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Sample records for a2 regulates phagocytosis

  1. Regulation of phagocytosis by Rho GTPases.

    PubMed

    Mao, Yingyu; Finnemann, Silvia C

    2015-01-01

    Phagocytosis is defined as a cellular uptake pathway for particles of greater than 0.5 μm in diameter. Particle clearance by phagocytosis is of critical importance for tissue health and homeostasis. The ultimate goal of anti-pathogen phagocytosis is to destroy engulfed bacteria or fungi and to stimulate cell-cell signaling that mount an efficient immune defense. In contrast, clearance phagocytosis of apoptotic cells and cell debris is anti-inflammatory. High capacity clearance phagocytosis pathways are available to professional phagocytes of the immune system and the retina. Additionally, a low capacity, so-called bystander phagocytic pathway is available to most other cell types. Different phagocytic pathways are stimulated by particle ligation of distinct surface receptors but all forms of phagocytosis require F-actin recruitment beneath tethered particles and F-actin re-arrangement promoting engulfment, which are controlled by Rho family GTPases. The specificity of Rho GTPase activity during the different forms of phagocytosis by mammalian cells is the subject of this review.

  2. Bis-Retinoid A2E Induces an Increase of Basic Fibroblast Growth Factor via Inhibition of Extracellular Signal-Regulated Kinases 1/2 Pathway in Retinal Pigment Epithelium Cells and Facilitates Phagocytosis

    PubMed Central

    Balmer, Delphine; Bapst-Wicht, Linda; Pyakurel, Aswin; Emery, Martine; Nanchen, Natacha; Bochet, Christian G.; Roduit, Raphael

    2017-01-01

    Age-related macular degeneration (ARMD) is the leading cause of vision loss in developed countries. Hallmarks of the disease are well known; indeed, this pathology is characterized by lipofuscin accumulation, is principally composed of lipid-containing residues of lysosomal digestion. The N-retinyl-N-retinylidene ethanolamine (A2E) retinoid which is thought to be a cytotoxic component for RPE is the best-characterized component of lipofuscin so far. Even if no direct correlation between A2E spatial distribution and lipofuscin fluorescence has been established in aged human RPE, modified forms or metabolites of A2E could be involved in ARMD pathology. Mitogen-activated protein kinase (MAPK) pathways have been involved in many pathologies, but not in ARMD. Therefore, we wanted to analyze the effects of A2E on MAPKs in polarized ARPE19 and isolated mouse RPE cells. We showed that long-term exposure of polarized ARPE19 cells to low A2E dose induces a strong decrease of the extracellular signal-regulated kinases' (ERK1/2) activity. In addition, we showed that A2E, via ERK1/2 decrease, induces a significant decrease of the retinal pigment epithelium-specific protein 65 kDa (RPE65) expression in ARPE19 cells and isolated mouse RPE. In the meantime, we showed that the decrease of ERK1/2 activity mediates an increase of basic fibroblast growth factor (bFGF) mRNA expression and secretion that induces an increase in phagocytosis via a paracrine effect. We suggest that the accumulation of deposits coming from outer segments (OS) could be explained by both an increase of bFGF-induced phagocytosis and by the decrease of clearance by A2E. The bFGF angiogenic protein may therefore be an attractive target to treat ARMD. PMID:28298893

  3. ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis

    PubMed Central

    Guo, Feiye; Ding, Ying; Caberoy, Nora; Alvarado, Gabriela; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis of shed photoreceptor outer segments (POSs) by retinal pigment epithelial (RPE) cells is critical to retinal homeostasis and shares many conserved signaling pathways with other phagocytes, including extrinsic regulations. Phagocytotic ligands are the key to cargo recognition, engulfment initiation, and activity regulation. In this study, we identified intracellular protein ATP-binding cassette subfamily F member 1 (ABCF1) as a novel RPE phagocytotic ligand by a new approach of functional screening. ABCF1 was independently verified to extrinsically promote phagocytosis of shed POSs by D407 RPE cells. This finding was further corroborated with primary RPE cells and RPE explants. Internalized POS vesicles were colocalized with a phagosome marker, suggesting that ABCF1-mediated engulfment is through a phagocytic pathway. ABCF1 was released from apoptotic cells and selectively bound to shed POS vesicles and apoptotic cells, possibly via externalized phosphatidylserine. ABCF1 is predominantly expressed in POSs and colocalized with the POS marker rhodopsin, providing geographical convenience for regulation of RPE phagocytosis. Collectively these results suggest that ABCF1 is released from and binds to shed POSs in an autocrine manner to facilitate RPE phagocytosis through a conserved pathway. Furthermore, the new approach is broadly applicable to many other phagocytes and will enable systematic elucidation of their ligands to understand extrinsic regulation and cargo recognition. PMID:25904329

  4. Feed-forward regulation of phagocytosis by Entamoeba histolytica.

    PubMed

    Sateriale, Adam; Vaithilingam, Archana; Donnelly, Liam; Miller, Peter; Huston, Christopher D

    2012-12-01

    The parasitic protozoan Entamoeba histolytica is aptly named for its capacity to destroy host tissue. When E. histolytica trophozoites invade the lamina propria of a host colon, extracellular matrices are degraded while host cells are killed and phagocytosed. The ability of E. histolytica to phagocytose host cells correlates with virulence in vivo. In order to better understand the mechanism of phagocytosis, we used an E. histolytica Affymetrix microarray chip to measure the total gene expression of phagocytic and nonphagocytic subpopulations. Using paramagnetic beads coated with a known host ligand that stimulates phagocytosis, phagocytic and nonphagocytic amoebae from a single culture were purified. Microarray analysis of the subpopulations identified 121 genes with >2-fold higher expression in phagocytic than in nonphagocytic amoebae. Functional annotation identified genes encoding proteins involved in actin binding and cytoskeletal organization as highly enriched gene clusters. Post hoc analyses of selected genes showed that the gene expression profile identified in the microarray experiment did not exist prior to cell sorting but rather was stimulated through phagocytosis. Further, these expression profiles correlated with an increase in phagocytic ability, as E. histolytica cultures exposed to an initial stimulus of phagocytosis showed increased phagocytic ability upon a second stimulus. To our knowledge, this is the first description of such feed-forward regulation of gene expression and phagocytic ability in a phagocyte.

  5. Regulation of phagocytosis by TAM receptors and their ligands

    PubMed Central

    Lu, Qingxian; Li, Qiutang; Lu, Qingjun

    2010-01-01

    The TAM family of receptors is preferentially expressed by professional and non-professional phagocytes, including macrophages, dendritic cells and natural killer cells in the immune system, osteoclasts in bone, Sertoli cells in testis, and retinal pigmental epithelium cells in the retina. Mutations in the Mertk single gene or in different combinations of the double or triple gene mutations in the same cell cause complete or partial impairment in phagocytosis of their preys; and as a result, either the normal apoptotic cells cannot be efficiently removed or the tissue neighbor cells die by apoptosis. This scenario of TAM regulation represents a widely adapted model system used by phagocytes in all different tissues. The present review will summarize current known functional roles of TAM receptors and their ligands, Gas 6 and protein S, in the regulation of phagocytosis. PMID:21057587

  6. Synaptotagmin XI regulates phagocytosis and cytokine secretion in macrophages.

    PubMed

    Arango Duque, Guillermo; Fukuda, Mitsunori; Descoteaux, Albert

    2013-02-15

    Synaptotagmins (Syts) are a group of type I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. All Syts possess a single transmembrane domain, and two conserved tandem Ca(2+)-binding C2 domains. However, Syts IV and XI possess a conserved serine in their C2A domain that precludes these Syts from binding Ca(2+) and phospholipids, and from mediating vesicle fusion. Given the importance of vesicular trafficking in macrophages, we investigated the role of Syt XI in cytokine secretion and phagocytosis. We demonstrated that Syt XI is expressed in murine macrophages, localized in recycling endosomes, lysosomes, and recruited to phagosomes. Syt XI had a direct effect on phagocytosis and on the secretion of TNF and IL-6. Whereas small interfering RNA-mediated knockdown of Syt XI potentiated secretion of these cytokines and particle uptake, overexpression of an Syt XI construct suppressed these processes. In addition, Syt XI knockdown led to decreased recruitment of gp91(phox) and lysosomal-associated membrane protein-1 to phagosomes, suggesting attenuated microbicidal activity. Remarkably, knockdown of Syt XI ensued in enhanced bacterial survival. Our data reveal a novel role for Syt XI as a regulator of cytokine secretion, particle uptake, and macrophage microbicidal activity.

  7. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells

    SciTech Connect

    Orlando, Kelly A.; Stone, Nicole L.; Pittman, Randall N. . E-mail: pittman@pharm.med.upenn.edu

    2006-01-01

    During the execution phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the execution phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other execution phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells.

  8. Microglial beclin 1 regulates retromer trafficking and phagocytosis and is impaired in Alzheimer's disease.

    PubMed

    Lucin, Kurt M; O'Brien, Caitlin E; Bieri, Gregor; Czirr, Eva; Mosher, Kira I; Abbey, Rachelle J; Mastroeni, Diego F; Rogers, Joseph; Spencer, Brian; Masliah, Eliezer; Wyss-Coray, Tony

    2013-09-04

    Phagocytosis controls CNS homeostasis by facilitating the removal of unwanted cellular debris. Accordingly, impairments in different receptors or proteins involved in phagocytosis result in enhanced inflammation and neurodegeneration. While various studies have identified extrinsic factors that modulate phagocytosis in health and disease, key intracellular regulators are less understood. Here we show that the autophagy protein beclin 1 is required for efficient phagocytosis in vitro and in mouse brains. Furthermore, we show that beclin 1-mediated impairments in phagocytosis are associated with dysfunctional recruitment of retromer to phagosomal membranes, reduced retromer levels, and impaired recycling of phagocytic receptors CD36 and Trem2. Interestingly, microglia isolated from human Alzheimer's disease (AD) brains show significantly reduced beclin 1 and retromer protein levels. These findings position beclin 1 as a link between autophagy, retromer trafficking, and receptor-mediated phagocytosis and provide insight into mechanisms by which phagocytosis is regulated and how it may become impaired in AD.

  9. The involvement of MiR-1-clathrin pathway in the regulation of phagocytosis.

    PubMed

    Liu, Cuilian; Wang, Jiajia; Zhang, Xiaobo

    2014-01-01

    Phagocytosis, one of the most powerful immune responses, is a complicated process regulated by many factors. However the regulation of phagocytosis mediated by microRNAs has not been extensively investigated. To address this issue, the regulation of phagocytosis by miR-1 was characterized in this study. The results showed that miR-1 played an important role in the phagocytosis regulation in shrimp in vivo. The sequence analysis indicated that miR-1 was highly conserved from invertebrates to mammals, suggesting that miR-1 might share the similar or same functions in phagocytosis of shrimp hemocytes and mammalian macrophages. The data presented that miR-1 was significantly downregulated in cancerous macrophage RAW264.7 cells compared with those in the isolated murine macrophage and in the immortalized macrophage ANA-1. The findings showed that miR-1 had a great effect on the regulation of phagocytosis in cancerous macrophage by the inhibition of clathrin heavy chain 1 (CLTC1) gene. Therefore our study presented a novel miR-1-mediated regulation of phagocytosis both in invertebrate and in vertebrate.

  10. Involvement of Ran in the regulation of phagocytosis against virus infection in S2 cells.

    PubMed

    Ye, Ting; Zhang, Xiaobo

    2013-12-01

    Phagocytosis plays important roles in innate and adaptive immunity in animals. Some small G proteins are found to be related to phagocytosis. However, the Ran GTPase has not been intensively characterized in immunity. In this paper, the sequence analysis showed that the Ran was highly conserved in animals, suggesting that its function was preserved during animal evolution. The results showed that Ran was upregulated in S2 cells in response to DCV infection. It was further revealed that the antiviral phagocytosis could be mediated by Ran in S2 cells. By comparison with the early marker and late marker of phagosomes, the results showed that the Ran protein played an essential role at the early stage of phagocytosis or throughout the entire phagocytic process. Therefore our findings enlarged our limited knowledge about the phagocytosis regulation by small G proteins concerning to the nucleus.

  11. RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis.

    PubMed

    Medraño-Fernandez, Iria; Reyes, Raquel; Olazabal, Isabel; Rodriguez, Elena; Sanchez-Madrid, Francisco; Boussiotis, Vassiliki A; Reche, Pedro A; Cabañas, Carlos; Lafuente, Esther M

    2013-07-01

    Phagocytosis mediated by the complement receptor CR3 (also known as integrin αMß2 or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß2 integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However, the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work, we examined the role of the Rap1 effector RIAM, a talin-binding protein, in the regulation of complement-mediated phagocytosis. Using the human myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired αMß2 integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of IgG-opsonized RBC particles, was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2'-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to ß2 integrin during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to the phagocytic complement receptors.

  12. Anion Exchanger 2 Regulates Dectin-1-Dependent Phagocytosis and Killing of Candida albicans

    PubMed Central

    Urso, Katia; Charles, Julia F.; Shull, Gary E.; Aliprantis, Antonios O.; Balestrieri, Barbara

    2016-01-01

    Anion exchanger 2 (Ae2; gene symbol, Slc4a2) is a plasma membrane Cl-/HCO3- exchanger expressed in the gastrointestinal tract, kidney and bone. We have previously shown that Ae2 is required for the function of osteoclasts, bone resorbing cells of the macrophage lineage, to maintain homeostatic cytoplasmic pH and electroneutrality during acid secretion. Macrophages require endosomal acidification for pathogen killing during the process known as phagocytosis. Chloride is thought to be the principal ion responsible for maintaining electroneutrality during organelle acidification, but whether Cl-/HCO3- exchangers such as Ae2 contribute to macrophage function is not known. In this study we investigated the role of Ae2 in primary macrophages during phagocytosis. We find that Ae2 is expressed in macrophages where it regulates intracellular pH and the binding of Zymosan, a fungal cell wall derivative. Surprisingly, the transcription and surface expression of Dectin-1, the major phagocytic receptor for Candida albicans (C. albicans) and Zymosan, is reduced in the absence of Ae2. As a consequence, Zymosan-induced Tnfα expression is also impaired in Ae2-deficient macrophages. Similar to Ae2 deficiency, pharmacological alkalinization of lysosomal pH with bafilomycin A decreases both Dectin-1 mRNA and cell surface expression. Finally, Ae2-deficient macrophages demonstrate defective phagocytosis and killing of the human pathogenic fungus C. albicans. Our results strongly suggest that Ae2 is a critical factor in the innate response to C. albicans. This study represents an important contribution to a better understanding of how Dectin-1 expression and fungal clearance is regulated. PMID:27391897

  13. Abl family kinases regulate FcγR-mediated phagocytosis in murine macrophages.

    PubMed

    Greuber, Emileigh K; Pendergast, Ann Marie

    2012-12-01

    Phagocytosis of Ab-coated pathogens is mediated through FcγRs, which activate intracellular signaling pathways to drive actin cytoskeletal rearrangements. Abl and Arg define a family of nonreceptor tyrosine kinases that regulate actin-dependent processes in a variety of cell types, including those important in the adaptive immune response. Using pharmacological inhibition as well as dominant negative and knockout approaches, we demonstrate a role for the Abl family kinases in phagocytosis by macrophages and define a mechanism whereby Abl kinases regulate this process. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Arg kinase is present at the phagocytic cup, and Abl family kinases are activated by FcγR engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the spleen tyrosine kinase (Syk). Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to FcγR ligation. The link between Abl family kinases and Syk may be direct, as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis. Together, these findings reveal that Abl family kinases control the efficiency of phagocytosis in part through the regulation of Syk function.

  14. Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

    NASA Technical Reports Server (NTRS)

    Beningo, Karen A.; Wang, Yu-li

    2002-01-01

    Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

  15. Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

    NASA Technical Reports Server (NTRS)

    Beningo, Karen A.; Wang, Yu-li

    2002-01-01

    Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

  16. Regulation of phagocytosis in macrophages by neuraminidase 1.

    PubMed

    Seyrantepe, Volkan; Iannello, Alexandre; Liang, Feng; Kanshin, Evgeny; Jayanth, Preethi; Samarani, Suzanne; Szewczuk, Myron R; Ahmad, Ali; Pshezhetsky, Alexey V

    2010-01-01

    The differentiation of monocytes into macrophages and dendritic cells is accompanied by induction of cell-surface neuraminidase 1 (Neu1) and cathepsin A (CathA), the latter forming a complex with and activating Neu1. To clarify the biological importance of this phenomenon we have developed the gene-targeted mouse models of a CathA deficiency (CathA(S190A)) and a double CathA/Neu1 deficiency (CathA(S190A-Neo)). Macrophages of CathA(S190A-Neo) mice and their immature dendritic cells showed a significantly reduced capacity to engulf Gram-positive and Gram-negative bacteria and positively and negatively charged polymer beads as well as IgG-opsonized beads and erythrocytes. Properties of the cells derived from CathA(S190A) mice were indistinguishable from those of wild-type controls, suggesting that the absence of Neu1, which results in the increased sialylation of the cell surface proteins, probably affects multiple receptors for phagocytosis. Indeed, treatment of the cells with purified mouse Neu1 reduced surface sialylation and restored phagocytosis. Because Neu1-deficient cells showed reduced internalization of IgG-opsonized sheep erythrocytes whereas binding of the erythrocytes to the cells at 4 degrees C persisted, we speculate that the absence of Neu1 in particular affected transduction of signals from the Fc receptors for immunoglobulin G (FcgammaR). Indeed the macrophages from the Neu1-deficient mice showed increased sialylation and impaired phosphorylation of FcgammaR as well as markedly reduced phosphorylation of Syk kinase in response to treatment with IgG-opsonized beads. Altogether our data suggest that the cell surface Neu1 activates the phagocytosis in macrophages and dendritic cells through desialylation of surface receptors, thus, contributing to their functional integrity.

  17. Group V secreted phospholipase A2 is upregulated by IL-4 in human macrophages and mediates phagocytosis via hydrolysis of ethanolamine phospholipids.

    PubMed

    Rubio, Julio M; Rodríguez, Juan P; Gil-de-Gómez, Luis; Guijas, Carlos; Balboa, María A; Balsinde, Jesús

    2015-04-01

    Studies on the heterogeneity and plasticity of macrophage populations led to the identification of two major polarization states: classically activated macrophages or M1, induced by IFN-γ plus LPS, and alternatively activated macrophages, induced by IL-4. We studied the expression of multiple phospholipase A2 enzymes in human macrophages and the effect that polarization of the cells has on their levels. At least 11 phospholipase A2 genes were found at significant levels in human macrophages, as detected by quantitative PCR. None of these exhibited marked changes after treating the cells with IFN-γ plus LPS. However, macrophage treatment with IL-4 led to strong upregulation of the secreted group V phospholipase A2 (sPLA2-V), both at the mRNA and protein levels. In parallel with increasing sPLA2-V expression levels, IL-4-treated macrophages exhibited increased phagocytosis of yeast-derived zymosan and bacteria, and we show that both events are causally related, because cells deficient in sPLA2-V exhibited decreased phagocytosis, and cells overexpressing the enzyme manifested higher rates of phagocytosis. Mass spectrometry analyses of lipid changes in the IL-4-treated macrophages suggest that ethanolamine lysophospholipid (LPE) is an sPLA2-V-derived product that may be involved in regulating phagocytosis. Cellular levels of LPE are selectively maintained by sPLA2-V. By supplementing sPLA2-V-deficient cells with LPE, phagocytosis of zymosan or bacteria was fully restored in IL-4-treated cells. Collectively, our results show that sPLA2-V is required for efficient phagocytosis by IL-4-treated human macrophages and provide evidence that sPLA2-V-derived LPE is involved in the process.

  18. MAP1S Protein Regulates the Phagocytosis of Bacteria and Toll-like Receptor (TLR) Signaling.

    PubMed

    Shi, Ming; Zhang, Yifan; Liu, Leyuan; Zhang, Tingting; Han, Fang; Cleveland, Joseph; Wang, Fen; McKeehan, Wallace L; Li, Yu; Zhang, Dekai

    2016-01-15

    Phagocytosis is a critical cellular process for innate immune defense against microbial infection. The regulation of phagocytosis process is complex and has not been well defined. An intracellular molecule might regulate cell surface-initiated phagocytosis, but the underlying molecular mechanism is poorly understood (1). In this study, we found that microtubule-associated protein 1S (MAP1S), a protein identified recently that is involved in autophagy (2), is expressed primarily in macrophages. MAP1S-deficient macrophages are impaired in the phagocytosis of bacteria. Furthermore, we demonstrate that MAP1S interacts directly with MyD88, a key adaptor of Toll-like receptors (TLRs), upon TLR activation and affects the TLR signaling pathway. Intriguingly, we also observe that, upon TLR activation, MyD88 participates in autophagy processing in a MAP1S-dependent manner by co-localizing with MAP1 light chain 3 (MAP1-LC3 or LC3). Therefore, we reveal that an intracellular autophagy-related molecule of MAP1S controls bacterial phagocytosis through TLR signaling.

  19. The Circadian Clock Protein Timeless Regulates Phagocytosis of Bacteria in Drosophila

    PubMed Central

    Ayres, Janelle S.; Pham, Linh N.; Ziauddin, Junaid; Shirasu-Hiza, Mimi M.

    2012-01-01

    Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection. PMID:22253593

  20. A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    PubMed Central

    Mansuri, M. Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-01-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics. PMID:25299184

  1. A novel alpha kinase EhAK1 phosphorylates actin and regulates phagocytosis in Entamoeba histolytica.

    PubMed

    Mansuri, M Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-10-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.

  2. Molecular imaging analysis of Rab GTPases in the regulation of phagocytosis and macropinocytosis.

    PubMed

    Egami, Youhei

    2016-01-01

    Phagocytosis and macropinocytosis, actin-dependent endocytic pathways that mediate the uptake of particles and fluid, respectively, are fundamental routes that enable cells to sample their environment, eliminate pathogens and endogenous cell debris, and contribute to immunoprotection and the maintenance of tissue homeostasis. These processes require a well-organized network of actin cytoskeletal remodeling and membrane transport, which are spatiotemporally regulated by small GTPases. The Rab family of small GTPases, which functions as molecular switches, plays central roles in intracellular membrane trafficking. Although multiple Rab proteins are localized to phagosomes and regulate phagosome maturation, the precise role of each Rab family member in Fcγ receptor (FcγR)-mediated phagocytosis is not fully characterized. Recently, we revealed that Rab35 and Rab20 are important regulators of phagosome formation and maturation, respectively. This review summarizes the functional implication of these Rab GTPases during FcγR-mediated phagocytosis in macrophages. Currently, compared with our knowledge of the regulatory mechanisms of receptor-mediated endocytosis including phagocytosis, the molecular components and signaling cascades of macropinocytosis remain poorly elucidated. Our time-lapse imaging showed that several Rab GTPases are sequentially recruited to the membrane of macropinosomes. Based on our observations, these findings regarding the spatiotemporal localization of Rab GTPases during macropinocytosis are introduced.

  3. Identification of low density lipoprotein as a regulator of Fc receptor-mediated phagocytosis.

    PubMed Central

    Bigler, R D; Khoo, M; Lund-Katz, S; Scerbo, L; Esfahani, M

    1990-01-01

    Optimal expression of the high-affinity Fc receptor for IgG (FcRI) by the human monocyte cell line U-937 requires the presence of low density lipoprotein (LDL), and neither cholesterol nor high density lipoprotein can provide the component necessary for optimal FcRI expression. Here we show that FcR-mediated phagocytosis also requires LDL. U-937 cells were cultured in medium containing interferon gamma and either fetal calf serum (FCS) or delipidated FCS (DLFCS). The phagocytosis of IgG-coated erythrocytes was measured by a colorimetric assay. U-937 cells cultured in DLFCS medium had less than 16% of the phagocytic activity of cells cultured in normal FCS medium. Phagocytosis of IgG-coated erythrocytes could be inhibited 85% by the addition of murine IgG2a myeloma protein (5 micrograms/ml). U-937 cells cultured in DLFCS medium supplemented with pure cholesterol in ethanol (10 micrograms/ml) had only 30% of the phagocytic activity of cells grown in FCS medium. Addition of very low density lipoprotein (0.2 mg of protein per ml) to DLFCS medium also failed to increase phagocytosis. However, the addition of LDL (0.2 mg of protein per ml) to DLFCS medium restored 90% of the phagocytic activity. Since neither pure cholesterol nor very low density lipoprotein restored normal phagocytic function to U-937 cells despite a normalization of cellular cholesterol content, the restoration of phagocytosis observed with LDL replacement cannot be explained by mere delivery of cholesterol by LDL. Thus, LDL is required for the expression of FcRI and FcR-mediated phagocytosis by U-937 cells and may be an important regulator of phagocytic activity of monocytes and macrophages in vivo. PMID:2367519

  4. Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils.

    PubMed

    Wofford, Jessica A; Wright, Jo Rae

    2007-12-01

    Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.

  5. Inhibition of phagocytosis by Haemophilus ducreyi requires expression of the LspA1 and LspA2 proteins.

    PubMed

    Vakevainen, Merja; Greenberg, Steven; Hansen, Eric J

    2003-10-01

    Haemophilus ducreyi previously has been shown to inhibit the phagocytosis of both secondary targets and itself by certain cells in vitro. Wild-type H. ducreyi strain 35000HP contains two genes, lspA1 and lspA2, whose encoded protein products are predicted to be 456 and 543 kDa, respectively. An isogenic mutant of H. ducreyi 35000HP with inactivated lspA1 and lspA2 genes has been shown to exhibit substantially decreased virulence in the temperature-dependent rabbit model for chancroid. This lspA1 lspA2 mutant was tested for its ability to inhibit phagocytosis of immunoglobulin G-opsonized particles by differentiated HL-60 and U-937 cells and by J774A.1 cells. The wild-type strain H. ducreyi 35000HP readily inhibited phagocytosis, whereas the lspA1 lspA2 mutant was unable to inhibit phagocytosis. Similarly, the wild-type strain was resistant to phagocytosis, whereas the lspA1 lspA2 mutant was readily engulfed by phagocytes. This inhibitory effect of wild-type H. ducreyi on phagocytic activity was primarily associated with live bacterial cells but could also be found, under certain conditions, in concentrated H. ducreyi culture supernatant fluids that lacked detectable outer membrane fragments. Both the wild-type strain and the lspA1 lspA2 mutant attached to phagocytes at similar levels. These results indicate that the LspA1 and LspA2 proteins of H. ducreyi are involved, directly or indirectly, in the antiphagocytic activity of this pathogen, and they provide a possible explanation for the greatly reduced virulence of the lspA1 lspA2 mutant.

  6. TRPV4 Mechanosensitive Ion Channel Regulates LPS-Stimulated Macrophage Phagocytosis

    PubMed Central

    Scheraga, Rachel G.; Abraham, Susamma; Niese, Kathryn A.; Southern, Brian D.; Grove, Lisa M.; Hite, R. Duncan; McDonald, Christine; Hamilton, Thomas A.; Olman, Mitchell A.

    2015-01-01

    Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens and the extracellular matrix. In the present report we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the lipopolysaccharide (LPS) and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of non-opsonized particles (E. coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/pro-resolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis. PMID:26597012

  7. Regulation of Rab5 Function during Phagocytosis of Live Pseudomonas aeruginosa in Macrophages

    PubMed Central

    Mustafi, Sushmita; Rivero, Nathalie; Olson, Joan C.; Stahl, Philip D.

    2013-01-01

    Pseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment of P. aeruginosa infection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages of P. aeruginosa invasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated, P. aeruginosa inhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of live P. aeruginosa. These studies provide evidence that live P. aeruginosa cells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5. PMID:23630954

  8. Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis.

    PubMed

    Egami, Youhei; Araki, Nobukazu

    2012-01-01

    Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

  9. miR-15a/16 regulates macrophage phagocytosis after bacterial infection.

    PubMed

    Moon, Hyung-Geun; Yang, Jincheng; Zheng, Yijie; Jin, Yang

    2014-11-01

    Bacterial infection and its associated sepsis are devastating clinical entities that lead to high mortality and morbidity in critically ill patients. Phagocytosis, along with other innate immune responses, exerts crucial impacts on the outcomes of these patients. MicroRNAs (miRNAs) are a novel class of regulatory noncoding RNAs that target specific mRNAs for modulation of translation and expression of a targeted protein. The roles of miRNAs in host defense against bacterial sepsis remain unclear. We found that bacterial infections and/or bacterial-derived LPS enhanced the level of miR-15a/16 in bone marrow-derived macrophages (BMDMs). Deletion of miR-15a/16 (miR-15a/16(-/-)) in myeloid cells significantly decreased the bacterial infection-associated mortality in sepsis mouse models. Moreover, miR-15a/16 deficiency (miR-15a/16(-/-)) resulted in augmented phagocytosis and generation of mitochondrial reactive oxygen species in BMDMs. Supportively, overexpression of miR-15a/16 using miRNA mimics led to decreased phagocytosis and decreased generation of mitochondrial reactive oxygen species. Mechanistically, deletion of miR-15a/16 upregulated the expression of TLR4 via targeting the principle transcriptional regulator PU.1 locating on the promoter region of TLR4, and further modulated the downstream signaling molecules of TLR4, including Rho GTPase Cdc 42 and TRAF6. In addition, deficiency of miR-15a/16 also facilitated TLR4-mediated proinflammatory cytokine/chemokine release from BMDMs at the initial phase of infections. Taken together, miR-15a/16 altered phagocytosis and bacterial clearance by targeting, at least partially, on the TLR4-associated pathways, subsequently affecting the survival of septic mice.

  10. Elk-3 is a KLF4-regulated gene that modulates the phagocytosis of bacteria by macrophages

    PubMed Central

    Tsoyi, Konstantin; Geldart, Adriana M.; Christou, Helen; Liu, Xiaoli; Chung, Su Wol; Perrella, Mark A.

    2015-01-01

    ETS family proteins play a role in immune responses. A unique member of this family, Elk-3, is a transcriptional repressor that regulates the expression of HO-1. Elk-3 is very sensitive to the effects of inflammatory mediators and is down-regulated by bacterial endotoxin (LPS). In the present study, exposure of mouse macrophages to Escherichia coli LPS resulted in decreased, full-length, and splice-variant isoforms of Elk-3. We isolated the Elk-3 promoter and demonstrated that LPS also decreased promoter activity. The Elk-3 promoter contains GC-rich regions that are putative binding sites for zinc-finger transcription factors, such as Sp1 and KLFs. Mutation of the GC-rich region from bp –613 to –603 blunted LPS-induced down-regulation of the Elk-3 promoter. Similar to the LPS response, coexpression of KLF4 led to repression of Elk-3 promoter activity, whereas coexpression of Sp1 increased activity. ChIP assays revealed that KLF4 binding to the Elk-3 promoter was increased by LPS exposure, and Sp1 binding was decreased. Thus, down-regulation of Elk-3 by bacterial LPS is regulated, in part, by the transcriptional repressor KLF4. Overexpression of Elk-3, in the presence of E. coli bacteria, resulted in decreased macrophage phagocytosis. To determine whether limited expression of HO-1 may contribute to this response, we exposed HO-1-deficient bone marrow-derived macrophages to E. coli and found a comparable reduction in bacterial phagocytosis. These data suggest that down-regulation of Elk-3 and the subsequent induction of HO-1 are important for macrophage function during the inflammatory response to infection. PMID:25351511

  11. Elk-3 is a KLF4-regulated gene that modulates the phagocytosis of bacteria by macrophages.

    PubMed

    Tsoyi, Konstantin; Geldart, Adriana M; Christou, Helen; Liu, Xiaoli; Chung, Su Wol; Perrella, Mark A

    2015-01-01

    ETS family proteins play a role in immune responses. A unique member of this family, Elk-3, is a transcriptional repressor that regulates the expression of HO-1. Elk-3 is very sensitive to the effects of inflammatory mediators and is down-regulated by bacterial endotoxin (LPS). In the present study, exposure of mouse macrophages to Escherichia coli LPS resulted in decreased, full-length, and splice-variant isoforms of Elk-3. We isolated the Elk-3 promoter and demonstrated that LPS also decreased promoter activity. The Elk-3 promoter contains GC-rich regions that are putative binding sites for zinc-finger transcription factors, such as Sp1 and KLFs. Mutation of the GC-rich region from bp -613 to -603 blunted LPS-induced down-regulation of the Elk-3 promoter. Similar to the LPS response, coexpression of KLF4 led to repression of Elk-3 promoter activity, whereas coexpression of Sp1 increased activity. ChIP assays revealed that KLF4 binding to the Elk-3 promoter was increased by LPS exposure, and Sp1 binding was decreased. Thus, down-regulation of Elk-3 by bacterial LPS is regulated, in part, by the transcriptional repressor KLF4. Overexpression of Elk-3, in the presence of E. coli bacteria, resulted in decreased macrophage phagocytosis. To determine whether limited expression of HO-1 may contribute to this response, we exposed HO-1-deficient bone marrow-derived macrophages to E. coli and found a comparable reduction in bacterial phagocytosis. These data suggest that down-regulation of Elk-3 and the subsequent induction of HO-1 are important for macrophage function during the inflammatory response to infection.

  12. A NPxY-independent {beta}5 integrin activation signal regulates phagocytosis of apoptotic cells

    SciTech Connect

    Singh, Sukhwinder; D'mello, Veera; Henegouwen, Paul van Bergen en; Birge, Raymond B.

    2007-12-21

    Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the {beta} chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 ({beta}5) integrin cDNA was expressed in {alpha}v positive, {beta}5 and {beta}3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, {beta}5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing {beta}5 mutant (Y750A) abrogated adhesion and {beta}5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of {beta}5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a {beta}5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 {mu}m diameter microspheres developed as apoptotic cell mimetics. The critical sequences in {beta}5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of {beta}5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the {beta}5 cytoplasmic tail for adhesion and phagocytosis.

  13. Minichromosome maintenance protein 7 regulates phagocytosis in kuruma shrimp Marsupenaeus japonicas against white spot syndrome virus.

    PubMed

    Wang, Zhi; Zhu, Fei

    2016-08-01

    Minichromosome maintenance protein (MCM7) belongs to the MCM protein family and participates in the MCM complex by playing a role in the cell replication cycle and chromosome initiation of eukaryotes. Previously, we found that several genes, including MCM7, were over-expressed in Drosophila melanogaster after white spot syndrome virus (WSSV) infection. In this study, we aimed to further research the MCM7 of kuruma shrimp, Marsupenaeus japonicus (mjMCM7) and determine its role in the innate immune system. To this end, we cloned the entire 2307-bp mjMCM7 sequence, including a 1974-bp open reading frame (ORF) encoding a 658-aa-long protein. Real-time PCR showed that the gene was primarily expressed in the hemolymph and hepatopancreas and over-expressed in shrimp challenged with WSSV. Gene function study was carried out by knocking down the expression of MCM7 using small interference RNA (siRNA). The results revealed that β-actin, hemocyanin, prophenoloxidase (proPO) and tumor necrosis factor-α (TNF-α) were up-regulated while the cytoskeleton proteins such as myosin and Rho were significantly down-regulated at 24 h after treatment. The results indicate a possible relationship between mjMCM7 and the innate immune system, and suggest that mjMCM7 may play a role in phagocytosis. After WSSV challenge, WSSV copies and mortality count were both higher in the MCM7-siRNA-treated groups at 60 h after treatment, and the mortality count approached that of the control groups over time. The phagocytosis rate was significantly lower in the MCM7-siRNA-treated group than in the WSSV group. The findings of this study confirm that mjMCM7 positively regulates phagocytosis and plays an important role against WSSV. These results could help researchers to further understand the function of the MCM7 protein and reveal its potential role in the innate immunity of invertebrates.

  14. Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.

    PubMed

    Marey, Mohamed A; Liu, Jinghui; Kowsar, Rasoul; Haneda, Shingo; Matsui, Motozumi; Sasaki, Motoki; Takashi, Shimizu; Hayakawa, Hiroyuki; Wijayagunawardane, Missaka P B; Hussein, Fekry M; Miyamoto, Akio

    2014-02-01

    This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E2 (PGE2), which dose-dependently (10(-6), 10(-7), and 10(-8) M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP2 receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE2 or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE2 released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP2 receptors. Thus, the bovine oviduct provides a PGE2-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1.

  15. Cleavage of Mer tyrosine kinase (MerTK) from the cell surface contributes to the regulation of retinal phagocytosis.

    PubMed

    Law, Ah-Lai; Parinot, Célia; Chatagnon, Jonathan; Gravez, Basile; Sahel, José-Alain; Bhattacharya, Shomi S; Nandrot, Emeline F

    2015-02-20

    Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Myeloperoxidase deficiency enhances zymosan phagocytosis associated with up-regulation of surface expression of CD11b in mouse neutrophils.

    PubMed

    Fujimoto, Kenta; Motowaki, Takehiro; Tamura, Naoya; Aratani, Yasuaki

    2016-12-01

    Myeloperoxidase (MPO), a major component of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion. Phagocytosis is a critical event induced by neutrophils for host defense and inflammation. Interestingly, we found that MPO-deficient (MPO(-/-)) neutrophils engulfed larger amounts of zymosan than wild-type neutrophils. Blocking of the CD11b subunit of complement receptor 3 (CR3) as well as inhibition of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) dramatically reduced zymosan phagocytosis. In contrast, blocking of dectin-1, toll-like receptor 2 (TLR2), or spleen tyrosine kinase (Syk) had no significant effects on phagocytosis. Western blotting analysis showed that inhibition of FAK decreased the phosphorylation of ERK1/2, indicating that ERK1/2 is a downstream regulator of FAK in neutrophils. Importantly, we found that cell surface expression of CD11b and phosphorylation of ERK1/2 was significantly higher in zymosan-stimulated MPO(-/-) neutrophils than in zymosan-stimulated wild-type neutrophils. Pretreatment with the MPO inhibitor 4-aminobenzoic acid hydrazide dramatically enhanced both zymosan phagocytosis and the surface expression of CD11b in wild-type neutrophils, but not in MPO(-/-) neutrophils. Collectively, these results strongly suggest that up-regulation of the CD11b/FAK/ERK signaling pathway due to absence of MPO enhances the zymosan phagocytic activity of mouse neutrophils.

  17. A2B adenosine receptor blockade enhances macrophage-mediated bacterial phagocytosis and improves polymicrobial sepsis survival in mice.

    PubMed

    Belikoff, Bryan G; Hatfield, Stephen; Georgiev, Peter; Ohta, Akio; Lukashev, Dmitriy; Buras, Jon A; Remick, Daniel G; Sitkovsky, Michail

    2011-02-15

    Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.

  18. Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans

    PubMed Central

    Pueyo, José I.; Amin, Unum; Evans, Iwan R.; Bishop, Sarah A.; Couso, Juan P.

    2016-01-01

    Translation of hundreds of small ORFs (smORFs) of less than 100 amino acids has recently been revealed in vertebrates and Drosophila. Some of these peptides have essential and conserved cellular functions. In Drosophila, we have predicted a particular smORF class encoding ~80 aa hydrophobic peptides, which may function in membranes and cell organelles. Here, we characterise hemotin, a gene encoding an 88aa transmembrane smORF peptide localised to early endosomes in Drosophila macrophages. hemotin regulates endosomal maturation during phagocytosis by repressing the cooperation of 14-3-3ζ with specific phosphatidylinositol (PI) enzymes. hemotin mutants accumulate undigested phagocytic material inside enlarged endo-lysosomes and as a result, hemotin mutants have reduced ability to fight bacteria, and hence, have severely reduced life span and resistance to infections. We identify Stannin, a peptide involved in organometallic toxicity, as the Hemotin functional homologue in vertebrates, showing that this novel regulator of phagocytic processing is widely conserved, emphasizing the significance of smORF peptides in cell biology and disease. PMID:27015288

  19. Prostaglandin E2 Receptor Subtype 2 Regulation of Scavenger Receptor CD36 Modulates Microglial Aβ42 Phagocytosis

    PubMed Central

    Li, Xianwu; Melief, Erica; Postupna, Nadia; Montine, Kathleen S.; Keene, C. Dirk; Montine, Thomas J.

    2016-01-01

    Recent studies underline the potential relevance of microglial innate immune activation in Alzheimer disease. Primary mouse microglia that lack prostaglandin E2 receptor subtype 2 (EP2) show decreased innate immune-mediated neurotoxicity and increased amyloid β (Aβ) peptide phagocytosis, features that were replicated in vivo. Here, we tested the hypothesis that scavenger receptor CD36 is an effector of EP2-regulatedphagocytosis. CD36 expression was 143-fold greater in mouse primary microglia than in primary astrocytes. Three different means of suppressing EP2 signaling increased and an agonist of EP2 decreased CD36 expression in primary wild-type microglia. Activation of Toll-like receptor (TLR) 3, TLR4, and TLR7, but not TLR2 or TLR9, reduced primary microglial CD36 transcription and cell surface CD36 protein and reduced Aβ42 phagocytosis as well. At each step, the effects of innate immune activation on CD36 were reversed by at least 50% by an EP2 antagonist, and this partial rescue of microglia Aβ42 phagocytosis was largely mediated by CD36 activity. Finally, we showed in hippocampus of wild-type mice that innate immune activation suppressed CD36 expression by an EP2-dependent mechanism. Taken together with results of others that found brain clearance of Aβ peptides and behavioral improvements mediated by CD36 in mice, regulation of CD36-mediated Aβ phagocytosis by suppression of EP2 signaling may provide a new approach to suppressing some aspects of Alzheimer disease pathogenesis. PMID:25452117

  20. Contribution of Ion Channels in Calcium Signaling Regulating Phagocytosis: MaxiK, Cav1.3 and Bestrophin-1.

    PubMed

    Strauß, Olaf; Reichhart, Nadine; Gomez, Nestor Mas; Müller, Claudia

    2016-01-01

    Mutations in the BEST1 gene lead to a variety of retinal degenerations including Best's vitelliforme macular degeneration. The BEST1 gene product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE). It is likely that mutant bestrophin-1 impairs functions of the RPE which support photoreceptor function and will thus lead to retinal degeneration. However, the RPE function which is influenced by bestrophin-1 is so far not identified. Previously we showed that bestrophin-1 interacts with L-type Ca²⁺ channels of the CaV1.3 subtype and that the endogenously expressed bestrophin-1 is required for intracellular Ca²⁺ regulation. A hallmark of Best's disease is the fast lipofuscin accumulation occurring already at young ages. Therefore, we addressed the hypothesis that bestrophin-1 might influence phagocytosis of photoreceptor outer segments (POS) by the RPE. Here, siRNA knock-down of bestrophin-1 expression as well as inhibition of L-type Ca²⁺ channel activity modulated the POS phagocytosis in vitro. In vivo CaV1.3 expression appeared to be diurnal regulated with a higher expression rate in the afternoon. Compared to wild-type littermates, Ca V 1.3 (-/-) mice showed a shift in the circadian POS phagocytosis with an increased activity in the afternoon. Thus we suggest that mutant bestrophin-1 leads to an impaired regulation of the POS phagocytosis by the RPE which would explain the fast lipofuscin accumulation in Best patients.

  1. Quantitative Phagocytosis.

    ERIC Educational Resources Information Center

    McCallister, Zane Gary; McCallister, Gary Loren

    1996-01-01

    Presents a model experiment for quantifying phagocytosis using earthworm coelomocytes and determining the optimum length of time necessary to obtain maximum phagocytosis. Involves incubating coelomocytes from invertebrates with an antigen, staining the cells, counting the number of antigen particles ingested, and measuring the effect of different…

  2. Quantitative Phagocytosis.

    ERIC Educational Resources Information Center

    McCallister, Zane Gary; McCallister, Gary Loren

    1996-01-01

    Presents a model experiment for quantifying phagocytosis using earthworm coelomocytes and determining the optimum length of time necessary to obtain maximum phagocytosis. Involves incubating coelomocytes from invertebrates with an antigen, staining the cells, counting the number of antigen particles ingested, and measuring the effect of different…

  3. Activated αvβ3 Integrin Regulates αvβ5 Integrin–Mediated Phagocytosis in Trabecular Meshwork Cells

    PubMed Central

    Gagen, Debjani; Filla, Mark S.; Clark, Ross; Liton, Paloma; Peters, Donna M.

    2013-01-01

    Purpose. To investigate the roles of αvβ3 and αvβ5 integrins in phagocytosis in human trabecular meshwork (HTM) cells. Methods. Immunofluorescence microscopy and FACS analysis were used to determine levels of αvβ3 and αvβ5 integrins in TM tissue and cultures of normal and immortalized TM cells. Phagocytosis was measured using pHrodo-labeled S. aureus bioparticles followed by FACS analysis. The role of αvβ5 integrin in phagocytosis was evaluated by knocking down αvβ5 integrin expression with siRNA against the human β5 gene. Signaling from focal adhesion kinase (FAK) was blocked using FAK inhibitor 14. The role of αvβ3 integrins in phagocytosis was determined by treating HTM cells with dexamethasone (DEX) or ethanol (EtOH) and by generating stable cell lines that overexpressed either wild type (WT) or constitutively active (CA) β3 integrin subunit. Results. Both TM tissue and cell lines expressed αvβ3 and αvβ5 integrins. Knockdown of αvβ5 integrin reduced phagocytosis by ∼60% and FAK inhibition significantly reduced phagocytosis up to 84%, in a dose-dependent manner. DEX treatment increased αvβ3 integrin expression in HTM cells but reduced phagocytosis by ∼50% compared with untreated and EtOH-treated cells. The CA β3 integrin–expressing cell line showed increased αvβ3 integrin levels and decreased phagocytosis by ∼50% compared with the control. Conclusions. The αvβ5 integrin-FAK–mediated pathway regulates phagocytosis in TM cells and this pathway is inhibited by activation of αvβ3 integrins. This suggests that changes in integrin expression and activity may be responsible for alterations in phagocytosis observed in steroid induced glaucoma. PMID:23821196

  4. The virulence regulator Sae of Staphylococcus aureus: promoter activities and response to phagocytosis-related signals.

    PubMed

    Geiger, Tobias; Goerke, Christiane; Mainiero, Markus; Kraus, Dirk; Wolz, Christiane

    2008-05-01

    The two-component system SaeRS of Staphylococcus aureus is closely involved in the regulation of major virulence factors. However, little is known about the signals leading to saeRS activation. A total of four overlapping transcripts (T1 to T4) from three different transcription starting points are expressed in the sae operon. We used a beta-galactosidase reporter assay to characterize the putative promoter regions within the saeRS upstream region. The main transcript T2 is probably generated by endoribonucleolytic processing of the T1 transcript. Only two distinct promoter elements (P1 and P3) could be detected within the saeRS upstream region. The P3 promoter, upstream of saeRS, generates the T3 transcript, includes a cis-acting enhancer element and is repressed by saeRS. The most distal P1 promoter is strongly autoregulated, activated by agr, and repressed by sigma factor B. In strain Newman a mutation within the histidine kinase SaeS leads to a constitutively activated sae system. Evaluation of different external signals revealed that the P1 promoter in strain ISP479R and strain UAMS-1 is inhibited by low pH and high NaCl concentrations but activated by hydrogen peroxide. The most prominent induction of P1 was observed at subinhibitory concentrations of alpha-defensins in various S. aureus strains, with the exception of strain ISP479R and strain COL. P1 was not activated by the antimicrobial peptides LL37 and daptomycin. In summary, the results indicate that the sensor molecule SaeS is activated by alteration within the membrane allowing the pathogen to react to phagocytosis related effector molecules.

  5. Lipoprotein lipase regulates Fc receptor-mediated phagocytosis by macrophages maintained in glucose-deficient medium.

    PubMed Central

    Yin, B; Loike, J D; Kako, Y; Weinstock, P H; Breslow, J L; Silverstein, S C; Goldberg, I J

    1997-01-01

    During periods of intense activity such as phagocytosis, macrophages are thought to derive most of their energy from glucose metabolism under both aerobic and anaerobic conditions. To determine whether fatty acids released from lipoproteins by macrophage lipoprotein lipase (LPL) could substitute for glucose as a source of energy for phagocytosis, we cultured peritoneal macrophages from normal and LPL knockout (LPL-KO) mice that had been rescued from neonatal demise by expression of human LPL via the muscle creatine kinase promoter. Normal and LPL-KO macrophages were cultured in medium containing normal (5 mM) or low (1 mM) glucose, and were tested for their capacity to phagocytose IgG-opsonized sheep erythrocytes. LPL-KO macrophages maintained in 1 and 5 mM glucose phagocytosed 67 and 79% fewer IgG-opsonized erythrocytes, respectively, than macrophages from normal mice. Addition of VLDL to LPL-expressing macrophages maintained in 1 mM glucose enhanced the macrophages' phagocytosis of IgG-opsonized erythrocytes, but did not stimulate phagocytosis by LPL-KO macrophages. Inhibition of secreted LPL with a monoclonal anti-LPL antibody or with tetrahydrolipstatin blocked the ability of VLDL to enhance phagocytosis by LPL-expressing macrophages maintained in 1 mM glucose. Addition of oleic acid significantly enhanced phagocytosis by both LPL-expressing and LPL-KO macrophages maintained in 1 mM glucose. Moreover, oleic acid stimulated phagocytosis in cells cultured in non-glucose-containing medium, and increased the intracellular stores of creatine phosphate. Inhibition of oxidative phosphorylation, but not of glycolysis, blocked the capacity of oleic acid to stimulate phagocytosis. Receptor-mediated endocytosis of acetyl LDL by macrophages from LPL-expressing and LPL-KO mice was similar whether the cells were maintained in 5 or 1 mM glucose, and was not augmented by VLDL. We postulate that fatty acids derived from macrophage LPL-catalyzed hydrolysis of triglycerides and

  6. The inositol phosphatase SHIP-2 down-regulates FcγR-mediated phagocytosis in murine macrophages independently of SHIP-1

    PubMed Central

    Ai, Jing; Maturu, Amita; Johnson, Wesley; Wang, Yijie; Marsh, Clay B.; Tridandapani, Susheela

    2006-01-01

    FcγR-mediated phagocytosis of IgG-coated particles is a complex process involving the activation of multiple signaling enzymes and is regulated by the inositol phosphatases PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP-1 (Src homology [SH2] domain-containing inositol phosphatase). In a recent study we have demonstrated that SHIP-2, an inositol phosphatase with high-level homology to SHIP-1, is involved in FcγR signaling. However, it is not known whether SHIP-2 plays a role in modulating phagocytosis. In this study we have analyzed the role of SHIP-2 in FcγR-mediated phagocytosis using independent cell models that allow for manipulation of SHIP-2 function without influencing the highly homologous SHIP-1. We present evidence that SHIP-2 translocates to the site of phagocytosis and down-regulates FcγR-mediated phagocytosis. Our data indicate that SHIP-2 must contain both the N-terminal SH2 domain and the C-terminal proline-rich domain to mediate its inhibitory effect. The effect of SHIP-2 is independent of SHIP-1, as overexpression of dominant-negative SHIP-2 in SHIP-1-deficient primary macrophages resulted in enhanced phagocytic efficiency. Likewise, specific knockdown of SHIP-2 expression using siRNA resulted in enhanced phagocytosis. Finally, analysis of the molecular mechanism of SHIP-2 down-regulation of phagocytosis revealed that SHIP-2 down-regulates upstream activation of Rac. Thus, we conclude that SHIP-2 is a novel negative regulator of FcγR-mediated phagocytosis independent of SHIP-1. (Blood. 2006;107:813-820) PMID:16179375

  7. miR-24, miR-30b, and miR-142-3p regulate phagocytosis in myeloid inflammatory cells.

    PubMed

    Naqvi, Afsar Raza; Fordham, Jezrom B; Nares, Salvador

    2015-02-15

    Micro-RNAs (miRNAs) are small noncoding RNAs that regulate various biological pathways. As their role in phagocytosis remains poorly understood, we investigated their impact on phagocytosis in myeloid inflammatory cells. Seven miRNAs (miR-24, -30b, -101, 142-3p, -652-3p, -652-5p, and -1275) that were differentially expressed during monocyte to macrophage (Mφ) and monocyte to dendritic cell (DC) differentiation were screened for their potential role in phagocytosis. Among these, overexpression of miR-24, miR-30b, and miR-142-3p in human monocyte-derived Mφ, DC, monocytes, and PBMCs significantly attenuate phagocytosis of Escherichia coli and Staphylococcus aureus, as well as the secretion of inflammatory mediators, including TNF-α, IL-6, and IL-12p40. miRNA-mediated changes in cytokine profiles were observed at transcriptional and/or posttranscriptional levels and importantly exhibit miRNA-specific impact. To examine the underlying mechanism, we monitored the expression of phagocytosis pathway-associated genes and identified several genes that were altered in Mφ and DC transfected with miR-24, miR-30b, and miR-142-3p mimics. Some of these genes with altered expression also harbor putative miRNA binding sites. We show that miR-142-3p directly regulates protein kinase Cα (PKCα), a key gene involved in phagocytosis. Interestingly, miR-142-3p and PKCα exhibit antagonistic expression during Mφ and DC differentiation. Short interfering RNA-mediated knockdown of PKCα in Mφ leads to reduced bacterial uptake, further highlighting the role of the gene in phagocytosis. Overall, these results demonstrate that miR-24, miR-30b, and miR-142-3p regulate phagocytosis and associated cytokine production in myeloid inflammatory cells through modulation of various genes involved in the pathway.

