge A; Zuñiga-Traslaviña, Constanza; Reyes, Ariel E; Feijóo, Carmen G

    2014-07-01

    G-CSF is an essential cytokine that regulates proliferation and differentiation of granulocytes from hematopoietic stem and progenitor cells. In mammals G-CSF has been identified as a key factor that promotes the release of neutrophils from the bone marrow into the blood circulation. In silico analysis indicates that zebrafish has two gcsf genes, gcsf-chr12 in chromosome 12 and gcsf-chr19 in chromosome 19. Gcsf-Chr12 participates in emergency myelopoiesis, but, in contrast to its mammalian orthologue, is not involved in neutrophil migration toward damaged tissue. In turn, the function of Gcsf-Chr19 has not been examined yet. In this study, we analyzed the role of Gcsf-Chr19 in regulating neutrophil migration toward the wound. Our results indicated that during the first h after caudal fin transection, neutrophils migrate from the hematopoietic tissue toward the injury, using the extracellular matrix as a substrate. Later, between 3 and 4 h postdamage, the recruitment mainly occurs through the bloodstream, and only a few neutrophils still use the extracellular matrix to migrate. During this process, the transcriptional levels of gcsf-chr19 are considerably increased, reaching a peak 1 h postdamage. The knockdown of Gcsf-chr19 indicated that the percentage of neutrophils that reach the wound decreased after the first h postinjury, suggesting that the knockdown specifically affects neutrophils that travel to the wound through blood vessels. Together, our data provide novel information about the regulation of neutrophil migration in zebrafish, positioning Gcsf-Chr19 as a key signal during the course of an inflammatory process triggered by severe damage.

  3. Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase.

    PubMed

    Jansen, P M; Pixley, R A; Brouwer, M; de Jong, I W; Chang, A C; Hack, C E; Taylor, F B; Colman, R W

    1996-03-15

    In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in

  4. Real-time detection of implant-associated neutrophil responses using a formyl peptide receptor-targeting NIR nanoprobe

    PubMed Central

    Zhou, Jun; Tsai, Yi-Ting; Weng, Hong; Tang, Ewin N; Nair, Ashwin; Davé, Digant P; Tang, Liping

    2012-01-01

    Neutrophils play an important role in implant-mediated inflammation and infection. Unfortunately, current methods which monitor neutrophil activity, including enzyme measurements and histological evaluation, require many animals and cannot be used to accurately depict the dynamic cellular responses. To understand the neutrophil interactions around implant-mediated inflammation and infection it is critical to develop methods which can monitor in vivo cellular activity in real time. In this study, formyl peptide receptor (FPR)-targeting near-infrared nanoprobes were fabricated. This was accomplished by conjugating near-infrared dye with specific peptides having a high affinity to the FPRs present on activated neutrophils. The ability of FPR-targeting nanoprobes to detect and quantify activated neutrophils was assessed both in vitro and in vivo. As expected, FPR-targeting nanoprobes preferentially accumulated on activated neutrophils in vitro. Following transplantation, FPR-targeting nanoprobes preferentially accumulated at the biomaterial implantation site. Equally important, a strong relationship was observed between the extent of fluorescence intensity in vivo and the number of recruited neutrophils at the implantation site. Furthermore, FPR-targeting nanoprobes may be used to detect and quantify the number of neutrophils responding to a catheter-associated infection. The results show that FPR-targeting nanoprobes may serve as a powerful tool to monitor and measure the extent of neutrophil responses to biomaterial implants in vivo. PMID:22619542

  5. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils.

    PubMed

    Hasan, Md Ashraful; Ahn, Won-Gyun; Song, Dong-Keun

    2016-09-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca(2+) signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca(2+)]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca(2+)]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca(2+)]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca(2+)]i in human neutrophils was observed. In Ca(2+)-free buffer, NAC- and cysteine-induced [Ca(2+)]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca(2+)]i in human neutrophils occur through Ca(2+) influx. NAC- and cysteine-induced [Ca(2+)]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na(+)-free HEPES, both NAC and cysteine induced a marked increase in [Ca(2+)]i in human neutrophils, arguing against the possibility that Na(+)-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca(2+)]i increasing activity. Our results show that NAC and cysteine induce [Ca(2+)]i increase through Ca(2+) influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  6. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

    PubMed Central

    Hasan, Md. Ashraful; Ahn, Won-Gyun

    2016-01-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca2+]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]i increasing activity. Our results show that NAC and cysteine induce [Ca2+]i increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  7. G Protein-Coupled Receptor 43 Modulates Neutrophil Recruitment during Acute Inflammation

    PubMed Central

    Nicholls, Alyce J.; Oliveira, Ana Carolina; Mason, Linda J.; Binge, Lauren; Mackay, Charles R.; Wong, Connie H. Y.

    2016-01-01

    Fermentation of dietary fibre in the gut yields large amounts of short chain fatty acids (SCFAs). SCFAs can impart biological responses in cells through their engagement of ‘metabolite-sensing’ G protein-coupled receptors (GPCRs). One of the main SCFA receptors, GPR43, is highly expressed by neutrophils, which suggests that the actions of GPR43 and dietary fibre intake may affect neutrophil recruitment during inflammatory responses in vivo. Using intravital imaging of the small intestine, we found greater intravascular neutrophil rolling and adhesion in Gpr43−/−mice in response to LPS at 1 h. After 4 h of LPS challenge, the intravascular rolling velocity of GPR43-deficient neutrophils was reduced significantly and increased numbers of neutrophils were found in the lamina propria of Gpr43−/−mice. Additionally, GPR43-deficient leukocytes demonstrated exacerbated migration into the peritoneal cavity following fMLP challenge. The fMLP-induced neutrophil migration was significantly suppressed in wildtype mice that were treated with acetate, but not in Gpr43−/−mice, strongly suggesting a role for SCFAs in modulating neutrophil migration via GPR43. Indeed, neutrophils of no fibre-fed wildtype mice exhibited elevated migratory behaviour compared to normal chow-fed wildtype mice. Interestingly, this elevated migration could also be reproduced through simple transfer of a no fibre microbiota into germ-free mice, suggesting that the composition and function of microbiota stemming from a no fibre diet mediated the changes in neutrophil migration. Therefore, GPR43 and a microbiota composition that allows for SCFA production function to modulate neutrophil recruitment during inflammatory responses. PMID:27658303

  8. Mechanisms of Interferon-γ Production by Neutrophils and Its Function during Streptococcus pneumoniae Pneumonia

    PubMed Central

    Gomez, John C.; Yamada, Mitsuhiro; Martin, Jessica R.; Dang, Hong; Brickey, W. June; Bergmeier, Wolfgang; Dinauer, Mary C.

    2015-01-01

    Bacterial pneumonia is a common public health problem associated with significant mortality, morbidity, and cost. Neutrophils are usually the earliest leukocytes to respond to bacteria in the lungs. Neutrophils rapidly sequester in the pulmonary microvasculature and migrate into the lung parenchyma and alveolar spaces, where they perform numerous effector functions for host defense. Previous studies showed that migrated neutrophils produce IFN-γ early during pneumonia induced by Streptococcus pneumoniae and that early production of IFN-γ regulates bacterial clearance. IFN-γ production by neutrophils requires Rac2, Hck/Lyn/Fgr Src family tyrosine kinases, and NADPH oxidase. Our current studies examined the mechanisms that regulate IFN-γ production by lung neutrophils during acute S. pneumoniae pneumonia in mice and its function. We demonstrate that IFN-γ production by neutrophils is a tightly regulated process that does not require IL-12. The adaptor molecule MyD88 is critical for IFN-γ production by neutrophils. The guanine nucleotide exchange factor CalDAG-GEFI modulates IFN-γ production. The CD11/CD18 complex, CD44, Toll-like receptors 2 and 4, TRIF, and Nrf2 are not required for IFN-γ production by neutrophils. The recently described neutrophil–dendritic cell hybrid cell, identified by its expression of Ly6G and CD11c, is present at low numbers in pneumonic lungs and is not a source of IFN-γ. IFN-γ produced by neutrophils early during acute S. pneumoniae pneumonia induces transcription of target genes in the lungs, which are critical for host defense. These studies underline the complexity of the neutrophil responses during pneumonia in the acute inflammatory response and in subsequent resolution or initiation of immune responses. PMID:25100610

  9. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

    PubMed Central

    Hasan, Md. Ashraful; Ahn, Won-Gyun

    2016-01-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca2+]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]i increasing activity. Our results show that NAC and cysteine induce [Ca2+]i increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

  10. Peripheral circulation.

    PubMed

    Laughlin, M Harold; Davis, Michael J; Secher, Niels H; van Lieshout, Johannes J; Arce-Esquivel, Arturo A; Simmons, Grant H; Bender, Shawn B; Padilla, Jaume; Bache, Robert J; Merkus, Daphne; Duncker, Dirk J

    2012-01-01

    Blood flow (BF) increases with increasing exercise intensity in skeletal, respiratory, and cardiac muscle. In humans during maximal exercise intensities, 85% to 90% of total cardiac output is distributed to skeletal and cardiac muscle. During exercise BF increases modestly and heterogeneously to brain and decreases in gastrointestinal, reproductive, and renal tissues and shows little to no change in skin. If the duration of exercise is sufficient to increase body/core temperature, skin BF is also increased in humans. Because blood pressure changes little during exercise, changes in distribution of BF with incremental exercise result from changes in vascular conductance. These changes in distribution of BF throughout the body contribute to decreases in mixed venous oxygen content, serve to supply adequate oxygen to the active skeletal muscles, and support metabolism of other tissues while maintaining homeostasis. This review discusses the response of the peripheral circulation of humans to acute and chronic dynamic exercise and mechanisms responsible for these responses. This is accomplished in the context of leading the reader on a tour through the peripheral circulation during dynamic exercise. During this tour, we consider what is known about how each vascular bed controls BF during exercise and how these control mechanisms are modified by chronic physical activity/exercise training. The tour ends by comparing responses of the systemic circulation to those of the pulmonary circulation relative to the effects of exercise on the regional distribution of BF and mechanisms responsible for control of resistance/conductance in the systemic and pulmonary circulations.

  11. Subcellular fractionation of human neutrophils and analysis of subcellular markers.

    PubMed

    Clemmensen, Stine Novrup; Udby, Lene; Borregaard, Niels

    2014-01-01

    The neutrophil has long been recognized for its impressive number of cytoplasmic granules that harbor proteins indispensable for innate immunity. Analysis of isolated granules has provided important information on how the neutrophil grades its response to match the challenges it meets on its passage from blood to tissues. Nitrogen cavitation was developed as a method for disruption of cells on the assumption that sudden reduction of the partial pressure of nitrogen would lead to aeration of nitrogen dissolved in the lipid bilayer of plasma membranes. We find that cells are broken by the shear stress that is associated with passage through the outlet valve under high pressure and that this results in disruption of the neutrophil cell membrane while granules remain intact. The unique properties of Percoll as a sedimentable density medium with no inherent tonicity or viscosity are used for creation of continuous density gradients with shoulders in the density profile created to optimize the physical separation of granule subsets and light membranes. Immunological methods (sandwich enzyme-linked immunosorbent assays) are used for quantitation of proteins that are characteristic constituents of the granule subsets of neutrophils. PMID:24504946

  12. Nicotine is Chemotactic for Neutrophils and Enhances Neutrophil Responsiveness to Chemotactic Peptides

    NASA Astrophysics Data System (ADS)

    Totti, Noel; McCusker, Kevin T.; Campbell, Edward J.; Griffin, Gail L.; Senior, Robert M.

    1984-01-01

    Neutrophils contribute to chronic bronchitis and pulmonary emphysema associated with cigarette smoking. Nicotine was found to be chemotactic for human neutrophils but not monocytes, with a peak activity at ~ 31 micromolar. In lower concentrations (comparable to those in smokers' plasma), nicotine enhanced the response of neutrophils to two chemotactic peptides. In contrast to most other chemoattractants for neutrophils, however, nicotine did not affect degranulation or superoxide production. Nicotine thus may promote inflammation and consequent lung injury in smokers.

  13. Biomaterial surface-dependent neutrophil mobility.

    PubMed

    Zhou, Yue; Doerschuk, Claire M; Anderson, James M; Marchant, Roger E

    2004-06-15

    Compromised neutrophil function in the presence of an implanted biomaterial may represent an important mechanism that allows for the development of implant-associated infections. Here, human neutrophil mobility has been investigated on a polyurethane (ChronoFlex AR), a hydrophobic surface consisting of an octadecyltrichlorosilane (OTS) self-assembled monolayer, and a glass reference material. Neutrophil mobility was quantified, based on cell movement speed and persistence time obtained from time-lapse optical microscopy, while neutrophil cytoskeletal structures and morphology were visualized using confocal microscopy and atomic force microscopy. Our results show that material surface properties affect neutrophil-surface interactions, as reflected by morphological changes, and the mobility of neutrophils stimulated by N-formylmethionyl-leucyl-phenylalanine (fMLP). In the absence of adsorbed plasma proteins, the mobility of stimulated neutrophils increased with increasing material hydrophobicity from glass, to polyurethane, to OTS. The opposite trend was observed in the presence of adsorbed plasma proteins, such that neutrophil mobility increased with decreasing material hydrophobicity. Analysis of the results showed that the mobility of fMLP-stimulated neutrophils cells was inversely related to the extent of cell spreading on the materials. PMID:15162402

  14. Coagulation induced by C3aR-dependent NETosis drives protumorigenic neutrophils during small intestinal tumorigenesis

    PubMed Central

    Guglietta, Silvia; Chiavelli, Andrea; Zagato, Elena; Krieg, Carsten; Gandini, Sara; Ravenda, Paola Simona; Bazolli, Barbara; Lu, Bao; Penna, Giuseppe; Rescigno, Maria

    2016-01-01

    Excessive activation of blood coagulation and neutrophil accumulation have been described in several human cancers. However, whether hypercoagulation and neutrophilia are linked and involved in cancer development is currently unknown. Here we show that spontaneous intestinal tumorigenesis correlates with the accumulation of low-density neutrophils with a pro-tumorigenic N2 phenotype and unprompted neutrophil extracellular traps (NET) formation. We find that increased circulating lipopolysaccharide induces upregulation of complement C3a receptor on neutrophils and activation of the complement cascade. This leads to NETosis, induction of coagulation and N2 polarization, which prompts tumorigenesis, showing a novel link between coagulation, neutrophilia and complement activation. Finally, in a cohort of patients with small but not large intestinal cancer, we find a correlation between neutrophilia and hypercoagulation. This study provides a mechanistic explanation for the tumour-promoting effects of hypercoagulation, which could be used as a new biomarker or as a therapeutic target. PMID:26996437

  15. In-vivo blockage of neutrophil migration by LPS is mimicked by a factor released from LPS-stimulated macrophages.

    PubMed Central

    Cunha, F. Q.; Souza, G. E.; Souza, C. A.; Cerqueira, B. C.; Ferreira, S. H.

    1989-01-01

    The present study was performed to determine the effect of an intravenous injection of the macrophage-derived neutrophil chemotactic factor (MNCF) (Cunha & Ferreira 1986) on neutrophil migration to rat peritoneal cavities, which were challenged with chemotactic stimuli. Macrophage monolayers stimulated by LPS release a factor (MW greater than 10,000 D) which, when injected intravenously, blocked neutrophil migration in carrageenin-induced peritonitis. This inhibition was dependent on dose and lasted more than 2 h. It was not due to neutropaenia, hypotension or LPS contamination. Neutrophil migration induced by LPS, MNCF, the Gram-negative bacterium Pseudomonas aeruginosa was also blocked by intravenous administration of the factor. Intravenous injection of recombinant interleukin 1 beta or tumour necrosis factor-alpha, present in the samples of the factor, failed to reproduce the described inhibitory effect on neutrophil migration. The release of this factor by LPS-stimulated macrophage monolayers was inhibited by dexamethasone but not by indomethacin. It is suggested that the failure of neutrophils to migrate during septicaemia may be the result of a continuous release of chemotactic factors in the circulation, particularly of the macrophage-derived neutrophil chemotactic factor(s). PMID:2647118

  16. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with spec...

  17. Rat, mouse and human neutrophils stimulated by a variety of activating agents produce much less nitrite than rodent macrophages.

    PubMed Central

    Padgett, E L; Pruett, S B

    1995-01-01

    The role of reactive nitrogen intermediates (RNI) in the antimicrobial activities of neutrophils from various mammalian species is unclear. However, it has been reported that rodent neutrophils possess the inducible form of nitric oxide synthase and that inflammatory neutrophils from rats produce potentially antimicrobial levels of RNI. In the present study, neutrophils from humans, rats and mice were evaluated for production of nitrite, a stable end-product of RNI. Human neutrophil preparations (> 95% neutrophils) isolated from peripheral blood were stimulated for 2-24 hr with agents known to trigger the Ca(2+)-dependent constitutive nitric oxide synthase, or to stimulate synthesis of the inducible nitric oxide synthase. Superoxide dismutase was added to some cultures to decrease the levels of superoxide, a compound reported to react with RNI and yield products other than nitrite. Even though the cells were viable and responsive to stimuli, they did not produce nitrite concentrations indicative of antimicrobial potential. Preparations of inflammatory (casein-elicited) mouse neutrophils also failed to produce high concentrations of nitrite. Inflammatory rat neutrophils (2.5 x 10(6)/ml) produced nitrite concentrations of approximately 40 microM in 24-hr cultures, but plots of nitrite production versus cell number for neutrophil and macrophage preparations indicated that contaminating macrophages could account for all the nitrite production in the neutrophil preparations. Thus, neutrophils from rats, mice and humans seem comparable in their inability to produce high levels of nitrite in response to a variety of stimuli. This suggests that in most circumstances the constitutive nitric oxide synthase known to be present in these cells is limited to the production of low levels of nitric oxide for intercellular signalling. In addition, this raises questions about the presence or functional status of inducible nitric oxide synthase in rodent neutrophils. PMID:7534260

  18. Neutrophil Crawling in Capillaries; A Novel Immune Response to Staphylococcus aureus

    PubMed Central

    Harding, Mark Geoffrey; Zhang, Kunyan; Conly, John; Kubes, Paul

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA), particularly the USA300 strain, is a highly virulent pathogen responsible for an increasing number of skin and soft tissue infections globally. Furthermore, MRSA-induced soft tissue infections can rapidly progress into life-threatening conditions, such as sepsis and necrotizing fasciitis. The importance of neutrophils in these devastating soft tissue infections remains ambiguous, partly because of our incomplete understanding of their behaviour. Spinning disk confocal microscopy was used to visualize the behaviour of GR1-labelled neutrophils in subcutaneous tissue in response to GFP-expressing MRSA attached to a foreign particle (agarose bead). We observed significant directional neutrophil recruitment towards the S. aureus agarose bead but not a control agarose bead. A significant increase in neutrophil crawling within the capillaries surrounding the infectious nidus was noted, with impaired capillary perfusion in these vessels and increased parenchymal cell death. No neutrophils were able to emigrate from capillaries. The crawling within these capillaries was mediated by the β2 and α4 integrins and blocking these integrins 2 hours post infection eliminated neutrophil crawling, improved capillary perfusion, reduced cell death and reduced lesion size. Blocking prior to infection increased pathology. Neutrophil crawling within capillaries during MRSA soft tissue infections, while potentially contributing to walling off or preventing early dissemination of the pathogen, resulted in impaired perfusion and increased tissue injury with time. PMID:25299673

  19. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    PubMed

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

  20. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    PubMed Central

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  1. Chemotactic response of human neutrophils to N-acetyl chitohexaose in vitro.

    PubMed

    Tokoro, A; Suzuki, K; Matsumoto, T; Mikami, T; Suzuki, S; Suzuki, M

    1988-01-01

    N-Acetyl chitohexaose (NACOS-6) was able to display chemotactic response of human neutrophils in vitro. In order to analyze the mechanism, a series of chemotaxis studies by means of neutrophils treated with inhibitors of phospholipase A2, cyclooxygenase, or lipoxygenase to NACOS-6 was conducted. The treatment of neutrophils with inhibitors of phospholipase A2 or cyclooxygenase resulted in decrease of number of migrated cells. However, the lipoxygenase inhibitors did not exhibit the same effect. On the other hand, the treatment of neutrophils with inhibitors of phospholipase A2 or lipoxygenase resulted in decrease of chemotactic response to Formyl-Met-Leu-Phe (FMLP), although the cyclooxygenase inhibitors did not inhibit chemotaxis of neutrophils. Neutrophils added to exogenous prostaglandin E2 (PGE2) caused an enhanced chemotactic response to NACOS-6. These results indicate that the mechanism of chemotactic response to NACOS-6 was different from that of FMLP, and that the response was enhanced by PGE2 released from the neutrophils with stimulation of NACOS-6.

  2. The effects of extracellular matrix proteins on neutrophil-endothelial interaction--a roadway to multiple therapeutic opportunities.

    PubMed

    Padmanabhan, Jagannath; Gonzalez, Anjelica L

    2012-06-01

    Polymorphoneuclear leukocytes or neutrophils, a major component of white blood cells, contribute to the innate immune response in humans. Upon sensing changes in the microenvironment, neutrophils adhere to the vascular wall, migrate through the endothelial cell (EC)-pericyte bilayer, and subsequently through the extracellular matrix to reach the site of inflammation. These cells are capable of destroying microbes, cell debris, and foreign proteins by oxidative and non-oxidative processes. While primarily mediators of tissue homeostasis, there are an increasing number of studies indicating that neutrophil recruitment and transmigration can also lead to host-tissue injury and subsequently inflammation-related diseases. Neutrophil-induced tissue injury is highly regulated by the microenvironment of the infiltrated tissue, which includes cytokines, chemokines, and the provisional extracellular matrix, remodeled through increased vascular permeability and other cellular infiltrates. Thus, investigation of the effects of matrix proteins on neutrophil-EC interaction and neutrophil transmigration may help identify the proteins that induce pro- or anti-inflammatory responses. This area of research presents an opportunity to identify therapeutic targets in inflammation-related diseases. This review will summarize recent literature on the role of neutrophils and the effects of matrix proteins on neutrophil-EC interactions, with focus on three different disease models: 1) atherosclerosis, 2) COPD, and 3) tumor growth and progression. For each disease model, inflammatory molecules released by neutrophils, important regulatory matrix proteins, current anti-inflammatory treatments, and the scope for further research will be summarized.

  3. A Radical Break: Restraining Neutrophil Migration.

    PubMed

    Renkawitz, Jörg; Sixt, Michael

    2016-09-12

    When neutrophils infiltrate a site of inflammation, they have to stop at the right place to exert their effector function. In this issue of Developmental Cell, Wang et al. (2016) show that neutrophils sense reactive oxygen species via the TRPM2 channel to arrest migration at their target site. PMID:27623379

  4. Mechanotransduction in neutrophil activation and deactivation.

    PubMed

    Ekpenyong, Andrew E; Toepfner, Nicole; Chilvers, Edwin R; Guck, Jochen

    2015-11-01

    Mechanotransduction refers to the processes through which cells sense mechanical stimuli by converting them to biochemical signals and, thus, eliciting specific cellular responses. Cells sense mechanical stimuli from their 3D environment, including the extracellular matrix, neighboring cells and other mechanical forces. Incidentally, the emerging concept of mechanical homeostasis,long term or chronic regulation of mechanical properties, seems to apply to neutrophils in a peculiar manner, owing to neutrophils' ability to dynamically switch between the activated/primed and deactivated/deprimed states. While neutrophil activation has been known for over a century, its deactivation is a relatively recent discovery. Even more intriguing is the reversibility of neutrophil activation and deactivation. We review and critically evaluate recent findings that suggest physiological roles for neutrophil activation and deactivation and discuss possible mechanisms by which mechanical stimuli can drive the oscillation of neutrophils between the activated and resting states. We highlight several molecules that have been identified in neutrophil mechanotransduction, including cell adhesion and transmembrane receptors, cytoskeletal and ion channel molecules. The physiological and pathophysiological implications of such mechanically induced signal transduction in neutrophils are highlighted as a basis for future work. This article is part of a Special Issue entitled: Mechanobiology. PMID:26211453

  5. Neutrophil survival in the death zone.

    PubMed

    Croker, Ben A

    2014-01-16

    In this issue of Blood, Thompson et al reveal a key role for hypoxia-inducible factor (HIF)-2a in the adaptation of neutrophils to hypoxia. Tissue hypoxia is a common feature of trauma and inflammation. Infiltrating neutrophils must adapt to this low-oxygen environment to satisfy the metabolic and functional demands of an immune response.

  6. Chronic neutrophilic leukaemia and plasma cell-related neutrophilic leukaemoid reactions.

    PubMed

    Bain, Barbara J; Ahmad, Shahzaib

    2015-11-01

    Many cases reported as 'chronic neutrophilic leukaemia' have had an associated plasma cell neoplasm. Recent evidence suggests that the great majority of such cases represent a neutrophilic leukaemoid reaction to the underlying multiple myeloma or monoclonal gammopathy of undetermined significance. We have analysed all accessible reported cases to clarify the likely diagnosis and to ascertain whether toxic granulation, Döhle bodies and an increased neutrophil alkaline phosphatase score were useful in making a distinction between chronic neutrophilic leukaemia and a neutrophilic leukaemoid reaction. We established that all these changes occur in both conditions. Toxic granulation and Döhle bodies are more consistently present in leukaemoid reactions but also occur quite frequently in chronic neutrophilic leukaemia. The neutrophil alkaline phosphatase score is increased in both conditions and is of no value in making a distinction.

  7. IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa.

    PubMed

    Altmeier, Simon; Toska, Albulena; Sparber, Florian; Teijeira, Alvaro; Halin, Cornelia; LeibundGut-Landmann, Salomé

    2016-09-01

    Mucosal infections with Candida albicans belong to the most frequent forms of fungal diseases. Host protection is conferred by cellular immunity; however, the induction of antifungal immunity is not well understood. Using a mouse model of oropharyngeal candidiasis (OPC) we show that interleukin-1 receptor (IL-1R) signaling is critical for fungal control at the onset of infection through its impact on neutrophils at two levels. We demonstrate that both the recruitment of circulating neutrophils to the site of infection and the mobilization of newly generated neutrophils from the bone marrow depended on IL-1R. Consistently, IL-1R-deficient mice displayed impaired chemokine production at the site of infection and defective secretion of granulocyte colony-stimulating factor (G-CSF) in the circulation in response to C. albicans. Strikingly, endothelial cells were identified as the primary cellular source of G-CSF during OPC, which responded to IL-1α that was released from keratinocytes in the infected tissue. The IL-1-dependent crosstalk between two different cellular subsets of the nonhematopoietic compartment was confirmed in vitro using a novel murine tongue-derived keratinocyte cell line and an established endothelial cell line. These data establish a new link between IL-1 and granulopoiesis in the context of fungal infection. Together, we identified two complementary mechanisms coordinating the neutrophil response in the oral mucosa, which is critical for preventing fungal growth and dissemination, and thus protects the host from disease. PMID:27632536

  8. IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa

    PubMed Central

    Altmeier, Simon; Toska, Albulena; Sparber, Florian; Teijeira, Alvaro; Halin, Cornelia; LeibundGut-Landmann, Salomé

    2016-01-01

    Mucosal infections with Candida albicans belong to the most frequent forms of fungal diseases. Host protection is conferred by cellular immunity; however, the induction of antifungal immunity is not well understood. Using a mouse model of oropharyngeal candidiasis (OPC) we show that interleukin-1 receptor (IL-1R) signaling is critical for fungal control at the onset of infection through its impact on neutrophils at two levels. We demonstrate that both the recruitment of circulating neutrophils to the site of infection and the mobilization of newly generated neutrophils from the bone marrow depended on IL-1R. Consistently, IL-1R-deficient mice displayed impaired chemokine production at the site of infection and defective secretion of granulocyte colony-stimulating factor (G-CSF) in the circulation in response to C. albicans. Strikingly, endothelial cells were identified as the primary cellular source of G-CSF during OPC, which responded to IL-1α that was released from keratinocytes in the infected tissue. The IL-1-dependent crosstalk between two different cellular subsets of the nonhematopoietic compartment was confirmed in vitro using a novel murine tongue-derived keratinocyte cell line and an established endothelial cell line. These data establish a new link between IL-1 and granulopoiesis in the context of fungal infection. Together, we identified two complementary mechanisms coordinating the neutrophil response in the oral mucosa, which is critical for preventing fungal growth and dissemination, and thus protects the host from disease. PMID:27632536

  9. The regulatory role of serum response factor pathway in neutrophil inflammatory response

    PubMed Central

    Taylor, Ashley; Halene, Stephanie

    2015-01-01

    Purpose of review Neutrophils rapidly migrate to sites of injury and infection. Egress of neutrophils from the circulation into tissues is a highly regulated process involving several distinct steps. Cell–cell interactions mediated by selectins and integrins and reorganization of the actin cytoskeleton are key mechanisms facilitating appropriate neutrophil recruitment. Neutrophil function is impaired in inherited and acquired disorders, such as leukocyte adhesion deficiency and myelodysplasia. Since the discovery that deletion of all or part of chromosome 5 is the most common genetic aberration in myelodysplasia, the roles of several of the deleted genes have been investigated in hematopoiesis. Several genes encoding proteins of the serum response factor (SRF) pathway are located on 5q. This review focuses, in particular, on the role of SRF in myeloid maturation and neutrophil function. Recent findings SRF and its pathway fulfill multiple complex roles in the regulation of the innate and adaptive immune system. Loss of SRF leads to defects in B-cell and T-cell development. SRF-deficient macrophages fail to spread, transmigrate, and phagocytose bacteria, and SRF-deficient neutrophils show defective chemotaxis in vitro and in vivo with failure of inside-out activation and trafficking of the Mac1 integrin complex. Loss of the formin mammalian Diaphanous 1, a regulator of linear actin polymerization and mediator of Ras homolog family member A signaling to SRF, results in aberrant myeloid differentiation and hyperactivity of the immune system. Summary SRF is an essential transcription factor in hematopoiesis and mature myeloid cell function. SRF regulates neutrophil migration, integrin activation, and trafficking. Disruption of the SRF pathway results in myelodysplasia and immune dysfunction. PMID:25402621

  10. Role of Transient Receptor Potential Vanilloid 4 in Neutrophil Activation and Acute Lung Injury.

    PubMed

    Yin, Jun; Michalick, Laura; Tang, Christine; Tabuchi, Arata; Goldenberg, Neil; Dan, Qinghong; Awwad, Khader; Wang, Liming; Erfinanda, Lasti; Nouailles, Geraldine; Witzenrath, Martin; Vogelzang, Alexis; Lv, Lu; Lee, Warren L; Zhang, Haibo; Rotstein, Ori; Kapus, Andras; Szaszi, Katalin; Fleming, Ingrid; Liedtke, Wolfgang B; Kuppe, Hermann; Kuebler, Wolfgang M

    2016-03-01

    The cation channel transient receptor potential vanilloid (TRPV) 4 is expressed in endothelial and immune cells; however, its role in acute lung injury (ALI) is unclear. The functional relevance of TRPV4 was assessed in vivo, in isolated murine lungs, and in isolated neutrophils. Genetic deficiency of TRPV4 attenuated the functional, histological, and inflammatory hallmarks of acid-induced ALI. Similar protection was obtained with prophylactic administration of the TRPV4 inhibitor, GSK2193874; however, therapeutic administration of the TRPV4 inhibitor, HC-067047, after ALI induction had no beneficial effect. In isolated lungs, platelet-activating factor (PAF) increased vascular permeability in lungs perfused with trpv4(+/+) more than with trpv4(-/-) blood, independent of lung genotype, suggesting a contribution of TRPV4 on blood cells to lung vascular barrier failure. In neutrophils, TRPV4 inhibition or deficiency attenuated the PAF-induced increase in intracellular calcium. PAF induced formation of epoxyeicosatrienoic acids by neutrophils, which, in turn, stimulated TRPV4-dependent Ca(2+) signaling, whereas inhibition of epoxyeicosatrienoic acid formation inhibited the Ca(2+) response to PAF. TRPV4 deficiency prevented neutrophil responses to proinflammatory stimuli, including the formation of reactive oxygen species, neutrophil adhesion, and chemotaxis, putatively due to reduced activation of Rac. In chimeric mice, however, the majority of protective effects in acid-induced ALI were attributable to genetic deficiency of TRPV4 in parenchymal tissue, whereas TRPV4 deficiency in circulating blood cells primarily reduced lung myeloperoxidase activity. Our findings identify TRPV4 as novel regulator of neutrophil activation and suggest contributions of both parenchymal and neutrophilic TRPV4 in the pathophysiology of ALI.

  11. Role of Transient Receptor Potential Vanilloid 4 in Neutrophil Activation and Acute Lung Injury.

    PubMed

    Yin, Jun; Michalick, Laura; Tang, Christine; Tabuchi, Arata; Goldenberg, Neil; Dan, Qinghong; Awwad, Khader; Wang, Liming; Erfinanda, Lasti; Nouailles, Geraldine; Witzenrath, Martin; Vogelzang, Alexis; Lv, Lu; Lee, Warren L; Zhang, Haibo; Rotstein, Ori; Kapus, Andras; Szaszi, Katalin; Fleming, Ingrid; Liedtke, Wolfgang B; Kuppe, Hermann; Kuebler, Wolfgang M

    2016-03-01

    The cation channel transient receptor potential vanilloid (TRPV) 4 is expressed in endothelial and immune cells; however, its role in acute lung injury (ALI) is unclear. The functional relevance of TRPV4 was assessed in vivo, in isolated murine lungs, and in isolated neutrophils. Genetic deficiency of TRPV4 attenuated the functional, histological, and inflammatory hallmarks of acid-induced ALI. Similar protection was obtained with prophylactic administration of the TRPV4 inhibitor, GSK2193874; however, therapeutic administration of the TRPV4 inhibitor, HC-067047, after ALI induction had no beneficial effect. In isolated lungs, platelet-activating factor (PAF) increased vascular permeability in lungs perfused with trpv4(+/+) more than with trpv4(-/-) blood, independent of lung genotype, suggesting a contribution of TRPV4 on blood cells to lung vascular barrier failure. In neutrophils, TRPV4 inhibition or deficiency attenuated the PAF-induced increase in intracellular calcium. PAF induced formation of epoxyeicosatrienoic acids by neutrophils, which, in turn, stimulated TRPV4-dependent Ca(2+) signaling, whereas inhibition of epoxyeicosatrienoic acid formation inhibited the Ca(2+) response to PAF. TRPV4 deficiency prevented neutrophil responses to proinflammatory stimuli, including the formation of reactive oxygen species, neutrophil adhesion, and chemotaxis, putatively due to reduced activation of Rac. In chimeric mice, however, the majority of protective effects in acid-induced ALI were attributable to genetic deficiency of TRPV4 in parenchymal tissue, whereas TRPV4 deficiency in circulating blood cells primarily reduced lung myeloperoxidase activity. Our findings identify TRPV4 as novel regulator of neutrophil activation and suggest contributions of both parenchymal and neutrophilic TRPV4 in the pathophysiology of ALI. PMID:26222277

  12. Volatile organic compounds discriminate between eosinophilic and neutrophilic inflammation in vitro.

    PubMed

    Schleich, Florence N; Dallinga, Jan W; Henket, Monique; Wouters, Emiel F M; Louis, Renaud; Van Schooten, Frederik J

    2016-03-01

    Inflammation associated oxidative stress leads to peroxidation of polyunsaturated fatty acids thereby generating volatile organic compounds (VOCs). The integrative analysis of the total amount of VOCs released by eosinophils and neutrophils in vitro enables the search for those compounds that discriminates between various inflammatory conditions. The approach comprises isolating eosinophils and neutrophils from 30 ml of blood of healthy non-smoking volunteers by gradient centrifugation, using lymphoprep. Eosinophils are separated from neutrophils by immunomagnetic cell separation using anti-CD16. Cells are activated with phorbol 12-myristate 13-acetate and VOCs from the headspace are collected at time 0', 30', 60' and 90' by introduction of ultra-pure nitrogen in the closed flasks at a flow rate of 200 ml min(-1) during 10 min. The gases are trapped onto a sorption tube and analyzed by gas chromatography-time-of-flight-mass spectometry (GC-TOF-MS) in order to identify VOCs released in the headspace by activated neutrophils and eosinophils. Eosinophils and neutrophils were isolated from 26 healthy non-smoking volunteers. The average absolute number of eosinophils and neutrophils upon isolation was 3.5  ×  10(6) and 19.4  ×  10(6), respectively. The volatome in headspace consisted of 2116 compounds and those compounds present in at least 8% of the samples (1123 compounds) were used for further discriminant analysis. Discriminant analysis showed that two VOCs were able to distinguish between eosinophilic and neutrophilic cultures in the unactivated state with 100% correct classification of the entire data set and upon cross validation while five VOCs were able to discriminate between activated eosinophils and neutrophils with 96% correct classification in the original set and upon cross-validation. Analysis of VOCs seems to be a very promising approach in identifying eosinophilic and neutrophilic inflammation but it needs further development

  13. Volatile organic compounds discriminate between eosinophilic and neutrophilic inflammation in vitro.

    PubMed

    Schleich, Florence N; Dallinga, Jan W; Henket, Monique; Wouters, Emiel F M; Louis, Renaud; Van Schooten, Frederik J

    2016-02-01

    Inflammation associated oxidative stress leads to peroxidation of polyunsaturated fatty acids thereby generating volatile organic compounds (VOCs). The integrative analysis of the total amount of VOCs released by eosinophils and neutrophils in vitro enables the search for those compounds that discriminates between various inflammatory conditions. The approach comprises isolating eosinophils and neutrophils from 30 ml of blood of healthy non-smoking volunteers by gradient centrifugation, using lymphoprep. Eosinophils are separated from neutrophils by immunomagnetic cell separation using anti-CD16. Cells are activated with phorbol 12-myristate 13-acetate and VOCs from the headspace are collected at time 0', 30', 60' and 90' by introduction of ultra-pure nitrogen in the closed flasks at a flow rate of 200 ml min(-1) during 10 min. The gases are trapped onto a sorption tube and analyzed by gas chromatography-time-of-flight-mass spectometry (GC-TOF-MS) in order to identify VOCs released in the headspace by activated neutrophils and eosinophils. Eosinophils and neutrophils were isolated from 26 healthy non-smoking volunteers. The average absolute number of eosinophils and neutrophils upon isolation was 3.5  ×  10(6) and 19.4  ×  10(6), respectively. The volatome in headspace consisted of 2116 compounds and those compounds present in at least 8% of the samples (1123 compounds) were used for further discriminant analysis. Discriminant analysis showed that two VOCs were able to distinguish between eosinophilic and neutrophilic cultures in the unactivated state with 100% correct classification of the entire data set and upon cross validation while five VOCs were able to discriminate between activated eosinophils and neutrophils with 96% correct classification in the original set and upon cross-validation. Analysis of VOCs seems to be a very promising approach in identifying eosinophilic and neutrophilic inflammation but it needs further development

  14. Febrile-range hyperthermia augments pulmonary neutrophil recruitment and amplifies pulmonary oxygen toxicity.

    PubMed

    Hasday, Jeffrey D; Garrison, Allen; Singh, Ishwar S; Standiford, Theodore; Ellis, Garrettson S; Rao, Srinivas; He, Ju-Ren; Rice, Penny; Frank, Mariah; Goldblum, Simeon E; Viscardi, Rose M

    2003-06-01

    Febrile-range hyperthermia (FRH) improves survival in experimental infections by accelerating pathogen clearance, but may also increase collateral tissue injury. We hypothesized that FRH would worsen the outcome of inflammation stimulated by a non-replicating agonist and tested this hypothesis in a murine model of pulmonary oxygen toxicity. Using a conscious, temperature-controlled mouse model, we showed that maintaining a core temperature at FRH (39 degrees C to 40 degrees C) rather than at euthermic levels (36.5 degrees C to 37 degrees C) during hyperoxia exposure accelerated lethal pulmonary vascular endothelial injury, reduced the inspired oxygen threshold for lethality, induced expression of granulocyte-colony stimulating factor, and expanded the circulating neutrophil pool. In these same mice, FRH augmented pulmonary expression of the ELR(+) CXC chemokines, KC and LPS-induced CXC chemokine, enhanced recruitment of neutrophils, and changed the histological pattern of lung injury to a neutrophilic interstitial pneumonitis. Immunoblockade of CXC receptor-2 abrogated neutrophil recruitment, reduced pulmonary vascular injury, and delayed death. These combined data demonstrate that FRH may enlist distinct mediators and effector cells to profoundly shift the host response to a defined injurious stimulus, in part by augmenting delivery of neutrophils to sites of inflammation, such as may occur in infections. In certain conditions, such as in the hyperoxic lung, this process may be deleterious.