  8. Generation of membrane structures during phagocytosis and chemotaxis of macrophages: role and regulation of the actin cytoskeleton

    PubMed Central

    Rougerie, Pablo; Miskolci, Veronika; Cox, Dianne

    2013-01-01

    Summary Macrophages are best known for their protective search and destroy functions against invading micro-organisms. These processes are commonly known as chemotaxis and phagocytosis. Both of these processes require actin cytoskeletal remodeling to produce distinct F-actin rich membrane structures called lamellipodia and phagocytic cups. This review will focus on the mechanisms by which macrophages regulate actin polymerization through initial receptor signaling and subsequent Arp2/3 activation by nucleation promoting factors like the WASP/WAVE family, followed by remodeling of actin networks to produce these very distinct structures. PMID:24117824

  9. Regulation of Microglial Phagocytosis by RhoA/ROCK-Inhibiting Drugs.

    PubMed

    Scheiblich, Hannah; Bicker, Gerd

    2017-04-01

    Inflammation within the central nervous system (CNS) is a major component of many neurodegenerative diseases. The underlying mechanisms of neuronal loss are not fully understood, but the activation of CNS resident phagocytic microglia seems to be a significant element contributing to neurodegeneration. At the onset of inflammation, high levels of microglial phagocytosis may serve as an essential prerequisite for creating a favorable environment for neuronal regeneration. However, the excessive and long-lasting activation of microglia and the augmented engulfment of neurons have been suggested to eventually govern widespread neurodegeneration. Here, we investigated in a functional assay of acute inflammation how the small GTPase RhoA and its main target the Rho kinase (ROCK) influence microglial phagocytosis of neuronal debris. Using BV-2 microglia and human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA activation and microglial phagocytosis of neuronal cell fragments. Inhibition of the downstream effector ROCK with the small-molecule agents Y-27632 and Fasudil reduces the engulfment of neuronal debris and attenuates the production of the inflammatory mediator nitric oxide during stimulation with lipopolysaccharide. Our results support a therapeutic potential for RhoA/ROCK-inhibiting agents as an effective treatment of excessive inflammation and the resulting progression of microglia-mediated neurodegeneration in the CNS.

  10. Emerging roles for the BAI1 protein family in the regulation of phagocytosis, synaptogenesis, neurovasculature, and tumor development

    PubMed Central

    Cork, Sarah M.

    2011-01-01

    While G-protein-coupled receptors (GPCRs) have received considerable attention for their biological activity in a diversity of physiological functions and have become targets for therapeutic intervention in many diseases, the function of the cell adhesion subfamily of GPCRs remains poorly understood. Within this group, the family of brain angiogenesis inhibitor molecules (BAI1-3) has become increasingly appreciated for their diverse roles in biology and disease. In particular, recent findings suggest emerging roles for BAI1 in the regulation of phenomena including phagocytosis, synaptogenesis, and the inhibition of tumor growth and angiogenesis via the processing of its extracellular domain into secreted vasculostatins. Here we summarize the known biological features of the BAI proteins, including their structure, proteolysis events, and interacting partners, and their recently identified ability to regulate certain signaling pathways. Finally, we discuss the potential of the BAIs as therapeutics or targets for diseases as varied as cancer, stroke, and schizophrenia. PMID:21509575

  11. Syk activation is a leukotriene B4-regulated event involved in macrophage phagocytosis of IgG-coated targets but not apoptotic cells.

    PubMed

    Canetti, Claudio; Hu, Bin; Curtis, Jeffrey L; Peters-Golden, Marc

    2003-09-01

    Macrophages are called upon to ingest both IgG-coated targets and apoptotic cells. Important roles for tyrosine kinase Syk and leukotriene B4 (LTB4) are recognized in FcgammaR-mediated phagocytosis. Here we evaluated the roles of Syk and LTB4 in macrophage phagocytosis of apoptotic thymocytes versus IgG-coated erythrocytes. Macrophage ingestion of apoptotic thymocytes was not influenced by exogenous or endogenous LTB4 nor associated with Syk activation (phosphorylation). By contrast, LTB4 dose-dependently amplified FcgammaR-mediated phagocytosis as well as Syk activation. Furthermore, a role for endogenous LTB4 in Syk activation during FcgammaR-mediated phagocytosis was demonstrated using pharmacologic and genetic abrogation of 5-lipoxygenase. LTB4 was unique among 5-lipoxygenase products in this regard, since LTD4 and 5-hydroxyeicosatetraenoic acid (HETE) were unable to amplify Syk activation in response to FcgammaR engagement. Ca2+ chelation studies revealed that FcgammaR-mediated Syk activation as well as LTB4 amplification thereof was Ca2+ regulated. These 2 parallel phagocytic processes therefore exhibit initial divergence in signal transduction events, with Syk activation being an LTB4-regulated event in FcgammaR-mediated but not apoptotic cell ingestion. As LTB4 is an important proinflammatory product of macrophages, we speculate that this divergence evolved to permit FcgammaR-mediated phagocytosis to proceed in an inflammatory milieu, while apoptotic cell clearance is noninflammatory.

  12. Downregulation of an Entamoeba histolytica rhomboid protease reveals roles in regulating parasite adhesion and phagocytosis.

    PubMed

    Baxt, Leigh A; Rastew, Elena; Bracha, Rivka; Mirelman, David; Singh, Upinder

    2010-08-01

    Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

  13. Specialized Pro-Resolving Mediators from Omega-3 Fatty Acids Improve Amyloid-β Phagocytosis and Regulate Inflammation in Patients with Minor Cognitive Impairment.

    PubMed

    Fiala, Milan; Terrando, Niccolo; Dalli, Jesmond

    2015-01-01

    In this review we discuss the immunopathology of Alzheimer's disease (AD) and recent advances in the prevention of minor cognitive impairment (MCI) by nutritional supplementation with omega-3 fatty acids. Defective phagocytosis of amyloid-β (Aβ) and abnormal inflammatory activation of peripheral blood mononuclear cells (PBMCs) are the two key immune pathologies of MCI and AD patients. The phagocytosis of Aβ by PBMCs of MCI and AD patients is universally defective and the inflammatory gene transcription is heterogeneously deregulated in comparison to normal subjects. Recent studies have discovered a cornucopia of beneficial anti-inflammatory and pro-resolving effects of the specialized proresolving mediators (SPMs) resolvins, protectins, maresins, and their metabolic precursors. Resolvin D1 and other mediators switch macrophages from an inflammatory to a tissue protective/pro-resolving phenotype and increase phagocytosis of Aβ. In a recent study of AD and MCI patients, nutritional supplementation by omega-3 fatty acids individually increased resolvin D1, improved Aβ phagocytosis, and regulated inflammatory genes toward a physiological state, but only in MCI patients. Our studies are beginning to dissect positive factors (adherence to Mediterranean diet with omega-3 and exercise) and negative factors (high fat diet, infections, cancer, and surgeries) in each patient. The in vitro and in vivo effects of omega-3 fatty acids and SPMs suggest that defective phagocytosis and chronic inflammation are related to defective production and/or defective signaling by SPMs in immune cells.

  14. Monoacylglycerol lipase promotes Fcγ receptor-mediated phagocytosis in microglia but does not regulate LPS-induced upregulation of inflammatory cytokines.

    PubMed

    Kouchi, Zen

    2015-08-21

    Monoacylglycerol lipase (MAGL) is important for neuroinflammation. However, the regulatory mechanisms underlying its expression and function remain unknown. Lipopolysaccharide (LPS) treatment post-translationally upregulated MAGL expression, whereas it downregulated MAGL transcription through a Stat6-mediated mechanism in microglia. Neither MAGL knockdown nor JZL-184, a selective MAGL inhibitor, suppressed LPS-induced upregulation of inflammatory cytokines in microglia. Moreover, exogenous expression of MAGL in BV-2 microglial cell line, which lacks endogenous MAGL, did not promote the induction of inflammatory cytokines by LPS treatment. Interestingly, MAGL knockdown reduced Fcγ receptor-mediated phagocytosis in primary microglia, and introduction of MAGL into the BV-2 cells increased Fcγ receptor-mediated phagocytosis. Collectively, these results suggest that MAGL regulates phagocytosis, but not LPS-mediated cytokine induction in microglia.

  15. An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

    PubMed Central

    LIU, Jinghui; MAREY, Mohamed A.; KOWSAR, Rasoul; HAMBRUCH, Nina; SHIMIZU, Takashi; HANEDA, Shingo; MATSUI, Motozumi; SASAKI, Motoki; HAYAKAWA, Hiroyuki; PFARRER, Christiane; MIYAMOTO, Akio

    2014-01-01

    We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct. PMID:24931131

  16. Small-conductance Cl- channels contribute to volume regulation and phagocytosis in microglia.

    PubMed

    Ducharme, Guillaume; Newell, Evan W; Pinto, Crystal; Schlichter, Lyanne C

    2007-10-01

    The shape and volume of microglia (brain immune cells) change when they activate during brain inflammation and become migratory and phagocytic. Swollen rat microglia express a large Cl(-) current (I(Clswell)), whose biophysical properties and functional roles are poorly understood and whose molecular identity is unknown. We constructed a fingerprint of useful biophysical properties for comparison with I(Clswell) in other cell types and with cloned Cl(-) channels. The microglial I(Clswell) was rapidly activated by cell swelling but not by voltage, and showed no time-dependence during voltage-clamp steps. Like I(Clswell) in many cell types, the halide selectivity sequence was I(-) > Br(-) > Cl(-) > F(-). However, it differed in lacking inactivation, even at +100 mV with high extracellular Mg(2+), and in having a much lower single-channel conductance: 1-3 pS. Based on these fundamental differences, the microglia channel is apparently a different gene product than the more common intermediate-conductance I(Clswell). Microglia express several candidate genes, with relative mRNA expression levels of: CLIC1 > ClC3 > I(Cln) > or = ClC2 > Best2 > Best1 > or = Best3 > Best4. Using a pharmacological toolbox, we show that all drugs that reduced the microglia current (NPPB, IAA-94, flufenamic acid and DIOA) increased the resting cell volume in isotonic solution and inhibited the regulatory volume decrease that followed cell swelling in hypotonic solution. Both channel blockers tested (NPPB and flufenamic acid) dose-dependently inhibited microglia phagocytosis of E. coli bacteria. Because I(Clswell) is involved in microglia functions that involve shape and volume changes, it is potentially important for controlling their ability to migrate to damage sites and phagocytose dead cells and debris.

  17. Stimulation of phagocytosis by sulforaphane

    SciTech Connect

    Suganuma, Hiroyuki; Fahey, Jed W.; Bryan, Kelley E.; Healy, Zachary R.; Talalay, Paul

    2011-02-04

    Research highlights: {yields} Sulforaphane stimulates the phagocytosis of RAW 264.7 macrophages under conditions of serum deprivation. {yields} This effect does not require Nrf2-dependent induction of phase 2 genes. {yields} Inactivation of macrophage migration inhibitory factor (MIF) by sulforaphane may be involved in stimulation of phagocytosis by sulforaphane. -- Abstract: Sulforaphane, a major isothiocyanate derived from cruciferous vegetables, protects living systems against electrophile toxicity, oxidative stress, inflammation, and radiation. A major protective mechanism is the induction of a network of endogenous cytoprotective (phase 2) genes that are regulated by transcription factor Nrf2. To obtain a more detailed understanding of the anti-inflammatory and immunomodulatory effects of sulforaphane, we evaluated its effect on the phagocytosis activity of RAW 264.7 murine macrophage-like cells by measuring the uptake of 2-{mu}m diameter polystyrene beads. Sulforaphane raised the phagocytosis activity of RAW 264.7 cells but only in the absence or presence of low concentrations (1%) of fetal bovine serum. Higher serum concentrations depressed phagocytosis and abolished its stimulation by sulforaphane. This stimulation did not depend on the induction of Nrf2-regulated genes since it occurred in peritoneal macrophages of nrf2{sup -/-} mice. Moreover, a potent triterpenoid inducer of Nrf2-dependent genes did not stimulate phagocytosis, whereas sulforaphane and another isothiocyanate (benzyl isothiocyanate) had comparable inducer potencies. It has been shown recently that sulforaphane is a potent and direct inactivator of macrophage migration inhibitory factor (MIF), an inflammatory cytokine. Moreover, the addition of recombinant MIF to RAW 264.7 cells attenuated phagocytosis, but sulforaphane-inactivated MIF did not affect phagocytosis. The inactivation of MIF may therefore be involved in the phagocytosis-enhancing activity of sulforaphane.

  18. Reticulocalbin-1 facilitates microglial phagocytosis.

    PubMed

    Ding, Ying; Caberoy, Nora B; Guo, Feiye; LeBlanc, Michelle E; Zhang, Chenming; Wang, Weiwen; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis is critical to the clearance of apoptotic cells, cellular debris and deleterious metabolic products for tissue homeostasis. Phagocytosis ligands directly recognizing deleterious cargos are the key to defining the functional roles of phagocytes, but are traditionally identified on a case-by-case basis with technical challenges. As a result, extrinsic regulation of phagocytosis is poorly defined. Here we demonstrate that microglial phagocytosis ligands can be systematically identified by a new approach of functional screening. One of the identified ligands is reticulocalbin-1 (Rcn1), which was originally reported as a Ca2+-binding protein with a strict expression in the endoplasmic reticulum. Our results showed that Rcn1 can be secreted from healthy cells and that secreted Rcn1 selectively bound to the surface of apoptotic neurons, but not healthy neurons. Independent characterization revealed that Rcn1 stimulated microglial phagocytosis of apoptotic but not healthy neurons. Ingested apoptotic cells were targeted to phagosomes and co-localized with phagosome marker Rab7. These data suggest that Rcn1 is a genuine phagocytosis ligand. The new approach described in this study will enable systematic identification of microglial phagocytosis ligands with broad applicability to many other phagocytes.

  19. Reticulocalbin-1 Facilitates Microglial Phagocytosis

    PubMed Central

    Ding, Ying; Caberoy, Nora B.; Guo, Feiye; LeBlanc, Michelle E.; Zhang, Chenming; Wang, Weiwen; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis is critical to the clearance of apoptotic cells, cellular debris and deleterious metabolic products for tissue homeostasis. Phagocytosis ligands directly recognizing deleterious cargos are the key to defining the functional roles of phagocytes, but are traditionally identified on a case-by-case basis with technical challenges. As a result, extrinsic regulation of phagocytosis is poorly defined. Here we demonstrate that microglial phagocytosis ligands can be systematically identified by a new approach of functional screening. One of the identified ligands is reticulocalbin-1 (Rcn1), which was originally reported as a Ca2+-binding protein with a strict expression in the endoplasmic reticulum. Our results showed that Rcn1 can be secreted from healthy cells and that secreted Rcn1 selectively bound to the surface of apoptotic neurons, but not healthy neurons. Independent characterization revealed that Rcn1 stimulated microglial phagocytosis of apoptotic but not healthy neurons. Ingested apoptotic cells were targeted to phagosomes and co-localized with phagosome marker Rab7. These data suggest that Rcn1 is a genuine phagocytosis ligand. The new approach described in this study will enable systematic identification of microglial phagocytosis ligands with broad applicability to many other phagocytes. PMID:25992960

  20. Hydrogen peroxide is produced by E. coli challenged haemocytes and regulates phagocytosis, in the medfly Ceratitis capitata. The active role of superoxide dismutase.

    PubMed

    Arbi, Marina; Pouliliou, Stamatia; Lampropoulou, Maria; Marmaras, Vassilis J; Tsakas, Sotiris

    2011-08-01

    Hydrogen peroxide (H(2)O(2)) participates as a second messenger in cell signaling. In this paper, the role of H(2)O(2) was investigated, in Escherichia coli phagocytosis by the haemocytes of the medfly Ceratitis capitata. Block of H(2)O(2) synthesis by specific enzymic inhibitors, namely N-ethylmaleimide (NEM) for NADPH oxidase and diethyldithiocarbamate (DDC) for SOD, resulted in the increase of E. coli phagocytosis. Immunoblot analysis, flow cytometry and confocal microscopy, revealed the constitutive expression of SOD, in the medfly haemocytes. Phagocytosis increased by small interfering RNA (siRNA) for SOD, revealing the active involvement of SOD and H(2)O(2). Immunoblot analysis showed an increase of the ERK1/2 phosphorylation, in the presence of the above H(2)O(2) synthesis enzymic inhibitors. In addition, confocal microscopy showed no co-localization of SOD with β integrin subunit. It appears that SOD participates in the regulation of bacterial phagocytosis, due to involvement of the produced H(2)O(2) in the differential phosphorylation of MAP kinases.

  1. Short-Term Regulation of FcγR-Mediated Phagocytosis by TLRs in Macrophages: Participation of 5-Lipoxygenase Products.

    PubMed

    Pinheiro, Carla da S; Monteiro, Ana Paula T; Dutra, Fabiano F; Bozza, Marcelo T; Peters-Golden, Marc; Benjamim, Claudia F; Canetti, Claudio

    2017-01-01

    TLRs recognize a broad spectrum of microorganism molecules, triggering a variety of cellular responses. Among them, phagocytosis is a critical process for host defense. Leukotrienes (LTs), lipid mediators produced from 5-lipoxygenase (5-LO) enzyme, increase FcγR-mediated phagocytosis. Here, we evaluated the participation of TLR2, TLR3, TLR4, and TLR9 in FcγR-mediated phagocytosis and whether this process is modulated by LTs. Rat alveolar macrophages (AMs), murine bone marrow-derived macrophages (BMDMs), and peritoneal macrophages (PMs) treated with TLR2, TLR3, and TLR4 agonists, but not TLR9, enhanced IgG-opsonized sheep red blood cell (IgG-sRBC) phagocytosis. Pretreatment of AMs or BMDMs with drugs that block LT synthesis impaired the phagocytosis promoted by TLR ligands, and TLR potentiation was also abrogated in PMs and BMDMs from 5-LO(-/-) mice. LTB4 production induced by IgG engagement was amplified by TLR ligands, while cys-LTs were amplified by activation of TLR2 and TLR4, but not by TLR3. We also noted higher ERK1/2 phosphorylation in IgG-RBC-challenged cells when preincubated with TLR agonists. Furthermore, ERK1/2 inhibition by PD98059 reduced the phagocytic activity evoked by TLR agonists. Together, these data indicate that TLR2, TLR3, and TLR4 ligands, but not TLR9, amplify IgG-mediated phagocytosis by a mechanism which requires LT production and ERK-1/2 pathway activation.

  2. The Src homology 2 domain-containing inositol 5-phosphatase negatively regulates Fcgamma receptor-mediated phagocytosis through immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors.

    PubMed

    Nakamura, Koji; Malykhin, Alexander; Coggeshall, K Mark

    2002-11-01

    Molecular mechanisms by which the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) negatively regulates phagocytosis in macrophages are unclear. We addressed the issue using bone marrow-derived macrophages from FcgammaR- or SHIP-deficient mice. Phagocytic activities of macrophages from FcgammaRII(b)(-/-) and SHIP(-/-) mice were enhanced to a similar extent, relative to those from wild type. However, calcium influx was only marginally affected in FcgammaRII(b)(-/-), but greatly enhanced in SHIP(-/-) macrophages. Furthermore, SHIP was phosphorylated on tyrosine residues upon FcgammaR aggregation even in macrophages from FcgammaRII(b)(-/-) mice or upon clustering of a chimeric receptor containing CD8 and the immunoreceptor tyrosine-based activation motif (ITAM)-bearing gamma-chain or human-restricted FcgammaRIIa. These findings indicate that, unlike B cells, SHIP is efficiently phosphorylated in the absence of an immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptor. We further demonstrate that SHIP directly bound to phosphorylated peptides derived from FcgammaRIIa with a high affinity, comparable to that of FcgammaRII(b). Lastly, FcgammaRIIa-mediated phagocytosis was significantly enhanced in THP-1 cells overexpressing dominant-negative form of SHIP in the absence of FcgammaRII(b). These results indicate that SHIP negatively regulates FcgammaR-mediated phagocytosis through all ITAM-containing IgG receptors using a molecular mechanism distinct from that in B cells.

  3. Genetic control of macrophage functions. I. Polygenic regulation of phagocytosis stimulation produced by Glyceryl Trioleate.

    PubMed

    Mouton, D; Bouthillier, Y; Feingold, N; Feingold, J; Decreusefond, C; Stiffel, C; Biozzi, G

    1975-02-01

    The phagocytic index K, established from the rate of blood clearance of colloidal carbon, measures the phagocytic activity of RE macrophages in contact with the circulating blood. The intravenous injection of glyceryl trioleate (triolein) produces a marked stimulation of the phagocytic activity of RE macrophages. This response is higher in the female than in the male mice. The phenotypic character "responsiveness of macrophage to triolein" presents large individual variants in population of random bred albinos mice. This character is submitted to polygenic regulation. Starting from a foundation population of 25 males and 25 females random bred albinos, mice, two lines were separated by selective breeding for the character "responsiveness to triolein": a "high" responder line, KTH, and a "low" responder line, KTL. After 26 consecutive generations of selective breeding, KTH mice present a very high response to triolein while KTL mice are almost irresponsive. The heritability of this character (h2) calculated from the interline divergence is of 12% plus or minus 1. This value of h2 indicates that the character investigated is determined by the cumulative effect of a group of about 27 independently segregating loci. The distribution of the character in (KTH plus KTL)F1 and their backcrosses with parental lines suggests that low responsiveness is dominant over high responsiveness. The genetic regulation of responsiveness to triolein is independent from the dose administered. These results are discussed in relation to the importance of genetic factors controlling macrophage functions involved in lipid metabolism and in the specific mechanisms of immunity.

  4. Genetic control of macrophage functions. I. Polygenic regulation of phagocytosis stimulation produced by Glyceryl Trioleate

    PubMed Central

    1975-01-01

    The phagocytic index K, established from the rate of blood clearance of colloidal carbon, measures the phagocytic activity of RE macrophages in contact with the circulating blood. The intravenous injection of glyceryl trioleate (triolein) produces a marked stimulation of the phagocytic activity of RE macrophages. This response is higher in the female than in the male mice. The phenotypic character "responsiveness of macrophage to triolein" presents large individual variants in population of random bred albinos mice. This character is submitted to polygenic regulation. Starting from a foundation population of 25 males and 25 females random bred albinos, mice, two lines were separated by selective breeding for the character "responsiveness to triolein": a "high" responder line, KTH, and a "low" responder line, KTL. After 26 consecutive generations of selective breeding, KTH mice present a very high response to triolein while KTL mice are almost irresponsive. The heritability of this character (h2) calculated from the interline divergence is of 12% plus or minus 1. This value of h2 indicates that the character investigated is determined by the cumulative effect of a group of about 27 independently segregating loci. The distribution of the character in (KTH plus KTL)F1 and their backcrosses with parental lines suggests that low responsiveness is dominant over high responsiveness. The genetic regulation of responsiveness to triolein is independent from the dose administered. These results are discussed in relation to the importance of genetic factors controlling macrophage functions involved in lipid metabolism and in the specific mechanisms of immunity. PMID:1113063

  5. CaV1.3 L-type channels, maxiK Ca(2+)-dependent K(+) channels and bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells.

    PubMed

    Müller, Claudia; Más Gómez, Néstor; Ruth, Peter; Strauss, Olaf

    2014-05-01

    Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca(2+) regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Cav1.3 L-type Ca(2+) channels (Ca1.3(-/-)) and maxiK Ca(2+)-dependent K(+) channels (BK(-/-)). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca(2+) homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (-)BayK8644 had no impact. The expression rate of Cav1.3, the predominant L-type Ca(2+) channel in RPE cells, varied at different times of day. CaV1.3(-/-) RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK(-/-) RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK(-/-) mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca(2+) channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Short-Term Regulation of FcγR-Mediated Phagocytosis by TLRs in Macrophages: Participation of 5-Lipoxygenase Products

    PubMed Central

    Pinheiro, Carla da S.; Monteiro, Ana Paula T.; Dutra, Fabiano F.; Bozza, Marcelo T.; Peters-Golden, Marc; Benjamim, Claudia F.

    2017-01-01

    TLRs recognize a broad spectrum of microorganism molecules, triggering a variety of cellular responses. Among them, phagocytosis is a critical process for host defense. Leukotrienes (LTs), lipid mediators produced from 5-lipoxygenase (5-LO) enzyme, increase FcγR-mediated phagocytosis. Here, we evaluated the participation of TLR2, TLR3, TLR4, and TLR9 in FcγR-mediated phagocytosis and whether this process is modulated by LTs. Rat alveolar macrophages (AMs), murine bone marrow-derived macrophages (BMDMs), and peritoneal macrophages (PMs) treated with TLR2, TLR3, and TLR4 agonists, but not TLR9, enhanced IgG-opsonized sheep red blood cell (IgG-sRBC) phagocytosis. Pretreatment of AMs or BMDMs with drugs that block LT synthesis impaired the phagocytosis promoted by TLR ligands, and TLR potentiation was also abrogated in PMs and BMDMs from 5-LO−/− mice. LTB4 production induced by IgG engagement was amplified by TLR ligands, while cys-LTs were amplified by activation of TLR2 and TLR4, but not by TLR3. We also noted higher ERK1/2 phosphorylation in IgG-RBC-challenged cells when preincubated with TLR agonists. Furthermore, ERK1/2 inhibition by PD98059 reduced the phagocytic activity evoked by TLR agonists. Together, these data indicate that TLR2, TLR3, and TLR4 ligands, but not TLR9, amplify IgG-mediated phagocytosis by a mechanism which requires LT production and ERK-1/2 pathway activation. PMID:28894350

  7. A novel phospholipase D2-Grb2-WASp heterotrimer regulates leukocyte phagocytosis in a two-step mechanism.

    PubMed

    Kantonen, Samuel; Hatton, Nathaniel; Mahankali, Madhu; Henkels, Karen M; Park, Haein; Cox, Dianne; Gomez-Cambronero, Julian

    2011-11-01

    Phagocytosis is a primary innate response of both macrophages and neutrophils involving the formation of filamentous actin (F-actin)-rich protrusions that are extended around opsonized pathogens to form a phagocytic cup, resulting in their subsequent internalization. The molecular mechanism for this is still not completely understood. We now show for the first time that phospholipase D2 (PLD2) binds to growth factor receptor-bound protein 2 (Grb2) and to the Wiskott-Aldrich syndrome protein (WASp) to form a heterotrimer complex, PLD2-Grb2-WASp, and present the mechanism of interaction. Grb2 binds to the Y169/Y179 residues of PLD2 using its only SH2 domain, and it interacts with the poly-proline region of WASp using its two SH3 domains. The PLD2-Grb2-WASp heterotrimer can be visualized in early phagocytic cups of macrophages ingesting opsonized red blood cells, where it associates with polymerized actin. Cup colocalization and phagocytosis are disrupted with mutants that alter binding at either of the two proteins or by silencing Grb2 with RNA interference (RNAi). WASp association to PLD2-K758R, a lipase-inactive mutant, still occurs, albeit at lower levels, indicating that PLD2 plays a second role in phagocytosis, which is the production of phosphatidic acid (PA) and activation of phosphatidylinositol 5-kinase (PI5K) with subsequent synthesis of phosphatidylinositol 4,5-bisphosphate (PIP(2)). The latter can be blocked with RNAi, which negates phagocytosis. Lastly, a constitutively "open" active form of WASp (WASp-L270P) brings phagocytosis to its maximum level, which can be mimicked with WASp-WT plus PLD2 or plus PA. Since neither a protein-protein disruption nor lack of PLD activity completely negates cup formation or phagocytosis, we posit a two-step mechanism: PLD2 anchors WASp at the phagocytic cup through Grb2 following protein-protein interactions and also activates it, making key lipids available locally. The heterotrimer PLD2-Grb2-WASp then enables actin

  8. Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

    PubMed Central

    Park, Mijeong; Raftery, Mark J.; Thomas, Paul S.; Geczy, Carolyn L.; Bryant, Katherine; Tedla, Nicodemus

    2016-01-01

    FcγRI cross-linking on monocytes may trigger clathrin-mediated endocytosis, likely through interaction of multiple intracellular molecules that are controlled by phosphorylation and dephosphorylation events. However, the identity of phospho-proteins and their regulation are unknown. We proposed the leukocyte immunoglobulin-like receptor B4 (LILRB4) that inhibits FcγRI-mediated cytokine production via Tyr dephosphorylation of multiple kinases, may also regulate endocytosis/phagocytosis through similar mechanisms. FcγRI and/or LILRB4 were antibody-ligated on THP-1 cells, lysates immunoprecipitated using anti-pTyr antibody and peptides sequenced by mass spectrometry. Mascot Search identified 25 Tyr phosphorylated peptides with high confidence. Ingenuity Pathway Analysis revealed that the most significantly affected pathways were clathrin-mediated endocytosis and Fc-receptor dependent phagocytosis. Tyr phosphorylation of key candidate proteins in these pathways included common γ-chain of the Fc receptors, Syk, clathrin, E3 ubiquitin protein ligase Cbl, hepatocyte growth factor-regulated tyrosine kinase substrate, tripartite motif-containing 21 and heat shock protein 70. Importantly, co-ligation of LILRB4 with FcγRI caused significant dephosphorylation of these proteins and was associated with suppression of Fc receptor-dependent uptake of antibody-opsonised bacterial particles, indicating that LILRB4. These results suggest that Tyr phosphorylation may be critical in FcγRI-dependent endocytosis/phagocytosis that may be regulated by LILRB4 by triggering dephosphorylation of key signalling proteins. PMID:27725776

  9. Antibiotics regulate the immune response in both presence and absence of lipopolysaccharide through modulation of Toll-like receptors, cytokine production and phagocytosis in vitro.

    PubMed

    Bode, Christian; Diedrich, Britta; Muenster, Stefan; Hentschel, Viktoria; Weisheit, Christina; Rommelsheim, Kuno; Hoeft, Andreas; Meyer, Rainer; Boehm, Olaf; Knuefermann, Pascal; Baumgarten, Georg

    2014-01-01

    The inflammatory response to pathogen-associated molecular patterns such as lipopolysaccharide (LPS) in sepsis is mediated via Toll-like receptors (TLRs). Since TLRs also trigger various immune functions, including phagocytosis, their modulation is a promising strategy in the treatment of sepsis. As antibiotics have immunomodulatory properties, this study examined the effect of commonly used classes of antibiotics on i) the expression of TLRs and cytokines and ii) the phagocytic activity under sepsis-like conditions in vitro. This was achieved by incubating THP-1 monocytes and peripheral blood mononuclear cells (PBMCs) obtained from patients after open-heart surgery with the addition of LPS and six key antibiotics (piperacillin, doxycycline, erythromycin, moxifloxacin or gentamicin). After 24h, mRNA levels of both cytokines (IL-1β, IL-6) and TLRs (1, 2, 4, and 6) were monitored and phagocytosis was determined following coincubation with Escherichia coli. Each antibiotic differentially regulated the gene expression of the investigated TLRs and cytokines in monocytes. Erythromycin, moxifloxacin and doxycyclin displayed the strongest effects and changed mRNA-levels of the investigated genes up to 5.6-fold. Consistent with this, antibiotics and, in particular, moxifloxacin, regulated the TLR-and cytokine expression in activated PBMCs obtained from patients after open-heart surgery. Furthermore, piperacillin, doxycyclin and moxifloxacin inhibited the phagocytic activity of monocytes. Our results suggest that antibiotics regulate the immune response by modulating TLR- and cytokine expression as well as phagocytosis under septic conditions. Moxifloxacin, doxycycline and erythromycin were shown to possess the strongest immunomodulatory effects and these antibiotic classes should be considered for future immunomodulatory studies in sepsis.

  10. Differential regulation by leukotrienes and calcium of Fc gamma receptor-induced phagocytosis and Syk activation in dendritic cells versus macrophages.

    PubMed

    Canetti, Claudio; Aronoff, David M; Choe, Mun; Flamand, Nicolas; Wettlaufer, Scott; Toews, Galen B; Chen, Gwo-Hsiao; Peters-Golden, Marc

    2006-06-01

    Macrophage (MØ) phagocytosis via the Fc receptor for immunoglobulin G (Fc gammaR) requires the spleen tyrosine kinase (Syk) and serves an important antimicrobial function. We have reported previously that Fc gammaR-mediated ingestion and Syk activation in MØ are amplified by and depend on the proinflammatory lipid mediator leukotriene B4 (LTB4). Although Fc gammaR-mediated ingestion is also important for antigen uptake, there is no information about LTB4 regulation of these processes in dendritic cells (DCs). In this study, we compared murine bone marrow (BM)-derived DCs to MØ from BM, peritoneum, and the pulmonary alveolar space. Neither phagocytosis nor Syk activation in DCs was influenced by exogenous LTB4. Unlike the various MØ populations, Syk activation in DCs was likewise unaffected by pharmacologic or genetic strategies to inhibit endogenous LTB4 synthesis or to block the high-affinity LTB4 receptor BLT1. DCs were refractory to regulation by LTB4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB4 in DCs instead reflected the fact that in contrast to MØ, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in Fc gammaR signaling between DCs and MØ, which may relate to the divergent, functional consequences of target ingestion in the two cell types.

  11. Inositol Polyphosphate-4-Phosphatase Type I Negatively Regulates Phagocytosis via Dephosphorylation of Phagosomal PtdIns(3,4)P2.

    PubMed

    Nigorikawa, Kiyomi; Hazeki, Kaoru; Sasaki, Junko; Omori, Yumio; Miyake, Mikiko; Morioka, Shin; Guo, Ying; Sasaki, Takehiko; Hazeki, Osamu

    2015-01-01

    Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P.

  12. Inositol Polyphosphate-4-Phosphatase Type I Negatively Regulates Phagocytosis via Dephosphorylation of Phagosomal PtdIns(3,4)P2

    PubMed Central

    Nigorikawa, Kiyomi; Hazeki, Kaoru; Sasaki, Junko; Omori, Yumio; Miyake, Mikiko; Morioka, Shin; Guo, Ying; Sasaki, Takehiko; Hazeki, Osamu

    2015-01-01

    Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P. PMID:26535897

  13. Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis ▿ †

    PubMed Central

    Baxt, Leigh A.; Rastew, Elena; Bracha, Rivka; Mirelman, David; Singh, Upinder

    2010-01-01

    Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis. PMID:20581296

  14. Selenoprotein K regulation of palmitoylation and calpain cleavage of ASAP2 is required for efficient FcγR-mediated phagocytosis.

    PubMed

    Norton, Robert L; Fredericks, Gregory J; Huang, Zhi; Fay, Jeffrey D; Hoffmann, FuKun W; Hoffmann, Peter R

    2017-02-01

    Effective activation of macrophages through phagocytic Fcγ receptors (FcγR) has been shown to require selenoprotein K (Selk). We set out to determine whether the FcγR-mediated uptake process itself also requires Selk and potential underlying mechanisms. Macrophages from Selk knockout (KO) mice were less efficient compared with wild-type (WT) controls in engulfing IgG-coated fluorescent beads. Using LC-MS/MS to screen for Selk-binding partners involved in FcγR-mediated phagocytosis, we identified Arf-GAP with SH3 domain, ANK repeat, and PH domain-containing protein 2 (ASAP2). Coimmunoprecipitation assays confirmed interactions between Selk and ASAP2. Selk was required for ASAP2 to be cleaved by calpain-2 within the Bin/Amphiphysin/Rvs (BAR) domain of ASAP2. BAR domains promote membrane association, which was consistent with our data showing that Selk deficiency led to retention of ASAP2 within the phagocytic cup. Because Selk was recently identified as a cofactor for the palmitoylation of certain proteins, we investigated whether ASAP2 was palmitoylated and whether this was related to its cleavage by calpain-2. Acyl/biotin exchange assays and MALDI-TOF analysis showed that cysteine-86 in ASAP2 was palmitoylated in WT, but to a much lesser extent in KO, mouse macrophages. Inhibitors of either palmitoylation or calpain-2 cleavage and rescue experiments with different versions of Selk demonstrated that Selk-dependent palmitoylation of ASAP2 leads to cleavage by calpain-2 within the BAR domain, which releases this protein from the maturing phagocytic cup. Overall, these findings identify ASAP2 as a new target of Selk-dependent palmitoylation and reveal a new mechanism regulating the efficiency of FcγR-mediated phagocytosis. © Society for Leukocyte Biology.

  15. Effect of a 2.45-GHz radiofrequency electromagnetic field on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells

    PubMed Central

    Koyama, Shin; Narita, Eijiro; Suzuki, Yoshihisa; Taki, Masao; Shinohara, Naoki; Miyakoshi, Junji

    2015-01-01

    The potential public health risks of radiofrequency (RF) fields have been discussed at length, especially with the use of mobile phones spreading extensively throughout the world. In order to investigate the properties of RF fields, we examined the effect of 2.45-GHz RF fields at the specific absorption rate (SAR) of 2 and 10 W/kg for 4 and 24 h on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells. Neutrophil chemotaxis was not affected by RF-field exposure, and subsequent phagocytosis was not affected either compared with that under sham exposure conditions. These studies demonstrated an initial immune response in the human body exposed to 2.45-GHz RF fields at the SAR of 2 W/kg, which is the maximum value recommended by the International Commission for Non-Ionizing Radiation Protection (ICNIRP) guidelines. The results of our experiments for RF-field exposure at an SAR under 10 W/kg showed very little or no effects on either chemotaxis or phagocytosis in neutrophil-like human HL-60 cells. PMID:25194051

  16. Effect of a 2.45-GHz radiofrequency electromagnetic field on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells.

    PubMed

    Koyama, Shin; Narita, Eijiro; Suzuki, Yoshihisa; Taki, Masao; Shinohara, Naoki; Miyakoshi, Junji

    2015-01-01

    The potential public health risks of radiofrequency (RF) fields have been discussed at length, especially with the use of mobile phones spreading extensively throughout the world. In order to investigate the properties of RF fields, we examined the effect of 2.45-GHz RF fields at the specific absorption rate (SAR) of 2 and 10 W/kg for 4 and 24 h on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells. Neutrophil chemotaxis was not affected by RF-field exposure, and subsequent phagocytosis was not affected either compared with that under sham exposure conditions. These studies demonstrated an initial immune response in the human body exposed to 2.45-GHz RF fields at the SAR of 2 W/kg, which is the maximum value recommended by the International Commission for Non-Ionizing Radiation Protection (ICNIRP) guidelines. The results of our experiments for RF-field exposure at an SAR under 10 W/kg showed very little or no effects on either chemotaxis or phagocytosis in neutrophil-like human HL-60 cells.

  17. Information processing during phagocytosis

    PubMed Central

    Underhill, David M.; Goodridge, Helen S.

    2017-01-01

    Phagocytosis, the process by which macrophages, dendritic cells and other myeloid phagocytes internalize diverse particulate targets, is a key mechanism of innate immunity. The molecular and cellular events underlying the binding and engulfment of targets in phagosomes have been extensively studied. More recent data suggest that the process of phagocytosis itself provides information to myeloid phagocytes about the nature of the targets they are eating and that this helps tailor inflammatory responses. In this Review, we discuss how such information is acquired during phagocytosis, and how it is processed to coordinate an immune response. PMID:22699831

  18. Plasminogen promotes macrophage phagocytosis in mice.

    PubMed

    Das, Riku; Ganapathy, Swetha; Settle, Megan; Plow, Edward F

    2014-07-31

    The phagocytic function of macrophages plays a pivotal role in eliminating apoptotic cells and invading pathogens. Evidence implicating plasminogen (Plg), the zymogen of plasmin, in phagocytosis is extremely limited with the most recent in vitro study showing that plasmin acts on prey cells rather than on macrophages. Here, we use apoptotic thymocytes and immunoglobulin opsonized bodies to show that Plg exerts a profound effect on macrophage-mediated phagocytosis in vitro and in vivo. Plg enhanced the uptake of these prey by J774A.1 macrophage-like cells by 3.5- to fivefold Plg receptors and plasmin proteolytic activity were required for phagocytosis of both preys. Compared with Plg(+/+) mice, Plg(-/-) mice exhibited a 60% delay in clearance of apoptotic thymocytes by spleen and an 85% reduction in uptake by peritoneal macrophages. Phagocytosis of antibody-mediated erythrocyte clearance by liver Kupffer cells was reduced by 90% in Plg(-/-) mice compared with Plg(+/+) mice. A gene array of splenic and hepatic tissues from Plg(-/-) and Plg(+/+) mice showed downregulation of numerous genes in Plg(-/-) mice involved in phagocytosis and regulation of phagocytic gene expression was confirmed in macrophage-like cells. Thus, Plg may play an important role in innate immunity by changing expression of genes that contribute to phagocytosis.

  19. Glycogen synthase kinase-3 (GSK3) regulates TNF production and haemocyte phagocytosis in the immune response of Chinese mitten crab Eriocheir sinensis.

    PubMed

    Li, Xiaowei; Jia, Zhihao; Wang, Weilin; Wang, Lingling; Liu, Zhaoqun; Yang, Bin; Jia, Yunke; Song, Xiaorui; Yi, Qilin; Qiu, Limei; Song, Linsheng

    2017-03-29

    Glycogen synthase kinase-3 (GSK3) is a serine/threonine protein kinase firstly identified as a regulator of glycogen synthesis. Recently, it has been proved to be a key regulator of the immune reaction. In the present study, a GSK3 homolog gene (designated as EsGSK3) was cloned from Chinese mitten crab, Eriocheir sinensis. The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3-β from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas. And EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge (p < 0.05) in comparison with control group, and then gradually decreased to the initial level at 48 h (p > 0.05). The mRNA expression of lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (EsLITAF) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and TNF-α production was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF-α production and inhibiting haemocyte phagocytosis.

  20. OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL.

    PubMed

    Howard, Jeffrey C; Florentinus-Mefailoski, Angelique; Bowden, Peter; Trimble, William; Grinstein, Sergio; Marshall, John G

    2016-11-15

    The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to heart attack and stroke. Microbeads coated with oxLDL were used to activate, capture and isolate the oxLDL receptor complex from the surface of live cells. Analysis of the resulting tryptic peptides by liquid chromatography and tandem mass spectrometry revealed the Spleen Tyrosine Kinase (SYK), and many of SYK's known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), receptor kinases like EGFRs, as well as RNA binding and metabolism proteins. High-intensity precursor ions (∼9*E3 to 2*E5 counts) were correlated to peptides and specific phosphopeptides from long isoform of SYK (SYK-L) by the SEQUEST, OMSSA and X!TANDEM algorithms. Peptides or phosphopeptides from SYK were observed with the oxLDL-microbeads. Pharmacological inhibitors of SYK activity significantly reduced the engulfment of oxLDL microbeads in the presence of serum factors, but had little effect on IgG phagocytosis. Anti SYK siRNA regulated oxLD engulfment in the context of serum factors and or SYK-L siRNA significantly inhibited engulfment of oxLDL microbeads, but not IgG microbeads. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Phagocytosis: An Immunobiologic Process.

    PubMed

    Gordon, Siamon

    2016-03-15

    It has been a century since the death of Élie Metchnikoff, who championed the role of phagocytosis in cellular immunity. Whereas others had observed the uptake of particles by cells from simple to complex organisms, he grasped its significance in the host response to injury and infection and established a firm basis for our understanding of inflammation and tissue homeostasis. The past century has brought improved tools of cellular and molecular biology to the study of phagocytosis and its contribution to physiological and pathological processes, including receptor function in innate and acquired immunity. In this review, I assess our present knowledge and consider opportunities for future research and therapeutic targeting.

  2. Autophagy meets phagocytosis.

    PubMed

    Cadwell, Ken; Philips, Jennifer A

    2013-09-19

    Autophagy can degrade intracellular bacteria, but how this pathway contributes to phagocytosis is unclear. In this issue of Immunity, Bonilla et al. (2013) demonstrate an additional role for autophagy in Mycobacterium tuberculosis internalization by macrophages. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Endo180 and MT1-MMP are involved in the phagocytosis of collagen scaffolds by macrophages and is regulated by interferon-gamma.

    PubMed

    Ye, Q; Xing, Q; Ren, Y; Harmsen, M C; Bank, R A

    2010-10-07

    Subcutaneously implanted disks of hexamethylenediisocyanate or glutaraldehyde cross-linked sheep collagen (referred to as HDSC and GDSC, respectively) in mice show large differences in degradation rate. Although comparable numbers of macrophages are seen in HDSC and GDSC, phagocytosis of collagen by macrophages occurred only in GDSC. The molecular mechanisms involved in the phagocytosis of collagen by macrophages are essentially unknown. Immunofluorescence and RT-PCR showed that Endo180 was expressed in GDSC only. TissueFaxs showed that Endo180 co-localized with MT1-MMP on F4÷80 positive cells, which is likely responsible for the phagocytosis in GDSC. RT-PCR further showed that Endo180 expression correlated with high levels of IFN-γ mRNA. In vitro, IFN-γ induced the expression Endo180 and MT1-MMP in murine macrophages cultured on collagen type I (although too high levels of IFN-γ dampened the expression of Endo180 and MT1-MMP). Moreover, the expression of Endo180 and MT1-MMP induced by IFN-γ can be inhibited through IL-10. The differences in microenvironment between GDSC and HDSC (high IFN-γ and low IL-10 levels in GDSC, low IFN-γ and high IL-10 levels in HDSC) provide an explanation why phagocytosis of collagen by macrophages is only seen in GDSC. In summary, we show for the first time that the IFN-γ dependent co-expression of Endo180 and MT1-MMP on macrophages coincides with collagen phagocytosis, thus providing evidence that the mechanism of collagen phagocytosis operating in the foreign body reaction by macrophages is comparable with the mechanism of intracellular collagen degradation by fibroblasts seen under physiological conditions.

  4. The origins of phagocytosis and eukaryogenesis.

    PubMed

    Yutin, Natalya; Wolf, Maxim Y; Wolf, Yuri I; Koonin, Eugene V

    2009-02-26

    Phagocytosis, that is, engulfment of large particles by eukaryotic cells, is found in diverse organisms and is often thought to be central to the very origin of the eukaryotic cell, in particular, for the acquisition of bacterial endosymbionts including the ancestor of the mitochondrion. Comparisons of the sets of proteins implicated in phagocytosis in different eukaryotes reveal extreme diversity, with very few highly conserved components that typically do not possess readily identifiable prokaryotic homologs. Nevertheless, phylogenetic analysis of those proteins for which such homologs do exist yields clues to the possible origin of phagocytosis. The central finding is that a subset of archaea encode actins that are not only monophyletic with eukaryotic actins but also share unique structural features with actin-related proteins (Arp) 2 and 3. All phagocytic processes are strictly dependent on remodeling of the actin cytoskeleton and the formation of branched filaments for which Arp2/3 are responsible. The presence of common structural features in Arp2/3 and the archaeal actins suggests that the common ancestors of the archaeal and eukaryotic actins were capable of forming branched filaments, like modern Arp2/3. The Rho family GTPases that are ubiquitous regulators of phagocytosis in eukaryotes appear to be of bacterial origin, so assuming that the host of the mitochondrial endosymbiont was an archaeon, the genes for these GTPases come via horizontal gene transfer from the endosymbiont or in an earlier event. The present findings suggest a hypothetical scenario of eukaryogenesis under which the archaeal ancestor of eukaryotes had no cell wall (like modern Thermoplasma) but had an actin-based cytoskeleton including branched actin filaments that allowed this organism to produce actin-supported membrane protrusions. These protrusions would facilitate accidental, occasional engulfment of bacteria, one of which eventually became the mitochondrion. The acquisition of

  5. Vitellogenin mediates phagocytosis through interaction with FcγR.

    PubMed

    Liu, Min; Pan, Junli; Ji, Hongfang; Zhao, Bosheng; Zhang, Shicui

    2011-10-01

    Vitellogenin (Vg), once reported to be a female-specific protein, has been identified in both male and juvenile fishes. However, the biological significance of the production of Vg in the male and juvenile fishes is elusive. Our previous studies showed that Vg is an opsonin capable of enhancing phagocytosis, but the mechanism by which Vg mediates phagocytosis is unknown. In this study we demonstrated that Vg-opsonized phagocytosis was characterized by pseudopod extension and depended upon tyrosine kinase. In contrast, inhibition of Rho family proteins and microtubule depolymerization had little effects on Vg-opsonized phagocytosis. Besides, Vg-opsonized phagocytosis was substantially blocked by monoclonal antibodies against FcγRs but not by CR3 antibody. Moreover, theoretical prediction analysis further revealed that Vg had the potency to interact with Fcγ receptors. Finally, the expression of proinflammatory cytokine genes tnf-α and il-1β was significantly up-regulated by Vg, and this up-regulation was inhibited by selective inhibitors of FcR signaling pathways, wortmannin and piceatannol. Taken together, these results suggest that Vg plays an IgG-like role in that it activates FcγR-mediated phagocytosis, thus establishing an antibody-like function for Vg for the first time. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

    PubMed Central

    Distel, Jesús S.; Aguilera, Milton O.; Colombo, María I.; Berón, Walter

    2015-01-01

    The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that process. PMID:26674774

  7. Ginseng (Panax ginseng Meyer) oligopeptides regulate innate and adaptive immune responses in mice via increased macrophage phagocytosis capacity, NK cell activity and Th cells secretion.

    PubMed

    He, Li-Xia; Ren, Jin-Wei; Liu, Rui; Chen, Qi-He; Zhao, Jian; Wu, Xin; Zhang, Zhao-Feng; Wang, Jun-Bo; Pettinato, Giuseppe; Li, Yong

    2017-09-06

    Traditionally used as a restorative medicine, ginseng (Panax ginseng Meyer) has been the most widely used and acclaimed herb in Chinese communities for thousands of years. To investigate the immune-modulating activity of ginseng oligopeptides (GOP), 420 healthy female BALB/c mice were intragastrically administered distilled water (control), whey protein (0.15 g per kg body weight (BW)), and GOP 0.0375, 0.075, 0.15, 0.3 and 0.6 g per kg BW for 30 days. Blood samples from mice were collected from the ophthalmic venous plexus and then sacrificed by cervical dislocation. Seven assays were conducted to determine the immunomodulatory effects of GOP on innate and adaptive immune responses, followed by flow cytometry to investigate spleen T lymphocyte sub-populations, multiplex sandwich immunoassays to investigate serum cytokine and immunoglobulin levels, and ELISA to investigate intestinally secreted immunoglobulin to study the mechanism of GOP affecting the immune system. Our results showed that GOP was able to enhance innate and adaptive immune responses in mice by improving cell-mediated and humoral immunity, macrophage phagocytosis capacity and NK cell activity. Notably, the use of GOP revealed a better immune-modulating activity compared to whey protein. We conclude that the immune-modulating activity might be due to the increased macrophage phagocytosis capacity and NK cell activity, and the enhancement of T and Th cells, as well as IL-2, IL-6 and IL-12 secretion and IgA, IgG1 and IgG2b production. These results indicate that GOP could be considered a good candidate that may improve immune functions if used as a dietary supplement, with a dosage that ranges from 0.3 to 0.6 g per kg BW.

  8. Mechanisms regulating the positioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on cellular membranes: retargeting to plasma membrane induced by phagocytosis.

    PubMed

    Martens, Sascha; Sabel, Katja; Lange, Rita; Uthaiah, Revathy; Wolf, Eva; Howard, Jonathan C

    2004-08-15

    The recently identified p47 GTPases are one of the most effective cell-autonomous resistance systems known against intracellular pathogens in the mouse. One member of the family, LRG-47, has been shown to be essential for immune control in vivo of Listeria monocytogenes, Toxoplasma gondii, Mycobacterium tuberculosis, and Mycobacterium avium, possibly by promoting acidification of the phagosome. However, the intracellular localization of LRG-47, and the nature of its association with the phagosomal or any other membrane system is unknown. In this study, we show that LRG-47 is a Golgi-associated protein in the IFN-stimulated cell, which is rapidly recruited to active plasma membrane upon phagocytosis and remains associated with phagosomes as they mature. We show that the Golgi localization of LRG-47 is dependent on the integrity of an amphipathic helix near the C terminus, whereas the plasma membrane localization depends on an unidentified signal associated with the G domain. Unlike LRG-47, but like the published p47 resistance GTPase, IGTP, a further p47 GTPase, IIGP1, is associated with the endoplasmic reticulum. However, unlike IGTP, IIGP1 is associated with the endoplasmic reticulum by an N-terminal myristoylation modification. Thus, the p47 GTPases are a diverse battery of intracellular defense factors dynamically associated with different membrane systems.

  9. PHAGOCYTOSIS INHIBITION AND REVERSAL I.

    PubMed Central

    Sbarra, Anthony J.; Shirley, William

    1963-01-01

    Sbarra, Anthony J. (St. Margaret's Hospital, Boston, Mass.) and William Shirley. Phagocytosis inhibition and reversal. I. Effect of glycolytic intermediates and nucleotides on particle uptake. J. Bacteriol. 86:259–265. 1963.—By microscopically monitoring phagocytosis and following the biochemical changes associated with this process, the inhibition of phagocytosis by fluoride or iodoacetate was shown to be partially reversed by pyruvate. This reversal occurred with both inhibitors, either aerobically or anaerobically. Nicotinamide adenine dinucleotide (NAD) apparently increased the degree of pyruvate reversal when fluoride, but not iodoacetate, was the inhibitory agent. Lactate under some conditions was also shown to reverse the inhibition. It is suggested that pyruvate and NAD are key compounds for the phagocytic process. PMID:14058950

  10. Temperature regulation in rats exposed to a 2 G field

    NASA Technical Reports Server (NTRS)

    Ishihama, Linda M.; Murakami, Dean M.; Fuller, Charles A.

    1989-01-01

    The regulation of body temperature involves both homeostatic and circadian control systems. Both systems are influenced by exposure to hyperdynamic fields and demonstrate acute responses that eventually recover to an adapted level. This experiment examined both the homeostatic and circadian responses of body temperature to a separate environmental challenge (high frequency light/dark cycles) during exposure to a 2 G hyperdynamic field.

  11. Temperature regulation in rats exposed to a 2 G field

    NASA Technical Reports Server (NTRS)

    Ishihama, Linda M.; Murakami, Dean M.; Fuller, Charles A.

    1989-01-01

    The regulation of body temperature involves both homeostatic and circadian control systems. Both systems are influenced by exposure to hyperdynamic fields and demonstrate acute responses that eventually recover to an adapted level. This experiment examined both the homeostatic and circadian responses of body temperature to a separate environmental challenge (high frequency light/dark cycles) during exposure to a 2 G hyperdynamic field.