  15. [The intracellular metabolism of neutrophils under different forms of ischemic heart disease].

    PubMed

    Kratnov, A E

    2012-12-01

    The article deals with the results of study of the intracellular metabolism of neutrophils in 96 patients including 56 (58.3%) patients with ischemic heart disease. It is established that in patients with ischemic heart disease as compared with patients without ischemic heart disease occurs the increase of neutrophils producing of active forms of oxygen against the background of decrease of intracellular activity of catalase and increase of concentration of lactate in phagocytes. Under severing of ischemic heart disease from unstable stenocardia to cardiac infarction the activation of oxygen-depending metabolism of neutrophils conditioned by increase of concentration of immune complexes circulating in blood was not compensated by increasing of intracellular activity of enzymes of system of antioxidant defense. The maximum activation of NAD(F)N-oxidase of neutrophils (production of superoxide anion-radical) was detected in patients with cardiac infarction with Q-wave. This condition was accompanied by maximal increase of concentration of lactate in phagocytes up to twice higher exceed the level of indicators in patients without ischemic heart disease. It can be assumed that in conditions of acute hypoxia occurring under myocardium ischemia in patients with ischemic heart disease, the lactate production increases in neutrophils hence the triggering of development of intracellular acidosis, impact of oxygen-depending factors and phagocytes destruction.

  16. Superoxide Induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-Dependent Mechanism

    PubMed Central

    Al-Khafaji, Ahmed B; Tohme, Samer; Yazdani, Hamza Obaid; Miller, David; Huang, Hai; Tsung, Allan

    2016-01-01

    Neutrophils constitute the early innate immune response to perceived infectious and sterile threats. Neutrophil extracellular traps (NETs) are a novel mechanism to counter pathogenic invasion and sequelae of ischemia, including cell death and oxidative stress. Superoxide is a radical intermediate of oxygen metabolism produced by parenchymal and nonparenchymal hepatic cells, and is a hallmark of oxidative stress after liver ischemia-reperfusion (I/R). While extracellular superoxide recruits neutrophils to the liver and initiates sterile inflammatory injury, it is unknown whether superoxide induces the formation of NETs. We hypothesize that superoxide induces NET formation through a signaling cascade involving Toll-like receptor 4 (TLR-4) and neutrophil NADPH oxidase (NOX). We treated neutrophils with extracellular superoxide and observed NET DNA release, histone H3 citrullination and increased levels of MPO-DNA complexes occurring in a TLR-4–dependent manner. Inhibition of superoxide generation by allopurinol and inhibition of NOX by diphenyleneiodonium prevented NET formation. When mice were subjected to warm liver I/R, we found significant NET formation associated with liver necrosis and increased serum ALT in TLR-4 WT but not TLR-4 KO mice. To reduce circulating superoxide, we pretreated mice undergoing I/R with allopurinol and N-acetylcysteine, which resulted in decreased NETs and ameliorated liver injury. Our study demonstrates a requirement for TLR-4 and NOX in superoxide-induced NETs, and suggests involvement of superoxide-induced NETs in pathophysiologic settings. PMID:27453505

  17. Neutrophil uptake of vaccinia virus in vitro

    SciTech Connect

    West, B.C.; Eschete, M.L.; Cox, M.E.; King, J.W.

    1987-10-01

    We studied human neutrophils for uptake of vaccinia virus. Uptake was determined radiometrically and by electron microscopy. Vaccinia virus was labeled with /sup 14/C or /sup 3/H, incubated with neutrophils, and quantified in neutrophil pellets in a new radiometric phagocytosis assay. Better results were obtained from assays of (/sup 3/H)thymidine-labeled virus; uptake increased through 1 hr and then plateaued. Phagocytosis of 3H-labeled Staphylococcus aureus was normal. Uptake of virus was serum dependent. Hexose monophosphate shunt activity was measured by two methods. No /sup 14/CO/sub 2/ from (/sup 14/C)1-glucose accompanied uptake of vaccinia virus, in contrast to the respiratory burst accompanying bacterial phagocytosis. Electron microscopy showed intact to slightly digested intraphagolysosomal vaccinia virus. Pock reduction assay showed a decrease in viral content due to neutrophils until 6 hr of incubation, when a modest but significant increase was observed. Thus, neutrophil uptake of vaccinia virus is distinguished from bacterial phagocytosis.

  18. Dynamic interactions of neutrophils and biofilms

    PubMed Central

    Hirschfeld, Josefine

    2014-01-01

    Background The majority of microbial infections in humans are biofilm-associated and difficult to treat, as biofilms are highly resistant to antimicrobial agents and protect themselves from external threats in various ways. Biofilms are tenaciously attached to surfaces and impede the ability of host defense molecules and cells to penetrate them. On the other hand, some biofilms are beneficial for the host and contain protective microorganisms. Microbes in biofilms express pathogen-associated molecular patterns and epitopes that can be recognized by innate immune cells and opsonins, leading to activation of neutrophils and other leukocytes. Neutrophils are part of the first line of defense and have multiple antimicrobial strategies allowing them to attack pathogenic biofilms. Objective/design In this paper, interaction modes of neutrophils with biofilms are reviewed. Antimicrobial strategies of neutrophils and the counteractions of the biofilm communities, with special attention to oral biofilms, are presented. Moreover, possible adverse effects of neutrophil activity and their biofilm-promoting side effects are discussed. Results/conclusion Biofilms are partially, but not entirely, protected against neutrophil assault, which include the processes of phagocytosis, degranulation, and formation of neutrophil extracellular traps. However, virulence factors of microorganisms, microbial composition, and properties of the extracellular matrix determine whether a biofilm and subsequent microbial spread can be controlled by neutrophils and other host defense factors. Besides, neutrophils may inadvertently contribute to the physical and ecological stability of biofilms by promoting selection of more resistant strains. Moreover, neutrophil enzymes can degrade collagen and other proteins and, as a result, cause harm to the host tissues. These parameters could be crucial factors in the onset of periodontal inflammation and the subsequent tissue breakdown. PMID:25523872

  19. A Novel Mechanism of Neutrophil Recruitment in a Coculture Model of the Rheumatoid Synovium

    PubMed Central

    Lally, Frank; Smith, Emily; Filer, Andrew; Stone, Michael A.; Shaw, John S.; Nash, Gerard B.; Buckley, Christopher D.; Rainger, G. Ed

    2011-01-01

    Objective Rheumatoid arthritis (RA) is classically thought of as a Th1, T lymphocyte–driven disease of the adaptive immune system. However, cells of the innate immune system, including neutrophils, are prevalent within the diseased joint, and accumulate in large numbers. This study was undertaken to determine whether cells of the rheumatoid stromal microenvironment could establish an inflammatory environment in which endothelial cells are conditioned in a disease-specific manner to support neutrophil recruitment. Methods Human umbilical vein endothelial cells (ECs) and fibroblasts isolated from the synovium or skin of RA patients were established in coculture on opposite sides of porous transwell filters. After 24 hours of EC conditioning, the membranes were incorporated into a parallel-plate, flow-based adhesion assay and levels of neutrophil adhesion to ECs were measured. Results ECs cocultured with synovial, but not skin, fibroblasts could recruit neutrophils in a manner that was dependent on the number of fibroblasts. Antibody blockade of P-selectin or E-selectin reduced neutrophil adhesion, and an antibody against CD18 (the β2 integrin) abolished adhesion. Blockade of CXCR2, but not CXCR1, also greatly inhibited neutrophil recruitment. Interleukin-6 (IL-6) was detectable in coculture supernatants, and both IL-6 and neutrophil adhesion were reduced in a dose-dependent manner by hydrocortisone added to cocultures. Antibody blockade of IL-6 also effectively abolished neutrophil adhesion. Conclusion Synovial fibroblasts from the rheumatoid joint play an important role in regulating the recruitment of inflammatory leukocytes during active disease. This process may depend on a previously unsuspected route of IL-6–mediated crosstalk between fibroblasts and endothelial cells. PMID:16255036

  20. Cytokines Induced Neutrophil Extracellular Traps Formation: Implication for the Inflammatory Disease Condition

    PubMed Central

    Keshari, Ravi S.; Jyoti, Anupam; Dubey, Megha; Kothari, Nikhil; Kohli, Monica; Bogra, Jaishri; Barthwal, Manoj K.; Dikshit, Madhu

    2012-01-01

    Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects. PMID:23110185

  1. Cigarette smoke-induced damage-associated molecular pattern release from necrotic neutrophils triggers proinflammatory mediator release.

    PubMed

    Heijink, Irene H; Pouwels, Simon D; Leijendekker, Carin; de Bruin, Harold G; Zijlstra, G Jan; van der Vaart, Hester; ten Hacken, Nick H T; van Oosterhout, Antoon J M; Nawijn, Martijn C; van der Toorn, Marco

    2015-05-01

    Cigarette smoking, the major causative factor for the development of chronic obstructive pulmonary disease, is associated with neutrophilic airway inflammation. Cigarette smoke (CS) exposure can induce a switch from apoptotic to necrotic cell death in airway epithelium. Therefore, we hypothesized that CS promotes neutrophil necrosis with subsequent release of damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1), alarming the innate immune system. We studied the effect of smoking two cigarettes on sputum neutrophils in healthy individuals and of 5-day CS or air exposure on neutrophil counts, myeloperoxidase, and HMGB1 levels in bronchoalveolar lavage fluid of BALB/c mice. In human peripheral blood neutrophils, mitochondrial membrane potential, apoptosis/necrosis markers, caspase activity, and DAMP release were studied after CS exposure. Finally, we assessed the effect of neutrophil-derived supernatants on the release of chemoattractant CXCL8 in normal human bronchial epithelial cells. Cigarette smoking caused a significant decrease in sputum neutrophil numbers after 3 hours. In mice, neutrophil counts were significantly increased 16 hours after repeated CS exposure but reduced 2 hours after an additional exposure. In vitro, CS induced necrotic neutrophil cell death, as indicated by mitochondrial dysfunction, inhibition of apoptosis, and DAMP release. Supernatants from CS-treated neutrophils significantly increased the release of CXCL8 in normal human bronchial epithelial cells. Together, these observations show, for the first time, that CS exposure induces neutrophil necrosis, leading to DAMP release, which may amplify CS-induced airway inflammation by promoting airway epithelial proinflammatory responses. PMID:25192219

  2. Proliferating cell nuclear antigen in neutrophil fate.

    PubMed

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  3. Candida albicans escapes from mouse neutrophils.

    PubMed

    Ermert, David; Niemiec, Maria J; Röhm, Marc; Glenthøj, Andreas; Borregaard, Niels; Urban, Constantin F

    2013-08-01

    Candida albicans, the most commonly isolated human fungal pathogen, is able to grow as budding yeasts or filamentous forms, such as hyphae. The ability to switch morphology has been attributed a crucial role for the pathogenesis of C. albicans. To mimic disseminated candidiasis in humans, the mouse is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed that murine neutrophils exhibited a significantly lower ability to kill C. albicans than their human counterparts. Strikingly, C. albicans yeast cells formed germ tubes upon internalization by murine neutrophils, eventually rupturing the neutrophil membrane and thereby, killing the phagocyte. On the contrary, growth and subsequent escape of C. albicans are blocked inside human neutrophils. According to our findings, this blockage in human neutrophils might be a result of higher levels of MPO activity and the presence of α-defensins. We therefore outline differences in antifungal immune defense between humans and mouse strains, which facilitates a more accurate interpretation of in vivo results.

  4. In vivo glucocorticoid effects on porcine natural killer cell activity and circulating leukocytes.

    PubMed

    Salak-Johnson, J L; McGlone, J J; Norman, R L

    1996-03-01

    Porcine natural killer (NK) cell cytotoxicity, plasma cortisol, total white blood cells (WBC), neutrophil:lymphocyte ratio (N:L), and circulating blood leukocytes were examined from pigs injected i.v. with either saline, ACTH, cortisol, or treated with metyrapone. Plasma cortisol increased (P < .05) after ACTH and cortisol treatments and decreased (P < .05) after metyrapone treatment; thus, treatments had the intended effects on in vivo cortisol concentrations. In Exp. 1, pigs were injected with either saline or ACTH at 0600 after the initial blood samples were taken (time 0). The ACTH had no effect (P > .10) on NK cytotoxicity. Pigs injected i.v. with ACTH had fewer lymphocytes and more neutrophils (P < .05) than control pigs. The N:L ratio was greater (P < .05) among ACTH-injected than among control pigs. In Exp. 2, pigs were injected i.v. with either saline or 40 or 400 micrograms of cortisol at 0600 after the initial blood samples were obtained (time 0). Cortisol at 40 micrograms had no effect (P > .10) on NK cytotoxicity. However, a 400-micrograms bolus of cortisol reduced (P < .05) NK cytotoxicity (control = 39.5, cortisol = 28.3% cytotoxicity, SEM = 3.7). Each dose of cortisol reduced (P < .05) circulating blood lymphocyte numbers. In Exp. 3, pigs were fed 1 g of metyrapone or no metyrapone the night before sampling. Blood samples were obtained at 0600, 0700, and 0800. Metyrapone reduced (P < .05) NK cytotoxicity (control = 28.6, metyrapone = 11.8%, SEM = 1.9). Pigs treated with metyrapone had greater (P < .05) numbers of neutrophils than control pigs. Numbers of lymphocytes were greater (P < .05) among control than among treated pigs. Pigs treated with metyrapone had a greater (P < .05) N:L ratio than control pigs. In conclusion, normal physiological concentrations or moderately increased blood cortisol concentrations did not influence NK activity, although leukocyte distributions were changed. We conclude that greatly increased or greatly decreased

  5. Combined analysis of copy number alterations by single-nucleotide polymorphism array and MYC status in non-metastatic breast cancer patients: comparison according to the circulating tumor cell status.

    PubMed

    Nadal, R; Salido, M; Nonell, L; Rodríguez-Rivera, M; Puigdecanet, E; Garcia-Puche, J L; Macià, M; Corominas, J M; Serrano, M J; Lorente, J A; Solé, F

    2015-02-01

    Recent technological advances have made it possible to detect circulating tumor cells (CTCs) as a prognostic marker in operable breast cancer patients. Whether the presence of CTCs in cancer patients correlates with molecular alterations in the primary tumor has not been widely explored. We identified 14 primary breast cancer specimens with known CTC status, in order to evaluate the presence of differential genetic aberrations by using SNP array assay. There was a global increase of altered genome, CNA, and copy-neutral loss of heterozygosity (cn-LOH) observed in the CTC-positive (CTC(+)) versus CTC-negative (CTC(-)) cases. As the preliminary results showed a higher proportion of copy number alteration (CNA) at 8q24 (MYC loci) and the available evidence supporting the role of MYC in the processes cancer metastases is conflicting, MYC status was determined in tissue microarray sections in a larger series of patients (n = 49) with known CTC status using FISH. MYC was altered in 62% (16/26) CTC(+) patients and in 43% (6/14) CTC(-) patients (p = 0.25). Based on the observation in our study, future studies involving a larger number of patients should be performed in order to definitively define if this correlation exists.

  6. Combined analysis of copy number alterations by single-nucleotide polymorphism array and MYC status in non-metastatic breast cancer patients: comparison according to the circulating tumor cell status.

    PubMed

    Nadal, R; Salido, M; Nonell, L; Rodríguez-Rivera, M; Puigdecanet, E; Garcia-Puche, J L; Macià, M; Corominas, J M; Serrano, M J; Lorente, J A; Solé, F

    2015-02-01

    Recent technological advances have made it possible to detect circulating tumor cells (CTCs) as a prognostic marker in operable breast cancer patients. Whether the presence of CTCs in cancer patients correlates with molecular alterations in the primary tumor has not been widely explored. We identified 14 primary breast cancer specimens with known CTC status, in order to evaluate the presence of differential genetic aberrations by using SNP array assay. There was a global increase of altered genome, CNA, and copy-neutral loss of heterozygosity (cn-LOH) observed in the CTC-positive (CTC(+)) versus CTC-negative (CTC(-)) cases. As the preliminary results showed a higher proportion of copy number alteration (CNA) at 8q24 (MYC loci) and the available evidence supporting the role of MYC in the processes cancer metastases is conflicting, MYC status was determined in tissue microarray sections in a larger series of patients (n = 49) with known CTC status using FISH. MYC was altered in 62% (16/26) CTC(+) patients and in 43% (6/14) CTC(-) patients (p = 0.25). Based on the observation in our study, future studies involving a larger number of patients should be performed in order to definitively define if this correlation exists. PMID:25286758

  7. Neutrophils and lymphoid chimerism after adult living-related liver transplantation from a homozygous donor.

    PubMed

    Hajeer, A H; Issa, S; Alaskar, A; Abdullah, K; Awad, M; Tbakhi, A; Alabdulkareem, A

    2005-12-01

    Chimerism and graft-versus-host disease (GVHD) pose significant risks to liver transplant patients. The risk of chimerism and GVHD is higher among cases of living-related liver transplant (LRLT). Donors homozygous at all HLA loci carry a higher risk for GVHD. Herein we present a case of LRLT. The recipient suffered from end-stage liver disease and received a right lobe graft from his son. After 8 months posttransplant, the patient developed profound bone marrow depression. The patient was negative for CMV, Brucella, HHV6, HHV8, HBV, HCV, and parvovirus. No skin or GI signs of GVHD were noted. The patient and donor were HLA typed by SSP. The donor was homozygous for all HLA loci while the patient shared the class II homozygosity and was class I heterozygous. Chimerism studies were prompted after noting that the neutrophil compartment of the patient was homozygous for all HLA loci. This initiated further studies of the PMN and lymphocytes by microsatellite analysis. A total 15 microsatellites were analyzed. The results suggest that the majority (75%) of the PMNs and 45% of the lymphocytes were of donor origin. The patient was treated with G-CSF; his WBC counts returned to normal. At 2.5 years posttransplant the patient had not developed GVHD, despite the large number of donor lymphocytes circulating in his bloodstream. The only complaint he had was severe arthritis, which was treated with steroids. It must be investigated whether this was the result of GVHD.

  8. Adhesion molecules involved in hepoxilin A3-mediated neutrophil transepithelial migration

    PubMed Central

    Hurley, B P; Sin, A; McCormick, B A

    2008-01-01

    A common feature underlying active states of inflammation is the migration of neutrophils (PMNs) from the circulation and across a number of tissue barriers in response to chemoattractant stimuli. Although our group has recently established a discreet role for the PMN chemoattractant, hepoxilin A3 (HXA3) in the process of PMN recruitment, very little is known regarding the interaction of HXA3 with PMNs. To characterize further the event of HXA3-induced PMN transepithelial migration, we sought to determine the adhesion molecules required for migration across different epithelial surfaces (T84 intestinal and A549 airway cells) relative to two well-studied PMN chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4). Our findings reveal that the adhesion interaction profile of PMN transepithelial migration in response to HXA3 differs from the adhesion interaction profile exhibited by the structurally related eicosanoid LTB4. Furthermore, unique to PMN transepithelial migration induced by gradients of HXA3 was the critical dependency of all four major surface adhesion molecules examined (i.e. CD18, CD47, CD44 and CD55). Our results suggest that the particular chemoattractant gradient imposed, as well as the type of epithelial cell monolayer, each plays a role in determining the adhesion molecules involved in transepithelial migration. Given the complexities of these interactions, our findings are important to consider with respect to adhesion molecules that may be targeted for potential drug development. PMID:18005361

  9. Platelet–neutrophil interactions under thromboinflammatory conditions

    PubMed Central

    Li, Jing; Kim, Kyungho; Barazia, Andrew; Tseng, Alan

    2015-01-01

    Platelets primarily mediate hemostasis and thrombosis, whereas leukocytes are responsible for immune responses. Since platelets interact with leukocytes at the site of vascular injury, thrombosis and vascular inflammation are closely intertwined and occur consecutively. Recent studies using real-time imaging technology demonstrated that platelet–neutrophil interactions on the activated endothelium are an important determinant of microvascular occlusion during thromboinflammatory disease in which inflammation is coupled to thrombosis. Although the major receptors and counter receptors have been identified, it remains poorly understood how heterotypic platelet–neutrophil interactions are regulated under disease conditions. This review discusses our current understanding of the regulatory mechanisms of platelet– neutrophil interactions in thromboinflammatory disease. PMID:25650236

  10. Roscovitine ameliorates endotoxin-induced uveitis through neutrophil apoptosis

    PubMed Central

    Jiang, Zhao-Xin; Qiu, Suo; Lou, Bing-Sheng; Yang, Yao; Wang, Wen-Cong; Lin, Xiao-Feng

    2016-01-01

    Neutrophils have been recognized as critical response cells during the pathogenesis of endotoxin-induced uveitis (EIU). Apoptosis of neutrophils induced by roscovitine has previously been demonstrated to ameliorate inflammation in several in vivo models. The present study aimed to assess whether roscovitine ameliorates EIU. EIU was induced in female C57BL/6 mice by a single intravitreal injection of lipopolysaccharide (LPS; 250 ng). The mice were divided into three groups as follows: LPS alone, LPS plus vehicle, LPS plus roscovitine (50 mg/kg). The mice were euthanized 12, 24, 48 and 72 h after LPS-induced uveitis. Accumulation of inflammatory cells in the vitreous body was confirmed by immunohistochemistry, and quantified following hematoxylin and eosin staining. Terminal deoxynucleotidyl transferase dUTP nick-end labeling was performed to detect of apoptotic cells. The mRNA levels of inflammatory cytokines were analyzed by reverse transcription-quantitative polymerase chain reaction and the changes in protein levels were analyzed by western blotting. Inflammatory cells accumulated in the vitreous near the optic nerve head and the quantity peaked at 24 h after LPS injection. Immunohistochemistry revealed that the majority of the inflammatory cells were neutrophils. The number of infiltrating cells was similar in the LPS and LPS plus vehicle groups, while there were significantly less in the roscovitine group at 24 h. Apoptosis of neutrophils was observed between 12 and 48 h after roscovitine injection, while no apoptosis was observed in the other groups. The mRNA expression levels of GMCSF, CINC-1 and ICAM-1 peaked at 12 h after LPS injection, and decreased to normal levels at 72 h. This trend in mRNA expression was similar in the LPS and LPS plus vehicle groups; however, the expression levels decreased more quickly in the roscovitine group at 24 and 48 h. Following roscovitine administration, upregulated cleaved caspase 3 expression levels and downregulated Mcl-1

  11. Myristoylated Alanine Rich C Kinase Substrate (MARCKS) is essential to β2-integrin dependent responses of equine neutrophils

    PubMed Central

    Sheats, Mary K.; Pescosolido, Kimberly C.; Hefner, Ethan M.; Sung, Eui Jae; Adler, Kenneth B.; Jones, Samuel L.

    2014-01-01

    Neutrophil infiltration is a prominent feature in a number of pathologic conditions affecting horses including recurrent airway obstruction, ischemia-reperfusion injury, and laminitis. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. In order to migrate into tissue, neutrophils must respond to chemoattractant signals in their external environment through activation of adhesion receptors (i.e. integrins) and reorganization of the actin cytoskeleton. Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a highly conserved actin-binding protein, has a well demonstrated role in cytoskeletal dependent cellular functions (i.e. adhesion, spreading, and migration), but the details of MARCKS involvement in these processes remain vague. We hypothesized that MARCKS serves as a link between the actin cytoskeleton and integrin function in neutrophils. Using a MARCKS-specific inhibitor peptide known as MANS on equine neutrophils in vitro, we demonstrate that inhibition of MARCKS function significantly attenuates β2-integrin-dependent neutrophil functions including migration, adhesion, and immune complex-mediated respiratory burst. The MANS peptide did not, however, inhibit the β2-integrin-independent PMA mediated respiratory burst. These results attest to the essential role of MARCKS function in regulating neutrophil responses, and strongly implicate MARCKS as a potential regulator of β2-integrins in neutrophils. PMID:24857637

  12. S. aureus blocks efferocytosis of neutrophils by macrophages through the activity of its virulence factor alpha toxin

    PubMed Central

    Cohen, Taylor S.; Jones-Nelson, Omari; Hotz, Meghan; Cheng, Lily; Miller, Lloyd S.; Suzich, JoAnn; Stover, C. Kendall; Sellman, Bret R.

    2016-01-01

    Bacterial pneumonia, such as those caused by Staphylococcus aureus, is associated with an influx of inflammatory neutrophils into the lung tissue and airways. Regulation and clearance of recruited neutrophils is essential for preventing tissue damage by “friendly fire”, a responsibility of macrophages in a process called efferocytosis. We hypothesized that S. aureus impairs efferocytosis by alveolar macrophages (AMs) through the activity of the secreted virulence factor alpha toxin (AT), which has been implicated in altering the antimicrobial function of AMs. Infection of mice lacking AMs resulted in significantly increased numbers of neutrophils in the lung, while clearance of neutrophils delivered intranasally into uninfected mice was reduced in AM depleted animals. In vitro, sublytic levels of AT impaired uptake of apoptotic neutrophils by purified AMs. In vivo, the presence of AT reduced uptake of neutrophils by AMs. Differential uptake of neutrophils was not due to changes in either the CD47/CD172 axis or CD36 levels. AT significantly reduced lung expression of CCN1 and altered AM surface localization of DD1α, two proteins known to influence efferocytosis. We conclude that AT may contribute to tissue damage during S. aureus pneumonia by inhibiting the ability of AM to clear neutrophils at the site of infection. PMID:27739519

  13. Myristoylated Alanine Rich C Kinase Substrate (MARCKS) is essential to β2-integrin dependent responses of equine neutrophils.

    PubMed

    Sheats, Mary K; Pescosolido, Kimberly C; Hefner, Ethan M; Sung, Eui Jae; Adler, Kenneth B; Jones, Samuel L

    2014-08-15

    Neutrophil infiltration is a prominent feature in a number of pathologic conditions affecting horses including recurrent airway obstruction, ischemia-reperfusion injury, and laminitis. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. In order to migrate into tissue, neutrophils must respond to chemoattractant signals in their external environment through activation of adhesion receptors (i.e. integrins) and reorganization of the actin cytoskeleton. Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a highly conserved actin-binding protein, has a well demonstrated role in cytoskeletal dependent cellular functions (i.e. adhesion, spreading, and migration), but the details of MARCKS involvement in these processes remain vague. We hypothesized that MARCKS serves as a link between the actin cytoskeleton and integrin function in neutrophils. Using a MARCKS-specific inhibitor peptide known as MANS on equine neutrophils in vitro, we demonstrate that inhibition of MARCKS function significantly attenuates β2-integrin-dependent neutrophil functions including migration, adhesion, and immune complex-mediated respiratory burst. The MANS peptide did not, however, inhibit the β2-integrin-independent PMA mediated respiratory burst. These results attest to the essential role of MARCKS function in regulating neutrophil responses, and strongly implicate MARCKS as a potential regulator of β2-integrins in neutrophils. PMID:24857637

  14. Depression of immunity to Naegleria fowleri in mice by selective depletion of neutrophils with a monoclonal antibody.

    PubMed

    Ferrante, A; Carter, R F; Lopez, A F; Rowan-Kelly, B; Hill, N L; Vadas, M A

    1988-09-01

    In an attempt to define the role of neutrophils in immunity to Naegleria fowleri in vivo, we examined the effects of treating immunized (with amoeba culture supernatant antigen) mice with the monoclonal antibody NIMP-R10, which binds to neutrophil complement receptor type 3bi (CR3) and causes selective neutrophil depletion in mice. Mice in the nonimmunized group challenged with amoebae all died by day 12, while 97% in the immunized group survived. By contrast, the immunized group treated with NIMP-R10 showed only 25% survival. The immunized group treated with "control" mouse ascites, WEM-G11, was highly resistant (90% survival). There was a significant neutrophil response in the nasal mucosa and olfactory lobes of immunized, NIMP-R10-treated mice, despite a marked degree of neutropenia similar to that seen in immunized, untreated mice. Nonimmunized mice showed virtually no neutrophil response. Despite this response in the NIMP-R10-treated mice, amoebic proliferation was not depressed, and there was no evidence of neutrophil degranulation or amoebic killing, despite the close apposition of large numbers of neutrophils to amoebae. The results indicate that neutrophils are necessary for the expression of immunity to N. fowleri.

  15. Augmentation of platelet and endothelial cell eNOS activity decreases sepsis-related neutrophil-endothelial cell interactions.

    PubMed

    Khan, Raymond; Kirschenbaum, Linda A; LaRow, Catherine; Berna, Gioiamaria; Griffin, Kelly; Astiz, Mark E

    2010-03-01

    NO is an important mediator of microvascular patency and blood flow. The purpose of this study was to examine the role of enhanced eNOS activity in attenuating sepsis-induced neutrophil-endothelial cell interactions. Microslides coated with human umbilical vein endothelial cells were stimulated with plasma from patients with septic shock. Neutrophil and platelets from control subjects were also stimulated with plasma from patients in septic shock and perfused over stimulated endothelial cells. l-Arginine (LA) with and without NG-monomethyl l-arginine (LNMMA), a nonselective NOS inhibitor, and N-(3-(aminomethyl) benzyl acetamide) ethanimidamide dihydrochloride (1400W), a highly selective iNOS inhibitor, were added to the septic plasma. The number of neutrophils adherent to endothelial cells, neutrophil rolling velocity, and the number of neutrophil aggregates were determined. Cell activation and the formation of platelet-neutrophil aggregates were assessed by flow cytometry. Separate experiments were done with isolated platelets using platelet aggregometry. l-Arginine significantly decreased sepsis-related neutrophil adhesion and aggregation and increased rolling velocity. The addition of LNMMA to LA and cell suspensions reversed the effects of LA on these parameters, whereas the addition of 1400W had no effect on LA-related changes. Platelet-neutrophil aggregation, platelet aggregation, platelet activation, and neutrophil activation induced by septic plasma were also significantly decreased by LA. Again, the addition of LNMMA reversed the effects of LA on these parameters, whereas 1400W had no effect on LA-related changes. These data suggest that enhancement of platelet and endothelial cell eNOS activity decreases sepsis-induced neutrophil-endothelial cell interactions and may play a role in maintaining microvascular patency in septic shock.

  16. Early infection during burn-induced inflammatory response results in increased mortality and p38-mediated neutrophil dysfunction.

    PubMed

    Adediran, Samuel G; Dauplaise, Derrick J; Kasten, Kevin R; Tschöp, Johannes; Dattilo, Jonathan; Goetzman, Holly S; England, Lisa G; Cave, Cindy M; Robinson, Chad T; Caldwell, Charles C

    2010-09-01

    Following burn injury, the host is susceptible to bacterial infections normally cleared by healthy patients. We hypothesized that during the systemic immune response that follows scald injury, the host's altered immune status increases infection susceptibility. Using a murine model of scald injury under inhaled anesthesia followed by intraperitoneal infection, we observed increased neutrophil numbers and function at postburn day (PBD) 1 compared with sham-burned and PBD4 mice. Further, increased mortality, bacteremia, and serum IL-6 were observed in PBD1 mice after Pseudomonas aeruginosa (PA) infection compared with sham-burned and PBD4 mice infected with PA. To examine these disparate responses, we investigated neutrophils isolated at 5 and 24 h following PA infection from PBD1 and sham-burned mice. Five hours after infection, there was no significant difference in number of recruited neutrophils; however, neutrophils from injured mice had decreased activation, active-p38, and oxidative burst compared with sham-burned mice. In direct contrast, 24 h after infection, we observed increased numbers, active-p38, and oxidative burst of neutrophils from PBD1 mice. Finally, we demonstrated that in neutrophils isolated from PBD1 mice, the observed increase in oxidative burst was p38 dependent. Altogether, neutrophil activation and function from thermally injured mice are initially delayed and later exacerbated by a p38-dependent mechanism. This mechanism is likely key to the observed increase in bacterial load and mortality of PBD1 mice infected with PA.

  17. Inhibition of Nicotinamide Phosphoribosyltransferase Reduces Neutrophil-Mediated Injury in Myocardial Infarction

    PubMed Central

    Bauer, Inga; Braunersreuther, Vincent; Bruzzone, Santina; Akhmedov, Alexander; Lüscher, Thomas F.; Speer, Timo; Poggi, Alessandro; Mannino, Elena; Pelli, Graziano; Galan, Katia; Bertolotto, Maria; Lenglet, Sébastien; Garuti, Anna; Montessuit, Christophe; Lerch, René; Pellieux, Corinne; Vuilleumier, Nicolas; Dallegri, Franco; Mage, Jacqueline; Sebastian, Carlos; Mostoslavsky, Raul; Gayet-Ageron, Angèle; Patrone, Franco; Mach, François; Nencioni, Alessio

    2013-01-01

    Abstract Aims: Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and recent evidence indicates its role in inflammatory processes. Here, we investigated the potential effects of pharmacological Nampt inhibition with FK866 in a mouse myocardial ischemia/reperfusion model. In vivo and ex vivo mouse myocardial ischemia/reperfusion procedures were performed. Results: Treatment with FK866 reduced myocardial infarct size, neutrophil infiltration, and reactive oxygen species (ROS) generation within infarcted hearts in vivo in a mouse model of ischemia and reperfusion. The benefit of FK866 was not shown in the Langendorff model (ex vivo model of working heart without circulating leukocytes), suggesting a direct involvement of these cells in cardiac injury. Sera from FK866-treated mice showed reduced circulating levels of the neutrophil chemoattractant CXCL2 and impaired capacity to prime migration of these cells in vitro. The release of CXCL8 (human homolog of murine chemokine CXCL2) by human peripheral blood mononuclear cells (PBMCs) and Jurkat cells was also reduced by FK866, as well as by sirtuin (SIRT) inhibitors and SIRT6 silencing, implying a pivotal role for this NAD+-dependent deacetylase in the production of this chemokine. Innovation: The pharmacological inhibition of Nampt might represent an effective approach to reduce neutrophilic inflammation- and oxidative stress-mediated tissue damage in early phases of reperfusion after a myocardial infarction. Conclusions: Nampt inhibition appears as a new strategy to dampen CXCL2-induced neutrophil recruitment and thereby reduce neutrophil-mediated tissue injury in mice. Antioxid. Redox Signal. 18, 630–641. PMID:22452634

  18. Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice.

    PubMed

    Yao, Yi; Liu, Yun; Takashima, Akira

    2016-01-01

    Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal - used as a measure of priming 2) in vivo neutrophil labeling - achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models. PMID:27403648

  19. Anti-neutrophil cytoplasmic antibodies in rheumatoid arthritis: two case reports and review of literature

    PubMed Central

    2012-01-01

    Background Anti-neutrophil cytoplasmic antibodies are typically detected in anti-neutrophil cytoplasmic antibody associated vasculitis, but are also present in a number of chronic inflammatory non-vasculitic conditions like rheumatoid arthritis. Rare cases of granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis, a vasculitic disorder frequently associated with the presence of anti-neutrophil cytoplasmic antibodies) in patients with rheumatoid arthritis have been described in literature. Case presentation We report two middle-aged female patients with rheumatoid arthritis who developed anti-neutrophil cytoplasmic antibodies and symptoms reminiscent of granulomatosis with polyangiitis. Despite the lack of antibodies specific for proteinase 3 and the absence of a classical histology, we report a probable case of granulomatosis with polyangiitis in the first patient, and consider rheumatoid vasculitis in the second patient. Conclusion Taken together with previous reports, these cases highlight that anti-neutrophil cytoplasmic antibodies have to be evaluated very carefully in patients with rheumatoid arthritis. In this context, anti-neutrophil cytoplasmic antibodies detected by indirect immunofluorescence appear to have a low diagnostic value for granulomatosis with polyangiitis. Instead they may have prognostic value for assessing the course of rheumatoid arthritis. PMID:23253567

  20. Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro.

    PubMed Central

    Whyte, M K; Hardwick, S J; Meagher, L C; Savill, J S; Haslett, C

    1993-01-01

    Elevation of cytosolic calcium ([Ca2+]i) has been reported to induce apoptosis in a number of cell types. However, in the neutrophil, which undergoes apoptosis constitutively during aging in vitro, activation by inflammatory mediators elevates [Ca2+]i and prolongs lifespan via inhibition of apoptosis. To examine this paradox, we investigated the effects of modulation of [Ca2+]i upon apoptosis of neutrophils in vitro. Calcium ionophores (A23187, ionomycin) retarded apoptosis in neutrophil populations after 20 h (P < 0.001). Conversely, intracellular Ca(2+)-chelation, using bis-(o-aminophenoxy)-N,N,N'N'-tetraacetic acid (BAPTA) acetoxymethyl ester (AM) promoted apoptosis (P < 0.02). W-7 (an inhibitor of calmodulin) also promoted apoptosis (P < 0.05). Measurements of [Ca2+]i, using fura-2, showed (a) increased apoptosis in neutrophil populations was not associated with elevated [Ca2+]i, (b) neutrophils cultured with ionophore at concentrations inhibiting apoptosis exhibited transient (< 1 h) elevations of [Ca2+]i, to levels previously reported with receptor-mediated stimuli, and (c) BAPTA was able to prevent the elevation of [Ca2+]i and the inhibition of apoptosis produced by ionophore. Modulation of apoptosis occurred without alterations in intracellular pH. Thus, in the neutrophil, unlike lymphoid cells, elevation of [Ca2+]i exerts an inhibitory effect upon apoptosis. Furthermore, these data suggest that transient elevation of [Ca2+]i elicits signaling events leading to prolonged inhibition of apoptosis. Images PMID:8392090

  1. Characterization of functions of neutrophils from bone marrow of cattle with leukocyte adhesion deficiency.

    PubMed

    Nagahata, H; Nochi, H; Tamoto, K; Yamashita, K; Noda, H; Kociba, G J

    1995-02-01

    Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired. PMID:7717579

  2. Neutrophils are required for 3-methylcholanthrene-initiated, butylated hydroxytoluene-promoted lung carcinogenesis.

    PubMed

    Vikis, Haris G; Gelman, Andrew E; Franklin, Andrew; Stein, Lauren; Rymaszewski, Amy; Zhu, Jihong; Liu, Pengyuan; Tichelaar, Jay W; Krupnick, Alexander S; You, Ming

    2012-12-01

    Multiple studies have shown a link between chronic inflammation and lung tumorigenesis. Inbred mouse strains vary in their susceptibility to methylcholanthrene (MCA)-initiated butylated hydroxytoluene (BHT)-promoted lung carcinogenesis. In the present study we investigated whether neutrophils play a role in strain dependent differences in susceptibility to lung tumor promotion. We observed a significant elevation in homeostatic levels of neutrophils in the lungs of tumor-susceptible BALB/cByJ (BALB) mice compared to tumor-resistant C57BL/6J (B6) mice. Additionally, BHT treatment further elevated neutrophil numbers as well as neutrophil chemoattractant keratinocyte-derived cytokine (KC)/chemokine (C-X-C motif) ligand 1 (Cxcl1) levels in BALB lung airways. Lung CD11c+ cells were a major source of KC expression and depletion of neutrophils in BALB mice resulted in a 71% decrease in tumor multiplicity. However, tumor multiplicity did not depend on the presence of T cells, despite the accumulation of T cells following BHT treatment. These data demonstrate that neutrophils are essential to promote tumor growth in the MCA/BHT two-step lung carcinogenesis model.