  12. Annexin A2 Regulates TRPA1-Dependent Nociception

    PubMed Central

    Avenali, Luca; Narayanan, Pratibha; Rouwette, Tom; Cervellini, Ilaria; Sereda, Michael

    2014-01-01

    The transient receptor potential A1 (TRPA1) channel is essential for vertebrate pain. Even though TRPA1 activation by ligands has been studied extensively, the molecular machinery regulating TRPA1 is only poorly understood. Using an unbiased proteomics-based approach we uncovered the physical association of Annexin A2 (AnxA2) with native TRPA1 in mouse sensory neurons. AnxA2 is enriched in a subpopulation of sensory neurons and coexpressed with TRPA1. Furthermore, we observe an increase of TRPA1 membrane levels in cultured sensory neurons from AnxA2-deficient mice. This is reflected by our calcium imaging experiments revealing higher responsiveness upon TRPA1 activation in AnxA2-deficient neurons. In vivo these findings are associated with enhanced nocifensive behaviors specifically in TRPA1-dependent paradigms of acute and inflammatory pain, while heat and mechanical sensitivity as well as TRPV1-mediated pain are preserved in AnxA2-deficient mice. Our results support a model whereby AnxA2 limits the availability of TRPA1 channels to regulate nociceptive signaling in vertebrates. PMID:25355205

  13. Annexin A2 regulates TRPA1-dependent nociception.

    PubMed

    Avenali, Luca; Narayanan, Pratibha; Rouwette, Tom; Cervellini, Ilaria; Sereda, Michael; Gomez-Varela, David; Schmidt, Manuela

    2014-10-29

    The transient receptor potential A1 (TRPA1) channel is essential for vertebrate pain. Even though TRPA1 activation by ligands has been studied extensively, the molecular machinery regulating TRPA1 is only poorly understood. Using an unbiased proteomics-based approach we uncovered the physical association of Annexin A2 (AnxA2) with native TRPA1 in mouse sensory neurons. AnxA2 is enriched in a subpopulation of sensory neurons and coexpressed with TRPA1. Furthermore, we observe an increase of TRPA1 membrane levels in cultured sensory neurons from AnxA2-deficient mice. This is reflected by our calcium imaging experiments revealing higher responsiveness upon TRPA1 activation in AnxA2-deficient neurons. In vivo these findings are associated with enhanced nocifensive behaviors specifically in TRPA1-dependent paradigms of acute and inflammatory pain, while heat and mechanical sensitivity as well as TRPV1-mediated pain are preserved in AnxA2-deficient mice. Our results support a model whereby AnxA2 limits the availability of TRPA1 channels to regulate nociceptive signaling in vertebrates.

  14. The Mechanism of Phagocytosis: Two Stages of Engulfment

    NASA Astrophysics Data System (ADS)

    Richards, David M.; Endres, Robert G.

    2014-10-01

    Despite being of vital importance to the immune system, the mechanism by which cells engulf relatively large solid particles during phagocytosis is still poorly understood. From movies of neutrophil phagocytosis of polystyrene beads, we measure the fractional engulfment as a function of time and demonstrate that phagocytosis occurs in two distinct stages. During the first stage, engulfment is relatively slow and progressively slows down as phagocytosis proceeds. However, at approximately half-engulfment, the rate of engulfment increases dramatically, with complete engulfment attained soon afterwards. By studying simple mathematical models of phagocytosis, we suggest that the first stage is due to a passive mechanism, determined by receptor diffusion and capture, whereas the second stage is more actively controlled, perhaps with receptors being driven towards the site of engulfment. We then consider a more advanced model that includes signaling and captures both stages of engulfment. This model predicts that there is an optimum ligand density for quick engulfment. Further, we show how this model explains why non-spherical particles engulf quickest when presented tip-first. Our findings suggest that active regulation may be a later evolutionary innovation, allowing fast and robust engulfment even for large particles.

  15. Effects of Microparticle Size and Fc Density on Macrophage Phagocytosis

    PubMed Central

    Pacheco, Patricia; White, David; Sulchek, Todd

    2013-01-01

    Controlled induction of phagocytosis in macrophages offers the ability to therapeutically regulate the immune system as well as improve delivery of chemicals or biologicals for immune processing. Maximizing particle uptake by macrophages through Fc receptor-mediated phagocytosis could lead to new delivery mechanisms in drug or vaccine development. Fc ligand density and particle size were examined independently and in combination in order to optimize and tune the phagocytosis of opsonized microparticles. We show the internalization efficiency of small polystyrene particles (0.5 µm to 2 µm) is significantly affected by changes in Fc ligand density, while particles greater than 2 µm show little correlation between internalization and Fc density. We found that while macrophages can efficiently phagocytose a large number of smaller particles, the total volume of phagocytosed particles is maximized through the non-specific uptake of larger microparticles. Therefore, larger microparticles may be more efficient at delivering a greater therapeutic payload to macrophages, but smaller opsonized microparticles can deliver bio-active substances to a greater percentage of the macrophage population. This study is the first to treat as independent variables the physical and biological properties of Fc density and microparticle size that initiate macrophage phagocytosis. Defining the physical and biological parameters that affect phagocytosis efficiency will lead to improved methods of microparticle delivery to macrophages. PMID:23630577

  16. Regulation of expression of the Haemophilus ducreyi LspB and LspA2 proteins by CpxR.

    PubMed

    Labandeira-Rey, Maria; Mock, Jason R; Hansen, Eric J

    2009-08-01

    The LspA1, LspA2, and LspB proteins of Haemophilus ducreyi comprise a two-partner secretion system that has been shown to be necessary for H. ducreyi to inhibit phagocytosis by immune cells in vitro. Inactivation of lspA1 resulted in increased levels of LspA2, suggesting that these two proteins are differentially controlled (C. J. Ward et al., Infect. Immun. 71:2478-2486, 2003). Expression of LspA2 but not LspA1 was shown to be both growth phase dependent and affected by the presence of fetal calf serum (FCS) in the growth medium. In addition, neither LspA1 nor LspA2 could be detected in culture supernatant fluid in the absence of FCS. DNA microarray analysis revealed that 324 H. ducreyi genes were differentially regulated after growth in the presence of FCS. Among these, the CpxRA two-component sensory transduction system was downregulated by the presence of FCS. Inactivation of cpxR resulted in increased expression of both LspB and LspA2. Electrophoretic mobility shift assays showed that a recombinant H. ducreyi CpxR protein bound the promoter region of the lspB-lspA2 operon. The cpxR and cpxA genes were shown to be part of an operon containing two additional genes in H. ducreyi 35000HP. This is the first description of a two-component sensory transduction system regulating a proven virulence factor of H. ducreyi.

  17. Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture.

    PubMed

    Mazzoni, Francesca; Safa, Hussein; Finnemann, Silvia C

    2014-09-01

    RPE cells are the most actively phagocytic cells in the human body. In the eye, RPE cells face rod and cone photoreceptor outer segments at all times but contribute to shedding and clearance phagocytosis of distal outer segment tips only once a day. Analysis of RPE phagocytosis in situ has succeeded in identifying key players of the RPE phagocytic mechanism. Phagocytic processes comprise three distinct phases, recognition/binding, internalization, and digestion, each of which is regulated separately by phagocytes. Studies of phagocytosis by RPE cells in culture allow specifically analyzing and manipulating these distinct phases to identify their molecular mechanisms. Here, we compare similarities and differences of primary, immortalized, and stem cell-derived RPE cells in culture to RPE cells in situ with respect to phagocytic function. We discuss in particular potential pitfalls of RPE cell culture phagocytosis assays. Finally, we point out considerations for phagocytosis assay development for future studies.

  18. Role of target geometry in phagocytosis

    PubMed Central

    Champion, Julie A.; Mitragotri, Samir

    2006-01-01

    Phagocytosis is a principal component of the body’s innate immunity in which macrophages internalize targets in an actin-dependent manner. Targets vary widely in shape and size and include particles such as pathogens and senescent cells. Despite considerable progress in understanding this complicated process, the role of target geometry in phagocytosis has remained elusive. Previous studies on phagocytosis have been performed using spherical targets, thereby overlooking the role of particle shape. Using polystyrene particles of various sizes and shapes, we studied phagocytosis by alveolar macrophages. We report a surprising finding that particle shape, not size, plays a dominant role in phagocytosis. All shapes were capable of initiating phagocytosis in at least one orientation. However, the local particle shape, measured by tangent angles, at the point of initial contact dictates whether macrophages initiate phagocytosis or simply spread on particles. The local shape determines the complexity of the actin structure that must be created to initiate phagocytosis and allow the membrane to move over the particle. Failure to create the required actin structure results in simple spreading and not internalization. Particle size primarily impacts the completion of phagocytosis in cases where particle volume exceeds the cell volume. PMID:16549762

  19. Differential regulation of acid sphingomyelinase in macrophages stimulated with oxidized low-density lipoprotein (LDL) and oxidized LDL immune complexes: role in phagocytosis and cytokine release.

    PubMed

    Truman, Jean-Philip; Al Gadban, Mohammed M; Smith, Kent J; Jenkins, Russell W; Mayroo, Nalini; Virella, Gabriel; Lopes-Virella, Maria F; Bielawska, Alicja; Hannun, Yusuf A; Hammad, Samar M

    2012-05-01

    Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to the formation of lipid-laden macrophages (foam cells). Fcγ receptors mediate uptake of oxLDL-IC, whereas scavenger receptors internalize oxLDL. We have previously reported that oxLDL-IC, but not free oxLDL, activate macrophages and prolong their survival. Sphingomyelin is a major constituent of cell membranes and lipoprotein particles and acid sphingomyelinase (ASMase) hydrolyses sphingomyelin to generate the bioactive lipid ceramide. ASMase exists in two forms: lysosomal (L-ASMase) and secretory (S-ASMase). In this study we examined whether oxLDL and oxLDL-IC regulate ASMase differently, and whether ASMase mediates monocyte/macrophage activation and cytokine release. The oxLDL-IC, but not oxLDL, induced early and consistent release of catalytically active S-ASMase. The oxLDL-IC also consistently stimulated L-ASMase activity, whereas oxLDL induced a rapid transient increase in L-ASMase activity before it steadily declined below baseline. Prolonged exposure to oxLDL increased L-ASMase activity; however, activity remained significantly lower than that induced by oxLDL-IC. Further studies were aimed at defining the function of the activated ASMase. In response to oxLDL-IC, heat-shock protein 70B' (HSP70B') was up-regulated and localized with redistributed ASMase in the endosomal compartment outside the lysosome. Treatment with oxLDL-IC induced the formation and release of HSP70-containing and IL-1β-containing exosomes via an ASMase-dependent mechanism. Taken together, the results suggest that oxLDL and oxLDL-IC differentially regulate ASMase activity, and the pro-inflammatory responses to oxLDL-IC are mediated by prolonged activation of ASMase. These findings may contribute to increased understanding of mechanisms mediating macrophage involvement in atherosclerosis.

  20. Methamphetamine inhibits antigen processing, presentation, and phagocytosis.

    PubMed

    Tallóczy, Zsolt; Martinez, Jose; Joset, Danielle; Ray, Yonaton; Gácser, Attila; Toussi, Sima; Mizushima, Noboru; Nosanchuk, Joshua D; Nosanchuk, Josh; Goldstein, Harris; Loike, John; Sulzer, David; Santambrogio, Laura

    2008-02-08

    Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal-lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.

  1. Methamphetamine Inhibits Antigen Processing, Presentation, and Phagocytosis

    PubMed Central

    Joset, Danielle; Ray, Yonaton; Gácser, Attila; Toussi, Sima; Mizushima, Noboru; Nosanchuk, Josh; Goldstein, Harris; Loike, John; Sulzer, David; Santambrogio, Laura

    2008-01-01

    Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal–lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology. PMID:18282092

  2. A2 noradrenergic neurons regulate forced swim test immobility.

    PubMed

    Nam, Hyungwoo; Kerman, Ilan A

    2016-10-15

    The Wistar-Kyoto (WKY) rat is a widely used animal model of depression, which is characterized by dysregulation of noradrenergic signaling. We previously demonstrated that WKY rats show a unique behavioral profile on the forced swim test (FST), characterized by high levels of immobility upon initial exposure and a greater learning-like response by further increasing immobility upon re-exposure than the genetically related Wistar rats. In the current study we aimed to determine whether altered activation of brainstem noradrenergic cell groups contributes to this behavioral profile. We exposed WKY and Wistar rats, to either 5min of forced swim or to the standard two-day FST (i.e. 15min forced swim on Day 1, followed by 5min on Day 2). We then stained their brains for FOS/tyrosine hydroxylase double-immunocytochemistry to determine potential differences in the activation of the brainstem noradrenergic cell groups. We detected a relative hyperactivation in the locus coeruleus of WKY rats when compared to Wistars in response to both one- and two-day forced swim. In contrast, within the A2 noradrenergic cell group, WKY rats exhibited diminished levels of FOS across both days of the FST, suggesting their lesser activation. We followed up these observations by selectively lesioning the A2 neurons, using anti-dopamine-β-hydroxylase-conjugated saporin, in Wistar rats, which resulted in increased FST immobility on both days of the test. Together these data indicate that the A2 noradrenergic cell group regulates FST behavior, and that its hypoactivation may contribute to the unique behavioral phenotype of WKY rats.

  3. Response gene to complement 32 protein promotes macrophage phagocytosis via activation of protein kinase C pathway.

    PubMed

    Tang, Rui; Zhang, Gui; Chen, Shi-You

    2014-08-15

    Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.

  4. Phagocytosis

    MedlinePlus

    Macrophages are scavenger cells that can ingest dead tissue and foreign cells. Macrophages form tentacles called pseudopods to surround an invader. Once inside the macrophage, the invader is walled off and then digested ...

  5. Teaching phagocytosis using flow cytometry.

    PubMed

    Boothby, John T; Kibler, Ruthann; Rech, Sabine; Hicks, Robert

    2004-05-01

    Investigative microbiology on protists in a basic teaching laboratory environment is limited by student skill level, ease of microbial culture and manipulation, instrumentation, and time. The flow cytometer is gaining use as a mainstream instrument in research and clinical laboratories, but has had minimal application in teaching laboratories. Although the cost of a flow cytometer is currently prohibitive for many microbiology teaching environments and the number of trained instructors and teaching materials is limited, in many ways the flow cytometer is an ideal instrument for teaching basic microbiology. We report here on a laboratory module to study phagocytosis in Tetrahymena sp. using flow cytometry in a basic microbiology teaching laboratory. Students and instructors found the flow cytometry data analysis program, Paint-AGate(PRO-TM), to be very intuitive and easy to learn within a short period of time. Assessment of student learning about Tetrahymena sp., phagocytosis, flow cytometry, and investigative microbiology using an inquiry-based format demonstrated an overall positive response from students.

  6. Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities.

    PubMed

    Castro, D P; Figueiredo, M B; Genta, F A; Ribeiro, I M; Tomassini, T C B; Azambuja, P; Garcia, E S

    2009-06-01

    The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A(2) (PLA(2)) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1mug/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10microg/insect) or PAF (1microg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA(2) activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.

  7. Attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts.

    PubMed

    Moerke, Caroline; Mueller, Petra; Nebe, Barbara

    2016-01-01

    Cells are sensitive to their underlying micro- and nano-topography, but the complex interplay is not completely understood especially if sharp edges and ridges of stochastically modified surfaces interfere with an attached cell body. Micro-topography offers cues that evoke a large range of cell responses e.g. altered adhesion behavior and integrin expression resulting in disturbed cell functions. In this study, we analyzed why osteoblastic cells mimic the underlying geometrical micro-pillar structure (5 × 5 × 5 μm, spacing of 5 μm) with their actin cytoskeleton. Interestingly, we discovered an attempted caveolae-mediated phagocytosis of each micro-pillar beneath the cells, which was accompanied by increased intracellular reactive oxygen species (ROS) production and reduced intracellular ATP levels. This energy consuming process hampered the cells in their function as osteoblasts at the interface. The raft-dependent/caveolae-mediated phagocytic pathway is regulated by diverse cellular components including caveolin-1 (Cav-1), cholesterol, actin cytoskeleton as well as actin-binding proteins like annexin A2 (AnxA2). Our results show a new aspect of osteoblast-material interaction and give insight into how cells behave on extraordinary micro-structures. We conclude that stochastically structured implants used in orthopedic surgery should avoid any topographical heights which induce phagocytosis to prevent their successful ingrowth.

  8. Ceramide-1-Phosphate in Phagocytosis and Calcium Homeostasis

    PubMed Central

    Hinkovska-Galcheva, Vania; Shayman, James A.

    2013-01-01

    Sphingolipids are well established sources of important signaling molecules. For example, ceramide (Cer) has been described as a potent inhibitor of cell growth and inducer of apoptosis. In contrast, ceramide 1-phosphate (C1P) has been reported to have mitogenic properties and to inhibit apoptosis. Our understanding of the distinct biological roles of C1P in the regulation of DNA synthesis, inflammation, membrane fusion, and intracellular Ca2+ increase has rapidly expanded. C1P is a bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). This chapter specifically focuses on the role of C1P in phagocytosis and Ca2+ homeostasis. Studies of the metabolism of C1P during phagocytosis, may lead to a better understanding of its role in signaling. Potentially, the inhibition of CERK and C1P formation may be a therapeutic target for inflammation. PMID:20919651

  9. Rab17 mediates differential antigen sorting following efferocytosis and phagocytosis

    PubMed Central

    Yin, Charles; Kim, Yohan; Argintaru, Dean; Heit, Bryan

    2016-01-01

    Macrophages engulf and destroy pathogens (phagocytosis) and apoptotic cells (efferocytosis), and can subsequently initiate adaptive immune responses by presenting antigens derived from engulfed materials. Both phagocytosis and efferocytosis share a common degradative pathway in which the target is engulfed into a membrane-bound vesicle, respectively, termed the phagosome and efferosome, where they are degraded by sequential fusion with endosomes and lysosomes. Despite this shared maturation pathway, macrophages are immunogenic following phagocytosis but not efferocytosis, indicating that differential processing or trafficking of antigens must occur. Mass spectrometry and immunofluorescence microscopy of efferosomes and phagosomes in macrophages demonstrated that efferosomes lacked the proteins required for antigen presentation and instead recruited the recycling regulator Rab17. As a result, degraded materials from efferosomes bypassed the MHC class II loading compartment via the recycling endosome – a process not observed in phagosomes. Combined, these results indicate that macrophages prevent presentation of apoptotic cell-derived antigens by preferentially trafficking efferocytosed, but not phagocytosed, materials away from the MHC class II loading compartment via the recycling endosome pathway. PMID:28005073

  10. Raft aggregation with specific receptor recruitment is required for microglial phagocytosis of Aβ42

    PubMed Central

    Persaud-Sawin, Dixie-Ann; Banach, Lynna; Harry, G. Jean

    2009-01-01

    Microglial phagocytosis contributes to the maintenance of brain homeostasis. Mechanisms involved however, remain unclear. Using Aβ42 solely as a stimulant, we provide novel insight into regulation of microglial phagocytosis by rafts. We demonstrate the existence of an Aβ42 threshold level of 250pg/ml, above which microglial phagocytic function is impaired. Low levels of Aβ42 facilitate fluorescent bead uptake, whereas phagocytosis is inhibited when Aβ42 accumulates. We also show that region-specific raft clustering occurs prior to microglial phagocytosis. Low Aβ42 levels stimulated this type of raft aggregation, but high Aβ42 levels inhibited it. Additionally, treatment with high Aβ42 concentrations caused a redistribution of the raft structural protein flotillin1 from low to higher density fractions along a sucrose gradient. This suggests a loss of raft structural integrity. Certain non-steroidal anti-inflammatory drugs, e.g. the COX-2-specific NSAID, celecoxib, raise Aβ42 levels. We demonstrated that prolonged celecoxib exposure can disrupt rafts in a manner similar to that seen in an elevated Aβ42 environment: abnormal raft aggregation and Flot1 distribution. This resulted in aberrant receptor recruitment to rafts and impaired receptor-mediated phagocytosis by microglial cells. Specifically, recruitment of the scavenger receptor CD36 to rafts during active phagocytosis was affected. Thus, we propose that maintaining raft integrity is crucial to determining microglial phagocytic outcomes and disease progression. PMID:18756527

  11. Endoplasmic reticulum‐resident Rab8A GTPase is involved in phagocytosis in the protozoan parasite Entamoeba histolytica

    PubMed Central

    Hanadate, Yuki; Saito‐Nakano, Yumiko; Nakada‐Tsukui, Kumiko

    2016-01-01

    Summary Phagocytosis is indispensable for the pathogenesis of the intestinal protozoan parasite Entamoeba histolytica. Here, we showed that in E. histolytica Rab8A, which is generally involved in trafficking from the trans‐Golgi network to the plasma membrane in other organisms but was previously identified in phagosomes of the amoeba in the proteomic analysis, primarily resides in the endoplasmic reticulum (ER) and participates in phagocytosis. We demonstrated that down‐regulation of EhRab8A by small antisense RNA‐mediated transcriptional gene silencing remarkably reduced adherence and phagocytosis of erythrocytes, bacteria and carboxylated latex beads. Surface biotinylation followed by SDS‐PAGE analysis revealed that the surface expression of several proteins presumably involved in target recognition was reduced in the EhRab8A gene‐silenced strain. Further, overexpression of wild‐type EhRab8A augmented phagocytosis, whereas expression of the dominant‐negative form of EhRab8A resulted in reduced phagocytosis. These results indicated that EhRab8A regulates transport of surface receptor(s) for the prey from the ER to the plasma membrane. To our knowledge, this is the first report that the ER‐resident Rab GTPase is involved in phagocytosis through the regulation of trafficking of a surface receptor, supporting a premise of direct involvement of the ER in phagocytosis. PMID:26807810

  12. 29 CFR 2530.200a-2 - Treasury regulations for purposes of the Act.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Treasury regulations for purposes of the Act. 2530.200a-2... RETIREMENT INCOME SECURITY ACT OF 1974 RULES AND REGULATIONS FOR MINIMUM STANDARDS FOR EMPLOYEE PENSION BENEFIT PLANS Scope and General Provisions § 2530.200a-2 Treasury regulations for purposes of the...

  13. Phagocytosis: A Fundamental Process in Immunity

    PubMed Central

    2017-01-01

    One hundred years have passed since the death of Élie Metchnikoff (1845–1916). He was the first to observe the uptake of particles by cells and realized the importance of this process for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this he gave us the basis for our modern understanding of inflammation and the innate and acquired immune responses. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In recent years, the use of new tools of molecular biology and microscopy has provided new insights into the cellular mechanisms of phagocytosis. In this review, we present a general view of our current knowledge on phagocytosis. We emphasize novel molecular findings, particularly on phagosome formation and maturation, and discuss aspects that remain incompletely understood. PMID:28691037

  14. Entamoeba histolytica. Phagocytosis as a virulence factor

    PubMed Central

    1983-01-01

    In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica. PMID:6313842

  15. Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

    PubMed

    Bloomfield, Gareth; Traynor, David; Sander, Sophia P; Veltman, Douwe M; Pachebat, Justin A; Kay, Robert R

    2015-03-27

    Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

  16. Rate and extent of Helicobacter pylori phagocytosis.

    PubMed

    Allen, Lee-Ann H

    2008-01-01

    Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric epithelium and plays a causative role in the development of peptic ulcers and gastric cancer. Phagocytosis is an element of innate defense used by macrophages and neutrophils to engulf microorganisms. We and others have shown that strains of H. pylori that contain the cag pathogenicity island actively retard their entry into phagocytes. Consequently, there is a lag of several minutes between bacterial binding and the onset of engulfment, and relative to other particles and microbes, the rate of internalization is slow. Herein, we describe in detail the use of synchronized phagocytosis and indirect immunofluorescence microscopy to quantify the rate and extent of H. pylori phagocytosis. This method is appropriate for primary phagocytes as well as transformed cell lines. More importantly, the effects of opsonins, virulence factors, and other agents on infection can be measured independent of bacterial viability or intracellular locale.

  17. Neutrophil-Mediated Phagocytosis of Staphylococcus aureus

    PubMed Central

    van Kessel, Kok P. M.; Bestebroer, Jovanka; van Strijp, Jos A. G.

    2014-01-01

    Initial elimination of invading Staphylococcus aureus from the body is mediated by professional phagocytes. The neutrophil is the major phagocyte of the innate immunity and plays a key role in the host defense against staphylococcal infections. Opsonization of the bacteria with immunoglobulins and complement factors enables efficient recognition by the neutrophil that subsequently leads to intracellular compartmentalization and killing. Here, we provide a review of the key processes evolved in neutrophil-mediated phagocytosis of S. aureus and briefly describe killing. As S. aureus is not helpless against the professional phagocytes, we will also highlight its immune evasion arsenal related to phagocytosis. PMID:25309547

  18. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient

    NASA Technical Reports Server (NTRS)

    Sun, E.; Gao, Y.; Chen, J.; Roberts, A. I.; Wang, X.; Chen, Z.; Shi, Y.

    2004-01-01

    Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance.

  19. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient

    NASA Technical Reports Server (NTRS)

    Sun, E.; Gao, Y.; Chen, J.; Roberts, A. I.; Wang, X.; Chen, Z.; Shi, Y.

    2004-01-01

    Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance.

  20. Small molecule phagocytosis inhibitors for immune cytopenias.

    PubMed

    Neschadim, Anton; Kotra, Lakshmi P; Branch, Donald R

    2016-08-01

    Immune cytopenias are conditions characterized by low blood cell counts, such as platelets in immune thrombocytopenia (ITP) and red blood cells in autoimmune hemolytic anemia (AIHA). Chronic ITP affects approximately 4 in 100,000 adults annually while AIHA is much less common. Extravascular phagocytosis and massive destruction of autoantibody-opsonized blood cells by macrophages in the spleen and liver are the hallmark of these conditions. Current treatment modalities for ITP and AIHA include the first-line use of corticosteroids; whereas, IVIg shows efficacy in ITP but not AIHA. One main mechanism of action by which IVIg treatment leads to the reduction in platelet destruction rates in ITP is thought to involve Fcγ receptor (FcγR) blockade, ultimately leading to the inhibition of extravascular platelet phagocytosis. IVIg, which is manufactured from the human plasma of thousands of donors, is a limited resource, and alternative treatments, particularly those based on bioavailable small molecules, are needed. In this review, we overview the pathophysiology of ITP, the role of Fcγ receptors, and the mechanisms of action of IVIg in treating ITP, and outline the efforts and progress towards developing novel, first-in-class inhibitors of phagocytosis as synthetic, small molecule substitutes for IVIg in ITP and other conditions where the pathobiology of the disease involves phagocytosis.

  1. Involvement of Tiam1, RhoG and ELMO2/ILK in Rac1-mediated phagocytosis in human trabecular meshwork cells.

    PubMed

    Peotter, Jennifer L; Phillips, Jenny; Tong, Tiegang; Dimeo, Kaylee; Gonzalez, Jose M; Peters, Donna M

    2016-10-01

    We previously demonstrated that an αvβ5 integrin/FAK- mediated pathway regulated the phagocytic properties of human trabecular meshwork (HTM) cells. Here we demonstrate that this process is mediated by Rac-1 and a previously unreported signaling pathway that utilizes the Tiam1 as well as a novel ILK/RhoG/ELMO2 signaling pathway. Phagocytosis in both a TM-1 cell line and normal HTM cells was mediated by Rac1 and could be significantly decreased by >75% using the Rac1 inhibitor EHop-016. Knockdown of Rac1 in TM-1 cells also inhibited phagocytosis by 40% whereas overexpression of a constitutively active Rac1 or stimulation with PDGF increased phagocytosis by 83% and 32% respectively. Tiam1 was involved in regulating phagocytosis. Knockdown of Tiam1 inhibited phagocytosis by 72% while overexpression of Tiam1 C1199 increased phagocytosis by 75%. Other upstream effectors of Rac1 found to be involved included ELMO2, RhoG, and ILK. Knockdowns of ELMO2, ILK, and RhoG caused a reduction in phagocytosis by 51%, 55% and 46% respectively. In contrast, knockdown of Vav2 and Dock1 or overexpression of Vav2 Y159/172F did not cause a significant change in phagocytosis. These data suggest a novel link between Tiam1 and RhoG/ILK /ELMO2 pathway as upstream effectors of the Rac1-mediated phagocytic process in TM cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Drosophila Rab14 mediates phagocytosis in the immune response to Staphylococcus aureus

    PubMed Central

    Garg, Aprajita; Wu, Louisa P.

    2014-01-01

    Drosophila hemocytes are essential for the animal to resist Staphylococcus aureus infections. Phagocytosis is a central component of the hemocyte-mediated immune response. It involves regulated interaction between the phagocytic and the endocytic compartments. Rab GTPases are pivotal for the membrane trafficking and fusion events, and thus are often targets of intracellular pathogens that subvert phagocytosis. An in vivo screen identified Rab2 and Rab14 as candidates for proteins regulating phagosome maturation. Since Rab14 is often targeted by intracellular pathogens, an understanding of its function during phagocytosis and the overall immune response can give insight into the pathogenesis of intracellular microbes. We generated a Drosophila Rab14 mutant and characterized the resulting immune defects in animals and specifically in hemocytes in response to an S. aureus infection. Hemocyte based immunofluorescence studies indicate that Rab14 is recruited to the phagosome and like Rab7, a well-characterized regulator of the phagocytic pathway, is essential for progression of phagosome maturation. Rab14 mutant hemocytes show impaired recruitment of Rab7 and of a lysosomal marker onto S. aureus phagosomes. The defect in phagocytosis is associated with higher bacterial load and increased susceptibility to S. aureus in the animal. PMID:24119134

  3. Group V secretory phospholipase A2 reveals its role in house dust mite-induced allergic pulmonary inflammation by regulation of dendritic cell function

    PubMed Central

    Giannattasio, Giorgio; Fujioka, Daisuke; Xing, Wei; Katz, Howard R.; Boyce, Joshua A.; Balestrieri, Barbara

    2010-01-01

    We have previously shown that group V secretory phospholipase A2 (sPLA2) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. Here we report that group V sPLA2 (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae (Df) had markedly reduced pulmonary inflammation and goblet cell metaplasia compared to wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to Df compared to WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by Df had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of Df-challenged mice. Adoptively transferred Df-loaded Pla2g5-null BMDCs were less able than Df-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null Df-loaded BMDCs exhibited significantly reduced local inflammatory responses to Df, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA2 in APC regulates Ag processing and maturation of dendritic cells, and contributes to pulmonary inflammation and immune response against Df. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA2 is upregulated by Df and whose function is also regulated by group V sPLA2. PMID:20817863

  4. Serotonin upregulates the activity of phagocytosis through 5-HT1A receptors

    PubMed Central

    Freire-Garabal, M; Núñez, M J; Balboa, J; López-Delgado, P; Gallego, R; García-Caballero, T; Fernández-Roel, M D; Brenlla, J; Rey-Méndez, M

    2003-01-01

    In this study, we investigated whether serotonin could regulate the in vitro activity of phagocytosis through 5-hydroxytryptamine or serotonin (5-HT1A) receptors. Mouse peritoneal macrophages were cultured with serotonin and the activity of phagocytosis was assessed by the uptake of zymosan and latex particles added to the culture media. Specific binding of [3H]8-OH-DPAT and immunohistochemistry using an affinity-purified anti-5-HT1A-receptor antibody were assayed in the macrophages. In addition, we took advantage of the availability of pharmacological inhibitors of nuclear factor-κB (NF-κB) to explore its role in the regulation of the 5-HT1A receptor. Serotonin increased the in vitro activity of phagocytosis in a dose-dependent manner. The 5-HT1A receptor agonist (±)-8-hydroxy-2-(di-n-propyl-amino)-tetralin (R(+)-8-OH-DPAT) reproduced these effects. Serotonin- or R(+)-8-OH-DPAT-induced increases in phagocytosis were blocked by the 5-HT1A receptor antagonist WAY100635 and the NF-κB inhibitor pyrrolidinedithiocarbamate. Moreover, mouse peritoneal macrophages expressed specific binding sites for [3H]8-OH-DPAT when cultivated in the presence of zymosan or latex beads. Immunohistochemistry confirmed the expression of the 5-HT1A receptor protein in the macrophages. These results show that serotonin can upregulate the activity of peritoneal macrophages through 5-HT1A receptors. PMID:12770951

  5. Phagocytosis of dying cells: influence of smoking and static magnetic fields.

    PubMed

    Dini, Luciana

    2010-09-01

    It is becoming evident that failure in the removal of dying cells causes and/or promotes the onset of chronic diseases. Impairment of phagocytosis of apoptotic cells can be due not only to genetic or molecular malfunctioning but also to external/environmental factors. Two of these environmental factors have been recently reported to down regulate the clearance of apoptotic cells: cigarette smoke and static magnetic fields. Cigarette smoke contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. Human macrophages interacting with carbonyl or cigarette smoke modified extracellular matrix (ECM) proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. It was postulated that changes in the ECM environment as a result of cigarette smoke affect the ability of macrophages to remove apoptotic cells. This decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion. Downregulation of the phagocytosis of apoptotic cells was also described when performed in presence of static magnetic fields (SMFs) of moderate intensity. SMFs have been reported to perturb distribution of membrane proteins and glycoproteins, receptors, cytoskeleton and trans-membrane fluxes of different ions, especially calcium [Ca(2+)]i, that in turn, interfere with many different physiological activities, including phagocytosis. The effects of cigarette smoke and SMF on the phagocytosis of dying cells will be here discussed.

  6. HMG-CoA reductase inhibitors enhance phagocytosis by upregulating ATP-binding cassette transporter A7

    PubMed Central

    Tanaka, Nobukiyo; Abe-Dohmae, Sumiko; Iwamoto, Noriyuki; Fitzgerald, Michael L.; Yokoyama, Shinji

    2011-01-01

    We recently reported that the endogenous ATP-binding cassette transporter (ABC) A7 strongly associates with phagocytosis, being regulated by sterol regulatory element binding protein 2. We therefore examined the effect of statins on phagocytosis in vitro and in vivo through the SREBP-ABCA7. Phagocytosis was found to be enhanced by pravastatin, rosuvastatin and simvastatin and cyclodextrin in J774 macrophages, as cellular cholesterol was reduced and expressions of the cholesterol-related genes were modulated, including an increase of ABCA7 mRNA and decrease of ABCA1 mRNA. Conversely, knock-down of ABCA7 expression by siRNA ablated enhancement of phagocytosis by statins. In vivo, pravastatin enhanced phagocytosis in wild-type mice, but not in ABCA7-knockout mice. We thus concluded that statins enhance phagocytosis through the SREBP-ABCA7 pathway. These findings provide a molecular basis for enhancement of the host-defense system by statins showing that one of their “pleiotropic” effects is in fact achieved through their reaction to a primary target. PMID:21762915

  7. Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium

    PubMed Central

    Bloomfield, Gareth; Traynor, David; Sander, Sophia P; Veltman, Douwe M; Pachebat, Justin A; Kay, Robert R

    2015-01-01

    Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures. DOI: http://dx.doi.org/10.7554/eLife.04940.001 PMID:25815683

  8. A role for connexin43 in macrophage phagocytosis and host survival after bacterial peritoneal infection.

    PubMed

    Anand, Rahul J; Dai, Shipan; Gribar, Steven C; Richardson, Ward; Kohler, Jeff W; Hoffman, Rosemary A; Branca, Maria F; Li, Jun; Shi, Xiao-Hua; Sodhi, Chhinder P; Hackam, David J

    2008-12-15

    The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection.

  9. The Macrophage A2b Adenosine Receptor Regulates Tissue Insulin Sensitivity

    PubMed Central

    Koupenova, Milka; Carroll, Shannon; Ravid, Katya

    2014-01-01

    High fat diet (HFD)-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies. Previously, we identified the A2b adenosine receptor (A2bAR), an established regulator of inflammation, as a regulator of HFD-induced insulin resistance. In particular, HFD was associated with vast upregulation of liver A2bAR in control mice, and while mice lacking this receptor showed augmented liver inflammation and tissue insulin resistance. As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptor's influence on tissue insulin sensitivity is mediated via its expression in macrophages. This was shown using a newly generated transgenic mouse model expressing the A2bAR gene in the macrophage lineage on an otherwise A2bAR null background. Reinstatement of macrophage A2bAR expression in A2bAR null mice fed HFD restored insulin tolerance and tissue insulin signaling to the level of control mice. The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2. Thus, our results illustrate that macrophage A2bAR signaling is needed and sufficient for relaying the protective effect of the A2bAR against HFD-induced tissue inflammation and insulin resistance in mice. PMID:24892847

  10. Regulation of LH/FSH expression by secretoglobin 3A2 in the mouse pituitary gland.

    PubMed

    Miyano, Yuki; Tahara, Shigeyuki; Sakata, Ichiro; Sakai, Takafumi; Abe, Hiroyuki; Kimura, Shioko; Kurotani, Reiko

    2014-04-01

    Secretoglobin (SCGB) 3A2 was originally identified as a downstream target for the homeodomain transcription factor NKX2-1 in the lung. NKX2-1 plays a role in the genesis and expression of genes in the thyroid, lung and ventral forebrain; Nkx2-1-null mice have no thyroid and pituitary and severely hypoplastic lungs and hypothalamus. To demonstrate whether SCGB3A2 plays any role in pituitary hormone production, NKX2-1 and SCGB3A2 expression in the mouse pituitary gland was examined by immunohistochemical analysis and RT-PCR. NKX2-1 was localized in the posterior pituitary lobe, whereas SCGB3A2 was observed in both anterior and posterior lobes as shown by immunohistochemistry and RT-PCR. Expression of CCAAT-enhancer binding proteins (C/EBPs), which regulate mouse Scgb3a2 transcription, was also examined by RT-PCR. C/EBPβ, γ, δ and ζ were expressed in the adult mouse pituitary gland. SCGB3A2 was expressed in the anterior and posterior lobes from postnatal days 1 and 5, respectively and the areas where SCGB3A2 expression was found coincided with the area where FSH-secreting cells were found. Double-staining for SCGB3A2 and pituitary hormones revealed that SCGB3A2 was mainly localized in gonadotrophs in 49 % of FSH-secreting cells and 47 % of LH-secreting cells. In addition, SCGB3A2 dramatically inhibited LH and FSH mRNA expression in rat pituitary primary cell cultures. These results suggest that SCGB3A2 regulates FSH/LH production in the anterior pituitary lobe and that transcription factors other than NKX2-1 may regulate SCGB3A2 expression.

  11. Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63

    PubMed Central

    Matte, Christine; Casgrain, Pierre-André; Séguin, Olivier; Moradin, Neda; Hong, Wan Jin; Descoteaux, Albert

    2016-01-01

    The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery. PMID:27280768

  12. MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage

    PubMed Central

    Thuong, N T T; Tram, T T B; Dinh, T D; Thai, P V K; Heemskerk, D; Bang, N D; Chau, T T H; Russell, D G; Thwaites, G E; Hawn, T R; Caws, M; Dunstan, S J

    2016-01-01

    Macrophage receptor with collagenous structure (MARCO) has an important role in the phagocytosis of Mycobacterium tuberculosis (M. tuberculosis). We hypothesized that MARCO polymorphisms are associated with phagocytosis, tuberculosis (TB) disease susceptibility and presentation, and infecting lineage. We used a human cellular model to examine how MARCO genotype mediates the immune response; a case–control study to investigate tuberculosis host genetic susceptibility; and a host–pathogen genetic analysis to study host–pathogen interactions. Two MARCO heterozygous (AG) genotypes (single-nucleotide polymorphisms rs2278589 and rs6751745) were associated with impaired phagocytosis of M. tuberculosis trehalose 6,6'-dimycolate-cord factor and β-glucan-coated beads in macrophages. The heterozygous genotypes of rs2278589 and rs6751745 were also associated with increased risk of pulmonary TB (PTB; rs2278589, P=0.001, odds ratio (OR)=1.6; rs6751745, P=0.009, OR=1.4), and with severe chest X-ray abnormalities (P=0.007, OR=1.6). These two genotypes were also associated with the Beijing lineage (rs2278589, P=0.001, OR=1.7; rs6751745, P=0.01, OR=1.5). Together, these results suggest that MARCO polymorphisms may regulate phagocytosis of M. tuberculosis and susceptibility and severity of PTB. They also suggest MARCO genotype and Beijing strains may interact to increase the risk of PTB. PMID:27853145

  13. Phagocytosis in Teleosts. Implications of the New Cells Involved

    PubMed Central

    Esteban, María Ángeles; Cuesta, Alberto; Chaves-Pozo, Elena; Meseguer, José

    2015-01-01

    Phagocytosis is the process by which cells engulf some solid particles to form internal vesicles known as phagosomes. Phagocytosis is in fact a specific form of endocytosis involving the vesicular interiorization of particles. Phagocytosis is essentially a defensive reaction against infection and invasion of the body by foreign substances and, in the immune system, phagocytosis is a major mechanism used to remove pathogens and/or cell debris. For these reasons, phagocytosis in vertebrates has been recognized as a critical component of the innate and adaptive immune responses to pathogens. Furthermore, more recent studies have revealed that phagocytosis is also crucial for tissue homeostasis and remodeling. Professional phagocytes in teleosts are monocyte/macrophages, granulocytes and dendritic cells. Nevertheless, in recent years phagocytic properties have also been attributed to teleost lymphocytes and thrombocytes. The possible implications of such cells on this important biological process, new factors affecting phagocytosis, evasion of phagocytosis or new forms of phagocytosis will be considered and discussed. PMID:26690236

  14. Energy metabolism of human neutrophils during phagocytosis.

    PubMed

    Borregaard, N; Herlin, T

    1982-09-01

    Detailed quantitative studies were performed on the generation and utilization of energy by resting and phagocytosing human neutrophils. The ATP content was 1.9 fmol/cell, was constant during rest, and was not influenced by the presence or absence of glucose in the medium. The intracellular content of phosphocreatine was less than 0.2 fmol/cell. In the presence of glucose, ATP was generated almost exclusively from lactate produced from glucose taken up from the surrounding medium. The amount of lactate produced could account for 85% of the glucose taken up by the cells, and the intracellular glycosyl store, glycogen, was not drawn upon. The rate of ATP generation as calculated from the rate of lactate production was 1.3 fmol/cell/min. During phagocytosis, there was no measurable increase in glucose consumption or lactate production, and the ATP content fell rapidly to 0.8 fmol/cell. This disappearance of ATP was apparently irreversible since no corresponding increase in ADP or AMP was observed. It therefore appears that this phagocytosis-induced fall in ATP concentration represents all the extra energy utilized in human neutrophils in the presence of glucose. In the absence of glucose, the rate of ATP generation in the resting cell was considerably smaller, 0.75 fmol/cell per min, as calculated from the rate of glycolysis, which is sustained exclusively by glycogenolysis. Under this condition, however, phagocytosis induces significant enhancement of glycogenolysis and the rate of lactate production is increased by 60%, raising the rate of ATP generation to 1.2 fmol/cell per min. Nonetheless, the ATP content drops significantly from 1.9 to 1.0 fmol/cell. Neutrophils from patients with chronic granulomatous disease have the same rate of glycolysis and the same ATP content as normal cells, thus confirming that the defective respiration of these cells does not affect their energy metabolism.

  15. Energy Metabolism of Human Neutrophils during Phagocytosis

    PubMed Central

    Borregaard, Niels; Herlin, Troels

    1982-01-01

    Detailed quantitative studies were performed on the generation and utilization of energy by resting and phagocytosing human neutrophils. The ATP content was 1.9 fmol/cell, was constant during rest, and was not influenced by the presence or absence of glucose in the medium. The intracellular content of phosphocreatine was less than 0.2 fmol/cell. In the presence of glucose, ATP was generated almost exclusively from lactate produced from glucose taken up from the surrounding medium. The amount of lactate produced could account for 85% of the glucose taken up by the cells, and the intracellular glycosyl store, glycogen, was not drawn upon. The rate of ATP generation as calculated from the rate of lactate production was 1.3 fmol/cell/min. During phagocytosis, there was no measurable increase in glucose consumption or lactate production, and the ATP content fell rapidly to 0.8 fmol/cell. This disappearance of ATP was apparently irreversible since no corresponding increase in ADP or AMP was observed. It therefore appears that this phagocytosis-induced fall in ATP concentration represents all the extra energy utilized in human neutrophils in the presence of glucose. In the absence of glucose, the rate of ATP generation in the resting cell was considerably smaller, 0.75 fmol/cell per min, as calculated from the rate of glycolysis, which is sustained exclusively by glycogenolysis. Under this condition, however, phagocytosis induces significant enhancement of glycogenolysis and the rate of lactate production is increased by 60%, raising the rate of ATP generation to 1.2 fmol/cell per min. Nonetheless, the ATP content drops significantly from 1.9 to 1.0 fmol/cell. Neutrophils from patients with chronic granulomatous disease have the same rate of glycolysis and the same ATP content as normal cells, thus confirming that the defective respiration of these cells does not affect their energy metabolism. PMID:7107894

  16. Phagocytosis as a biomarker for stress responses

    NASA Astrophysics Data System (ADS)

    Huber, K.; Krotz-Fahning, M.; Hock, B.

    2005-08-01

    An in vitro test has been developed for the detection of immunotoxic events. It will be used within the project "TRIPLE LUX" on the International Space Station to investigate the effects of single and combined space flight conditions on mammalian phagocytes. The intensity of the respiratory burst during phagocytosis can be followed by the luminol-based chemiluminescence response after stimulation with zymosan. We adapted this test system for polymorphonuclear leukocytes, purified from sheep blood and stored by cryoconservation. In this report we show the immunostimulating effect of hydrocortisone and the immunosuppressive impact of cadmium as an example for alterations that can be detected by this test.

  17. NR4A2 is regulated by gastrin and influences cellular responses of gastric adenocarcinoma cells.

    PubMed

    Misund, Kristine; Selvik, Linn-Karina Myrland; Rao, Shalini; Nørsett, Kristin; Bakke, Ingunn; Sandvik, Arne K; Lægreid, Astrid; Bruland, Torunn; Prestvik, Wenche S; Thommesen, Liv

    2013-01-01

    The peptide hormone gastrin is known to play a role in differentiation, growth and apoptosis of cells in the gastric mucosa. In this study we demonstrate that gastrin induces Nuclear Receptor 4A2 (NR4A2) expression in the adenocarcinoma cell lines AR42J and AGS-GR, which both possess the gastrin/CCK2 receptor. In vivo, NR4A2 is strongly expressed in the gastrin responsive neuroendocrine ECL cells in normal mucosa, whereas gastric adenocarcinoma tissue reveals a more diffuse and variable expression in tumor cells. We show that NR4A2 is a primary early transient gastrin induced gene in adenocarcinoma cell lines, and that NR4A2 expression is negatively regulated by inducible cAMP early repressor (ICER) and zinc finger protein 36, C3H1 type-like 1 (Zfp36l1), suggesting that these gastrin regulated proteins exert a negative feedback control of NR4A2 activated responses. FRAP analyses indicate that gastrin also modifies the nucleus-cytosol shuttling of NR4A2, with more NR4A2 localized to cytoplasm upon gastrin treatment. Knock-down experiments with siRNA targeting NR4A2 increase migration of gastrin treated adenocarcinoma AGS-GR cells, while ectopically expressed NR4A2 increases apoptosis and hampers gastrin induced invasion, indicating a tumor suppressor function of NR4A2. Collectively, our results uncover a role of NR4A2 in gastric adenocarcinoma cells, and suggest that both the level and the localization of NR4A2 protein are of importance regarding the cellular responses of these cells.

  18. NR4A2 Is Regulated by Gastrin and Influences Cellular Responses of Gastric Adenocarcinoma Cells

    PubMed Central

    Misund, Kristine; Selvik, Linn-Karina Myrland; Rao, Shalini; Nørsett, Kristin; Bakke, Ingunn; Sandvik, Arne K.; Lægreid, Astrid; Bruland, Torunn; Prestvik, Wenche S.; Thommesen, Liv

    2013-01-01

    The peptide hormone gastrin is known to play a role in differentiation, growth and apoptosis of cells in the gastric mucosa. In this study we demonstrate that gastrin induces Nuclear Receptor 4A2 (NR4A2) expression in the adenocarcinoma cell lines AR42J and AGS-GR, which both possess the gastrin/CCK2 receptor. In vivo, NR4A2 is strongly expressed in the gastrin responsive neuroendocrine ECL cells in normal mucosa, whereas gastric adenocarcinoma tissue reveals a more diffuse and variable expression in tumor cells. We show that NR4A2 is a primary early transient gastrin induced gene in adenocarcinoma cell lines, and that NR4A2 expression is negatively regulated by inducible cAMP early repressor (ICER) and zinc finger protein 36, C3H1 type-like 1 (Zfp36l1), suggesting that these gastrin regulated proteins exert a negative feedback control of NR4A2 activated responses. FRAP analyses indicate that gastrin also modifies the nucleus-cytosol shuttling of NR4A2, with more NR4A2 localized to cytoplasm upon gastrin treatment. Knock-down experiments with siRNA targeting NR4A2 increase migration of gastrin treated adenocarcinoma AGS-GR cells, while ectopically expressed NR4A2 increases apoptosis and hampers gastrin induced invasion, indicating a tumor suppressor function of NR4A2. Collectively, our results uncover a role of NR4A2 in gastric adenocarcinoma cells, and suggest that both the level and the localization of NR4A2 protein are of importance regarding the cellular responses of these cells. PMID:24086717

  19. Male fertility and apoptosis in normal spermatogenesis are regulated by vacuolar-ATPase isoform a2.

    PubMed

    Jaiswal, Mukesh K; Agrawal, Varkha; Katara, Gajendra K; Pamarthy, Sahithi; Kulshrestha, Arpita; Chaouat, Gerard; Gilman-Sachs, Alice; Beaman, Kenneth D

    2015-11-01

    The a2 isoform of vacuolar-ATPase (ATP6V0A2, referred to as a2V) is required for normal spermatogenesis and maturation of sperm. Treatment of male mice with anti-a2V disturbs the testicular cytokine/chemokine balance and leads to severe deficiencies of spermatogenesis. The aim of the present study was to investigate the role of a2V in male fertility and in the regulation of apoptotic pathways required for normal spermatogenesis in mice. To study the role of a2V single dose of anti-a2V monoclonal antibody or mouse IgG isotype (3μg/animal) was injected i.p. into males on alternate days for 10 days. The expression of sperm maturation-related molecules and pro-apoptotic molecules was measured by real-time PCR or immunohistochemistry in control and anti-a2V-treated testes. The caspase levels and their activity were measured by western blot and fluorometry. We found that the expression of the sperm maturation-related molecules SPAM1, ADAM1, and ADAM2 was significantly decreased in testes from anti-a2V-treated males. The expression of pro-apoptotic molecules (Bax, p53, and p21) and molecules involved in the intrinsic pathway of apoptosis (caspase-9, caspase-3, and PARP), which are crucial for normal spermatogenesis was significantly reduced in testes from anti-a2V-treated males compared with the control. The total ATP level was significantly lower in anti-a2V-treated testes. The data provide novel evidence showing that a2V can regulate the apoptotic pathways, an essential testicular feature, and is necessary for efficient spermatogenesis.

  20. EphA2 Expression Regulates Inflammation and Fibroproliferative Remodeling in Atherosclerosis.

    PubMed

    Finney, Alexandra C; Funk, Steven D; Green, Jonette M; Yurdagul, Arif; Rana, Mohammad Atif; Pistorius, Rebecca; Henry, Miriam; Yurochko, Andrew; Pattillo, Christopher B; Traylor, James G; Chen, Jin; Woolard, Matthew D; Kevil, Christopher G; Orr, A Wayne

    2017-08-08

    Atherosclerotic plaque formation results from chronic inflammation and fibroproliferative remodeling in the vascular wall. We previously demonstrated that both human and mouse atherosclerotic plaques show elevated expression of EphA2, a guidance molecule involved in cell-cell interactions and tumorigenesis. Here, we assessed the role of EphA2 in atherosclerosis by deleting EphA2 in a mouse model of atherosclerosis (Apoe(-)(/-)) and by assessing EphA2 function in multiple vascular cell culture models. After 8 to 16 weeks on a Western diet, male and female mice were assessed for atherosclerotic burden in the large vessels, and plasma lipid levels were analyzed. Despite enhanced weight gain and plasma lipid levels compared with Apoe(-/-) controls, EphA2(-/-)Apoe(-/-) knockout mice show diminished atherosclerotic plaque formation, characterized by reduced proinflammatory gene expression and plaque macrophage content. Although plaque macrophages express EphA2, EphA2 deletion does not affect macrophage phenotype, inflammatory responses, and lipid uptake, and bone marrow chimeras suggest that hematopoietic EphA2 deletion does not affect plaque formation. In contrast, endothelial EphA2 knockdown significantly reduces monocyte firm adhesion under flow. In addition, EphA2(-/-)Apoe(-/-) mice show reduced progression to advanced atherosclerotic plaques with diminished smooth muscle and collagen content. Consistent with this phenotype, EphA2 shows enhanced expression after smooth muscle transition to a synthetic phenotype, and EphA2 depletion reduces smooth muscle proliferation, mitogenic signaling, and extracellular matrix deposition both in atherosclerotic plaques and in vascular smooth muscle cells in culture. Together, these data identify a novel role for EphA2 in atherosclerosis, regulating both plaque inflammation and progression to advanced atherosclerotic lesions. Cell culture studies suggest that endothelial EphA2 contributes to atherosclerotic inflammation by

  1. Diclofenac enhances proinflammatory cytokine-induced phagocytosis of cultured microglia via nitric oxide production

    SciTech Connect

    Kakita, Hiroki; Aoyama, Mineyoshi; Nagaya, Yoshiaki; Asai, Hayato; Hussein, Mohamed Hamed; Suzuki, Mieko; Kato, Shin; Saitoh, Shinji; Asai, Kiyofumi

    2013-04-15

    Influenza-associated encephalopathy (IAE) is a central nervous system complication with a high mortality rate, which is increased significantly by the non-steroidal anti-inflammatory drug diclofenac sodium (DCF). In the present study, we investigated the effects of DCF on brain immune cells (i.e. microglia) stimulated with three proinflammatory cytokines, namely tumor necrosis factor-α, interleukin-1β, and interferon-γ. Similar to previous findings in astrocytes, all three cytokines induced the expression of inducible NO synthase (iNOS), as well as NO production, in microglia. The addition of DCF to the culture system augmented iNOS expression and NO production. Immunocytochemical analysis and the phagocytosis assay revealed that cytokine treatment induced morphological changes to and phagocytosis by the microglia. The addition of DCF to the culture system enhanced microglial activation, as well as the phagocytic activity of cytokine-stimulated microglia. Inhibitors of nuclear factor (NF)-κB inhibited iNOS gene expression in cytokine-stimulated microglia with or without DCF, suggesting that the NF-κB pathway is one of the main signaling pathways involved. The iNOS inhibitor N{sup G}-monomethyl-L-arginine (L-NMMA) reduced both cytokine-induced phagocytosis and phagocytosis induced by the combination of cytokines plus DCF. Furthermore, the NO donor sodium nitroprusside induced phagocytosis, indicating that NO production is a key regulator of microglial phagocytosis. In conclusion, DCF acts synergistically with proinflammatory cytokines to increase the production of NO in microglia, leading to phagocytic activity of the activated microglia. These findings, together with previous observations regarding astrocytes, may explain the significant increase in mortality of IAE patients treated with DCF. - Highlights: ► Influenza-associated encephalopathy (IAE) is associated with a high mortality rate. ► Hyperimmunization in the brain is believed to be responsible for

  2. Influenza-Specific Antibody-Dependent Phagocytosis

    PubMed Central

    Ana-Sosa-Batiz, Fernanda; Vanderven, Hillary; Jegaskanda, Sinthujan; Johnston, Angus; Rockman, Steven; Laurie, Karen; Barr, Ian; Reading, Patrick; Lichtfuss, Marit; Kent, Stephen J.

    2016-01-01

    Background Immunity to human influenza A virus (IAV) infection is only partially understood. Broadly non-neutralizing antibodies may assist in reducing disease but have not been well characterized. Methods We measured internalization of opsonized, influenza protein-coated fluorescent beads and live IAV into a monocytic cell line to study antibody-dependent phagocytosis (ADP) against multiple influenza hemagglutinin (HA) subtypes. We analyzed influenza HA-specific ADP in healthy human donors, in preparations of intravenous immunoglobulin (IVIG), and following IAV infection of humans and macaques. Results We found that both sera from healthy adults and IVIG preparations had broad ADP to multiple seasonal HA proteins and weak cross-reactive ADP to non-circulating HA proteins. The ADP in experimentally influenza-infected macaque plasma and naturally influenza-infected human sera mediated phagocytosis of both homologous and heterologous IAVs. Further, the IAV phagocytosed in an antibody-mediated manner had reduced infectivity in vitro. Conclusion We conclude that IAV infections in humans and macaques leads to the development of influenza-specific ADP that can clear IAV infection in vitro. Repeated exposure of humans to multiple IAV infections likely leads to the development of ADP that is cross-reactive to strains not previously encountered. Further analyses of the protective capacity of broadly reactive influenza-specific ADP is warranted. PMID:27124730

  3. Phagocytosis Affects Biguanide Sensitivity of Acanthamoeba spp.

    PubMed Central

    Noble, Judith A.; Ahearn, Donald G.; Avery, Simon V.; Crow Jr., Sidney A.

    2002-01-01

    The incidence of Acanthamoeba keratitis, a disease associated with contact lens wear, has been in apparent decline with the advent of multipurpose contact lens solutions. The concentrations of the biguanides chlorhexidine digluconate (CHX) and particularly polyhexamethylene biguanide (PHMB) included in multipurpose solutions (MPSs) are sublethal for amoebae. We evaluated by flow cytometry the effects of these two biguanides on phagocytosis of particles and the survival of trophozoites of Acanthamoeba castellanii and A. polyphaga. Trophozoites of A. castellanii and A. polyphaga (106/ml) were exposed to solutions of 5 and 50 μg of PHMB and CHX per ml in the presence and absence of particles (i.e., heat-killed yeasts and bacteria and latex beads). In addition, trophozoites were exposed to particles treated with these concentrations of the two biguanides. In the absence of particles, trophozoites of A. polyphaga appeared to be more resistant to the biguanides than those of A. castellanii. In the presence of particles, the rates of survival of both species were decreased. In most instances, particles treated with sublethal concentrations of both biguanides that were adsorbed onto the particles reduced the incidence of phagocytosis. Particles present in MPSs in contact lens cases may be involved in the decreased incidence of Acanthamoeba keratitis. PMID:12069957

  4. Connexin43 is dispensable for phagocytosis.

    PubMed

    Glass, Aaron M; Wolf, Benjamin J; Schneider, Karin M; Princiotta, Michael F; Taffet, Steven M

    2013-05-01

    Macrophages that lack connexin43 (Cx43), a gap junction protein, have been reported to exhibit dramatic deficiencies in phagocytosis. In this study, we revisit these findings using well-characterized macrophage populations. Cx43 knockout (Cx43(-/-)) mice die soon after birth, making the harvest of macrophages from adult Cx43(-/-) mice problematic. To overcome this obstacle, we used several strategies: mice heterozygous for the deletion of Cx43 were crossed to produce Cx43(+/+) (wild type [WT]) and Cx43(-/-) fetuses. Cells isolated from 12- to 14-d fetal livers were used to reconstitute irradiated recipient animals. After reconstitution, thioglycollate-elicited macrophages were collected by peritoneal lavage and bone marrow was harvested. Bone marrow cells and, alternatively, fetal liver cells were cultured in media containing M-CSF for 7-10 d, resulting in populations of cells that were >95% macrophages based on flow cytometry. Phagocytic uptake was detected using flow cytometric and microscopic techniques. Quantification of phagocytic uptake of IgG-opsonized sheep erythrocytes, zymosan particles, and Listeria monocytogenes failed to show any significant difference between WT and Cx43(-/-) macrophages. Furthermore, the use of particles labeled with pH-sensitive dyes showed equivalent acidification of phagosomes in both WT and Cx43(-/-) macrophages. Our findings suggest that modulation of Cx43 levels in cultured macrophages does not have a significant impact on phagocytosis.

  5. Phagocytosis affects biguanide sensitivity of Acanthamoeba spp.

    PubMed

    Noble, Judith A; Ahearn, Donald G; Avery, Simon V; Crow, Sidney A

    2002-07-01

    The incidence of Acanthamoeba keratitis, a disease associated with contact lens wear, has been in apparent decline with the advent of multipurpose contact lens solutions. The concentrations of the biguanides chlorhexidine digluconate (CHX) and particularly polyhexamethylene biguanide (PHMB) included in multipurpose solutions (MPSs) are sublethal for amoebae. We evaluated by flow cytometry the effects of these two biguanides on phagocytosis of particles and the survival of trophozoites of Acanthamoeba castellanii and A. polyphaga. Trophozoites of A. castellanii and A. polyphaga (10(6)/ml) were exposed to solutions of 5 and 50 microg of PHMB and CHX per ml in the presence and absence of particles (i.e., heat-killed yeasts and bacteria and latex beads). In addition, trophozoites were exposed to particles treated with these concentrations of the two biguanides. In the absence of particles, trophozoites of A. polyphaga appeared to be more resistant to the biguanides than those of A. castellanii. In the presence of particles, the rates of survival of both species were decreased. In most instances, particles treated with sublethal concentrations of both biguanides that were adsorbed onto the particles reduced the incidence of phagocytosis. Particles present in MPSs in contact lens cases may be involved in the decreased incidence of Acanthamoeba keratitis.

  6. A2A adenosine receptors are up-regulated in lymphocytes from amyotrophic lateral sclerosis patients.

    PubMed

    Vincenzi, Fabrizio; Corciulo, Carmen; Targa, Martina; Casetta, Ilaria; Gentile, Mauro; Granieri, Enrico; Borea, Pier Andrea; Popoli, Patrizia; Varani, Katia

    2013-09-01

    Adenosine, a purine nucleoside interacting with A1, A2A, A2B and A3 adenosine receptors (ARs), is a potent endogenous modulator of inflammatory and neuronal processes involved in the pathophysiology of several neurodegenerative diseases. In the present study, ARs were investigated in lymphocytes from patients with amyotrophic lateral sclerosis (ALS) and compared with age-matched healthy subjects. In ALS patients A2AARs were analysed by using RT-PCR, Western blotting and saturation binding experiments. The effect of A2AAR stimulation on cyclic AMP levels was evaluated in lymphocytes from ALS patients and healthy subjects. An up-regulation of A2AARs was observed in ALS patients with respect to healthy subjects while A1, A2B and A3AR affinity and density did not change. In ALS patients, the A2AAR density values correlated with the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) scores. Furthermore, the stimulation of A2AARs mediated a significant increase in cyclic AMP levels in lymphocytes from ALS patients, with a higher potency than in lymphocytes from healthy subjects. In conclusion, the positive correlation between A2AAR density and ALSFRS-R scores could indicate a possible protective effect of this receptor subtype, representing an interesting starting point for the study of alternative therapeutic approaches for ALS based on A2AAR modulation.

  7. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

    PubMed Central

    Watson, Michael J.; Lee, Shernita L.; Marklew, Abigail J.; Gilmore, Rodney C.; Gentzsch, Martina; Sassano, Maria F.; Gray, Michael A.; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  8. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor.

    PubMed

    Watson, Michael J; Lee, Shernita L; Marklew, Abigail J; Gilmore, Rodney C; Gentzsch, Martina; Sassano, Maria F; Gray, Michael A; Tarran, Robert

    2016-06-09

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR's function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR's PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs.

  9. Influence of Stress-Induced Catecholamines on Macrophage Phagocytosis

    DTIC Science & Technology

    1989-04-01

    levels of adenosine-3’, 5"-cyclic monophosphate on phagocytosis: effects on macrophage- Trypanosoma cruzi interaction. J. Immunol. 129:2757. 11 7. Lima...decreased phagocytosis of Trgpanosoma cruzi (6) and IgG-coated erythrocytes (7,B) by mouse macrophages. Alterations in cAMP concentrations influence

  10. High-throughput quantification of early stages of phagocytosis.

    PubMed

    Yeo, Jeremy Changyu; Wall, Adam Alexander; Stow, Jennifer Lea; Hamilton, Nicholas Ahti

    2013-09-01

    Phagocytosis--the engulfment of cells and foreign bodies--is an important cellular process in innate immunity, development, and disease. Quantification of various stages of phagocytosis, especially in a rapid screening fashion, is an invaluable tool for elucidating protein function during this process. However, current methods for assessing phagocytosis are largely limited to flow cytometry and manual image-based assays, providing limited information. Here, we present an image-based, semi-automated phagocytosis assay to rapidly quantitate three distinct stages during the early engulfment of opsonized beads. Captured images are analyzed using the image-processing software ImageJ and quantified using a macro. Modifications to this method allowed quantification of phagocytosis only in fluorescently labeled transfected cells. Additionally, the time course of bead internalization could be measured using this approach. The assay could discriminate perturbations to stages of phagocytosis induced by known pharmacological inhibitors of filamentous actin and phosphoinositol-3-kinase. Our methodology offers the ability to automatically categorize large amounts of image data into the three early stages of phagocytosis within minutes, clearly demonstrating its potential value in investigating aberrant phagocytosis when manipulating proteins of interest in drug screens and disease.

  11. Heat production as a quantitative parameter of phagocytosis.

    PubMed

    Hayatsu, H; Miyamae, T; Yamamura, M

    1988-05-09

    Microcalorimetry was applied to measure phagocytosis by human peripheral blood neutrophils and monocytes. Heat production was 9.1 +/- 2.6 microW by 1 X 10(6) unstimulated neutrophils and increased to 28.4 +/- 3.2 microW in association with phagocytosis. The increase in heat production was directly proportional to the number of Saccharomyces cerevisiae particles phagocytosed as well as to the concentration of opsonizing serum. No heat increase was observed in the absence of phagocytosis. An increase in heat production by monocytes was also observed in association with phagocytosis, but it was much less obvious than that by neutrophils. Heat production can thus be used as a quantitative measure of phagocytosis.

  12. A novel real time imaging platform to quantify macrophage phagocytosis.

    PubMed

    Kapellos, Theodore S; Taylor, Lewis; Lee, Heyne; Cowley, Sally A; James, William S; Iqbal, Asif J; Greaves, David R

    2016-09-15

    Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM® real time imaging platform for macrophage phagocytosis based on pHrodo® pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyte™ Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20× less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro.

  13. Estrogen binding by leukocytes during phagocytosis,

    PubMed Central

    1977-01-01

    Estradiol binds covalently to normal leukocytes during phagocytosis. The binding involves three cell types, neutrophils, eosinophils, and monocytes and at least two reaction mechanisms, one involving the peroxidase of neutrophils and monocytes (myeloperoxidase [MPO]) and possibly the eosinophil peroxidase, and the second involving catalase. Binding is markedly reduced when leukocytes from patients with chronic granulomatous disease (CGD), severe leukocytic glucose 6-phosphate dehydrogenase deficiency, and familial lipochrome histiocytosis are employed and two populations of neutrophils, one which binds estradiol and one which does not, can be demonstrated in the blood of a CGD carrier. Leukocytes from patients with hereditary MPO deficiency also bind estradiol poorly although the defect is not as severe as in CGD. These findings are discussed in relation to the inactivation of estrogens during infection and the possible role of estrogens in neutrophil function. PMID:858996

  14. Perfluorochemical emulsions decrease Kupffer cell phagocytosis

    SciTech Connect

    Bottalico, L.A.; Betensky, H.T.; Min, Y.B.; Weinstock, S.B. )

    1991-07-01

    One drawback to using perfluorochemical emulsions as blood substitutes is that perfluorochemical particles are cleared from the blood by the reticuloendothelial system, primarily liver and spleen. The authors measured the impact of two perfluorochemical emulsions on clearance of colloidal carbon (less than 1 microns) and 51Cr-sheep red blood cells (about 8 microns) by the reticuloendothelial system in vivo and in the isolated perfused liver. Male rats were injected with 2 ml/100 gm body wt of Fluosol-DA or Oxypherol-ET for 4 consecutive days. Carbon (1 ml/100 gm body wt) or sheep red blood cells (0.05 ml of 5% vol/vol/100 gm body wt) were then injected intravenously (in vivo) or added to perfusate. Samples were taken at several time points for 1 hr. In the isolated perfused liver, carbon clearance was depressed by 25% 1 day after treatment. Rates returned to control levels by 12 days in Fluosol-DA-treated rats but remained depressed by 67% in Oxypherol-ET-treated rats. Sheep red blood cell (8 microns) clearance was two to five times slower than carbon clearance and depressed by 40% in livers from Fluosol-DA rats 1 day and 12 days after treatment. Added serum did not improve phagocytosis. In vivo carbon clearance remained normal in Fluosol-DA-treated rats but decreased by 74% in Oxypherol-ET-treated rats 1 day after treatment, returning to normal by 12 days. Clearance rates were similar in control rats in vivo and in the perfused liver. They conclude that the isolated perfused liver is a good model to measure liver clearance function. Although low doses of perfluorochemical emulsions may depress Kupffer cell phagocytosis, general reticuloendothelial system function is not significantly compromised.

  15. Cdc42, Rac1, and Rac2 Display Distinct Patterns of Activation during PhagocytosisV⃞

    PubMed Central

    Hoppe, Adam D.; Swanson, Joel A.

    2004-01-01

    The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcγ receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes. PMID:15169870

  16. Dictyostelium discoideum Nucleoside Diphosphate Kinase C Plays a Negative Regulatory Role in Phagocytosis, Macropinocytosis and Exocytosis

    PubMed Central

    Annesley, Sarah J.; Bago, Ruzica; Bosnar, Maja Herak; Filic, Vedrana; Marinović, Maja; Weber, Igor; Mehta, Anil; Fisher, Paul R.

    2011-01-01

    Nucleoside diphosphate kinases (NDPKs) are ubiquitous phosphotransfer enzymes responsible for producing most of the nucleoside triphosphates except for ATP. This role is important for the synthesis of nucleic acids and proteins and the metabolism of sugars and lipids. Apart from this housekeeping role NDPKs have been shown to have many regulatory functions in diverse cellular processes including proliferation and endocytosis. Although the protein has been shown to have a positive regulatory role in clathrin- and dynamin-mediated micropinocytosis, its roles in macropinocytosis and phagocytosis have not been studied. The additional non-housekeeping roles of NDPK are often independent of enzyme activity but dependent on the expression level of the protein. In this study we altered the expression level of NDPK in the model eukaryotic organism Dictyostelium discoideum through antisense inhibition and overexpression. We demonstrate that NDPK levels affect growth, endocytosis and exocytosis. In particular we find that Dictyostelium NDPK negatively regulates endocytosis in contrast to the positive regulatory role identified in higher eukaryotes. This can be explained by the differences in types of endocytosis that have been studied in the different systems - phagocytosis and macropinocytosis in Dictyostelium compared with micropinocytosis in mammalian cells. This is the first report of a role for NDPK in regulating macropinocytosis and phagocytosis, the former being the major fluid phase uptake mechanism for macrophages, dendritic cells and other (non dendritic) cells exposed to growth factors. PMID:21991393

  17. Dictyostelium discoideum nucleoside diphosphate kinase C plays a negative regulatory role in phagocytosis, macropinocytosis and exocytosis.

    PubMed

    Annesley, Sarah J; Bago, Ruzica; Bosnar, Maja Herak; Filic, Vedrana; Marinović, Maja; Weber, Igor; Mehta, Anil; Fisher, Paul R

    2011-01-01

    Nucleoside diphosphate kinases (NDPKs) are ubiquitous phosphotransfer enzymes responsible for producing most of the nucleoside triphosphates except for ATP. This role is important for the synthesis of nucleic acids and proteins and the metabolism of sugars and lipids. Apart from this housekeeping role NDPKs have been shown to have many regulatory functions in diverse cellular processes including proliferation and endocytosis. Although the protein has been shown to have a positive regulatory role in clathrin- and dynamin-mediated micropinocytosis, its roles in macropinocytosis and phagocytosis have not been studied. The additional non-housekeeping roles of NDPK are often independent of enzyme activity but dependent on the expression level of the protein. In this study we altered the expression level of NDPK in the model eukaryotic organism Dictyostelium discoideum through antisense inhibition and overexpression. We demonstrate that NDPK levels affect growth, endocytosis and exocytosis. In particular we find that Dictyostelium NDPK negatively regulates endocytosis in contrast to the positive regulatory role identified in higher eukaryotes. This can be explained by the differences in types of endocytosis that have been studied in the different systems - phagocytosis and macropinocytosis in Dictyostelium compared with micropinocytosis in mammalian cells. This is the first report of a role for NDPK in regulating macropinocytosis and phagocytosis, the former being the major fluid phase uptake mechanism for macrophages, dendritic cells and other (non dendritic) cells exposed to growth factors.

  18. Rab31 and APPL2 enhance FcγR-mediated phagocytosis through PI3K/Akt signaling in macrophages.

    PubMed

    Yeo, Jeremy C; Wall, Adam A; Luo, Lin; Stow, Jennifer L

    2015-03-01

    Membrane remodeling in the early stages of phagocytosis enables the engulfment of particles or pathogens and receptor signaling to activate innate immune responses. Members of the Rab GTPase family and their disparate effectors are recruited sequentially to regulate steps throughout phagocytosis. Rab31 (Rab22b) is known for regulating post-Golgi trafficking, and here we show in macrophages that Rab31-GTP is additionally and specifically recruited to early-stage phagosomes. At phagocytic cups, Rab31 is first recruited during the phosphoinositide transition from PI(4,5)P2 to PI(3,4,5)P3, and it persists on PI(3)P-enriched phagosomes. During early phagocytosis, we find that Rab31 recruits the signaling adaptor APPL2. siRNA depletion of either Rab31 or APPL2 reduces FcγR-mediated phagocytosis. Mechanistically, this corresponds with a delay in the transition to PI(3,4,5)P3 and phagocytic cup closure. APPL2 depletion also reduced PI3K/Akt signaling and enhanced p38 signaling from FcγR. We thus conclude that Rab31/APPL2 is required for key roles in phagocytosis and prosurvival responses of macrophages. Of interest, in terms of localization and function, this Rab31/APPL2 complex is distinct from the Rab5/APPL1 complex, which is also involved in phagocytosis and signaling.

  19. Lyar Is a New Ligand for Retinal Pigment Epithelial Phagocytosis.

    PubMed

    Guo, Feiye; Ding, Ying; Caberoy, Nora B; Alvarado, Gabriela; Liu, Robert; Shen, Chen; Yu, Jisu; Zhou, Yixiong; Salero, Enrique; LeBlanc, Michelle E; Wang, Weiwen; Li, Wei

    2015-10-01

    Phagocytosis is critical to tissue homeostasis, as highlighted by phagocytosis defect of retinal pigment epithelial (RPE) cells with debris accumulation, photoreceptor degeneration and blindness. Phagocytosis ligands are the key to delineating molecular mechanisms and functional roles of phagocytes, but are traditionally identified in individual cases with technical challenges. We recently developed open reading frame phage display (OPD) for phagocytosis-based functional cloning (PFC) to identify unknown ligands. One of the identified ligands was Ly-1 antibody reactive clone (Lyar) with functions poorly defined. Herein, we characterized Lyar as a new ligand to stimulate RPE phagocytosis. In contrast to its reported nucleolar expression, immunohistochemistry showed that Lyar was highly expressed in photoreceptor outer segments (POSs) of the retina. Cytoplasmic Lyar was released from apoptotic cells, and selectively bound to shed POSs and apoptotic cells, but not healthy cells. POS vesicles engulfed through Lyar-dependent pathway were targeted to phagosomes and colocalized with phagosome marker Rab7. These results suggest that Lyar is a genuine RPE phagocytosis ligand, which in turn supports the validity of OPD/PFC as the only available approach for unbiased identification of phagocytosis ligands with broad applicability to various phagocytes.

  20. Cytosolic phospholipase A2α regulates G1 progression through modulating FOXO1 activity

    PubMed Central

    Naini, Said Movahedi; Choukroun, Gabriel J.; Ryan, James R.; Hentschel, Dirk M.; Shah, Jagesh V.; Bonventre, Joseph V.

    2016-01-01

    Group IVA phospholipase A2 [cytosolic phospholipase A2α (cPLA2α)] is a key mediator of inflammation and tumorigenesis. In this study, by using a combination of chemical inhibition and genetic approaches in zebrafish and murine cells, we identify a mechanism by which cPLA2α promotes cell proliferation. We identified 2 cpla2α genes in zebrafish, cpla2αa and cpla2αb, with conserved phospholipase activity. In zebrafish, loss of cpla2α expression or inhibition of cpla2α activity diminished G1 progression through the cell cycle. This phenotype was also seen in both mouse embryonic fibroblasts and mesangial cells. G1 progression was rescued by the addition of arachidonic acid or prostaglandin E2 (PGE2), indicating a phospholipase-dependent mechanism. We further show that PGE2, through PI3K/AKT activation, promoted Forkhead box protein O1 (FOXO1) phosphorylation and FOXO1 nuclear export. This led to up-regulation of cyclin D1 and down-regulation of p27Kip1, thus promoting G1 progression. Finally, using pharmacologic inhibitors, we show that cPLA2α, rapidly accelerated fibrosarcoma (RAF)/MEK/ERK, and PI3K/AKT signaling pathways cooperatively regulate G1 progression in response to platelet-derived growth factor stimulation. In summary, these data indicate that cPLA2α, through its phospholipase activity, is a critical effector of G1 phase progression through the cell cycle and suggest that pharmacological targeting of this enzyme may have important therapeutic benefits in disease mechanisms that involve excessive cell proliferation, in particular, cancer and proliferative glomerulopathies.—Naini, S. M., Choukroun, G. J., Ryan, J. R., Hentschel, D. M., Shah, J. V., Bonventre, J. V. Cytosolic phospholipase A2α regulates G1 progression through modulating FOXO1 activity. PMID:26644349

  1. Effect of Trace Metals on Phagocytosis by Alveolar Macrophages

    PubMed Central

    Graham, Judith A.; Gardner, Donald E.; Waters, Michael D.; Coffin, David L.

    1975-01-01

    Experiments were performed to measure the effect of trace metals on a vital function of the alveolar macrophage (AM), phagocytosis. Since certain trace metals were found to reduce the viability of AMs, a technique was developed to permit examination of live cells only for phagocytosis. Evidence is presented that Ni2+ selectively altered the phagocytic activity of AMs at concentrations lower than those which caused cell death. It is further shown that a level of VO3− that caused extensive lysis and death did not reduce phagocytosis in surviving cells. The effects of Cd2+, Cr3+, and Mn2+ on AMs were also examined. PMID:49299

  2. microRNA-34a-Mediated Down-Regulation of the Microglial-Enriched Triggering Receptor and Phagocytosis-Sensor TREM2 in Age-Related Macular Degeneration

    PubMed Central

    Dua, Prerna; Rogaev, Evgeny I.; Lukiw, Walter J.

    2016-01-01

    The aggregation of Aβ42-peptides and the formation of drusen in age-related macular degeneration (AMD) are due in part to the inability of homeostatic phagocytic mechanisms to clear self-aggregating Aβ42-peptides from the extracellular space. The triggering receptor expressed in myeloid/microglial cells-2 (TREM2), a trans-membrane-spanning, sensor-receptor of the immune-globulin/lectin-like gene superfamily is a critical component of Aβ42-peptide clearance. Here we report a significant deficit in TREM2 in AMD retina and in cytokine- or oxidatively-stressed microglial (MG) cells. RT-PCR, miRNA-array, LED-Northern and Western blot studies indicated up-regulation of a microglial-enriched NF-кB-sensitive miRNA-34a coupled to a down-regulation of TREM2 in the same samples. Bioinformatics/transfection-luciferase reporter assays indicated that miRNA-34a targets the 299 nucleotide TREM2-mRNA-3’UTR, resulting in TREM2 down-regulation. C8B4-microglial cells challenged with Aβ42 were able to phagocytose these peptides, while miRNA-34a down-regulated both TREM2 and the ability of microglial-cells to phagocytose. Treatment of TNFα-stressed MG cells with phenyl-butyl nitrone (PBN), caffeic-acid phenethyl ester (CAPE), the NF-B-inhibitor/resveratrol analog CAY10512 or curcumin abrogated these responses. Incubation of anti-miRNA-34a (AM-34a) normalized miRNA-34a abundance and restored TREM2 back to homeostatic levels. These data support five novel observations: (i) that a ROS- and NF-B-sensitive, miRNA-34a-mediated modulation of TREM2 may in part regulate the phagocytic response; (ii) that gene products encoded on two different chromosomes (miRNA-34a at chr1q36.22 and TREM2 at chr6p21.1) orchestrate a phagocytic-Aβ42-peptide clearance-system; (iii) that this NF-kB-mediated-miRNA-34a-TREM2 mechanism is inducible from outside of the cell; (iv) that when operating normally, this pathway can clear Aβ42 peptide monomers from the extracellular medium; and (v) that anti

  3. Hydrogen peroxide signals E. coli phagocytosis by human polymorphonuclear cells; up-stream and down-stream pathway.

    PubMed

    Petropoulos, Michalis; Karamolegkou, Georgia; Rosmaraki, Eleftheria; Tsakas, Sotiris

    2015-12-01

    Hydrogen peroxide (Η2Ο2) is produced during a variety of cellular procedures. In this paper, the regulatory role of Η2Ο2, in Escherichia coli phagocytosis by the human polymorphonuclears, was investigated. White blood cells were incubated with dihydrorhodamine (DHR) in order to study H2O2 synthesis and E. coli-FITC to study phagocytosis. Flow cytometry revealed increased synthesis of H2O2 in polymorphonuclears which incorporated E. coli-FITC. The blocking of H2O2 synthesis by specific inhibitors, N-ethylmaleimide (ΝΕΜ) for NADPH oxidase and diethyldithiocarbamate (DDC) for superoxide dismutase (SOD), decreased E. coli phagocytosis, as well. Immunoblot analysis of white blood cell protein extracts revealed that the blocking of NADPH oxidase and SOD decreased ERK-1/2 phosphorylation, while it had no effect on JNK and p38. Confocal microscopy showed that phosphorylation of MAPKs and phagocytosis solely occur in the polymorphonuclear and not in mononuclear cells. The use of specific MAPKs inhibitors showed that all of them are necessary for phagocytosis, but only phospho-p38 affects H2O2 synthesis. The blocking of JNK phosphorylation, in the presence of E. coli, evoked a further decrease of cytoplasmic p47 thus increasing its translocation onto the plasma membrane for the assembly of NADPH oxidase. It appears that newly synthesised H2O2 invigorates the phosphorylation and action of ERK-1/2 in E. coli phagocytosis, while phospho-JNK and phospho-p38 appear to regulate H2O2 production.

  4. Metabolites of Aliphatic Alcohols Detected in Alcoholic Beverages Inhibit Phagocytosis.

    PubMed

    Árnyas, Ervin M; Pál, László; Baranyi, Gergő; Bujdosó, Orsolya; Rácz, Gábor; Ádány, Róza; McKee, Martin; Szűcs, Sándor

    2016-07-01

    The aim of our study was to measure granulocyte and monocyte phagocytosis following treatment of cells with some metabolites of aliphatic alcohols alone and in combination with acetaldehyde. The cells were separated from human peripheral blood prior to determination of phagocytosis of opsonized zymosan particles by granulocytes and monocytes treated individually with metabolites of aliphatic alcohols including formaldehyde, 1-propanal, acetone, 1-butanal, and 2-butanone and in combination with acetaldehyde. The findings revealed that metabolites of aliphatic alcohols inhibited phagocytosis by granulocytes and monocytes in a concentration-dependent manner and when combined with acetaldehyde, they caused a further decrease in phagocytic activity. Due to their additive effects, it is possible that, in combination with acetaldehyde, metabolites of aliphatic alcohols may inhibit phagocytosis at physiologically realistic concentrations in episodic heavy drinkers, thereby contributing to their increased susceptibility to infectious diseases. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  5. Resistance of Enterococcus faecium to neutrophil-mediated phagocytosis.

    PubMed Central

    Arduino, R C; Jacques-Palaz, K; Murray, B E; Rakita, R M

    1994-01-01

    During a previous study of the opsonic requirements for neutrophil (polymorphonuclear leukocyte [PMN])-mediated killing of enterococci, we identified two strains of Enterococcus faecium (TX0015 and TX0016) that were resistant to PMN-mediated killing. To better define the mechanism of this resistance, we examined phagocytosis with a fluorescence assay and found that TX0016 was completely resistant to phagocytosis by PMNs; this finding was confirmed by electron microscopy. Examination of multiple strains of enterococci revealed that all 20 strains of Enterococcus faecalis tested were readily phagocytosed (mean, 18 intracellular organisms per PMN; range, 7 to 28). In contrast, only 13 (50%) of 26 strains of E. faecium tested were susceptible to phagocytosis (> or = 7 organisms per PMN); the other 13 strains showed < or = 3 organisms per PMN. Enterococcus casseliflavus ATCC 25788 and one strain of Enterococcus hirae were also resistant to phagocytosis, while two strains of Enterococcus durans, Enterococcus mundtii ATCC 43186, and one strain each of Enterococcus raffinosus and Enterococcus solitarius were readily phagocytosed. Exposure of E. faecium TX0016 to sodium periodate, but not to the protease trypsin or pronase or to phospholipase C, eliminated resistance to phagocytosis. Sialic acid, a common periodate-sensitive structure used by microorganisms to resist opsonization, could not be demonstrated in E. faecium TX0016 by the thiobarbituric acid method, nor was phagocytosis of TX0016 altered by neuraminidase treatment. This study suggests that there is a difference in susceptibility to phagocytosis by PMNs between different species of enterococci and that a carbohydrate-containing moiety which is not sialic acid may be involved in the resistance of E. faecium TX0016 to phagocytosis. Images PMID:7960141

  6. Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry.

    PubMed

    Fine, Noah; Barzilay, Oriyah; Glogauer, Michael

    2017-01-01

    Neutrophils are primary phagocytes that recognize their targets through surface chemistry, either through Pattern Recognition Receptor (PPR) interaction with Pathogen-Associated Molecular Patterns (PAMPs) or through immunoglobulin (Ig) or complement mediated recognition. Opsonization can be important for target recognition, and phagocytosis by neutrophils in whole blood can be greatly enhanced due to the presence of blood serum components and platelets. Powerful and sensitive flow cytometry based methods are presented to measure phagocytosis by human blood neutrophils and mouse peritoneal neutrophils.

  7. Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages.

    PubMed

    Singh, Mahavir; Kapoor, Aniruddh; McCracken, James; Hill, Bradford; Bhatnagar, Aruni

    2017-03-01

    Macrophages are critical drivers of the immune response during infection and inflammation. The pathogenesis of several inflammatory conditions, such as diabetes, cancer and sepsis has been linked with aldose reductase (AR), a member of the aldo-keto reductase (AKR) superfamily. However, the role of AR in the early stages of innate immunity such as phagocytosis remains unclear. In this study, we examined the role of AR in regulating the growth and the phagocytic activity of bone marrow-derived mouse macrophages (BMMs) from AR-null and wild-type (WT) mice. We found that macrophages derived from AR-null mice were larger in size and had a slower growth rate than those derived from WT mice. The AR-null macrophages also displayed higher basal, and lipopolysaccharide (LPS) stimulated phagocytic activity than WT macrophages. Moreover, absence of AR led to a marked increase in cellular levels of both ATP and NADPH. These data suggest that metabolic pathways involving AR suppress macrophage energy production, and that inhibition of AR could induce a favorable metabolic state that promotes macrophage phagocytosis. Hence, modulation of macrophage metabolism by inhibition of AR might represent a novel strategy to modulate host defense responses and to modify metabolism to promote macrophage hypertrophy and phagocytosis under inflammatory conditions.

  8. SIRPα polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells

    PubMed Central

    Nuvolone, Mario; Kana, Veronika; Hutter, Gregor; Sakata, Daiji; Mortin-Toth, Steven M.; Russo, Giancarlo

    2013-01-01

    Prnp−/− mice lack the prion protein PrPC and are resistant to prion infections, but variable phenotypes have been reported in Prnp−/− mice and the physiological function of PrPC remains poorly understood. Here we examined a cell-autonomous phenotype, inhibition of macrophage phagocytosis of apoptotic cells, previously reported in Prnp−/− mice. Using formal genetic, genomic, and immunological analyses, we found that the regulation of phagocytosis previously ascribed to PrPC is instead controlled by a linked locus encoding the signal regulatory protein α (Sirpa). These findings indicate that control of phagocytosis was previously misattributed to the prion protein and illustrate the requirement for stringent approaches to eliminate confounding effects of flanking genes in studies modeling human disease in gene-targeted mice. The plethora of seemingly unrelated functions attributed to PrPC suggests that additional phenotypes reported in Prnp−/− mice may actually relate to Sirpa or other genetic confounders. PMID:24145514

  9. FcγRIIB mediates the inhibitory effect of aggregated α-synuclein on microglial phagocytosis.

    PubMed

    Choi, Yu Ree; Kang, Seo-Jun; Kim, Jin-Mo; Lee, Seung-Jae; Jou, Ilo; Joe, Eun-Hye; Park, Sang Myun

    2015-11-01

    Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. Although the etiology of PD has not yet been fully understood, accumulating evidence indicates that neuroinflammation plays a critical role in the progression of PD. α-Synuclein (α-Syn) has been considered to be a key player of the pathogenesis of PD, and recent reports that prion-like propagation of misfolded α-syn released from neurons may play an important role in the progression of PD have led to increased attention to the studies elucidating the roles of extracellular α-syn in the CNS. Extracellular α-syn has also been reported to regulate microglial inflammatory response. In this study, we demonstrated that aggregated α-syn inhibited microglial phagocytosis by activating SHP-1. SHP-1 activation was also observed in A53T α-syn transgenic mice. In addition, aggregated α-syn bound to FcγRIIB on microglia, inducing SHP-1 activation, further inhibiting microglial phagocytosis. Aggregated α-syn upregulated FcγRIIB expression in microglia and upregulated FcγRIIB was also observed in A53T α-syn transgenic mice. These data suggest that aggregated α-syn released from neurons dysregulates microglial immune response through inhibiting microglial phagocytosis, further causing neurodegeneration observed in PD. The interaction of aggregated α-syn and FcγRIIB and further SHP-1 activation can be a new therapeutic target against PD.

  10. Phagocytosis in vitro of polyethylene glycol-modified liposome-encapsulated hemoglobin by human peripheral blood monocytes plus macrophages through scavenger receptors.

    PubMed

    Shibuya-Fujiwara, N; Hirayama, F; Ogata, Y; Ikeda, H; Ikebuchi, K

    2001-12-07

    Liposome-encapsulated hemoglobin (LEH), a candidate for red blood cell substitute, is cleared from circulation primarily by the phagocytic system, most likely after opsonization of the vesicles by immunoproteins, particularly complement components. Although modification of LEH by polyethylene glycol (PEG) derivatives prolongs its half-life by blocking the opsonization, the half-life is still short as compared with that of red blood cell components. Therefore, this study was performed to elucidate the opsonin-independent mechanisms that regulate phagocytosis of Neo Red Cell (NRC), a PEG-modified LEH, in culture. PKH67 was used as a fluorescence marker, allowing the quantitation of the phagocytosis of NRC by peripheral blood monocytes plus macrophages. The phagocytosis of PKH67-labeled NRC was inhibited by the addition of an excess of unlabeled NRC, indicating that the phagocytosis of PKH67-labeled NRC is specific to NRC, but not to PKH67. The phagocytosis of NRC was blocked about 70% by anti-CD14, 60% by anti-CD36 and 30% by anti-CD51/61 (vitronectin receptor, alpha(v)beta3). These results provided evidence of an opsonin-independent pathway for the phagocytosis of PEG-modified LEH.

  11. Antimicrobial peptide LL-37 promotes bacterial phagocytosis by human macrophages.

    PubMed

    Wan, Min; van der Does, Anne M; Tang, Xiao; Lindbom, Lennart; Agerberth, Birgitta; Haeggström, Jesper Z

    2014-06-01

    LL-37/hCAP-18 is the only human member of the cathelicidin family and plays an important role in killing various pathogens, as well as in immune modulation. In this study, we investigated the effect of LL-37 on bacterial phagocytosis by macrophages and demonstrate that LL-37 enhances phagocytosis of IgG-opsonized Gram-negative and Gram-positive bacteria in a dose- and time-dependent manner by dTHP-1 cells. In addition, LL-37 enhanced phagocytosis of nonopsonized Escherichia coli by human macrophages. Consistently, LL-37 elevated the expression of FcγRs on macrophages but not the complement receptors CD11b and -c. Further studies revealed that the expression of TLR4 and CD14 is also increased on LL-37-treated macrophages. Several lines of evidence indicated that the FPR2/ALX receptor mediated LL-37-induced phagocytosis. However, TLR4 signaling was also coupled to the phagocytic response, as a specific TLR4 antibody significantly suppressed phagocytosis of IgG-opsonized E. coli and nonopsonized E. coli by dTHP-1 cells. Finally, macrophages from Cnlp(-/-) mice exhibited diminished bacterial phagocytosis compared with macrophages from their WT littermates. In conclusion, we demonstrate a novel, immune-modulatory mechanism of LL-37, which may contribute to bacterial clearance.

  12. Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos.

    PubMed

    Nonaka, Saori; Hori, Aki; Nakanishi, Yoshinobu; Kuraishi, Takayuki

    2017-08-03

    The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

  13. Ticagrelor potentiates adenosine-induced stimulation of neutrophil chemotaxis and phagocytosis

    PubMed Central

    Alsharif, Khalaf F.; Thomas, Mark R.; Judge, Heather M.; Khan, Haroon; Prince, Lynne R.; Sabroe, Ian; Ridger, Victoria C.; Storey, Robert F.

    2015-01-01

    In the PLATO study, ticagrelor was associated with fewer pulmonary infections and subsequent deaths than clopidogrel. Neutrophils are a first-line defence against bacterial lung infection; ticagrelor inhibits cellular uptake of adenosine, a known regulator of neutrophil chemotaxis and phagocytosis. We assessed whether the inhibition of adenosine uptake by ticagrelor influences neutrophil chemotaxis and phagocytosis. Neutrophils and erythrocytes were isolated from healthy volunteers. Concentration-dependent effects of adenosine on IL-8-induced neutrophil chemotaxis were investigated and the involved receptors identified using adenosine receptor antagonists. The modulatory effects of ticagrelor on adenosine-mediated changes in neutrophil chemotaxis and phagocytosis of Streptococcus pneumoniae were determined in the presence of erythrocytes to replicate physiological conditions of cellular adenosine uptake. Low-concentration adenosine (10− 8 M) significantly increased IL-8-induced neutrophil chemotaxis (% neutrophil chemotaxis: adenosine 28.7% ± 4.4 vs. control 22.6% ± 2.4; p < 0.01) by acting on the high-affinity A1 receptor. Erythrocytes attenuated the effect of adenosine, although this was preserved by ticagrelor and dipyridamole (another inhibitor of adenosine uptake) but not by control or by cangrelor. Similarly, in the presence of erythrocytes, a low concentration of adenosine (10− 8 M) significantly increased neutrophil phagocytic index compared to control when ticagrelor was present (37.6 ± 6.6 vs. 28.0 ± 6.6; p = 0.028) but had no effect in the absence of ticagrelor. We therefore conclude that the inhibition of cellular adenosine reuptake by ticagrelor potentiates the effects of a nanomolar concentration of adenosine on neutrophil chemotaxis and phagocytosis. This represents a potential mechanism by which ticagrelor could influence host defence against bacterial lung infection. PMID:25869515

  14. Ticagrelor potentiates adenosine-induced stimulation of neutrophil chemotaxis and phagocytosis.

    PubMed

    Alsharif, Khalaf F; Thomas, Mark R; Judge, Heather M; Khan, Haroon; Prince, Lynne R; Sabroe, Ian; Ridger, Victoria C; Storey, Robert F

    2015-08-01

    In the PLATO study, ticagrelor was associated with fewer pulmonary infections and subsequent deaths than clopidogrel. Neutrophils are a first-line defence against bacterial lung infection; ticagrelor inhibits cellular uptake of adenosine, a known regulator of neutrophil chemotaxis and phagocytosis. We assessed whether the inhibition of adenosine uptake by ticagrelor influences neutrophil chemotaxis and phagocytosis. Neutrophils and erythrocytes were isolated from healthy volunteers. Concentration-dependent effects of adenosine on IL-8-induced neutrophil chemotaxis were investigated and the involved receptors identified using adenosine receptor antagonists. The modulatory effects of ticagrelor on adenosine-mediated changes in neutrophil chemotaxis and phagocytosis of Streptococcus pneumoniae were determined in the presence of erythrocytes to replicate physiological conditions of cellular adenosine uptake. Low-concentration adenosine (10(-8)M) significantly increased IL-8-induced neutrophil chemotaxis (% neutrophil chemotaxis: adenosine 28.7%±4.4 vs. control 22.6%±2.4; p<0.01) by acting on the high-affinity A1 receptor. Erythrocytes attenuated the effect of adenosine, although this was preserved by ticagrelor and dipyridamole (another inhibitor of adenosine uptake) but not by control or by cangrelor. Similarly, in the presence of erythrocytes, a low concentration of adenosine (10(-8)M) significantly increased neutrophil phagocytic index compared to control when ticagrelor was present (37.6±6.6 vs. 28.0±6.6; p=0.028) but had no effect in the absence of ticagrelor. We therefore conclude that the inhibition of cellular adenosine reuptake by ticagrelor potentiates the effects of a nanomolar concentration of adenosine on neutrophil chemotaxis and phagocytosis. This represents a potential mechanism by which ticagrelor could influence host defence against bacterial lung infection.

  15. Endothelial Cells Use a Formin-Dependent Phagocytosis-Like Process to Internalize the Bacterium Listeria monocytogenes

    PubMed Central

    Rengarajan, Michelle; Hayer, Arnold; Theriot, Julie A.

    2016-01-01

    Vascular endothelial cells act as gatekeepers that protect underlying tissue from blood-borne toxins and pathogens. Nevertheless, endothelial cells are able to internalize large fibrin clots and apoptotic debris from the bloodstream, although the precise mechanism of such phagocytosis-like uptake is unknown. We show that cultured primary human endothelial cells (HUVEC) internalize both pathogenic and non-pathogenic Listeria bacteria comparably, in a phagocytosis-like process. In contrast with previously studied host cell types, including intestinal epithelial cells and hepatocytes, we find that endothelial internalization of Listeria is independent of all known pathogenic bacterial surface proteins. Consequently, we exploited the internalization and intracellular replication of L. monocytogenes to identify distinct host cell factors that regulate phagocytosis-like uptake in HUVEC. Using siRNA screening and subsequent genetic and pharmacologic perturbations, we determined that endothelial infectivity was modulated by cytoskeletal proteins that normally modulate global architectural changes, including phosphoinositide-3-kinase, focal adhesions, and the small GTPase Rho. We found that Rho kinase (ROCK) is acutely necessary for adhesion of Listeria to endothelial cells, whereas the actin-nucleating formins FHOD1 and FMNL3 specifically regulate internalization of bacteria as well as inert beads, demonstrating that formins regulate endothelial phagocytosis-like uptake independent of the specific cargo. Finally, we found that neither ROCK nor formins were required for macrophage phagocytosis of L. monocytogenes, suggesting that endothelial cells have distinct requirements for bacterial internalization from those of classical professional phagocytes. Our results identify a novel pathway for L. monocytogenes uptake by human host cells, indicating that this wily pathogen can invade a variety of tissues by using a surprisingly diverse suite of distinct uptake mechanisms that

  16. Solute Carrier Family 26 Member a2 (slc26a2) Regulates Otic Development and Hair Cell Survival in Zebrafish.

    PubMed

    Liu, Fei; Xia, Wenjun; Hu, Jiongjiong; Wang, Yingzhi; Yang, Fan; Sun, Shaoyang; Zhang, Jin; Jiang, Nan; Wang, Huijun; Tian, Weidong; Wang, Xu; Ma, Duan

    2015-01-01

    Hearing loss is one of the most prevalent human birth defects. Genetic factors contribute to the pathogenesis of deafness. It is estimated that one-third of deafness genes have already been identified. The current work is an attempt to find novel genes relevant to hearing loss using guilt-by-profiling and guilt-by-association bioinformatics analyses of approximately 80 known non-syndromic hereditary hearing loss (NSHL) genes. Among the 300 newly identified candidate deafness genes, slc26a2 were selected for functional studies in zebrafish. The slc26a2 gene was knocked down using an antisense morpholino (MO), and significant defects were observed in otolith patterns, semicircular canal morphology, and lateral neuromast distributions in morphants. Loss-of-function defects are caused primarily by apoptosis, and morphants are insensitive to sound stimulation and imbalanced swimming behaviours. Morphant defects were found to be partially rescued by co-injection of human SLC26A2 mRNA. All the results suggest that bioinformatics is capable of predicting new deafness genes and this showed slc26a2 is to be a critical otic gene whose dysfunction may induce hearing impairment.

  17. Solute Carrier Family 26 Member a2 (slc26a2) Regulates Otic Development and Hair Cell Survival in Zebrafish

    PubMed Central

    Wang, Yingzhi; Yang, Fan; Sun, Shaoyang; Zhang, Jin; Jiang, Nan; Wang, Huijun; Tian, Weidong; Wang, Xu; Ma, Duan

    2015-01-01

    Hearing loss is one of the most prevalent human birth defects. Genetic factors contribute to the pathogenesis of deafness. It is estimated that one-third of deafness genes have already been identified. The current work is an attempt to find novel genes relevant to hearing loss using guilt-by-profiling and guilt-by-association bioinformatics analyses of approximately 80 known non-syndromic hereditary hearing loss (NSHL) genes. Among the 300 newly identified candidate deafness genes, slc26a2 were selected for functional studies in zebrafish. The slc26a2 gene was knocked down using an antisense morpholino (MO), and significant defects were observed in otolith patterns, semicircular canal morphology, and lateral neuromast distributions in morphants. Loss-of-function defects are caused primarily by apoptosis, and morphants are insensitive to sound stimulation and imbalanced swimming behaviours. Morphant defects were found to be partially rescued by co-injection of human SLC26A2 mRNA. All the results suggest that bioinformatics is capable of predicting new deafness genes and this showed slc26a2 is to be a critical otic gene whose dysfunction may induce hearing impairment. PMID:26375458

  18. Arx together with FoxA2, regulates Shh floor plate expression.

    PubMed

    Cho, Ginam; Lim, Youngshin; Cho, Il-Taeg; Simonet, Jacqueline C; Golden, Jeffrey A

    2014-09-01

    Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.

  19. Casein Kinase 2 Is a Novel Regulator of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Trafficking.

    PubMed

    Chan, Ting; Cheung, Florence Shin Gee; Zheng, Jian; Lu, Xiaoxi; Zhu, Ling; Grewal, Thomas; Murray, Michael; Zhou, Fanfan

    2016-01-04

    Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.

  20. The Phagocytosis and Toxicity of Amorphous Silica

    PubMed Central

    Costantini, Lindsey M.; Gilberti, Renée M.; Knecht, David A.

    2011-01-01

    cases. However, the result suggests a mechanistic difference between FcγRIIA receptor-mediated and non-opsonized silica particle phagocytosis. PMID:21311600

  1. Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2*

    PubMed Central

    Cavazzini, Davide; Meschi, Francesca; Corsini, Romina; Bolchi, Angelo; Rossi, Gian Luigi; Einsle, Oliver; Ottonello, Simone

    2013-01-01

    Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A2 (sPLA2s). TbSP1, the sPLA2 primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A2, whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA2 overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation. PMID:23192346

  2. Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2.

    PubMed

    Cavazzini, Davide; Meschi, Francesca; Corsini, Romina; Bolchi, Angelo; Rossi, Gian Luigi; Einsle, Oliver; Ottonello, Simone

    2013-01-18

    Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.

  3. KIM-1-/TIM-1-mediated phagocytosis links ATG5-/ULK1-dependent clearance of apoptotic cells to antigen presentation

    PubMed Central

    Brooks, Craig R; Yeung, Melissa Y; Brooks, Yang S; Chen, Hui; Ichimura, Takaharu; Henderson, Joel M; Bonventre, Joseph V

    2015-01-01

    Phagocytosis of apoptotic cells by both professional and semi-professional phagocytes is required for resolution of organ damage and maintenance of immune tolerance. KIM-1/TIM-1 is a phosphatidylserine receptor that is expressed on epithelial cells and can transform the cells into phagocytes. Here, we demonstrate that KIM-1 phosphorylation and association with p85 results in encapsulation of phagosomes by lipidated LC3 in multi-membrane organelles. KIM-1-mediated phagocytosis is not associated with increased ROS production, and NOX inhibition does not block LC3 lipidation. Autophagy gene expression is required for efficient clearance of apoptotic cells and phagosome maturation. KIM-1-mediated phagocytosis leads to pro-tolerogenic antigen presentation, which suppresses CD4 T-cell proliferation and increases the percentage of regulatory T cells in an autophagy gene-dependent manner. Taken together, these data reveal a novel mechanism of epithelial biology linking phagocytosis, autophagy and antigen presentation to regulation of the inflammatory response. PMID:26282792

  4. Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

    PubMed Central

    Rieger, Daniela; Assinger, Alice; Einfinger, Katrin; Sokolikova, Barbora; Geiger, Margarethe

    2014-01-01

    Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10–30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal. PMID:25000564

  5. Histamine H3 receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion.

    PubMed

    Iida, Tomomitsu; Yoshikawa, Takeo; Matsuzawa, Takuro; Naganuma, Fumito; Nakamura, Tadaho; Miura, Yamato; Mohsen, Attayeb S; Harada, Ryuichi; Iwata, Ren; Yanai, Kazuhiko

    2015-07-01

    Histamine is a physiological amine which initiates a multitude of physiological responses by binding to four known G-protein coupled histamine receptor subtypes as follows: histamine H1 receptor (H1 R), H2 R, H3 R, and H4 R. Brain histamine elicits neuronal excitation and regulates a variety of physiological processes such as learning and memory, sleep-awake cycle and appetite regulation. Microglia, the resident macrophages in the brain, express histamine receptors; however, the effects of histamine on critical microglial functions such as chemotaxis, phagocytosis, and cytokine secretion have not been examined in primary cells. We demonstrated that mouse primary microglia express H2 R, H3 R, histidine decarboxylase, a histamine synthase, and histamine N-methyltransferase, a histamine metabolizing enzyme. Both forskolin-induced cAMP accumulation and ATP-induced intracellular Ca(2+) transients were reduced by the H3 R agonist imetit but not the H2 R agonist amthamine. H3 R activation on two ubiquitous second messenger signalling pathways suggests that H3 R can regulate various microglial functions. In fact, histamine and imetit dose-dependently inhibited microglial chemotaxis, phagocytosis, and lipopolysaccharide (LPS)-induced cytokine production. Furthermore, we confirmed that microglia produced histamine in the presence of LPS, suggesting that H3 R activation regulate microglial function by autocrine and/or paracrine signalling. In conclusion, we demonstrate the involvement of histamine in primary microglial functions, providing the novel insight into physiological roles of brain histamine.