  3. Generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants.

    PubMed Central

    Kalyanaraman, B; Sohnle, P G

    1985-01-01

    A large number of foreign compounds, including many drugs, industrial pollutants, and environmental chemicals, can be oxidized under appropriate conditions to potentially toxic free radical intermediates. We evaluated the ability of the oxidants produced by the neutrophil myeloperoxidase system to generate free radical intermediates from several such compounds. Sodium hypochlorite or hypochlorous acid produced by human peripheral blood neutrophils and trapped in the form of taurine chloramine were both found to be capable of producing free radicals from chlorpromazine, aminopyrine, and phenylhydrazine. These radical intermediates were demonstrated by visible light spectroscopy and by direct electron spin resonance (for the chlorpromazine and aminopyrine radicals) or by spin-trapping (for the phenyl radical generated from phenylhydrazine). Stable oxidants produced by the neutrophils (i.e., those present in the supernatants of stimulated neutrophils in the absence of added taurine) also were found to be capable of generating free radical intermediates. The production of the oxidants and the ability of neutrophil supernatants to generate these radicals were almost completely eliminated by sodium azide, a myeloperoxidase inhibitor. We suggest that the oxidation by neutrophils of certain chemical compounds to potentially damaging electrophilic free radical forms may represent a new metabolic pathway for these substances and could be important in the processes of drug toxicity and chemical carcinogenesis. PMID:2987307

  4. Neutrophils play an important role in protective immunity against Coxiella burnetii infection.

    PubMed

    Elliott, Alexandra; Schoenlaub, Laura; Freches, Danielle; Mitchell, William; Zhang, Guoquan

    2015-08-01

    Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes the zoonotic disease Q fever. Although Q fever is mainly transmitted by aerosol infection, study of the immune responses in the lung following pulmonary C. burnetii infection is lacking. Neutrophils are considered the first immune cell to migrate into the lung and play an important role in host defense against aerosol infection with microbial pathogens. However, the role of neutrophils in the host defense against C. burnetii infection remains unclear. To determine the role of neutrophils in protective immunity against C. burnetii infection, the RB6-8C5 antibody was used to deplete neutrophils in mice before intranasal infection with C. burnetii. The results indicated that neutrophil-depleted mice developed more severe disease than their wild-type counterparts, suggesting that neutrophils play an important role in host defense against C. burnetii pulmonary infection. We also found that neither CXC chemokine receptor 2 (CXCR2) nor interleukin-17 (IL-17) receptor (IL-17R) deficiency changed the severity of disease following intranasal C. burnetii challenge, suggesting that keratinocyte-derived chemokine and IL-17 may not play essential roles in the response to C. burnetii infection. However, significantly higher C. burnetii genome copy numbers were detected in the lungs of IL-1R(-/-) mice at 14 days postinfection. This indicates that IL-1 may be important for the clearance of C. burnetii from the lungs following intranasal infection. Our results also suggest that neutrophils are involved in protecting vaccinated mice from C. burnetii challenge-induced disease. This is the first study to demonstrate an important role for neutrophils in protective immunity against C. burnetii infection.

  5. Store-operated calcium signaling in neutrophils.

    PubMed

    Clemens, Regina A; Lowell, Clifford A

    2015-10-01

    Calcium signals in neutrophils are initiated by a variety of cell-surface receptors, including formyl peptide and other GPCRs, FcRs, and integrins. The predominant pathway by which calcium enters immune cells is termed SOCE, whereby plasma membrane CRAC channels allow influx of extracellular calcium into the cytoplasm when intracellular ER stores are depleted. The identification of 2 key families of SOCE regulators, STIM calcium "sensors" and ORAI calcium channels, has allowed for genetic manipulation of SOCE pathways and provided valuable insight into the molecular mechanism of calcium signaling in immune cells, including neutrophils. This review focuses on our current knowledge of the molecules involved in neutrophil SOCE and how study of these molecules has further informed our understanding of the role of calcium signaling in neutrophil activation.

  6. [Effect of erythromycin on neutrophil adhesion molecules].

    PubMed

    Kusano, S; Mukae, H; Morikawa, T; Asai, T; Sawa, H; Morikawa, N; Oda, H; Sakito, O; Shukuwa, C; Senju, R

    1993-01-01

    The mechanisms of erythromycin (EM) in chronic lower respiratory tract diseases including diffuse panbronchiolitis (DPB) has been reported. In this study we investigated the effect of EM on peripheral neutrophil adhesion molecules such as LFA-1 and Mac-1 obtained from six healthy subjects. Pretreatment of neutrophils with each concentration (10 ng/ml approximately 100 micrograms/ml) of EM resulted in no significant reduction in the expression of LFA-1 alpha, beta and Mac-1. Moreover, EM had no capability of reducing these expressions even when neutrophils were pretreated with 1 microgram/ml of EM at time from 0 to 60 min. These findings indicate that EM does not directly reduce the expression of LFA-1 alpha, beta and Mac-1 on peripheral neutrophil obtained from healthy subjects. PMID:8450276

  7. Elevated Neutrophil Lymphocyte Ratio in Recurrent Optic Neuritis

    PubMed Central

    Guclu, Hande; Ozal, Sadık Altan; Pelitli Gurlu, Vuslat; Birgul, Ramazan

    2015-01-01

    Purpose. To demonstrate the relation between optic neuritis (ON) and systemic inflammation markers as neutrophil lymphocyte ratio (N/L ratio), platelet count, mean platelet volume (MPV), and red cell distribution width (RDW) and furthermore to evaluate the utilization of these markers to predict the frequency of the ON episodes. Methods. Forty-two patients with acute ON and forty healthy subjects were enrolled into the study. The medical records were reviewed for age, sex, hemoglobin (Hb), Haematocrit (Htc), RDW, platelet count, MPV, white blood cell count (WBC), neutrophil and lymphocyte count, and neutrophil lymphocyte ratio (N/L ratio). Results. The mean N/L ratio, platelet counts, and RDW were significantly higher in ON group (p = 0.000, p = 0.048, and p = 0.002). There was a significant relation between N/L ratio and number of episodes (r = 0.492, p = 0.001). There was a statistically significant difference for MPV between one episode group and recurrent ON group (p = 0.035). Conclusions. Simple and inexpensive laboratory methods could help us show systemic inflammation and monitor ON patients. Higher N/L ratio can be a useful marker for predicting recurrent attacks. PMID:26060578

  8. Neutrophil Extracellular Traps in Periodontitis: A Web of Intrigue.

    PubMed

    White, P C; Chicca, I J; Cooper, P R; Milward, M R; Chapple, I L C

    2016-01-01

    Neutrophil extracellular traps (NETs) represent a novel paradigm in neutrophil-mediated immunity. NETs are believed to constitute a highly conserved antimicrobial strategy comprising decondensed nuclear DNA and associated histones that are extruded into the extracellular space. Associated with the web-like strands of DNA is an array of antimicrobial peptides (AMPs), which facilitate the extracellular destruction of microorganisms that become entrapped within the NETs. NETs can be released by cells that remain viable or following a unique form of programmed cell death known as NETosis, which is dependent on the production of reactive oxygen species (ROS) and the decondensing of the nuclear DNA catalyzed by peptidyl arginine deiminase-4. NETs are produced in response to a range of pathogens, including bacteria, viruses, fungi, and protozoa, as well as host-derived mediators. NET release is, however, not without cost, as the concomitant release of cytotoxic molecules can also cause host tissue damage. This is evidenced by a number of immune-mediated diseases, in which excess or dysfunctional NET production, bacterial NET evasion, and decreased NET removal are associated with disease pathogenesis. Periodontitis is the most prevalent infectious-inflammatory disease of humans, characterized by a dysregulated neutrophilic response to specific bacterial species within the subgingival plaque biofilm. Neutrophils are the predominant inflammatory cell involved in periodontitis and have previously been found to exhibit hyperactivity and hyperreactivity in terms of ROS production in chronic periodontitis patients. However, the contribution of ROS-dependent NET formation to periodontal health or disease remains unclear. In this focused review, we discuss the mechanisms, stimuli, and requirements for NET production; the ability of NET-DNA and NET-associated AMPs to entrap and kill pathogens; and the potential immunogenicity of NETs in disease. We also speculate on the potential

  9. Neutrophils in asthma--a review.

    PubMed

    Ciepiela, Olga; Ostafin, Magdalena; Demkow, Urszula

    2015-04-01

    Asthma is a chronic inflammatory disease, with an array of cells involved in the pathogenesis of the disease. The role of neutrophils in the development of bronchial asthma is found to be complex, as they may trigger activation of immunocompetent cells and are a potent source of free oxygen radicals and enzymes participating in airway remodeling. The review highlights the role of neutrophils in bronchial asthma.

  10. Clinical and Laboratory Differences between Lymphocyte- and Neutrophil-Predominant Pleural Tuberculosis

    PubMed Central

    Kim, Kang; Kim, Sukyeon; Oh, Ki-Jong; Jeong, Suk Hyeon; Jung, Woo Jin; Shin, Beomsu; Jhun, Byung Woo; Lee, Hyun; Park, Hye Yun; Koh, Won-Jung

    2016-01-01

    Pleural tuberculosis (TB), a form of extrapulmonary TB, can be difficult to diagnose. High numbers of lymphocytes in pleural fluid have been considered part of the diagnostic criteria for pleural TB; however, in many cases, neutrophils rather than lymphocytes are the predominant cell type in pleural effusions, making diagnosis more complicated. Additionally, there is limited information on the clinical and laboratory characteristics of neutrophil-predominant pleural effusions caused by Mycobacterium tuberculosis (MTB). To investigate clinical and laboratory differences between lymphocyte- and neutrophil-predominant pleural TB, we retrospectively analyzed 200 patients with the two types of pleural TB. Of these patients, 9.5% had neutrophil-predominant pleural TB. Patients with lymphocyte-predominant and neutrophil-predominant pleural TB showed similar clinical signs and symptoms. However, neutrophil-predominant pleural TB was associated with significantly higher inflammatory serum markers, such as white blood cell count (P = 0.001) and C-reactive protein (P = 0.001). Moreover, MTB was more frequently detected in the pleural fluid from patients in the neutrophil-predominant group than the lymphocyte-predominant group, with the former group exhibiting significantly higher rates of positive results for acid-fast bacilli in sputum (36.8 versus 9.4%, P = 0.003), diagnostic yield of MTB culture (78.9% versus 22.7%, P < 0.001) and MTB detected by polymerase chain reaction (31.6% versus 5.0%, P = 0.001). Four of seven patients with repeated pleural fluid analyses revealed persistent neutrophil-predominant features, which does not support the traditional viewpoint that neutrophil-predominant pleural TB is a temporary form that rapidly develops into lymphocyte-predominant pleural TB. In conclusion, neutrophil-predominant pleural TB showed a more intense inflammatory response and a higher positive rate in microbiological testing compared to lymphocyte-predominant pleural TB

  11. NETosis in Cancer – Platelet–Neutrophil Crosstalk Promotes Tumor-Associated Pathology

    PubMed Central

    Olsson, Anna-Karin; Cedervall, Jessica

    2016-01-01

    It has become increasingly clear that circulating immune cells in the body have a major impact on cancer development, progression, and outcome. The role of both platelets and neutrophils as independent regulators of various processes in cancer has been known for long, but it has quite recently emerged that the platelet–neutrophil interplay is yet a critical component to take into account during malignant disease. It was reported a few years ago that neutrophils in mice with cancer have increased propensity to form neutrophil extracellular traps (NETs) – web-like structures formed by externalized chromatin and secreted proteases. The initial finding describing this as a cell death-associated process has been followed by reports of additional mechanisms for NET formation (NETosis), and it has been shown that similar structures can be formed also without lysis and neutrophil cell death as a consequence. Furthermore, presence of NETs in humans with cancer has been verified in a few recent studies, indicating that tumor-induced NETosis is clinically relevant. Several reports have also described that NETs contribute to cancer-associated pathology, by promoting processes responsible for cancer-related death such as thrombosis, systemic inflammation, and relapse of the disease. This review summarizes current knowledge about NETosis in cancer, including the role of platelets as regulators of tumor-induced NETosis. It has been shown that platelets can serve as inducers of NETosis, and the platelet–neutrophil interface can therefore be an important issue to consider when designing therapies targeting cancer-associated pathology in the future. PMID:27708646

  12. On the maturation rate of the neutrophil.

    PubMed

    Zajicek, G; Shohat, M; Polliack, A

    1984-05-01

    Fifty-three maturing bone marrow cells of the granulocyte cell series stained with Giemsa stain and magnified 1,000 times were scanned by a "computerized microscope" consisting of a LSI-11/23 microprocessor and a black-and-white video camera attached to a "frame grabber ." Each sampled cell was digitized into 70 X 70 pixels, each pixel representing 0.04 micron of the real image. The pixel gray values ranged between 0 and 255. Zero stood for white, 255 represented black, while the numbers in between stood for the various shades of gray. The cells represented six different stages of granulocytic maturation: myeloblast, promyelocyte, myelocyte, metamyelocyte , band form, and polymorphonuclear granulocyte. A discriminant analysis program selected 19 features best distinguishing between the six different cell types and computed five canonical discriminant functions defining a Space in which maturation was studied. In the Space, distance between two cells serves as a measure of similarity. The closer two cells are, the more similar they are and vice versa. This measure was applied here to express the degree of similarity between the neutrophil maturation classes, and since they represent states in the neutrophil life history, it is applicable also as a yardstick for the quantitation of differentiation. In the Space, the life history of a cell is represented by a trajectory originating in the myeloblast and terminating in the granulocyte state. Displacement along the trajectory represents cell maturation that is expressed relatively to the least differentiated state of the myeloblast. The further a cell from this state the more mature it is. The same yardstick also serves for differentiation rate estimates represented in the Space by displacement velocities that are derived from the known "transit times" of a cell in each state. The methodology is also applied for cell production estimates. Unlike other "computerized microscopes" serving for cell classification, the

  13. Differential expression of pentraxin 3 in neutrophils.

    PubMed

    Razvina, Olga; Jiang, Shuying; Matsubara, Koichi; Ohashi, Riuko; Hasegawa, Go; Aoyama, Takashi; Daigo, Kenji; Kodama, Tatsuhiko; Hamakubo, Takao; Naito, Makoto

    2015-02-01

    Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense.

  14. Elevated neutrophil membrane expression of proteinase 3 is dependent upon CD177 expression

    PubMed Central

    Abdgawad, M; Gunnarsson, L; Bengtsson, A A; Geborek, P; Nilsson, L; Segelmark, M; Hellmark, T

    2010-01-01

    Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte–macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177–mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene. PMID:20491791

  15. Neutrophilic inflammatory response and oxidative stress in premenopausal women chronically exposed to indoor air pollution from biomass burning.

    PubMed

    Banerjee, Anirban; Mondal, Nandan Kumar; Das, Debangshu; Ray, Manas Ranjan

    2012-04-01

    The possibility of inflammation and neutrophil activation in response to indoor air pollution (IAP) from biomass fuel use has been investigated. For this, 142 premenopausal, never-smoking women (median age, 34 years) who cook exclusively with biomass (wood, dung, crop wastes) and 126 age-matched control women who cook with cleaner fuel liquefied petroleum gas (LPG) were enrolled. The neutrophil count in blood and sputum was significantly higher (p < 0.05) in biomass users than the control group. Flow cytometric analysis revealed marked increase in the surface expression of CD35 (complement receptor-1), CD16 (F(C)γ receptor III), and β(2) Mac-1 integrin (CD11b/CD18) on circulating neutrophils of biomass users. Besides, enzyme-linked immunosorbent assay showed that they had 72%, 67%, and 54% higher plasma levels of the proinflammatory cytokines tumor necrosis factor-alpha, interleukin-6, and interleukin-12, respectively, and doubled neutrophil chemoattractant interleukin-8. Immunocytochemical study revealed significantly higher percentage of airway neutrophils expressing inducible nitric oxide synthase, while the serum level of nitric oxide was doubled in women who cooked with biomass. Spectrophotometric analysis documented higher myeloperoxidase activity in circulating neutrophils of biomass users, suggesting neutrophil activation. Flow cytometry showed excess generation of reactive oxygen species (ROS) by leukocytes of biomass-using women, whereas their erythrocytes contained a depleted level of antioxidant enzyme superoxide dismutase (SOD). Indoor air of biomass-using households had two to four times more particulate matter with diameters of <10 μm (PM(10)) and <2.5 μm (PM(2.5)) as measured by real-time laser photometer. After controlling potential confounders, rise in proinflammatory mediators among biomass users were positively associated with PM(10) and PM(2.5) in indoor air, suggesting a close relationship between IAP and neutrophil activation. Besides

  16. MBL-Mediated Opsonophagocytosis of Candida albicans by Human Neutrophils Is Coupled with Intracellular Dectin-1-Triggered ROS Production

    PubMed Central

    Tong, Zhongsheng; Wang, Qinning; Liu, Weihuang; Wang, Yan; Liu, Wei; Chen, Jinbo; Xu, Li; Chen, Liuqing; Duan, Yiqun

    2012-01-01

    Mannan-binding lectin (MBL), a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans) by human neutrophils, and has the capacity to bind to CR1 (CD35) expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS) generation through recognition of β-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The β-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the β-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production. PMID:23239982

  17. Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

    PubMed Central

    Song, Hyun-Ouk

    2013-01-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2.-) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis. PMID:24039294

  18. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    PubMed

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis. PMID:24039294

  19. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    PubMed

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

  20. Neutrophils in Cancer: Two Sides of the Same Coin

    PubMed Central

    Uribe-Querol, Eileen; Rosales, Carlos

    2015-01-01

    Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs) have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions. PMID:26819959

  1. Neutrophils and neutrophil extracellular traps orchestrate initiation and resolution of inflammation.

    PubMed

    Hahn, Jonas; Knopf, Jasmin; Maueröder, Christian; Kienhöfer, Deborah; Leppkes, Moritz; Herrmann, Martin

    2016-01-01

    Neutrophils, the most abundant leukocytes in the human body, are considered to be the first line of defense in the fight against microorganisms. In this fight neutrophils employ weaponry such as reactive oxygen species produced via the NADPH oxidase complex 2 together with the release of intracellular granules containing antimicrobial agents. The discovery that activated neutrophils release decondensed chromatin as DNase-sensitive neutrophil extracellular traps (NETs) lead to a renewed interest in these leukocytes and the function of NETs in vivo. In this review, we will focus on desirable as well as detrimental features of NETs by the example of gout and pancreatitis. In our models we observed that neutrophils drive the initiation of inflammation and are required for the resolution of inflammation. PMID:27586795

  2. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes

    SciTech Connect

    Vlasova, Irina I.; Vakhrusheva, Tatyana V.; Sokolov, Alexey V.; Kostevich, Valeria A.; Gusev, Alexandr A.; Gusev, Sergey A.; Melnikova, Viktoriya I.; Lobach, Anatolii S.

    2012-10-01

    Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H{sub 2}O{sub 2} system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes. -- Highlights: ► Myeloperoxidase (MPO) product hypochlorous acid is able to degrade CNTs. ► PEGylated SWCNTs stimulate isolated neutrophils to produce hypochlorous acid. ► SWCNTs are capable of activating neutrophils in blood samples. ► Activation of

  3. Gene Expression during the Generation and Activation of Mouse Neutrophils: Implication of Novel Functional and Regulatory Pathways

    PubMed Central

    Ericson, Jeffrey A.; Duffau, Pierre; Yasuda, Kei; Ortiz-Lopez, Adriana; Rothamel, Katherine; Rifkin, Ian R.; Monach, Paul A.

    2014-01-01

    As part of the Immunological Genome Project (ImmGen), gene expression was determined in unstimulated (circulating) mouse neutrophils and three populations of neutrophils activated in vivo, with comparison among these populations and to other leukocytes. Activation conditions included serum-transfer arthritis (mediated by immune complexes), thioglycollate-induced peritonitis, and uric acid-induced peritonitis. Neutrophils expressed fewer genes than any other leukocyte population studied in ImmGen, and down-regulation of genes related to translation was particularly striking. However, genes with expression relatively specific to neutrophils were also identified, particularly three genes of unknown function: Stfa2l1, Mrgpr2a and Mrgpr2b. Comparison of genes up-regulated in activated neutrophils led to several novel findings: increased expression of genes related to synthesis and use of glutathione and of genes related to uptake and metabolism of modified lipoproteins, particularly in neutrophils elicited by thioglycollate; increased expression of genes for transcription factors in the Nr4a family, only in neutrophils elicited by serum-transfer arthritis; and increased expression of genes important in synthesis of prostaglandins and response to leukotrienes, particularly in neutrophils elicited by uric acid. Up-regulation of genes related to apoptosis, response to microbial products, NFkB family members and their regulators, and MHC class II expression was also seen, in agreement with previous studies. A regulatory model developed from the ImmGen data was used to infer regulatory genes involved in the changes in gene expression during neutrophil activation. Among 64, mostly novel, regulatory genes predicted to influence these changes in gene expression, Irf5 was shown to be important for optimal secretion of IL-10, IP-10, MIP-1α, MIP-1β, and TNF-α by mouse neutrophils in vitro after stimulation through TLR9. This data-set and its analysis using the ImmGen regulatory

  4. Imaging early pathogenesis of bubonic plague: are neutrophils commandeered for lymphatic transport of bacteria?

    PubMed

    Bland, David M; Anderson, Deborah M

    2013-11-05

    Vector-borne infections begin in the dermis when a pathogen is introduced by an arthropod during a blood meal. Several barriers separate an invading pathogen from its replicative niche, including phagocytic cells in the dermis that activate immunity by engulfing would-be pathogens and migrating to the lymph node. In addition, neutrophils circulating in the blood are rapidly recruited when the dermal barriers are penetrated. For flea-borne disease, no insect-encoded immune-suppressive molecules have yet been described that might influence the establishment of infection, leaving the bacteria on their own to defend against the mammalian immune system. Shortly after a flea transmits Yersinia pestis to a mammalian host, the bacteria are transported to the lymph node, where they grow logarithmically and later spread systemically. Even a single cell of Y. pestis can initiate a lethal case of plague. In their article, J. G. Shannon et al. [mBio 4(5):e00170-13, 2013, doi:10.1128/mBio.00170-13] used intravital microscopy to visualize trafficking of Y. pestis in transgenic mice in vivo, which allowed them to examine interactions between bacteria and specific immune cells. Bacteria appeared to preferentially interact with neutrophils but had no detectable interactions with dendritic cells. These findings suggest that Y. pestis infection of neutrophils not only prevents their activation but may even result in their return to circulation and migration to distal sites.

  5. Proteins derived from neutrophil extracellular traps may serve as self-antigens and mediate organ damage in autoimmune diseases.

    PubMed

    Knight, Jason S; Carmona-Rivera, Carmelo; Kaplan, Mariana J

    2012-01-01

    Neutrophils are the most abundant leukocytes in circulation and represent one of the first lines of defense against invading pathogens. Neutrophils possess a vast arsenal of antimicrobial proteins, which can be released from the cell by a death program termed NETosis. Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin decorated with granular and cytosolic proteins. Both exuberant NETosis and impaired clearance of NETs have been implicated in the organ damage of autoimmune diseases, such as systemic lupus erythematosus (SLE), small vessel vasculitis (SVV), and psoriasis. NETs may also represent an important source of modified autoantigens in SLE and SVV. Here, we review the autoimmune diseases linked to NETosis, with a focus on how modified proteins externalized on NETs may trigger loss of immune tolerance and promote organ damage.

  6. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence.

    PubMed

    Allen, Robert C

    2015-01-01

    Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2) facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen ((1)O2(*)) is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism.

  7. Exercise, training and neutrophil microbicidal activity.

    PubMed

    Smith, J A; Telford, R D; Mason, I B; Weidemann, M J

    1990-06-01

    The concentration in human plasma of putative neutrophil-"priming" cytokines like endogenous pyrogens is known to increase significantly in response to moderate exercise (11). This is characteristic of an acute-phase response. The ability of blood neutrophils isolated from both trained and untrained human subjects (n = 11, 9) to produce microbicidal reactive oxygen species was determined using luminol-enhanced chemiluminescence both before and after one hour of aerobic exercise at 60% VO2max. Irrespective of training and stimulus concentration, exercise nearly always caused significant "priming" of the capacity of neutrophils to produce H2O2 and HOCl upon stimulation with opsonized zymosan (P less than 0.01); however, compared to their untrained counterparts, the activity of cells isolated from trained individuals was depressed about 50% at unit stimulus concentration, both before and after exercise (P less than 0.075), whilst remaining unaltered at saturating concentrations. Although neutrophil oxygenation activity is only one parameter that contributes to immunological status, regular episodes of moderate exercise may increase resistance to infection by priming the "killing capacity" of neutrophils. In contrast, prolonged periods of intensive training may lead to increased susceptibility to common infections by diminishing this activity. PMID:2115507

  8. Methoxatin (PQQ) in guinea-pig neutrophils.

    PubMed

    Bishop, A; Paz, M A; Gallop, P M; Karnovsky, M L

    1994-10-01

    PQQ, also called methoxatin, has been isolated from guinea-pig neutrophils. The organic cations diphenyleneiodonium (DPI) and diphenyliodonium (BPI) and the aromatic o-diamine 4,5-dimethylphenylenediamine (DIMPDA) sequester synthetic PQQ and inhibit its redox-cycling activity in a model system. Standards were made of adducts of tritiated PQQ with unlabeled DIMPDA and of unlabeled PQQ with tritiated DPI or DIMPDA. PQQ adducts were isolated from guinea-pig neutrophils with each of the tritiated inhibitors. They were separated and defined by high-performance liquid chromatography (HPLC). Tiron, a disodium benzene disulphonic acid, broke the DPI-PQQ adduct isolated from neutrophils and released free PQQ. Both DPI and DIMPDA, as well as BPI, blocked O2.- release by stimulated neutrophils. The blockade exerted by these inhibitors was released by the addition of PQQ to the cell suspensions. The data demonstrate the presence of PQQ in guinea-pig neutrophils and suggest that it has a possible role, direct or indirect, in the O2.(-)-producing respiratory burst.

  9. Stimulation of neutrophils by tumor necrosis factor

    SciTech Connect

    Klebanoff, S.J.; Vadas, M.A.; Harlan, J.M.; Sparks, L.H.; Gamble, J.R.; Agosti, J.M.; Waltersdorph, A.M.

    1986-06-01

    Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H/sub 2/O/sub 2/ production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.

  10. Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia

    PubMed Central

    Hillgruber, Carina; Pöppelmann, Birgit; Weishaupt, Carsten; Steingräber, Annika Kathrin; Wessel, Florian; Berdel, Wolfgang E.; Gessner, J. Engelbert; Ho-Tin-Noé, Benoît

    2015-01-01

    Spontaneous organ hemorrhage is the major complication in thrombocytopenia with a potential fatal outcome. However, the exact mechanisms regulating vascular integrity are still unknown. Here, we demonstrate that neutrophils recruited to inflammatory sites are the cellular culprits inducing thrombocytopenic tissue hemorrhage. Exposure of thrombocytopenic mice to UVB light provokes cutaneous petechial bleeding. This phenomenon is also observed in immune-thrombocytopenic patients when tested for UVB tolerance. Mechanistically, we show, analyzing several inflammatory models, that it is neutrophil diapedesis through the endothelial barrier that is responsible for the bleeding defect. First, bleeding is triggered by neutrophil-mediated mechanisms, which act downstream of capturing, adhesion, and crawling on the blood vessel wall and require Gαi signaling in neutrophils. Second, mutating Y731 in the cytoplasmic tail of VE-cadherin, known to selectively affect leukocyte diapedesis, but not the induction of vascular permeability, attenuates bleeding. Third, and in line with this, simply destabilizing endothelial junctions by histamine did not trigger bleeding. We conclude that specifically targeting neutrophil diapedesis through the endothelial barrier may represent a new therapeutic avenue to prevent fatal bleeding in immune-thrombocytopenic patients. PMID:26169941

  11. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence

    PubMed Central

    Allen, Robert C.

    2015-01-01

    Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2) facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen (1O2*) is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism. PMID:26783542

  12. Mitochondrial DNA released by trauma induces neutrophil extracellular traps.

    PubMed

    Itagaki, Kiyoshi; Kaczmarek, Elzbieta; Lee, Yen Ting; Tang, I Tien; Isal, Burak; Adibnia, Yashar; Sandler, Nicola; Grimm, Melissa J; Segal, Brahm H; Otterbein, Leo E; Hauser, Carl J

    2015-01-01

    Neutrophil extracellular traps (NETs) are critical for anti-bacterial activity of the innate immune system. We have previously shown that mitochondrial damage-associated molecular patterns (mtDAMPs), including mitochondrial DNA (mtDNA), are released into the circulation after injury. We therefore questioned whether mtDNA is involved in trauma-induced NET formation. Treatment of human polymorphoneutrophils (PMN) with mtDNA induced robust NET formation, though in contrast to phorbol myristate acetate (PMA) stimulation, no NADPH-oxidase involvement was required. Moreover, formation of mtDNA-induced NETs was completely blocked by TLR9 antagonist, ODN-TTAGGG. Knowing that infective outcomes of trauma in elderly people are more severe than in young people, we measured plasma mtDNA and NET formation in elderly and young trauma patients and control subjects. MtDNA levels were significantly higher in the plasma of elderly trauma patients than young patients, despite lower injury severity scores in the elderly group. NETs were not visible in circulating PMN isolated from either young or old control subjects. NETs were however, detected in PMN isolated from young trauma patients and to a lesser extent from elderly patients. Stimulation by PMA induced widespread NET formation in PMN from both young volunteers and young trauma patients. NET response to PMA was much less pronounced in both elderly volunteers' PMN and in trauma patients' PMN. We conclude that mtDNA is a potent inducer of NETs that activates PMN via TLR9 without NADPH-oxidase involvement. We suggest that decreased NET formation in the elderly regardless of higher mtDNA levels in their plasma may result from decreased levels of TLR9 and/or other molecules, such as neutrophil elastase and myeloperoxidase that are involved in NET generation. Further study of the links between circulating mtDNA and NET formation may elucidate the mechanisms of trauma-related organ failure as well as the greater susceptibility to

  13. Enhanced neutrophil activity is associated with shorter time to tumor progression in glioblastoma patients

    PubMed Central

    Rahbar, Afsar; Cederarv, Madeleine; Wolmer-Solberg, Nina; Tammik, Charlotte; Stragliotto, Giuseppe; Peredo, Inti; Fornara, Olesja; Xu, Xinling; Dzabic, Mensur; Taher, Chato; Skarman, Petra; Söderberg-Nauclér, Cecilia

    2016-01-01

    ABSTRACT Glioblastoma multiforme (GBM) is a highly malignant tumor with a poor outcome that is often positive for human cytomegalovirus (HCMV). GBM patients often have excessive numbers of neutrophils and macrophages near and within the tumor. Here, we characterized the cytokine patterns in the blood of GBM patients with and without Valganciclovir treatment. Furthermore, we determined whether neutrophil activation is related to HCMV status and patient outcome. Blood samples for analyses of cytokines and growth factors were collected from 42 GBM patients at the time of diagnosis (n = 42) and at weeks 12 and 24 after surgery. Blood neutrophils of 28 GBM patients were examined for CD11b expression. The levels of pro- and anti-inflammatory cytokines and chemokines—including interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, transforming growth factor (TGF)-β1, interferon-γ, interferon-α, tumor necrosis factor α, and monocyte chemoattractant protein (MCP)-1were analyzed with a bead-based flow cytometry assay. During the first six months after surgery, neutrophil activity was increased in 12 patients and was unchanged or decreased in 16. Patients with increased neutrophil activity had enhanced IL-12p70, high grade HCMV and a shorter time to tumor progression (TTP) than patients without or decreased neutrophil activity (median TTP; 5.4 vs. 12 months, 95% confidence interval; 1.6–10 vs. 0.1–0.6, hazard ratio = 3 vs. 0.4, p = 0.004). The levels of IL-12p70 were significantly decreased in Valganciclovir treated patients (n = 22, T 12W vs. T 24W, p = 0.03). In conclusion, our findings suggest that neutrophil activation is an early sign of tumor progression in GBM patients. PMID:27057448

  14. Essential Role for Neutrophils in Pathogenesis and Adaptive Immunity in Chlamydia caviae Ocular Infections ▿

    PubMed Central

    Lacy, H. Marie; Bowlin, Anne K.; Hennings, Leah; Scurlock, Amy M.; Nagarajan, Uma M.; Rank, Roger G.

    2011-01-01

    Trachoma, the world's leading cause of preventable blindness, is produced by chronic ocular infection with Chlamydia trachomatis, an obligate intracellular bacterium. While many studies have focused on immune mechanisms for trachoma during chronic stages of infection, less research has targeted immune mechanisms in primary ocular infections, events that could impact chronic responses. The goal of this study was to investigate the function of neutrophils during primary chlamydial ocular infection by using the guinea pig model of Chlamydia caviae inclusion conjunctivitis. We hypothesized that neutrophils help modulate the adaptive response and promote host tissue damage. To test these hypotheses, guinea pigs with primary C. caviae ocular infections were depleted of neutrophils by using rabbit antineutrophil antiserum, and immune responses and immunopathology were evaluated during the first 7 days of infection. Results showed that neutrophil depletion dramatically decreased ocular pathology, both clinically and histologically. The adaptive response was also altered, with increased C. caviae-specific IgA titers in tears and serum and decreased numbers of CD4+ and CD8+ T cells in infected conjunctivae. Additionally, there were changes in conjunctival chemokines and cytokines, such as increased expression of IgA-promoting interleukin-5 and anti-inflammatory transforming growth factor β, along with decreased expression of T cell-recruiting CCL5 (RANTES). This study, the first to investigate the role of neutrophils in primary chlamydial ocular infection, indicates a previously unappreciated role for neutrophils in modulating the adaptive response and suggests a prominent role for neutrophils in chlamydia-associated ocular pathology. PMID:21402767

  15. Essential role for neutrophils in pathogenesis and adaptive immunity in Chlamydia caviae ocular infections.

    PubMed

    Lacy, H Marie; Bowlin, Anne K; Hennings, Leah; Scurlock, Amy M; Nagarajan, Uma M; Rank, Roger G

    2011-05-01

    Trachoma, the world's leading cause of preventable blindness, is produced by chronic ocular infection with Chlamydia trachomatis, an obligate intracellular bacterium. While many studies have focused on immune mechanisms for trachoma during chronic stages of infection, less research has targeted immune mechanisms in primary ocular infections, events that could impact chronic responses. The goal of this study was to investigate the function of neutrophils during primary chlamydial ocular infection by using the guinea pig model of Chlamydia caviae inclusion conjunctivitis. We hypothesized that neutrophils help modulate the adaptive response and promote host tissue damage. To test these hypotheses, guinea pigs with primary C. caviae ocular infections were depleted of neutrophils by using rabbit antineutrophil antiserum, and immune responses and immunopathology were evaluated during the first 7 days of infection. Results showed that neutrophil depletion dramatically decreased ocular pathology, both clinically and histologically. The adaptive response was also altered, with increased C. caviae-specific IgA titers in tears and serum and decreased numbers of CD4(+) and CD8(+) T cells in infected conjunctivae. Additionally, there were changes in conjunctival chemokines and cytokines, such as increased expression of IgA-promoting interleukin-5 and anti-inflammatory transforming growth factor β, along with decreased expression of T cell-recruiting CCL5 (RANTES). This study, the first to investigate the role of neutrophils in primary chlamydial ocular infection, indicates a previously unappreciated role for neutrophils in modulating the adaptive response and suggests a prominent role for neutrophils in chlamydia-associated ocular pathology. PMID:21402767

  16. Decreased apoptosis of beta 2- integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, Hajime; Higuchi, Hidetoshi; Teraoka, Hiroki; Takahashi, Kenji; Takahashi, Kensi; Kuwabara, Mikinori; Inanami, Osamu; Kuwabara, Mikwori

    2004-02-01

    Stimulant-induced viability of neutrophils, nuclear-fragmentation, increase in intracellular calcium ([Ca2+]i), expression of annexin V on neutrophils and proteolysis of a fluorogenic peptide substrate Ac-DEVD-MCA (acetyl Asp-Glu-Val-Asp alpha-[4-methyl-coumaryl-7-amide]) by neutrophil lysates from five normal calves and three calves with leucocyte adhesion deficiency were determined to evaluate the apoptosis of normal and CD18-deficient neutrophils. Viability was markedly decreased in control neutrophils stimulated with opsonized zymosan (OPZ), compared to CD18-deficient neutrophils at 37 degrees C after incubation periods of 6 and 24 hours. The rate of apoptosis of control neutrophils stimulated with OPZ increased significantly depending on the incubation time, whereas no apparent increase in apoptosis was found in CD18-deficient neutrophils under the same conditions. Aggregated bovine (Agg) IgG-induced apoptosis of control neutrophils was not significantly different from that of CD18-deficient neutrophils. The expression of annexin V on OPZ-stimulated control neutrophils was greater than that of unstimulated ones 6 h after stimulation. No apparent increase in annexin V expression on CD18-deficient neutrophils was found with OPZ stimulation. A delay in apoptosis was demonstrated in CD18-deficient bovine neutrophils and this appeared to be closely associated with lowered signalling via [Ca2+]i, diminished annexin V expression on the cell surface, and decreased caspase 3 activity in lysates. PMID:14984592

  17. Intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion

    PubMed Central

    Chang, Jungshan; Shi, Patricia A.; Chiang, Elaine Y.

    2008-01-01

    Previous studies using intravital microscopy in a sickle cell disease (SCD) mouse model suggest that adherent white blood cells (WBCs) play a key role in vaso-occlusion by capturing circulating red blood cells (RBCs) in venules. Commercial intravenous immunoglobulin (IVIG) given before the inflammatory stimuli increased microcirculatory blood flow and survival. To mimic the clinical situation in which SCD patients seek medical attention after the onset of symptoms, we developed an in vivo model in which the therapeutic intervention (eg, IVIG) was administered after in the inflammatory challenge. In this setting, IVIG rapidly (< 10 minutes) reduced adherent leukocyte numbers and dramatically inhibited interactions between RBCs and WBCs, resulting in improved microcirculatory blood flow and survival of sickle cell “Berkeley” mice. Longer survival correlated positively with blood flow (P = .001) and negatively with the number of adherent leukocytes (P = .001) and RBC-WBC interactions (P = .002). Using multichannel digital fluorescence videomicroscopy, we found that IVIG affected specifically the recruitment of neutrophils. Moreover, further analyses of leukocyte behavior revealed that IVIG significantly increased rolling velocities, indicating that it alters adhesion pathways involved in slow rolling. These data suggest that the potential therapeutic benefits of IVIG in SCD crises should be evaluated in a clinical trial. PMID:17932253

  18. Clinical Microfluidics for Neutrophil Genomics and Proteomics

    PubMed Central

    Kotz, Kenneth T.; Xiao, Wenzong; Miller-Graziano, Carol; Qian, Wei-Jun; Russom, Aman; Warner, Elizabeth A.; Moldawer, Lyle L.; De, Asit; Bankey, Paul E.; Petritis, Brianne O.; Camp, David G.; Rosenbach, Alan E.; Goverman, Jeremy; Fagan, Shawn P.; Brownstein, Bernard H.; Irimia, Daniel; Xu, Weihong; Wilhelmy, Julie; Mindrinos, Michael N.; Smith, Richard D.; Davis, Ronald W.; Tompkins, Ronald G.; Toner, Mehmet

    2010-01-01

    Neutrophils play critical roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood and develop ‘on-chip’ processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Lastly, we implement this tool as part of a near patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of patients in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting. PMID:20802500

  19. Clinical microfluidics for neutrophil genomics and proteomics.

    PubMed

    Kotz, Kenneth T; Xiao, Wenzong; Miller-Graziano, Carol; Qian, Wei-Jun; Russom, Aman; Warner, Elizabeth A; Moldawer, Lyle L; De, Asit; Bankey, Paul E; Petritis, Brianne O; Camp, David G; Rosenbach, Alan E; Goverman, Jeremy; Fagan, Shawn P; Brownstein, Bernard H; Irimia, Daniel; Xu, Weihong; Wilhelmy, Julie; Mindrinos, Michael N; Smith, Richard D; Davis, Ronald W; Tompkins, Ronald G; Toner, Mehmet

    2010-09-01

    Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.

  20. Granulopoiesis and granules of human neutrophils.

    PubMed

    Cowland, Jack B; Borregaard, Niels

    2016-09-01

    Granules are essential for the ability of neutrophils to fulfill their role in innate immunity. Granule membranes contain proteins that react to environmental cues directing neutrophils to sites of infection and initiate generation of bactericidal oxygen species. Granules are densely packed with proteins that contribute to microbial killing when liberated to the phagosome or extracellularly. Granules are, however, highly heterogeneous and are traditionally subdivided into azurophil granules, specific granules, and gelatinase granules in addition to secretory vesicles. This review will address issues pertinent to formation of granules, which is a process intimately connected to maturation of neutrophils from their precursors in the bone marrow. We further discuss possible mechanisms by which decisions are made regarding sorting of proteins to constitutive secretion or storage in granules and how degranulation of granule subsets is regulated. PMID:27558325

  1. Defective neutrophil chemotaxis in juvenile periodontitis.

    PubMed Central

    Clark, R A; Page, R C; Wilde, G

    1977-01-01

    Neutrophil chemotaxis was evaluated in nine patients with juvenile periodontitis, with normal subjects and patients with the adult form of periodontitis as controls. Defective chemotactic responses were observed in neutrophils from seven of nine juvenile patients, and a reduced level of complement-derived chemotactic activity was demonstrated in serum from four patients. These determinations were normal in all the patients with adult periodontitis. Serum from five of the juvenile patients contained a heat-stable, non-dialyzable factor that markedly inhibited the chemotaxis of normal neutrophils. Thus the characteristic tissue destruction seen in juvenile periodontitis may be, at least in part, a consequence of a failure of host defense mechanisms. PMID:591063

  2. [Ultrastructural location of enzymes in peripheral blood neutrophils and in cerebrospinal fluid neutrophils in neuroinfections].

    PubMed

    Skotarczak, B

    1993-01-01

    Using cytochemical methods the location and activity were determined of alkaline phosphatase, ATP-ase and succinate dehydrogenase as representative enzymes for the metabolic processes in neutrophils isolated from blood and cerebrospinal fluid (CSF) of patients with meningococcal meningoencephalitis as compared with peripheral blood neutrophils in a control group. The study showed presence of phosphatase on the membranes of many intracellular structures. The activity of the enzymes was higher than in the control group in the membranes of neutrophils in blood and CSF. This is explained as an effect of action of the chemotactic factor on the cell membrane and activation of the cell to movements and phagocytosis. ATP-ase activity in peripheral blood neutrophils in controls was found in all membranous structures in the cell. However, in peripheral blood neutrophils and CSF neutrophils in the acute stage of the disease the active enzyme was noted, in the first place, in cell membranes and digesting vacuoles, which reflected probably the direction of metabolic processes for phagocytosis and destroying of bacteria. The activity of succinate dehydrogenase was found in mitochondrial membranes. Peripheral blood and CSF neutrophils showed a high activity of the enzyme. In the CSF cells in acute phase atypical sites of succinate dehydrogenase activity were noted, which was explained as a sign of cell destruction.

  3. Mannheimia haemolytica and its leukotoxin cause neutrophil extracellular trap formation by bovine neutrophils.

    PubMed

    Aulik, Nicole A; Hellenbrand, Katrina M; Klos, Heather; Czuprynski, Charles J

    2010-11-01

    Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of bovine respiratory disease. PMID:20823211

  4. Attachment and ingestion of gonococci human neutrophils.

    PubMed

    Dilworth, J A; Hendley, J O; Mandell, G L

    1975-03-01

    Previous studies have indirectly shown that type 1 gonococci are more resistant to phagocytosis by human neutrophils (PMN) than type 3 gonococci. Using phase contrast, fluorescent, and light microscopy, we directly quantitated PMN-gonococcal interaction, with emphasis on separating ingestion from attachment. PMN monolayers were incubated on slides with type 1 or type 3 gonococcal fluorescent antibody (FA). After methanol fixation, the FA-stained gonococci associated with PMN were cointed. Since the live PMN excludes FA, the FA-stained gonococci represent only extracellular gonococci. Methylene blue was then added to the smae slide to stain both ingested and surface attached gonococci. Using these methods, intracellular and extracellular cell-associated gonococci were quantitated under varying conditions. The numbers of methylene blue-stained cell-associated gonococci that were ingested were: with normal serum, 3.7 plus or minus 4.1 per cent for type 1 and 56.2 plus or minus 3.7 percent for type 3 (P smaller than 0.001); with heat-inactivated serum, 1.0 plus or minus 3.0 per cent for type 1 and 52.6 plus or minus 3.7 per cent for type 3 (P smaller than 0.001); with higher-titer anti-gonococcal antibody serum, 4.8 plus or minus 4.3 percent for type 1 and 64.0 plus or minus 1.6 per cent for type 3 (P smaller than 0.001). Thus, most type 3 organisms were ingested, but most type 1 gonococci were bound on the PMN surface.

  5. Attachment and ingestion of gonococci human neutrophils.

    PubMed Central

    Dilworth, J A; Hendley, J O; Mandell, G L

    1975-01-01

    Previous studies have indirectly shown that type 1 gonococci are more resistant to phagocytosis by human neutrophils (PMN) than type 3 gonococci. Using phase contrast, fluorescent, and light microscopy, we directly quantitated PMN-gonococcal interaction, with emphasis on separating ingestion from attachment. PMN monolayers were incubated on slides with type 1 or type 3 gonococcal fluorescent antibody (FA). After methanol fixation, the FA-stained gonococci associated with PMN were cointed. Since the live PMN excludes FA, the FA-stained gonococci represent only extracellular gonococci. Methylene blue was then added to the smae slide to stain both ingested and surface attached gonococci. Using these methods, intracellular and extracellular cell-associated gonococci were quantitated under varying conditions. The numbers of methylene blue-stained cell-associated gonococci that were ingested were: with normal serum, 3.7 plus or minus 4.1 per cent for type 1 and 56.2 plus or minus 3.7 percent for type 3 (P smaller than 0.001); with heat-inactivated serum, 1.0 plus or minus 3.0 per cent for type 1 and 52.6 plus or minus 3.7 per cent for type 3 (P smaller than 0.001); with higher-titer anti-gonococcal antibody serum, 4.8 plus or minus 4.3 percent for type 1 and 64.0 plus or minus 1.6 per cent for type 3 (P smaller than 0.001). Thus, most type 3 organisms were ingested, but most type 1 gonococci were bound on the PMN surface. Images PMID:46842

  6. Metabolic requirements for neutrophil extracellular traps formation

    PubMed Central

    Rodríguez-Espinosa, Oscar; Rojas-Espinosa, Oscar; Moreno-Altamirano, María Maximina Bertha; López-Villegas, Edgar Oliver; Sánchez-García, Francisco Javier

    2015-01-01

    As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 1010 to 1011 cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis. PMID:25545227

  7. Neutrophil haptotaxis induced by the lectin KM+.

    PubMed

    Ganiko, L; Martins, A R; Espreáfico, E M; Roque-Barreira, M C

    1998-05-01

    KM+ is a D-mannose binding lectin from Artocarpus integrifolia that induces neutrophil migration in vitro and in vivo. This attractant activity was shown to be caused by haptotaxis rather than chemotaxis. The inhibition by D-mannose of the neutrophil attraction exerted by KM+, both in vitro and in vivo, supports the idea that haptotaxis is triggered in vivo by the sugar binding sites interacting with glycoconjugates located on the neutrophil surface and in the extracellular matrix. In the present study an in vivo haptotaxis assay was performed by intradermally (i.d.) injecting 125I-KM+ (200 ng), which led to a selective staining of loose connective tissue and vascular endothelium. The radiolabelled area exhibited a maximum increase (five-fold) in neutrophil infiltration 3 h after injection, relative to i.d. 200 ng 125I-BSA. We characterized the ex vivo binding of KM+ to tissue elements by immunohistochemistry, using paraformaldehyde-fixed, paraffin-embedded, untreated rat skin. Bound KM+ was detected with an affinity-purified rabbit IgG anti-KM+ and visualized with an alkaline phosphatase based system. KM+ binding to connective tissue and vascular endothelium was inhibited by preincubating KM+ with 0.4 mM D-mannose and was potentiated by heparan sulfate (100 microg ml(-1)). An in vitro assay carried out in a Boyden microchamber showed that heparan sulfate potentiated the attractant effect of 10 microg KM+ by 34%. The present data suggest that KM+ induces neutrophil migration in vivo by haptotaxis and that the haptotactic gradient could be provided by the interaction of the KM+ carbohydrate recognition site(s) with mannose-containing glycoconjugate(s) in vascular endothelium and connective tissue. Heparan sulfate would act as an accessory molecule, enhancing the KM+ tissue binding and potentiating the induced neutrophil haptotaxis.

  8. Dynamics of neutrophil migration in lymph nodes during infection

    PubMed Central

    Chtanova, Tatyana; Schaeffer, Marie; Han, Seong-Ji; van Dooren, Giel G.; Nollmann, Marcelo; Herzmark, Paul; Chan, Shiao Wei; Satija, Harshita; Camfield, Kristin; Aaron, Holly; Striepen, Boris; Robey, Ellen A.

    2008-01-01

    Summary While the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes, or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning laser microscopy (TPSLM) to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We find that neutrophils form both small, transient or large, persistent swarms via a strikingly coordinated migration pattern. We provide evidence that cooperative action of neutrophils and parasite egress from host cells can trigger swarm formation. Neutrophil swarm formation coincides in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses. PMID:18718768

  9. Streptococcal M1 protein triggers chemokine formation, neutrophil infiltration, and lung injury in an NFAT-dependent manner.

    PubMed

    Zhang, Songen; Zhang, Su; Garcia-Vaz, Eliana; Herwald, Heiko; Gomez, Maria F; Thorlacius, Henrik

    2015-06-01

    Streptococcus pyogenes of the M1 serotype can cause STSS, which is associated with significant morbidity and mortality. The purpose of the present study was to examine the role of NFAT signaling in M1 protein-induced lung injury. NFAT-luc mice were treated with the NFAT inhibitor A-285222 before administration of the M1 protein. Neutrophil infiltration, edema, and CXC chemokines were quantified in the lung, 4 h after challenge with the M1 protein. Flow cytometry was used to determine Mac-1 expression. Challenge with the M1 protein increased NFAT-dependent transcriptional activity in the lung, spleen, and liver in NFAT-luc mice. Administration of the NFAT inhibitor A-285222 abolished M1 protein-evoked NFAT activation in the lung, spleen, and liver. M1 protein challenge induced neutrophil recruitment, edema, and CXC chemokine production in the lung, as well as up-regulation of Mac-1 on circulating neutrophils. Inhibition of NFAT activity attenuated M1 protein-induced neutrophil infiltration by 77% and edema formation by 50% in the lung. Moreover, administration of A-285222 reduced M1 protein-evoked pulmonary formation of CXC chemokine >80%. In addition, NFAT inhibition decreased M1 protein-triggered Mac-1 up-regulation on neutrophils. These findings indicate that NFAT signaling controls pulmonary infiltration of neutrophils in response to streptococcal M1 protein via formation of CXC chemokines and neutrophil expression of Mac-1. Thus, the targeting of NFAT activity might be a useful way to ameliorate lung injury in streptococcal infections.

  10. Identification of CD177 as the most dysregulated parameter in a microarray study of purified neutrophils from septic shock patients.

    PubMed

    Demaret, Julie; Venet, Fabienne; Plassais, Jonathan; Cazalis, Marie-Angélique; Vallin, Hélène; Friggeri, Arnaud; Lepape, Alain; Rimmelé, Thomas; Textoris, Julien; Monneret, Guillaume

    2016-10-01

    Sepsis represents the host's systemic inflammatory response to an infection. In this condition, immune response associates a marked inflammatory response and the delayed development of severe dysfunctions affecting both innate and adaptive responses. As neutrophils are the first line of defense against infection, they are central to the pathophysiology of sepsis in first hours. Nevertheless, their role during immunosuppression phase remains elusive. The main objective of the current work was to perform a transcriptomic study on purified neutrophils from septic shock patients (n=9) so as to identify genes that are differentially expressed during the first week after disease onset both (3-4 and 6-8days) versus healthy donors. Then, 45 septic shock patients were prospectively enrolled to confirm results at the protein level using flow cytometry. Twenty healthy volunteers (HV) were also included for the whole study. By comparing the transcriptome of purified neutrophils, we identified 364 up-regulated and 328 down-regulated genes differentially expressed. Of them, CD177 mRNA, coding for an activation molecule in chemotaxis, had the highest fold change modulation between patients and HV. This increase was then confirmed at the protein level. There was a constant subset of neutrophils that did not express CD177. However, when positive, septic neutrophils presented with significantly increased CD177 expression. Of note, no association between CD177 overexpression and features of immunosuppression has been highlighted. In addition, this up-regulation was negatively correlated with a decreased expression of CD10, a characteristic of immature myeloid cells. In conclusion, in this exploratory work, we shed light on the increased CD177 mRNA and protein expressions in circulating neutrophils after septic shock. Considering the potential dual roles of CD177 neutrophil (i.e., maturation/chemotaxis), negatively correlated in this study, its participation in septic shock

  11. Adaptation to Resistance Training Is Associated with Higher Phagocytic (but Not Oxidative) Activity in Neutrophils of Older Women

    PubMed Central

    Bartholomeu-Neto, João; Brito, Ciro José; Nóbrega, Otávio Toledo; Sousa, Vinícius Carolino; Oliveira Toledo, Juliana; Silva Paula, Roberta; Alves, David Junger Fonseca; Ferreira, Aparecido Pimentel; Franco Moraes, Clayton; Córdova, Cláudio

    2015-01-01

    Failure in antimicrobial activity contributes to high morbidity and mortality in the geriatric population. Little is known about the potential effect of resistance training (RT) on the functional properties of the innate immunity. This study aimed to investigate the influence of long-term RT on the endocytic and oxidative activities of neutrophils and monocytes in healthy older women. Our results indicate that the phagocytosis index (PhI) of neutrophils (but not of monocytes) in the RT-adapted group was significantly higher (P < 0.001; effect size, (d) = 0.90, 95% CI: [0.75–1.04]) compared to that in sedentary subjects. In contrast, the oxidative activity of either neutrophils or monocytes was not significantly influenced by RT. Also, total energy and carbohydrate intake as well as serum IL6 levels had a significant influence on the phagocytic activity of neutrophils (P = 0.04), being considered in the model. Multivariate regression identified the physical condition of the subject (β = 0.425; P = 0.01) as a significant predictor of PhI. In conclusion, circulating neutrophils of older women adapted to a long-term RT program expressed higher phagocytic activity. PMID:26524964

  12. DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps.

    PubMed

    Buchanan, John T; Simpson, Amelia J; Aziz, Ramy K; Liu, George Y; Kristian, Sascha A; Kotb, Malak; Feramisco, James; Nizet, Victor

    2006-02-21

    The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF. Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo. The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS infection.

  13. Decreased phagocytic function in neutrophils and monocytes from peripheral blood in periodontal disease

    PubMed Central

    CARNEIRO, Valéria Martins Araújo; BEZERRA, Ana Cristina Barreto; GUIMARÃES, Maria do Carmo Machado; MUNIZ-JUNQUEIRA, Maria Imaculada

    2012-01-01

    Phagocytosis by neutrophils and monocytes constitutes the main defense mechanism against bacterial challenges in periodontitis. Phagocytosis by neutrophils has already been evaluated, whereas phagocytic function of monocytes has hardly been addressed so far. Objectives The aim of this study was to assess phagocytosis by neutrophils and monocytes in periodontitis. Material and Methods The sample included 30 subjects with severe periodontitis and 27 control subjects without periodontal disease. The phagocytic index (PhI) was calculated as the mean number of adhered/ingested Saccharomyces cerevisiae per phagocytozing monocyte or neutrophil multiplied by the percentage of phagocytes involved in phagocytosis. Results A significant reduction in phagocyte functions was observed in individuals with periodontitis. The median of PhI of neutrophils using non-sensitized S. cerevisiae was 3 for the control group, and 1.5 for the periodontitis group (p=0.01, Mann-Whitney test). The median of PhI of monocytes with non-sensitized S. cerevisiae was 26.13 for the control group, and 13.23 for the periodontitis group (p=0.03, Mann Whitney test). The median of PhI of monocytes assessed with sensitized S. cerevisiae was 97.92 for the control group and 60.1 for the periodontitis group (p=0.005, t-test). Conclusion The data demonstrated a reduction in the function of phagocytes, suggesting a decrease in immune defenses in periodontitis. PMID:23138734

  14. A Morphological and Cytochemical Study of the Interaction between Paracoccidiodes brasiliensis and Neutrophils

    NASA Astrophysics Data System (ADS)

    Dias, Maria Fernanda R. G.; Filgueira, Absalom L.; de Souza, Wanderley

    2004-04-01

    Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the most prevalent systemic mycosis of Latin America and 80% of the reported cases are from Brazil. Because of the great number of neutrophils found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. Scanning and transmission electron microscopy of thin sections showed that the neutrophils ingest yeast cells through a typical phagocytic process with the formation of pseudopodes. The pseudopodes even disrupt the connection established between the mother and the bud cells. Neutrophils also associate to each other, forming a kind of extracellular vacuole where large yeast cells are encapsulated. Cytochemical studies showed that once P. brasiliensis attaches to the neutrophil surface, it triggers a respiratory burst with release of oxygen-derived products. Attachment also triggers neutrophils' degranulation, with release of endogenous peroxidase localized in cytoplasmic granules. Together, these processes lead to killing of both ingested and extracellular P. brasiliensis.

  15. Altered neutrophil immunophenotypes in childhood B-cell precursor acute lymphoblastic leukemia

    PubMed Central

    Oliveira, Elen; Bacelar, Thiago S.; Ciudad, Juana; Ribeiro, Maria Cecília M.; Garcia, Daniela R.N.; Sedek, Lukasz; Maia, Simone F.; Aranha, Daniel B.; Machado, Indyara C.; Ikeda, Arissa; Baglioli, Bianca F.; Lopez-Duarte, Nathalia; Teixeira, Lisandra A. C.; Szczepanski, Tomasz; Silva, Maria Luiza M.; Land, Marcelo G.P.

    2016-01-01

    An increasing number of evidences suggest a genetic predisposition in acute lymphoblastic leukemia (ALL) that might favor the occurrence of the driver genetic alterations. Such genetic background might also translate into phenotypic alterations of residual hematopoietic cells. Whether such phenotypic alterations are present in bone marrow (BM) cells from childhood B-cell precursor (BCP)-ALL remains to be investigated. Here we analyzed the immunophenotypic profile of BM and peripheral blood (PB) maturing/matured neutrophils from 118 children with BCP-ALL and their relationship with the features of the disease. Our results showed altered neutrophil phenotypes in most (77%) BCP-ALL cases. The most frequently altered marker was CD10 (53%), followed by CD33 (34%), CD13 (15%), CD15/CD65 (10%) and CD123 (7%). Of note, patients with altered neutrophil phenotypes had younger age (p = 0.03) and lower percentages of BM maturing neutrophils (p = 0.004) together with greater BM lymphocyte (p = 0.04), and mature B-cell (p = 0.03) counts. No significant association was found between an altered neutrophil phenotype and other disease features. These findings point out the potential existence of an altered residual hematopoiesis in most childhood BCP-ALL cases. PMID:27028865

  16. Immunostimulation of sugar cane extract on neutrophils to Salmonella typhimurium infection in mice.

    PubMed

    Chen, Ming-Hua; Lo, Dan-Yuan; Liao, Jiunn-Wang; Hsuan, Shih-Ling; Chien, Maw-Sheng; Lin, Cheng-Chung; Chen, Ter-Hsin; Lee, Wei-Cheng

    2012-07-01

    The aim of this study was to evaluate the immunomodulatory effects of sugar cane extract (SCE) on the biological activities of neutrophils in mice. Six-week-old BALB/c mice were fed 1250 mg/kg of SCE once. The generation, migration and biological functions of neutrophils and the survival rates of the mice in response to Salmonella typhimurium infection were evaluated. The results show that the numbers of both bone marrow cells and neutrophils were significantly increased in response to SCE administration (p < 0.05) compared with controls. The migration, phagocytosis and H₂O₂ generation of neutrophils were all significantly enhanced in SCE-treated mice (p < 0.05). After challenge with S. typhimurium (lethal dose, 50% (LD₅₀), SCE-treated mice had a 19.2% higher survival rate and milder hepatic lesions than the controls. Additionally, fewer invasive bacteria were recovered from the spleens of SCE-treated mice. In conclusion, our results suggest that SCE has a positive regulatory effect on the biological function of mouse neutrophils that may increase host resistance against bacterial infections.

  17. Alpha-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase.

    PubMed

    Manna, Sunil K; Sarkar, Abira; Sreenivasan, Yashin

    2006-03-01

    Considering the role of interleukin-8 (IL-8) in a large number of acute and chronic inflammatory diseases, the regulation of IL-8-mediated biological responses is important. Alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that alpha-MSH interacts predominantly with melanocortin-1 receptors and inhibits several IL-8-induced biological responses in macrophages and neutrophils. It down-regulated receptors for IL-8 but not for TNF, IL-4, IL-13 or TNF-related apoptosis-inducing ligand (TRAIL) in neutrophils. It down-regulated CXCR type 1 and 2 but not mRNA levels. alpha-MSH did not inhibit IL-8 binding in purified cell membrane or affinity-purified CXCR. IL-8 or anti-CXCR Ab protected against alpha-MSH-mediated inhibition of IL-8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in alpha-MSH-treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in alpha-MSH-induced down-regulation of CXCR. These studies suggest that alpha-MSH inhibits IL-8-mediated biological responses by down-regulating CXCR through induction of serine protease and that alpha-MSH acts as a potent immunomodulator in neutrophil-driven inflammatory distress. PMID:16479540

  18. Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporeal perfusion.

    PubMed

    Wachtfogel, Y T; Kucich, U; Hack, C E; Gluszko, P; Niewiarowski, S; Colman, R W; Edmunds, L H

    1993-07-01

    Aprotinin reduces blood loss after cardiac operations and decreases the bleeding time. The mechanism of action of aprotinin that produces these effects is not clear. During simulated extracorporeal circulation the contact and complement systems, platelets, and neutrophils are activated. We investigated the effect of aprotinin on kallikrein-C1-inhibitor complex and C1-C1-inhibitor complex formation, neutrophil degranulation, and platelet release and aggregation during simulated extracorporeal circulation. Fresh heparinized human blood was recirculated at 37 degrees C for 2 hours in a spiral coil membrane oxygenator-roller pump perfusion circuit. Changes in platelet count, leukocyte count, platelet response to adenosine diphosphate, and plasma levels of beta-thromboglobulin, kallikrein-C1-inhibitor complexes, C1-C1-inhibitor complexes, and neutrophil elastase were measured before and at 5, 30, 60, and 120 minutes of recirculation at 0, 0.015, 0.03, 0.06, and 0.12 mg/ml doses of aprotinin. Platelet counts decreased to 36% +/- 12% of control values at 5 minutes and increased to 56% +/- 13% at 120 minutes without aprotinin. Aprotinin did not affect platelet counts, but it did prevent the decrease in sensitivity of platelets to adenosine diphosphate and it attenuated beta-thromboglobulin release. In the absence of aprotinin, kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively to 0.53 +/- 0.14 U/ml and 2.38 +/- 0.33 U/ml, respectively, at 120 minutes. Kallikrein-C1-inhibitor complexes were completely inhibited and C1-C1-inhibitor complexes were partially inhibited at aprotinin concentrations of 0.03 mg/ml or greater. Release of neutrophil elastase was partially but not completely inhibited at the highest dose of aprotinin and was 50% inhibited at a dose of 0.03 mg/ml. Because activation of the fibrinolytic system does not occur in this system, the changes were independent of the inhibition of plasmin. We conclude that aprotinin in high doses

  19. Aberrant “Barbed-Wire” Nuclear Projections of Neutrophils in Trisomy 18 (Edwards Syndrome)

    PubMed Central

    Kahwash, Basil M.; Nowacki, Nicholas B.; Kahwash, Samir B.

    2015-01-01

    We discuss the significance of neutrophils with increased, aberrant nuclear projections mimicking “barbed-wire” in a newborn child with trisomy 18 (T18). Increased, aberrant nuclear projections have been previously reported in trisomy of the D group of chromosomes (chromosomes 13, 14, and 15), and we report similar findings in a patient with T18. The peripheral blood smear showed relative neutrophilia with the majority (37%) of neutrophils showing two or more thin, rod-shaped or spike-shaped, and often pedunculated aberrant nuclear projections. The number of projections ranged from 2 to 6 per cell, averaged 2 per affected neutrophil, and ranged in length from 0.22 μm to 0.83 μm. This case confirms that the morphologic finding described is not restricted to trisomy of one of the chromosomes in group D, as implied in the literature. PMID:26770846

  20. Exposure to Leishmania braziliensis Triggers Neutrophil Activation and Apoptosis

    PubMed Central

    Hurrell, Benjamin P.; Celes, Fabiana S.; Curvelo, Rebecca P.; Prates, Deboraci B.; Barral, Aldina; Borges, Valeria M.; Tacchini-Cottier, Fabienne; de Oliveira, Camila I.

    2015-01-01

    Background Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate. Methods and Findings Inoculation of serum-opsonized L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis. Conclusions We show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection. PMID:25756874

  1. Staphylococcus epidermidis and biofilm-associated neutrophils in chronic rhinosinusitis. A pilot study.

    PubMed

    Marcinkiewicz, Janusz; Stręk, Paweł; Strus, Magdalena; Głowacki, Roman; Ciszek-Lenda, Marta; Zagórska-Świeży, Katarzyna; Gawda, Anna; Tomusiak, Anna

    2015-12-01

    A key role of bacterial biofilm in the pathogenesis of chronic rhinosinusitis (CRS) with (CRSwNP) and without nasal polyps (CRSsNP) is commonly accepted. However, the impact of some bacterial species isolated from inflamed sinus mucosa on biofilm formation is unclear. In particular, the role of Staphylococcus epidermidis as aetiological agents of CRS is controversial. Moreover, the effect of biofilm formation on neutrophil infiltration and activity in CRSwNP calls for explanation. In this study, biofilms were found in three of 10 patients (mean age = 46 ± 14) with CRS undergoing endoscopic sinus surgery by means of scanning electron microscopy. Unexpectedly, S. epidermidis was the primary isolated bacteria and was also found to be present in all biofilm-positive mucosa specimens, indicating its pivotal role in the pathogenesis of severe chronic infections associated with biofilm formation. We have also measured the activity of myeloperoxidase (MPO), the most abundant neutrophil enzyme, to demonstrate the presence of neutrophils in the samples tested. Our present results show that the level of MPO in CRS associated with biofilm is lower than that without biofilm. It may suggest either a low number of neutrophils or the presence of a type of neutrophils with compromised antimicrobial activity, described as biofilm-associated neutrophils (BAN). Finally, we conclude that further studies with a large number of CRS cases should be performed to establish the association between S. epidermidis and other frequently isolated bacterial species from paranasal sinuses, with the severity of CRS, biofilm formation and the infiltration of BAN.

  2. Staphylococcus epidermidis and biofilm-associated neutrophils in chronic rhinosinusitis. A pilot study.

    PubMed

    Marcinkiewicz, Janusz; Stręk, Paweł; Strus, Magdalena; Głowacki, Roman; Ciszek-Lenda, Marta; Zagórska-Świeży, Katarzyna; Gawda, Anna; Tomusiak, Anna

    2015-12-01

    A key role of bacterial biofilm in the pathogenesis of chronic rhinosinusitis (CRS) with (CRSwNP) and without nasal polyps (CRSsNP) is commonly accepted. However, the impact of some bacterial species isolated from inflamed sinus mucosa on biofilm formation is unclear. In particular, the role of Staphylococcus epidermidis as aetiological agents of CRS is controversial. Moreover, the effect of biofilm formation on neutrophil infiltration and activity in CRSwNP calls for explanation. In this study, biofilms were found in three of 10 patients (mean age = 46 ± 14) with CRS undergoing endoscopic sinus surgery by means of scanning electron microscopy. Unexpectedly, S. epidermidis was the primary isolated bacteria and was also found to be present in all biofilm-positive mucosa specimens, indicating its pivotal role in the pathogenesis of severe chronic infections associated with biofilm formation. We have also measured the activity of myeloperoxidase (MPO), the most abundant neutrophil enzyme, to demonstrate the presence of neutrophils in the samples tested. Our present results show that the level of MPO in CRS associated with biofilm is lower than that without biofilm. It may suggest either a low number of neutrophils or the presence of a type of neutrophils with compromised antimicrobial activity, described as biofilm-associated neutrophils (BAN). Finally, we conclude that further studies with a large number of CRS cases should be performed to establish the association between S. epidermidis and other frequently isolated bacterial species from paranasal sinuses, with the severity of CRS, biofilm formation and the infiltration of BAN. PMID:26765504

  3. Migration of neutrophils across endothelial monolayers is stimulated by treatment of the monolayers with beta-endorphin.

    PubMed

    Wiedermann, C J; Schratzberger, P; Kähler, C M

    1994-09-01

    To study the effects of the cytokine and neuroendocrine hormone beta-endorphin on the transendothelial migration of neutrophils, bovine pulmonary artery endothelial cells were grown to confluence on PVP-free polycarbonate filters coated with gelatin. Pretreatment of endothelial cell cultures with 1 to 10 mumol/liter of beta-endorphin for 60 min resulted in significantly stimulated migration of subsequently added neutrophils across the endothelial monolayer. The number of neutrophils that migrated across beta-endorphin-treated endothelial cells was similar to the number that traversed untreated monolayers in response to gradients of formylpeptide. Consistently, an additive effect was seen when migration was induced by both beta-endorphin pretreatment of the endothelial cells and a formylpeptide chemotactic gradient. When used at optimal concentration, beta-endorphin was equally effective in stimulating neutrophil migration as was tumor necrosis factor-alpha. In the absence of formylpeptide the effect of apical surface exposure of endothelial cells to beta-endorphin versus basal surface exposure was comparable. Stimulation of neutrophil transendothelial migration in this system appeared to be specific and mediated by opiate receptors, since excess concentration of naloxone completely abolished the effect of beta-endorphin but not of tumor necrosis factor-alpha. These results suggest that beta-endorphin, released during stress, may act upon the endothelium to promote emigration of neutrophils from the vasculature.

  4. [Perfluorocarbon emulsions and other corpuscular systems influence on neutrophil activity].

    PubMed

    Shekhtman, D G; Safronova, V G; Sklifas, A N; Alovskaia, A A; Gapeev, A B; Obraztsov, V V; Chemeris, N K

    1997-01-01

    Influence of perfluorodecalin, perfluoromethilcyclohexylpiperidine, perfluorotributylamine emulsions on active oxygen form (AOF) generation by neutrophils has been studied. All investigated emulsions stabilized both proxanol 268 and egg yolk phospholipids inhibited PMA-stimulated neutrophil activity. Castor oil emulsion also inhibited the neutrophil activity. Neutrophil response for chemotactic peptide, was unchanged in the presence of all tested emulsions. We suppose that fast hydrophobic attachment of inert submicrone emulsion particles to cell surface provokes alteration of neutrophil plasma membrane function resulting in a decrease of AOF generation. PMID:9490112

  5. Inhibition of Neutrophil Exocytosis Ameliorates Acute Lung Injury in Rats

    PubMed Central

    Uriarte, Silvia M.; Rane, Madhavi J.; Merchant, Michael L.; Jin, Shunying; Lentsch, Alex B.; Ward, Richard A.; McLeish, Kenneth R.

    2013-01-01

    Exocytosis of neutrophil granules contributes to acute lung injury (ALI) induced by infection or inflammation, suggesting that inhibition of neutrophil exocytosis in vivo could be a viable therapeutic strategy. This study was conducted to determine the effect of a cell-permeable fusion protein that inhibits neutrophil exocytosis (TAT-SNAP-23) on ALI using an immune complex deposition model in rats. The effect of inhibition of neutrophil exocytosis by intravenous administration of TAT-SNAP-23 on ALI was assessed by albumin leakage, neutrophil infiltration, lung histology, and proteomic analysis of bronchoalveolar lavage fluid (BALf). Administration of TAT-SNAP-23, but not TAT-Control, significantly reduced albumin leakage, total protein levels in the BALf, and intra-alveolar edema and hemorrhage. Evidence that TAT-SNAP-23 inhibits neutrophil exocytosis included a reduction in plasma membrane CD18 expression by BALf neutrophils and a decrease in neutrophil granule proteins in BALf. Similar degree of neutrophil accumulation in the lungs and/or BALf suggests that TAT-SNAP-23 did not alter vascular endothelial cell function. Proteomic analysis of BALf revealed that components of the complement and coagulation pathways were significantly reduced in BALf from TAT-SNAP-23-treated animals. Our results indicate that administration of a TAT-fusion protein that inhibits neutrophil exocytosis reduces in vivo ALI. Targeting neutrophil exocytosis is a potential therapeutic strategy to ameliorate ALI. PMID:23364427

  6. Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity.

    PubMed

    Gao, Chunyan; Xie, Rui; Li, Wen; Zhou, Jin; Liu, Shuchuan; Cao, Fenglin; Liu, Yue; Ma, Ruishuang; Si, Yu; Liu, Yan; Bi, Yayan; Gilbert, Gary E; Shi, Jialan

    2013-06-01

    Abundant senescent neutrophils traverse the vascular compartment and may contribute to pathologic conditions. For example, they become procoagulant when undergoing apoptosis and may contribute to thrombosis or inflammation. Our previous studies demonstrated a dominant clearance pathway in which the neutrophils can be phagocytosed by liver macrophages. The aim of this study was to explore an alternate pathway of neutrophil clearance by endothelial cells. Phagocytosis of the neutrophils by endothelial cells was performed using various experimental approaches includingflow cytometry, confocal microscopy and electron microscopy assays in vitro and in vivo. Procoagulant activity of cultured neutrophils was evaluated by coagulation time, factor Xase and prothrombinase assays. Lactadherin functioned as a novel probe for the detection of phosphatidylserine on apoptotic cells, an opsonin (bridge) between apoptotic cell and phagocyte for promoting phagocytosis, and an efficient anticoagulant for inhibition of factor Xase and thrombin formation. When cultured, purified human neutrophils spontaneously entered apoptosis and developed procoagulant activity that was directly related to the degree of phosphatidylserine exposure. Co-culture of aged neutrophils and endothelial cells resulted in phagocytosis of the neutrophils and prolonged coagulation time. Lactadherin diminished the procoagulant activity and increased the rate of neutrophil clearance. In vivo, neutrophils were sequestered by endothelial cells after blockade of Kupffer cells, a process that was dependent upon both phosphatidylserine exposure and P-selectin expression. Thus, the ability of endothelial cells to clear senescent neutrophils may limit the procoagulant and/or inflammatory impact of these cells.

  7. Neutrophil extracellular traps: Their role in periodontal disease

    PubMed Central

    Kolaparthy, Lakshmi Kanth; Sanivarapu, Sahitya; Swarna, Chakrapani; Devulapalli, Narasimha Swamy

    2014-01-01

    Neutrophils are the first line of innate immune defense against infectious diseases. Since their discovery, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during infections. Extensive research in the field of neutrophil cell biology and their role skewing the immune response in various infections or inflammatory disorders revealed their importance in the regulation of immune response. Neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation along with other antimicrobial molecules. Activated neutrophils provide signals for the activation and maturation of macrophages as well as dendritic cells. Neutrophils are also involved in the regulation of T-cell immune response against various pathogens and tumor antigens. Thus, the present review is intended to highlight the emerging role of neutrophil extracellular trap production in the regulation of immune response and its role in periodontal disease. PMID:25624623

  8. Neutrophil extracellular traps: Their role in periodontal disease.

    PubMed

    Kolaparthy, Lakshmi Kanth; Sanivarapu, Sahitya; Swarna, Chakrapani; Devulapalli, Narasimha Swamy

    2014-01-01

    Neutrophils are the first line of innate immune defense against infectious diseases. Since their discovery, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during infections. Extensive research in the field of neutrophil cell biology and their role skewing the immune response in various infections or inflammatory disorders revealed their importance in the regulation of immune response. Neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation along with other antimicrobial molecules. Activated neutrophils provide signals for the activation and maturation of macrophages as well as dendritic cells. Neutrophils are also involved in the regulation of T-cell immune response against various pathogens and tumor antigens. Thus, the present review is intended to highlight the emerging role of neutrophil extracellular trap production in the regulation of immune response and its role in periodontal disease. PMID:25624623

  9. Time-dependent recovery of passive neutrophils after large deformation.

    PubMed Central

    Tran-Son-Tay, R; Needham, D; Yeung, A; Hochmuth, R M

    1991-01-01

    Experiments are performed in which a passive human neutrophil is deformed into an elongated "sausage" shape by aspirating it into a small glass pipette. When expelled from the pipette the neutrophil recovers its natural spherical shape in approximately 1 minute. This recovery process is analyzed according to a Newtonian, liquid-drop model in which a variational method is used to simultaneously solve the hydrodynamic equations for low Reynolds-number flow and the equations for membrane equilibrium with a constant membrane tension. The theoretical model gives a good fit to the experimental data for a ratio of membrane cortical tension to cytoplasmic viscosity of approximately 1.7 x 10(-5) cm/s (0.17 micron/s). However, when the cell is held in the pipette for only a short time period of 5 s or less, and then expelled, the cell undergoes an initial, rapid elastic rebound suggesting that the cell behaves in this instance as a Maxwell viscoelastic liquid rather than a Newtonian liquid with constant cortical tension. PMID:1742456

  10. Neutrophil extracellular traps involvement in corneal fungal infection

    PubMed Central

    Zhao, Yingying; Zhang, Fan; Wan, Ting; Fan, Fangli; Xie, Xin; Lin, Zhenyun

    2016-01-01

    Purpose Neutrophils release neutrophil extracellular traps (NETs) when defending against invading microorganisms. We investigated the existence of NETs in fungal keratitis. Methods Fourteen patients with unilateral fungal keratitis were included. Detailed information about each patient was recorded, including (1) patient history (onset of symptoms and previous therapy), (2) ocular examination findings by slit-lamp biomicroscopy, (3) laboratory findings from direct smear examination and culture of corneal scrapings, (4) NET formation, and (5) treatment strategy and prognosis. Immunofluorescence staining was used to evaluate the existence of NETs on corneal scrapings. The relationship between the quantification of NETs and the clinical character of the fungal keratitis was identified. Results NETs were identified in all 14 patients. Patients with a higher grade of NET formation and fewer fungal hyphae always showed a good treatment response and a short course of infection. NETs were consistently found mixed with fungal hyphae in the corneal scrapings from infected patients. No statistical significance was found between the grade of NETs formed and the course of infection before presentation, and no relationship between the quantification of NETs and the size of the ulcer was found. Conclusions The results suggest that NETs are involved in fungal keratitis. The number of NETs in infected corneas may provide a tool for evaluating the prognosis for fungal keratitis. PMID:27559290

  11. Effects of Space Flight on Neutrophil Functions in Astronauts

    NASA Technical Reports Server (NTRS)

    Kaur, Indreshpal; Valadez, Victoria A.; Simons, Elizabeth R.; Pierson, Duane L.

    2000-01-01

    Neutrophil phagocytosis, oxidative burst, degranulation, and the expression of selected surface markers were studied in 25 astronauts following 4 space shuttle missions. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch, immediately after landing, and again at 3 days after landing. The number of neutrophils increased at landing by 85%. Phagocytosis of Escherichia coli (E. coli) and oxidative burst following the medium length (9 to 11 days) missions were lower than the control mean values. Whereas, following the short-duration (5 days) mission, these functions were unchanged from control values. No consistent changes in degranulation were observed following either short or medium length space missions. The expression of CD16, CD32, CD11a, CD11b, CD11c, L-selectin and CD36 were measured and found to be variable. Specifically, CD16 and CD32 did not correlate with the changes in oxidative burst. Mission duration appears to be a factor in phagocytic and oxidative functions.