  6. Regulation of human eosinophil degranulation and activation by endogenous phospholipase A2.

    PubMed Central

    White, S R; Strek, M E; Kulp, G V; Spaethe, S M; Burch, R A; Neeley, S P; Leff, A R

    1993-01-01

    The unique granular proteins of eosinophils may have a pathogenetic role in asthma and in the defense against parasitic infestations. However, the mechanisms regulating eosinophil degranulation are largely unknown. We examined the hypothesis that release of these proteins is regulated by endogenous activation of phospholipase A2. Human eosinophils (HE) were isolated from the peripheral blood of 42 subjects either by Percoll density separation or by negative-selection immunomagnetic fractionation. Eosinophil activation was initiated in vitro with 10(-6) M FMLP and 5 micrograms/ml cytochalasin B and was assessed by measurement of eosinophil peroxidase (EPO), leukotriene C4 (LTC4) and superoxide radical (.O2-) secretion. Treatment of HE with 100 microM mepacrine before activation blocked EPO release (2.0 +/- 0.2 vs 10.2 +/- 2.1% cell content for activated HE, P < 0.004, n = 9), .O2- generation (2.6 +/- 0.9 vs 44.2 +/- 10.8 nmol/ml per 10(6) HE, P < 0.002, n = 5), and LTC4 secretion (68.2 +/- 32.2 vs 1,125.2 +/- 526.8 pg/ml per 10(6) HE, P < 0.04, n = 8). Pretreatment of HE with 100 microM 4-bromophenacyl bromide before activation similarly blocked EPO release, .O2- generation and LTC4 secretion. Addition of AA to HE after treatment with 100 microM mepacrine and before subsequent activation reversed the inhibition of both EPO (10.4 +/- 2.2% with 1 microM AA vs 2.0 +/- 0.2% for mepacrine, n = 5, P < 0.02) and LTC4 secretion (695.1 +/- 412.9 with 10 microM AA vs 68.2 +/- 32.2 pg/ml per 10(6) HE for mepacrine, n = 8, P < 0.04), but did not reverse inhibition of .O2- generation by mepacrine. We demonstrate that secretion of preformed cytotoxic proteins and .O2- by eosinophils is regulated endogenously by phospholipase A2. PMID:8387540

  7. AMPKα1 controls hepatocyte proliferation independently of energy balance by regulating Cyclin A2 expression.

    PubMed

    Merlen, Grégory; Gentric, Géraldine; Celton-Morizur, Séverine; Foretz, Marc; Guidotti, Jacques-Emmanuel; Fauveau, Véronique; Leclerc, Jocelyne; Viollet, Benoit; Desdouets, Chantal

    2014-01-01

    AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status that contributes to restoration of energy homeostasis by slowing down ATP-consuming pathways and activating ATP-producing pathways. Unexpectedly, in different systems, AMPK is also required for proper cell division. In the current study, we evaluated the potential effect of the AMPK catalytic subunit, AMPKα1, on hepatocyte proliferation. Hepatocyte proliferation was determined in AMPKα1 knockout and wild-type mice in vivo after two thirds partial hepatectomy, and in vitro in primary hepatocyte cultures. The activities of metabolic and cell cycle-related signaling pathways were measured. After partial hepatectomy, hepatocytes proliferated rapidly, correlating with increased AMPK phosphorylation. Deletion of AMPKα1 delayed liver regeneration by impacting on G1/S transition phase. The proliferative defect of AMPKα1-deficient hepatocytes was cell autonomous, and independent of energy balance. The priming phase, lipid droplet accumulation, protein anabolic responses and growth factor activation after partial hepatectomy occurred normally in the absence of AMPKα1 activity. By contrast, mRNA and protein expression of cyclin A2, a key driver of S phase progression, were compromised in the absence of AMPK activity. Importantly, AMPKα1 controlled cyclin A2 transcription mainly through the ATF/CREB element. Our study highlights a novel role for AMPKα1 as a positive regulator of hepatocyte division occurring independently of energy balance. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  8. A2A adenosine receptor regulates the human blood brain barrier permeability

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2015-01-01

    The blood brain barrier (BBB) symbolically represents the gateway to the central nervous system. It is a single layer of specialized endothelial cells that coats the central nervous system (CNS) vasculature and physically separates the brain environment from the blood constituents, to maintain the homeostasis of the CNS. However, this protective measure is a hindrance to the delivery of therapeutics to treat neurological diseases. Here, we show that activation of A2A adenosine receptor (AR) with an FDA-approved agonist potently permeabilizes an in vitro primary human brain endothelial barrier (hBBB) to the passage of chemotherapeutic drugs and T cells. T cell migration under AR signaling occurs primarily by paracellular transendothelial route. Permeabilization of the hBBB is rapid, time-dependent and reversible and is mediated by morphological changes in actin-cytoskeletal reorganization induced by RhoA signaling and a potent down-regulation of Claudin-5 and VE-Cadherin. Moreover, the kinetics of BBB permeability in mice closely overlaps with the permeability kinetics of the hBBB. These data suggest that activation of A2A AR is an endogenous mechanism that may be used for CNS drug delivery in human. PMID:25262373

  9. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

    PubMed Central

    Schmidt, Florian I.; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L.

    2015-01-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. PMID:26431038

  10. Imaging the spatial distribution of membrane receptors during neutrophil phagocytosis.

    PubMed

    Kindzelskii, A L; Xue, W; Todd, R F; Petty, H R

    1994-01-01

    Optical microscopy and image processing have been employed to study the distribution of several cell surface receptors on living human neutrophils during opsonin-dependent and opsonin-independent phagocytosis. Receptors were labeled using fluorescein-, rhodamine-, or AMCA-conjugated F(ab')2 fragments of anti-Fc gamma RIIIB (CD16), anti-CR3 (CD11b/CD18), and anti-uPAR (urokinase-type plasminogen activator receptor) antibodies, intact phycoerythrin-labeled interleukin 8, and fluorescein- or rhodamine-labeled Con A (concanavalin A), Boc-PLPLP (tert-butyl-oxycarbonyl-Phe(D)-Leu-Phe(D)-Leu-Phe-OH), and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys. Labeled neutrophils were observed during the phagocytosis of IgG-opsonized erythrocytes and nonopsonized latex beads, Escherichia coli, and Staphylococcus aureus. To quantitate receptor distribution, cells were divided into four quadrants with the first being the point of attachment and the fourth being opposite the point of attachment. Ligated formyl peptide receptors, and to a lesser extent CR3, accumulated at the sites of target internalization for all forms of phagocytosis examined. However, Fc gamma RIIIB, uPAR, IL-8, Con A, and the FPR antagonist FBoc-PLPLP were not polarized on cells during phagocytosis. These data suggest that agonist-labeled formyl peptide receptors may play a broader role in leukocyte function than previously suggested, including possible participation in phagocytosis.

  11. Aliphatic alcohols in spirits inhibit phagocytosis by human monocytes.

    PubMed

    Pál, László; Árnyas, Ervin M; Bujdosó, Orsolya; Baranyi, Gergő; Rácz, Gábor; Ádány, Róza; McKee, Martin; Szűcs, Sándor

    2015-04-01

    A large volume of alcoholic beverages containing aliphatic alcohols is consumed worldwide. Previous studies have confirmed the presence of ethanol-induced immunosuppression in heavy drinkers, thereby increasing susceptibility to infectious diseases. However, the aliphatic alcohols contained in alcoholic beverages might also impair immune cell function, thereby contributing to a further decrease in microbicidal activity. Previous research has shown that aliphatic alcohols inhibit phagocytosis by granulocytes but their effect on human monocytes has not been studied. This is important as they play a crucial role in engulfment and killing of pathogenic microorganisms and a decrease in their phagocytic activity could lead to impaired antimicrobial defence in heavy drinkers. The aim of this study was to measure monocyte phagocytosis following their treatment with those aliphatic alcohols detected in alcoholic beverages. Monocytes were separated from human peripheral blood and phagocytosis of opsonized zymosan particles by monocytes treated with ethanol and aliphatic alcohols individually and in combination was determined. It was shown that these alcohols could suppress the phagocytic activity of monocytes in a concentration-dependent manner and when combined with ethanol, they caused a further decrease in phagocytosis. Due to their additive effects, it is possible that they may inhibit phagocytosis in a clinically meaningful way in alcoholics and episodic heavy drinkers thereby contribute to their increased susceptibility to infectious diseases. However, further research is needed to address this question.

  12. Norepinephrine-mediated Suppression of Phagocytosis by Wound Neutrophils

    PubMed Central

    Gosain, Ankush; Gamelli, Richard L.; DiPietro, Luisa A.

    2009-01-01

    Background The systemic response to injury is characterized by massive release of norepinephrine (NE) into the circulation as a result of global sympathetic activation. Multiple authors have demonstrated NE-mediated alterations in migration of circulating neutrophils to wounds. We hypothesized that NE further alters wound neutrophil phagocytic function through adrenergic signaling pathways. Materials and Methods A standard subcutaneous sponge wound model was employed. Murine wound neutrophils were harvested at 24 and 120 hours after injury and treated with physiologic (10−9M) and pharmacologic (10−6M) doses of norepinephrine. Phagocytosis of green fluorescent protein-labeled E. coli was assayed by flow cytometry. The signaling pathways mediating NE modulation of phagocytosis by wound neutrophils were defined by pharmacologic manipulation of alpha- and beta-adrenorecptors (ARs) and protein kinase A (PKA). Results Pharmacologic-dose NE, but not-physiologic-dose NE, suppressed the phagocytic efficiency of 120-hour wound neutrophils. This alteration in phagocytic efficiency appears to be mediated through alpha- and beta-ARs and downstream PKA. Phagocytosis by 24-hour wound neutrophils was not impacted by NE treatment. Conclusions The present study is the first to demonstrate NE-mediated alterations in the process of phagocytosis by wound neutrophils. We conclude that NE plays a temporally- and dose-defined immunomodulatory role in cutaneous wound healing through alterations in phagocytosis by wound neutrophils, and may represent a target for therapeutic manipulation of the innate immune response. PMID:18952237

  13. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

    PubMed

    Tafesse, Fikadu G; Rashidfarrokhi, Ali; Schmidt, Florian I; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L

    2015-10-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

  14. Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase.

    PubMed

    Chandak, Prakash G; Radovic, Branislav; Aflaki, Elma; Kolb, Dagmar; Buchebner, Marlene; Fröhlich, Eleonore; Magnes, Christoph; Sinner, Frank; Haemmerle, Guenter; Zechner, Rudolf; Tabas, Ira; Levak-Frank, Sanja; Kratky, Dagmar

    2010-06-25

    Macrophage phagocytosis is an essential biological process in host defense and requires large amounts of energy. To date, glucose is believed to represent the prime substrate for ATP production in macrophages. To investigate the relative contribution of free fatty acids (FFAs) in this process, we determined the phagocytosis rates in normal mouse macrophages and macrophages of adipose triglyceride lipase (ATGL)-deficient mice. ATGL was shown to be the rate-limiting enzyme for the hydrolysis of lipid droplet-associated triacylglycerol (TG) in many tissues. Here, we demonstrate that Atgl(-/-) macrophages fail to efficiently hydrolyze cellular TG stores leading to decreased cellular FFA concentrations and concomitant accumulation of lipid droplets, even in the absence of exogenous lipid loading. The reduced availability of FFAs results in decreased cellular ATP concentrations and impaired phagocytosis suggesting that fatty acids must first go through a cycle of esterification and re-hydrolysis before they are available as energy substrate. Exogenously added glucose cannot fully compensate for the phagocytotic defect in Atgl(-/-) macrophages. Hence, phagocytosis was also decreased in vivo when Atgl(-/-) mice were challenged with bacterial particles. These findings imply that phagocytosis in macrophages depends on the availability of FFAs and that ATGL is required for their hydrolytic release from cellular TG stores. This novel mechanism links ATGL-mediated lipolysis to macrophage function in host defense and opens the way to explore possible roles of ATGL in immune response, inflammation, and atherosclerosis.

  15. Bin2 Is a Membrane Sculpting N-BAR Protein That Influences Leucocyte Podosomes, Motility and Phagocytosis

    PubMed Central

    Sánchez-Barrena, María José; Vallis, Yvonne; Clatworthy, Menna R.; Doherty, Gary J.; Veprintsev, Dmitry B.; Evans, Philip R.; McMahon, Harvey T.

    2012-01-01

    Cell motility, adhesion and phagocytosis are controlled by actin and membrane remodelling processes. Bridging integrator-2 (Bin2) also called Breast cancer-associated protein 1 (BRAP1) is a predicted N-BAR domain containing protein with unknown function that is highly expressed in leucocytic cells. In the present study we solved the structure of Bin2 BAR domain and studied its membrane binding and bending properties in vitro and in vivo. Live-cell imaging experiments showed that Bin2 is associated with actin rich structures on the plasma membrane, where it was targeted through its N-BAR domain. Pull-down experiments and immunoprecipitations showed that Bin2 C-terminus bound SH3 domain containing proteins such as Endophilin A2 and α-PIX. siRNA of endogenous protein led to decreased cell migration, increased phagocytosis and reduced podosome density and dynamics. In contrast, overexpression of Bin2 led to decreased phagocytosis and increased podosome density and dynamics. We conclude that Bin2 is a membrane-sculpting protein that influences podosome formation, motility and phagocytosis in leucocytes. Further understanding of this protein may be key to understand the behaviour of leucocytes under physiological and pathological conditions. PMID:23285027

  16. Ethylene-Mediated Regulation of A2-Type CYCLINs Modulates Hyponastic Growth in Arabidopsis.

    PubMed

    Polko, Joanna K; van Rooij, Jop A; Vanneste, Steffen; Pierik, Ronald; Ammerlaan, Ankie M H; Vergeer-van Eijk, Marleen H; McLoughlin, Fionn; Gühl, Kerstin; Van Isterdael, Gert; Voesenek, Laurentius A C J; Millenaar, Frank F; Beeckman, Tom; Peeters, Anton J M; Marée, Athanasius F M; van Zanten, Martijn

    2015-09-01

    Upward leaf movement (hyponastic growth) is frequently observed in response to changing environmental conditions and can be induced by the phytohormone ethylene. Hyponasty results from differential growth (i.e. enhanced cell elongation at the proximal abaxial side of the petiole relative to the adaxial side). Here, we characterize Enhanced Hyponasty-d, an activation-tagged Arabidopsis (Arabidopsis thaliana) line with exaggerated hyponasty. This phenotype is associated with overexpression of the mitotic cyclin CYCLINA2;1 (CYCA2;1), which hints at a role for cell divisions in regulating hyponasty. Indeed, mathematical analysis suggested that the observed changes in abaxial cell elongation rates during ethylene treatment should result in a larger hyponastic amplitude than observed, unless a decrease in cell proliferation rate at the proximal abaxial side of the petiole relative to the adaxial side was implemented. Our model predicts that when this differential proliferation mechanism is disrupted by either ectopic overexpression or mutation of CYCA2;1, the hyponastic growth response becomes exaggerated. This is in accordance with experimental observations on CYCA2;1 overexpression lines and cyca2;1 knockouts. We therefore propose a bipartite mechanism controlling leaf movement: ethylene induces longitudinal cell expansion in the abaxial petiole epidermis to induce hyponasty and simultaneously affects its amplitude by controlling cell proliferation through CYCA2;1. Further corroborating the model, we found that ethylene treatment results in transcriptional down-regulation of A2-type CYCLINs and propose that this, and possibly other regulatory mechanisms affecting CYCA2;1, may contribute to this attenuation of hyponastic growth. © 2015 American Society of Plant Biologists. All Rights Reserved.

  17. Ethylene-Mediated Regulation of A2-Type CYCLINs Modulates Hyponastic Growth in Arabidopsis1[OPEN

    PubMed Central

    Polko, Joanna K.; van Rooij, Jop A.; Vanneste, Steffen; Pierik, Ronald; Ammerlaan, Ankie M.H.; Vergeer-van Eijk, Marleen H.; McLoughlin, Fionn; Gühl, Kerstin; Van Isterdael, Gert; Voesenek, Laurentius A.C.J.; Millenaar, Frank F.; Beeckman, Tom; Peeters, Anton J.M.; Marée, Athanasius F.M.; van Zanten, Martijn

    2015-01-01

    Upward leaf movement (hyponastic growth) is frequently observed in response to changing environmental conditions and can be induced by the phytohormone ethylene. Hyponasty results from differential growth (i.e. enhanced cell elongation at the proximal abaxial side of the petiole relative to the adaxial side). Here, we characterize Enhanced Hyponasty-d, an activation-tagged Arabidopsis (Arabidopsis thaliana) line with exaggerated hyponasty. This phenotype is associated with overexpression of the mitotic cyclin CYCLINA2;1 (CYCA2;1), which hints at a role for cell divisions in regulating hyponasty. Indeed, mathematical analysis suggested that the observed changes in abaxial cell elongation rates during ethylene treatment should result in a larger hyponastic amplitude than observed, unless a decrease in cell proliferation rate at the proximal abaxial side of the petiole relative to the adaxial side was implemented. Our model predicts that when this differential proliferation mechanism is disrupted by either ectopic overexpression or mutation of CYCA2;1, the hyponastic growth response becomes exaggerated. This is in accordance with experimental observations on CYCA2;1 overexpression lines and cyca2;1 knockouts. We therefore propose a bipartite mechanism controlling leaf movement: ethylene induces longitudinal cell expansion in the abaxial petiole epidermis to induce hyponasty and simultaneously affects its amplitude by controlling cell proliferation through CYCA2;1. Further corroborating the model, we found that ethylene treatment results in transcriptional down-regulation of A2-type CYCLINs and propose that this, and possibly other regulatory mechanisms affecting CYCA2;1, may contribute to this attenuation of hyponastic growth. PMID:26041787

  18. Learning to learn self-regulation in practice: a 2 cohort evaluation.

    PubMed

    Sharples, Kath; Moseley, Laurence G

    2011-11-01

    The transfer of nurse training into higher education following 'Project 2000' dramatically changed the status of student nurses. While the majority received a grant or bursary, students were no longer regarded as belonging to the hospital staffing complement. Elcock et al. (2007), argue that the intended advantage of supernumerary status following the move of nurse education into higher education has not been reflected in the student learning experience. Students have reported difficulties in being accepted into the community of practice, as they are no longer viewed as belonging in the professional sense to the practice environment (Cope et al., 2000). Students must therefore be prepared for their supernumerary role through the development of appropriate skills for learning in practice (Elcock et al., 2007). The Thames Valley University pre-registration nursing curriculum promotes the effective socialisation of students into the professional role (Fitzpatrick et al., 1996) through a 35-day practice orientation programme in the Common Foundation Programme. The results of a 2 cohort evaluation of a 35-day programme facilitated in the Brent and Harrow learning community indicate that novice pre-registration students will not always possess the self-directed ability to spontaneously engage with nursing care in practice. Students must be provided the opportunity to develop the skills that define the self-regulated learner (Langendyk, 2006). Students must be empowered to self-assess what they know and what they do not know. It is argued that in order to learn during practice experiences, the student nurse of today must first learn how to be self-regulated. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. A phagocytosis-enhancing factor in human plasma.

    PubMed Central

    Gigli, I; Wintroub, B U; Goetzl, E J

    1976-01-01

    A phagocytosis-enhancing factor (PEF) with the capacity to stimulate the ingestion of sensitized sheep erythrocytes by human polymorphonuclear and mononuclear leucocytes has been isolated from human plasma by chromatography on DEAE-cellulose and filtration on Sephadex G-150 and Sephadex G-100. PEF is a protein of approximately 70,000 molecular weight which is susceptible to inactivation by heating at 60 degrees or by tryptic digestion. PEF promotes phagocytosis of erythrocytes sensitized with intact 7S antibody or bearing the C3b complement fragment, but not of unsensitized erythrocytes or erythrocytes sensitized with 19S antibody. The specificity of PEF interaction with target erythrocytes and the persistence of its stimulatory effect after the target cells are washed suggest that it promotes phagocytosis by an action on the erythrocytes. PMID:1027715

  20. Does tuftsin alter phagocytosis by human polymorphonuclear neutrophils

    SciTech Connect

    Cooper, M.R.; DeChatelet, L.R.; Shirley, P.S.; Cooper, M.R.

    1982-03-01

    The physiological significance of the putative phagocytosis-promoting peptide, tuftsin, was investigated by measurement of chemiluminescence generated during phagocytosis and by assay of the uptake of radiolabeled bacteria. Researchers found no differences in either assay when reasearchers compared serum from splenectomized patients (which purportedly lacks tuftsin) with normal serum. Further, there was no difference when serum from splenectomized patients was employed in the presence of absence of exogenous tuftsin. Similar results were obtained under a variety of conditions, utilizing three different challenge particles with varying particle-cell ratios and serum from 20 different splenectomized patients. These results do not agree with the hypothesis that tuftsin plays a major role in promoting phagocytosis.

  1. Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes.

    PubMed

    Nagasawa, Takahiro; Somamoto, Tomonori; Nakao, Miki

    2015-10-01

    Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb(+) thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb(-) leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb(-) leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair.

  2. Investigations of biomechanical activity of macrophages during phagocytosis

    NASA Astrophysics Data System (ADS)

    Kovari, Daniel; Curtis, Jennifer

    2012-02-01

    Phagocytosis has traditionally been investigated in terms of the relevant biochemical signaling pathways that trigger the process and lead to the deformation of the cell as it engulfs a target. Physical changes in the cell include rearrangement and polymerization of actin in the phagocytic cup, large membrane deformations, increased membrane area via exocytosis, and closure of the phagocytic cup through membrane fusion. Hence, phagocytosis is a fine-tuned balance between biophysical cellular events and chemical signaling, which are responsible for driving these materials and mechanical changes. We present a series of assays designed to probe the physical/mechanical parameters that govern a cell during phagocytosis. Custom built micropipette manipulators are used to manipulate individual cells, facilitating high-resolution microscopy of individual phagocytic events. This work has been supported by NSF PoLS #0848797.

  3. Measuring actin dynamics during phagocytosis using photo-switchable fluorescence

    NASA Astrophysics Data System (ADS)

    Kovari, Daniel T.; Curtis, Jennifer E.

    2013-03-01

    Phagocytosis has traditionally been investigated in terms of the relevant biochemical signaling pathways. However, a growing number of studies investigating the physical aspects of phagocytosis have demonstrated that several distinct forces are exerted throughout particle ingestion. We use variations on FRAP (Fluorescence Recovery After Photobleaching) in combination with photo-switchable fluorescent protein to investigate actin dynamics as a phagocyte attempts to engulf its prey. The goal of our actin studies are to determine the recruitment and polymerization rate of actin in the forming phagosome and whether an organized contractile actin ring is present and responsible for phagosome closure, as proposed in the literature. These experiments are ongoing and contribute to our long term effort of developing a physics based model of phagocytosis.

  4. Effect of biogenic amines on phagocytosis in Tetrahymena thermophila.

    PubMed

    Quiñones-Maldonado, V; Renaud, F L

    1987-11-01

    Stimulation of phagocytosis by serotonin and catecholamines in Tetrahymena grown in proteose-peptone medium proved to be concentration dependent, the optimal concentrations being approximately 0.1 to 1.0 microM. The serotonergic antagonists, spiperone, and metergoline, also stimulated the process, whereas the beta- and alpha-adrenergic antagonists, propranolol, alprenolol, and ergocryptine, had no effect or inhibited phagocytosis. A wide variety of derivatives of the biogenic amines had no effect on phagocytosis, demonstrating the specificity of recognition mechanism for neurohormones in Tetrahymena. Such hormones act by at least two independent mechanisms, one for adrenergic agonists, another for dopamine. Presumably, recognition mechanisms for hormones in protozoa resemble in some respects those in multicellular organisms, therefore bespeaking a common origin.

  5. PDZK1 and NHERF1 regulate the function of human organic anion transporting polypeptide 1A2 (OATP1A2) by modulating its subcellular trafficking and stability.

    PubMed

    Zheng, Jian; Chan, Ting; Cheung, Florence Shin Gee; Zhu, Ling; Murray, Michael; Zhou, Fanfan

    2014-01-01

    The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (V(max): 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)⁻¹ in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.

  6. PDZK1 and NHERF1 Regulate the Function of Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) by Modulating Its Subcellular Trafficking and Stability

    PubMed Central

    Zheng, Jian; Chan, Ting; Cheung, Florence Shin Gee; Zhu, Ling; Murray, Michael; Zhou, Fanfan

    2014-01-01

    The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (Vmax: 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)−1 in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability. PMID:24728453

  7. Circadian-clock driven cone-like photoreceptor phagocytosis in the neural retina leucine zipper gene knockout mouse.

    PubMed

    Krigel, Arthur; Felder-Schmittbuhl, Marie-Paule; Hicks, David

    2010-12-28

    Whereas much information is available on rod outer segment phagocytosis by the retinal pigmented epithelium (RPE), corresponding data for cones are quite limited, especially in laboratory models of normal rats and mice with very low cone numbers. To characterize the light and circadian control of cone photoreceptor phagocytosis in mice, we capitalized on the blue cone-like phenotype of neural retina leucine zipper gene (Nrl) null mice (Nrl(-/-)). Nrl(-/-) mice were maintained under standard cyclic light (12h:12h light-dark [LD] cycle; light=300 lux) for one month, then divided into two groups: 1) continued maintenance in LD (36 mice); or 2) transferred to constant darkness (DD; 21 mice) for 36 h. Animals were sacrificed every 3 h over 24 h, and their eyes were rapidly enucleated and fixed. Cryosections were stained using specific cone short-wavelength opsin antibodies. Phagosome numbers in the RPE were quantified with a morphometric system. We monitored the expression of c-mer proto-oncogene tyrosine kinase (MerTK) in wild-type and knockout mice using a specific MerTK antibody. In LD, cone phagocytosis showed a statistically significant peak of activity 1 h after light onset, 2-3 fold higher than at other times. In constant darkness, the temporal phagocytic profile resembled that of LD (significant peak at 1 h of subjective day), but the number of phagosomes was decreased at all time points. Immunostaining of MerTK in wild-type and Nrl(-/-) mice showed expression at the apical surface of the RPE. Cone-like outer segment phagocytosis in Nrl(-/-) mice shows a similar profile to that of rods in normal mice and other species. These data are the first to quantify blue cone-like photoreceptor phagocytosis under different lighting conditions in mice, and suggest this model may constitute a valuable system for investigating circadian regulation of cone function.

  8. CD13 mediates phagocytosis in human monocytic cells.

    PubMed

    Licona-Limón, Ileana; Garay-Canales, Claudia A; Muñoz-Paleta, Ofelia; Ortega, Enrique

    2015-07-01

    CD13 is a membrane-bound ectopeptidase, highly expressed on monocytes, macrophages, and dendritic cells. CD13 is involved in diverse functions, including degradation of peptide mediators, cellular adhesion, migration, viral endocytosis, signaling, and positive modulation of phagocytosis mediated by FcγRs and other phagocytic receptors. In this work, we explored whether besides acting as an accessory receptor, CD13 by itself is a primary phagocytic receptor. We found that hCD13 mediates efficient phagocytosis of large particles (erythrocytes) modified so as to interact with the cell only through CD13 in human macrophages and THP-1 monocytic cells. The extent of this phagocytosis is comparable with the phagocytosis mediated through the canonical phagocytic receptor FcγRI. Furthermore, we demonstrated that hCD13 expression in the nonphagocytic cell line HEK293 is sufficient to enable these cells to internalize particles bound through hCD13. CD13-mediated phagocytosis is independent of other phagocytic receptors, as it occurs in the absence of FcγRs, CR3, and most phagocytic receptors. Phagocytosis through CD13 is independent of its enzymatic activity but is dependent on actin rearrangement and activation of PI3K and is partially dependent on Syk activation. Moreover, the cross-linking of CD13 with antibodies rapidly induced pSyk in human macrophages. Finally, we observed that antibody-mediated cross-linking of hCD13, expressed in the murine macrophage-like J774 cell line, induces production of ROS. These results demonstrate that CD13 is a fully competent phagocytic receptor capable of mediating internalization of large particles.

  9. Kinase-Dependent and -Independent Roles of EphA2 in the Regulation of Prostate Cancer Invasion and Metastasis

    PubMed Central

    Taddei, Maria Letizia; Parri, Matteo; Angelucci, Adriano; Onnis, Barbara; Bianchini, Francesca; Giannoni, Elisa; Raugei, Giovanni; Calorini, Lido; Rucci, Nadia; Teti, Anna; Bologna, Mauro; Chiarugi, Paola

    2009-01-01

    Ligand-activated Eph tyrosine kinases regulate cellular repulsion, morphology, adhesion, and motility. EphA2 kinase is frequently up-regulated in several different types of cancers, including prostate, breast, colon, and lung carcinomas, as well as in melanoma. The existing data do not clarify whether EphA2 receptor phosphorylation or its simple overexpression, which likely leads to Eph kinase-independent responses, plays a role in the progression of malignant prostate cancer. In this study, we address the role of EphA2 tyrosine phosphorylation in prostate carcinoma cell adhesion, motility, invasion, and formation of metastases. Tumor cells expressing kinase-deficient EphA2 mutants, as well as an EphA2 variant lacking the cytoplasmic domain, are defective in ephrinA1-mediated cell rounding, retraction fiber formation, de-adhesion from the extracellular matrix, RhoA and Rac1 GTPase regulation, three-dimensional matrix invasion, and in vivo metastasis, suggesting a key role for EphA2 kinase activity. Nevertheless, EphA2 regulation of cell motility and invasion, as well as the formation of bone and visceral tumor colonies, reveals a component of both EphA2 kinase-dependent and -independent features. These results uncover a differential requirement for EphA2 kinase activity in the regulation of prostate carcinoma metastasis outcome, suggesting that although the kinase activity of EphA2 is required for the regulation of cell adhesion and cytoskeletal rearrangement, some distinct kinase-dependent and -independent pathways likely cooperate to drive cancer cell migration, invasion, and metastasis outcome. PMID:19264906

  10. An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity.

    PubMed

    Jia, Zhihao; Zhang, Tao; Jiang, Shuai; Wang, Mengqiang; Cheng, Qi; Sun, Mingzhe; Wang, Lingling; Song, Linsheng

    2015-11-01

    Integrins are a family of cell adhesion molecules which play important roles in the regulation of cell adhesion, migration, proliferation, apoptosis and phagocytosis. In the present study, the immune function of an integrin from the oyster Crassostrea gigas (designated CgIntegrin) was characterized to understand the regulatory mechanism of hemocyte phagocytosis toward different microbes. The full-length cDNA of CgIntegrin was 2571 bp with an open reading frame (ORF) of 2397 bp, encoding a polypeptide of 799 amino acids. The mRNA transcripts of CgIntegrin were predominantly detected in hemocytes, gonad and adductor muscle, while lowly in hepatopancreas, mantle and gill. The mRNA expression level was up-regulated at 6 h post lipopolysaccharide (LPS) stimulation (p < 0.01), while no significant change was observed after peptidoglycan (PGN) stimulation. The oyster hemocytes with relative high CgIntegrin expression level exhibited different phagocytic abilities towards different microorganism and particles, such as Gram-positive bacteria Vibrio splendidus, Gram-negative bacteria Staphylococcus aureus and latex beads. Moreover, the phagocytic rate towards V. splendidus was significantly decreased after the blockade of CgIntegrin using the polyclonal antibody. The recombinant CgIntegrin (rCgIntegrin) displayed agglutinating activity towards V. splendidus but not S. aureus and Y. lipolytica. It also exhibited a higher binding affinity towards LPS (compared to rTrx group) in a dose-dependent manner with the apparent dissociation constant (Kd) of 5.53 × 10(-6) M. The results indicated that CgIntegrin served as a pattern recognition receptor with LPS binding activity, which could directly bind to V. splendidus and enhance the phagocytosis of oyster hemocytes.

  11. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid.

    PubMed Central

    Lin, L L; Lin, A Y; Knopf, J L

    1992-01-01

    Cytosolic phospholipase A2 (cPLA2) binds to natural membrane vesicles in a Ca(2+)-dependent fashion, resulting in the selective release of arachidonic acid, thus implicating cPLA2 in the hormonally regulated production of eicosanoids. Here we report that the treatment of Chinese hamster ovary (CHO) cells overexpressing cPLA2 with ATP or thrombin resulted in an increased release of arachidonic acid as compared with parental CHO cells, demonstrating the hormonal coupling of cPLA2. In contrast, CHO cells overexpressing a secreted form of mammalian PLA2 (sPLA2-II) failed to show any increased hormonal responsiveness. Interestingly, we have noted that the activation of cPLA2 with a wide variety of agents stimulates the phosphorylation of cPLA2 on serine residues. Pretreatment of cells with staurosporin blocked the ATP-mediated phosphorylation of cPLA2 and strongly inhibited the activation of the enzyme. Increased cPLA2 activity was also observed in lysates prepared from ATP-treated cells and was sensitive to phosphatase treatment. These results suggest that in addition to Ca2+, the phosphorylation of cPLA2 plays an important role in the agonist-induced activation of cPLA2. Images PMID:1631101

  12. Failure of Immune Sera to Enhance Significantly Phagocytosis of Staphylococus aureus: Nonspecific Adsorption of Phagocytosis-Promoting Factors.

    PubMed

    Shayegani, M

    1970-12-01

    Serum from rabbits immunized with either heat-killed or live nonencapsulated Staphylococcus aureus failed further to enhance phagocytosis and intracellular killing of the homologous organism by either normal rabbit polymorphonuclear leukocytes or monocytes, when compared with normal rabbit serum. These immune sera did, however, show an increase in agglutinating and precipitating antibody level. Adsorption of normal human serum with some gram-positive and gram-negative bacteria, yeast, and some inert particles significantly reduced the phagocytosis-promoting factors of the serum. It would seem, then, that nonencapsulated S. aureus differs from other pathogenic bacteria in that the humoral antibacterial factors promoting its phagocytosis and intracellular killing are not significantly enhanced by infection or immunization.

  13. Quantitative Studies of Phagocytosis by Polymorphonuclear Leukocytes: Use of Emulsions to Measure the Initial Rate of Phagocytosis

    PubMed Central

    Stossel, Thomas P.; Mason, Robert J.; Hartwig, John; Vaughan, Martha

    1972-01-01

    Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red O emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by N-ethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested

  14. Direct suppression of phagocytosis by amphipathic polymeric surfactants.

    PubMed

    Watrous-Peltier, N; Uhl, J; Steel, V; Brophy, L; Merisko-Liversidge, E

    1992-09-01

    Recent studies have demonstrated that phagocytosis of colloidal particles by the mononuclear phagocytes of the liver and spleen can be controlled by either coating or stabilizing particulate carriers with the amphipathic polymeric surfactants, F108 and T908. These surfactants consist of copolymers of polypropylene oxide (PPO) and polyethylene oxide (PEO) and, when adsorbed to particulate surfaces, significantly decrease sequestration of particulates by the mononuclear phagocytes (MPS) of the liver. To evaluate these observations further, murine peritoneal macrophages were incubated for varying periods with surfactant-coated and noncoated polystyrene particles (PSPs). Phagocytosis was monitored using gamma counting and quantitative fluorescence microscopy. The data show that phagocytosis is decreased when PSPs are coated with F108 and T908. In addition, suppression of phagocytic activity was observed when cells were pretreated with the surfactant and then challenged with noncoated particles. The data confirm previous observations that polymeric surfactants consisting of PEO and PPO protect particulate carriers from rapid uptake by the MPS of the liver. Further, F108 and T908 suppress phagocytosis directly without affecting the integrity, viability, or functional state of the cell.

  15. Carbocisteine promotes phagocytosis of apoptotic cells by alveolar macrophages.

    PubMed

    Inoue, Masako; Ishibashi, Yuji; Nogawa, Hisashi; Yasue, Tokutaro

    2012-02-29

    Clearance of apoptotic cells, so-called efferocytosis, by alveolar macrophages (AMs) is important for lung homeostasis and is impaired in pulmonary inflammatory diseases, such as chronic obstructive pulmonary disease and asthma. Carbocisteine, a mucoregulatory drug, corrects the contents of fucose in airway mucus and has anti-inflammatory properties in airway inflammation. Thus, we conducted the present study to better understand the anti-inflammatory properties of carbocisteine. First, we induced airway inflammation in mice with lipopolysaccharide intratracheally. Carbocisteine significantly decreased neutrophil numbers in bronchoalveolar lavage fluid at the resolution phase of inflammation, implying the promotion of neutrophil clearance. Then, we investigated whether carbocisteine would enhance the efferocytosis by AMs isolated from mice and found that this drug promoted not only the phagocytosis but also the binding of apoptotic cells to AMs in vitro. Furthermore, carbocisteine decreased the fucose residues stained with fluorescent fucose-binding lectin, Lens culinaris agglutinin, on the cell surface of AMs. We found here that removing fucose residues from cell surfaces of AMs by fucosidase markedly enhanced both the binding and phagocytosis of apoptotic cells. Finally, AMs from mice orally given carbocisteine also promoted both the binding and phagocytosis ex vivo similarly to in vitro. These results suggest that carbocisteine could promote the clearance of apoptotic cells by AMs in airway. In addition, the present findings suggest that the binding and phagocytosis of apoptotic cells may be modulated by fucose residues on the cell surface of AMs.

  16. Preventing phagocytosis takes more than a sweet disposition.

    PubMed

    Hull, Christina M

    2011-03-17

    While a polysaccharide capsule is known to be important for preventing phagocytosis of the human pathogen Cryptococcus neoformans, other antiphagocytic pathways have been generally elusive. Now, a capsule-independent pathway has been identified that prevents macrophages from ingesting the fungus, contributing to evasion of the host immune response. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. ACTIVATED NEUTROPHILS INHIBIT PHAGOCYTOSIS BY HUMAN MONOCYTE CELLS IN VITRO

    EPA Science Inventory

    We have previously reported the correlation of decreased phagocytosis of opsonized zymosan by sputum monocytic cells with the increase in sputum neutrophils in volunteers 6h after inhalation of endotoxin (20,000 EU) (Alexis, et al. JACI, 2003;112:353). To define whether an intrin...

  18. ACTIVATED NEUTROPHILS INHIBIT PHAGOCYTOSIS BY HUMAN MONOCYTE CELLS IN VITRO

    EPA Science Inventory

    We have previously reported the correlation of decreased phagocytosis of opsonized zymosan by sputum monocytic cells with the increase in sputum neutrophils in volunteers 6h after inhalation of endotoxin (20,000 EU) (Alexis, et al. JACI, 2003;112:353). To define whether an intrin...

  19. Endothelin-1 downregulates sperm phagocytosis by neutrophils in vitro: A physiological implication in bovine oviduct immunity

    PubMed Central

    MAREY, Mohamed Ali; YOUSEF, Mohamed Samy; LIU, Jinghui; MORITA, Kazuhiro; SASAKI, Motoki; HAYAKAWA, Hiroyuki; SHIMIZU, Takashi; ELSHAHAWY, Ibrahim I.; MIYAMOTO, Akio

    2016-01-01

    The oviduct is an active contractile tube that provides the proper environment for sperm transport, capacitation and survival. Oviductal contractions are regulated by autocrine/paracrine secretion of several factors, such as prostaglandins (PGs) and endothelin-1 (EDN-1). We have previously shown that during the preovulatory stage, sperm are exposed to polymorphonuclear neutrophils (PMNs) in the bovine oviduct, and the bovine oviduct epithelial cells (BOECs) secrete molecules including PGE2 that suppress sperm phagocytosis by PMNs in vitro. In this study, we investigated the possible effects of EDN-1 on the phagocytic activity of PMNs toward sperm. The local concentrations of EDN-1 in oviduct fluid and BOEC culture medium ranged from 10–10 to 10–11 M as determined by EIA. Phagocytosis and superoxide production were assayed by co-incubation of sperm pretreated to induce capacitation with PMNs exposed to EDN-1 (0, 10–11, 10–10, 10–9, and 10–8 M) for 2 h. EDN-1 suppressed dose dependently (10–11 to 10–8 M) the phagocytic activity for sperm and superoxide production of PMNs in response to capacitated sperm. Moreover, this suppression was eliminated by an ETB receptor antagonist (BQ-788). EDN-1 suppressed mRNA expression of EDN-1 and ETB but not ETA receptors in PMNs, suggesting the ETB receptor-mediated pathway. Scanning electron microscopic observation revealed that incubation of PMNs with EDN-1 (10–9 M) completely suppressed the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results provide evidence indicating that EDN-1 may be involved in the protection of sperm from phagocytosis by PMNs in the bovine oviduct, supporting sperm survival until fertilization. PMID:26781611

  20. Calmodulin and Ca2+/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis.

    PubMed

    Gonda, K; Komatsu, M; Numata, O

    2000-08-01

    The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.

  1. Role of MicroRNA-26b in Glioma Development and Its Mediated Regulation on EphA2

    PubMed Central

    Wu, Ning; Zhao, Xiangzhong; Liu, Ming; Liu, Haizhou; Yao, Weicheng; Zhang, Yuyan; Cao, Shousong; Lin, Xiukun

    2011-01-01

    Background MicroRNAs (miRNAs) are short, non-coding RNAs that regulate the expression of multiple target genes. Deregulation of miRNAs is common in human tumorigenesis. Low level expression of miR-26b has been found in glioma cells. However, its underlying mechanism of action has not been determined. Methodology/Principal Findings Real-time PCR was employed to measure the expression level of miR-26b in glioma patients and cells. The level of miR-26b was inversely correlated with the grade of glioma. Ectopic expression of miR-26b inhibited the proliferation, migration and invasion of human glioma cells. A binding site for miR-26b was identified in the 3′UTR of EphA2. Over-expression of miR-26b in glioma cells repressed the endogenous level of EphA2 protein. Vasculogenic mimicry (VM) experiments were performed to further confirm the effects of miR-26b on the regulation of EphA2, and the results showed that miR-26b inhibited the VM processes which regulated by EphA2. Significance This study demonstrated that miR-26b may act as a tumor suppressor in glioma and it directly regulates EphA2 expression. EphA2 is a direct target of miR-26b, and the down-regulation of EphA2 mediated by miR-26b is dependent on the binding of miR-26b to a specific response element of microRNA in the 3′UTR region of EphA2 mRNA. PMID:21264258

  2. The nuclear factor kappa B (NF-κB) activation is required for phagocytosis of staphylococcus aureus by RAW 264.7 cells

    SciTech Connect

    Zhu, Fei Yue, Wanfu; Wang, Yongxia

    2014-10-01

    Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor which controls the expression of various genes involved in immune responses. However, it is not clear whether NF-κB activation is critical for phagocytosis when Staphylococcus aureus is the pathogen. Using oligonucleotide microarrays, we investigated whether NF-κB cascade genes are altered in a mouse leukemic monocyte macrophage cell line (RAW 264.7) when the cells were stimulated to activate a host innate immune response against live S. aureus or heat-inactivated S. aureus (HISA). NF-κB cascade genes such as Nfκb1, Nfκbiz, Nfκbie, Rel, Traf1 and Tnfaip3 were up-regulated by all treatments at one hour after incubation. NF-κB play an important role in activating phagocytosis in RAW 264.7 cells infected with S. aureus. Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus and decreased the expression of NFκB1, IL1α, IL1β and TLR2 in this cell line. Our results demonstrate that S. aureus may activate the NF-κB pathway and that NF-κB activation is required for phagocytosis of S. aureus by macrophages. - Highlights: • NF-κB cascade genes such as Nfκb1 and Traf1 were up-regulated by heat-inactivated S. aureus. • Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus. • NF-κB activation is required for phagocytosis of S. aureus by macrophages.

  3. Particulate matter phagocytosis induces tissue factor in differentiating macrophages.

    PubMed

    Milano, M; Dongiovanni, P; Artoni, A; Gatti, S; Rosso, L; Colombo, F; Bollati, V; Maggioni, M; Mannucci, P M; Bertazzi, P A; Fargion, S; Valenti, L

    2016-01-01

    Airborne exposure to particulate matter with diameter < 10 mcM (PM10) has been linked to an increased risk of thromboembolic events, but the mechanisms are not completely understood. The aim of this study was to evaluate the effect of PM10 phagocytosis on the release of procoagulant molecules in human differentiating macrophages, and that of PM10 inhalation in an experimental model in rats. Human monocytes were separated from the peripheral blood by the lymphoprep method, differentiated in vitro and treated with standard PM10 or vehicle. Sprague-Dawley rats were instilled intratracheally with PM10 or vehicle alone. The outcome was expression of proinflammatory genes and of tissue factor (TF). In human differentiating macrophages, PM10 exposure upregulated inflammatory genes, but most consistently induced TF mRNA and protein levels, but not TF protein inhibitor, resulting in increased TF membrane expression and a procoagulant phenotype. Differentiation towards the anti-inflammatory M2 phenotype inhibited PM10 -mediated TF expression. TF induction required phagocytosis of PM10 , whereas phagocytosis of inert particles was less effective. PM10 phagocytosis was associated with a gene expression profile consistent with intracellular retention of iron, inducing oxidative stress. Both PM10 and iron activated the stress kinases ERK1/2 pathway, involved in the induction of TF expression. In rats, alveolar exposure to PM10 was associated with pulmonary recruitment of inflammatory cells and resulted in local, but not systemic, induction of TF expression, which was sufficient to increase circulating TF levels. In conclusion, TF induction by differentiating lung macrophages, activated following phagocytosis, contributes to the increased risk of thromboembolic complications associated with PM10 exposure. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Regulation of fear responses by striatal and extrastriatal adenosine A2A receptors in forebrain.

    PubMed

    Wei, Catherine J; Augusto, Elisabete; Gomes, Catarina A; Singer, Philipp; Wang, Yumei; Boison, Detlev; Cunha, Rodrigo A; Yee, Benjamin K; Chen, Jiang-Fan

    2014-06-01

    Adenosine A2A receptors (A2ARs) are enriched in the striatum but are also present at lower levels in the extrastriatal forebrain (i.e., hippocampus, cortex), integrating dopamine, glutamate, and brain-derived neurotrophic factor (BDNF) signaling, and are thus essential for striatal neuroplasticity and fear and anxiety behavior. We tested two brain region-specific A2AR knockout lines with A2ARs selectively deleted either in the striatum (st-A2AR KO) or the entire forebrain (striatum, hippocampus, and cortex [fb-A2AR KO]) on fear and anxiety-related responses. We also examined the effect of hippocampus-specific A2AR deletion by local injection of adeno-associated virus type 5 (AAV5)-Cre into floxed-A2AR knockout mice. Selectively deleting A2ARs in the striatum increased Pavlovian fear conditioning (both context and tone) in st-A2AR KO mice, but extending the deletion to the rest of the forebrain apparently spared context fear conditioning and attenuated tone fear conditioning in fb-A2AR KO mice. Moreover, focal deletion of hippocampal A2ARs by AAV5-Cre injection selectively attenuated context (but not tone) fear conditioning. Deletion of A2ARs in the entire forebrain in fb-A2AR KO mice also produced an anxiolytic phenotype in both the elevated plus maze and open field tests, and increased the startle response. These extrastriatal forebrain A2AR behavioral effects were associated with reduced BDNF levels in the fb-A2AR KO hippocampus. This study provides evidence that inactivation of striatal A2ARs facilitates Pavlovian fear conditioning, while inactivation of extrastriatal A2ARs in the forebrain inhibits fear conditioning and also affects anxiety-related behavior. Copyright © 2014. Published by Elsevier Inc.

  5. The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway.

    PubMed

    Bento, Carla F; Ashkenazi, Avraham; Jimenez-Sanchez, Maria; Rubinsztein, David C

    2016-06-09

    Forms of Parkinson's disease (PD) are associated with lysosomal and autophagic dysfunction. ATP13A2, which is mutated in some types of early-onset Parkinsonism, has been suggested as a regulator of the autophagy-lysosome pathway. However, little is known about the ATP13A2 effectors and how they regulate this pathway. Here we show that ATP13A2 depletion negatively regulates another PD-associated gene (SYT11) at both transcriptional and post-translational levels. Decreased SYT11 transcription is controlled by a mechanism dependent on MYCBP2-induced ubiquitination of TSC2, which leads to mTORC1 activation and decreased TFEB-mediated transcription of SYT11, while increased protein turnover is regulated by SYT11 ubiquitination and degradation. Both mechanisms account for a decrease in the levels of SYT11, which, in turn, induces lysosomal dysfunction and impaired degradation of autophagosomes. Thus, we propose that ATP13A2 and SYT11 form a new functional network in the regulation of the autophagy-lysosome pathway, which is likely to contribute to forms of PD-associated neurodegeneration.

  6. The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

    PubMed Central

    Bento, Carla F.; Ashkenazi, Avraham; Jimenez-Sanchez, Maria; Rubinsztein, David C.

    2016-01-01

    Forms of Parkinson's disease (PD) are associated with lysosomal and autophagic dysfunction. ATP13A2, which is mutated in some types of early-onset Parkinsonism, has been suggested as a regulator of the autophagy–lysosome pathway. However, little is known about the ATP13A2 effectors and how they regulate this pathway. Here we show that ATP13A2 depletion negatively regulates another PD-associated gene (SYT11) at both transcriptional and post-translational levels. Decreased SYT11 transcription is controlled by a mechanism dependent on MYCBP2-induced ubiquitination of TSC2, which leads to mTORC1 activation and decreased TFEB-mediated transcription of SYT11, while increased protein turnover is regulated by SYT11 ubiquitination and degradation. Both mechanisms account for a decrease in the levels of SYT11, which, in turn, induces lysosomal dysfunction and impaired degradation of autophagosomes. Thus, we propose that ATP13A2 and SYT11 form a new functional network in the regulation of the autophagy–lysosome pathway, which is likely to contribute to forms of PD-associated neurodegeneration. PMID:27278822

  7. PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity

    PubMed Central

    Ramonet, David; Podhajska, Agata; Stafa, Klodjan; Sonnay, Sarah; Trancikova, Alzbeta; Tsika, Elpida; Pletnikova, Olga; Troncoso, Juan C.; Glauser, Liliane; Moore, Darren J.

    2012-01-01

    Mutations in the ATP13A2 gene (PARK9, OMIM 610513) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome and early-onset parkinsonism. ATP13A2 is an uncharacterized protein belonging to the P5-type ATPase subfamily that is predicted to regulate the membrane transport of cations. The physiological function of ATP13A2 in the mammalian brain is poorly understood. Here, we demonstrate that ATP13A2 is localized to intracellular acidic vesicular compartments in cultured neurons. In the human brain, ATP13A2 is localized to pyramidal neurons within the cerebral cortex and dopaminergic neurons of the substantia nigra. ATP13A2 protein levels are increased in nigral dopaminergic and cortical pyramidal neurons of Parkinson's disease brains compared with normal control brains. ATP13A2 levels are increased in cortical neurons bearing Lewy bodies (LBs) compared with neurons without LBs. Using short hairpin RNA-mediated silencing or overexpression to explore the function of ATP13A2, we find that modulating the expression of ATP13A2 reduces the neurite outgrowth of cultured midbrain dopaminergic neurons. We also find that silencing of ATP13A2 expression in cortical neurons alters the kinetics of intracellular pH in response to cadmium exposure. Furthermore, modulation of ATP13A2 expression leads to reduced intracellular calcium levels in cortical neurons. Finally, we demonstrate that silencing of ATP13A2 expression induces mitochondrial fragmentation in neurons. Oppositely, overexpression of ATP13A2 delays cadmium-induced mitochondrial fragmentation in neurons consistent with a neuroprotective effect. Collectively, this study reveals a number of intriguing neuronal phenotypes due to the loss- or gain-of-function of ATP13A2 that support a role for this protein in regulating intracellular cation homeostasis and neuronal integrity. PMID:22186024

  8. Rapamycin-insensitive up-regulation of adipocyte phospholipase A2 in tuberous sclerosis and lymphangioleiomyomatosis.

    PubMed

    Li, Chenggang; Zhang, Erik; Sun, Yang; Lee, Po-Shun; Zhan, Yongzhong; Guo, Yanan; Osorio, Juan C; Rosas, Ivan O; Xu, Kai-Feng; Kwiatkowski, David J; Yu, Jane J

    2014-01-01

    Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/- mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2-/-MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived "mesenchymal" cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role

  9. Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration.

    PubMed

    Moreland, Jessica G; Davis, A Paige; Bailey, Gail; Nauseef, William M; Lamb, Fred S

    2006-05-05

    NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.

  10. HtrA2 suppresses autoimmune arthritis and regulates activation of STAT3

    PubMed Central

    Lee, Seung Hoon; Moon, Young-Mee; Seo, Hyeon-Beom; Kim, Se-Young; Kim, Eun-Kyung; Yi, Junyeong; Nam, Min-Kyung; Min, Jun-Ki; Park, Sung-Hwan; Rhim, Hyangshuk; Cho, Mi-La

    2016-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease that is related to the induction of T helper (Th)17 cells, which secrete interleukin-17, and activation of the signal transducer and activator of transcription (STAT) 3. The expression of high-temperature requirement protein A (HtrA) 2, a serine protease involved in apoptosis, was decreased in RA patients nonresponsive to drug treatment of RA. The aim of this study was to determine whether overexpression of HtrA2 has a therapeutic effect on RA. Th17 differentiation, osteoclastogenesis, and lymphocyte activation are increased in motor neuron degeneration (mnd)2 mice, which lack HtrA2 activity because of a missense mutation (Ser276Cys) in the protease domain of HtrA2. The inhibitor of HtrA2 also increased Th17 differentiation. On the other hand, HtrA2 induced cleavage of STAT3 and overexpression of HtrA2 attenuated CIA in a mouse model. HtrA2 overexpression inhibited plaque development as well as the differentiation of Th17 in ApoE−/− mice after immunization with proteoglycans to induce a hyperlipidemia-based RA animal model. The therapeutic function of HtrA2 in inflammatory diseases is linked with Th17 development and the STAT3 pathway in splenocytes. These results suggest that HtrA2 participates in immunomodulatory activity where the upregulation of HtrA2 may shed light on therapeutic approaches to RA and hyperlipidemia. PMID:28008946

  11. Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development

    PubMed Central

    Nissim, Sahar; Weeks, Olivia; Talbot, Jared C.; Hedgepeth, John W.; Wucherpfennig, Julia; Schatzman-Bone, Stephanie; Swinburne, Ian; Cortes, Mauricio; Alexa, Kristen; Megason, Sean; North, Trista E.; Amacher, Sharon L.; Goessling, Wolfram

    2016-01-01

    The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic versus pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease. PMID:27474396

  12. Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development.