  12. Distinct Oral Neutrophil Subsets Define Health and Periodontal Disease States.

    PubMed

    Fine, N; Hassanpour, S; Borenstein, A; Sima, C; Oveisi, M; Scholey, J; Cherney, D; Glogauer, M

    2016-07-01

    Neutrophils exit the vasculature and swarm to sites of inflammation and infection. However, these cells are abundant in the healthy, inflammation-free human oral environment, suggesting a unique immune surveillance role within the periodontium. We hypothesize that neutrophils in the healthy oral cavity occur in an intermediary parainflammatory state that allows them to interact with and contain the oral microflora without eliciting a marked inflammatory response. Based on a high-throughput screen of neutrophil CD (cluster of differentiation) marker expression and a thorough literature review, we developed multicolor flow cytometry panels to determine the surface marker signatures of oral neutrophil subsets in periodontal health and disease. We define here 3 distinct neutrophil subsets: resting/naive circulatory neutrophils, parainflammatory neutrophils found in the healthy oral cavity, and proinflammatory neutrophils found in the oral cavity during chronic periodontal disease. Furthermore, parainflammatory neutrophils manifest as 2 distinct subpopulations-based on size, granularity, and expression of specific CD markers-and exhibit intermediate levels of activation as compared with the proinflammatory oral neutrophils. These intermediately activated parainflammatory populations occur in equal proportions in the healthy oral cavity, with a shift to one highly activated proinflammatory neutrophil population in chronic periodontal disease. This work is the first to identify and characterize oral parainflammatory neutrophils that interact with commensal biofilms without inducing an inflammatory response, thereby demonstrating that not all neutrophils trafficking through periodontal tissues are fully activated. In addition to establishing possible diagnostic and treatment monitoring biomarkers, this oral neutrophil phenotype model builds on existing literature suggesting that the healthy periodontium may be in a parainflammatory state. PMID:27270666

  13. Neutrophil elastase processing of Gelatinase A is mediated by extracellular matrix

    SciTech Connect

    Rice, A.; Banda, M.J.

    1995-07-18

    Gelatinase A (72-kDa type IV collagenase) is a metalloproteinase that is expressed by many cells in culture and is overexpressed by some tumor cells. It has been suggested that the serine proteinase neutrophil elastase might play a role iii the posttranslational processing of gelatinase A and that noncatalytic interactions between gelatinase A and components of the extracellular matrix might alter potential processing pathways. These questions were addressed with the use of gelatin substrate zymography, gelatinolytic activity assays, and amino acid sequence analysis. We found that neutrophil elastase does proteolytically modify gelatinase A by cleaving at a number of sites within gelatinase A. Sequential treatment of gelatinase A with 4-aminophenylmercuric acetate (APMA) and neutrophil elastase yielded an active gelatinase with a 4-fold increase in gelatinolytic activity. The increased gelatinolytic activity correlated with that of a 40-kDa fragment of gelatinase A. Matrix components altered the proteolytic modifications in gelatinase A that were mediated by neutrophil elastase. In the absence of gelatin, neutrophil elastase destructively degraded gelatinase A by hydrolyzing at least two bonds within the fibronectin-like gelatin-binding domain of gelatinase A. In the presence of gelatin, these two inactivating cleavage sites were protected, and cleavage at a site within the hemopexin-like carboxyl-terminal domain resulted in a truncated yet active gelatinase. The results suggest a regulatory role for extracellular matrix molecules in stabilizing gelatinase A fragments and in altering the availability of sites susceptible to destructive proteolysis by neutrophil elastase. 32 refs., 10 figs.

  14. Neutrophil functions and cytokines expression profile in buffaloes with impending postpartum reproductive disorders.

    PubMed

    Patra, Manas Kumar; Kumar, Harendra; Nandi, Sukdeb

    2013-10-01

    The study was conducted to correlate the periparturient immune status in terms of neutrophil functions and cytokine expression in peripheral blood mononuclear cell culture with impending postpartum reproductive disorders in buffaloes. Forty pregnant buffaloes were observed for occurrence of postpartum reproductive disorders (PRD), i.e., metritis, endometritis and delayed uterine involution etc., during one week prepartum to four weeks postpartum period. A representative number (n = 6) of buffaloes that did not develop any PRD were included in group I (healthy, control), while the animals which experienced PRD were assigned into group II (PRD, n = 8). The blood samples were collected at weekly interval from one week prepartum to four weeks postpartum period considering the day of calving as 'd 0'. Differential leucocytes counts, superoxide and hydrogen peroxide production activity in isolated neutrophils and the mRNA expression profile of cytokines i.e., IL-2, IL-4 and IFN-γ in PBMC culture were studied in all the samples. A higher total leucocytes, neutrophil and band cells count along with impaired neutrophil functions i.e., lowered level of production of superoxide and hydrogen peroxide before parturition and during early postpartum period were observed in buffaloes developing PRD. Further, a lower expression of IL-2, IFN-γ and IL-4 mRNA in PBMC culture was observed at calving in buffaloes that subsequently developed PRD at later postpartum. Thus, suppression in neutrophil function and cytokine expression at prepartum to early postpartum period predisposes the buffaloes to develop postpartum reproductive disorders. Hence, monitoring of neutrophils function and cytokine expression profile would be effective to predict certain reproductive disorders at late pregnancy or immediately after parturition in buffaloes. In future, this may be a novel approach for determining suitable management and therapeutic decisions for prevention of commonly occurring reproductive

  15. Detection of circulating tumor cells.

    PubMed

    de Wit, Sanne; van Dalum, Guus; Terstappen, Leon W M M

    2014-01-01

    The increasing number of treatment options for patients with metastatic carcinomas has created an accompanying need for methods to determine if the tumor will be responsive to the intended therapy and to monitor its effectiveness. Ideally, these methods would be noninvasive and provide quantitative real-time analysis of tumor activity in a variety of carcinomas. Assessment of circulating tumor cells shed into the blood during metastasis may satisfy this need. Here we review the CellSearch technology used for the detection of circulating tumor cells and discuss potential future directions for improvements.

  16. Detection of Circulating Tumor Cells

    PubMed Central

    Terstappen, Leon W. M. M.

    2014-01-01

    The increasing number of treatment options for patients with metastatic carcinomas has created an accompanying need for methods to determine if the tumor will be responsive to the intended therapy and to monitor its effectiveness. Ideally, these methods would be noninvasive and provide quantitative real-time analysis of tumor activity in a variety of carcinomas. Assessment of circulating tumor cells shed into the blood during metastasis may satisfy this need. Here we review the CellSearch technology used for the detection of circulating tumor cells and discuss potential future directions for improvements. PMID:25133014

  17. Neutrophil extracellular traps entrap and kill Borrelia burgdorferi sensu stricto spirochetes and are not affected by Ixodes ricinus tick saliva.

    PubMed

    Menten-Dedoyart, Catherine; Faccinetto, Céline; Golovchenko, Maryna; Dupiereux, Ingrid; Van Lerberghe, Pierre-Bernard; Dubois, Sophie; Desmet, Christophe; Elmoualij, Benaissa; Baron, Frédéric; Rudenko, Nataliia; Oury, Cécile; Heinen, Ernst; Couvreur, Bernard

    2012-12-01

    Lyme disease is caused by spirochetes of the Borrelia burgdorferi sensu lato complex. They are transmitted mainly by Ixodes ricinus ticks. After a few hours of infestation, neutrophils massively infiltrate the bite site. They can kill Borrelia via phagocytosis, oxidative burst, and hydrolytic enzymes. However, factors in tick saliva promote propagation of the bacteria in the host even in the presence of a large number of neutrophils. The neutrophil extracellular trap (NET) consists in the extrusion of the neutrophil's own DNA, forming traps that can retain and kill bacteria. The production of reactive oxygen species is apparently associated with the onset of NETs (NETosis). In this article, we describe NET formation at the tick bite site in vivo in mice. We show that Borrelia burgdorferi sensu stricto spirochetes become trapped and killed by NETs in humans and that the bacteria do not seem to release significant nucleases to evade this process. Saliva from I. ricinus did not affect NET formation by human neutrophils or its stability. However, it greatly decreased neutrophil reactive oxygen species production, suggesting that a strong decrease of hydrogen peroxide does not affect NET formation. Finally, round bodies trapped in NETs were observed, some of them staining as live bacteria. This observation could help contribute to a better understanding of the early steps of Borrelia invasion and erythema migrans formation after tick bite. PMID:23109724

  18. Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected with Paracoccidioides brasiliensis

    PubMed Central

    Lopera, Damaris; Urán-Jiménez, Martha Eugenia

    2016-01-01

    Neutrophils predominate during the acute phase of the Paracoccidioides brasiliensis infection. Herein, we determined the role of the neutrophil during the early stages of experimental pulmonary paracoccidioidomycosis using a monoclonal antibody (mAb) specific for neutrophils. Male BALB/c mice were inoculated intranasally with 1.5 × 106 or 2 × 106 P. brasiliensis yeast cells. The mAb was administered 24 h before infection, followed by doses every 48 h until mice were sacrificed. Survival time was evaluated and mice were sacrificed at 48 h and 96 h after inoculation to assess cellularity, fungal load, cytokine/chemokine levels, and histopathological analysis. Neutrophils from mAb-treated mice were efficiently depleted (99.04%). Eighty percent of the mice treated with the mAb and infected with 1.5 × 106 yeast cells died during the first two weeks after infection. When mice were treated and infected with 2 × 106 yeast cells, 100% of them succumbed by the first week after infection. During the acute inflammatory response significant increases in numbers of eosinophils, fungal load and levels of proinflammatory cytokines/chemokines were observed in the mAb-treated mice. We also confirmed that neutrophils are an important source of IFN-γ and IL-17. These results indicate that neutrophils are essential for protection as well as being important for regulating the early inflammatory immune response in experimental pulmonary paracoccidioidomycosis.

  19. Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected with Paracoccidioides brasiliensis

    PubMed Central

    Lopera, Damaris; Urán-Jiménez, Martha Eugenia

    2016-01-01

    Neutrophils predominate during the acute phase of the Paracoccidioides brasiliensis infection. Herein, we determined the role of the neutrophil during the early stages of experimental pulmonary paracoccidioidomycosis using a monoclonal antibody (mAb) specific for neutrophils. Male BALB/c mice were inoculated intranasally with 1.5 × 106 or 2 × 106 P. brasiliensis yeast cells. The mAb was administered 24 h before infection, followed by doses every 48 h until mice were sacrificed. Survival time was evaluated and mice were sacrificed at 48 h and 96 h after inoculation to assess cellularity, fungal load, cytokine/chemokine levels, and histopathological analysis. Neutrophils from mAb-treated mice were efficiently depleted (99.04%). Eighty percent of the mice treated with the mAb and infected with 1.5 × 106 yeast cells died during the first two weeks after infection. When mice were treated and infected with 2 × 106 yeast cells, 100% of them succumbed by the first week after infection. During the acute inflammatory response significant increases in numbers of eosinophils, fungal load and levels of proinflammatory cytokines/chemokines were observed in the mAb-treated mice. We also confirmed that neutrophils are an important source of IFN-γ and IL-17. These results indicate that neutrophils are essential for protection as well as being important for regulating the early inflammatory immune response in experimental pulmonary paracoccidioidomycosis. PMID:27642235

  20. Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.

    PubMed

    Reich, Kristian; Papp, Kim A; Matheson, Robert T; Tu, John H; Bissonnette, Robert; Bourcier, Marc; Gratton, David; Kunynetz, Rodion A; Poulin, Yves; Rosoph, Les A; Stingl, Georg; Bauer, Wolfgang M; Salter, Janeen M; Falk, Thomas M; Blödorn-Schlicht, Norbert A; Hueber, Wolfgang; Sommer, Ulrike; Schumacher, Martin M; Peters, Thomas; Kriehuber, Ernst; Lee, David M; Wieczorek, Grazyna A; Kolbinger, Frank; Bleul, Conrad C

    2015-07-01

    The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil-keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

  1. Administration of C1 inhibitor reduces neutrophil activation in patients with sepsis.

    PubMed

    Zeerleder, Sacha; Caliezi, Christoph; van Mierlo, Gerard; Eerenberg-Belmer, Anke; Sulzer, Irmela; Hack, C Erik; Wuillemin, Walter A

    2003-07-01

    Forty patients with severe sepsis or septic shock recently received C1 inhibitor. In the present study we studied the effect of C1 inhibitor therapy on circulating elastase-alpha(1)-antitrypsin complex (EA) and lactoferrin (LF) levels in these patients to gain further insight about agonists involved in the activation of neutrophils in human sepsis. Elevated levels of EA and LF were found in 65 and 85% of the septic patients, respectively. Patients with elevated EA levels had higher organ dysfunction scores, higher levels of cytokines, and higher levels of complement activation products than patients with normal EA levels. C1 inhibitor therapy reduced EA as well as complement activation and IL-8 release in the patients with elevated EA on admission. We conclude that neutrophil activation in human sepsis correlates with the severity of organ dysfunction and involves complement and interleukin-8 as agonists. The effect of C1 inhibitor therapy on neutrophils may provide an explanation for the beneficial, although mild, effects of this treatment on organ dysfunction in sepsis.

  2. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis

    PubMed Central

    Schmidt, Eric P; Yang, Yimu; Janssen, William J; Gandjeva, Aneta; Perez, Mario J; Barthel, Lea; Zemans, Rachel L; Bowman, Joel C; Koyanagi, Dan E; Yunt, Zulma X; Smith, Lynelle P; Cheng, Sara S; Overdier, Katherine H; Thompson, Kathy R; Geraci, Mark W; Douglas, Ivor S; Pearse, David B; Tuder, Rubin M

    2013-01-01

    Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α–responsive, heparan sulfate–specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue. PMID:22820644

  3. In vivo characterization of neutrophil extracellular traps in various organs of a murine sepsis model.

    PubMed

    Tanaka, Koji; Koike, Yuhki; Shimura, Tadanobu; Okigami, Masato; Ide, Shozo; Toiyama, Yuji; Okugawa, Yoshinaga; Inoue, Yasuhiro; Araki, Toshimitsu; Uchida, Keiichi; Mohri, Yasuhiko; Mizoguchi, Akira; Kusunoki, Masato

    2014-01-01

    Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model in vivo and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.

  4. Induction of Neutrophil Extracellular Traps in Shiga Toxin-Associated Hemolytic Uremic Syndrome.

    PubMed

    Ramos, Maria Victoria; Mejias, Maria Pilar; Sabbione, Florencia; Fernandez-Brando, Romina Jimena; Santiago, Adriana Patricia; Amaral, Maria Marta; Exeni, Ramon; Trevani, Analia Silvina; Palermo, Marina Sandra

    2016-01-01

    Hemolytic uremic syndrome (HUS), a vascular disease characterized by hemolytic anemia, thrombocytopenia, and acute renal failure, is caused by enterohemorrhagic Shiga toxin (Stx)-producing bacteria, which mainly affect children. Besides Stx, the inflammatory response mediated by neutrophils (PMN) is essential to HUS evolution. PMN can release neutrophil extracellular traps (NET) composed of DNA, histones, and other proteins. Since NET are involved in infectious and inflammatory diseases, the aim of this work was to investigate the contribution of NET to HUS. Plasma from HUS patients contained increased levels of circulating free-DNA and nucleosomes in comparison to plasma from healthy children. Neutrophils from HUS patients exhibited a greater capacity to undergo spontaneous NETosis. NET activated human glomerular endothelial cells, stimulating secretion of the proinflammatory cytokines IL-6 and IL-8. Stx induced PMN activation as judged by its ability to trigger reactive oxygen species production, increase CD11b and CD66b expression, and induce NETosis in PMN from healthy donors. During HUS, NET can contribute to the inflammatory response and thrombosis in the microvasculature and thus to renal failure. Intervention strategies to inhibit inflammatory mechanisms mediated by PMN, such as NETosis, could have a potential therapeutic impact towards amelioration of the severity of HUS.

  5. β2 integrin mediates hantavirus-induced release of neutrophil extracellular traps.

    PubMed

    Raftery, Martin J; Lalwani, Pritesh; Krautkrӓmer, Ellen; Peters, Thorsten; Scharffetter-Kochanek, Karin; Krüger, Renate; Hofmann, Jörg; Seeger, Karl; Krüger, Detlev H; Schönrich, Günther

    2014-06-30

    Rodent-borne hantaviruses are emerging human pathogens that cause severe human disease. The underlying mechanisms are not well understood, as hantaviruses replicate in endothelial and epithelial cells without causing any cytopathic effect. We demonstrate that hantaviruses strongly stimulated neutrophils to release neutrophil extracellular traps (NETs). Hantavirus infection induced high systemic levels of circulating NETs in patients and this systemic NET overflow was accompanied by production of autoantibodies to nuclear antigens. Analysis of the responsible mechanism using neutrophils from β2 null mice identified β2 integrin receptors as a master switch for NET induction. Further experiments suggested that β2 integrin receptors such as complement receptor 3 (CR3) and 4 (CR4) may act as novel hantavirus entry receptors. Using adenoviruses, we confirmed that viral interaction with β2 integrin induced strong NET formation. Collectively, β2 integrin-mediated systemic NET overflow is a novel viral mechanism of immunopathology that may be responsible for characteristic aspects of hantavirus-associated disease such as kidney and lung damage.

  6. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.

    PubMed

    Schmidt, Eric P; Yang, Yimu; Janssen, William J; Gandjeva, Aneta; Perez, Mario J; Barthel, Lea; Zemans, Rachel L; Bowman, Joel C; Koyanagi, Dan E; Yunt, Zulma X; Smith, Lynelle P; Cheng, Sara S; Overdier, Katherine H; Thompson, Kathy R; Geraci, Mark W; Douglas, Ivor S; Pearse, David B; Tuder, Rubin M

    2012-08-01

    Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

  7. Immunofluorescence and Confocal Microscopy of Neutrophils

    PubMed Central

    Allen, Lee-Ann H.

    2015-01-01

    Rapid recruitment of neutrophils to sites of infection and their ability to phagocytose and kill microbes is an important aspect of the innate immune response. Challenges associated with imaging of these cells include their short lifespan and small size and the fact that unstimulated cells are nonadherent. In addition, although cytoplasmic granules are plentiful, the abundance of many other organelles is diminished. Here we reprise methods for analysis of resting and activated cells using immunofluorescence and confocal microscopy, including kinetic analysis of phagosome maturation and degranulation, and detection of intraphagosomal superoxide accumulation. We describe approaches for rapid cell fixation and permeabilization that maximize antigen detection and discuss other variables that also affect data interpretation and image quality (such as cell spreading, degranulation, and phagocytosis). Finally, we show that these methods are also applicable to studies of neutrophil interactions with the extracellular matrix. PMID:24504957

  8. Network crosstalk dynamically changes during neutrophil polarization.

    PubMed

    Ku, Chin-Jen; Wang, Yanqin; Weiner, Orion D; Altschuler, Steven J; Wu, Lani F

    2012-05-25

    How complex signaling networks shape highly coordinated, multistep cellular responses is poorly understood. Here, we made use of a network-perturbation approach to investigate causal influences, or "crosstalk," among signaling modules involved in the cytoskeletal response of neutrophils to chemoattractant. We quantified the intensity and polarity of cytoskeletal marker proteins over time to characterize stereotyped cellular responses. Analyzing the effects of network disruptions revealed that, not only does crosstalk evolve rapidly during polarization, but also that intensity and polarity responses are influenced by different patterns of crosstalk. Interestingly, persistent crosstalk is arranged in a surprisingly simple circuit: a linear cascade from front to back to microtubules influences intensities, and a feed-forward network in the reverse direction influences polarity. Our approach provided a rational strategy for decomposing a complex, dynamically evolving signaling system and revealed evolving paths of causal influence that shape the neutrophil polarization response.

  9. [Inhibition of neutrophil adhesion by pectic galacturonans].

    PubMed

    Popov, S V; Ovodova, R G; Popova, G Iu; Nikitina, I R; Ovodov, Iu S

    2007-01-01

    The inhibition of the adhesion of neutrophils to fibronectin by the fragments of the main galacturonan chain of the following pectins was demonstrated: comaruman from the marsh cinquefoil Comarum polustre, bergenan from the Siberian tea Bergenia crassifolia, lemnan from the duckweed Lemna minor, zosteran from the seagrass Zostera marina, and citrus pectin. The parent pectins, except for comaruman, did not affect the cell adhesion. Galacturonans prepared from the starting pectins by acidic hydrolysis were shown to reduce the neutrophil adhesion stimulated by phorbol 12-myristate 13-acetate (1.625 microM) and dithiothreitol (0.5 mM) at a concentration of 50-200 microg/ml. The presence of carbohydrate chains with molecular masses higher than 300, from 100 to 300, and from 50 to 100 kDa in the galacturonan fractions was proved by membrane ultrafiltration. PMID:17375675

  10. Neutrophil myeloperoxidase destruction by ultraviolet irradiation

    SciTech Connect

    Hanker, J.; Giammara, B.; Strauss, G.

    1988-01-01

    The peroxidase activity of enriched leukocyte preparations on coverslips was determined cytochemically with a newly developed method. The techniques utilizes diaminobenzidine medium and cupric nitrate intensification and is suitable for analysis with light microscopy, SEM, and TEM. Blood specimens from control individuals were studied with and without in vitro UV irradiation and compared with those from psoriasis patients exposed therapeutically to various types of UV in phototherapy. All UV irradiated samples showed diminished neutrophil myeloperoxidase (MP) activity although that of the principal eosinophil peroxidase was unaffected. The SEMs supported the contention that decreased neutrophil MP activity might be related to UV induced degranulation. It is believed to be possible, eventually, to equate the observed MP degranulation effect after UV irradiation with diminished ability to fight bacterial infections.

  11. Blood neutrophil bactericidal activity against methicillin-resistant and methicillin-sensitive Staphylococcus aureus during cardiac surgery.

    PubMed

    Mekontso-Dessap, Armand; Honoré, Stéphanie; Kirsch, Matthias; Plonquet, Anne; Fernandez, Eric; Touqui, Lhousseine; Farcet, Jean-Pierre; Soussy, Claude-James; Loisance, Daniel; Delclaux, Christophe

    2005-08-01

    Whether methicillin-resistant Staphylococcus aureus (MRSA) constitutes per se an independent risk factor for morbidity and mortality after surgery as compared with methicillin-sensitive Staphylococcus aureus (MSSA) remains a subject of debate. The aim of this study was to assess whether innate defenses against MRSA and MSSA strains are similarly impaired after cardiac surgery. Both intracellular (isolated neutrophil functions) and extracellular (plasma) defenses of 12 patients undergoing scheduled cardiac surgery were evaluated preoperatively (day 0) and postoperatively (day 3) against two MSSA strains with a low level of catalase secretion and two MRSA strains with a high level of catalase secretion, inasmuch as SA killing by neutrophils relies on oxygen-dependent mechanisms. After surgery, an increase in plasma concentration of IL-10, an anti-inflammatory cytokine able to inhibit reactive oxygen species secretion and bactericidal activity of neutrophils, was evidenced. Despite the fact that univariate analysis suggested a specific impairment of neutrophil functions against MRSA strains, two-way repeated-measures ANOVA failed to demonstrate that the effect of S. aureus phenotype was significant. On the other hand, an increase in type-II secretory phospholipase A2 activity, a circulating enzyme involved in SA lysis, was evidenced and was associated with an enhancement of extracellular defenses (bactericidal activity of plasma) against MRSA. Overall, cardiac surgery and S. aureus phenotype had a significant effect on plasma bactericidal activity. Cardiac surgery was characterized by enhanced antibacterial defenses of plasma, whereas neutrophil killing properties were reduced. The overall effect of S. aureus phenotype on neutrophil functions did not seem significant.

  12. Photoimmunological properties of borage in bovine neutrophil in vitro model.

    PubMed

    Asadollahi, Firouzeh; Mehrzad, Jalil; Chaichi, Mohammad Javad; Taghavi Razavizadeh, Alireza

    2015-10-01

    Borage (Echium amoenum fisch) is one of the most commonly used medicinal plants, and has long been used as a traditional herbal medicine for many (non)infectious diseases in Iran. Study on photoredox and photoimmunology of borage is little. Natural immunomodulatory plants with minimal adverse/toxic effects could help boost animal health and, ultimately, public health. To determine the effect of borage on the functions of key circulating innate immune cells, effects of borage extract (BE) on bovine neutrophils (PMN) photoredox and phagocytosis events were evaluated using an in vitro model system. Blood PMN isolated from healthy high yielding dairy cows (n = 8/treatment) were pre-incubated with BE and the impact on phagocytosis-dependent-and-independent cellular chemiluminescence (CL), phagocytosis, killing of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), fluorescence-based PMN H2O2 production and necrosis were assessed. Relative to control (no BE) PMN, treatment with BE significantly increased phagocytosis-dependent-and-independent PMN CL (>10-15% increase). While BE also led to increased PMN H2O2 production, necrosis was also surprisingly higher in these cells. Phagocytosis and killing of both E. coli and S. aureus by PMN treated with BE was substantially higher than that by control PMN. The increased photoimmunobiological events especially intracellular CL, intracellular H2O2 formation, and phagocytic capacity of BE-treated PMN support the potential immunotherapeutic implications of borage and its components for particularly immunocompromised animals and humans. PMID:26334939

  13. Photoimmunological properties of borage in bovine neutrophil in vitro model.

    PubMed

    Asadollahi, Firouzeh; Mehrzad, Jalil; Chaichi, Mohammad Javad; Taghavi Razavizadeh, Alireza

    2015-10-01

    Borage (Echium amoenum fisch) is one of the most commonly used medicinal plants, and has long been used as a traditional herbal medicine for many (non)infectious diseases in Iran. Study on photoredox and photoimmunology of borage is little. Natural immunomodulatory plants with minimal adverse/toxic effects could help boost animal health and, ultimately, public health. To determine the effect of borage on the functions of key circulating innate immune cells, effects of borage extract (BE) on bovine neutrophils (PMN) photoredox and phagocytosis events were evaluated using an in vitro model system. Blood PMN isolated from healthy high yielding dairy cows (n = 8/treatment) were pre-incubated with BE and the impact on phagocytosis-dependent-and-independent cellular chemiluminescence (CL), phagocytosis, killing of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), fluorescence-based PMN H2O2 production and necrosis were assessed. Relative to control (no BE) PMN, treatment with BE significantly increased phagocytosis-dependent-and-independent PMN CL (>10-15% increase). While BE also led to increased PMN H2O2 production, necrosis was also surprisingly higher in these cells. Phagocytosis and killing of both E. coli and S. aureus by PMN treated with BE was substantially higher than that by control PMN. The increased photoimmunobiological events especially intracellular CL, intracellular H2O2 formation, and phagocytic capacity of BE-treated PMN support the potential immunotherapeutic implications of borage and its components for particularly immunocompromised animals and humans.

  14. Prognostic value of neutrophil distribution in cholangiocarcinoma

    PubMed Central

    Mao, Zhi-Yuan; Zhu, Guang-Qing; Xiong, Mei; Ren, Li; Bai, Li

    2015-01-01

    AIM: To explore the relationship of clinicopathological features and the distribution of neutrophils in the tumor microenvironment with the prognosis of cholangiocarcinoma. METHODS: Two hundred and fifty-four formalin-fixed and paraffin embedded tissue blocks were analyzed, including tissues from cholangiocarcinoma (n = 254), and tumor adjacent tissues (n = 238). Tissue sections were stained for CD15 using immunohistochemical staining. CD15 expression was detected to identify the distribution of neutrophils in the local tumor microenvironment. The neutrophil density of the tumor tissues and the adjacent tumor tissues was detected to reflect their inflammatory status. Clinical data and follow-up information of cholangiocarcinoma patients who underwent surgery from January 2004 to December 2010 were analyzed retrospectively. The relationship between clinicopathological features and the distribution of neutrophils with prognosis of the patients were analyzed. RESULTS: The positive expression level of CD15 was only significantly related to the TNM stage. CD15 expression was higher in tumor tissues than in adjacent tissues (73.6% vs 54.6%), with significant differences. Patients with high expression of CD15 had significantly shorter overall survival (OS) than those with low expression of CD15 (median overall survival time 39.77 mo vs 16.87 mo, P = 0.008). Patients with high CD15 expression had significantly shorter disease free survival time (DFS) than those with low expression of CD15 (median DFS 38.27 mo vs 16.83 mo, P = 0.029). COX multivariate analysis indicated that high CD15 expression in tumor tissues was an independent risk factor for predicting OS for patients with cholangiocarcinoma [P = 0.012, relative risk (RR) = 1.601], but it was not an independent risk factor for predicting DFS (P = 0.073, RR = 1.462). CONCLUSION: Patients with high CD15 expression in cancer tissues had shorter DFS and OS. High expression of CD15 is an independent risk factor for OS. PMID

  15. Characterization of prostanoid receptors on rat neutrophils.

    PubMed Central

    Wise, H; Jones, R L

    1994-01-01

    1. The effects of various prostanoid agonists have been compared on the increase in intracellular free calcium ([Ca2+]i) and the aggregation reaction of rat peritoneal neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). 2. Prostaglandin E2 (PGE2) and the specific IP-receptor agonist, cicaprost, both inhibited the FMLP-induced increase in [Ca2+]i (IC50 33 nM and 18 nM respectively) and the FMLP-induced aggregation reaction (IC50 5.6 nM and 7.9 nM respectively). PGD2, PGF2 alpha, and the TP-receptor agonist, U 46619, were inactive at the highest concentration tested (1 microM). 3. The EP1-receptor agonist, 17-phenyl-omega-trinor PGE2, and the EP3-receptor agonists, GR 63799X and sulprostone, had no inhibitory effect on FMLP-stimulated rat neutrophils. 4. PGE1 (EP/IP-receptor agonist) and iloprost (IP-receptor agonist) inhibited the FMLP-induced increase in [Ca2+]i with IC50 values of 34 nM and 38 nM respectively. The EP2-receptor agonists, butaprost and misoprostol (1 microM), inhibited both FMLP-stimulated [Ca2+]i and aggregation. However another EP2-receptor agonist, AH 13205, was inactive in both assays. 5. Prostanoid receptors present on rat neutrophils were further characterized by measuring [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) accumulation. Only those agonists capable of stimulating [3H]-cyclic AMP accumulation were able to inhibit both FMLP-stimulated [Ca2+]i and aggregation. 6. These results indicate that rat neutrophils possess inhibitory IP and EP-receptors; the relative potencies of PGE2, misoprostol and butaprost are those expected for the EP2-receptor subtype. No evidence for DP, FP, TP or EP1 and EP3-receptors was obtained. PMID:7834211

  16. Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils.

    PubMed

    Rocha, Juliana D B; Nascimento, Michelle T C; Decote-Ricardo, Debora; Côrte-Real, Suzana; Morrot, Alexandre; Heise, Norton; Nunes, Marise P; Previato, José Osvaldo; Mendonça-Previato, Lucia; DosReis, George A; Saraiva, Elvira M; Freire-de-Lima, Célio G

    2015-01-01

    In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen. PMID:25620354

  17. Delayed human neutrophil apoptosis by Trichomonas vaginalis lysate.

    PubMed

    Song, Hyun-Ouk; Lim, Young-Su; Moon, Sun-Joo; Ahn, Myoung-Hee; Ryu, Jae-Sook

    2010-03-01

    Neutrophils play an important role in the human immune system for protection against such microorganisms as a protozoan parasite, Trichomonas vaginalis; however, the precise role of neutrophils in the pathogenesis of trichomoniasis is still unknown. Moreover, it is thought that trichomonal lysates and excretory-secretory products (ESP), as well as live T. vaginalis, could possibly interact with neutrophils in local tissues, including areas of inflammation induced by T. vaginalis in humans. The aim of this study was to investigate the influence of T. vaginalis lysate on the fate of neutrophils. We found that T. vaginalis lysate inhibits apoptosis of human neutrophils as revealed by Giemsa stain. Less altered mitochondrial membrane potential (MMP) and surface CD16 receptor expression also supported the idea that neutrophil apoptosis is delayed after T. vaginalis lysate stimulation. In contrast, ESP stimulated-neutrophils were similar in apoptotic features of untreated neutrophils. Maintained caspase-3 and myeloid cell leukemia-1 (Mcl-1) in neutrophils co-cultured with trichomonad lysate suggest that an intrinsic mitochondrial pathway of apoptosis was involved in T. vaginalis lysate-induced delayed neutrophil apoptosis; this phenomenon may contribute to local inflammation in trichomoniasis. PMID:20333279

  18. Phagocytosis and killing of Staphylococcus aureus by human neutrophils.

    PubMed

    Lu, Thea; Porter, Adeline R; Kennedy, Adam D; Kobayashi, Scott D; DeLeo, Frank R

    2014-01-01

    Neutrophils are essential for host defense against Staphylococcus aureus infections. Although significant progress has been made, our understanding of neutrophil interactions with S. aureus remains incomplete. To provide a more comprehensive view of this process, we investigated phagocytosis and killing of S. aureus by human neutrophils using varied assay conditions in vitro. A greater percentage of bacteria were internalized by adherent neutrophils compared to those in suspension, and, unexpectedly, uptake of S. aureus by adherent neutrophils occurred efficiently in the absence of opsonins. An antibody specific for S. aureus promoted uptake of unopsonized bacteria in suspension, but had little or no capacity to enhance phagocytosis of S. aureus opsonized with normal human serum or by adherent neutrophils. Collectively, these results indicate that assay conditions can have a significant influence on the phagocytosis and killing of S. aureus by neutrophils. More importantly, the results suggest a vaccine approach directed to enhance opsonophagocytosis alone is not sufficient to promote increased killing of S. aureus by human neutrophils. With the emergence and reemergence of antibiotic-resistant microorganisms, establishing parameters that are optimal for studying neutrophil-S. aureus interactions will pave the way towards developing immune-directed strategies for anti-staphylococcal therapies.

  19. Acetaminophen prevents oxidative burst and delays apoptosis in human neutrophils.

    PubMed

    Freitas, Marisa; Costa, Vera M; Ribeiro, Daniela; Couto, Diana; Porto, Graça; Carvalho, Félix; Fernandes, Eduarda

    2013-05-23

    Acetaminophen is a frequently prescribed over-the-counter drug to reduce fever and pain in the event of inflammatory process. As neutrophils are relevant cells in inflammatory processes, the putative interaction of acetaminophen with these cells, if present, would be of paramount importance. The present study was undertaken to evaluate the effect of acetaminophen in human neutrophils' oxidative burst and lifespan in vitro. The obtained results demonstrate that acetaminophen efficiently modulates neutrophils' oxidative burst in phorbol myristate acetate-activated neutrophils, in a concentration-dependent manner, at in vivo relevant concentrations. It was clearly demonstrated that acetaminophen is a strong scavenger of HOCl and H2O2, which probably contributed to the effect observed in neutrophils. Acetaminophen also induced the depletion of glutathione in stimulated neutrophils, suggesting its transformation into a reactive intermediate. Obtained results further revealed that acetaminophen affects programmed cell death of human neutrophils, resulting in a delay of previously stimulated neutrophils-mediated apoptosis. Overall, our data suggested that acetaminophen has considerable potential to be included in anti-inflammatory therapeutic strategies, by preventing biological damage induced by an excessive production of reactive species generated in activated neutrophils and by extending the lifespan of neutrophils, favoring the elimination of pathogens, thus contributing to tissue healing and resolution of inflammation. PMID:23518321

  20. Predominant role of neutrophils in the inactivation of alpha 2-macroglobulin in arthritic joints.

    PubMed

    Abbink, J J; Kamp, A M; Nieuwenhuys, E J; Nuijens, J H; Swaak, A J; Hack, C E

    1991-09-01

    We studied the state of alpha 2-macroglobulin (alpha 2M), an important inhibitor of cartilage-degrading proteinases, in relation to activation of neutrophils in 82 patients with several types of arthritis, including 52 with rheumatoid arthritis and 11 with osteoarthritis. Levels of total inactive alpha 2M (i alpha 2M), which comprises alpha 2M complexed to proteinases and alpha 2M inactivated by oxidation or hydrolysis, were measured with a monoclonal antibody specific for i alpha 2M. In addition, levels of alpha 2M complexed to proteinases were quantitated with specific assays. Neutrophil activation was assessed by measuring elastase-alpha 1-antitrypsin complexes and lactoferrin. In 83% of the 82 patients tested, the synovial fluid (SF) to plasma ratio of i alpha 2M exceeded 1, indicating an intraarticular generation. Levels of i alpha 2M significantly correlated with neutrophil numbers (P less than 0.0005) and with levels of elastase-alpha 1-antitrypsin complexes and of lactoferrin (P less than 0.00001 for both). Moreover, part of i alpha 2M consisted of alpha 2M complexed to elastase-like and chymotrypsin-like proteinases, presumably, neutrophil elastase and cathepsin G, respectively. However, the amount of i alpha 2M was approximately 10-fold larger than the amount complexed to these proteinases. In vitro inactivation of alpha 2M by activated neutrophils was only partly inhibitable by eglin C, a specific inhibitor of both elastase and cathepsin G. Release of reactive oxygen species was presumably responsible for the additional inactivation of alpha 2M, because eglin C completely abolished the inactivation of alpha 2M by cell-free supernatant of activated neutrophils. Thus, our results suggest a predominant role of neutrophils in the inactivation of alpha 2M in the SF of patients with inflammatory joint diseases. However, this inactivation could be explained only in part by the release of neutrophilic proteinases. We propose that the inactivation of alpha 2M in

  1. Neutrophil activator of matrix metalloproteinase-2 (NAM).

    PubMed

    Rollo, Ellen E; Hymowitz, Michelle; Schmidt, Cathleen E; Montana, Steve; Foda, Hussein; Zucker, Stanley

    2006-01-01

    We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

  2. Differentiating neutrophils using the optical coulter counter

    NASA Astrophysics Data System (ADS)

    Schonbrun, E.; Di Caprio, G.

    2015-03-01

    We present an opto-fluidic measurement system that quantifies cell volume, dry mass and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell's refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.

  3. Differentiating neutrophils using the optical coulter counter

    NASA Astrophysics Data System (ADS)

    Schonbrun, Ethan; Di Caprio, Giuseppe

    2015-11-01

    We present an optofluidic measurement system that quantifies cell volume, dry mass, and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell's refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.

  4. Role of neutrophilic inflammation in ozone-induced epithelial alterations in the nasal airways of rats

    NASA Astrophysics Data System (ADS)

    Cho, Hye Youn

    Ozone is a principal oxidant air pollutant in photochemical smog. Epithelial cells lining the centriacinar region of lung and the proximal aspects of nasal passage are primary target sites for ozone-induced injury in laboratory animals. Acute exposure of rats to high ambient concentrations of ozone (e.g., 0.5 ppm) results in neutrophilic inflammation, epithelial hyperplasia and mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE) lining the proximal nasal airways. The principal purpose of the present study was to investigate the role of pre-metaplastic cellular responses, especially neutrophilic inflammation, in the pathogenesis of ozone-induced MCM in rat NTE. For this purpose, three specific hypotheses-based whole-animal inhalation studies were conducted. Male F344/N rats were exposed in whole-body inhalation chambers to 0 (filtered air) or 0.5 ppm ozone for 1-3 days (8 h/day). Histochemical, immunochemical, molecular and morphometric techniques were used to investigate the ozone-induced cellular and molecular events in the NTE. Two in vitro studies were also conducted to examine the effects of ozone-inducible cytokines (i.e., tumor necrosis factor-alpha; TNF- a, and interleukin-6; IL-6) on mucin gene (rMuc-5AC) expression. Ozone induced a rapid increase of rMuc-5AC mRNA in nasal tissues within hours after the start of exposure. It preceded the appearance of MCM, and persisted with MCM. Ozone-induced neutrophilic inflammation accompanied the mucin gene upregulation, but was resolved when MCM first appeared in the NTE. Antibody-mediated depletion of circulating neutrophils attenuated ozone-induced MCM, although it did not affect the ozone-induced epithelial hyperplasia and mucin mRNA upregulation. In another study, it was found that preexisting neutrophilic rhinitis induced by endotoxin augmented the ozone-induced MCM. However, pre-existing rhinitis did not alter the severity of ozone-induced epithelial hyperplasia and mucin gene upregulation

  5. In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions.

    PubMed

    Akenhead, Michael L; Horrall, Nolan M; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y

    2015-09-01

    Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s(-1) shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia).

  6. Inflammatory mediators in dengue virus infection in children: interleukin-8 and its relationship to neutrophil degranulation.

    PubMed

    Juffrie, M; van Der Meer, G M; Hack, C E; Haasnoot, K; Sutaryo; Veerman, A J; Thijs, L G

    2000-02-01

    The chemokine interleukin-8 (IL-8) has chemoattractant activity for neutrophils and is able to activate and degranulate these cells. We investigated whether IL-8 may exert these effects in children with dengue virus infection. Circulating levels of IL-8, neutrophilic elastase (a constituent of the azurophilic granula of neutrophils), and lactoferrin, released from specific granula, were measured in 186 children with dengue virus infection, 33 healthy children as negative controls and 11 children with bacterial infections as positive controls. Levels of IL-8 on admission were elevated in 71% of the dengue patients, while the elastase and lactoferrin levels were increased in 68 and 17% of patients, respectively. These levels were significantly higher than in healthy children (P < 0.05) for IL-8 and elastase but not for lactoferrin (by the Wilcoxon-Mann-Whitney [WMW] U test). Similar levels of IL-8 were found in patients with bacterial infections. Levels of IL-8 and elastase in patients with shock were significantly higher than in patients without shock (P = 0.02; WMW), but those of lactoferrin were not. IL-8 correlated with elastase and lactoferrin (r = 0.19 and P = 0.009 versus r = 0.24 and P = 0.001, respectively; two-tailed Spearman rank correlation). Thus, IL-8 levels are increased in most patients with dengue virus infection and correlate with degranulation of neutrophils as well as with some clinical and hemodynamic variables. These findings suggest a role for IL-8 in the pathogenesis of dengue virus infection.

  7. Interleukin 8-related neutrophil elastase and the severity of the adult respiratory distress syndrome.

    PubMed

    Groeneveld, A B; Raijmakers, P G; Hack, C E; Thijs, L G

    1995-10-01

    The interaction between activated neutrophils and pulmonary endothelium is thought to contribute to the pathogenesis of the adult respiratory distress syndrome (ARDS), but its relation to ARDS severity, which may support a pathogenetic role, is unclear. Therefore, circulating inflammatory mediators, including the neutrophil chemoattractant and activator interleukin 8 (IL-8), the acute phase cytokine IL-6, and the neutrophil product elastase complexed to alpha 1-antitrypsin (alpha 1-AT), were measured prospectively, together with gas exchange, ventilatory and radiographic variables, in 13 mechanically ventilated patients with ARDS, mostly owing to sepsis, at admission into the intensive care unit. Measurements were repeated in the eight improving patients at the time that positive end-expiratory pressure could be reduced to 0 cm H2O. From the gas exchange, ventilatory and radiographic abnormalities, a lung injury score (LIS) was calculated. For pooled data, the LIS and the arterial PO2/inspiratory O2 fraction, the oxygenation ratio, correlated with plasma levels of IL-8 (rs = 0.60, P < 0.01 and rs = -0.65, P < 0.005, respectively), with levels of IL-6 (rs = 0.60, P < 0.01, and rs = -0.68, P < 0.005, respectively), and the oxygenation ratio related to elastase-alpha 1-AT (rs = -0.70, P < 0.005). Levels of IL-8 and IL-6 interrelated (rs = 0.61, P < 0.01) and related to the elastase complexes (rs = 0.45, P < 0.05). Hence, our data support a role of cytokine-induced activation of neutrophils in the clinical severity of ARDS.

  8. Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives.

    PubMed

    Lahoz-Beneytez, Julio; Elemans, Marjet; Zhang, Yan; Ahmed, Raya; Salam, Arafa; Block, Michael; Niederalt, Christoph; Asquith, Becca; Macallan, Derek

    2016-06-30

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. PMID:27136946

  9. Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives

    PubMed Central

    Lahoz-Beneytez, Julio; Elemans, Marjet; Zhang, Yan; Ahmed, Raya; Salam, Arafa; Block, Michael; Niederalt, Christoph; Macallan, Derek

    2016-01-01

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. PMID:27136946

  10. Effect of phenolic acids of microbial origin on production of reactive oxygen species in mitochondria and neutrophils

    PubMed Central

    2012-01-01

    Background Several low-molecular-weight phenolic acids are present in the blood of septic patients at high levels. The microbial origin of the most of phenolic acids in the human body was shown previously, but pathophysiological role of the phenolic acids is not clear. Sepsis is associated with the excessive production of reactive oxygen species (ROS) in both the circulation and the affected organs. In this work the influence of phenolic acids on ROS production in mitochondria and neutrophils was investigated. Methods ROS production in mitochondria and neutrophils was determined by MCLA- and luminol-dependent chemiluminescence. The rate of oxygen consumption by mitochondria was determined polarographically. The difference of electric potentials on the inner mitochondrial membrane was registered using a TPP+-selective electrode. The formation of phenolic metabolites in monocultures by the members of the main groups of the anaerobic human microflora and aerobic pathogenic bacteria was investigated by the method of gas chromatography–mass spectrometry. Results All phenolic acids had impact on mitochondria and neutrophils, the main producers of ROS in tissues and circulation. Phenolic acids (benzoic and cinnamic acids) producing the pro-oxidant effect on mitochondria inhibited ROS formation in neutrophils. Their effect on mitochondria was abolished by dithiothreitol (DTT). Phenyllactate and p-hydroxyphenyllactate decreased ROS production in both mitochondria and neutrophils. Bifidobacteria and lactobacilli produced in vitro considerable amounts of phenyllactic and p-hydroxyphenyllactic acids, Clostridia s. produced great quantities of phenylpropionic and p-hydroxyphenylpropionic acids, p-hydroxyphenylacetic acid was produced by Pseudomonas aeruginosa and Acinetobacter baumanii; and benzoic acid, by Serratia marcescens. Conclusions The most potent activators of ROS production in mitochondria are phenolic acids whose effect is mediated via the interaction with thiol

  11. Neutrophils in host defense: new insights from zebrafish

    PubMed Central

    Harvie, Elizabeth A.; Huttenlocher, Anna

    2015-01-01

    Neutrophils are highly motile phagocytic cells that play a critical role in the immune response to infection. Zebrafish (Danio rerio) are increasingly used to study neutrophil function and host-pathogen interactions. The generation of transgenic zebrafish lines with fluorescently labeled leukocytes has made it possible to visualize the neutrophil response to infection in real time by use of optically transparent zebrafish larvae. In addition, the genetic tractability of zebrafish has allowed for the generation of models of inherited neutrophil disorders. In this review, we discuss several zebrafish models of infectious disease, both in the context of immunocompetent, as well as neutrophil-deficient hosts and how these models have shed light on neutrophil behavior during infection. PMID:25717145

  12. The role of tissue resident cells in neutrophil recruitment

    PubMed Central

    Kim, Nancy D.; Luster, Andrew D.

    2015-01-01

    Neutrophils are first responders of the immune system, rapidly migrating into affected tissues in response to injury or infection. To effectively call in this first line of defense, strategically placed cells within the vasculature and tissue respond to noxious stimuli by sending out coordinated signals that recruit neutrophils. Regulation of organ-specific neutrophil entry occurs at two levels. First, the vasculature supplying the organ provides cues for neutrophil egress out of the bloodstream in a manner dependent upon its unique cellular composition and architectural features. Second, resident immune cells and stromal cells within the organ send coordinated signals that guide neutrophils to their final destination. Here, we review recent findings that highlight the importance of these tissue-specific responses in the regulation of neutrophil recruitment and the initiation and resolution of inflammation. PMID:26297103

  13. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection

    PubMed Central

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J.; Roediger, Ben; Brzoska, Anthony J.; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L.; von Andrian, Ulrich H.; Hickey, Michael J.; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in post-capillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we show that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor alpha-hemolysin lyses perivascular macrophages leading to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin, and indicate that Staphylococcus aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  14. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection.

    PubMed

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J; Roediger, Ben; Brzoska, Anthony J; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L; von Andrian, Ulrich H; Hickey, Michael J; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  15. Circulating glioma biomarkers

    PubMed Central

    Kros, Johan M.; Mustafa, Dana M.; Dekker, Lennard J.M.; Sillevis Smitt, Peter A.E.; Luider, Theo M.; Zheng, Ping-Pin

    2015-01-01

    Validated biomarkers for patients suffering from gliomas are urgently needed for standardizing measurements of the effects of treatment in daily clinical practice and trials. Circulating body fluids offer easily accessible sources for such markers. This review highlights various categories of tumor-associated circulating biomarkers identified in blood and cerebrospinal fluid of glioma patients, including circulating tumor cells, exosomes, nucleic acids, proteins, and oncometabolites. The validation and potential clinical utility of these biomarkers is briefly discussed. Although many candidate circulating protein biomarkers were reported, none of these have reached the required validation to be introduced for clinical practice. Recent developments in tracing circulating tumor cells and their derivatives as exosomes and circulating nuclear acids may become more successful in providing useful biomarkers. It is to be expected that current technical developments will contribute to the finding and validation of circulating biomarkers. PMID:25253418

  16. Benefits of heat treatment to the protease packed neutrophil for proteome analysis: halting protein degradation.

    PubMed

    Kennedy, Susan A; Scaife, Caitríona; Dunn, Michael J; Wood, Alfred E; Watson, R William G

    2011-06-01

    Neutrophils, cells of the innate immune system, contain an array of proteases and reactive oxygen species-generating enzymes that assist in controlling the invasion of bacteria and pathogens. The high content of intracellular proteolytic enzymes makes them difficult cells to work with as they can degrade proteins of potential interest. Here, we describe the benefits of heat treatment of neutrophils in reducing protein degradation for subsequent proteome analysis. Neutrophils isolated from four healthy volunteers were each divided into three aliquots and subjected to different preparation methods for 2-DE: (i) Heat treatment, (ii) resuspension in NP40 lysis buffer and (iii) resuspension in standard 2-DE lysis buffer. Representative spots found to be statistically significant between groups (p<0.01) were excised and identified by LC-MS/MS, three of which were validated by immunoblotting. Heat-treated samples contained proteins in the high-molecular-weight range that were absent from NP40-treated samples. Moreover, NP40-treated samples showed an increase in spot number and volume at lower molecular weights suggestive of protein degradation. Incorporating heat treatment into sample preparation resulted in the identification of proteins that may not have previously been detected due to sample degradation, thus leading to a more comprehensive 2-DE map of the human neutrophil proteome.

  17. In search of neutrophil granule proteins of the tammar wallaby (Macropus eugenii).

    PubMed

    Ambatipudi, Kiran; Deane, Elizabeth M

    2008-02-01

    Two approaches have been used to isolate and identify proteins of the granules of neutrophils of the tammar wallaby, Macropus eugenii. Stimulation with PMA, Ionomycin and calcium resulted in exocytosis of neutrophil granules as demonstrated with electron microscopy. However proteomic analysis using two dimensional gel electrophoresis, in-gel trypsin digestion followed by nano liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) failed to identify any anticipated granule proteins in the reaction supernatants. Subsequent use of differential centrifugation and lysis followed by the application of the same proteomic analysis approach resulted in the isolation and confident identification of 39 proteins, many of which are known to be present in the granules of neutrophils of eutherian mammals or play a role in degranulation. These proteins notably consisted of the known antimicrobials, myeloperoxidase (MPO), serine proteinase, dermcidin, lysozyme and alkaline phosphatase. A number of important known antimicrobials, however, were not detected and these include defensins and cathelicidins. This is the first report of the neutrophil granule proteins of any marsupial and complements previous reports on the cytosolic proteins. PMID:17706783

  18. Changes in several neutrophil functions in basketball players before, during and after the sports season.

    PubMed

    Benoni, G; Bellavite, P; Adami, A; Chirumbolo, S; Lippi, G; Brocco, G; Giulini, G M; Cuzzolin, L

    1995-01-01

    Neutrophils play an important role in the immune system, forming the "first line of defence" against invading microorganisms and there are few data available concerning neutrophil functions in relation to exercise. We investigated in 7 basketball professional players possible changes before, during and after the sports season, in some haematological parameters and in several aspects of the phagocytic process of neutrophils, such as adhesion, superoxide anion release and bactericidal activity. Training and competitions produced a significant rise in the number of total leukocytes and differential counts, but the values returned to the pre-start levels 3 weeks after the end of the championship. The bactericidal activity and the superoxide anion released were significantly greater during the sports season, while the percentage of cellular adhesion significantly decreased during the championship; after the sports season the values returned to the control levels. As in the literature data concerning neutrophil functions in relation to exercise are non-convergent, it is important in our opinion, to understand whether the alterations induced by exercise can persist after repeated stimuli.

  19. Mediators of neutrophil recruitment in human abdominal aortic aneurysms

    PubMed Central

    Houard, Xavier; Touat, Ziad; Ollivier, Véronique; Louedec, Liliane; Philippe, Monique; Sebbag, Uriel; Meilhac, Olivier; Rossignol, Patrick; Michel, Jean-Baptiste

    2009-01-01

    Aims Neutrophils/platelet interactions are involved in abdominal aortic aneurysm (AAA). The intraluminal thrombus (ILT) is a human model of platelet/neutrophil interactions. The present study focused on mediators involved in neutrophil recruitment in AAA. Methods and results Conditioned media from luminal, intermediate, and abluminal layers of 29 human ILTs were analysed for neutrophil markers [elastase/α1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase (MPO), and α-defensin peptides], RANTES, platelet factor 4 (PF4), and interleukin-8 (IL-8). Their time-dependent release into serum from clots generated in vitro and their plasma concentrations in AAA patients and controls were determined. Immunohistochemistry for neutrophils, platelets, IL-8, PF4, and RANTES on AAA sections was performed; and molecules involved in ILT neutrophil chemotactic function were analysed in vitro. Neutrophils and platelets colocalized in the luminal layer of the thrombus. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were released predominantly by the luminal thrombus pole, where their concentrations were significantly correlated. The luminal ILT layer was also the main source of IL-8, whose immunostaining colocalized with neutrophils. All were also released time dependently from clots and were increased in plasma of AAA patients. Luminal ILT layers displayed potent neutrophil chemotactic activity in vitro, which was inhibited by RANTES- and IL-8-blocking antibodies as well as by reparixin, an antagonist of the IL-8 receptors CXCR1 and CXCR2. Conclusion Taken together, these results suggest that platelet-derived RANTES and neutrophil-derived IL-8 are involved in attracting neutrophils to the luminal layer of AAA ILT. PMID:19201759

  20. A brief etymology of the collateral circulation.

    PubMed

    Faber, James E; Chilian, William M; Deindl, Elisabeth; van Royen, Niels; Simons, Michael

    2014-09-01

    It is well known that the protective capacity of the collateral circulation falls short in many individuals with ischemic disease of the heart, brain, and lower extremities. In the past 15 years, opportunities created by molecular and genetic tools, together with disappointing outcomes in many angiogenic trials, have led to a significant increase in the number of studies that focus on: understanding the basic biology of the collateral circulation; identifying the mechanisms that limit the collateral circulation's capacity in many individuals; devising methods to measure collateral extent, which has been found to vary widely among individuals; and developing treatments to increase collateral blood flow in obstructive disease. Unfortunately, accompanying this increase in reports has been a proliferation of vague terms used to describe the disposition and behavior of this unique circulation, as well as the increasing misuse of well-ensconced ones by new (and old) students of collateral circulation. With this in mind, we provide a brief glossary of readily understandable terms to denote the formation, adaptive growth, and maladaptive rarefaction of collateral circulation. We also propose terminology for several newly discovered processes that occur in the collateral circulation. Finally, we include terms used to describe vessels that are sometimes confused with collaterals, as well as terms describing processes active in the general arterial-venous circulation when ischemic conditions engage the collateral circulation. We hope this brief review will help unify the terminology used in collateral research.

  1. Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro.

    PubMed

    Tamarozzi, F; Wright, H L; Johnston, K L; Edwards, S W; Turner, J D; Taylor, M J

    2014-10-01

    The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism. PMID:24909063

  2. Neutrophils: Between Host Defence, Immune Modulation, and Tissue Injury

    PubMed Central

    Kruger, Philipp; Saffarzadeh, Mona; Weber, Alexander N. R.; Rieber, Nikolaus; Radsak, Markus; von Bernuth, Horst; Benarafa, Charaf; Roos, Dirk; Skokowa, Julia; Hartl, Dominik

    2015-01-01

    Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage. PMID:25764063

  3. Neutrophilic Skin Lesions in Autoimmune Connective Tissue Diseases

    PubMed Central

    Hau, Estelle; Vignon Pennamen, Marie-Dominique; Battistella, Maxime; Saussine, Anne; Bergis, Maud; Cavelier-Balloy, Benedicte; Janier, Michel; Cordoliani, Florence; Bagot, Martine; Rybojad, Michel; Bouaziz, Jean-David

    2014-01-01

    Abstract The pathophysiology of neutrophilic dermatoses (NDs) and autoimmune connective tissue diseases (AICTDs) is incompletely understood. The association between NDs and AICTDs is rare; recently, however, a distinctive subset of cutaneous lupus erythematosus (LE, the prototypical AICTD) with neutrophilic histological features has been proposed to be included in the spectrum of lupus. The aim of our study was to test the validity of such a classification. We conducted a monocentric retrospective study of 7028 AICTDs patients. Among these 7028 patients, a skin biopsy was performed in 932 cases with mainly neutrophilic infiltrate on histology in 9 cases. Combining our 9 cases and an exhaustive literature review, pyoderma gangrenosum, Sweet syndrome (n = 49), Sweet-like ND (n = 13), neutrophilic urticarial dermatosis (n = 6), palisaded neutrophilic granulomatous dermatitis (n = 12), and histiocytoid neutrophilic dermatitis (n = 2) were likely to occur both in AICTDs and autoinflammatory diseases. Other NDs were specifically encountered in AICTDs: bullous LE (n = 71), amicrobial pustulosis of the folds (n = 28), autoimmunity-related ND (n = 24), ND resembling erythema gyratum repens (n = 1), and neutrophilic annular erythema (n = 1). The improvement of AICTDS neutrophilic lesions under neutrophil targeting therapy suggests possible common physiopathological pathways between NDs and AICTDs. PMID:25546688

  4. Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis.

    PubMed

    Schwartz, Justin T; Bandyopadhyay, Sarmistha; Kobayashi, Scott D; McCracken, Jenna; Whitney, Adeline R; Deleo, Frank R; Allen, Lee-Ann H

    2013-01-01

    We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24-hour time period relative to the uninfected controls. Of approximately 47,000 genes analyzed, 3,435 were significantly up- or downregulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes asso-ciated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2 and CASP8) and genes that act via the NFĸB pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2 and RELA, IL1B, CAST, CDK2,GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that the X-linked inhibitor of apoptosis protein remained abundant in polymorphonuclear leukocytes over 48 h of LVS infection, whereas BAX mRNA and protein were progressively downregulated. These data strongly suggest that antiapoptotic and prosurvival mechanisms collaborate to sustain the viability of F. tularensis--infected neutrophils. PMID:22986450

  5. Leukocyte numbers and function in subjects eating n-3 enriched foods: selective depression of natural killer cell levels

    PubMed Central

    Mukaro, Violet R; Costabile, Maurizio; Murphy, Karen J; Hii, Charles S; Howe, Peter R; Ferrante, Antonio

    2008-01-01

    Introduction While consumption of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) has been recommended for those at risk of inflammatory disease such as rheumatoid arthritis, the mechanism of their anti-inflammatory effect remains to be clearly defined, particularly in relation to the dose and type of n-3 LCPUFA. The objective of this study was to determine whether varying the levels of n-3 LCPUFA in erythrocyte membrane lipids, following dietary supplementation, is associated with altered numbers and function of circulating leukocytes conducive to protection against inflammation. Methods In a double-blind and placebo-controlled study, 44 healthy subjects aged 23 to 63 years consumed either standard or n-3 LCPUFA-enriched versions of typical processed foods, the latter allowing a target daily consumption of 1 gram n-3 LCPUFA. After six months, peripheral blood leukocyte and subpopulation proportions and numbers were assessed by flow cytometry. Leukocytes were also examined for lymphoproliferation and cytokine production, neutrophil chemotaxis, chemokinesis, bactericidal, adherence and iodination activity. Erythrocytes were analyzed for fatty-acid content. Results Erythrocyte n-3 LCPUFA levels were higher and absolute leukocyte and lymphocyte numbers were lower in subjects consuming n-3 enriched foods than in controls. There were no changes in the number of neutrophils, monocytes, T cells (CD3+), T-cell subsets (CD4+, CD8+) and B cells (CD19+). However, natural killer (NK) (CD3-CD16+CD56+) cell numbers were lower in n-3 supplemented subjects than in controls and were inversely related to the amount of eicosapentaenoic acid or docosahexaenoic acid in erythrocytes. No significant correlations were found with respect to lymphocyte lymphoproliferation and production of IFN-γ and IL-2, but lymphotoxin production was higher with greater n-3 LCPUFA membrane content. Similarly, neutrophil chemotaxis, chemokinesis, bactericidal activity and adherence did not

  6. Characterization of C1 inhibitor binding to neutrophils.

    PubMed Central

    Chang, N S; Boackle, R J; Leu, R W

    1991-01-01

    In a previous study we have isolated neutrophil membrane proteins that non-covalently bind to native C1-INH (105,000 MW) and a non-functional, degraded C1-INH (88,000 MW; C1-INH-88). To further characterize the binding nature, we have designed a novel kinetic C1 titration assay which enables not only a quantification of the removal of fluid-phase C1-INH by neutrophils, but also a concomitant measure of residual C1-INH function. Native C1-INH, when adsorbed to EDTA-pretreated neutrophils, lost its function in the inhibition of fluid-phase C1. The non-functional C1-INH-88, which is probably devoid of a reactive centre, was found to block the binding of native C1-INH to neutrophils. Pretreatment of neutrophils with serine esterase inhibitors did not abrogate binding capacity of the cells for C1-INH, whereas the binding affinity for C1-INH was lost when the cells were pretreated with trypsin. An array of human peripheral blood leucocytes and several lymphoid cell lines has surface binding sites for C1-INH, but not on human erythrocytes and U937 cells. Binding was further confirmed using (i) C1-INH-microsphere beads to neutrophils, in which the binding was blocked when pretreating neutrophils with excess C1-INH or with trypsin, and (ii) radiolabelled C1-INH to neutrophils, which was competitively blocked by unlabelled non-functional C1-INH-88. Desialylation of C1-INH significantly reduced its binding affinity for neutrophils, indicating that the membrane receptor sites on neutrophils could be specific for the binding of sialic acid residues on C1-INH. Overall, our studies indicate that neutrophils or other leucocytes possess specific surface binding sites for the sialic acid-containing portion of C1-INH. PMID:2045131

  7. LFA-1 and Mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ.

    PubMed

    Sumagin, Ronen; Prizant, Hen; Lomakina, Elena; Waugh, Richard E; Sarelius, Ingrid H

    2010-12-01

    To exit blood vessels, most (∼80%) of the lumenally adhered monocytes and neutrophils crawl toward locations that support transmigration. Using intravital confocal microscopy of anesthetized mouse cremaster muscle, we separately examined the crawling and emigration patterns of monocytes and neutrophils in blood-perfused unstimulated or TNF-α-activated venules. Most of the interacting cells in microvessels are neutrophils; however, in unstimulated venules, a greater percentage of the total monocyte population is adherent compared with neutrophils (58.2 ± 6.1% versus 13.6 ± 0.9%, adhered/total interacting), and they crawl for significantly longer distances (147.3 ± 13.4 versus 61.8 ± 5.4 μm). Intriguingly, after TNF-α activation, monocytes crawled for significantly shorter distances (67.4 ± 9.6 μm), resembling neutrophil crawling. Using function-blocking Abs, we show that these different crawling patterns were due to CD11a/CD18 (LFA-1)- versus CD11b/CD18 (Mac-1)-mediated crawling. Blockade of either Mac-1 or LFA-1 revealed that both LFA-1 and Mac-1 contribute to monocyte crawling; however, the LFA-1-dependent crawling in unstimulated venules becomes Mac-1 dependent upon inflammation, likely due to increased expression of Mac-1. Mac-1 alone was responsible for neutrophil crawling in both unstimulated and TNF-α-activated venules. Consistent with the role of Mac-1 in crawling, Mac-1 block (compared with LFA-1) was also significantly more efficient in blocking TNF-α-induced extravasation of both monocytes and neutrophils in cremaster tissue and the peritoneal cavity. Thus, mechanisms underlying leukocyte crawling are important in regulating the inflammatory responses by regulating the numbers of leukocytes that transmigrate.

  8. Are Neutrophil Extracellular Traps Playing a Role in the Parasite Control in Active American Tegumentary Leishmaniasis Lesions?

    PubMed Central

    Morgado, Fernanda Nazaré; Nascimento, Michelle T. C.; Saraiva, Elvira M.; de Oliveira-Ribeiro, Carla; Madeira, Maria de Fátima; da Costa-Santos, Marcela; Vasconcellos, Erica C. F.; F. Pimentel, Maria Ines; Rosandiski Lyra, Marcelo; Schubach, Armando de Oliveira; Conceição-Silva, Fátima

    2015-01-01

    Neutrophil extracellular traps (NETs) have been described as a network of extracellular fibers composed by DNA, histones and various proteins/enzymes. Studies have demonstrated that NETs could be responsible for the trapping and elimination of a variety of infectious agents. In order to verify the presence of NETs in American tegumentary leishmaniasis (ATL) and their relationship with the presence of amastigotes we evaluated active cutaneous lesions of 35 patients before treatment by the detection of parasites, neutrophils (neutrophil elastase) and histones through immunohistochemistry and confocal immunofluorescence. Intact neutrophils could be detected in all ATL lesions. NETs were present in 27 patients (median 1.1; range from 0.1 to 23.5/mm2) with lesion duration ranging from one to seven months. NETs were in close proximity with neutrophils (r = 0.586; p = 0.0001) and amastigotes (r = 0.710; p = 0.0001). Two patterns of NET formation were detected: small homogeneously distributed networks observed in all lesions; and large structures that could be visualized at a lower magnification in lesions presenting at least 20% of neutrophils. Lesions presenting the larger NET formation showed high parasite detection. A correlation between NET size and the number of intact amastigotes was observed (p=0.02). As we detected an association between NET and amastigotes, our results suggest that neutrophil migration and NET formation could be stimulated and maintained by stimuli derived from the parasite burden/parasite antigen in the extracellular environment. The observation of areas containing only antigens not intermingled with NETs (elastase and histone) suggests that the involvement of these structures in the control of parasite burden is a dynamic process in which the formation of NETs is exhausted with the destruction of the parasites. Since NETs were also associated with granulomas, this trapping would favor the activity of macrophages in order to control the parasite

  9. The Inflammatory Cytokine IL-21 is Expressed by Splenic Neutrophils in Response to Transplantation of Allogeneic Cells

    PubMed Central

    Thompson, John S.; Hardin, Debra L.; Glass, Judy F.; Dziba, Joshua; Campion, Jeffrey; Brown, Stephen A.

    2016-01-01

    We have previously reported that GR-1 neutrophil/monocytes rose dramatically in the spleen, peaked by day 7 and declined through day 14. This period corresponded to the peak of acute Graft-Versus-Host Disease (aGVHD) in BALB/c mice transplanted with allogeneic donor cells. We now asked: what cytokines did these splenic neutrophil/monocytes express on day 7 and 14 post transplant? BALB/c mice were transplanted with allogeneic B6 or syngeneic BALB/c donor cells. Long term survival was recorded through day 31. Other groups were sacrificed on days 3, 5, 7, 14, 21 and 31 days post transplant to record the total number of cells in the spleens and their phenotypes. Neutrophils were isolated from the spleens of mice transplanted with B6 and BALB/c cells on days 7 and 14. Daily body weight demonstrated a transient drop in the syngeneic transplants on day 2 but a much greater drop with its nadir at day 7 and never fully recovering through 31 days. CD8/CD4 T lymphocytes peaked in the spleen on day 5 and were followed on day 7 by GR-I cells in all of the allogeneic transplants. In syngeneic transplants this early rise in lymphocytes did not occur and GR-1 cells peaked on day 14. Highly purified neutrophils were isolated in two separate experiments from the spleens on days 7 and 14 post transplant. In both experiments day 7 allogeneic neutrophils expressed significantly elevated levels of Interleukin-21 (IL-21) mRNA whereas the day 7 and 14 syngeneic cells expressed lower but significant levels of TNFα. Intracellular IL-21 was demonstrated in the allogeneic neutrophils on day 7 before and after in vitro stimulation. In conclusion Purified neutrophils isolated from the spleen on day 7, the early peak of allogeneic transplantation a GVHD, express high levels of IL-21 message and intracellular IL-21. PMID:27774526

  10. Migration of neutrophils across endothelial monolayers is stimulated by treatment of the monolayers with interleukin-1 or tumor necrosis factor-alpha.

    PubMed

    Furie, M B; McHugh, D D

    1989-11-15

    To study the effects of the cytokines IL-1 and TNF-alpha on the transendothelial migration of neutrophils, human umbilical vein endothelial cells (HUVEC) were grown to confluence on connective tissue prepared from human amniotic membrane. Pretreatment of HUVEC-amnion cultures with rIL-1 beta (7.5 ng/ml) or rTNF-alpha (5 ng/ml) for 4 h resulted in rapid migration of from 20 to 50% of subsequently added neutrophils across the endothelial monolayer. In contrast, only 3 +/- 3% of added neutrophils penetrated the HUVEC monolayer in the absence of any stimulus. The number of neutrophils that migrated across cytokine-treated HUVEC was similar to the number that traversed untreated monolayers in response to gradients of FMLP; in addition, it was only 35% less than the number of neutrophils that migrated in response to leukotriene B4. No consistent additive effect was seen when migration was induced by both cytokine pretreatment of the HUVEC and a chemotactic gradient. The number of neutrophils that migrated across IL-1-treated cultures was proportional to the number added over the range of 2.5 x 10(5) to 4 x 10(6) neutrophils. When used at optimal concentrations, IL-1 and TNF-alpha were equally effective in stimulating neutrophil migration; no additive effect was seen when HUVEC were pretreated with optimal doses of both cytokines together. Direct addition of IL-1 or TNF-alpha to a 1-h migration assay had no effect on neutrophil adhesion to or migration across HUVEC, either in the presence or absence of a chemotactic gradient. Stimulation of neutrophil transendothelial migration in this system did not appear to be caused by adsorption of cytokine by the amniotic tissue, nor was it due to contamination of the cytokine preparations by LPS. These results suggest that IL-1 and TNF-alpha, generated at sites of inflammation, may act upon the endothelium to promote emigration of neutrophils from the vasculature.

  11. Xanthine oxidase, but not neutrophils, contributes to activation of cardiac sympathetic afferents during myocardial ischaemia in cats

    PubMed Central

    Tjen-A-Looi, Stephanie C; Fu, Liang-Wu; Longhurst, John C

    2002-01-01

    Activation of cardiac sympathetic afferents during myocardial ischaemia causes angina and induces important cardiovascular reflex responses. Reactive oxygen species (ROS) are important chemical stimuli of cardiac afferents during and after ischaemia. Iron-catalysed Fenton chemistry constitutes one mechanism of production of hydroxyl radicals. Another potential source of these species is xanthine oxidase-catalysed oxidation of purines. Polymorphonuclear leukocytes (PMNs) also contribute to the production of ROS in some conditions. The present study tested the hypothesis that both xanthine oxidase-catalysed oxidation of purines and neutrophils provide a source of ROS sufficient to activate cardiac afferents during ischaemia. We recorded single-unit activity of cardiac afferents innervating the ventricles recorded from the left thoracic sympathetic chain (T1-5) of anaesthetized cats to identify the afferents' responses to ischaemia. The role of xanthine oxidase in activation of these afferents was determined by infusion of oxypurinol (10 mg kg−1, i.v.), an inhibitor of xanthine oxidase. The importance of neutrophils as a potential source of ROS in the activation of cardiac afferents during ischaemia was assessed by the infusion of a polyclonal antibody (3 mg ml−1 kg−1, i.v.) raised in rabbits immunized with cat PMNs. This antibody decreased the number of circulating PMNs and, to a smaller extent, platelets. Since previous data suggest that platelets release serotonin (5-HT), which activates cardiac afferents through a serotonin receptor (subtype 3,5-HT3 receptor) mechanism, before treatment with the antibody in another group, we blocked 5-HT3 receptors on sensory nerve endings with tropisetron (300 μg kg−1, i.v.). We observed that oxypurinol significantly decreased the activity of cardiac afferents during myocardial ischaemia from 1.5 ± 0.4 to 0.8 ± 0.4 impulses s−1. Similarly, the polyclonal antibody significantly reduced the discharge frequency of

  12. Apparatus Circulates Sterilizing Gas

    NASA Technical Reports Server (NTRS)

    Cross, John H.; Schwarz, Ray P.

    1991-01-01

    Apparatus circulates sterilizing gas containing ethylene oxide and chlorofluorocarbon through laboratory or medical equipment. Confines sterilizing gas, circulating it only through parts to be treated. Consists of two units. One delivers ethylene oxide/chlorofluorocarbon gas mixture and removes gas after treatment. Other warms, humidifies, and circulates gas through equipment to be treated. Process provides reliable sterilization with negligible residual toxicity from ethylene oxide. Particularly suitable for sterilization of interiors of bioreactors, heart/lung machines, dialyzers, or other equipment including complicated tubing.

  13. CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis

    PubMed Central

    Hurtado, Plinio R; Jeffs, Lisa; Nitschke, Jodie; Patel, Mittal; Sarvestani, Ghafar; Cassidy, John; Hissaria, Pravin; Gillis, David; Au Peh, Chen

    2008-01-01

    Background Wegener's Granulomatosis and Microscopic Polyangiitis are life-threatening systemic necrotizing vasculitides of unknown aetiology. The appearance of circulating antibodies to neutrophil cytoplasmic antigens (ANCA) is strongly associated with the development of the disease. A link between infection and disease has long been suspected, and the appearance of ANCA antibodies has been reported following bacterial and viral infections. The depletion of circulating B cells with monoclonal antibody therapy can induce remission, and this observation suggests a pathogenic role for B cells in this disease. As bacterial DNA is known to induce B cell proliferation and antibody production via TLR-9 stimulation, we have explored the possibility that unmethylated CpG oligodeoxynucleotide, as found in bacterial and viral DNA, may play a role in stimulating circulating autoreactive B cells to produce ANCA in patients with vasculitis. Results We have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC in vitro. The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls. In spite of this, PR3 ANCA+ patients synthesised significantly higher amount of IgG ANCA than normal controls. In MPO ANCA+ patients, there was a tendency for patients to produce higher amount of ANCA than controls, however, the difference did not reach significance. Furthermore, we were able to detect circulating MPO-reactive B cells by ELISpot assay from the peripheral blood of 2 MPO+ ANCA vasculitis patients. Together, this indicates that circulating anti-neutrophil autoreactive B cells are present in ANCA+ vasculitis patients, and they are capable of producing antibodies in response to CpG stimulation. Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases. Conclusion Circulating ANCA autoreactive B

  14. Neutrophil transmigration mediated by the neutrophil-specific antigen CD177 is influenced by the endothelial S536N dimorphism of platelet endothelial cell adhesion molecule-1.

    PubMed

    Bayat, Behnaz; Werth, Silke; Sachs, Ulrich J H; Newman, Debra K; Newman, Peter J; Santoso, Sentot

    2010-04-01

    The human neutrophil-specific adhesion molecule CD177 (also known as the NB1 alloantigen) becomes upregulated on the cell surface in a number of inflammatory settings. We recently showed that CD177 functions as a novel heterophilic counterreceptor for the endothelial junctional protein PECAM-1 (CD31), an interaction that is mediated by membrane-proximal PECAM-1 IgD 6, which is known to harbor an S(536)N single nucleotide polymorphism of two major isoforms V(98)N(536)G(643) and L(98)S(536)R(643) and a yet-to-be-determined region on CD177. In vitro transendothelial migration experiments revealed that CD177(+) neutrophils migrated significantly faster through HUVECs expressing the LSR, compared with the VNG, allelic variant of PECAM-1 and that this correlated with the decreased ability of anti-PECAM-1 Ab of ITIM tyrosine phosphorylation in HUVECs expressing the LSR allelic variant relative to the VNG allelic variant. Moreover, engagement of PECAM-1 with rCD177-Fc (to mimic heterophilic CD177 binding) suppressed Ab-induced tyrosine phosphorylation to a greater extent in cells expressing the LSR isoform compared with the VNG isoform, with a corresponding increased higher level of beta-catenin phosphorylation. These data suggest that heterophilic PECAM-1/CD177 interactions affect the phosphorylation state of PECAM-1 and endothelial cell junctional integrity in such a way as to facilitate neutrophil transmigration in a previously unrecognized allele-specific manner. PMID:20194726

  15. How Neutrophil Extracellular Traps Become Visible

    PubMed Central

    2016-01-01

    Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one hand in vitro live-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital, in vivo, and in situ microscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages. PMID:27294157

  16. Pondering neutrophil extracellular traps with healthy skepticism.

    PubMed

    Nauseef, William M; Kubes, Paul

    2016-10-01

    The authors engage in a dialogue that evaluates critically the state of the study of neutrophil extracellular traps (NETs), a phenomenon currently the object of considerable interest, with the goal of identifying those aspects that merit clarification in order to assign the process its proper place in our current understanding of cell biology. Since the seminal observations in the Zychlinsky laboratory that described the extrusion of filaments of nuclear DNA associated with histones and granule proteins from neutrophils stimulated in vitro, many investigators have examined the phenomenon of NET formation in numerous and diverse settings. However, an overview of work in this rapidly growing field prompts several fundamental questions about NETs, including their precise composition, the mechanisms by which they arise, their clinical relevance, and the interrelationship of those observed in vitro and in vivo. In this discussion, the authors challenge interpretation of data from some experimental settings and provide recommendations for specific studies that would address the concerns raised, improve understanding of the biological relevance of NETs, and strengthen the field. PMID:27470975

  17. Pondering neutrophil extracellular traps with healthy skepticism.

    PubMed

    Nauseef, William M; Kubes, Paul

    2016-10-01

    The authors engage in a dialogue that evaluates critically the state of the study of neutrophil extracellular traps (NETs), a phenomenon currently the object of considerable interest, with the goal of identifying those aspects that merit clarification in order to assign the process its proper place in our current understanding of cell biology. Since the seminal observations in the Zychlinsky laboratory that described the extrusion of filaments of nuclear DNA associated with histones and granule proteins from neutrophils stimulated in vitro, many investigators have examined the phenomenon of NET formation in numerous and diverse settings. However, an overview of work in this rapidly growing field prompts several fundamental questions about NETs, including their precise composition, the mechanisms by which they arise, their clinical relevance, and the interrelationship of those observed in vitro and in vivo. In this discussion, the authors challenge interpretation of data from some experimental settings and provide recommendations for specific studies that would address the concerns raised, improve understanding of the biological relevance of NETs, and strengthen the field.