    PubMed

    Nissim, Sahar; Weeks, Olivia; Talbot, Jared C; Hedgepeth, John W; Wucherpfennig, Julia; Schatzman-Bone, Stephanie; Swinburne, Ian; Cortes, Mauricio; Alexa, Kristen; Megason, Sean; North, Trista E; Amacher, Sharon L; Goessling, Wolfram

    2016-10-01

    The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease. Copyright © 2016

  13. Mechanisms of glucocorticoid induced suppression of phagocytosis in murine peritoneal macrophage cultures

    SciTech Connect

    Becker, J.L.

    1986-01-01

    Glucocorticoids suppress phagocytosis of heat killed Saccharomyces cerevisiae in macrophage cultures. In order to determine the mechanisms by which this response occurs, this investigation was initiated to examine whether the suppression of phagocytosis is mediated by a steroid induced phagocytosis inhibitory protein (PIP). Furthermore, it is postulated that these suppressive effects may be associated with alterations in macrophage phospholipid metabolism. To assess the association between phospholipid metabolism and phagocytosis, control and 1 ..mu..M dexamethasone treated macrophages were exposed to the phospholipase inhibitor bromophenacylbromide. The enzyme inhibitor suppressed phagocytosis in a time and dose dependent manner. However, supplying dexamethasone treated cultures with arachidonate did not reverse the steroid induced suppression of phagocytosis, whether the arachidonate was supplied alone or together with indomethacin and nordihydroguaiaretic acid. Control cells, prelabeled with /sup 3/H-arachidonate, exhibited an increased percentage of the radiolabeled fatty acid in neutral lipids following phagocytosis, with a corresponding decrease in the percentage associated with phosphatidylcholine.

  14. Annexin A2 and S100A10 regulate human papillomavirus type 16 entry and intracellular trafficking in human keratinocytes.

    PubMed

    Dziduszko, Agnieszka; Ozbun, Michelle A

    2013-07-01

    Human papillomaviruses (HPVs) cause benign and malignant tumors of the mucosal and cutaneous epithelium. The initial events regulating HPV infection impact the establishment of viral persistence, which is requisite for malignant progression of HPV-infected lesions. However, the precise mechanisms involved in HPV entry into host cells, including the cellular factors regulating virus uptake, are not clearly defined. We show that HPV16 exposure to human keratinocytes initiates epidermal growth factor receptor (EGFR)-dependent Src protein kinase activation that results in phosphorylation and extracellular translocation of annexin A2 (AnxA2). HPV16 particles interact with AnxA2 in association with S100A10 as a heterotetramer at the cell surface in a Ca(2+)-dependent manner, and the interaction appears to involve heparan-sulfonated proteoglycans. We show multiple lines of evidence that this interaction promotes virus uptake into host cells. An antibody to AnxA2 prevents HPV16 internalization, whereas an antibody to S100A10 blocks infection at a late endosomal/lysosomal site. These results suggest that AnxA2 and S100A10 have separate roles during HPV16 binding, entry, and trafficking. Our data additionally imply that AnxA2 and S100A10 may be involved in regulating the intracellular trafficking of virus particles prior to nuclear delivery of the viral genome.

  15. Annexin A2 and S100A10 Regulate Human Papillomavirus Type 16 Entry and Intracellular Trafficking in Human Keratinocytes

    PubMed Central

    Dziduszko, Agnieszka

    2013-01-01

    Human papillomaviruses (HPVs) cause benign and malignant tumors of the mucosal and cutaneous epithelium. The initial events regulating HPV infection impact the establishment of viral persistence, which is requisite for malignant progression of HPV-infected lesions. However, the precise mechanisms involved in HPV entry into host cells, including the cellular factors regulating virus uptake, are not clearly defined. We show that HPV16 exposure to human keratinocytes initiates epidermal growth factor receptor (EGFR)-dependent Src protein kinase activation that results in phosphorylation and extracellular translocation of annexin A2 (AnxA2). HPV16 particles interact with AnxA2 in association with S100A10 as a heterotetramer at the cell surface in a Ca2+-dependent manner, and the interaction appears to involve heparan-sulfonated proteoglycans. We show multiple lines of evidence that this interaction promotes virus uptake into host cells. An antibody to AnxA2 prevents HPV16 internalization, whereas an antibody to S100A10 blocks infection at a late endosomal/lysosomal site. These results suggest that AnxA2 and S100A10 have separate roles during HPV16 binding, entry, and trafficking. Our data additionally imply that AnxA2 and S100A10 may be involved in regulating the intracellular trafficking of virus particles prior to nuclear delivery of the viral genome. PMID:23637395

  16. Thrombocytopenia in Plasmodium vivax Malaria Is Related to Platelets Phagocytosis

    PubMed Central

    Coelho, Helena Cristina C.; Lopes, Stefanie C. P.; Pimentel, João Paulo D.; Nogueira, Paulo A.; Costa, Fábio T. M.; Siqueira, André M.; Melo, Gisely C.; Monteiro, Wuelton M.; Malheiro, Adriana; Lacerda, Marcus V. G.

    2013-01-01

    Background Although thrombocytopenia is a hematological disorder commonly reported in malarial patients, its mechanisms are still poorly understood, with only a few studies focusing on the role of platelets phagocytosis. Methods and Findings Thirty-five malaria vivax patients and eight healthy volunteers (HV) were enrolled in the study. Among vivax malaria patients, thrombocytopenia (<150,000 platelets/µL) was found in 62.9% (22/35). Mean platelet volume (MPV) was higher in thrombocytopenic patients as compared to non- thrombocytopenic patients (p = 0.017) and a negative correlation was found between platelet count and MPV (r = −0.483; p = 0.003). Platelets from HV or patients were labeled with 5-chloromethyl fluorescein diacetate (CMFDA), incubated with human monocytic cell line (THP-1) and platelet phagocytosis index was analyzed by flow cytometry. The phagocytosis index was higher in thrombocytopenic patients compared to non-thrombocytopenic patients (p = 0.042) and HV (p = 0.048). A negative correlation was observed between platelet count and phagocytosis index (r = −0.402; p = 0.016). Platelet activation was assessed measuring the expression of P-selectin (CD62-P) in platelets’ surface by flow cytometry. No significant difference was found in the expression of P-selectin between thrombocytopenic patients and HV (p = 0.092). After evaluating the cytokine profile (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17) in the patients’ sera, levels of IL-6, IL-10 and IFN-γ were elevated in malaria patients compared to HV. Moreover, IL-6 and IL-10 values were higher in thrombocytopenic patients than non-thrombocytopenic ones (p = 0.044 and p = 0.017, respectively. In contrast, TNF-α levels were not different between the three groups, but a positive correlation was found between TNF-α and phagocytosis index (r = −0.305; p = 0.037). Conclusion/Significance Collectively, our findings indicate that platelet

  17. Retinoid X receptor activation reverses age-related deficiencies in myelin debris phagocytosis and remyelination

    PubMed Central

    Natrajan, Muktha S.; de la Fuente, Alerie G.; Crawford, Abbe H.; Linehan, Eimear; Nuñez, Vanessa; Johnson, Kory R.; Wu, Tianxia; Fitzgerald, Denise C.; Ricote, Mercedes; Bielekova, Bibiana

    2015-01-01

    The efficiency of central nervous system remyelination declines with age. This is in part due to an age-associated decline in the phagocytic removal of myelin debris, which contains inhibitors of oligodendrocyte progenitor cell differentiation. In this study, we show that expression of genes involved in the retinoid X receptor pathway are decreased with ageing in both myelin-phagocytosing human monocytes and mouse macrophages using a combination of in vivo and in vitro approaches. Disruption of retinoid X receptor function in young macrophages, using the antagonist HX531, mimics ageing by reducing myelin debris uptake. Macrophage-specific RXRα (Rxra) knockout mice revealed that loss of function in young mice caused delayed myelin debris uptake and slowed remyelination after experimentally-induced demyelination. Alternatively, retinoid X receptor agonists partially restored myelin debris phagocytosis in aged macrophages. The agonist bexarotene, when used in concentrations achievable in human subjects, caused a reversion of the gene expression profile in multiple sclerosis patient monocytes to a more youthful profile and enhanced myelin debris phagocytosis by patient cells. These results reveal the retinoid X receptor pathway as a positive regulator of myelin debris clearance and a key player in the age-related decline in remyelination that may be targeted by available or newly-developed therapeutics. PMID:26463675

  18. Up-regulation of eEF1A2 promotes proliferation and inhibits apoptosis in prostate cancer

    SciTech Connect

    Sun, Yue; Du, Chengli; Wang, Bo; Zhang, Yanling; Liu, Xiaoyan; Ren, Guoping

    2014-07-18

    Highlights: • The expression of eEF1A2 is up-regulated in prostate cancer tissues. • Suppression of eEF1A2 inhibits the proliferation and promotes apoptosis. • Inhibition of eEF1A2 enhances the expression of apoptotic relevant proteins. • The expressions of eEF1A2 and cleavage-caspase3 are inversely correlated. - Abstract: Background: eEF1A2 is a protein translation factor involved in protein synthesis, which possesses important function roles in cancer development. This study aims at investigating the expression pattern of eEF1A2 in prostate cancer and its potential role in prostate cancer development. Methods: We examined the expression level of eEF1A2 in 30 pairs of prostate cancer tissues by using RT-PCR and immunohistochemical staining (IHC). Then we applied siRNA specifically targeting eEF1A2 to down-regulate its expression in DU-145 and PC-3 cells. Flow cytometer was used to explore apoptosis and Western-blot was used to detect the pathway proteins of apoptosis. Results: Our results showed that the expression level of eEF1A2 in prostate cancer tissues was significantly higher compared to their corresponding normal tissues. Reduction of eEF1A2 expression in DU-145 and PC-3 cells led to a dramatic inhibition of proliferation accompanied with enhanced apoptosis rate. Western blot revealed that apoptosis pathway proteins (caspase3, BAD, BAX, PUMA) were significantly up-regulated after suppression of eEF1A2. More importantly, the levels of eEF1A2 and caspase3 were inversely correlated in prostate cancer tissues. Conclusion: Our data suggests that eEF1A2 plays an important role in prostate cancer development, especially in inhibiting apoptosis. So eEF1A2 might serve as a potential therapeutic target in prostate cancer.

  19. EphA2 is a key effector of the MEK/ERK/RSK pathway regulating glioblastoma cell proliferation.

    PubMed

    Hamaoka, Yuho; Negishi, Manabu; Katoh, Hironori

    2016-08-01

    EphA2, a member of the Eph receptor tyrosine kinases, is frequently overexpressed in a variety of malignancies, including glioblastoma, and its expression is correlated with poor prognosis. EphA2 acts as a tumor promoter through a ligand ephrin-independent mechanism, which requires phosphorylation of EphA2 on serine 897 (S897), leading to increased cell migration and invasion. In this study, we show that ligand-independent EphA2 signaling occurs downstream of the MEK/ERK/RSK pathway and mediates epidermal growth factor (EGF)-induced cell proliferation in glioblastoma cells. Suppression of EphA2 expression by long-term exposure to ligand ephrinA1 or EphA2-targeted shRNA inhibited EGF-induced cell proliferation. Stimulation of the cells with EGF induced EphA2 S897 phosphorylation, which was suppressed by MEK and RSK inhibitors, but not by phosphatidylinositol 3-kinase (PI3K) and Akt inhibitors. The RSK inhibitor or RSK2-targeted shRNA also suppressed EGF-induced cell proliferation. Furthermore, overexpression of wild-type EphA2 promoted cell proliferation without EGF stimulation, whereas overexpression of EphA2-S897A mutant suppressed EGF- or RSK2-induced proliferation. Taken together, these results suggest that EphA2 is a key downstream target of the MEK/ERK/RSK signaling pathway in the regulation of glioblastoma cell proliferation.

  20. Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis

    PubMed Central

    Sierra, Amanda; Encinas, Juan M.; Deudero, Juan JP; Chancey, Jessica H.; Enikolopov, Grigori; Overstreet-Wadiche, Linda S.; Tsirka, Stella E.; Maletic-Savatic, Mirjana

    2010-01-01

    Summary In the adult hippocampus, neuroprogenitor cells in the subgranular zone (SGZ) of the dentate gyrus give rise to newborn neuroblasts. However, only a small subset of these cells integrates into the hippocampal circuitry as mature neurons at the end of a four-week period. Here, we show that the majority of the newborn cells undergo death by apoptosis in the first one to four days of their life, during the transition from amplifying neuroprogenitors to neuroblasts. These apoptotic newborn cells are rapidly cleared out through phagocytosis by unchallenged microglia present in the adult SGZ niche. Phagocytosis by the microglia is efficient and undeterred by increased age or inflammatory challenge. Our results suggest that the main critical period of newborn cell survival occurs within a few days of birth and reveal a new role for microglia in maintaining the homeostasis of the baseline neurogenic cascade. PMID:20887954

  1. Integrins and small GTPases as modulators of phagocytosis.

    PubMed

    Sayedyahossein, Samar; Dagnino, Lina

    2013-01-01

    Phagocytosis is the mechanism whereby cells engulf large particles. This process has long been recognized as a critical component of the innate immune response, which constitutes the organism's defense against microorganisms. In addition, phagocytic internalization of apoptotic cells or cell fragments plays important roles in tissue homeostasis and remodeling. Phagocytosis requires target interactions with receptors on the plasma membrane of the phagocytic cell. Integrins have been identified as important mediators of particle clearance, in addition to their well-established roles in cell adhesion, migration and mechanotransduction. Indeed, these ubiquitously expressed proteins impart phagocytic capacity to epithelial, endothelial and mesenchymal cell types. The importance of integrins in particle internalization is emphasized by the ability of microbial and viral pathogens to exploit their signaling pathways to invade host cells, and by the wide variety of disorders that arise from abnormalities in integrin-dependent phagocytic uptake.

  2. Adenosine A2 receptor-mediated regulation of renal hemodynamics and glomerular filtration rate is abolished in diabetes.

    PubMed

    Persson, Patrik; Hansell, Peter; Palm, Fredrik

    2013-01-01

    Alterations in glomerular filtration rate (GFR) are one of the earliest indications of altered kidney function in diabetes. Adenosine regulates GFR through tubuloglomerular feedback mechanism acting on adenosine A1 receptor. In addition, adenosine can directly regulate vascular tone by acting on A1 and A2 receptors expressed in afferent and efferent arterioles. Opposite to A1 receptors, A2 receptors mediate vasorelaxation. This study investigates the involvement of adenosine A2 receptors in regulation of renal blood flow (RBF) and GFR in control and diabetic kidneys. GFR was measured by inulin clearance and RBF by a transonic flow probe placed around the renal artery. Measurements were performed in isoflurane-anesthetized normoglycemic and alloxan-diabetic C57BL/6 mice during baseline and after acute administration of 3,7-dimethyl-1-propargylxanthine (DMPX), a selective A2 receptor antagonist. GFR and RBF were lower in diabetic mice compared to control (258 ± 61 vs. 443 ± 33 μl min(-1) and 1,083 ± 51 vs. 1,405 ± 78 μl min(-1)). In control animals, DMPX decreased RBF by -6%, whereas GFR increased +44%. DMPX had no effects on GFR and RBF in diabetic mice. Sodium excretion increased in diabetic mice after A2 receptor blockade (+78%). In conclusion, adenosine acting on A2 receptors mediates an efferent arteriolar dilatation which reduces filtration fraction (FF) and maintains GFR within normal range in normoglycemic mice. However, this regulation is absent in diabetic mice, which may contribute to reduced oxygen availability in the diabetic kidney.

  3. Computer Image Analysis Method for Rapid Quantitation of Macrophage Phagocytosis

    DTIC Science & Technology

    1990-01-01

    number of micro- spheres per cell. *Rapid Macrophage Phagocytosis Ouantitatlon 405 analysis system is a Kontron-Zeiss SEM-IPS ( Carl Zeiss, digitizing...re- croscope (Photomicroscope 1I, Carl Zeiss, Inc., Thorn- suiting image was scaled by making grey values from 0 wood, NY) equipped with phase...fidelity of the discrim- lease 4.4 ( Carl Zeiss, Inc., Thornwood, NY) was used to ination was evaluated by overlaying contour maps of the analyze the

  4. The Physiology of Phagocytosis in the Context of Mitochondrial Origin.

    PubMed

    Martin, William F; Tielens, Aloysius G M; Mentel, Marek; Garg, Sriram G; Gould, Sven B

    2017-09-01

    How mitochondria came to reside within the cytosol of their host has been debated for 50 years. Though current data indicate that the last eukaryote common ancestor possessed mitochondria and was a complex cell, whether mitochondria or complexity came first in eukaryotic evolution is still discussed. In autogenous models (complexity first), the origin of phagocytosis poses the limiting step at eukaryote origin, with mitochondria coming late as an undigested growth substrate. In symbiosis-based models (mitochondria first), the host was an archaeon, and the origin of mitochondria was the limiting step at eukaryote origin, with mitochondria providing bacterial genes, ATP synthesis on internalized bioenergetic membranes, and mitochondrion-derived vesicles as the seed of the eukaryote endomembrane system. Metagenomic studies are uncovering new host-related archaeal lineages that are reported as complex or phagocytosing, although images of such cells are lacking. Here we review the physiology and components of phagocytosis in eukaryotes, critically inspecting the concept of a phagotrophic host. From ATP supply and demand, a mitochondrion-lacking phagotrophic archaeal fermenter would have to ingest about 34 times its body weight in prokaryotic prey to obtain enough ATP to support one cell division. It would lack chemiosmotic ATP synthesis at the plasma membrane, because phagocytosis and chemiosmosis in the same membrane are incompatible. It would have lived from amino acid fermentations, because prokaryotes are mainly protein. Its ATP yield would have been impaired relative to typical archaeal amino acid fermentations, which involve chemiosmosis. In contrast, phagocytosis would have had great physiological benefit for a mitochondrion-bearing cell. Copyright © 2017 American Society for Microbiology.

  5. Insulin antagonizes the phagocytosis stimulating action of histamine in Tetrahymena.

    PubMed

    Csaba, G; Darvas, Z

    1992-02-01

    Histamine increased specifically the phagocytic activity of the unicellular Tetrahymena, whereas insulin had no influence on it. Insulin antagonized the phagocytosis stimulating action of histamine after simultaneous exposure and after preexposure two days earlier as well, although in the latter case to a lesser degree. Double exposure to a combination of histamine+insulin didn't influence the phagocytic activity at all, demonstrating the histamine antagonizing effect of insulin in this model.

  6. Phagocytosis in pup and adult harbour, grey and harp seals.

    PubMed

    Frouin, Héloïse; Lebeuf, Michel; Hammill, Mike; Fournier, Michel

    2010-04-15

    Knowledge on pinniped immunology is still in its infancy. For instance, age-related and developmental aspects of the immune system in pinnipeds need to be better described. The present study examined the phagocytic activity and efficiency of harbour, grey and harp seal leukocytes. In the first part of the study, peripheral blood was collected from captive female harbour seals of various ages. Data showed an age-related decrease in phagocytosis in female harbour seals from sub-adult to adulthood. In the second part of the study, changes in phagocytosis were quantified during lactation in wild newborn harbour, grey and harp seals and in their mothers (harp and grey seals). In newborns of the same age, leukocytes of harbour and harp seals phagocytosed less than those of grey seal pups. The phagocytic activity and efficiency increased significantly from early to mid-lactation in newborn harbour seals, and from early to late lactation in newborn grey seals, which could suggest that the transfer of phagocytosis-promoting factor(s) in colostrum is an important feature of temporary protection for pups. In contrast, no changes in phagocytic activity and efficiency were observed in lactating females of the two seal species, harp and grey, examined. At late lactation, phagocytic activity in both grey and harp seal pups and phagocytic efficiency in grey seal pups were significantly higher than in their mothers. These results could reflect either the capacity of phagocytes of the newborn harp and grey seals to respond to pathogens. Results from this study suggest that the phagocytosis of the seal species examined is not fully developed at birth as it generally increases in pups during lactation. Thereafter, the phagocytic activity of seals appears to decrease throughout adulthood.

  7. Phagocytosis, oxidative burst, and produced reactive species are affected by iron deficiency anemia and anemia of chronic diseases in elderly.

    PubMed

    Paino, I M M; Miranda, J C; Marzocchi-Machado, C M; Cesarino, E J; de Castro, F A; de Souza, A M

    2009-01-01

    Iron and oxidative stress have a regulatory interplay. During the oxidative burst, phagocytic cells produce free radicals such as hypochlorous acid (HOCl). Nevertheless, scarce studies evaluated the effect of either iron deficiency anemia (IDA) or anemia of chronic disease (ACD) on phagocyte function in the elderly. The aim of the present study was to determine the oxidative burst, phagocytosis, and nitric oxide (*NO) and HOCl, reactive species produced by monocytes and neutrophils in elderly with ACD or IDA. Soluble transferrin receptor, serum ferritin, and soluble transferrin receptor/log ferritin (TfR-F) index determined the iron status. The study was constituted of 39 patients aged over 60 (28 women and 11 men) recruited from the Brazilian Public Health System. Oxidative burst fluorescence intensity per neutrophil in IDA group and HOCl generation in both ACD and IDA groups were found to be lower (p < 0.05). The percentages of neutrophils and monocytes expressing phagocytosis in ACD group were found to be higher (p < 0.05). There was an overproduction of *NO from monocytes, whereas the fundamental generation of HOCl appeared to be lower. Phagocytosis, oxidative burst, and *NO and HOCl production are involved in iron metabolism regulation in elderly patients with ACD and IDA.

  8. Entamoeba histolytica Cell Surface Calreticulin Binds Human C1q and Functions in Amebic Phagocytosis of Host Cells

    PubMed Central

    Vaithilingam, Archana; Teixeira, Jose E.; Miller, Peter J.; Heron, Bradley T.

    2012-01-01

    Phagocytosis of host cells is characteristic of tissue invasion by the intestinal ameba Entamoeba histolytica, which causes amebic dysentery and liver abscesses. Entamoeba histolytica induces host cell apoptosis and uses ligands, including C1q, on apoptotic cells to engulf them. Two mass spectrometry analyses identified calreticulin in amebic phagosome preparations, and, in addition to its function as an endoplasmic reticulum chaperone, calreticulin is believed to be the macrophage receptor for C1q. The purpose of this study was to determine if calreticulin functions as an E. histolytica C1q receptor during phagocytosis of host cells. Calreticulin was localized to the surface of E. histolytica during interaction with both Jurkat lymphocytes and erythrocytes and was present in over 75% of phagocytic cups during amebic erythrophagocytosis. Presence of calreticulin on the cell surface was further demonstrated using a method that selectively biotinylated cell surface proteins and by flow cytometry using trophozoites overexpressing epitope-tagged calreticulin. Regulated overexpression of calreticulin increased E. histolytica's ability to phagocytose apoptotic lymphocytes and calcium ionophore-treated erythrocytes but had no effect on amebic adherence to or destruction of cell monolayers or surface expression of the GalNAc lectin and serine-rich E. histolytica protein (SREHP) receptors. Finally, E. histolytica calreticulin bound specifically to apoptotic lymphocytes and to human C1q. Collectively, these data implicate cell surface calreticulin as a receptor for C1q during E. histolytica phagocytosis of host cells. PMID:22473608

  9. HlSRB, a Class B scavenger receptor, is key to the granulocyte-mediated microbial phagocytosis in ticks.

    PubMed

    Aung, Kyaw Min; Boldbaatar, Damdinsuren; Umemiya-Shirafuji, Rika; Liao, Min; Tsuji, Naotoshi; Xuenan, Xuan; Suzuki, Hiroshi; Kume, Aiko; Galay, Remil Linggatong; Tanaka, Tetsuya; Fujisaki, Kozo

    2012-01-01

    Ixodid ticks transmit various pathogens of deadly diseases to humans and animals. However, the specific molecule that functions in the recognition and control of pathogens inside ticks is not yet to be identified. Class B scavenger receptor CD36 (SRB) participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins. Recently, we have reported on recombinant HlSRB, a 50-kDa protein with one hydrophobic SRB domain from the hard tick, Haemaphysalis longicornis. Here, we show that HlSRB plays vital roles in granulocyte-mediated phagocytosis to invading Escherichia coli and contributes to the first-line host defense against various pathogens. Data clearly revealed that granulocytes that up-regulated the expression of cell surface HlSRB are almost exclusively involved in hemocyte-mediated phagocytosis for E. coli in ticks, and post-transcriptional silencing of the HlSRB-specific gene ablated the granulocytes' ability to phagocytose E. coli and resulted in the mortality of ticks due to high bacteremia. This is the first report demonstrating that a scavenger receptor molecule contributes to hemocyte-mediated phagocytosis against exogenous pathogens, isolated and characterized from hematophagous arthropods.

  10. HlSRB, a Class B Scavenger Receptor, Is Key to the Granulocyte-Mediated Microbial Phagocytosis in Ticks

    PubMed Central

    Aung, Kyaw Min; Boldbaatar, Damdinsuren; Umemiya-Shirafuji, Rika; Liao, Min; Tsuji, Naotoshi; Xuenan, Xuan; Suzuki, Hiroshi; Kume, Aiko; Galay, Remil Linggatong; Tanaka, Tetsuya; Fujisaki, Kozo

    2012-01-01

    Ixodid ticks transmit various pathogens of deadly diseases to humans and animals. However, the specific molecule that functions in the recognition and control of pathogens inside ticks is not yet to be identified. Class B scavenger receptor CD36 (SRB) participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins. Recently, we have reported on recombinant HlSRB, a 50-kDa protein with one hydrophobic SRB domain from the hard tick, Haemaphysalis longicornis. Here, we show that HlSRB plays vital roles in granulocyte-mediated phagocytosis to invading Escherichia coli and contributes to the first-line host defense against various pathogens. Data clearly revealed that granulocytes that up-regulated the expression of cell surface HlSRB are almost exclusively involved in hemocyte-mediated phagocytosis for E. coli in ticks, and post-transcriptional silencing of the HlSRB-specific gene ablated the granulocytes' ability to phagocytose E. coli and resulted in the mortality of ticks due to high bacteremia. This is the first report demonstrating that a scavenger receptor molecule contributes to hemocyte-mediated phagocytosis against exogenous pathogens, isolated and characterized from hematophagous arthropods. PMID:22479406

  11. Mechanics of neutrophil phagocytosis: experiments and quantitative models.

    PubMed

    Herant, Marc; Heinrich, Volkmar; Dembo, Micah

    2006-05-01

    To quantitatively characterize the mechanical processes that drive phagocytosis, we observed the FcgammaR-driven engulfment of antibody-coated beads of diameters 3 mum to 11 mum by initially spherical neutrophils. In particular, the time course of cell morphology, of bead motion and of cortical tension were determined. Here, we introduce a number of mechanistic models for phagocytosis and test their validity by comparing the experimental data with finite element computations for multiple bead sizes. We find that the optimal models involve two key mechanical interactions: a repulsion or pressure between cytoskeleton and free membrane that drives protrusion, and an attraction between cytoskeleton and membrane newly adherent to the bead that flattens the cell into a thin lamella. Other models such as cytoskeletal expansion or swelling appear to be ruled out as main drivers of phagocytosis because of the characteristics of bead motion during engulfment. We finally show that the protrusive force necessary for the engulfment of large beads points towards storage of strain energy in the cytoskeleton over a large distance from the leading edge ( approximately 0.5 microm), and that the flattening force can plausibly be generated by the known concentrations of unconventional myosins at the leading edge.

  12. Signalling and phagocytosis in the orchestration of host defence.

    PubMed

    Blander, J Magarian

    2007-02-01

    Dendritic cells (DCs) orchestrate either tolerance or immunity. At the heart of this function lies phagocytosis, which allows DCs to sample the tissue microenvironment and deliver both its self and non-self constituents into endocytic compartments for clearance, degradation and presentation by major histocompatibility complex (MHC) molecules. Depending on the type of signalling pathways triggered during phagocytosis, DCs deliver appropriate signals to T cells that determine either their tolerance or activation and differentiation. Here I draw attention to the ability of DCs to read the contents of their phagosomes depending on the type of compartmentalized signalling pathways engaged during internalization. Toll-like receptors (TLRs) engaged during phagocytosis of microbial pathogens, but not syngeneic apoptotic cells exert phagosome autonomous control on both the kinetics and outcome of phagosome maturation. By bearing the assembly of signalling complexes on their membranes, individual phagosomes undergo separate programmes of maturation irrespective of the activation status of the DC carrying them. Phagosomes carrying microbial cargo are favoured for MHC class II presentation precluding phagosomes carrying self from contributing to the first signal delivered to T cells - the peptide-MHC complex. This mechanism prevents the potential presentation of peptides derived from self within the context of TLR-induced co-stimulatory signals.

  13. Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors

    PubMed Central

    Qi, Yi-xiang; Huang, Jia; Li, Meng-qi; Wu, Ya-su; Xia, Ren-ying; Ye, Gong-yin

    2016-01-01

    Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution. DOI: http://dx.doi.org/10.7554/eLife.12241.001 PMID:26974346

  14. Phagocytosis of boar spermatozoa in vitro and in vivo.

    PubMed

    Woelders, H; Matthijs, A

    2001-01-01

    For successful conception, fertilization-competent spermatozoa must be present at the site of fertilization in adequate numbers until ovulation has taken place. In pigs, a large volume of semen is delivered into the uterus. Most, if not all, of the inseminated liquid is voided from the vulva within a few hours after insemination and approximately 45% of the spermatozoa are lost. Large numbers of spermatozoa are also lost due to phagocytosis by polymorphonuclear leukocytes (PMNs). In pigs, the recruitment of PMNs to the uterine lumen appears to be triggered by insemination of a volume of liquid, rather than by specific components of that liquid or by spermatozoa or seminal plasma. However, persistence of large numbers of PMNs in the uterine lumen at > 12 h after insemination appears to depend on the presence of spermatozoa in the inseminate. In vitro studies have indicated that damaged, killed or capacitated spermatozoa are not phagocytosed preferentially, but that capacitation treatment strongly reduced phagocytosis of spermatozoa. Recent studies have also shown that PMN recruitment and phagocytosis of spermatozoa in vivo can be reduced by addition of caffeine plus CaCl2 to the inseminate, which appeared to have positive consequences for the longer term availability of spermatozoa at the site of fertilization.

  15. Ankyrin-repeat proteins from sponge symbionts modulate amoebal phagocytosis.

    PubMed

    Nguyen, Mary T H D; Liu, Michael; Thomas, Torsten

    2014-03-01

    Bacteria-eukaryote symbiosis occurs in all stages of evolution, from simple amoebae to mammals, and from facultative to obligate associations. Sponges are ancient metazoans that form intimate symbiotic interactions with complex communities of bacteria. The basic nutritional requirements of the sponge are in part satisfied by the phagocytosis of bacterial food particles from the surrounding water. How bacterial symbionts, which are permanently associated with the sponge, survive in the presence of phagocytic cells is largely unknown. Here, we present the discovery of a genomic fragment from an uncultured gamma-proteobacterial sponge symbiont that encodes for four proteins, whose closest known relatives are found in a sponge genome. Through recombinant approaches, we show that these four eukaryotic-like, ankyrin-repeat proteins (ARP) when expressed in Eschericha coli can modulate phagocytosis of amoebal cells and lead to accumulation of bacteria in the phagosome. Mechanistically, two ARPs appear to interfere with phagosome development in a similar way to reduced vacuole acidification, by blocking the fusion of the early phagosome with the lysosome and its digestive enzymes. Our results show that ARP from sponge symbionts can function to interfere with phagocytosis, and we postulate that this might be one mechanism by which symbionts can escape digestion in a sponge host.

  16. It is all about fluidity: Fatty acids and macrophage phagocytosis.

    PubMed

    Schumann, Julia

    2016-08-15

    Phagocytosis is an early and fundamental step for the effective clearance of disease causing agents. The ability to engulf and kill pathogens is considered as a major effector function of macrophages. In their phagocytic role macrophages are part of the first line of innate immune defense. A number of studies investigating fatty acid effects on macrophage phagocytosis have been conducted over many years. In vitro-data consistently report that alterations in macrophage membrane fatty acid composition are linked to an altered phagocytic capacity, i.e. an increase in membrane unsaturated fatty acid content is associated with an increase in engulfment and killing rate. The mode of action of fatty acids seems to be the modulation of the physical nature of the macrophage plasma membrane. It appears that the saturated-to-unsaturated fatty acid ratio of macrophage membrane phospholipids is of importance in determining macrophage phagocytic capacity. Available in vivo-data are less clear. At present, there is a lack of systematic studies elucidating key factors such as fatty acid efficacy, effective dose or dosing intervals. Without this knowledge the targeted modulation of macrophage phagocytosis in vivo by fatty acids is still a distant possibility.

  17. A simple assay to measure phagocytosis of live bacteria.

    PubMed

    Bicker, Heike; Höflich, Conny; Wolk, Kerstin; Vogt, Katrin; Volk, Hans-Dieter; Sabat, Robert

    2008-05-01

    The phagocytosis of pathogens is essential for fighting infections. No assay is available, however, to measure both engulfment and degradation of bacteria under conditions similar to those in vivo. We sought to develop a flow cytometric assay to measure the engulfment and degradation of live bacteria by human blood monocytes and granulocytes. We generated enhanced green fluorescent protein (EGFP)-expressing Eschericha coli by transforming E. coli with the plasmid vector pEGFP. We used these bacteria in a flow cytometric assay to measure both engulfment and degradation of living bacteria by monocytes and granulocytes in human whole blood from fresh, heparinized venous blood samples. To determine whether the test detected differences between healthy individuals and patients with secondary immunodeficiencies, we compared the phagocytosis of monocytes and granulocytes measured in blood samples from immunosuppressed kidney transplantation patients and from patients with postoperative sepsis in immunoparalysis with phagocytosis measured in samples from age-matched healthy individuals. In samples from healthy individuals, we found that in both monocytes and granulocytes bacterial degradation was negatively correlated with the age of the sample donor. Furthermore, we detected decreased bacterial engulfment in granulocytes from septic patients and decreased bacterial degradation in monocytes from immunosuppressed kidney transplantation patients. This flow cytometric assay measures the engulfment and degradation of live bacteria by human blood monocytes and granulocytes. By means of this assay we detected significant differences between healthy controls and patients with secondary immunodeficiencies that may contribute to the increased incidence of infection complications seen in these patients.

  18. IN VITRO INDUCTION OF LYSOSOMAL ENZYMES BY PHAGOCYTOSIS

    PubMed Central

    Axline, Stanton G.; Cohn, Zanvil A.

    1970-01-01

    The in vitro induction of lysosomal enzymes by phagocytosis was demonstrated in cultivated mouse peritoneal macrophages. The contribution of each of several steps in the endocytic process to enzyme induction was examined. The enzymatic response after the uptake of equal numbers of erythrocytes (RBC) and nondigestible particles were compared. Phagocytosis of RBC produced a marked increase in the levels of acid phosphatase, β-glucuronidase, and cathepsin D. Puromycin (1 µg/ml) inhibited the enzyme response. In contrast, phagocytosis of polyvinyl toluene, polystyrene, and insoluble starch particles produced no increase in macrophage lysosomal enzymes, although fusion of phagosomes with preexisting lysosomes occurred normally. The endocytic stimulus to synthesis of inducible lysosomal enzymes, therefore, occurred at or beyond the stage of digestion. Purified protein (bovine gamma globulin) aggregates and homopolymer coacervates of poly-l-glutamic acid: poly-l-lysine were effective inducers of lysosomal acid phosphatase, β-glucuronidase, and cathepsin D, whereas homopolymers of the same D-amino acids were ineffective as inducers. Both the quantity of phagocytized substrate and its rate of enzymatic hydrolysis appear to control the level and persistance of lysosomal hydrolases. PMID:4911552

  19. Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors.

    PubMed

    Qi, Yi-Xiang; Huang, Jia; Li, Meng-Qi; Wu, Ya-Su; Xia, Ren-Ying; Ye, Gong-Yin

    2016-03-14

    Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution.

  20. CD93 interacts with the PDZ domain-containing adaptor protein GIPC: implications in the modulation of phagocytosis.

    PubMed

    Bohlson, Suzanne S; Zhang, Mingyu; Ortiz, Christopher E; Tenner, Andrea J

    2005-01-01

    CD93 was originally identified as a myeloid cell-surface marker and subsequently associated with an ability to modulate phagocytosis of suboptimally opsonized immunoglobulin G and complement particles in vitro. Recent studies using mice deficient in CD93 have demonstrated that this molecule modulates phagocytosis of apoptotic cells in vivo. To investigate signal transduction mechanisms mediated by CD93, CD93 cytoplasmic tail (CYTO)-binding proteins were identified in a yeast two-hybrid screen. Fifteen of 34 positive clones contained a splice variant or a partial cDNA encoding GIPC, a PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein, shown previously to regulate cytoskeletal dynamics. A single clone of the N-terminal kinase-like protein p105 and an uncharacterized stem cell transcript also showed specificity for binding to the CYTO by yeast two-hybrid. Using the yeast two-hybrid system and an in vitro glutathione S-transferase fusion protein-binding assay, the binding of GIPC to the CYTO was shown to involve a newly identified class I PDZ-binding domain in the CD93 carboxyl terminus. Four positively charged amino acids in the juxtamembrane domain of CD93 were shown to be critical in stabilizing these interactions. Treatment of human monocytes with a cell-permeable peptide encoding the C-terminal 11 amino acids of CD93 resulted in an enhancement of phagocytosis, supporting the hypothesis that this protein-protein interaction domain is involved in the modulation of phagocytosis. These protein interactions may participate as molecular switches in modulating cellular phagocytic activity.

  1. Augmentation of human monocyte opsonin-independent phagocytosis by fragments of human plasma fibronectin.

    PubMed

    Czop, J K; Kadish, J L; Austen, K F

    1981-06-01

    Human plasma fibronectin isolated by gelatin-affinity chromatography increases in a dose-dependent fashion the number of human monocytes that ingest particulate activators of the human alternative complement pathway in a fully synthetic medium. The fibronectin effect is selective for these particulate activators, does not extend to particles whose ingestion is dependent upon opsonization with IgG, and is not observed with pretreatment of the monocytes. Affinity chromatography with monoclonal antibody to plasma fibronectin of 440,000 daltons reveals that only 12-53% of the protein in a phagocytically active gelatin-affinity-purified fibronectin preparations is bound to the antibody. The protein eluted after affinity chromatography with monoclonal antibody of active preparations, which represented 10-43% of the protein applied, exhibits a 2- to 10-fold increment of activity per microgram of protein above the starting gelatin-affinity-purified material. Thus, the activity that augments the percent of human monocytes ingesting particulate activators of the alternative pathway is antigenically defined as plasma fibronectin. Preparations containing only intact 440,000-dalton fibronectin are also bound to and eluted from the monoclonal antibody, but they fail to augment phagocytosis. When inactive 440,000-dalton plasma fibronectin is subjected to limited trypsin cleavage, phagocytosis-enhancing activity develops that is bound to and elutes from the affinity column prepared with monoclonal antibody, thereby indicating that the enhancing activity of plasma fibronectin resides in cleavage fragments.

  2. The Role of Adenosine A2A Receptor, CYP450s, and PPARs in the Regulation of Vascular Tone

    PubMed Central

    Khayat, Maan T.

    2017-01-01

    Adenosine is an endogenous mediator involved in a myriad of physiologic functions, including vascular tone regulation. It is also implicated in some pathologic conditions. Four distinct receptor subtypes mediate the effects of adenosine, such as its role in the regulation of the vascular tone. Vascular tone regulation is a complex and continuous process which involves many mechanisms and mediators that are not fully disclosed. The vascular endothelium plays a pivotal role in regulating blood flow to and from all body organs. Also, the vascular endothelium is not merely a physical barrier; it is a complex tissue with numerous functions. Among adenosine receptors, A2A receptor subtype (A2AAR) stands out as the primary receptor responsible for the vasodilatory effects of adenosine. This review focuses on important effectors of the vascular endothelium, including adenosine, adenosine receptors, EETs (epoxyeicosatrienoic acids), HETEs (hydroxyeicosatetraenoic acids), PPARs (peroxisome proliferator-activated receptors), and KATP channels. Given the impact of vascular tone regulation in cardiovascular physiology and pathophysiology, better understanding of the mechanisms affecting it could have a significant potential for developing therapeutic agents for cardiovascular diseases. PMID:28884118

  3. The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain

    PubMed Central

    Gehman, Lauren T.; Stoilov, Peter; Maguire, Jamie; Damianov, Andrey; Lin, Chia-Ho; Shiue, Lily; Ares, Manuel; Mody, Istvan; Black, Douglas L.

    2011-01-01

    The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts but their role in neuronal physiology is not clear1. We show here that central nervous system (CNS)-specific deletion of the Rbfox1 gene results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording reveals a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole transcriptome analyses identify multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation, some of which are implicated in human epilepsy. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function. PMID:21623373

  4. Annexin A2 binds to endosomes and negatively regulates TLR4-triggered inflammatory responses via the TRAM-TRIF pathway

    PubMed Central

    Zhang, Shuang; Yu, Min; Guo, Qiang; Li, Rongpeng; Li, Guobo; Tan, Shirui; Li, Xuefeng; Wei, Yuquan; Wu, Min

    2015-01-01

    Lipopolysaccharide (LPS) derived from Gram-negative bacteria activates plasma membrane signaling via Toll-like receptor 4 (TLR4) on host cells and triggers innate inflammatory responses, but the underlying mechanisms remain to be fully elucidated. Here we reveal a role for annexin A2 (AnxA2) in host defense against infection as anxa2−/− mice were highly susceptible to Gram-negative bacteria-induced sepsis with enhanced inflammatory responses. Computing analysis and biochemical experiments identified that constitutive AnxA2 expression facilitated TLR4 internalization and its subsequent translocation into early endosomal membranes. It activated the TRAM-dependent endosomal signaling, leading to the release of anti-inflammatory cytokines. Importantly, AnxA2 deficiency prolonged TLR4-mediated signaling from the plasma membrane, which was attributable to pro-inflammatory cytokine production (IL-6, TNFα and IL-1β). Thus, AnxA2 directly exerted negative regulation of inflammatory responses through TLR4-initiated TRAM-TRIF pathway occurring on endosomes. This study reveals AnxA2 as a critical regulator in infection-initiated inflammation, which protects the host from excessive inflammatory damage. PMID:26527544

  5. Cannabinoid receptor 1 but not 2 mediates macrophage phagocytosis by G(α)i/o /RhoA/ROCK signaling pathway.

    PubMed

    Mai, Ping; Tian, Lei; Yang, Le; Wang, Lin; Yang, Lin; Li, Liying

    2015-07-01

    Phagocytosis is critical to macrophages linking innate and adaptive immune reaction. Cannabinoid receptor 1 (CB1) and 2 (CB2) mediate immune modulation. However, the role of cannabinoid receptors in macrophage phagocytosis is undefined. In this study, we found that two murine macrophage lines (J774A.1 and RAW264.7) and peripheral blood macrophages all expressed CB1 and CB2 by immunofluorescence-staining, real time RT-PCR and Western blot. Macrophage phagocytic activity was determined by quantifying fluorescent intensity of the engulfed BioParticles or fluorescence-activated cell sorting. mAEA (CB1 agonist) enhanced phagocytosis of macrophages, but JWH133 (CB2 agonist) had no influence. Pharmacological or genetic ablation of CB1 inhibited mAEA-enhanced phagocytosis, while CB2 had no such effects. Meanwhile, activation of CB1 increased GTP-bounding active form of small GTPase RhoA, but not Rac1 or Cdc42. AM281 (CB1 antagonist) and pertussis toxin (PTX, G((α)i/o) protein inhibitor) decreased GTP-bound RhoA protein level with mAEA. In addition, PTX, C3 Transferase (RhoA inhibitor) or Y27632 (Rho-associated kinase ROCK inhibitor) attenuated CB1-mediated phagocytosis. These results confirm that activation of CB1 regulates macrophage phagocytosis through G((α)i/o)/RhoA/ROCK signaling pathway. Moreover, activation of CB1 induced significant up-regulation of CB1 expression by real time RT-PCR and Western blot analysis, but not CB2. It indicated the existence of a positive feedback between CB1 activation and CB1 expression. The up-regulation of CB1 was RhoA-independent but it may contribute to maintaining high phagocytic activity of macrophages for a longer time. In conclusion, CB1 mediates macrophage phagocytosis by G((α)i/o)/RhoA/ROCK signal axis. These data further underline the role of CB1 in macrophage phagocytic process.

  6. Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors.

    PubMed

    Forster, Brian M; Zemansky, Jason; Portnoy, Daniel A; Marquis, Hélène

    2011-11-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.

  7. Posttranslocation Chaperone PrsA2 Regulates the Maturation and Secretion of Listeria monocytogenes Proprotein Virulence Factors ▿

    PubMed Central

    Forster, Brian M.; Zemansky, Jason; Portnoy, Daniel A.; Marquis, Hélène

    2011-01-01

    PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface. PMID:21908675

  8. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

    PubMed Central

    Orr, Anna G.; Hsiao, Edward C.; Wang, Max M.; Ho, Kaitlyn; Kim, Daniel H.; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B.; Masliah, Eliezer; Conklin, Bruce R.; Mucke, Lennart

    2014-01-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer’s disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice, and increased the levels of Arc/Arg3.1, an immediate-early gene required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Similar to humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  9. Melanin targets LC3-associated phagocytosis (LAP): A novel pathogenetic mechanism in fungal disease.

    PubMed

    Chamilos, Georgios; Akoumianaki, Tonia; Kyrmizi, Irene; Brakhage, Axel; Beauvais, Anne; Latge, Jean-Paul

    2016-05-03

    Intracellular swelling of conidia of the major human airborne fungal pathogen Aspergillus fumigatus results in surface exposure of immunostimulatory pathogen-associated molecular patterns (PAMPs) and triggers activation of a specialized autophagy pathway called LC3-associated phagocytosis (LAP) to promote fungal killing. We have recently discovered that, apart from PAMPs exposure, cell wall melanin removal during germination of A. fumigatus is a prerequisite for activation of LAP. Importantly, melanin promotes fungal pathogenicity via targeting LAP, as a melanin-deficient A. fumigatus mutant restores its virulence upon conditional inactivation of Atg5 in hematopoietic cells of mice. Mechanistically, fungal cell wall melanin selectively excludes the CYBA/p22phox subunit of NADPH oxidase from the phagosome to inhibit LAP, without interfering with signaling regulating cytokine responses. Notably, inhibition of LAP is a general property of melanin pigments, a finding with broad physiological implications.

  10. HtrA2 regulates beta-amyloid precursor protein (APP) metabolism through endoplasmic reticulum-associated degradation.

    PubMed

    Huttunen, Henri J; Guénette, Suzanne Y; Peach, Camilla; Greco, Christopher; Xia, Weiming; Kim, Doo Yeon; Barren, Cory; Tanzi, Rudolph E; Kovacs, Dora M

    2007-09-21

    Alzheimer disease-associated beta-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Abeta secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.

  11. Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    SciTech Connect

    Seo, Young-Kyo; Zhu, Bing; Jeon, Tae-Il; Osborne, Timothy F.

    2009-11-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  12. Inducible CYP2J2 and its product 11,12-EET promotes bacterial phagocytosis: a role for CYP2J2 deficiency in the pathogenesis of Crohn's disease?

    PubMed

    Bystrom, Jonas; Thomson, Scott J; Johansson, Jörgen; Edin, Matthew L; Zeldin, Darryl C; Gilroy, Derek W; Smith, Andrew M; Bishop-Bailey, David

    2013-01-01

    The epoxygenase CYP2J2 has an emerging role in inflammation and vascular biology. The role of CYP2J2 in phagocytosis is not known and its regulation in human inflammatory diseases is poorly understood. Here we investigated the role of CYP2J2 in bacterial phagocytosis and its expression in monocytes from healthy controls and Crohns disease patients. CYP2J2 is anti-inflammatory in human peripheral blood monocytes. Bacterial LPS induced CYP2J2 mRNA and protein. The CYP2J2 arachidonic acid products 11,12-EET and 14,15-EET inhibited LPS induced TNFα release. THP-1 monocytes were transformed into macrophages by 48h incubation with phorbol 12-myristate 13-acetate. Epoxygenase inhibition using a non-selective inhibitor SKF525A or a selective CYP2J2 inhibitor Compound 4, inhibited E. coli particle phagocytosis, which could be specifically reversed by 11,12-EET. Moreover, epoxygenase inhibition reduced the expression of phagocytosis receptors CD11b and CD68. CD11b also mediates L. monocytogenes phagocytosis. Similar, to E. coli bioparticle phagocytosis, epoxygenase inhibition also reduced intracellular levels of L. monocytogenes, which could be reversed by co-incubation with 11,12-EET. Disrupted bacterial clearance is a hallmark of Crohn's disease. Unlike macrophages from control donors, macrophages from Crohn's disease patients showed no induction of CYP2J2 in response to E. coli. These results demonstrate that CYP2J2 mediates bacterial phagocytosis in macrophages, and implicates a defect in the CYP2J2 pathway may regulate bacterial clearance in Crohn's disease.

  13. CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain.

    PubMed

    Yan, Junpeng; Hao, Caili; DeLucia, Maria; Swanson, Selene; Florens, Laurence; Washburn, Michael P; Ahn, Jinwoo; Skowronski, Jacek

    2015-05-22

    SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain*

    PubMed Central

    Yan, Junpeng; Hao, Caili; DeLucia, Maria; Swanson, Selene; Florens, Laurence; Washburn, Michael P.; Ahn, Jinwoo; Skowronski, Jacek

    2015-01-01

    SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication. PMID:25847232

  15. The nuclear factor kappa B (NF-κB) activation is required for phagocytosis of staphylococcus aureus by RAW 264.7 cells.

    PubMed

    Zhu, Fei; Yue, Wanfu; Wang, Yongxia

    2014-10-01

    Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor which controls the expression of various genes involved in immune responses. However, it is not clear whether NF-κB activation is critical for phagocytosis when Staphylococcus aureus is the pathogen. Using oligonucleotide microarrays, we investigated whether NF-κB cascade genes are altered in a mouse leukemic monocyte macrophage cell line (RAW 264.7) when the cells were stimulated to activate a host innate immune response against live S. aureus or heat-inactivated S. aureus (HISA). NF-κB cascade genes such as Nfκb1, Nfκbiz, Nfκbie, Rel, Traf1 and Tnfaip3 were up-regulated by all treatments at one hour after incubation. NF-κB play an important role in activating phagocytosis in RAW 264.7 cells infected with S. aureus. Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus and decreased the expression of NFκB1, IL1α, IL1β and TLR2 in this cell line. Our results demonstrate that S. aureus may activate the NF-κB pathway and that NF-κB activation is required for phagocytosis of S. aureus by macrophages.

  16. Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

    PubMed Central

    Sato, Hiroyasu; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Yamamoto, Kei; Masuda, Seiko; Hosono, Tomohiko; Arata, Satoru; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Nakanishi, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Kudo, Ichiro; Murakami, Makoto

    2010-01-01

    Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction. PMID:20424323

  17. NcoA2-Dependent Inhibition of HIF-1α Activation Is Regulated via AhR.

    PubMed

    Tsai, Chi-Hao; Li, Ching-Hao; Liao, Po-Lin; Cheng, Yu-Wen; Lin, Cheng-Hui; Huang, Shih-Hsuan; Kang, Jaw-Jou

    2015-12-01

    High endogenous levels of aryl hydrocarbon receptor (AhR) contribute to hypoxia signaling pathway inhibition following exposure to the potent AhR ligand benzo[a]pyrene (B[a]P) and could alter cellular homeostasis and disease condition. Increasing evidence indicates that AhR might compete with AhR nuclear translocator (ARNT) for complex formation with hypoxia-inducible factor-1α (HIF-1α) for transactivation, which could alter several physiological variables. Nuclear receptor coactivator 2 (NcoA2) is a transcription coactivator that regulates transcription factor activation and inhibition of basic helix-loop-helix Per (Period)-ARNT-SIM (single-minded) (bHLH-PAS) family proteins, such as HIF-1α, ARNT, and AhR, through protein-protein interactions. In this study, we demonstrated that both hypoxia and hypoxia-mimic conditions decreased NcoA2 protein expression in HEK293T cells. Hypoxia response element (HRE) and xenobiotic-responsive element (XRE) transactivation also were downregulated with NcoA2 knockdown under hypoxic conditions. In addition, B[a]P significantly decreased NcoA2 protein expression be accompanied with AhR degradation. We next evaluated whether the absence of AhR could affect NcoA2 protein function under hypoxia-mimetic conditions. NcoA2 and HIF-1α nuclear localization decreased in both B[a]P-pretreated and AhR-knockdown HepG2 cells under hypoxia-mimic conditions. Interestingly, NcoA2 overexpression downregulated HRE transactivation by competing with HIF-1α and AhR to form protein complexes with ARNT. Both NcoA2 knockdown and overexpression inhibited endothelial cell tube formation in vitro. We also demonstrated using the in vivo plug assay that NcoA2-regulated vascularization decreased in mice. Taken together, these results revealed a biphasic role of NcoA2 between AhR and hypoxic conditions, thus providing a novel mechanism underlying the cross talk between AhR and hypoxia that affects disease development and progression.

  18. A novel opioid mechanism seems to modulate phagocytosis in Tetrahymena.

    PubMed

    Renaud, F L; Colon, I; Lebron, J; Ortiz, N; Rodriguez, F; Cadilla, C

    1995-01-01

    We have previously reported that a beta-endorphin-like substance inhibits phagocytosis in Tetrahymena perhaps by a mu-like opioid receptor. We now report a further characterization of the elements involved in the signal transduction mechanism of this opioid. Affinity chromatography followed by immunoblots of both intracellular extracts and extracellular medium reveal the presence of two main proteins of 64 and 75 kDa. These molecular weights are much higher than that of any known opioid peptide or precursor protein and suggest that we may be dealing with either a novel opioid or with proteins that by chance cross-react with anti-beta-endorphin antibody. Nevertheless, when the biological activity of these proteins was tested it was found that they had an effect similar to that of mammalian beta-endorphin, namely inhibition of phagocytosis by a naloxone-reversible mechanism. We have probed a size-selected Tetrahymena library with a pro-opiomelanocortin probe and have obtained several positive clones; the sequencing of their inserts should establish whether we are dealing with a bona fide member of the opioid family. Another aspect we have been studying is the G-proteins which appear to be involved in the modulation of phagocytosis. We have found, by means of Western blotting (using an antibody against the conserved GTP-binding region of the alpha-subunit), two bands of 51 and 59 kDa; no alpha-subunit of 59 kDa had been reported previously and may represent a novel G-protein. In spite of these differences, the opioid signal transduction mechanism appears to remarkably resemble that present in more complex organisms.

  19. Effect of chronic opioid treatment on phagocytosis in Tetrahymena.

    PubMed

    Salaman, A; Roman, M; Renaud, F L; Silva, W I

    1990-07-01

    Opioid inhibition of phagocytosis in the protozoan ciliate Tetrahymena is antagonized by naloxone and this antagonism can be surmounted by increasing agonist concentration, which suggests a receptor-mediated mechanism. Desensitization of the opioid effect is time dependent in addition to concentration dependent. Chronic exposure to opioids results in the development of tolerance to the inhibitory effect of the agonists, and withdrawal of the latter results in a decrease in phagocytic capacity, which suggests that a state akin to dependence has been developed in these cells. Naloxone appears to behave as a partial agonist in tolerant cells, and there seems to exist cross-tolerance to mu and delta agonists.

  20. Phagocytosis in Tetrahymena thermophila: naloxone-reversible inhibition by opiates.

    PubMed

    De Jesus, S; Renaud, F L

    1989-01-01

    1. Nanomolar concentrations of opiates inhibit phagocytosis in the ciliated protozoan Tetrahymena thermophila. 2. Naloxone and naltrexone counteract the effect of the opiate agonists tested. 3. The dose-response curves are U-shaped, with no detectable effect at low or high concentrations. 4. An increase in extracellular calcium and dopamine counteract the inhibition caused by metenkephalin. 5. The recognition mechanism for opiates in Tetrahymena cannot be classified as belonging to any of the mammalian opiate receptor subtypes and is perhaps a primitive receptor.

  1. Increased antibody production following depression of hepatic phagocytosis.

    PubMed Central

    Souhami, R L; Addison, I E; Bradfield, J W

    1975-01-01

    The influence of Kupffer cell blockade on the humoral immune response to suboptimal doses of intravenous sheep red blood cells has been measured in mice. Hepatic phagocytosis was suppressed using dextran sulphate. Direct (IgM) and indirect (IgG) plaque-forming cells were measured in spleens from treated and untreated mice at varying times after the antigen. The results show that after Kupffer cell blockade both IgM and IgG responses correspond to the responses seen after a 10-fold greater dose of cells in control animals. The implications of this are discussed. PMID:1106919

  2. CD98hc (SLC3A2) regulation of skin homeostasis wanes with age

    PubMed Central

    Boulter, Etienne; Estrach, Soline; Errante, Aurélia; Pons, Catherine; Cailleteau, Laurence; Tissot, Floriane; Meneguzzi, Guerrino

    2013-01-01

    Skin aging is linked to reduced epidermal proliferation and general extracellular matrix atrophy. This involves factors such as the cell adhesion receptors integrins and amino acid transporters. CD98hc (SLC3A2), a heterodimeric amino acid transporter, modulates integrin signaling in vitro. We unravel CD98hc functions in vivo in skin. We report that CD98hc invalidation has no appreciable effect on cell adhesion, clearly showing that CD98hc disruption phenocopies neither CD98hc knockdown in cultured keratinocytes nor epidermal β1 integrin loss in vivo. Instead, we show that CD98hc deletion in murine epidermis results in improper skin homeostasis and epidermal wound healing. These defects resemble aged skin alterations and correlate with reduction of CD98hc expression observed in elderly mice. We also demonstrate that CD98hc absence in vivo induces defects as early as integrin-dependent Src activation. We decipher the molecular mechanisms involved in vivo by revealing a crucial role of the CD98hc/integrins/Rho guanine nucleotide exchange factor (GEF) leukemia-associated RhoGEF (LARG)/RhoA pathway in skin homeostasis. Finally, we demonstrate that the deregulation of RhoA activation in the absence of CD98hc is also a result of impaired CD98hc-dependent amino acid transports. PMID:23296466

  3. Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels.

    PubMed

    Mayer, Gaétan; Poirier, Steve; Seidah, Nabil G

    2008-11-14

    The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a approximately 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.

  4. Membrane Restructuring by Phospholipase A2 Is Regulated by the Presence of Lipid Domains

    PubMed Central

    Leidy, Chad; Ocampo, Jackson; Duelund, Lars; Mouritsen, Ole G.; Jørgensen, Kent; Peters, Günther H.

    2011-01-01

    Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (Lα/Lβ) lipid domains. In this work, we explore whether lipid domains can also direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA2. Specifically, we show that the presence of Lα/Lβ and Lα/Pβ′ phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) system results in the preferential hydrolysis of the shorter-chained lipid component in the mixture, leading to an enrichment in the longer-chained component. The restructuring process is monitored by atomic force microscopy on supported single and double bilayers formed by vesicle fusion. We observe that during preferential hydrolysis of the DMPC-rich Lα regions, the Lβ and Pβ′ regions grow and reseal, maintaining membrane integrity. This result indicates that a sharp reorganization of the membrane structure can occur during sPLA2 hydrolysis without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary. Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity in the hydrolytic activity of the enzyme, resulting in marked differences in the physical properties of the membrane end-product. PMID:21723818

  5. Annexin A2 and PSF proteins interact with p53 IRES and regulate translation of p53 mRNA.

    PubMed

    Sharathchandra, Arandkar; Lal, Ridhima; Khan, Debjit; Das, Saumitra

    2012-12-01

    p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and ΔN-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

  6. Notch Balances Th17 and Induced Regulatory T Cell Functions in Dendritic Cells by Regulating Aldh1a2 Expression.

    PubMed

    Zaman, Taskia Sultana; Arimochi, Hideki; Maruyama, Satoshi; Ishifune, Chieko; Tsukumo, Shin-Ichi; Kitamura, Akiko; Yasutomo, Koji

    2017-09-15

    Dendritic cells (DCs) are important for adaptive immune responses through the activation of T cells. The molecular interplay between DCs and T cells determines the magnitude of T cell responses or outcomes of functional differentiation of T cells. In this study, we demonstrated that DCs in mice that are Rbpj deficient in CD11c(+) cells (Rbpj(-/-) mice) promoted the differentiation of IL-17A-producing Th17 cells. Rbpj-deficient DCs expressed little Aldh1a2 protein that is required for generating retinoic acid. Those DCs exhibited a reduced ability for differentiating regulatory T cells induced by TGF-β. Rbpj protein directly regulated Aldh1a2 transcription by binding to its promoter region. The overexpression of Aldh1a2 in Rbpj-deficient DCs negated their Th17-promoting ability. Transfer of naive CD4(+) T cells into Rag1-deficient Rbpj(-/-) mice enhanced colitis with increased Th17 and reduced induced regulatory T cells (iTreg) compared with control Rag1-deficient mice. The cotransfer of iTreg and naive CD4(+) T cells into Rag1-deficient Rbpj(-/-) mice improved colitis compared with transfer of naive CD4(+) T cell alone. Furthermore, cotransfer of DCs from Rbpj(-/-) mice that overexpressed Aldh1a2 or Notch-stimulated DCs together with naive CD4(+) T cells into Rbpj(-/-)Rag1-deficient mice led to reduced colitis with increased iTreg numbers. Therefore, our studies identify Notch signaling in DCs as a crucial balancer of Th17/iTreg, which depends on the direct regulation of Aldh1a2 transcription in DCs. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. Matrix Metalloproteinase‐2 Negatively Regulates Cardiac Secreted Phospholipase A2 to Modulate Inflammation and Fever

    PubMed Central

    Berry, Evan; Hernandez‐Anzaldo, Samuel; Ghomashchi, Farideh; Lehner, Richard; Murakami, Makoto; Gelb, Michael H.; Kassiri, Zamaneh; Wang, Xiang; Fernandez‐Patron, Carlos

    2015-01-01

    Background Matrix metalloproteinase (MMP)‐2 deficiency makes humans and mice susceptible to inflammation. Here, we reveal an MMP‐2–mediated mechanism that modulates the inflammatory response via secretory phospholipase A2 (sPLA2), a phospholipid hydrolase that releases fatty acids, including precursors of eicosanoids. Methods and Results Mmp2−/− (and, to a lesser extent, Mmp7−/− and Mmp9−/−) mice had between 10‐ and 1000‐fold elevated sPLA2 activity in plasma and heart, increased eicosanoids and inflammatory markers (both in the liver and heart), and exacerbated lipopolysaccharide‐induced fever, all of which were blunted by adenovirus‐mediated MMP‐2 overexpression and varespladib (pharmacological sPLA2 inhibitor). Moreover, Mmp2 deficiency caused sPLA2‐mediated dysregulation of cardiac lipid metabolic gene expression. Compared with liver, kidney, and skeletal muscle, the heart was the single major source of the Ca2+‐dependent, ≈20‐kDa, varespladib‐inhibitable sPLA2 that circulates when MMP‐2 is deficient. PLA2G5, which is a major cardiac sPLA2 isoform, was proinflammatory when Mmp2 was deficient. Treatment of wild‐type (Mmp2+/+) mice with doxycycline (to inhibit MMP‐2) recapitulated the Mmp2−/− phenotype of increased cardiac sPLA2 activity, prostaglandin E2 levels, and inflammatory gene expression. Treatment with either indomethacin (to inhibit cyclooxygenase‐dependent eicosanoid production) or varespladib (which inhibited eicosanoid production) triggered acute hypertension in Mmp2−/− mice, revealing their reliance on eicosanoids for blood pressure homeostasis. Conclusions A heart‐centric MMP‐2/sPLA2 axis may modulate blood pressure homeostasis, inflammatory and metabolic gene expression, and the severity of fever. This discovery helps researchers to understand the cardiovascular and systemic effects of MMP‐2 inhibitors and suggests a disease mechanism for human MMP‐2 gene deficiency. PMID:25820137

  8. Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells.

    PubMed

    Fanger, N A; Wardwell, K; Shen, L; Tedder, T F; Guyre, P M

    1996-07-15

    Three classes of Fc receptors for IgG, Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), are expressed on blood leukocytes. Although Fc gamma R are important phagocytic receptors on phagocytes, most reports suggest that dendritic cells lack Fc gamma R-mediated phagocytosis and express significant levels of only CD32. We now report that phagocytically active forms of both CD64 and CD32 are expressed significantly on at least one subset of human blood dendritic cells. Countercurrent elutriation and magnetic bead selection were used to rapidly enrich subsets of blood dendritic cells (CD33brightCD14-HLA-DRbrightCD83-) and monocytes (CD33brightCD14brightHLA-DRdimCD83-). Upon culture for 2 days, dendritic cells became CD83-positive and markedly increased HLA-DR expression, whereas monocytes did not express CD83 and exhibited reduced levels of HLA-DR. Constitutive CD64 expression was identified on this circulating dendritic cell population, but at a lower level than on monocytes. CD64 expression by dendritic cells and monocytes did not decrease during 2 days in culture, and was up-regulated on both cell types following incubation with IFN-gamma. Freshly isolated blood dendritic cells performed CD64- and CD32-mediated phagocytosis, although at a lower level than monocytes. Dendritic cells generated by culture of adherent mononuclear cells in granulocyte-macrophage CSF and IL-4 also up-regulated CD64 following IFN-gamma stimulation, and mediated CD64-dependent phagocytosis. These results indicate that both CD64 and CD32 expressed on blood dendritic cells may play a role in uptake of foreign particles and macromolecules through a phagocytic mechanism before trafficking to T cell-reactive areas.

  9. 3D visualization and quantitative analysis of human erythrocyte phagocytosis.

    PubMed

    Stachurska, Anna; Król, Teodora; Trybus, Wojciech; Szary, Karol; Fabijańska-Mitek, Jadwiga

    2016-11-01

    Since the erythrophagocytosis of opsonized erythrocytes is investigated mainly by calculating the phagocytic index using subjective light microscopy evaluation, we present methods for the quantitative and qualitative analysis of human cell erythrophagocytosis. Erythrocytes from two storage periods were used. Using Imaris software, we were able to create a three-dimensional model of erythrophagocytosis. The use of microscopy instead of cytometry revealed a significantly higher number of monocytes and erythrocytes that appeared active in phagocytosis. Spatial reconstruction allowed for detailed analysis of the process by precisely locating erythrocytes in phagocytes. Additionally, a technique of sequential image registration using Nis Elements software allowed for observation of the course of phagocytosis over a range of time intervals. This in vitro research may be helpful for understanding the cellular interactions between monocytes and erythrocytes. The cytometric method-being relatively rapid, sensitive, and specific-can serve as an alternative technique to microscopy in the quantitative analysis of erythrophagocytosis. This allows us to avoid counting the erythrocytes nonspecifically attached to monocytes and gives objective results. © 2016 International Federation for Cell Biology.

  10. Xpf suppresses the mutagenic consequences of phagocytosis in Dictyostelium

    PubMed Central

    Langenick, Judith; Zhang, Xiao-Yin; Traynor, David; Kay, Robert R.

    2016-01-01

    ABSTRACT As time passes, mutations accumulate in the genomes of all living organisms. These changes promote genetic diversity, but also precipitate ageing and the initiation of cancer. Food is a common source of mutagens, but little is known about how nutritional factors cause lasting genetic changes in the consuming organism. Here, we describe an unusual genetic interaction between DNA repair in the unicellular amoeba Dictyostelium discoideum and its natural bacterial food source. We found that Dictyostelium deficient in the DNA repair nuclease Xpf (xpf−) display a severe and specific growth defect when feeding on bacteria. Despite being proficient in the phagocytosis and digestion of bacteria, over time, xpf− Dictyostelium feeding on bacteria cease to grow and in many instances die. The Xpf nuclease activity is required for sustained growth using a bacterial food source. Furthermore, the ingestion of this food source leads to a striking accumulation of mutations in the genome of xpf− Dictyostelium. This work therefore establishes Dictyostelium as a model genetic system to dissect nutritional genotoxicity, providing insight into how phagocytosis can induce mutagenesis and compromise survival fitness. PMID:27872153

  11. Effects of endogenous antidiuretic hormone (ADH) on macrophage phagocytosis

    SciTech Connect

    Fernandez-Repollet, E.; Opava-Stitzer, S.; Tiffany, S.; Schwartz, A.

    1983-07-01

    Although several studies have indicated that antidiuretic hormone (ADH) enhances the phagocytic function of the reticuloendothelial system (RES) in shock syndromes, it remains unknown what influence ADH exerts upon the individual phagocytic components of this system. The present investigation was designed to evaluate the effects of endogenous ADH on the phagocytic activity of peritoneal macrophage cells. As a phagocytic stimuli, fluorescent methacrylate microbeads were injected intraperitoneally into Brattleboro (ADH deficient) and normal Long Evans rats in the presence and absence of exogenous ADH. Peritoneal cells were harvested 19-22 hr after the administration of the microbeads and the percent phagocytosis was determined in macrophage cells using a fluorescence-activated cell sorter (FACS II). Our results indicate that the percentage of peritoneal macrophages ingesting the fluorescent methacrylate microbeads was significantly reduced in the absence of ADH (Brattleboro rats: 5.4 +/- 0.6% versus Long Evans rats: 16.8 +/- 2.3%; p less than 0.001). In addition, our data demonstrate that exogenous administration of ADH significantly enhanced macrophage phagocytosis in Brattleboro (14.7 +/- 2.2%) and normal Long Evans (49.6 +/- 4.5%) rats. These data suggest, for the first time, that endogenous ADH might play a modulatory role in the phagocytic activity of a specific component of the RES, namely, the macrophage cell.

  12. Enhancement of Phagocytosis by Interferon-Containing Preparations

    PubMed Central

    Huang, Kun-Yen; Donahoe, Robert M.; Gordon, Francis B.; Dressler, Harry R.

    1971-01-01

    Exposure of mononuclear cells from the mouse peritoneal cavity to interferon (IF)-containing mouse sera enhanced phagocytosis of colloidal carbon particles by the cells. The same effect was observed when the cells were exposed to IF-containing cell culture harvest free of serum. The magnitude of this effect of IF-containing preparations paralleled the titer of IF and was not related to the dilution of various IF-containing serum specimens tested. The factor responsible for the enhancing effect was stable at pH 2, inactivated by trypsin, and nonsedimentable at 105,000 × g. Heating at 60 C for 1 hr destroyed it, and its kinetics of heat inactivation paralleled that of the antiviral activity of IF. A period of incubation of phagocytic cells with IF-containing serum was necessary before a maximum level of enhancement was reached, and once established was not removable by repeated washing of cells. The kinetics of the production of the enhancing factor in mice injected with Newcastle disease virus was essentially identical to that of the simultaneous production of IF as measured by antiviral activity. Contrary to the effect of mouse IF preparations, human IF preparation did not enhance the activity of mouse phagocytes. It appears, therefore, that the phagocytosis-enhancing factor falls within the present definition of IF. Images PMID:4343410

  13. The effect of bacterial endotoxin of phagocytosis of Tetrahymena and serotonin induced imprinting.

    PubMed

    Kovács, G; Nagy, S U; Csaba, G

    1986-01-01

    Endotoxin inhibited the phagocytosis of Tetrahymena pyriformis after a short exposure and, to a lesser degree, after repeated treatments during one week (about 35 generations). Endotoxin also prevented the development of serotonin imprinting. Detoxified endotoxin (Tolerin) affected the phagocytosis of Tetrahymena much less, indicating that the lipid-A part of the molecule may account for the membrane-toxic effect.

  14. Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

    PubMed

    Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao; Xiao, Xiubin; Zhang, Weijing

    2014-02-18

    Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

  15. Influence of Thromboxane A2 on the Regulation of Adenosine Triphosphate-Sensitive Potassium Channels in Mouse Ventricular Myocytes

    PubMed Central

    Jeong, In Seok; Cho, Hwa Jin; Cho, Jeong Gwan; Kim, Sang Hyung; Na, Kook Joo

    2016-01-01

    Background and Objectives Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels play an important role in myocardial protection. We examined the effects of thromboxane A2 on the regulation of KATP channel activity in single ventricular myocytes. Subjects and Methods Single ventricular myocytes were isolated from the hearts of adult Institute of Cancer Research (ICR) mice by enzymatic digestion. Single channel activity was recorded by excised inside-out and cell-attached patch clamp configurations at −60 mV holding potential during the perfusion of an ATP-free K-5 solution. Results In the excised inside-out patches, the thromboxane A2 analog, U46619, decreased the KATP channel activity in a dose-dependent manner; however, the thromboxane A2 receptor antagonist, SQ29548, did not significantly attenuate the inhibitory effect of U46619. In the cell-attached patches, U46619 inhibited dinitrophenol (DNP)-induced KATP channel activity in a dose-dependent manner, and SQ29548 attenuated the inhibitory effects of U46619 on DNP-induced KATP channel activity. Conclusion Thromboxane A2 may inhibit KATP channel activity, and may have a harmful effect on ischemic myocardium. PMID:27482267

  16. Altered apoptosis regulation in Kufor–Rakeb syndrome patients with mutations in the ATP13A2 gene

    PubMed Central

    Radi, Elena; Formichi, Patrizia; Maio, Giuseppe Di; Battisti, Carla; Federico, Antonio

    2012-01-01

    Abstract ATP13A2 gene encodes for a protein of the group 5 P-type ATPase family. ATP13A2 mutations are responsible for Kufor–Rakeb syndrome (KRS), a rare autosomal recessive juvenile parkinsonism characterized by the subacute onset of extrapyramidal, pyramidal and cognitive dysfunction with secondary nonresponsiveness to levodopa. FBXO7 protein is an F-box-containing protein. Recessive FBXO7 mutations are responsible for PARK15, a rare juvenile parkinsonism characterized by progressive neurodegeneration with extrapyramidal and pyramidal system involvement. Our aim was to evaluate apoptosis in cells from two KRS siblings carrying a homozygous ATP13A2 mutation and a heterozygous FBXO7 mutation. We also analysed apoptosis in the patients’ healthy parents. Peripheral blood lymphocytes from the KRS patients and parents were exposed to 2-deoxy-D-ribose; apoptosis was analysed by flow cytometry and fluorescence microscopy. Apoptosis was much higher in lymphocytes from the KRS patients and parents than in controls, both in standard conditions and after induction with a pro-apoptotic stimulus. The lack of correlation between increased apoptosis and the presence of the mutated FBXO7 gene rules out the involvement of FBXO7 in apoptosis regulation. The altered apoptotic pattern of subjects with mutated ATP13A2 suggests a correlation between apoptosis alteration and the mutated ATP13A2 protein. We hypothesize that ATP13A2 mutations may compromise protein function, disrupting cell cation balance and rendering cells prone to apoptosis. However, the deregulation of apoptosis in KRS patients displaying different disease severity suggested that the altered apoptotic pathway probably does not have a pathogenetic role in KRS by itself. PMID:22117566

  17. In vitro phagocytosis and intracellular survival of Campylobacter jejuni with phagocytes

    SciTech Connect

    Kiehlbauch, J.A.

    1986-01-01

    In vitro phagocytosis and intracellular survival of Campylobacter jejuni was studied using three types of mononuclear phagocytes: a J774G8 peritoneal macrophage line, resident BABL/c peritoneal macrophages and human peripheral blood monocytes. In phagocytosis assays using CFU determinations, phagocytosis increased steadily over an 8 hr time period. Results obtained using a /sup 51/Cr assay indicated no consistent significant difference between phagocytosis of C. jejuni between the three mononuclear phagocytes or PMN's and that maximum infection occurred prior to 0.5 hr and maintained throughout the 4 hr assay. Further investigation of the mechanism of attachment and entry of C. jejuni revealed this process required the expenditure of energy by the phagocyte, but was not inhibited by inhibitors of microfilament functions. In addition, phagocytosis was enhanced by the presence of 20% FCS,

  18. Effect of a trivalent vaccine against Staphylococcus aureus mastitis lymphocyte subpopulations, antibody production, and neutrophil phagocytosis.

    PubMed

    Lee, Jai-Wei; O'Brien, Celia N; Guidry, Albert J; Paape, Max J; Shafer-Weaver, Kimberley A; Zhao, X

    2005-01-01

    The effect of a novel bovine mastitis trivalent vaccine, containing Staphylococcus aureus capsular polysaccharide type 5 (T5), 8 (T8), and 336 (T336), on lymphocyte subpopulations, antibody production, and neutrophil phagocytosis was evaluated. Twenty pregnant heifers were immunized with either the trivalent alone, trivalent emulsified in Freund's incomplete adjuvant (FICA), trivalent in aluminum hydroxide, or adjuvant only (FICA). Immunization was done 30 d before the expected calving date followed by 2 boosts in a 2-week interval. Compared to FICA, serum antigen-specific immunoglobulin (Ig)G1 and IgG2 were significantly increased in all the vaccinated groups before parturition and sustained until 3 wk postpartum. In comparison with the trivalent alone, formulation with either adjuvant enhanced production of IgG2, but not IgG1. Immune sera, which contained the highest amount of antibodies, slightly increased neutrophil phagocytosis to the 3 serotypes of killed S. aureus, but most of the differences were not significant due to large variation between the cows. The percentage of CD4+ lymphocyte was significantly higher in vaccinated groups than that of FICA 4 wk after the primary immunization. In comparison with FICA, cows inoculated with trivalent vaccine and adjuvants had an increased percentage of CD8+ lymphocytes at 2 time points, 2 wk before and after calving. Our results indicated that the whole cell trivalent vaccine, with or without adjuvants, is able to elicit antibody responses specific to the 3 capsular polysaccharide antigens. The increase of T8-specific IgG2 was more noticeable when the vaccine was emulsified with adjuvants.

  19. Shrimp miR-12 Suppresses White Spot Syndrome Virus Infection by Synchronously Triggering Antiviral Phagocytosis and Apoptosis Pathways

    PubMed Central

    Shu, Le; Zhang, Xiaobo

    2017-01-01

    Growing evidence has indicated that the innate immune system can be regulated by microRNAs (miRNAs). However, the mechanism underlying miRNA-mediated simultaneous activation of multiple immune pathways remains unknown. To address this issue, the role of host miR-12 in shrimp (Marsupenaeus japonicus) antiviral immune responses was characterized in the present study. The results indicated that miR-12 participated in virus infection, host phagocytosis, and apoptosis in defense against white spot syndrome virus invasion. miR-12 could simultaneously trigger phagocytosis, apoptosis, and antiviral immunity through the synchronous downregulation of the expression of shrimp genes [PTEN (phosphatase and tensin homolog) and BI-1(transmembrane BAX inhibitor motif containing 6)] and the viral gene (wsv024). Further analysis showed that miR-12 could synchronously mediate the 5′–3′ exonucleolytic degradation of its target mRNAs, and this degradation terminated in the vicinity of the 3′ untranslated region sequence complementary to the seed sequence of miR-12. Therefore, the present study showed novel aspects of the miRNA-mediated simultaneous regulation of multiple immune pathways. PMID:28824612

  20. Developmental and heat stress-regulated expression of HsfA2 and small heat shock proteins in tomato anthers

    PubMed Central

    Giorno, Filomena; Wolters-Arts, Mieke; Grillo, Stefania; Scharf, Klaus-Dieter; Vriezen, Wim H.; Mariani, Celestina

    2010-01-01

    The high sensitivity of male reproductive cells to high temperatures may be due to an inadequate heat stress response. The results of a comprehensive expression analysis of HsfA2 and Hsp17-CII, two important members of the heat stress system, in the developing anthers of a heat-tolerant tomato genotype are reported here. A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR. The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis. Based on the analysis of the gene and protein expression profiles, HsfA2 and Hsp17-CII are finely regulated during anther development and are further induced under both short and prolonged heat stress conditions. These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures. PMID:19854799

  1. Phospholipase A(2) activation by poultry particulate matter is mediated through extracellular signal-regulated kinase in lung epithelial cells: regulation of interleukin-8 release.

    PubMed

    Kotha, Sainath R; Piper, Melissa G; Patel, Rishi B; Sliman, Sean; Malireddy, Smitha; Zhao, Lingying; Baran, Christopher P; Nana-Sinkam, Patrick S; Wewers, Mark D; Romberger, Debra; Marsh, Clay B; Parinandi, Narasimham L

    2013-11-01

    The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA2 in A549 cells, which was attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA2 serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA2. The poultry PM-induced cPLA2 serine phosphorylation and IL-8 release were attenuated by AACOCF3, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA2 knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA2 activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.

  2. Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis

    PubMed Central

    Levin, Roni; Hammond, Gerald R. V.; Balla, Tamas; De Camilli, Pietro; Fairn, Gregory D.; Grinstein, Sergio

    2017-01-01

    We analyzed the distribution, fate, and functional role of phosphatidylinositol 4-phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis. The phosphoinositide, which is present in the plasmalemma before engagement of the target particle, is transiently enriched in the phagosomal cup. Soon after the phagosome seals, PtdIns4P levels drop precipitously due to the hydrolytic activity of Sac2 and phospholipase C, becoming undetectable for ∼10 min. PtdIns4P disappearance coincides with the emergence of phagosomal PtdIns3P. Conversely, the disappearance of PtdIns3P that signals the transition from early to late phagosomes is accompanied by resurgence of PtdIns4P, which is associated with the recruitment of phosphatidylinositol 4-kinase 2A. The reacquisition of PtdIns4P can be prevented by silencing expression of the kinase and can be counteracted by recruitment of a 4-phosphatase with a heterodimerization system. Using these approaches, we found that the secondary accumulation of PtdIns4P is required for proper phagosomal acidification. Defective acidification may be caused by impaired recruitment of Rab7 effectors, including RILP, which were shown earlier to displace phagosomes toward perinuclear lysosomes. Our results show multimodal dynamics of PtdIns4P during phagocytosis and suggest that the phosphoinositide plays important roles during the maturation of the phagosome. PMID:28035045

  3. Group X Secretory Phospholipase A2 Negatively Regulates ABCA1 and ABCG1 expression and cholesterol efflux in macrophages

    PubMed Central

    Shridas, Preetha; Bailey, William M; Gizard, Florence; Oslund, Rob C; Gelb, Michael H; Bruemmer, Dennis; Webb, Nancy R

    2010-01-01

    Objectives Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids and has been implicated in inflammatory diseases including atherosclerosis. Here we identify a novel role for GX sPLA2 in modulating ABCA1 and ABCG1 expression and hence macrophage cholesterol efflux. Methods and Results Overexpression or exogenous addition of GX sPLA2 significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA2 deficiency in mouse peritoneal macrophages (MPMs) was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA2 overexpressing J774 cells, and increased efflux in GX sPLA2-deficient MPMs. Gene regulation was dependent on GX sPLA2 catalytic activity, mimicked by arachidonic acid, abrogated when LXRα/β expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA2 suppresses the ability of LXR to trans-activate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand binding domain. Conclusions GX sPLA2 modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA2 may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating genes critical for cellular cholesterol efflux. PMID:20844270

  4. Induction of Live Cell Phagocytosis by a Specific Combination of Inflammatory Stimuli.

    PubMed

    Ishidome, Takamasa; Yoshida, Takeshi; Hanayama, Rikinari

    2017-08-01

    Conditions of severe hyper-inflammation can lead to uncontrolled activation of macrophages, and the ensuing phagocytosis of live cells. However, relationships between inflammatory stimuli and uncontrolled phagocytosis of live cells by macrophages are poorly understood. To identify mediators of this process, we established phagocytosis assays of live cells by stimulating macrophages with CpG DNA, interferon-γ, and anti-interleukin-10 receptor antibody. In this model, various cell surface receptors were upregulated on macrophages, and phagocytosis of live cells was induced in a Rac1-dependent manner. Subsequent inhibition of the ICAM-1, VCAM-1, and both of these receptors abolished in vitro and in vivo phagocytosis of live T cells, myeloid cells, and B cells, respectively. Specifically, the reduction in lymphocyte numbers due to in vivo activation of macrophages was ameliorated in Icam-1-deficient mice. In addition, overexpression of ICAM-1 or VCAM-1 in non-phagocytic NIH3T3 cells led to active phagocytosis of live cells. These data indicate molecular mechanisms underlying live cell phagocytosis induced by hyper-inflammation, and this experimental model will be useful to clarify the pathophysiological mechanisms of hemophagocytosis and to indicate therapeutic targets. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis

    PubMed Central

    Sierra, Amanda; Abiega, Oihane; Shahraz, Anahita; Neumann, Harald

    2012-01-01

    Microglia are the resident brain macrophages and they have been traditionally studied as orchestrators of the brain inflammatory response during infections and disease. In addition, microglia has a more benign, less explored role as the brain professional phagocytes. Phagocytosis is a term coined from the Greek to describe the receptor-mediated engulfment and degradation of dead cells and microbes. In addition, microglia phagocytoses brain-specific cargo, such as axonal and myelin debris in spinal cord injury or multiple sclerosis, amyloid-β deposits in Alzheimer's disease, and supernumerary synapses in postnatal development. Common mechanisms of recognition, engulfment, and degradation of the different types of cargo are assumed, but very little is known about the shared and specific molecules involved in the phagocytosis of each target by microglia. More importantly, the functional consequences of microglial phagocytosis remain largely unexplored. Overall, phagocytosis is considered a beneficial phenomenon, since it eliminates dead cells and induces an anti-inflammatory response. However, phagocytosis can also activate the respiratory burst, which produces toxic reactive oxygen species (ROS). Phagocytosis has been traditionally studied in pathological conditions, leading to the assumption that microglia have to be activated in order to become efficient phagocytes. Recent data, however, has shown that unchallenged microglia phagocytose apoptotic cells during development and in adult neurogenic niches, suggesting an overlooked role in brain remodeling throughout the normal lifespan. The present review will summarize the current state of the literature regarding the role of microglial phagocytosis in maintaining tissue homeostasis in health as in disease. PMID:23386811

  6. Role of Yops in inhibition of phagocytosis and killing of opsonized Yersinia enterocolitica by human granulocytes.

    PubMed

    Visser, L G; Annema, A; van Furth, R

    1995-07-01

    The virulence plasmid of Yersinia enterocolitica codes for the production of the outer membrane protein YadA and the secretion of several proteins, called Yops, which may play a role in the interaction between granulocytes and this bacterium. We investigated whether the expression of YadA or the secretion of Yops affected the phagocytosis and killing of opsonized Y. enterocolitica by human granulocytes. The rates of phagocytosis and killing of Y. enterocolitica by granulocytes in suspension in the presence of rabbit Yersinia antibodies and complement were determined by microbiological assays. In addition, noningested cell-adherent bacteria were differentiated from ingested yersiniae by immunofluorescence microscopy. Plasmid-bearing opsonized Y. enterocolitica was able to inhibit phagocytosis and killing by human granulocytes. The inhibition of phagocytosis was specific for the plasmid-bearing strain of Y. enterocolitica, since granulocytes were still able to phagocytose and kill Staphylococcus aureus in the presence of Y. enterocolitica. Plasmid-cured Y. enterocolitica was readily phagocytosed and killed by these cells. To investigate the role of YadA or Yops in the inhibition of phagocytosis by granulocytes, the phagocytosis of mutant strains unable to express YadA or to secrete Yops was studied. A Y. enterocolitica mutant unable to secrete Yops lost its ability to inhibit phagocytosis; a mutant expressing only YadA was readily ingested by granulocytes. These results indicate that after attachment of opsonized Y. enterocolitica to granulocytes, Yops play an important role in inhibiting the ingestion of Y. enterocolitica by human granulocytes.

  7. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis.

    PubMed

    Hornik, Tamara C; Vilalta, Anna; Brown, Guy C

    2016-01-01

    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis.

  8. In vitro phagocytosis of boar spermatozoa by neutrophils from peripheral blood of sows.

    PubMed

    Matthijs, A; Harkema, W; Engel, B; Woelders, H

    2000-11-01

    A considerable number of spermatozoa are used in each sow in routine artificial insemination. However, within a few hours after insemination, many spermatozoa are phagocytosed by polymorphonuclear leucocytes. Some aspects of sperm transport in the female genital tract in the sow have been thoroughly investigated, whereas little is known about the mechanisms involved in the phagocytosis of spermatozoa, or about which spermatozoa (fresh, capacitated or dead) are the most susceptible to ingestion by polymorphonuclear leucocytes. In this study, phagocytosis was investigated by use of an in vitro phagocytosis assay. Polymorphonuclear leucocytes were challenged with either untreated, cold-shocked or frozen-thawed spermatozoa, or with spermatozoa that had been treated to induce capacitation in vitro. The influence of serum on phagocytosis was also investigated. Treatment of the semen to induce capacitation in vitro considerably reduced the phagocytosis of spermatozoa, whereas crude treatments like cold-shock or freezing and thawing reduced phagocytosis only in the first 15-30 min of incubation with polymorphonuclear leucocytes. Viable spermatozoa were phagocytosed mainly through a pathway that was independent of complement or other serum components (for example, antibodies). Complement had little effect on phagocytosis of spermatozoa, but did cause acrosomal exocytosis and cell death.

  9. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

    PubMed Central

    Hornik, Tamara C.; Vilalta, Anna; Brown, Guy C.

    2016-01-01

    ABSTRACT Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis. PMID:26567213

  10. Phagocytosis of gram-negative bacteria by a unique CD14-dependent mechanism.

    PubMed

    Schiff, D E; Kline, L; Soldau, K; Lee, J D; Pugin, J; Tobias, P S; Ulevitch, R J

    1997-12-01

    THP-1-derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI)-anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat-inactivated)-opsonized Escherichia coli (opEc). Both the GPI-anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide-binding protein (LBP) played a role in CD14-dependent phagocytosis as evidenced by inhibition of CD14-dependent phagocytosis of opEc with anti-LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coli opsonized with purified LBP. CD14-dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3-kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolyl-maleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti-LBP mAb 18G4 and anti-CD14 mAb 18E12 were used to differentiate between the pathways involved in CD14-dependent phagocytosis and CD14-dependent cell activation. F(ab')2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1-wtCD14 cells. 18E12 (an anti-CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14-dependent cell activation) did not inhibit phagocytosis of LBP-opEc by THP1-wtCD14 cells. Furthermore, CD14-dependent phagocytosis was not inhibited by anti-CD18 (CR3 and CR4 beta-chain) or anti-Fcgamma receptor mAb.

  11. Aging impairs peritoneal but not bone marrow-derived macrophage phagocytosis.

    PubMed

    Linehan, Eimear; Dombrowski, Yvonne; Snoddy, Rachel; Fallon, Padraic G; Kissenpfennig, Adrien; Fitzgerald, Denise C

    2014-08-01

    Aging results in deterioration of the immune system, which is associated with increased susceptibility to infection and impaired wound healing in the elderly. Phagocytosis is an essential process in both wound healing and immune defence. As such, age-related impairments in phagocytosis impact on the health of the elderly population. Phagocytic efficiency in peritoneal macrophages, bone marrow-derived macrophages and bone marrow monocytes from young and old mice was investigated. Aging significantly impaired phagocytosis by peritoneal macrophages, both in vitro and in vivo. However, bone marrow-derived macrophages and bone marrow monocytes did not exhibit age-related impairments in phagocytosis, suggesting no intrinsic defect in these cells. We sought to investigate underlying mechanisms in age-related impairments in phagocytosis by peritoneal macrophages. We hypothesized that microenvironmental factors in the peritoneum of old mice impaired macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. Proportions of peritoneal immune cells were characterized, and striking increases in numbers of T cells, B1 and B2 cells were observed in the peritoneum of old mice compared with young mice. In addition, B cell-derived IL-10 was increased in resting and LPS-activated peritoneal cell cultures from old mice. These data demonstrate that aging impairs phagocytosis by tissue-resident peritoneal macrophages, but not by bone marrow-derived macrophages/monocytes, and suggest that age-related defects in macrophage phagocytosis may be due to extrinsic factors in the tissue microenvironment. As such, defects may be reversible and macrophages could be targeted therapeutically in order to boost immune function in the elderly.

  12. A Diaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis

    PubMed Central

    Junemann, Alexander; Filić, Vedrana; Winterhoff, Moritz; Nordholz, Benjamin; Litschko, Christof; Schwellenbach, Helena; Stephan, Till; Weber, Igor; Faix, Jan

    2016-01-01

    Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas. PMID:27821733

  13. Targeting of Neutrophil Lewis X Blocks Transepithelial Migration and Increases Phagocytosis and Degranulation.

    PubMed

    Brazil, Jennifer C; Sumagin, Ronen; Cummings, Richard D; Louis, Nancy A; Parkos, Charles A

    2016-02-01

    Polymorphonuclear leukocytes (PMNs) are innate immune cells whose principal function is to migrate from the blood to sites of inflammation, where they exert crucial anti-infectious and immunomodulatory effects. However, dysregulated migration of PMNs into mucosal epithelial tissues is characteristic of chronic inflammatory disorders, including inflammatory bowel disease. Carbohydrate-mediated binding interactions between PMN Lewis glycans and endothelial glycan-binding proteins are critical for initial migration of PMN out of the vasculature. However, the role of Lewis glycans during transepithelial migration (TEM) has not been well characterized. Herein, we show that antibody blockade of Lewis X (Le(x)) displayed as terminal glycan residues on the PMN surface blocks chemotaxis and TEM while enhancing PMN-adhesive interactions with intestinal epithelia. Unexpectedly, targeting of subterminal Le(x) residues within glycan chains had no effect on PMN migration or adhesive interactions. There was increased surface expression of Le(x) on PMN after TEM, and blockade of terminal Le(x) regulated post-migratory PMN functions, increasing PMN phagocytosis and the surface mobilization of azurophilic (CD63, myeloperoxidase, and neutrophil elastase) and specific (CD66b and lactoferrin) granule markers. These findings suggest that terminal Le(x) represents a potential target for regulating PMN trafficking and function in inflamed mucosa. Furthermore, given its abundant expression on migrating PMN, Le(x) may be a rational target for modulating inflammation in diseases where dysregulated PMN influx is associated with host tissue damage.

  14. The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

    PubMed Central

    2013-01-01

    Background In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. Results The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. Conclusions The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic

  15. Dopamine modulates hemocyte phagocytosis via a D1-like receptor in the rice stem borer, Chilo suppressalis

    PubMed Central

    Wu, Shun-Fan; Xu, Gang; Stanley, David; Huang, Jia; Ye, Gong-Yin

    2015-01-01

    Dopamine (DA) is a signal moiety bridging the nervous and immune systems. DA dysregulation is linked to serious human diseases, including addiction, schizophrenia, and Parkinson’s disease. However, DA actions in the immune system remain incompletely understood. In this study, we found that DA modulates insect hemocyte phagocytosis using hemocytes prepared from the rice stem borer (RSB), Chilo suppressalis. We investigated whether insect hemocytes are capable of de novo DA production. Here we show that exposing hemocytes to lipopolysaccharide (LPS) led to induction of DA-generating enzymes. Exogenous DA induced rapid phosphorylation of extracellular signal-regulated kinase (ERK) in naïve hemocytes. Activation of ERK was inhibited by preincubating with a DOP1 receptor antagonist. Thus, DA signaling via the DOP1 receptor may contribute to early hemocyte activation. DA synthesized and released from hemocytes may act in an autocrine mechanism to stimulate or maintain phagocytic activity. Consistent with this hypothesis, we found that inhibition of DA synthesis with α-methyl-DL-tyrosine methyl ester hydrochloride or blockage of DOP1 receptor with antagonist SCH23390 impaired hemocyte phagocytosis. Topical DA application also significantly decreased RSB mortality following challenge with the insect pathogenic fungus, Beauveria bassiana. We infer that a DA-dependent signaling system operates in hemocytes to mediate phagocytotic functions. PMID:26179416

  16. Virulent and Vaccine Strains of Streptococcus equi ssp. zooepidemicus Have Different Influences on Phagocytosis and Cytokine Secretion of Macrophages.

    PubMed

    Jie, Peng; Zhe, Ma; Chengwei, Hua; Huixing, Lin; Hui, Zhang; Chengping, Lu; Hongjie, Fan

    2017-01-06

    Swine streptococcosis is a significant threat to the Chinese pig industry, and Streptococcus equi ssp. zooepidemicus (SEZ) is one of the major pathogens. SEZ ATCC35246 is a classical virulent strain, while SEZ ST171 is a Chinese attenuated vaccine strain. In this study, we employed stable isotope labeling by amino acids in cell culture and liquid chromatography-mass spectrometry (LC-MS) to determine the differential response of macrophages to infection by these two strains. Eighty-seven upregulated proteins and 135 downregulated proteins were identified. The proteomic results were verified by real-time polymerase chain reaction for 10 chosen genes and Western blotting for three proteins. All differentially abundant proteins were analyzed for their Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations. Certain downregulated proteins were associated with immunity functions, and the upregulated proteins were related to cytomembrane and cytoskeleton regulation. The phagocytosis rate and cytokine genes transcription in Raw264.7 cells during SEZ ATCC35246 and ST171 infection were detected to confirm the bioinformatics results. These results showed that different effects on macrophage phagocytosis and cytokine expression might explain the different phenotypes of SEZ ATCC35246 and ST171 infection. This research provided clues to the mechanisms of host immunity responses to SEZ ST171and SEZ ATCC35246, which could identify potential therapy and vaccine development targets.

  17. ROCK inhibition with fasudil promotes early functional recovery of spinal cord injury in rats by enhancing microglia phagocytosis.

    PubMed

    Fu, Pei-cai; Tang, Rong-hua; Wan, Yue; Xie, Min-jie; Wang, Wei; Luo, Xiang; Yu, Zhi-yuan

    2016-02-01

    Emerging evidence indicates that microglia activation plays an important role in spinal cord injury (SCI) caused by trauma. Studies have found that inhibiting the Rho/Rho-associated protein kinase (ROCK) signaling pathway can reduce inflammatory cytokine production by microglia. In this study, Western blotting was conducted to detect ROCK2 expression after the SCI; the ROCK Activity Assay kit was used for assay of ROCK pathway activity; microglia morphology was examined using the CD11b antibody; electron microscopy was used to detect microglia phagocytosis; TUNEL was used to detect tissue cell apoptosis; myelin staining was performed using an antibody against myelin basic protein (MBP); behavioral outcomes were evaluated according to the methods of Basso, Beattie, and Bresnahan (BBB). We observed an increase in ROCK activity and microglial activation after SCI. The microglia became larger and rounder and contained myelin-like substances. Furthermore, treatment with fasudil inhibited neuronal cells apoptosis, alleviated demyelination and the formation of cavities, and improved motor recovery. The experimental evidence reveals that the ROCK inhibitor fasudil can regulate microglial activation, promote cell phagocytosis, and improve the SCI microenvironment to promote SCI repair. Thus, fasudil may be useful for the treatment of SCI.

  18. Dopamine modulates hemocyte phagocytosis via a D1-like receptor in the rice stem borer, Chilo suppressalis.

    PubMed

    Wu, Shun-Fan; Xu, Gang; Stanley, David; Huang, Jia; Ye, Gong-Yin

    2015-07-16

    Dopamine (DA) is a signal moiety bridging the nervous and immune systems. DA dysregulation is linked to serious human diseases, including addiction, schizophrenia, and Parkinson's disease. However, DA actions in the immune system remain incompletely understood. In this study, we found that DA modulates insect hemocyte phagocytosis using hemocytes prepared from the rice stem borer (RSB), Chilo suppressalis. We investigated whether insect hemocytes are capable of de novo DA production. Here we show that exposing hemocytes to lipopolysaccharide (LPS) led to induction of DA-generating enzymes. Exogenous DA induced rapid phosphorylation of extracellular signal-regulated kinase (ERK) in naïve hemocytes. Activation of ERK was inhibited by preincubating with a DOP1 receptor antagonist. Thus, DA signaling via the DOP1 receptor may contribute to early hemocyte activation. DA synthesized and released from hemocytes may act in an autocrine mechanism to stimulate or maintain phagocytic activity. Consistent with this hypothesis, we found that inhibition of DA synthesis with α-methyl-DL-tyrosine methyl ester hydrochloride or blockage of DOP1 receptor with antagonist SCH23390 impaired hemocyte phagocytosis. Topical DA application also significantly decreased RSB mortality following challenge with the insect pathogenic fungus, Beauveria bassiana. We infer that a DA-dependent signaling system operates in hemocytes to mediate phagocytotic functions.

  19. Annexin A2 could enhance multidrug resistance by regulating NF-κB signaling pathway in pediatric neuroblastoma.

    PubMed

    Wang, Yi; Chen, Kai; Cai, Yihong; Cai, Yuanxia; Yuan, Xiaojun; Wang, Lifeng; Wu, Zhixiang; Wu, Yeming

    2017-08-16

    Chemotherapy is one of major therapeutic regimens for neuroblastoma (NB) in children. However, recurrence and metastasis associated with poor prognosis caused by acquired multidrug resistance remains a challenge. There is a great need to achieve new insight into the molecular mechanism of drug resistance in NB. The aim of this study is to identify novel drug sensitivity-related biomarkers as well as new therapeutic targets to overcome chemoresistance. We proteome-wide quantitatively compared protein expression of two NB cell lines with different drug sensitivities, isolated from the same patient prior to and following chemotherapy. Annexin A2 (ANXA2) emerged as a key factor contributing to drug resistance in NB. Then, we assessed the correlation of ANXA2 expression and clinical characteristics using a tissue microarray. Further, the roles of ANXA2 in chemoresistance for NB and the underlying mechanisms were studied by using short hairpin RNA (shRNA) in vitro and vivo. First in total, over 6000 proteins were identified, and there were about 460 significantly regulated proteins which were up- or down-regulated by greater than two folds. We screened out ANXA2 which was upregulated by more than 12-fold in the chemoresistant NB cell line, and it might be involved in the drug resistance of NB. Then, using a tissue chip containing 42 clinical NB samples, we found that strong expression of ANXA2 was closely associated with advanced stage, greater number of chemotherapy cycles, tumor metastasis and poor prognosis. Following knockdown of ANXA2 in NB cell line SK-N-BE(2) using shRNA, we demonstrate enhanced drug sensitivity for doxorubicin (2.77-fold) and etoposide (7.87-fold) compared with control. Pro-apoptotic genes such as AIF and cleaved-PARP were upregulated. Inhibiting ANXA2 expression attenuated transcriptional activity of NF-κB via down-regulated nuclear translocation of subunit p50. Finally, simulated chemotherapy in a xenograft NB nude mouse model suggests that

  20. Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro.

    PubMed

    Ducusin, Rio John T; Nishimura, Masakazu; Sarashina, Takao; Uzuka, Yuji; Tanabe, Shigeyuki; Otani, Masayuki

    2003-04-01

    To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.

  1. Free lung cell phagocytosis and the effect of cigarette smoke exposure

    SciTech Connect

    Fogelmark, B.; Rylander, R.; Sjoestrand, M.; Reininghaus, W.

    1980-06-01

    We report on a technique for studying phagocytosis in free lung cells with the use of fungal spores. Free lung cells were obtained from a bronchial lavage. They were incubated with fungal spores and the engulfment of these spores was studied at various time intervals and under different conditions. The phagocytosis process was found to occur from relatively stationary macrophages within the first hours after incubation. The number of engulfed spores was proportional to their number in the solution. Addition of serum or surfactant to the medium increased the phagocytosis rate. In hamsters and rats exposed to tobacco smoke under in vivo conditions, a dose-related increase in phagocytosis rate could be demonstrated.

  2. Real-time measurements of membrane surface dynamics on macrophages and the phagocytosis of Leishmania parasites.

    PubMed

    Coelho Neto, José; Agero, Ubirajara; Oliveira, Diogo C P; Gazzinelli, Ricardo T; Mesquita, Oscar N

    2005-02-15

    Defocusing microscopy was used for real-time observation and quantification of membrane surface dynamics in murine bone marrow macrophages. Small random membrane fluctuations (SRMF), possibly metabolic driven, were detected uniformly over all membrane surface. Morphological and dynamical parameters of ruffles, such as shape, dimensions, and velocity of propagation, were analyzed. Optical tweezers were used to promote phagocytosis of single Leishmania amazonensis amastigotes by selected macrophages. Analysis of ruffling activity on the macrophages before and during phagocytosis of the parasites indicated that increased ruffling response near forming phagosomes, most likely induced by the parasite, accelerates phagocytosis. The effects of temperature decrease on the dynamics of membrane surface fluctuations and on the phagocytosis of parasites were used to determine the overall activation energies involved in these processes. The values obtained support the existence of strong correlation between membrane motility and phagocytic capacity.

  3. Dectin-1 mediates in vitro phagocytosis of Candida albicans yeast cells by retinal microglia.

    PubMed

    Maneu, Victoria; Yáñez, Alberto; Murciano, Celia; Molina, Andrés; Gil, María Luisa; Gozalbo, Daniel

    2011-10-01

    We have investigated the expression of TLR2 and Dectin-1 in retinal microglia and their involvement in Candida albicans phagocytosis using a cytometric approach. The expression of both receptors has been demonstrated in CD11b(+) retinal cells. Phagocytosis of pHrodo-labelled C. albicans yeasts by microglial CD11b(+) cells of C57BL/6 mice was inhibited both by the Dectin-1 antagonist laminarin and anti-Dectin-1 antibodies, whereas phagocytosis of yeasts by retinal microglia of TLR2 KO mice was unaffected. These data indicate that phagocytosis of C. albicans yeasts by retinal microglia is mediated by Dectin-1, whereas TLR2 does not play a significant role in this process.

  4. Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

    PubMed Central

    Lewis, Leanne E.; Bain, Judith M.; Okai, Blessing; Gow, Neil A.R.; Erwig, Lars Peter

    2013-01-01

    Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10. In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a

  5. Genetic variation in Surfactant Protein-A2 (SP-A2) leads to differential binding to Mycoplasma pneumoniae membranes and regulation of host responses

    PubMed Central

    Ledford, Julie G.; Voelker, Dennis R.; Addison, Kenneth J.; Wang, Ying; Nikam, Vinayak; Degan, Simone; Kandasamy, Pitachaimani; Tanyaratsrisakul, Sasipa; Fischer, Bernard M.; Kraft, Monica; Hollingsworth, John W.