  18. Diverse novel functions of neutrophils in immunity, inflammation, and beyond

    PubMed Central

    Mócsai, Attila

    2013-01-01

    Neutrophils have long been considered simple suicide killers at the bottom of the hierarchy of the immune response. That view began to change 10–20 yr ago, when the sophisticated mechanisms behind how neutrophils locate and eliminate pathogens and regulate immunity and inflammation were discovered. The last few years witnessed a new wave of discoveries about additional novel and unexpected functions of these cells. Neutrophils have been proposed to participate in protection against intracellular pathogens such as viruses and mycobacteria. They have been shown to intimately shape the adaptive immune response at various levels, including marginal zone B cells, plasmacytoid dendritic cells and T cell populations, and even to control NK cell homeostasis. Neutrophils have been shown to mediate an alternative pathway of systemic anaphylaxis and to participate in allergic skin reactions. Finally, neutrophils were found to be involved in physiological and pathological processes beyond the immune system, such as diabetes, atherosclerosis, and thrombus formation. Many of those functions appear to be related to their unique ability to release neutrophil extracellular traps even in the absence of pathogens. This review summarizes those novel findings on versatile functions of neutrophils and how they change our view of neutrophil biology in health and disease. PMID:23825232

  19. Human neutrophil leukocyte elastase activity is inhibited by Phenol Red

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neutrophil elastase (NE) activity in urine, sputum and nasal mucous is used as an indicator of inflammation due to viral or bacterial infection. However, bovine nasal mucous neutrophils collected, lysed and stored in Dulbecco's minimal medium containing Phenol Red, showed no NE activity with methox...

  20. ATF3 is a novel regulator of mouse neutrophil migration

    PubMed Central

    Boespflug, Nicholas D.; Kumar, Sachin; McAlees, Jaclyn W.; Phelan, James D.; Grimes, H. Leighton; Hoebe, Kasper; Hai, Tsonwin; Karp, Christopher L.

    2014-01-01

    Expression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3−/− neutrophil recruitment to wild-type lungs. In vitro, ATF3−/− neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3−/− neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3−/− and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis. PMID:24470589

  1. Intergrin-dependent neutrophil migration in the injured mouse cornea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an early responder to an inflammatory stimulus, neutrophils must exit the vasculature and migrate through the extravascular tissue to the site of insult, which is often remote from the point of extravasation. Following a central epithelial corneal abrasion, neutrophils recruited from the peripher...

  2. Intracellular localization of VAMP-1 protein in human neutrophils.

    PubMed

    Nabokina, S M

    2001-02-01

    We studied the intracellular localization of vesicle-associated membrane protein VAMP-1 in human neutrophils. VAMP-1 was associated with membranes of gelatinase and specific secretory granules rapidly mobilized during exocytosis. VAMP-1 probably acts as a component of the SNARE complex during exocytosis of gelatinase and specific granules in human neutrophils.

  3. Swell activated chloride channel function in human neutrophils

    SciTech Connect

    Salmon, Michael D.; Ahluwalia, Jatinder

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  4. Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis

    PubMed Central

    Majumdar, Ritankar; Tavakoli Tameh, Aidin; Parent, Carole A.

    2016-01-01

    Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments. PMID:26741884

  5. Paradoxical Roles of the Neutrophil in Sepsis: Protective and Deleterious

    PubMed Central

    Sônego, Fabiane; Castanheira, Fernanda Vargas e Silva; Ferreira, Raphael Gomes; Kanashiro, Alexandre; Leite, Caio Abner Vitorino Gonçalves; Nascimento, Daniele Carvalho; Colón, David Fernando; Borges, Vanessa de Fátima; Alves-Filho, José Carlos; Cunha, Fernando Queiróz

    2016-01-01

    Sepsis, an overwhelming inflammatory response syndrome secondary to infection, is one of the costliest and deadliest medical conditions worldwide. Neutrophils are classically considered to be essential players in the host defense against invading pathogens. However, several investigations have shown that impairment of neutrophil migration to the site of infection, also referred to as neutrophil paralysis, occurs during severe sepsis, resulting in an inability of the host to contain and eliminate the infection. On the other hand, the neutrophil antibacterial arsenal contributes to tissue damage and the development of organ dysfunction during sepsis. In this review, we provide an overview of the main events in which neutrophils play a beneficial or deleterious role in the outcome of sepsis. PMID:27199981

  6. Spatial control of actin polymerization during neutrophil chemotaxis

    PubMed Central

    Weiner, Orion D.; Servant, Guy; Welch, Matthew D.; Mitchison, Timothy J.; Sedat, John W.; Bourne, Henry R.

    2010-01-01

    Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients. PMID:10559877

  7. The role of neutrophils in the immune system: an overview.

    PubMed

    Malech, Harry L; Deleo, Frank R; Quinn, Mark T

    2014-01-01

    Neutrophils, also known as polymorphonuclear leukocytes (PMNs), have long been considered as the short-lived, nonspecific white cells that form pus-and also happen to kill invading microbes. Indeed, neutrophils were often neglected (and largely not considered) as immune cells. This historic view of neutrophils has changed considerably over the past several decades, and we know now that, in addition to playing the predominant role in the clearance of bacteria and fungi, they play a major role in shaping the host response to infection and immune system homeostasis. The change in our view of the role of neutrophils in the immune system has been due in large part to the study of these cells in vitro. Such work has been made possible by new and/or improved methods and approaches used to investigate neutrophils. These methods are the focus of this volume.

  8. Interaction of natural killer cells with neutrophils exerts a significant antitumor immunity in hematopoietic stem cell transplantation recipients.

    PubMed

    Ueda, Ryosuke; Narumi, Kenta; Hashimoto, Hisayoshi; Miyakawa, Reina; Okusaka, Takuji; Aoki, Kazunori

    2016-01-01

    Autologous hematopoietic stem cell transplantation (HSCT) can induce a strong antitumor immunity by homeostatic proliferation (HP) of T cells and suppression of regulatory T cells following preconditioning-induced lymphopenia. However, the role of innate immunity including natural killer (NK) cells is still not understood. Here, first, we examined whether NK cells exert an antitumor effect after syngeneic HSCT in a murine colon cancer model. Flow cytometry showed that NK cells as well as T cells rapidly proliferated after HSCT, and the frequency of mature NK cells was increased in tumor during HP. Furthermore, NK cells undergoing HP were highly activated, which contributed to substantial tumor suppression. Then, we found that a large number of neutrophils accumulated in tumor early after syngeneic HSCT. It was recently reported that neutrophil-derived mediators modulate NK cell effector functions, and so we examined whether the neutrophils infiltrated in tumor are associated with NK cell-mediated antitumor effect. The depletion of neutrophils significantly impaired an activation of NK cells in tumor and increased the fraction of proliferative NK cells accompanied by a decrease in NK cell survival. The results suggested that neutrophils in tumor prevent NK cells from activation-induced cell death during HP, thus leading to a significant antitumor effect by NK cells. This study revealed a novel aspect of antitumor immunity induced by HSCT and may contribute to the development of an effective therapeutic strategy for cancer using HSCT.

  9. Human neutrophil elastase detection with fluorescent peptide sensors conjugated to cellulosic and nanocellulosic materials: part II, structure/function analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human neutrophil elastase (HNE) is one of a number of proteases that is receiving increased attention as a marker for inflammatory diseases and sensor-based point of care diagnostics. Integral to sensor-based detection is the transducer surface which is the platform of the sensor's signal transmitta...

  10. Human plasma kallikrein releases neutrophil elastase during blood coagulation.

    PubMed Central

    Wachtfogel, Y T; Kucich, U; James, H L; Scott, C F; Schapira, M; Zimmerman, M; Cohen, A B; Colman, R W

    1983-01-01

    Elastase is released from human neutrophils during the early events of blood coagulation. Human plasma kallikrein has been shown to stimulate neutrophil chemotaxis, aggregation, and oxygen consumption. Therefore, the ability of kallikrein to release neutrophil elastase was investigated. Neutrophils were isolated by dextran sedimentation, and elastase release was measured by both an enzyme-linked immunosorbent assay, and an enzymatic assay using t-butoxy-carbonyl-Ala-Ala-Pro-Val-amino methyl coumarin as the substrate. Kallikrein, 0.1-1.0 U/ml, (0.045-0.45 microM), was incubated with neutrophils that were preincubated with cytochalasin B (5 micrograms/ml). The release of elastase was found to be proportional to the kallikrein concentration. Kallikrein released a maximum of 34% of the total elastase content, as measured by solubilizing the neutrophils in the nonionic detergent Triton X-100. A series of experiments was carried out to determine if kallikrein was a major enzyme involved in neutrophil elastase release during blood coagulation. When 10 million neutrophils were incubated in 1 ml of normal plasma in the presence of 30 mM CaCl2 for 90 min, 2.75 micrograms of elastase was released. In contrast, neutrophils incubated in prekallikrein-deficient or Factor XII-deficient plasma released less than half of the elastase, as compared with normal plasma. The addition of purified prekallikrein to prekallikrein-deficient plasma restored neutrophil elastase release to normal levels. Moreover, release of elastase was enhanced in plasma deficient in C1-inhibitor, the major plasma inhibitor of kallikrein. This release was not dependent upon further steps in the coagulation pathway, or on C5a, since levels of elastase, released in Factor XI- or C5-deficient plasma, were similar to that in normal plasma, and an antibody to C5 failed to inhibit elastase release. These data suggest that kallikrein may be a major enzyme responsible for the release of elastase during blood

  11. Human resistin promotes neutrophil proinflammatory activation and neutrophil extracellular trap formation and increases severity of acute lung injury.

    PubMed

    Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Gregoire, Murielle; Deshane, Jessy; Pittet, Jean Francois; Abraham, Edward; Zmijewski, Jaroslaw W

    2014-05-15

    Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies also suggested that resistin has proinflammatory properties. We examined whether the human-specific variant of resistin affects neutrophil activation and the severity of LPS-induced acute lung injury. Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using humanized resistin mice that exclusively express human resistin (hRTN(+/-)(/-)) but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn(+/-/-) neutrophils compared with control Rtn(-/-/-) neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase, a major sensor and regulator of cellular bioenergetics that also is implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin enhanced neutrophil extracellular trap formation. In LPS-induced acute lung injury, humanized resistin mice demonstrated enhanced production of proinflammatory cytokines, more severe pulmonary edema, increased neutrophil extracellular trap formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4-induced inflammatory responses, and it may be a target for future therapies aimed at reducing the severity of acute lung injury and other inflammatory situations in which neutrophils play a major role.

  12. Eosinophils versus Neutrophils in Host Defense

    PubMed Central

    Bass, David A.; Szejda, Pamela

    1979-01-01

    Eosinophil leukocytes have been reported to have a major role in host defense against invasive, migratory phases of helminth infestations, yet the relative larvicidal abilities of eosinophils and neutrophils have not been thoroughly examined. This study examined the killing of newborn (migratory phase) larvae of Trichinella spiralis during incubation by human granulocytes in vitro. The assay employed cultue of larvae with cells, sera, and reagents in microtiter wells with direct counting of surviving larvae after incubation. Killed larvae appeared to be lysed. Verification of the microplate assay was obtained by demonstrating complete loss of infectivity of larvae incubated with leukocytes and immune serum. In the presence of optimal immune serum concentrations, purified neutrophils or eosinophils achieved ≥95% killing of larvae at cell:larva ratios of 2,000:1 or greater. Fresh normal serum prompted slight (19%) killing by leukocytes at a cell:larva ratio of 9,000:1. Cells plus heat-inactivated normal serum and all sera preparations in the absence of leukocytes killed <8% of the larvae. The activity of immune serum was opsonic. Cells adhered to larvae that had been preincubated in immune serum, and immunofluorescent studies indicated that such preopsonized larvae were coated with immunoglobulin (Ig)G. However, preopsonized larvae lost opsonic activity and surface IgG during incubation for 3 h in medium lacking immune serum. The rate of killing was dependent on the cell:larva ratio; at high leukocyte concentrations (4,200:1), 99% were killed within 7 h; at lower cell:larva ratios, killing increased steadily during a 20-h incubation period. Killing was inhibited by 20 μg catalase, 5 μg/ml cytochalasin B, or 5μM colchicine, but was unchanged by superoxide dismutase and was enhanced by azide or cyanide. Leukocytes from a patient with chronic granulomatous disease, lacking ability to mount a normal oxidative response, demonstrated a markedly suppressed larvicidal

  13. Circulating colony-forming units of granulocytes and monocytes/macrophages in systemic lupus erythematosus.

    PubMed Central

    López-Karpovitch, X; Cardiel, M; Cardenas, R; Piedras, J; Alarcón-Segovia, D

    1989-01-01

    In systemic lupus erythematosus (SLE) patients, in vitro bone marrow (BM) colony-forming units of granulocytes and monocytes/macrophages (CFU-GM) are decreased, suggesting that granulomonopoietic failure may play an important role in the mechanism of peripheral blood (PB) depletion of neutrophils and monocytes. No information concerning CFU-GM in PB of patients with SLE is available. The present study was undertaken in order to determine whether SLE itself and the inactive or active stage of disease would modify the number of GFU-GM in PB samples from 20 treatment-free SLE women, 12 inactive and eight active. CFU-GM growth was significantly decreased in both inactive (P = 0.018) and active (P = 0.008) SLE patients as compared with controls (n = 8). The difference in CFU-GM growth between SLE groups was not significant. These results indicate that the number of circulating CFU-GM is significantly reduced in patients with SLE regardless of disease activity or remission. PMID:2766577

  14. CXC Receptor 1 and 2 and Neutrophil Elastase Inhibitors Alter Radiation-induced Lung Disease in the Mouse

    SciTech Connect

    Fox, Jessica; Haston, Christina K.

    2013-01-01

    Purpose: We previously reported increased numbers of neutrophils to be associated with the development of the radiation-induced lung responses of alveolitis (pneumonitis) and fibrosis in mice. In the present study we investigated whether CXC receptor 1 and 2 antagonism with DF2156A, a small molecule inhibitor of neutrophil chemotaxis, or the neutrophil elastase inhibitor sivelestat decreases the lung response to irradiation. Methods and Materials: KK/HIJ mice received 14 Gy whole-thorax irradiation, and a subset of them received drug treatment 3 times per week from the day of irradiation until they were killed because of respiratory distress symptoms. Results: Irradiated mice receiving sivelestat survived 18% longer than did mice receiving radiation alone (73 vs 60 days for female mice, 91 vs 79 days for male mice), whereas postirradiation survival times did not differ between the group of mice receiving DF2156A and the radiation-only group. The numbers of neutrophils in lung tissue and in bronchoalveolar lavage fluid did not differ among groups of irradiated mice, but they significantly exceeded the levels in unirradiated control mice. The extent of alveolitis, assessed histologically, did not differ between irradiated mice treated with either drug and those receiving radiation alone, when assessed at the end of the experiment, but it was significantly reduced, as were the neutrophil measures, in sivelestat-treated mice at the common kill time of 60 days after irradiation. Mice treated with radiation and DF2156A developed significantly less fibrosis than did mice receiving radiation alone, and this difference was associated with decreased expression of interleukin-13 in lung tissue. Conclusions: We conclude that neutrophil elastase inhibition affects alveolitis and prolongs survival, whereas CXCR1/2 antagonism reduces radiation-induced fibrotic lung disease in mice without affecting the onset of distress.

  15. tPA-MMP-9 Axis Plays a Pivotal Role in Mobilization of Endothelial Progenitor Cells from Bone Marrow to Circulation and Ischemic Region for Angiogenesis.

    PubMed

    Leu, Steve; Day, Yuan-Ji; Sun, Cheuk-Kwan; Yip, Hon-Kan

    2016-01-01

    We examined the role of tissue plasminogen activator- (tPA-) matrix metalloproteinase- (MMP-) 9 in mobilizing endothelial progenitor cells (EPCs) from bone marrow to circulation and critical limb ischemia (CLI) region. Male C57BL/6J mice having been irradiated were categorized into wild-type mice (WT) receiving WT bone marrow cell (BMC) transfusion (group 1), WT mice receiving MMP-9 knockout (MMP-9(-/-)) BMC (group 2), MMP-9(-/-) receiving MMP-9(-/-) BMC (group 3), and MMP-9(-/-) receiving WT BMC (group 4), each of which was subdivided into sham control (SC), CLI, SC-tPA, and CLI-tPA. In groups 1 and 4, by post-CLI 18 h and day 14, circulating EPC (C-kit+/CD31+, Sca-1+/KDR+) levels were highest in CLI-tPA subgroup. In groups 2 and 3, EPC levels did not differ among all subgroups. The EPC levels in bone marrow were higher in groups 2 and 3 than those in groups 1 and 4. By day 14, in animals with CLI, expression levels of proangiogenic factors (CXCR4, SDF-1α, and VEGF) showed similar trends as circulating EPC levels. Moreover, the number of infiltrated neutrophils and macrophages in quadriceps was higher in groups 1 and 4 than groups in 2 and 3. In conclusion, tPA-MMP-9 axis plays a crucial role in EPC mobilization and angiogenesis in experimental CLI.

  16. tPA-MMP-9 Axis Plays a Pivotal Role in Mobilization of Endothelial Progenitor Cells from Bone Marrow to Circulation and Ischemic Region for Angiogenesis

    PubMed Central

    Day, Yuan-Ji

    2016-01-01

    We examined the role of tissue plasminogen activator- (tPA-) matrix metalloproteinase- (MMP-) 9 in mobilizing endothelial progenitor cells (EPCs) from bone marrow to circulation and critical limb ischemia (CLI) region. Male C57BL/6J mice having been irradiated were categorized into wild-type mice (WT) receiving WT bone marrow cell (BMC) transfusion (group 1), WT mice receiving MMP-9 knockout (MMP-9−/−) BMC (group 2), MMP-9−/− receiving MMP-9−/− BMC (group 3), and MMP-9−/− receiving WT BMC (group 4), each of which was subdivided into sham control (SC), CLI, SC-tPA, and CLI-tPA. In groups 1 and 4, by post-CLI 18 h and day 14, circulating EPC (C-kit+/CD31+, Sca-1+/KDR+) levels were highest in CLI-tPA subgroup. In groups 2 and 3, EPC levels did not differ among all subgroups. The EPC levels in bone marrow were higher in groups 2 and 3 than those in groups 1 and 4. By day 14, in animals with CLI, expression levels of proangiogenic factors (CXCR4, SDF-1α, and VEGF) showed similar trends as circulating EPC levels. Moreover, the number of infiltrated neutrophils and macrophages in quadriceps was higher in groups 1 and 4 than groups in 2 and 3. In conclusion, tPA-MMP-9 axis plays a crucial role in EPC mobilization and angiogenesis in experimental CLI.

  17. tPA-MMP-9 Axis Plays a Pivotal Role in Mobilization of Endothelial Progenitor Cells from Bone Marrow to Circulation and Ischemic Region for Angiogenesis

    PubMed Central

    Day, Yuan-Ji

    2016-01-01

    We examined the role of tissue plasminogen activator- (tPA-) matrix metalloproteinase- (MMP-) 9 in mobilizing endothelial progenitor cells (EPCs) from bone marrow to circulation and critical limb ischemia (CLI) region. Male C57BL/6J mice having been irradiated were categorized into wild-type mice (WT) receiving WT bone marrow cell (BMC) transfusion (group 1), WT mice receiving MMP-9 knockout (MMP-9−/−) BMC (group 2), MMP-9−/− receiving MMP-9−/− BMC (group 3), and MMP-9−/− receiving WT BMC (group 4), each of which was subdivided into sham control (SC), CLI, SC-tPA, and CLI-tPA. In groups 1 and 4, by post-CLI 18 h and day 14, circulating EPC (C-kit+/CD31+, Sca-1+/KDR+) levels were highest in CLI-tPA subgroup. In groups 2 and 3, EPC levels did not differ among all subgroups. The EPC levels in bone marrow were higher in groups 2 and 3 than those in groups 1 and 4. By day 14, in animals with CLI, expression levels of proangiogenic factors (CXCR4, SDF-1α, and VEGF) showed similar trends as circulating EPC levels. Moreover, the number of infiltrated neutrophils and macrophages in quadriceps was higher in groups 1 and 4 than groups in 2 and 3. In conclusion, tPA-MMP-9 axis plays a crucial role in EPC mobilization and angiogenesis in experimental CLI. PMID:27610138

  18. tPA-MMP-9 Axis Plays a Pivotal Role in Mobilization of Endothelial Progenitor Cells from Bone Marrow to Circulation and Ischemic Region for Angiogenesis.

    PubMed

    Leu, Steve; Day, Yuan-Ji; Sun, Cheuk-Kwan; Yip, Hon-Kan

    2016-01-01

    We examined the role of tissue plasminogen activator- (tPA-) matrix metalloproteinase- (MMP-) 9 in mobilizing endothelial progenitor cells (EPCs) from bone marrow to circulation and critical limb ischemia (CLI) region. Male C57BL/6J mice having been irradiated were categorized into wild-type mice (WT) receiving WT bone marrow cell (BMC) transfusion (group 1), WT mice receiving MMP-9 knockout (MMP-9(-/-)) BMC (group 2), MMP-9(-/-) receiving MMP-9(-/-) BMC (group 3), and MMP-9(-/-) receiving WT BMC (group 4), each of which was subdivided into sham control (SC), CLI, SC-tPA, and CLI-tPA. In groups 1 and 4, by post-CLI 18 h and day 14, circulating EPC (C-kit+/CD31+, Sca-1+/KDR+) levels were highest in CLI-tPA subgroup. In groups 2 and 3, EPC levels did not differ among all subgroups. The EPC levels in bone marrow were higher in groups 2 and 3 than those in groups 1 and 4. By day 14, in animals with CLI, expression levels of proangiogenic factors (CXCR4, SDF-1α, and VEGF) showed similar trends as circulating EPC levels. Moreover, the number of infiltrated neutrophils and macrophages in quadriceps was higher in groups 1 and 4 than groups in 2 and 3. In conclusion, tPA-MMP-9 axis plays a crucial role in EPC mobilization and angiogenesis in experimental CLI. PMID:27610138

  19. Inactivated pepsin inhibits neutrophil activation by Fcgamma-receptor-dependent and independent stimuli.

    PubMed

    Kustiawan, Iwan; Derksen, Ninotska; Rispens, Theo

    2016-08-01

    Pepsin is widely used to produce F(ab')2 fragments of immunoglobulin G (IgG). In many cases, at least part of the pepsin will remain present in the F(ab')2 preparation, albeit in (irreversibly) inactivated form. Here we report on a potent immunomodulatory effect of irreversibly inactivated pepsin on activated human neutrophils. Degranulation, induced by coated IgG or via cytochalasin B/N-formyl-Met-Leu-Phe, was measured by quantifying elastase release, and was found to be inhibited in a dose-dependent manner by inactivated pepsin. Since a number of intravenous immunoglobulin (IVIg) products are also treated by limited digestion with pepsin, we investigated if pepsin would be present in quantities large enough to inhibit neutrophil activation. The amounts of pepsin detected in three different pepsin-treated IVIg products were found to be too low to induce an effect, at least in an in vitro setting. PMID:27368805

  20. Interaction of Bacterial Exotoxins with Neutrophil Extracellular Traps: Impact for the Infected Host

    PubMed Central

    von Köckritz-Blickwede, Maren; Blodkamp, Stefanie; Nizet, Victor

    2016-01-01

    Since their discovery in 2004, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense against various pathogens. Released in response to infectious and pro-inflammatory stimuli, NETs can immobilize invading pathogens within a fibrous matrix consisting of DNA, histones, and antimicrobial peptides. Conversely, excessive or dysregulated NET release may hold a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during infectious challenge. In recent years, a number of microbial virulence factors have been shown to modulate formation of NETs, thereby facilitating colonization or spread within the host. In this mini-review we summarize the contemporary research on the interaction of bacterial exotoxins with neutrophils that modulate NET production, focusing particular attention on consequences for the host. Understanding host–pathogen dynamics in this extracellular battlefield of innate immunity may provide novel therapeutic approaches for infectious and inflammatory disorders. PMID:27064864

  1. Polymorphonuclear neutrophil function in systemic sclerosis.

    PubMed Central

    Czirják, L; Dankó, K; Sipka, S; Zeher, M; Szegedi, G

    1987-01-01

    In vitro functions of polymorphonuclear (PMN) neutrophils were studied in 20 patients with progressive systemic sclerosis (PSS). An increase in the basal chemiluminescence (CL) activity of peripheral blood PMNs was found, suggesting that these cells had been preactivated in vivo. Patients with more extensive skin disease or signs of disease progression tended to have higher basal CL values. Active oxygen products during the respiratory burst may increase the extent of inflammatory and fibrotic processes and could be involved in the endothelial injury in PSS. The stimulatory capacity of CL response was normal in our study. No alterations were found in the opsonised yeast phagocytic activity of granulocytes when compared with control values. The binding of erythrocyte-antibody particles was found also to be normal. A depressed chemotactic activity of PMN cells against zymosan activated serum was also shown. The cause of the decreased chemotaxis of PMNs remains to be elucidated. PMID:3592786

  2. Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection.

    PubMed

    Bass, D A; Olbrantz, P; Szejda, P; Seeds, M C; McCall, C E

    1986-02-01

    Stimulated human polymorphonuclear leukocytes (PMNL) have a marked increase in oxidative metabolism, producing reduced oxygen species (e.g., H2O2) that mediate bacterial killing. Previously, quantitation of metabolic responses of PMNL from patients with acute infections employed assays that measure mean activity of the entire PMNL population; such studies reported a modest and highly variable increase in oxidative metabolic responses of such "toxic" PMNL compared with normal cells. To assess metabolic capability of PMNL from 51 patients with acute bacterial infection, we employed a quantitative flow cytometric assay of H2O2-dependent oxidative product formation, the intracellular oxidation of 2',7'-dichlorofluorescin (DCFH). After stimulation by phorbol myristate acetate, the PMNL of patients demonstrated an increase in mean DCFH oxidation (315 +/- 14 and 180 +/- 4.5 amol/cell, patients and controls). Hexose monophosphate shunt activation was similarly increased in stimulated PMNL from bacteremic patients. These data are comparable with previous studies of mean metabolic activities of toxic PMNL. However, these mean values underestimate the quantitative responses of the hyperresponsive ("primed") PMNL within a mixture of normal and primed PMNL in the patients' blood. The flow cytometric assay demonstrated that the PMNL of the patients were composed of two populations. One population of PMNL had normal oxidative responses; the other "primed" population had up to 4.6 times the oxidative product formation of normal cells. Similar priming of circulating PMNL was caused by infection with gram-positive or gram-negative staining bacteria or by Candida species. The proportion and oxidative ability of the primed PMNL occurred independently of the number of juvenile neutrophil forms and independently of "toxic" morphologic changes of Wright's-stained PMNL. On the average, 40% of the PMNL of patients were primed, but the size of the primed PMNL population varied widely

  3. Mountains and Tropical Circulation

    NASA Astrophysics Data System (ADS)

    Naiman, Z.; Goodman, P. J.; Krasting, J. P.; Malyshev, S.; Russell, J. L.; Stouffer, R. J.

    2015-12-01

    Observed tropical convection exhibits zonal asymmetries that strongly influence spatial precipitation patterns. The drivers of changes to this zonally-asymmetric Walker circulation on decadal and longer timescales have been the focus of significant recent research. Here we use two state-of-the-art earth system models to explore the impact of earth's mountains on the Walker circulation. When all land-surface topography is removed, the Walker circulation weakens by 33-59%. There is a ~30% decrease in global, large-scale upward vertical wind velocities in the middle of the troposphere, but only minor changes in global average convective mass flux, precipitation, surface and sea-surface temperatures. The zonally symmetric Hadley circulation is also largely unchanged. Following the spatial pattern of changes to large-scale vertical wind velocities, precipitation becomes less focused over the tropics. The weakening of the Walker circulation, but not the Hadley circulation, is similar to the behavior of climate models during radiative forcing experiments: in our simulations, the weakening is associated with changes in vertical wind velocities, rather than the hydrologic cycle. These results indicate suggest that mountain heights may significantly influence the Walker circulation on geologic time scales, and observed changes in tropical precipitation over millions of years may have been forced by changes in tropical orography.

  4. Platelet-neutrophil interactions during hemodialysis: a proposed biocompatibility approach.

    PubMed

    Stuard, S; Bonomini, M; Settefrati, N; Albertazzi, A

    1998-02-01

    Platelet interaction with neutrophils may occur to a significant degree during hemodialysis (HD). We have recently shown that the enhanced neutrophil reactive oxygen species (ROS) production during the early phase of HD with cuprophan (CUP) is sustained by neutrophils which have bound platelets through P-selectin (CD62P). The evaluation of platelet-neutrophil interactions during dialysis offers the novel aspect of cell-cell interactions as a new parameter for studying the biocompatibility of dialyzer membranes. By the use of flow cytometry techniques, the present study was set up to analyze intradialytic platelet-neutrophil coaggregate formation and neutrophil ROS (hydrogen peroxide) production from 6 HD patients each dialysed with CUP, cellulose diacetate (CDA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN) in a cross-over clinical trial. Platelet-neutrophil coaggregate formation (percentage of neutrophil cells positive for CD62P) and ROS production by neutrophils (total population; CD62P+ cells; CD62P- cells) were determined before HD and after 10', 20'and 40'. CD62P+ neutrophils significantly increased during HD with CUP (10', 20', 40'), PMMA (20') and CDA (20), while no change was observed with PAN. The difference between CUP and the other membranes was significant at 10', 20' and 40'; at 20', PMMA vs PAN p<0.005. ROS production by total neutrophil population significantly increased with CUP (10', 20), PMMA (20) and CDA (20'). The increase with CUP was higher at 10' when compared to CDA (p<0.020) or PAN (p<0.005), and at 20' versus the other three membranes; at 20' PMMA vs PAN p<0.005. Only neutrophils gated in neutrophil-platelet coaggregate areas (CD26P+ neutrophils) produced hydrogen peroxide. ROS production by CD62P+ neutrophils significantly increased with CUP (10', 20), PMMA (20') and CDA (20'). The increase with CUP was significantly (p<0.0002) higher than the other three membranes at 10' and 20'; at 20', PMMA vs PAN p<0.02. With each

  5. Successful cord blood transplantation in a girl with monosomy 7 myelodysplastic syndrome and reduced numbers of B cells.

    PubMed

    Lee, Chien-Chung; Yang, Chao-Ping; Tsai, Ming Horng; Lee, Wen-I; Fang, En-Chen; Jaing, Tang-Her

    2010-05-01

    This report described unrelated umbilical cord blood transplantation for a 3-year-old girl with myelodysplastic syndrome and monosomy 7. The patient had a prolonged course characterized by recurrent infection and slowly progressive pancytopenia. She had reduced numbers of circulating B cells but no decline in immunoglobulin levels. Chemotherapy was not initially recommended because it was contraindicated due to intercurrent lower respiratory tract infection. After 10 months, the girl achieved hematologic remission after induction chemotherapy. The patient then underwent 2-loci HLA-mismatched unrelated donor cord blood transplantation. The time to neutrophil and platelet engraftment was 12 and 23 days post-transplantation, respectively. Acute graft-versus-host disease following transplantation was minimal. She was in continuing hematological remission with full donor chimerism 3 years after transplantation.

  6. Human intravenous immunoglobulin (IVIG) preparations degranulate human neutrophils in vitro.

    PubMed

    Teeling, J L; De Groot, E R; Eerenberg, A J; Bleeker, W K; Van Mierlo, G; Aarden, L A; Hack, C E

    1998-11-01

    IVIG preparations have biological effects in vivo that are not fully understood. Possible effects include the property to stimulate Fc receptors on various cell types. To study whether IVIG may interact with neutrophils we developed an in vitro system, in which neutrophils, in whole blood or purified, were incubated with IVIG and assessed for degranulation by measuring the release of elastase and lactoferrin in culture medium. All commercially available IVIG preparations tested induced degranulation of neutrophils when incubated for 2 h at therapeutically relevant concentrations. In studies with blocking antibodies against Fc receptors (FcR), this degranulation was shown to be dependent on Fc gammaRII, whereas Fc gammaRIII had no effect. Experiments with purified neutrophils as well as binding experiments with labelled IVIG preparations indicated that neutrophil degranulation resulted from a direct interaction of IVIG with neutrophils. Using gel filtration fractions, it was found that polymeric and dimeric IgG present in IVIG was mainly responsible for the degranulation. We suggest that degranulation of neutrophils may contribute to the (side)effects of IVIG treatment in vivo.

  7. Quantitative assessment of neutrophil phagocytosis using flow cytometry.

    PubMed

    Nordenfelt, Pontus

    2014-01-01

    Neutrophils have an incredible ability to find and eradicate intruders such as bacteria and fungi. They do this largely through the process of phagocytosis, where the target is internalized into a phagosome, and eventually destroyed by the hostile phagosomal environment. It is important to study phagocytosis in order to understand how neutrophils interact with various pathogens and how they respond to different stimuli. Here, I describe a method to study neutrophil phagocytosis of bacteria using flow cytometry. The bacteria are fluorescently labeled before being introduced to neutrophils. After phagocytosis, both any remaining extracellular bacteria and neutrophils are labeled using one-step staining before three-color analysis. To assess phagocytosis, first the average time it takes for the neutrophils to internalize all bound bacteria is determined. Experiments are then performed using that time point while varying the bacteria-to-neutrophil ratio for full control of the analysis. Due to the ease with which multiple samples can be analyzed, and the quantitative nature of flow cytometry, this approach is both reproducible and sensitive.

  8. Constitutive apoptosis in equine peripheral blood neutrophils in vitro

    PubMed Central

    Brazil, Timothy J.; Dixon, Padraic M.; Haslett, Christopher; Murray, Joanna; McGorum, Bruce C.

    2014-01-01

    The aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained responsiveness to phorbol myristate acetate (PMA). The constitutive rate of equine neutrophil apoptosis was promoted by lipopolysaccharide (LPS), tumour necrosis factor α and phagocytosis of opsonised ovine erythrocytes, while it was inhibited by dexamethasone and ZAS (a source of C5a). Formyl-Met-Leu-Phe, leukotriene B4, platelet activating factor and PMA had no demonstrable effect on equine neutrophil apoptosis. There was a difference between equine and human neutrophil apoptosis in response to LPS and the time-dependence of the response to dexamethasone. PMID:25239298

  9. Review of the neutrophil response to Bordetella pertussis infection.

    PubMed

    Eby, Joshua C; Hoffman, Casandra L; Gonyar, Laura A; Hewlett, Erik L

    2015-12-01

    The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10-14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28-35 days in a naïve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors.

  10. Neutrophil function in an experimental model of hemolytic uremic syndrome.

    PubMed

    Vedanarayanan, V V; Kaplan, B S; Fong, J S

    1987-03-01

    To understand the role of neutrophil leukocytosis in hemolytic uremic syndrome, we studied the changes in neutrophil function in the modified generalized Shwartzman reaction in rabbits. This model resembles hemolytic uremic syndrome associated with endotoxemia. At the end of an endotoxin infusion, we observed leukopenia, thrombocytopenia, and a decrease in hematocrit associated with schistocytosis. Plasma B-glucuronidase levels increased and this was associated with a decrease in neutrophil content of the enzyme. The chemotactic index and neutrophil aggregation to zymosan-activated serum were impaired compared to controls. The neutrophil procoagulant content increased after endotoxin infusion. The serum creatinine concentration and proteinuria increased in the endotoxin-treated animals. The changes returned to normal by 48 h. Renal cortical malondialdehyde, a reflection of lipid peroxidation, was higher in the endotoxin-treated animals than in the controls. We have shown enzyme release by neutrophils, impairment of chemotaxis and aggregation, increased procoagulant content in neutrophils, and evidence of lipid peroxidation in renal cortical tissue in this model. These observations raise the possibility that leukocytes may have a role in the pathogenesis of the hemolytic uremic syndrome. PMID:3550673

  11. Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages.

    PubMed

    Braian, Clara; Hogea, Valentin; Stendahl, Olle

    2013-01-01

    Neutrophils activated by Mycobacterium tuberculosis (Mtb) form neutrophil extracellular traps (NETs), containing DNA and several biologically active cytosolic and granular proteins. These NETs may assist in the innate immune defense against different pathogens. We investigated whether the NET-forming neutrophils mediate an activating signal to macrophages during the early multicellular inflammatory reaction and granuloma formation. Mtb-induced NETs were found to be reactive oxygen species dependent and phagocytosis dependent. A neutrophil elastase inhibitor also delayed NET formation. However, NET formation occurred independently of Mtb-induced apoptosis. We observed close interactions between macrophages and Mtb-activated neutrophils, where macrophages bound and phagocytosed NETs. Significant secretion of the cytokines interleukin (IL)-6, tumor necrosis factor-α, IL-1β and IL-10 were detected from macrophages cocultured with NETs from Mtb-activated but not phorbol myristate acetate-activated neutrophils. NETs binding heat shock protein 72 (Hsp72) or recombinant Hsp72 were able to trigger cytokine release from macrophages. Only Mtb-induced NETs contained Hsp72, suggesting that these NETs can transfer this danger signal to adjacent macrophages. We propose that Hsp72 sequestered in NETs plays an important role in the interaction between neutrophils and macrophages during the early innate immune phase of an Mtb infection. The immunomodulatory role of NETs and proteins derived from them may influence not only chronic inflammation during tuberculosis but also immune regulation and autoimmunity.

  12. Dermal neutrophil, macrophage and dendritic cell responses to Yersinia pestis transmitted by fleas.

    PubMed

    Shannon, Jeffrey G; Bosio, Christopher F; Hinnebusch, B Joseph

    2015-03-01

    Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.

  13. Dermal Neutrophil, Macrophage and Dendritic Cell Responses to Yersinia pestis Transmitted by Fleas

    PubMed Central

    Shannon, Jeffrey G.; Bosio, Christopher F.; Hinnebusch, B. Joseph

    2015-01-01

    Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis. PMID:25781984

  14. Neutrophil adhesion and chemotaxis depend on substrate mechanics

    NASA Astrophysics Data System (ADS)

    Jannat, Risat A.; Robbins, Gregory P.; Ricart, Brendon G.; Dembo, Micah; Hammer, Daniel A.

    2010-05-01

    Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micromachined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the KD of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against β2-integrins leads to a significant reduction, but not an elimination, of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

  15. Neutrophil adhesion and chemotaxis depend on substrate mechanics

    PubMed Central

    Jannat, Risat A.; Dembo, Micah; Hammer, Daniel A.

    2009-01-01

    Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma, and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micro-machined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the KD of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against β2-integrins leads to a significant reduction but not an elimination of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation. PMID:20473350

  16. Neutrophil localization in acute and chronic experimental abscesses

    SciTech Connect

    Bamberger, D.M.; Bettin, K.M.; Gerding, D.N.

    1987-04-01

    Abdominal abscesses are associated with a high mortality, and usually require surgical drainage for cure. A potential mechanism explaining the inability of the host to clear this infection may be in part a result of the inability of the neutrophil to localize at the site of an established infection. To study this question, either acute (4 hours old) or chronic (2 weeks old) abscesses caused by Staphylococcus aureus were created in perforated capsules implanted in the peritoneal cavity of rabbits. Homologous neutrophils were obtained from donor rabbits 4 hours after peritoneal glycogen stimulation and labeled with indium 111 oxine. Only 0.71% of injected /sup 111/In-labeled neutrophils localized in the chronic abscesses, compared with 1.77% in acute abscesses (P less than or equal to 0.01). Animals with chronic infections had a lower intravascular recovery of injected neutrophils (P less than 0.002). Failure of neutrophil localization was not associated with less chemotactic activity within the abscess, as measured by a chemotaxis-under-agarose assay, or caused by a barrier surrounding the abscess as detected by radionuclide imaging. Only 0.07% of injected neutrophils localized into acute abdominal abscesses in animals with a concomitant chronic subcutaneous abscess. These chronically infected animals also demonstrated a low peak intravascular recovery of injected neutrophils when compared with animals with only an acute infection (P less than 0.002). These data reveal that neutrophils localize to abscesses poorly in animals with chronic infections. The mechanism is possibly related to a systemic factor(s) associated with a lower intravascular recovery of injected neutrophils in chronically infected animals.

  17. Neutrophil Integrins and Matrix Ligands and NET Release

    PubMed Central

    O’Brien, Xian M.; Reichner, Jonathan S.

    2016-01-01

    Neutrophils are motile and responsive to tissue injury and infection. As neutrophils emigrate from the bloodstream and migrate toward a site of affliction, they encounter the tissue extracellular matrix (ECM) and thereby engage integrins. Our laboratory studies the neutrophilic response to the fungal pathogen Candida albicans either in the filamentous state of the microbe or to the purified pathogen-associated molecular pattern, β-glucan. We have gained an appreciation for the role of integrins in regulating the neutrophil anti-Candida response and how the presence or absence of ECM can drive experimental outcome. The β2 integrin CR3 (complement receptor 3; αMβ2; Mac-1; CD11b/CD18) plays an important role in fungal recognition by its ability to bind β-glucan at a unique lectin-like domain. The presence of ECM differentially regulates essential neutrophil anti-fungal functions, including chemotaxis, respiratory burst, homotypic aggregation, and the release of neutrophil extracellular traps (NETs). We have shown that NET release to C. albicans hyphae or immobilized β-glucan occurs rapidly and without the requirement for respiratory burst on ECM. This is in contrast to the more frequently reported mechanisms of NETosis to other pathogens without the context of ECM, which occur after a prolonged lag period and require respiratory burst. As expected for an ECM-dependent phenotype, NETosis and other neutrophil functions are dependent on specific integrins. The focus of this review is the role of ECM ligation by neutrophil integrins as it pertains to host defense functions with an emphasis on lessons we have learned studying the anti-Candida response of human neutrophils.

  18. Neutrophil Integrins and Matrix Ligands and NET Release

    PubMed Central

    O’Brien, Xian M.; Reichner, Jonathan S.

    2016-01-01

    Neutrophils are motile and responsive to tissue injury and infection. As neutrophils emigrate from the bloodstream and migrate toward a site of affliction, they encounter the tissue extracellular matrix (ECM) and thereby engage integrins. Our laboratory studies the neutrophilic response to the fungal pathogen Candida albicans either in the filamentous state of the microbe or to the purified pathogen-associated molecular pattern, β-glucan. We have gained an appreciation for the role of integrins in regulating the neutrophil anti-Candida response and how the presence or absence of ECM can drive experimental outcome. The β2 integrin CR3 (complement receptor 3; αMβ2; Mac-1; CD11b/CD18) plays an important role in fungal recognition by its ability to bind β-glucan at a unique lectin-like domain. The presence of ECM differentially regulates essential neutrophil anti-fungal functions, including chemotaxis, respiratory burst, homotypic aggregation, and the release of neutrophil extracellular traps (NETs). We have shown that NET release to C. albicans hyphae or immobilized β-glucan occurs rapidly and without the requirement for respiratory burst on ECM. This is in contrast to the more frequently reported mechanisms of NETosis to other pathogens without the context of ECM, which occur after a prolonged lag period and require respiratory burst. As expected for an ECM-dependent phenotype, NETosis and other neutrophil functions are dependent on specific integrins. The focus of this review is the role of ECM ligation by neutrophil integrins as it pertains to host defense functions with an emphasis on lessons we have learned studying the anti-Candida response of human neutrophils. PMID:27698655

  19. Impaired neutrophils in children with the typical form of hemolytic uremic syndrome.

    PubMed

    Fernández, Gabriela C; Gómez, Sonia A; Rubel, Carolina J; Bentancor, Leticia V; Barrionuevo, Paula; Alduncín, Marta; Grimoldi, Irene; Exeni, Ramón; Isturiz, Martín A; Palermo, Marina S

    2005-09-01

    Experimental and clinical evidence suggest that activated neutrophils (PMN) could contribute to endothelial damage in Hemolytic Uremic Syndrome (D+HUS). Additionally, while PMN-activating cytokines and PMN-derived products have been found in D+HUS sera, we have demonstrated phenotypic alterations in D+HUS PMN compatible with a deactivation state. Here, we investigated whether D+HUS PMN were actually hyporesponsive, and explored some of the mechanisms probably involved in their derangement. Twenty-two D+HUS children were bled in the acute period, and blood samples from healthy, acute uremic and neutrophilic children were obtained as controls. We evaluated degranulation markers in response to cytokines, intracellular granule content, and reactive oxygen species (ROS) generation in circulating D+HUS and control PMN. The influence of D+HUS-derived plasma and the direct effects of Stx in vitro were evaluated on healthy donors' PMN. We found that D+HUS PMN presented reduced degranulatory capacity in response to cytokines and intracellular granule content, and decreased ROS generation. D+HUS plasma or Stx did not affect the phenotype and function of healthy donors' PMN. These results suggest that upon hospitalization D+HUS PMN are functionally impaired and show features of previous degranulation, indicating a preceding process of activation with release of ROS and proteases involved in endothelial damage.

  20. Elevated neutrophil elastase and acrolein-protein adducts are associated with W256 regression.

    PubMed

    Jaganjac, M; Poljak-Blazi, M; Schaur, R J; Zarkovic, K; Borovic, S; Cipak, A; Cindric, M; Uchida, K; Waeg, G; Zarkovic, N

    2012-11-01

    The involvement of granulocytes in immune response against cancer is not well understood. Depending on the cytokine milieu in which they act and on their oxidative burst, granulocytes may play either an inhibitory or stimulatory role in tumour growth. Unsaturated fatty acids, essential components of cellular membranes and storage lipids, are susceptible to granulocyte-derived reactive oxygen species (ROS). ROS can induce lipid peroxidation (LPO) resulting in the destruction of biomembranes. Thus, murine W256 tumour progressing and tumour regressing animal models were used to study the involvement of plasma inflammatory mediators and oxidative burst of circulating granulocytes in malignant destruction and detrimental tumour growth. The involvement of LPO-derived aldehydes (i.e. acrolein, 4-hydroxy-2-nonenal and malondialdehyde) and myeloperoxidase (MPO) appearance in the granulocyte anti-cancer response were further evaluated. The results obtained revealed a significant increase in neutrophil elastase in animals with regressing tumour. Furthermore, the presence of MPO in tumour microenvironment was accompanied by the formation of acrolein only 5 h after tumour transplantation and its presence increased during tumour regression. Later, at an early stage of tumour regression, the presence of other LPO-derived aldehydes were also observed. The results obtained suggest that elevated neutrophil elastase and initiation of LPO may play an important role in the tumour development leading to tumour regression.

  1. Elevated neutrophil elastase and acrolein-protein adducts are associated with W256 regression

    PubMed Central

    Jaganjac, M; Poljak-Blazi, M; Schaur, R J; Zarkovic, K; Borovic, S; Cipak, A; Cindric, M; Uchida, K; Waeg, G; Zarkovic, N

    2012-01-01

    The involvement of granulocytes in immune response against cancer is not well understood. Depending on the cytokine milieu in which they act and on their oxidative burst, granulocytes may play either an inhibitory or stimulatory role in tumour growth. Unsaturated fatty acids, essential components of cellular membranes and storage lipids, are susceptible to granulocyte-derived reactive oxygen species (ROS). ROS can induce lipid peroxidation (LPO) resulting in the destruction of biomembranes. Thus, murine W256 tumour progressing and tumour regressing animal models were used to study the involvement of plasma inflammatory mediators and oxidative burst of circulating granulocytes in malignant destruction and detrimental tumour growth. The involvement of LPO-derived aldehydes (i.e. acrolein, 4-hydroxy-2-nonenal and malondialdehyde) and myeloperoxidase (MPO) appearance in the granulocyte anti-cancer response were further evaluated. The results obtained revealed a significant increase in neutrophil elastase in animals with regressing tumour. Furthermore, the presence of MPO in tumour microenvironment was accompanied by the formation of acrolein only 5 h after tumour transplantation and its presence increased during tumour regression. Later, at an early stage of tumour regression, the presence of other LPO-derived aldehydes were also observed. The results obtained suggest that elevated neutrophil elastase and initiation of LPO may play an important role in the tumour development leading to tumour regression. PMID:23039888

  2. Circulating serotonin in vertebrates.

    PubMed

    Maurer-Spurej, E

    2005-08-01

    The role of circulating serotonin is unclear and whether or not serotonin is present in the blood of non-mammalian species is not known. This study provides the first evidence for the presence of serotonin in thrombocytes of birds and three reptilian species, the endothermic leatherback sea turtle, the green sea turtle and the partially endothermic American alligator. Thrombocytes from a fresh water turtle, American bullfrog, Yellowfin tuna, and Chinook salmon did not contain serotonin. Serotonin is a vasoactive substance that regulates skin blood flow, a major mechanism for endothermic body temperature regulation, which could explain why circulating serotonin is present in warm-blooded species. The temperature sensitivity of human blood platelets with concomitant changes in serotonin content further supports a link between circulating serotonin and thermoregulation. Phylogenetic comparison of the presence of circulating serotonin indicated an evolutionary divergence within reptilian species that might coincide with the emergence of endothermy. PMID:16041566

  3. Structural Determination of Circulation.

    ERIC Educational Resources Information Center

    Blankenburg, William B.

    1981-01-01

    Analyzes the effects of both structural factors (demographics, economic conditions, and competition) and discretionary factors (content, design, and marketing techniques) and concludes that it is the former that determine a newspaper's circulation. (FL)

  4. Atmospheric Circulation and Dynamics

    NASA Astrophysics Data System (ADS)

    Limaye, Sanjay S.; Rengel, Miriam

    The deep atmosphere of Venus ( ˜ 180 km including the thermosphere) presents both observational and modeling challenges. Its thick, nearly uniform global cloud cover makes it difficult to fathom the vertical structure of the global circulation through available techniques that are applied to Earth's atmosphere. Further, the slow rotation of the planet and the consequential prevailing cyclostrophic balance restricts easy inferences about the meridional flow and circulation (Gierasch et al. 1997, Read 1986, Schubert et al. 2007).

  5. Complement factor H modulates the activation of human neutrophil granulocytes and the generation of neutrophil extracellular traps.

    PubMed

    Schneider, Andrea E; Sándor, Noémi; Kárpáti, Éva; Józsi, Mihály

    2016-04-01

    Factor H (FH) is a major inhibitor of the alternative pathway of complement activation in plasma and on certain host surfaces. In addition to being a complement regulator, FH can bind to various cells via specific receptors, including binding to neutrophil granulocytes through complement receptor type 3 (CR3; CD11b/CD18), and modulate their function. The cellular roles of FH are, however, poorly understood. Because neutrophils are important innate immune cells in inflammatory processes and the host defense against pathogens, we aimed at studying the effects of FH on various neutrophil functions, including the generation of extracellular traps. FH co-localized with CD11b on the surface of neutrophils isolated from peripheral blood of healthy individuals, and cell-bound FH retained its cofactor activity and enhanced C3b degradation. Soluble FH supported neutrophil migration and immobilized FH induced cell spreading. In addition, immobilized but not soluble FH enhanced IL-8 release from neutrophils. FH alone did not trigger the cells to produce neutrophil extracellular traps (NETs), but NET formation induced by PMA and by fibronectin plus fungal β-glucan were inhibited by immobilized, but not by soluble, FH. Moreover, in parallel with NET formation, immobilized FH also inhibited the production of reactive oxygen species induced by PMA and by fibronectin plus β-glucan. Altogether, these data indicate that FH has multiple regulatory roles on neutrophil functions. While it can support the recruitment of neutrophils, FH may also exert anti-inflammatory effects and influence local inflammatory and antimicrobial reactions, and reduce tissue damage by modulating NET formation. PMID:26938503

  6. Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected with Paracoccidioides brasiliensis.

    PubMed

    Pino-Tamayo, Paula Andrea; Puerta-Arias, Juan David; Lopera, Damaris; Urán-Jiménez, Martha Eugenia; González, Ángel

    2016-01-01

    Neutrophils predominate during the acute phase of the Paracoccidioides brasiliensis infection. Herein, we determined the role of the neutrophil during the early stages of experimental pulmonary paracoccidioidomycosis using a monoclonal antibody (mAb) specific for neutrophils. Male BALB/c mice were inoculated intranasally with 1.5 × 10(6) or 2 × 10(6) P. brasiliensis yeast cells. The mAb was administered 24 h before infection, followed by doses every 48 h until mice were sacrificed. Survival time was evaluated and mice were sacrificed at 48 h and 96 h after inoculation to assess cellularity, fungal load, cytokine/chemokine levels, and histopathological analysis. Neutrophils from mAb-treated mice were efficiently depleted (99.04%). Eighty percent of the mice treated with the mAb and infected with 1.5 × 10(6) yeast cells died during the first two weeks after infection. When mice were treated and infected with 2 × 10(6) yeast cells, 100% of them succumbed by the first week after infection. During the acute inflammatory response significant increases in numbers of eosinophils, fungal load and levels of proinflammatory cytokines/chemokines were observed in the mAb-treated mice. We also confirmed that neutrophils are an important source of IFN-γ and IL-17. These results indicate that neutrophils are essential for protection as well as being important for regulating the early inflammatory immune response in experimental pulmonary paracoccidioidomycosis. PMID:27642235

  7. Depletion of Neutrophils Promotes the Resolution of Pulmonary Inflammation and Fibrosis in Mice Infected with Paracoccidioides brasiliensis

    PubMed Central

    Arango, Julián Camilo

    2016-01-01

    Chronic stages of paracoccidioidomycosis (PCM) are characterized by granulomatous lesions which promote the development of pulmonary fibrosis leading to the loss of respiratory function in 50% of patients; in addition, it has been observed that neutrophils predominate during these chronic stages of P. brasiliensis infection. The goal of this study was to evaluate the role of the neutrophil during the chronic stages of experimental pulmonary PCM and during the fibrosis development and tissue repair using a monoclonal specific to this phagocytic cell. Male BALB/c mice were inoculated intranasally with 1.5x106 P. brasiliensis yeast cells. A monoclonal antibody specific to neutrophils was administered at 4 weeks post-inoculation followed by doses every 48h during two weeks. Mice were sacrificed at 8 and 12 weeks post-inoculation to assess cellularity, fungal load, cytokine/chemokine levels, histopathological analysis, collagen and expression of genes related to fibrosis development. Depletion of neutrophils was associated with a significant decrease in the number of eosinophils, dendritic cells, B cells, CD4-T cells, MDSCs and Treg cells, fungal load and levels of most of the pro-inflammatory cytokines/chemokines evaluated, including IL-17, TNF-α and TGF-β1. Recovery of lung architecture was also associated with reduced levels of collagen, high expression of TGF-β3, matrix metalloproteinase (MMP)-12 and -14, and decreased expression of tissue inhibitor metalloproteinase (TIMP)-2, and MMP-8. Depletion of neutrophils might attenuate lung fibrosis and inflammation through down-regulating TGF-β1, TNF-α, IL-17, MMP-8 and TIMP-2. These results suggest that neutrophil could be considered as a therapeutic target in pulmonary fibrosis induced by P. brasiliensis. PMID:27690127

  8. Contribution of complement-stimulated hepatic macrophages and neutrophils to endotoxin-induced liver injury in rats.

    PubMed

    Jaeschke, H; Farhood, A; Smith, C W

    1994-04-01

    The role of complement as potential activator for tissue macrophages and neutrophils was investigated in an experimental model of endotoxin-induced liver injury in male Fischer rats. Injection of Salmonella enteritidis endotoxin (1 mg/kg) into Corynebacterium parvum-pretreated animals (7 mg/kg; single dose 6 days before endotoxin) resulted in severe oxidant stress, as indicated by a 37-fold increase of plasma levels of glutathione disulfide (basal concentration, 0.36 +/- 14 mumol/L), accumulation of neutrophils in the liver (600 +/- 31 neutrophils/50 high-power fields) and liver injury (plasma ALT, 1184 +/- 185 U/l; necrosis; 19% +/- 3%) 10 hr after endotoxin. The oxidant stress induced by 1 mg/kg endotoxin in the C. parvum-treated animals was always significantly higher than that in control animals receiving the same dose of endotoxin. Inhibition of complement activation with the soluble complement receptor type 1 attenuated the oxidant stress and liver injury by 50% to 65% but had no effect on hepatic neutrophil accumulation or plasma tumor necrosis factor-alpha levels. Treatment with a monoclonal antibody directed against the alpha-chain of CD11b/CD18 adhesion proteins (clone 17), which was highly effective in attenuating ischemia-reperfusion injury in the liver by reducing the number of neutrophils and functionally inactivating these cells, neither protected against parenchymal cell injury nor affected hepatic neutrophil infiltration in the C. parvum model. We conclude that reactive oxygen derived from complement-stimulated macrophages is critical for the development of liver injury in the C. parvum/endotoxin model. PMID:8138272

  9. 3,4-methylenedioxymethamphetamine (MDMA--Ecstasy) decreases neutrophil activity through the glucocorticoid pathway and impairs host resistance to Listeria monocytogenes infection in mice.

    PubMed

    Ferraz-de-Paula, V; Ribeiro, A; Souza-Queiroz, J; Pinheiro, M L; Vecina, J F; Souza, D P M; Quinteiro-Filho, W M; Moreau, R L M; Queiroz, M L S; Palermo-Neto, J

    2014-12-01

    Ecstasy is the popular name of the abuse drug 3,4-methylenedioxymethamphetamine (MDMA) that decreases immunity in animals. The mechanisms that generate such alterations are still controversial. Seven independent pharmacological approaches were performed in mice to identify the possible mechanisms underlying the decrease of neutrophil activity induced by MDMA and the possible effects of MDMA on host resistance to Listeria monocytogenes. Our data showed that MDMA (10 mg kg(-1)) administration decreases NFκB expression in circulating neutrophils. Metyrapone or RU-486 administration prior to MDMA treatment abrogated MDMA effects on neutrophil activity and NFκB expression, while 6-OHDA or ICI-118,551 administration did not. As MDMA treatment increased the plasmatic levels of adrenaline and noradrenaline, propranolol pre-treatment effects were also evaluated. Propranolol suppressed both MDMA-induced increase in corticosterone serum levels and its effects on neutrophil activity. In a L. monocytogenes experimental infection context, we showed that MDMA: induced myelosuppression by decreasing granulocyte-macrophage hematopoietic progenitors (CFU-GM) in the bone marrow but increased CFU-GM in the spleen; decreased circulating leukocytes and bone marrow cellularity and increased spleen cellularity; decreased pro-inflammatory cytokine (IL-12p70, TNF, IFN-γ, IL-6) and chemokine (MCP-1) production 24 h after the infection; increased the production of pro-inflammatory cytokines and chemokines 72 h after infection and decreased IL-10 levels at all time points analyzed. It was proposed that MDMA immunosuppressive effects on neutrophil activity and host resistance to L monocytogenes rely on NFκB signaling, being mediated by HPA axis activity and corticosterone.

  10. Role of CD 11/CD 18 in neutrophil emigration during acute and recurrent Pseudomonas aeruginosa-induced pneumonia in rabbits.

    PubMed Central

    Kumasaka, T.; Doyle, N. A.; Quinlan, W. M.; Graham, L.; Doerschuk, C. M.

    1996-01-01

    This study examined CD11/CD18-mediated adhesion in neutrophil emigration during acute and recurrent Pseudomonas aeruginosa-induced pneumonia. Neutrophil emigration during acute pneumonia was studied in anti-CD18 antibody or murine-IgG-pretreated rabbits 4 hours after intrabronchial instillation of P. aeruginosa. To examine emigration in recurrent pneumonias, rabbits given P. aeruginosa on day 0 received anti-CD18 antibody or IgG on day 7. A second instillate was placed either at the initial site or in a separate lobe, and emigration into alveolar spaces was quantitated morphometrically after 4 hours. The results show that CD11/CD18 was required for neutrophil emigration in acute pneumonias and in recurrent pneumonias that occurred at a site distant from the initial infection. However, when the recurrent pneumonia occurred in the previously inflamed site, CD11/CD18 was not required. When the same number of organisms were instilled on days 0 and 7, emigration was reduced to 15 to 20 percent of the number that migrated initially and only CD18-independent adhesion pathways were used. Increasing the concentration of organisms threefold increased emigration through both CD18-dependent and CD18-independent pathways. These data indicate that P. aeruginosa induces CD11/CD18-dependent emigration during acute pneumonia and recurrent pneumonia at previously uninflamed sites. However, adhesion pathways are altered in regions of chronic inflammation, and a greater proportion of neutrophil emigration occurs through CD11/CD18-independent pathways. PMID:8644870

  11. Thalidomide enhances both primary and secondary host resistances to Listeria monocytogenes infection by a neutrophil-related mechanism in female B6C3F1 mice

    SciTech Connect

    Guo, Tai L. . E-mail: tlguo@hsc.vcu.edu; Chi, Rui P.; Karrow, Niel A.; Zhang, Ling X.; Pruett, Stephen B.; Germolec, Dori R.; White, Kimber L.

    2005-12-15

    Previously, we have reported that thalidomide can modulate the immune responses in female B6C3F1 mice. Furthermore, thalidomide immunomodulation increased primary host resistance to intravenously infected Listeria monocytogenes. The present study was intended to evaluate the mechanisms underlying the enhanced host resistance to L. monocytogenes by focusing on the neutrophils. Female B6C3F1 mice were treated intraperitoneally with thalidomide (100 mg/kg) for 15 days. Exposure to thalidomide increased the numbers of neutrophils in the spleens and livers of L. monocytogenes-infected mice when compared to the L. monocytogenes-infected control mice. Additionally, the percentage of neutrophils was also significantly increased after Thd treatment in L. monocytogenes-infected mice. Further studies using antibodies to deplete corresponding cells indicated that thalidomide-mediated increase in primary host resistance (both the moribundity and colony counts in the liver and spleen) to L. monocytogenes infection was due to its effect on neutrophils but not CD8{sup +} T cells or NK cells. Finally, Thd exposure also increased host resistance to secondary host resistance to L. monocytogenes infection, and depletion of neutrophils abolished the protective effect. In conclusion, thalidomide enhanced host resistance to both primary and secondary L. monocytogenes infections by a neutrophil-related mechanism in female B6C3F1 mice.

  12. Mitochondrial DNA is released by shock and activates neutrophils via p38 map kinase.

    PubMed

    Zhang, Qin; Itagaki, Kiyoshi; Hauser, Carl J

    2010-07-01

    Bacterial DNA (bDNA) can activate an innate-immune stimulatory "danger" response via toll-like receptor 9 (TLR9). Mitochondrial DNA (mtDNA) is unique among endogenous molecules in that mitochondria evolved from prokaryotic ancestors. Thus, mtDNA retains molecular motifs similar to bDNA. It is unknown, however, whether mtDNA is released by shock or is capable of eliciting immune responses like bDNA. We hypothesized shock-injured tissues might release mtDNA and that mtDNA might act as a danger-associated molecular pattern (or "alarmin") that can activate neutrophils (PMNs) and contribute to systemic inflammatory response syndrome. Standardized trauma/hemorrhagic shock caused circulation of mtDNA as well as nuclear DNA. Human PMNs were incubated in vitro with purified mtDNA or nuclear DNA, with or without pretreatment by chloroquine (an inhibitor of endosomal receptors like TLR9). Neutrophil activation was assessed as matrix metalloproteinase (MMP) 8 and MMP-9 release as well as p38 and p44/42 mitogen-activated protein kinase (MAPK) phosphorylation. Mitochondrial DNA induced PMN MMP-8/MMP-9 release and p38 phosphorylation but did not activate p44/42. Responses were inhibited by chloroquine. Nuclear DNA did not induce PMN activation. Intravenous injection of disrupted mitochondria (mitochondrial debris) into rats induced p38 MAPK activation and IL-6 and TNF-alpha accumulation in the liver. In summary, mtDNA is released into the circulation by shock. Mitochondrial DNA activates PMN p38 MAPK, probably via TLR9, inducing an inflammatory phenotype. Mitochondrial DNA may act as a danger-associated molecular pattern or alarmin after shock, contributing to the initiation of systemic inflammatory response syndrome.

  13. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae

    PubMed Central

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine

    2016-01-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4+/1013). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4+/1013 mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4+/1013 mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis. PMID:27111140

  14. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

    PubMed

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine; Bachelerie, Françoise; Martin, Coralie

    2016-04-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis. PMID:27111140

  15. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

    PubMed

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine; Bachelerie, Françoise; Martin, Coralie

    2016-04-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis.

  16. Oxidative burst of neutrophils against melanoma B16-F10.

    PubMed

    Zivkovic, Morana; Poljak-Blazi, Marija; Zarkovic, Kamelija; Mihaljevic, Danijela; Schaur, Rudolf Joerg; Zarkovic, Neven

    2007-02-01

    Intensive oxidative burst was determined by chemiluminescence of peripheral blood neutrophils of mice that were intramuscularly injected with melanoma B16-F10 and/or subcutaneously with Sephadex G-200. The neutrophils from papula developed at the site of Sephadex injection were cytotoxic for the B16-F10 cells in vitro. However, survival of Sephadex injected tumour-bearing mice was lower than of control animals bearing B16-F10, while their tumours grew faster and were less necrotic. Thus, it is likely that injection of Sephadex distracted the neutrophils from the tumour allowing faster progression of the tumour, indicating that neutrophils may have an important role in the host defence against malignant cells in the early stage of tumour development.

  17. Alterations of neutrophil functions in foundry and pottery workers.

    PubMed

    Başaran, N; Undeğer, U; Shubair, M

    2002-11-01

    To assess the immune competence of workers occupationally exposed to mainly silica, neutrophil functions such as the chemotactic and oxidative burst activity in foundry and pottery workers were evaluated. The chemotactic activity was examined in 22 foundry and 10 pottery workers and oxidative burst activity of neutrophils were determined in 22 foundry and 6 pottery workers. Healthy subjects of comparable age, sex, and smoking habits and with no history of silica exposure were used as the control groups. Chemotaxis was carried out in Boyden chambers using Zymosan activated serum as chemotactic stimulus. Oxidative burst activity was measured using nitroblue tetrazolium (NBT) dye reduction test. Both neutrophil functions were significantly reduced in silica-exposed foundry and pottery workers (p < 0.001) compared to controls suggesting that human chronic exposure mainly to silica and other chemicals originated from foundry and pottery settings may diminish neutrophil functions in humans. PMID:12510795

  18. [The phagocytosis of polymorphonuclear neutrophilic granulocytes in progressive periodontitis].

    PubMed

    Konopka, T; Zietek, M

    1995-01-01

    The aim of this paper was the evaluation of the phagocytic activity of neutrophils in blood and in gingival pocket fluid in patients suffering from rapidly progressive periodontitis (RPP) and postjuvenile periodontitis (PJP). Prior to periodontal treatment the authors evaluated the capacity to phagocytose latex particles of peripheral blood neutrophils from 21 patients with RPP, 51 with PJP and 59 healthy subjects (control group) as well as the phagocytic activity of neutrophils in pocket fluid from 21 patients with RPP, 14 with PJP and from 20 healthy subjects. This phagocytic activity was significantly lower in all examined groups in comparison with the control group. A similar evaluation executed 3 months after treatment revealed normal phagocytosis of blood neutrophils from patients with RPP. In patients receiving complementary pharmacotherapy (spiramycine combined with metronidazol), a better improvement of phagocytosis was noted, than that observed in patients treated only surgically. PMID:7481699

  19. Immunomodulatory activity of plant residues on ovine neutrophils.

    PubMed

    Farinacci, Maura; Colitti, Monica; Sgorlon, Sandy; Stefanon, Bruno

    2008-11-15

    Neutrophils play an essential role in host defense and inflammation. Plants have long been used to improve the immune function, but for most of them specific investigations on animal health are lacking. In the present study, water and hydroethanolic extracts from 11 plant wastes have been screened on immune responses of ovine neutrophils. Eight sheep clinically healthy, not lactating, non-pregnant were selected and used for the experiment. Freshly isolated neutrophils were incubated with the extracts of the residues at increasing doses, and then they were tested for adhesion and superoxide production induced with PMA. The residues of Larix decidua, Thymus vulgaris, Salix alba, Sinupret, Helianthus annuus, Mangifera indica modulated the neutrophil immune functions, moreover, Larix decidua, Thymus vulgaris and Salix alba presented the highest anti-inflammatory activity.

  20. Alterations of neutrophil functions in foundry and pottery workers.

    PubMed

    Başaran, N; Undeğer, U; Shubair, M

    2002-11-01

    To assess the immune competence of workers occupationally exposed to mainly silica, neutrophil functions such as the chemotactic and oxidative burst activity in foundry and pottery workers were evaluated. The chemotactic activity was examined in 22 foundry and 10 pottery workers and oxidative burst activity of neutrophils were determined in 22 foundry and 6 pottery workers. Healthy subjects of comparable age, sex, and smoking habits and with no history of silica exposure were used as the control groups. Chemotaxis was carried out in Boyden chambers using Zymosan activated serum as chemotactic stimulus. Oxidative burst activity was measured using nitroblue tetrazolium (NBT) dye reduction test. Both neutrophil functions were significantly reduced in silica-exposed foundry and pottery workers (p < 0.001) compared to controls suggesting that human chronic exposure mainly to silica and other chemicals originated from foundry and pottery settings may diminish neutrophil functions in humans.

  1. [The phagocytosis of polymorphonuclear neutrophilic granulocytes in progressive periodontitis].

    PubMed

    Konopka, T; Zietek, M

    1995-01-01

    The aim of this paper was the evaluation of the phagocytic activity of neutrophils in blood and in gingival pocket fluid in patients suffering from rapidly progressive periodontitis (RPP) and postjuvenile periodontitis (PJP). Prior to periodontal treatment the authors evaluated the capacity to phagocytose latex particles of peripheral blood neutrophils from 21 patients with RPP, 51 with PJP and 59 healthy subjects (control group) as well as the phagocytic activity of neutrophils in pocket fluid from 21 patients with RPP, 14 with PJP and from 20 healthy subjects. This phagocytic activity was significantly lower in all examined groups in comparison with the control group. A similar evaluation executed 3 months after treatment revealed normal phagocytosis of blood neutrophils from patients with RPP. In patients receiving complementary pharmacotherapy (spiramycine combined with metronidazol), a better improvement of phagocytosis was noted, than that observed in patients treated only surgically.

  2. Relationships between lifestyle factors and neutrophil functions in the elderly.

    PubMed

    Tsukamoto, Kazumasa; Suzuki, Katsuhiko; Machida, Kazuhiko; Saiki, Chinatsu; Murayama, Rumiko; Sugita, Minoru

    2002-01-01

    We investigated the relationships between neutrophil functions and lifestyle factors in the elderly. The subjects (84 males, 73.9+/-5.8 years old; and 63 females, 70.0+/-4.6 years old) belonged to a recreational seniors club in Japan. Investigations of the subjects' stress, exercise habits, smoking habits, and alcohol-drinking habits were performed. The phagocytosis and superoxide productivity of the neutrophils were measured with a nitroblue tetrazolium (NBT) reduction test. In addition, leukocyte counts and serum total protein (TP) levels were determined. The results revealed that aging, high serum levels, and stress-coping factors (e.g., having hobbies, keeping pets, and close links with friends or family) significantly correlated with preferable neutrophil functions. In addition, significant effects of lifestyle factors on the balance between phagocytosis and subsequent superoxide production were observed. Thus, the results of the present study suggest that there are correlations between neutrophil functions and lifestyle factors in the elderly. PMID:12357457

  3. The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

    PubMed Central

    1985-01-01

    Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl- phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C. PMID:2859292

  4. Staphylococcus aureus agr Genotypes with Enterotoxin Production Capabilities Can Resist Neutrophil Bactericidal Activity

    PubMed Central

    Mullarky, I. K.; Su, C.; Frieze, N.; Park, Y. H.; Sordillo, L. M.

    2001-01-01

    Staphylococcus aureus pathogenicity is mainly due to the production of a number of secreted and cell surface-associated proteins under the regulation of the agr gene. A region of the agr gene was used to subgroup S. aureus strains according to restriction fragment length polymorphisms. Additionally, strains were subtyped according to the coagulase gene in order to strengthen discriminatory power. Virulence capabilities of agr genotype subgroups were evaluated using an in vitro neutrophil bactericidal assay, which showed that prevalent genotypes were significantly better at evading this primary host defense. Multiplex PCR was then used to detect enterotoxin genes among the genotype subgroups in order to determine possible virulence candidates that enable strains to combat neutrophil killing. The prevalent genotype strains were found to possess higher production capabilities for enterotoxin A than did low-prevalence strains. The significance of enterotoxin A production capabilities in affecting pathogenicity of S. aureus strains was evaluated and found to have a profound effect on neutrophil killing abilities. The use of a large epidemiological database as a tool for subgrouping strains with varying degrees of pathogenicity has allowed the identification of relevant and previously undefined virulence factors that affect a pathogen's capability to overcome host immune defenses. PMID:11119487

  5. Genetic Mechanism of Human Neutrophil Antigen 2 Deficiency and Expression Variations

    PubMed Central

    Li, Yunfang; Mair, David C.; Schuller, Randy M.; Li, Ling; Wu, Jianming

    2015-01-01

    Human neutrophil antigen 2 (HNA-2) deficiency is a common phenotype as 3–5% humans do not express HNA-2. HNA-2 is coded by CD177 gene that associates with human myeloproliferative disorders. HNA-2 deficient individuals are prone to produce HNA-2 alloantibodies that cause a number of disorders including transfusion-related acute lung injury and immune neutropenia. In addition, the percentages of HNA-2 positive neutrophils vary significantly among individuals and HNA-2 expression variations play a role in human diseases such as myelodysplastic syndrome, chronic myelogenous leukemia, and gastric cancer. The underlying genetic mechanism of HNA-2 deficiency and expression variations has remained a mystery. In this study, we identified a novel CD177 nonsense single nucleotide polymorphism (SNP 829A>T) that creates a stop codon within the CD177 coding region. We found that all 829TT homozygous individuals were HNA-2 deficient. In addition, the SNP 829A>T genotypes were significantly associated with the percentage of HNA-2 positive neutrophils. Transfection experiments confirmed that HNA-2 expression was absent on cells expressing the CD177 SNP 829T allele. Our data clearly demonstrate that the CD177 SNP 829A>T is the primary genetic determinant for HNA-2 deficiency and expression variations. The mechanistic delineation of HNA-2 genetics will enable the development of genetic tests for diagnosis and prognosis of HNA-2-related human diseases. PMID:26024230

  6. Interleukin-8 in Hodgkin's disease. Preferential expression by reactive cells and association with neutrophil density.

    PubMed Central

    Foss, H. D.; Herbst, H.; Gottstein, S.; Demel, G.; Araujó, I.; Stein, H.

    1996-01-01

    Hodgkin's disease (HD) shows rare neoplastic Hodgkin and Reed-Sternberg cells embedded in an abundant reactive infiltrate containing, among other cell types, neutrophilic granulocytes. Interleukin (IL)-8 is chemotactic for neutrophils. The expression of IL-8 was tested by in situ hybridization with 35S-labeled IL-8-specific RNA probes on 38 cases of HD. Reactive lesions, non-Hodgkin's lymphomas of B and T phenotype, and Langerhans cell histiocytosis served as controls. IL-8 expression was observed in Hodgkin and Reed-Sternberg cells in 3 of 33 cases of classical HD and in reactive cells in 20 of 33 HD cases as evidenced by combined isotopic in situ hybridization and immunohistology for the demonstration of cell-type-characteristic antigens or enzyme histochemistry for chloroacetate esterase. IL-8-positive cells were more numerous in cases of nodular sclerosing HD as compared with the mixed cellularity histotype (P = 0.01). The number of IL-8-positive cells and the density of neutrophils were positively correlated (P < 0. 01). In 5 cases of lymphocyte-predominant HD, IL-8 expression was not displayed. Non-Hodgkin's lymphoma cases contained IL-8 transcripts only in 1 of 23 cases in sparse reactive cells. In 4 of 7 cases of Langerhans cell histiocytosis, IL-8-specific signals were displayed in S100-negative cells. In conclusion, IL-8 expression in HD is largely confined to reactive cells and associated with infiltration by neutrophils. Elaboration of other cytokines by Hodgkin and Reed-Sternberg cells and reactive cells may explain the frequent expression of this cytokine in HD, particularly in the nodular sclerosing type. Images Figure 1 Figure 2 PMID:8644863

  7. Disentangling the effects of tocilizumab on neutrophil survival and function.

    PubMed

    Gaber, Timo; Hahne, Martin; Strehl, Cindy; Hoff, Paula; Dörffel, Yvonne; Feist, Eugen; Burmester, Gerd-Rüdiger; Buttgereit, Frank

    2016-06-01

    The synovial tissue in rheumatoid arthritis (RA) represents a hypoxic environment with up-regulated pro-inflammatory cytokines and cellular infiltrates including neutrophils. Although inhibition of the interleukin (IL)6 receptor pathway by tocilizumab is a potent treatment option for RA, it may also cause adverse effects such as an occasionally high-grade neutropenia. We analysed the impact of tocilizumab on survival, mediator secretion, oxidative burst, phagocytosis and energy availability of high-dose toll-like receptor (TLR)2/4-stimulated neutrophils (to mimic an arthritis flare) under normoxic versus hypoxic conditions. Human neutrophils were purified, pre-treated with varying doses of tocilizumab, dexamethasone or human IgG1 and high-dose-stimulated with lipopolysaccharide (LPS) alone-triggering TLR2/4-, LPS plus IL6, or left unstimulated. Cells were then incubated under normoxic (18 % O2) or hypoxic (1 % O2) conditions and subsequently analysed. Neutrophil survival and energy availability were significantly decreased by tocilizumab in a dose-dependent manner in high-dose TLR2/4-stimulated cells, but to a greater extent under normoxia as compared to hypoxia. We also found high-dose LPS-stimulated oxidative burst and phagocytosis of neutrophils to be higher under hypoxic versus normoxic conditions, but this difference was reduced by tocilizumab. Finally, we observed that tocilizumab affected neutrophil mediator secretion as a function of oxygen availability. Tocilizumab is known for both beneficial effects and a higher incidence of neutropenia when treating RA patients. Our results suggest that both effects can at least in part be explained by a reduction in neutrophil survival, a dose-dependent inhibition of hypoxia-induced NADPH oxidase-mediated oxidative burst and phagocytosis of infiltrating hypoxic neutrophils and an alteration of mediator secretion.

  8. Matrix-free constructions of circulant and block circulant preconditioners

    SciTech Connect

    Yang, Chao; Ng, Esmond G.; Penczek, Pawel A.

    2001-12-01

    A framework for constructing circulant and block circulant preconditioners (C) for a symmetric linear system Ax=b arising from certain signal and image processing applications is presented in this paper. The proposed scheme does not make explicit use of matrix elements of A. It is ideal for applications in which A only exists in the form of a matrix vector multiplication routine, and in which the process of extracting matrix elements of A is costly. The proposed algorithm takes advantage of the fact that for many linear systems arising from signal or image processing applications, eigenvectors of A can be well represented by a small number of Fourier modes. Therefore, the construction of C can be carried out in the frequency domain by carefully choosing its eigenvalues so that the condition number of C{sup T} AC can be reduced significantly. We illustrate how to construct the spectrum of C in a way such that the smallest eigenvalues of C{sup T} AC overlaps with those of A extremely well while the largest eigenvalues of C{sup T} AC are smaller than those of A by several orders of magnitude. Numerical examples are provided to demonstrate the effectiveness of the preconditioner on accelerating the solution of linear systems arising from image reconstruction application.

  9. Dynamics of neutrophil rolling over stimulated endothelium in vitro.

    PubMed Central

    Goetz, D J; el-Sabban, M E; Pauli, B U; Hammer, D A

    1994-01-01

    Prior to extravasation at sites of acute inflammation, neutrophils roll over activated endothelium. Neutrophil rolling is often characterized by the average rolling velocity. An additional dynamic feature of rolling that has been identified but not extensively studied is the fluctuation in the rolling velocity about the average. To analyze this characteristic further, we have measured the instantaneous velocity of bovine neutrophils interacting with lipopolysaccharide-stimulated bovine aortic endothelium at shear stresses of 1, 2, 3, and 4 dynes/cm2. The average velocities are quantitative