    2015-01-01

    Mycoplasma pneumoniae (Mp) is an extracellular pathogen that colonizes mucosal surfaces of the respiratory tract and is associated with asthma exacerbations. Previous reports demonstrate that surfactant protein-A (SP-A) binds live Mp and mycoplasma membranes (MMF) with high affinity. Humans express a repertoire of single amino acid genetic variants of SP-A that may be associated with lung disease, and our findings demonstrate that allelic differences in SP-A2 (Gln223Lys) affect the binding to MMF. We show that SP-A−/− mice are more susceptible to MMF exposure and have significant increases in mucin production and neutrophil recruitment. Novel humanized-SP-A2 transgenic mice harboring the hSP-A2 223K allele exhibit reduced neutrophil influx and mucin production in the lungs, when challenged with MMF, compared to SP-A−/− mice. Conversely, mice expressing hSP-A2 223Q have increased neutrophil influx and mucin production that is similar to SP-A−/− mice. Using tracheal epithelial cell cultures, we show that enhanced mucin production to MMF occurs in the absence of SP-A, and is not dependent upon neutrophil recruitment. Increased phosphorylation of the epidermal growth factor receptor (EGFR) was evident in the lungs of MMF-challenged mice when SP-A was absent. Pharmacologic inhibition of EGFR prior to MMF challenge dramatically reduced mucin production in SP-A−/− mice. These findings suggest a protective role for SP-A in limiting MMF-stimulated mucin production that occurs through interference with EGFR mediated signaling. The SP-A interaction with the EGFR signaling pathway appears to occur in an allele specific manner that may have important implications for SP-A polymorphisms in human diseases. PMID:25957169

  6. Expression and regulation of phospholipase A2 in venom gland of the chinese honeybee, Apis cerana cerana.

    PubMed

    Li, Jiang-Hong; Zhang, Chuan-Xi; Shen, Li-rong; Tang, Zhen-Hua; Cheng, Jia-An

    2005-09-01

    Phospholipase A(2) (PLA(2)) is one of the components of bee venom with a wide range of pharmacological functions. It operates as a major allergen working with other venom components to defend the colony from intruder. In the present study, the cDNA sequence of the Ac-pla(2) gene from cDNA library of the venom gland of Apis cerana was compared with the amplified corresponding region of genomic DNA. The result showed that the Ac-pla(2) gene consisted of four exons and three introns. Southern blot showed that the Ac-pla(2) gene was a single copy per haploid genome. The most active transcription period was during the first 8 days of adults, which correspondingly was the period of sharp increase of PLA(2) protein. ELISA analysis revealed that the PLA(2) was undetectable in pupal stage and the newly eclosed adult, but increased sharply to a maximum of 10-12 mug per honeybee by 8-10 days of adult life, followed by a gradual decrease to 8 mug for the rest of adult life. Transcriptional or post transcriptional regulation is the key step for Ac-pla(2) expression. The early secreted Ac-PLA(2) showed a low degree of post-translational modification; with increasing age, glycosylation was detected by Western blot and glycoprotein staining analysis. Different post-translational modifications were found among different individuals in A. cerana when compared to A. mellifera.

  7. Effects of Schistosoma mansoni infection on phagocytosis and killing of Proteus vulgaris in Biomphalaria glabrata hemocytes.

    PubMed

    Douglas, J S; Hunt, M D; Sullivan, J T

    1993-04-01

    With the use of a fluorescence microassay, in vitro phagocytosis and killing of Proteus vulgaris were measured in hemocytes of NIH albino Biomphalaria glabrata infected with Schistosoma mansoni for 1, 2, 3, or 4 wk. Although hemocytes of infected snails displayed decreased phagocytosis, relative to hemocytes of uninfected snails, at 4 wk postinfection (PI), they exhibited enhanced microbicidal activity at 3 wk PI. No microbicidal activity was detected in the plasma of either infected or uninfected snails.

  8. The importance of surfactant proteins-New aspects on macrophage phagocytosis.

    PubMed

    Tschernig, Thomas; Veith, Nils T; Diler, Ebru; Bischoff, Markus; Meier, Carola; Schicht, Martin

    2016-11-01

    Surfactant and its components have multiple functions. The so called collectins are surfactant proteins which opsonize bacteria and improve pulmonary host defense via the phagocytosis and clearance of microorganisms and particles. In this special issue of the Annals of Anatomy a new surfactant protein, Surfactant Associated 3, is highlighted. As outlined in this mini review Surfactant Associated 3 is regarded as an enhancer of phagocytosis. In addition, the role played by SP-A is updated and open research questions raised.

  9. Anti-CD16 autoantibodies and delayed phagocytosis of apoptotic cells in primary biliary cirrhosis.

    PubMed

    Allina, Jorge; Stanca, Carmen M; Garber, John; Hu, Bin; Sautes-Fridman, Catherine; Bach, Nancy; Odin, Joseph A

    2008-06-01

    Primary biliary cirrhosis is characterized by chronic hepatic inflammation and immune mediated apoptosis of bile duct epithelial cells. Delayed macrophage phagocytosis of opsonized apoptotic cells, noted in other autoimmune diseases, may promote inflammation. Recent studies suggest serum anti-CD16 autoantibodies contribute to impaired macrophage phagocytosis by blocking complement receptor 3 (CR3) signaling via CD16. Therefore, serum anti-CD16 levels and the ability of monocyte derived macrophages from individuals with PBC to phagocytosis apoptotic cells were compared to controls. The mean level of anti-CD16 IgM autoantibodies (0.86+/-0.62 v. 0.35+/-0.22, respectively, p=0.031) was increased in PBC compared to control sera, and mean PBC phagocytosis of opsonized apoptotic cells was significantly decreased compared to controls (23.9+/-12.2% v. 43.9+/-14.4%, respectively, p=0.020). However, PBC phagocytosis of opsonized apoptotic cells was not significantly affected by the presence or absence of autologous serum (20.8+/-13.5% v. 23.9+/-12.2%, respectively, p=0.560). PBC phagocytosis of opsonized apoptotic cells inversely correlated with CD16 (and CR3) expression levels on Day 5 after culture in the presence or absence of autologous serum (r=-0.546, p=0.033 and r=-0.519, p=0.042, respectively). Phagocytosis of non-opsonized apoptotic cells did not correlate with CD16 or CR3 expression (p>0.050). In conclusion, PBC macrophage phagocytosis of opsonized apoptotic cells is impaired, irrespective of serum factors and may increase hepatic inflammation.

  10. Collagen remodeling by phagocytosis is determined by collagen substrate topology and calcium-dependent interactions of gelsolin with nonmuscle myosin IIA in cell adhesions

    PubMed Central

    Arora, P. D.; Wang, Y.; Bresnick, A.; Dawson, J.; Janmey, P. A.; McCulloch, C. A.

    2013-01-01

    We examine how collagen substrate topography, free intracellular calcium ion concentration ([Ca2+]i, and the association of gelsolin with nonmuscle myosin IIA (NMMIIA) at collagen adhesions are regulated to enable collagen phagocytosis. Fibroblasts plated on planar, collagen-coated substrates show minimal increase of [Ca2+]i, minimal colocalization of gelsolin and NMMIIA in focal adhesions, and minimal intracellular collagen degradation. In fibroblasts plated on collagen-coated latex beads there are large increases of [Ca2+]i, time- and Ca2+-dependent enrichment of NMMIIA and gelsolin at collagen adhesions, and abundant intracellular collagen degradation. NMMIIA knockdown retards gelsolin recruitment to adhesions and blocks collagen phagocytosis. Gelsolin exhibits tight, Ca2+-dependent binding to full-length NMMIIA. Gelsolin domains G4–G6 selectively require Ca2+ to interact with NMMIIA, which is restricted to residues 1339–1899 of NMMIIA. We conclude that cell adhesion to collagen presented on beads activates Ca2+ entry and promotes the formation of phagosomes enriched with NMMIIA and gelsolin. The Ca2+ -dependent interaction of gelsolin and NMMIIA in turn enables actin remodeling and enhances collagen degradation by phagocytosis. PMID:23325791

  11. The Normative Development of Emotion Regulation Strategy Use in Children and Adolescents: A 2-Year Follow-Up Study

    ERIC Educational Resources Information Center

    Gullone, Eleonora; Hughes, Elizabeth K.; King, Neville J.; Tonge, Bruce

    2010-01-01

    Background: Emotion regulation involves intrinsic and extrinsic processes responsible for managing one's emotions toward goal accomplishment. Research on emotion regulation has predominantly focused on early developmental periods and the majority of emotion regulation research examining the pre-adult years has lacked a comprehensive theoretical…

  12. Increased phagocytosis of platelets from patients with secondary dengue virus infection by human macrophages.

    PubMed

    Honda, Shoko; Saito, Mariko; Dimaano, Efren M; Morales, Philip A; Alonzo, Maria T G; Suarez, Lady-Anne C; Koike, Natsuki; Inoue, Shingo; Kumatori, Atsushi; Matias, Ronald R; Natividad, Filipinas F; Oishi, Kazunori

    2009-05-01

    The relationship between the percent phagocytosis of platelets by differentiated THP-1 cells was examined using flowcytometry and the peripheral platelet counts as well as platelet-associated IgG (PAIgG) in 36 patients with secondary dengue virus (DV) infections. The percent phagocytosis and the levels of PAIgG were significantly increased in these patients during the acute phase compared with the healthy volunteers. The increased percent phagocytosis and PAIgG found during the acute phase significantly decreased during the convalescent phase. An inverse correlation between platelet count and the percent phagocytosis (P = 0.011) and the levels of PAIgG (P = 0.041) was found among these patients during the acute phase. No correlation was found, however, between the percent phagocytosis and the levels of PAIgG. Our present data suggest that accelerated platelet phagocytosis occurs during the acute phase of secondary DV infections, and it is one of the mechanisms of thrombocytopenia in this disease.

  13. Mechanism involved in interleukin-21-induced phagocytosis in human monocytes and macrophages.

    PubMed

    Vallières, F; Girard, D

    2017-02-01

    The interleukin (IL)-21/IL-21 receptor (R) is a promising system to be exploited for the development of therapeutic strategies. Although the biological activities of IL-21 and its cell signalling events have been largely studied in immunocytes, its interaction with human monocytes and macrophages have been neglected. Previously, we reported that IL-21 enhances Fc gamma receptor (FcRγ)-mediated phagocytosis in human monocytes and in human monocyte-derived macrophages (HMDM) and identified Syk as a novel molecular target of IL-21. Here, we elucidate further how IL-21 promotes phagocytosis in these cells. Unlike its ability to enhance phagocytosis of opsonized sheep red blood cells (SRBCs), IL-21 did not promote phagocytosis of Escherichia coli and zymosan by monocytes and did not alter the cell surface expression of CD16, CD32 and CD64. In HMDM, IL-21 was found to enhance phagocytosis of zymosan. In addition, we found that IL-21 activates p38, protein kinase B (Akt), signal transducer and activator of transcription (STAT)-1 and STAT-3 in monocytes and HMDM. Using a pharmacological approach, we demonstrate that IL-21 enhances phagocytosis by activating some mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)-Akt and Janus kinase (JAK)-STAT pathways. These results obtained in human monocytes and macrophages have to be considered for a better exploitation of the IL-21/IL-21R system for therapeutic purposes. © 2016 British Society for Immunology.

  14. Aggregates of ultrafine particles impair phagocytosis of microorganisms by human alveolar macrophages.

    PubMed

    Lundborg, Margot; Dahlén, Sven-Erik; Johard, Urban; Gerde, Per; Jarstrand, Connie; Camner, Per; Låstbom, Lena

    2006-02-01

    We investigated whether exposure of alveolar macrophages to aggregates of ultrafine carbon particles affected subsequent phagocytosis of microorganisms. Human alveolar macrophages were obtained by bronchoalveolar lavage and exposed to aggregates of ultrafine carbon particles or diesel exhaust particles (DEP) for 20 h before measurements of phagocytosis. The particle loads were estimated to be comparable to those of air pollution exposure with established health effects in humans. Phagocytotic activity was measured as attachment and ingestion of four different test particles (amorphous silica particles, yeast cells from Candida albicans, and Cryptococcus neoformans opsonized with specific IgG or fresh serum) that bind to scavenger, mannose, Fc, and complement receptors, respectively. Carbon preloading significantly impaired the attachment and ingestion process (P<0.01) for all particles, except for yeast cells from C. neoformans opsonized with specific IgG. On the average, the accumulated attachment decreased by 30% and the ingested fraction decreased by 10%. Loading of alveolar macrophages with either aggregates of ultrafine DEP or carbon particles impaired the phagocytosis of silica test particles in a similar way. Exposure of human alveolar macrophages to aggregates of carbon or DEP, in concentrations relevant to human environmental exposures, caused significant impairment of phagocytosis of silica particles and microorganisms. The inhibitory effect on particle phagocytosis mediated by four different receptors suggests that air pollution particles cause a general inhibition of macrophage phagocytosis. Such an effect may contribute to increased susceptibility to infections and, for example, result in more exacerbations of asthma and chronic obstructive pulmonary disease.

  15. The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis.

    PubMed

    Jayaprakash, K; Demirel, I; Khalaf, H; Bengtsson, T

    2015-10-01

    Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.

  16. A galectin from shrimp Litopenaeus vannamei is involved in immune recognition and bacteria phagocytosis.

    PubMed

    Hou, Fujun; Liu, Yongjie; He, Shulin; Wang, Xianzong; Mao, Aitao; Liu, Zhigang; Sun, Chengbo; Liu, Xiaolin

    2015-06-01

    Galectins are conserved family members with β-galactosides affinity that play multiple functions in embryogenesis, development and regulation of innate and adaptive immunity. However, little functional studies were reported in crustaceans. Here, a shrimp Litopenaeus vannamei galectin (LvGal) cDNA was identified with an open reading frame of 1017 bp, which encodes a putative protein of 338 amino acids. A carbohydrate recognition domain (CRD) and several amino acids residues involved in dimerization were found in LvGal. LvGal mRNA was mainly expressed in gills and hemocytes and upregulated post Vibrio anguillarum challenge. Recombinant LvGal (rLvGal) was expressed in Escherichia coli BL21 (DE3) and the purified rLvGal could strongly bind G(-) bacteria V. anguillarum and G(+) bacteria Micrococcus lysodeikticus. Besides, rLvGal exhibited strong activity to agglutinate V. anguillarum and weak activity to agglutinate M. lysodeikticus but no obvious antibacterial activity was found with selected bacteria. In addition, in vivo experiments showed rLvGal could promote phagocytosis of bacteria by hemocytes. Thus, through these collective data we predicted LvGal is involved in immune recognition and functions as a potential pattern recognition receptor.

  17. Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages.

    PubMed

    Herate, Cecile; Ramdani, Ghania; Grant, Nancy J; Marion, Sabrina; Gasman, Stephane; Niedergang, Florence; Benichou, Serge; Bouchet, Jerome

    2016-01-01

    Phospholipid Scramblase 1 (PLSCR1) was initially characterized as a type II transmembrane protein involved in bilayer movements of phospholipids across the plasma membrane leading to the cell surface exposure of phosphatidylserine, but other cellular functions have been ascribed to this protein in signaling processes and in the nucleus. In the present study, expression and functions of PLSCR1 were explored in specialized phagocytic cells of the monocyte/macrophage lineage. The expression of PLSCR1 was found to be markedly increased in monocyte-derived macrophages compared to undifferentiated primary monocytes. Surprisingly, this 3-fold increase in PLSCR1 expression correlated with an apparent modification in the membrane topology of the protein at the cell surface of differentiated macrophages. While depletion of PLSCR1 in the monocytic THP-1 cell-line with specific shRNA did not inhibit the constitutive cell surface exposure of phosphatidylserine observed in differentiated macrophages, a net increase in the FcR-mediated phagocytic activity was measured in PLSCR1-depleted THP-1 cells and in bone marrow-derived macrophages from PLSCR1 knock-out mice. Reciprocally, phagocytosis was down-regulated in cells overexpressing PLSCR1. Since endogenous PLSCR1 was recruited both in phagocytic cups and in phagosomes, our results reveal a specific role for induced PLSCR1 expression in the modulation of the phagocytic process in differentiated macrophages.

  18. MOTION PICTURE STUDIES ON DEGRANULATION OF HORSE EOSINOPHILS DURING PHAGOCYTOSIS

    PubMed Central

    Archer, Gordon T.; Hirsch, James G.

    1963-01-01

    Horse eosinophil function has been studied in vitro by means of phase contrast cinemicrophotography. Locomotion of horse eosinophils was inhibited by serum factors reacting with glass surfaces. Under appropriate conditions which eliminated this inhibitory effect, eosinophils moved about and ingested some particles as rapidly as did neutrophils. Eosinophils were attracted to and readily engulfed such diverse materials as yeast cell walls, foreign erythrocytes, and antigen-antibody precipitates. Specific antibody was required for phagocytosis of red cells, and greatly accelerated the uptake of yeast cell walls. Horse eosinophil granules situated adjacent to material being engulfed disrupted with discharge of granule contents into or alongside the phagocytic vacuole. Granule disruption resulted in a clear zone and deposition of amorphous, phase-dense material. A heat-labile serum factor was required for degranulation of eosinophils ingesting foreign red cells, but not for degranulation during engulfment of yeast cell walls or antigen-antibody precipitates. Horse eosinophils were incapable under these conditions of engulfing an entire human red cell. The eosinophil commonly put out a large pseudopod to surround about half the red cell, and then appeared to constrict this pseudopod distally to cut the erythrocyte in half. It is concluded that eosinophils are phagocytic cells, resembling neutrophils in many of their properties. Any specific functions of eosinophils, distinguishing them from other phagocytes, remain to be discovered. PMID:14074392

  19. Integrins form an expanding diffusional barrier that coordinates phagocytosis

    PubMed Central

    Freeman, Spencer A.; Goyette, Jesse; Furuya, Wendy; Woods, Elliot C.; Bertozzi, Carolyn R.; Bergmeier, Wolfgang; Hinz, Boris; van der Merwe, P. Anton; Das, Raibatak; Grinstein, Sergio

    2015-01-01

    Summary Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the “zippering” of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup. PMID:26771488

  20. Characterization of alveolar macrophage receptors involved in opsonin independent phagocytosis

    SciTech Connect

    Godleski, J.J.; Parod, R.J.; Katler, M.; Brain, J.D.

    1986-03-05

    Hamster alveolar macrophages (AM) avidly ingest particles in the absence of serum. This process is calcium dependent and can be blocked by HAMM, a mouse monoclonal antibody specific for hamster AM's. The purpose of this study was to identify the membrane receptors involved. AM membranes were labeled with /sup 125/I, lysed with RIPA-PMSF, and then reacted with uncoated latex beads or with beads coated with BSA, gelatin, or poly-L-lysine. Lysed membranes were also reacted with zymosan particles. All of these reactions were done in the presence and absence of calcium and magnesium ions. After reaction, the particles were boiled in SDS to remove attached membrane constituents which were then separated by polyacrylamide gel electrophoresis. Only one AM membrane protein (37 kilodaltons (kd)) bound to BSA and gelatin coated latex, uncoated latex, and zymosan particles in the presence of Ca/sup + +/ and Mg/sup + +/. Proteins of 45 and 19 kd attached to all particles even in the absence of divalent cations. In contrast, HAMM reacted with a membrane constituent of 102 kd. The authors conclude that the Ca/sup + +/ dependent receptor for opsonin independent phagocytosis has a molecular weight of 37 kd and is different from the antigen identified by HAMM.

  1. Responses of macrophages against Salmonella infection compared with phagocytosis.

    PubMed

    Hu, Maozhi; Yang, Yun; Meng, Chuang; Pan, Zhiming; Jiao, Xinan

    2013-12-01

    To explore the responses of host cell after infection with live Salmonella compared with phagocytosis to dead bacteria, the responses of mouse macrophage after infection with Salmonella enteritidis C50041 and the fixed C50041 (C50041-d) were analyzed. Results indicated that the cytotoxicity induced by C50041 was stronger than C50041-d. Similar changing trends of mitochondrial membrane potential, intracellular concentration of calcium ions, reactive oxygen species and nitric oxide were found between C50041 and C50041-d infection. But the cell responses against C50041 were earlier and stronger than C50041-d. LC3 expression of macrophage induced by C50041 was lower than C50041-d. C50041 significantly inhibited the production of tumor necrosis factor and interleukin (IL)-6. Whereas intracellular caspase-1 activation and IL-1β release induced by C50041 were stronger than C50041-d, caspase-1 activation and IL-1β release are the innate defense responses of macrophage. Therefore, it will be beneficial to explore the use of this pathway in the control of Salmonella infection.

  2. Human CD14 mediates recognition and phagocytosis of apoptotic cells.

    PubMed

    Devitt, A; Moffatt, O D; Raykundalia, C; Capra, J D; Simmons, D L; Gregory, C D

    1998-04-02

    Cells undergoing programmed cell death (apoptosis) are cleared rapidly in vivo by phagocytes without inducing inflammation. Here we show that the glycosylphosphatidylinositol-linked plasma-membrane glycoprotein CD14 on the surface of human macrophages is important for the recognition and clearance of apoptotic cells. CD14 can also act as a receptor that binds bacterial lipopolysaccharide (LPS), triggering inflammatory responses. Overstimulation of CD14 by LPS can cause the often fatal toxic-shock syndrome. Here we show that apoptotic cells interact with CD14, triggering phagocytosis of the apoptotic cells. This interaction depends on a region of CD14 that is identical to, or at least closely associated with, a region known to bind LPS. However, apoptotic cells, unlike LPS, do not provoke the release of pro-inflammatory cytokines from macrophages. These results indicate that clearance of apoptotic cells is mediated by a receptor whose interactions with 'non-self' components (LPS) and 'self' components (apoptotic cells) produce distinct macrophage responses.

  3. Cytosolic Phospholipase A2α and Eicosanoids Regulate Expression of Genes in Macrophages Involved in Host Defense and Inflammation

    PubMed Central

    Suram, Saritha; Silveira, Lori J.; Mahaffey, Spencer; Brown, Gordon D.; Bonventre, Joseph V.; Williams, David L.; Gow, Neil A. R.; Bratton, Donna L.; Murphy, Robert C.; Leslie, Christina C.

    2013-01-01

    The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α+/+ than cPLA2α-/- macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α+/+ than cPLA2α-/- macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α+/+ and cPLA2α-/- macrophages (3 h) was compared by microarray. cPLA2α+/+ macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α-/- macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α+/+ macrophages. Representative genes expressed lower in cPLA2α+/+ macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation. PMID:23950842

  4. Transcriptional regulation of heat shock proteins and ascorbate peroxidase by CtHsfA2b from African bermudagrass conferring heat tolerance in Arabidopsis

    PubMed Central

    Wang, Xiuyun; Huang, Wanlu; Yang, Zhimin; Liu, Jun; Huang, Bingru

    2016-01-01

    Heat stress transcription factor A2s (HsfA2s) are key regulators in plant response to high temperature. Our objectives were to isolate an HsfA2 gene (CtHsfA2b) from a warm-season grass species, African bermudagrass (Cynodon transvaalensis Burtt-Davy), and to determine the physiological functions and transcriptional regulation of HsfA2 for improving heat tolerance. Gene expression analysis revealed that CtHsfA2b was heat-inducible and exhibited rapid response to increasing temperature. Ectopic expression of CtHsfA2b improved heat tolerance in Arabidopsis and restored heat-sensitive defects of Arabidopsis hsfa2 mutant, which was demonstrated by higher survival rate and photosynthetic parameters, and lower electrolyte leakage in transgenic plants compared to the WT or hsfa2 mutant. CtHsfA2b transgenic plants showed elevated transcriptional regulation of several downstream genes, including those encoding ascorbate peroxidase (AtApx2) and heat shock proteins [AtHsp18.1-CI, AtHsp22.0-ER, AtHsp25.3-P and AtHsp26.5-P(r), AtHsp70b and AtHsp101-3]. CtHsfA2b was found to bind to the heat shock element (HSE) on the promoter of AtApx2 and enhanced transcriptional activity of AtApx2. These results suggested that CtHsfA2b could play positive roles in heat protection by up-regulating antioxidant defense and chaperoning mechanisms. CtHsfA2b has the potential to be used as a candidate gene to genetically modify cool-season species for improving heat tolerance. PMID:27320381

  5. HsfA2 Controls the Activity of Developmentally and Stress-Regulated Heat Stress Protection Mechanisms in Tomato Male Reproductive Tissues.

    PubMed

    Fragkostefanakis, Sotirios; Mesihovic, Anida; Simm, Stefan; Paupière, Marine Josephine; Hu, Yangjie; Paul, Puneet; Mishra, Shravan Kumar; Tschiersch, Bettina; Theres, Klaus; Bovy, Arnaud; Schleiff, Enrico; Scharf, Klaus-Dieter

    2016-04-01

    Male reproductive tissues are more sensitive to heat stress (HS) compared to vegetative tissues, but the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection from HS In tomato (Solanum lycopersicum), HsfA2 acts as coactivator of HsfA1a and is one of the major Hsfs accumulating in response to elevated temperatures. The contribution of HsfA2 in heat stress response (HSR) and thermotolerance was investigated in different tissues of transgenic tomato plants with suppressed HsfA2 levels (A2AS). Global transcriptome analysis and immunodetection of two major Hsps in vegetative and reproductive tissues showed that HsfA2 regulates subsets of HS-induced genes in a tissue-specific manner. Accumulation of HsfA2 by a moderate HS treatment enhances the capacity of seedlings to cope with a subsequent severe HS, suggesting an important role for HsfA2 in regulating acquired thermotolerance. In pollen, HsfA2 is an important coactivator of HsfA1a during HSR HsfA2 suppression reduces the viability and germination rate of pollen that received the stress during the stages of meiosis and microspore formation but had no effect on more advanced stages. In general, pollen meiocytes and microspores are characterized by increased susceptibility to HS due to their lower capacity to induce a strong HSR This sensitivity is partially mitigated by the developmentally regulated expression of HsfA2 and several HS-responsive genes mediated by HsfA1a under nonstress conditions. Thereby, HsfA2 is an important factor for the priming process that sustains pollen thermotolerance during microsporogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  6. HsfA2 Controls the Activity of Developmentally and Stress-Regulated Heat Stress Protection Mechanisms in Tomato Male Reproductive Tissues1[OPEN

    PubMed Central

    Simm, Stefan; Paupière, Marine Josephine; Theres, Klaus; Bovy, Arnaud; Schleiff, Enrico; Scharf, Klaus-Dieter

    2016-01-01

    Male reproductive tissues are more sensitive to heat stress (HS) compared to vegetative tissues, but the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection from HS. In tomato (Solanum lycopersicum), HsfA2 acts as coactivator of HsfA1a and is one of the major Hsfs accumulating in response to elevated temperatures. The contribution of HsfA2 in heat stress response (HSR) and thermotolerance was investigated in different tissues of transgenic tomato plants with suppressed HsfA2 levels (A2AS). Global transcriptome analysis and immunodetection of two major Hsps in vegetative and reproductive tissues showed that HsfA2 regulates subsets of HS-induced genes in a tissue-specific manner. Accumulation of HsfA2 by a moderate HS treatment enhances the capacity of seedlings to cope with a subsequent severe HS, suggesting an important role for HsfA2 in regulating acquired thermotolerance. In pollen, HsfA2 is an important coactivator of HsfA1a during HSR. HsfA2 suppression reduces the viability and germination rate of pollen that received the stress during the stages of meiosis and microspore formation but had no effect on more advanced stages. In general, pollen meiocytes and microspores are characterized by increased susceptibility to HS due to their lower capacity to induce a strong HSR. This sensitivity is partially mitigated by the developmentally regulated expression of HsfA2 and several HS-responsive genes mediated by HsfA1a under nonstress conditions. Thereby, HsfA2 is an important factor for the priming process that sustains pollen thermotolerance during microsporogenesis. PMID:26917685

  7. UDP-Induced Phagocytosis and ATP-Stimulated Chemotactic Migration Are Impaired in STIM1(-/-) Microglia In Vitro and In Vivo.

    PubMed

    Lim, Hye Min; Woon, Heo; Han, Jung Woo; Baba, Yoshihiro; Kurosaki, Tomohiro; Lee, Min Goo; Kim, Joo Young

    2017-01-01

    STIM1 is the only currently known intracellular calcium sensor that functions as the calcium influx regulator controlling immune cell activation. STIM1 function in immune cell calcium signalling has been studied extensively; however, its role in microglia, innate immune cells in brain, has not been fully understood. Here, we report that STIM1(-/-) murine microglia lost store-operated calcium influx and displayed aberrant immunological functions. Microglial functions regulated by chronic and global [Ca(2+)]i changes were reduced significantly, including cytokine releases and opsonin-dependent phagocytosis. More dramatically, cellular functions governed by Ca(2+) regulation in local microdomains at the cell periphery, such as UDP-induced phagocytosis and ATP-stimulated chemotactic migration, were severely reduced in STIM1(-/-) microglia. Interestingly, UDP-induced Orai1 mobilization to the peripheral region was greatly attenuated in STIM1(-/-) microglia. Their chemotactic migration defect was reproduced in vivo in embryonic brain; the aggregated number of STIM1(-/-) microglia in LPS- (lipopolysaccharide-) injected lesions was much smaller than that in wild-type microglia. Furthermore, the neuron phagoptosis activities of activated microglia were significantly diminished in the STIM1(-/-) microglia. These in vitro and in vivo results suggest that STIM1-mediated store-operated calcium entry is important for the regulation of global [Ca(2+)]i changes which differentiates into active immune state of microglia, but it is more crucial for the regulation of local [Ca(2+)] microdomains which mediates the acute motility of murine microglia.

  8. Dietary antioxidants and behavioral enrichment enhance neutrophil phagocytosis in geriatric Beagles.

    PubMed

    Hall, Jean A; Picton, Rebecca A; Finneran, Phyllis S; Bird, Karyn E; Skinner, Monica M; Jewell, Dennis E; Zicker, Steven

    2006-09-15

    The study objective was to determine the effects of feeding food enriched in antioxidants and a program of environmental/cognitive enrichment on selected ex vivo assays of inflammatory and immune cells in healthy geriatric Beagle dogs (n=21). Four groups of dogs were tested using a 2 x 2 factorial design. The 2-year longitudinal study included both nutritional (control food or antioxidant-fortified food) and behavioral (normal level or cognitive enrichment) interventions. Behavior enrichment included increased exercise, environmental enrichment, and a series of learning tasks. Phagocytosis of opsonized latex-coated beads by peripheral blood neutrophils was measured by flow cytometry and found to be significantly increased in dogs receiving both dietary antioxidants and cognitive enrichment. Simultaneous stimulation of cells with Con A and suppression with Dex resulted in decreased lymphocyte proliferation in dogs receiving both dietary antioxidants and cognitive enrichment, compared to dogs receiving dietary antioxidants or cognitive enrichment alone. There were no significant differences between the groups of dogs for percentages of CD4 and CD8 T-lymphocyte subpopulations before or after lymphocyte stimulation with Con A. These results support our hypothesis that both dietary antioxidants and behavioral enrichment enhance host defense mechanisms.

  9. The Role of Behavioral Self-Regulation in Learning to Read: A 2-Year Longitudinal Study of Icelandic Preschool Children

    ERIC Educational Resources Information Center

    Birgisdóttir, Freyja; Gestsdóttir, Steinunn; Thorsdóttir, Fanney

    2015-01-01

    Research Findings: Research suggests that behavioral self-regulation skills are critical for early school success, including success in literacy, but few studies have explored the relations that behavioral self-regulation may have with different components of early literacy development. The present study investigated the longitudinal contribution…

  10. The Role of Behavioral Self-Regulation in Learning to Read: A 2-Year Longitudinal Study of Icelandic Preschool Children

    ERIC Educational Resources Information Center

    Birgisdóttir, Freyja; Gestsdóttir, Steinunn; Thorsdóttir, Fanney

    2015-01-01

    Research Findings: Research suggests that behavioral self-regulation skills are critical for early school success, including success in literacy, but few studies have explored the relations that behavioral self-regulation may have with different components of early literacy development. The present study investigated the longitudinal contribution…

  11. H2O2 Release from Human Granulocytes during Phagocytosis

    PubMed Central

    Root, Richard K.; Metcalf, Julia A.

    1977-01-01

    Normal and cytochalasin B-treated human granulocytes have been studied to determine some of the interrelationships between phagocytosis-induced respiration and superoxide and hydrogen peroxide formation and release into the extracellular medium by intact cells. By using the scopoletin fluorescent assay to continuously monitor extracellular hydrogen peroxide concentrations during contact of cells with opsonized staphylococci, it was demonstrated that the superoxide scavengers ferricytochrome c and nitroblue tetrazolium significantly reduced the amount of H2O2 released with time from normal cells but did not abolish it. This inhibitory effect was reversed by the simultaneous addition of superoxide dismutase (SOD), whereas the addition of SOD alone increased the amount of detectable H2O2 in the medium. The addition of sodium azide markedly inhibited myeloperoxidase-H2O2-dependent protein iodination and more than doubled H2O2 release, including the residual amount remaining after exposure of the cells to ferricytochrome c, suggesting its origin from an intracellular pool shared by several pathways for H2O2 catabolism. When cells were pretreated with cytochalasin B and opsonized bacteria added, reduced oxygen consumption was observed, but this was in parallel to a reduction in specific binding of organisms to the cells when compared to normal. Under the influence of inhibited phagosome formation by cytochalasin B, the cells released an increased amount of superoxide and peroxide into the extracellular medium relative to oxygen consumption, and all detectable peroxide release could be inhibited by the addition of ferricytochrome c. Decreased H2O2 production in the presence of this compound could not be ascribed to diminished bacterial binding, decreased oxidase activity, or increased H2O2 catabolism and was reversed by the simultaneous addition of SOD. Furthermore, SOD and ferricytochrome c had similar effects on another H2O2-dependent reaction, protein iodination, in

  12. Non-specific recognition in phagocytosis: ingestion of aldehyde-treated erythrocytes by rat peritoneal macrophages.

    PubMed Central

    Capo, C; Bongrand, P; Benoliel, A M; Depieds, R

    1979-01-01

    Particles were chemically modified with aldehydes and incubated with rat peritoneal cells for phagocytosis. All dialdehydes and lower monaldehydes tested (methanal, ethanal and propanal) made sheep erythrocytes phagocytosable. Failure of higher monaldehydes to induce phagocytosis of treated erythrocytes was not due to lack of reactivity with red cell membranes. All erythrocytes tested (bird and mammal red cells were used) and rat thymocytes were phagocytosed by rat macrophages after incubation with aldehyde. Treatment of Candida albicans did not induce phagocytosis: this failure was not due to lack of aldehyde binding (as demonstrated with [14C]-methanal) nor to anti-phagocytic properties of the parasite membrane. Sheep erythrocytes were submitted to enzymatic treatment (pronase, trypsin, neuraminidase) or incubated with succinic anhydride (to block free NH2 groups) or iodacetamide (to block free SH groups) before aldehyde treatment: phagocytosis was not decreased, which suggested that aldehydes did not act by altering some definite surface structure of the treated particles. Treatment of erythrocytes with cross-linking compounds such as tetraazotized o-dianisidine (coupling occurs mainly on tyrosine and histidine residues) or l-ethyl(3-dimethyl aminopropyl) carbodiimide (a bivalent reagent binding free COOH groups) did not induce any substantial phagocytosis of erythrocytes. Phagocytosis of aldehyde treated erythrocytes was partly correlated with hydrophobicity of these cells, as measured with a two-phase partition system. It is concluded that aldehyde-mediated phagocytosis of erythrocytes is mainly due to cross-linking of red cell membrane structures, probably involving free OH groups, which must increase local rigidity and thereby modify hydrophobicity of the red cell surface. Images Figure 1 PMID:437841

  13. Non-specific recognition in phagocytosis: ingestion of aldehyde-treated erythrocytes by rat peritoneal macrophages.

    PubMed

    Capo, C; Bongrand, P; Benoliel, A M; Depieds, R

    1979-03-01

    Particles were chemically modified with aldehydes and incubated with rat peritoneal cells for phagocytosis. All dialdehydes and lower monaldehydes tested (methanal, ethanal and propanal) made sheep erythrocytes phagocytosable. Failure of higher monaldehydes to induce phagocytosis of treated erythrocytes was not due to lack of reactivity with red cell membranes. All erythrocytes tested (bird and mammal red cells were used) and rat thymocytes were phagocytosed by rat macrophages after incubation with aldehyde. Treatment of Candida albicans did not induce phagocytosis: this failure was not due to lack of aldehyde binding (as demonstrated with [14C]-methanal) nor to anti-phagocytic properties of the parasite membrane. Sheep erythrocytes were submitted to enzymatic treatment (pronase, trypsin, neuraminidase) or incubated with succinic anhydride (to block free NH2 groups) or iodacetamide (to block free SH groups) before aldehyde treatment: phagocytosis was not decreased, which suggested that aldehydes did not act by altering some definite surface structure of the treated particles. Treatment of erythrocytes with cross-linking compounds such as tetraazotized o-dianisidine (coupling occurs mainly on tyrosine and histidine residues) or l-ethyl(3-dimethyl aminopropyl) carbodiimide (a bivalent reagent binding free COOH groups) did not induce any substantial phagocytosis of erythrocytes. Phagocytosis of aldehyde treated erythrocytes was partly correlated with hydrophobicity of these cells, as measured with a two-phase partition system. It is concluded that aldehyde-mediated phagocytosis of erythrocytes is mainly due to cross-linking of red cell membrane structures, probably involving free OH groups, which must increase local rigidity and thereby modify hydrophobicity of the red cell surface.

  14. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    PubMed

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.

  15. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

    PubMed Central

    Sateriale, Adam; Miller, Peter

    2016-01-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment. PMID:26810036

  16. Monocyte phagocytosis of viable Staphylococcus aureus is impaired by barbiturates, but not by propofol.

    PubMed

    Ploppa, A; Kiefer, R T; Nohé, B; Haeberle, Helene A; Dieterich, H J; Unertl, K E; Durieux, M E; Krueger, W A

    2008-06-01

    Barbiturates and propofol are used for deep sedation of patients with elevated intracranial pressure refractory to standard therapeutic regimens. Such patients often suffer from bacterial infections, which are most commonly caused by Staphylococcus aureus. Various interactions of anesthetics with components of the host defense have been documented, but very little is known about the influence on monocytes, which are a first-line defense against bacterial invasion. Therefore, we studied the effects of thiopental, methohexital, and propofol on monocyte phagocytosis using an in vitro whole blood model of viable S. aureus. Whole blood samples were preincubated with different concentrations of thiopental, methohexital, and propofol. Phagocytosis was stopped at different time points after addition of viable S. aureus. Monocytes then were stained with monoclonal antibodies for flow cytometric analysis of monocyte recruitment (ratio of ingesting monocytes). Furthermore, the fluorescence intensity of ingested bacteria served as semiquantitative measurement of phagocytosis activity. Both barbiturates inhibited monocyte recruitment and phagocytosis activity concentration-dependently, whereas propofol did not affect any of the investigated parameters. At concentrations of 7.6 x10(-3) M thiopental or 1.1 x 10(-3) M methohexital and greater, monocyte recruitment and phagocytosis activity were significantly inhibited. The calculated half-maximum inhibitory concentration (IC50) of thiopental was 8.4 x 10(-3) M for monocyte recruitment and 8.6 x 10(-3) M for phagocytosis activity. The corresponding values for methohexital were 4.1 x 10(-3) M and 1.1 x 10(-3) M, respectively. The two barbiturates induce concentration-dependent inhibition of monocyte phagocytosis, whereas propofol is without effect. In combination with previously described effects on granulocyte function, these findings suggest that defense against bacterial infection might be reduced by barbiturates.

  17. Phagocytosis of bacteria adhering to a biomaterial surface in a surface thermodynamic perspective.

    PubMed

    da Silva Domingues, Joana F; van der Mei, Henny C; Busscher, Henk J; van Kooten, Theo G

    2013-01-01

    Bacterial biofilms can increase the pathogenicity of infection and constitute a major problem in modern health-care, especially on biomaterial implants and devices. Biofilms are difficult to eradicate by the host immune system, even with antibiotics, and have been the number one cause of biomaterial implant and device failure for decades. Therefore, it is important to understand how immune cells interact with adhering pathogens. This study firstly aims to develop a simple method to quantify phagocytosis of six different strains of staphylococci adhering on a surface with phase-contrast-microscopy. Phagocytosis of adhering staphylococci to a glass surface by phagocytes was quantified in a parallel plate flow chamber, and expressed as a phagocytosis rate, accounting for the number of adhering staphylococci initially present and for the duration of phagocytosis. Murine macrophages were more effective in clearing staphylococci from a surface than human phagocytes, which require differentiation from their monocyte or promyelocytic state during an experiment. Direct visualization of internalization of a GFP-modified S. aureus strain inside phagocytes confirmed the validity of the method proposed. As a second aim, the differences in phagocytosis rates observed were investigated on a surface thermodynamic basis using measured contact angles of liquids on macroscopic lawns of staphylococci and phagocytes, confirming that phagocytosis of adhering pathogens can be regarded as a surface phenomenon. In addition, surface thermodynamics revealed that phagocytosis of adhering pathogens is determined by an interplay of physical attraction between pathogens and phagocytes and the influence of chemo-attractants. For future studies, these results will help to place in vitro experiments and murine infection models in better perspective with respect to human ones.

  18. CALCIUM OXALATE STONE FRAGMENT AND CRYSTAL PHAGOCYTOSIS BY HUMAN MACROPHAGES

    PubMed Central

    Kusmartsev, Sergei; Dominguez-Gutierrez, Paul R.; Canales, Benjamin K.; Bird, Vincent G.; Vieweg, Johannes; Khan, Saeed R.

    2015-01-01

    Purpose In murine and human hyperoxaluric conditions, macrophages can be seen surrounding renal calcium oxalate (CaOx) crystal deposits. We hypothesize that macrophages play a role in degrading and destroying these deposits and investigated inflammatory response and phagocytic mechanisms when macrophages are exposed to human kidney stones and inorganic crystals. Materials and Methods Human monocytes were differentiated into resting, fully-differentiated macrophages by treating with recombinant human M-CSF or GM-CSF for 6 days. After confirming phenotype by flow cytometry, macrophages were exposed for 20 hours to fragments of sterile human CaOx stones or CaOx crystals. Crystal uptake was determined, and supernatant cytokine and chemokine profiles were analyzed using antibody arrays. qRT-PCR was used to validate mRNA profile expression. Results Under direct-vision fluorescent microscopy, activated human macrophages were noted to surround both stone fragments and synthesized crystals and destroy them in a step-by-step process that involved clathrin-mediated endocytosis and phagocytosis. An inflammatory cascade was released by macrophages, including chemokines CCL2, CCL3, interleukin-1 receptor antagonist (IL-1ra), complement component C5/C5a and IL-8. The response patterns to stone and crystal material was dependent on macrophage phenotype and activation status. Conclusions In our in vitro study, macrophages differentiated with M-CSF displayed a greater ability to phagocytize crystal deposits than those treated with GM-CSF. Following clathrin-mediated endocytosis, macrophages released a number of cytokines crucial for inflammatory immune response, suggesting that tissue macrophages play an important role in preventing kidney stone disease by removing and digesting interstitial renal crystal deposits. PMID:26626217

  19. Molecular regulation and physiological functions of a novel FaHsfA2c cloned from tall fescue conferring plant tolerance to heat stress.

    PubMed

    Wang, Xiuyun; Huang, Wanlu; Liu, Jun; Yang, Zhimin; Huang, Bingru

    2017-02-01

    Heat stress transcription factors (HSFs) compose a large gene family, and different members play differential roles in regulating plant responses to abiotic stress. The objectives of this study were to identify and characterize an A2-type HSF, FaHsfA2c, in a cool-season perennial grass tall fescue (Festuca arundinacea Schreb.) for its association with heat tolerance and to determine the underlying physiological functions and regulatory mechanisms of FaHsfA2c imparting plant tolerance to heat stress. FaHsfA2c was localized in nucleus and exhibited a rapid transcriptional increase in leaves and roots during early phase of heat stress. Ectopic expression of FaHsfA2c improved basal and acquired thermotolerance in wild-type Arabidopsis and also restored heat-sensitive deficiency of hsfa2 mutant. Overexpression of FaHsfA2c in tall fescue enhanced plant tolerance to heat by triggering transcriptional regulation of heat-protective gene expression, improving photosynthetic capacity and maintaining plant growth under heat stress. Our results indicated that FaHsfA2c acted as a positive regulator conferring thermotolerance improvement in Arabidopsis and tall fescue, and it could be potentially used as a candidate gene for genetic modification and molecular breeding to develop heat-tolerant cool-season grass species. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  20. The Medicago Species A2-Type Cyclin Is Auxin Regulated and Involved in Meristem Formation But Dispensable for Endoreduplication-Associated Developmental Programs1

    PubMed Central

    Roudier, François; Fedorova, Elena; Lebris, Manuel; Lecomte, Phillippe; Györgyey, Janos; Vaubert, Daniele; Horvath, Gabor; Abad, Pierre; Kondorosi, Adam; Kondorosi, Eva

    2003-01-01

    Phytohormones as well as temporal and spatial regulation of the cell cycle play a key role in plant development. Here, we investigated the function and regulation of an alfalfa (Medicago sativa) A2-type cyclin in three distinct root developmental programs: in primary and secondary root development, nodule development, and nematode-elicited gall formation. Using transgenic plants carrying the Medsa;cycA2;2 promoter-β-glucuronidase gene fusion, in combination with other techniques, cycA2;2 expression was localized in meristems and proliferating cells in the lateral root and nodule primordia. Rapid induction of cycA2;2 by Nod factors demonstrated that this gene is implicated in cell cycle activation of differentiated cells developing to nodule primordia. Surprisingly, cycA2;2 was repressed in the endoreduplicating, division-arrested cells both during nodule development and formation of giant cells in nematode-induced galls, indicating that CycA2;2 was dispensable for S-phase in endoreduplication cycles. Overexpression of cycA2;2 in transgenic plants corresponded to wild type protein levels and had no apparent phenotype. In contrast, antisense expression of cycA2;2 halted regeneration of somatic embryos, suggesting a role for CycA2;2 in the formation or activity of apical meristems. Expression of cycA2;2 was up-regulated by auxins, as expected from the presence of auxin response elements in the promoter. Moreover, auxin also affected the spatial expression pattern of this cyclin by shifting the cycA2;2 expression from the phloem to the xylem poles. PMID:12644661

  1. Identification of different promoters in the absA1-absA2 two-component system, a negative regulator of antibiotic production in Streptomyces coelicolor.

    PubMed

    Santos-Beneit, Fernando; Rodríguez-García, Antonio; Martín, Juan F

    2013-02-01

    The absA1-absA2 genes encode a two-component system that negatively regulates the transcription of multiple antibiotic gene clusters of Streptomyces coelicolor. The microarray dataset time series of a S. coelicolor M145 bioreactor culture indicated that the transcription values of absA2 were approximately four times higher than those of absA1 throughout the time course of the culture. The co-transcription of absA1 and absA2 genes has been previously shown, although an independent absA2 promoter was not detected. In this study, we show by different technical approaches that the absA1-absA2 operon is transcribed from at least two promoters, the first producing a read-through transcript that includes both absA1 and absA2 genes and the second including only the absA2 gene. An absA2 mRNA 5' end was mapped by primer extension and confirmed as TSS by deep sequencing in combination with TEX. Promoter-probe analyses detected promoter activity in both the absA1 and absA2 upstream regions. The absA2 upstream region showed a higher promoter activity, at least sevenfold higher than that of absA1. Furthermore, the absA2 gene may contain at least two additional promoters as shown by deep sequencing analyses. All together this work contributes to the understanding of the complex transcriptional regulation of these antibiotic regulators genes in S. coelicolor.

  2. Cytosolic phospholipase A2 regulates alcohol-mediated astrocyte inflammatory responses in HIV-associated neurocognitive disorders

    PubMed Central

    Pandey, R; Ghorpade, A

    2015-01-01

    Alcohol (EtOH) abuse and HIV-1 infection remain leading public health problems not only in the United States but also across the world. Alcohol abusers have a significantly greater risk of HIV-1 infection than non-drinkers globally. In the United States, prevalence of EtOH abuse is over two-fold higher in HIV-1-positive individuals than that of the general population. Although alcohol abusers show neurodegeneration, exacerbated neuroinflammation and oxidative damage, the mechanism(s) by which EtOH regulates astrocyte inflammatory responses in HIV-associated neurocognitive disorders is unknown. Thus, we explored signaling pathway(s) involved in EtOH-mediated activation of human astrocytes with HIV-1 and subsequent alterations in their inflammatory functions. Alcohol exposure altered the morphology of astrocytes, proinflammatory responses and induced cytotoxicity in a dose-dependent manner. Time-dependent changes were also evaluated. EtOH and HIV-1 cotreatment decreased cell viability and proliferation, while increasing apoptosis and mitochondrial depolarization. EtOH and HIV-1 together increased the levels of proinflammatory molecules, interleukin-1β, tumor necrosis factor-α, CXCL8, tissue inhibitor of metalloproteinases-1 and more importantly, arachidonic acid, a known downstream target of cytosolic phospholipase A2 (cPLA2). Consistent with this observation, phospho-cPLA2 levels were augmented in HIV-1 and EtOH cotreatment as compared with HIV-1 or EtOH alone. Cyclooxygenase 2 was upregulated as measured by real-time PCR and western blot, whereas cotreatment of HIV-1 and EtOH decreased cytochrome P450-2E1 levels as compared with EtOH alone. Furthermore, we confirmed that blocking cPLA2 with arachidonyl tri floro methyl ketone, a cPLA2-specific inhibitor, effectively prevented cPLA2 phosphorylation and downstream outcomes. Thus, the present findings suggest that cPLA2 has a critical role in alcohol and HIV-induced astrocyte inflammation. In the future, cPLA2

  3. Analysis of the Relative Contribution of Phagocytosis, LC3-Associated Phagocytosis, and Canonical Autophagy During Helicobacter pylori Infection of Macrophages.

    PubMed

    Deen, Nadia S; Gong, Lan; Naderer, Thomas; Devenish, Rodney J; Kwok, Terry

    2015-12-01

    Previous findings have suggested that Helicobacter pylori induces autophagic processes and subsequently takes refuge in autophagosomes, thereby contributing to persistent infection. Recently, a noncanonical form of autophagy, LC3 (microtubule-associated protein 1 light chain 3)-associated phagocytosis (LAP), has been shown to be required for efficient clearance of some intracellular bacteria. Whether H. pylori infection induces LAP had not been examined previously. In this study, we determined the extent to which H. pylori infection induces canonical autophagy or LAP in macrophages, and the involvement of the H. pylori cag pathogenicity island (cagPAI) with these processes. Immunofluorescence confocal microscopy was used to analyze the formation of GFP-LC3 puncta and their colocalization with H. pylori. Transmission electron microscopy was used to detect the ultrastructure of H. pylori-containing compartments. The majority of intracellular bacteria (85-95%) were found in phagosomes that were LC3-negative, with a small proportion (4-14%) appearing "free" in the cytosol. Only a very small percentage (0.5-6%) of intracellular H. pylori was sequestered in autophagosomes. Furthermore, no statistically significant difference in the relative distribution of H. pylori in the various compartments was observed between wild-type and cagPAI-mutant bacteria. In macrophages, H. pylori infection does not induce LAP, but can induce canonical autophagy, which entraps a very small fraction of intracellular bacteria. We propose that this subpopulation of intracellular H. pylori might have escaped from phagosomes into the cytosol before being sequestered by autophagosomes. The cagPAI of H. pylori has only minor influence, if any, on the extent of these processes. © 2015 John Wiley & Sons Ltd.

  4. The Arabidopsis eukaryotic translation initiation factor eIF5A-2 regulates root protoxylem development by modulating cytokinin signaling.

    PubMed

    Ren, Bo; Chen, Qingguo; Hong, Sulei; Zhao, Wenming; Feng, Jian; Feng, Haizhong; Zuo, Jianru

    2013-10-01

    The phytohormone cytokinin regulates various aspects of plant growth and development, including root vascular development. In Arabidopsis thaliana, mutations in the cytokinin signaling components cause misspecification of protoxylem cell files. Auxin antagonizes cytokinin-regulated root protoxylem differentiation by inducing expression of Arabidopsis phosphotransfer protein6 (AHP6), a negative regulator of cytokinin signaling. However, the molecular mechanism of cytokinin-regulated protoxylem differentiation is not fully understood. Here, we show that a mutation in Arabidopsis fumonisin B1-resistant12 (FBR12), which encodes a eukaryotic translation initiation factor 5A, causes defective protoxylem development and reduced sensitivity to cytokinin. FBR12 genetically interacts with the cytokinin receptor cytokinin response1 (CRE1) and downstream AHP genes, as double mutants show enhanced phenotypes. FBR12 forms a protein complex with CRE1 and AHP1, and cytokinin regulates formation of this protein complex. Intriguingly, ahp6 partially suppresses the fbr12 mutant phenotype, and the fbr12 mutation causes increased expression of AHP6, indicating that FBR12 negatively regulates AHP6. Consistent with this, ectopic expression of FBR12 in the CRE1-expressing domain partially rescues defective protoxylem development in fbr12, and overexpression of AHP6 causes an fbr12-like phenotype. These results define a regulatory role of the highly conserved FBR12 in cytokinin-mediated root protoxylem specification.

  5. HMGB1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine

    PubMed Central

    Liu, Gang; Wang, Jing; Park, Young-Jun; Tsuruta, Yuko; Lorne, Emmanuel F; Zhao, Xia; Abraham, Edward

    2008-01-01

    Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical to resolution of inflammation. Impaired efferocytosis is associated with unfavorable outcome from inflammatory diseases, including acute lung injury and pulmonary manifestations of cystic fibrosis. HMGB1, a nuclear non-histone DNA-binding protein, has recently been found to be secreted by immune cells upon stimulation with LPS and cytokines. Plasma and tissue levels of HMGB1 are elevated for prolonged periods in chronic and acute inflammatory conditions, including sepsis, rheumatoid arthritis, acute lung injury, burns, and hemorrhage. In this study, we found that HMGB1 inhibits phagocytosis of apoptotic neutrophils by macrophages in vivo and in vitro. Phosphatidylserine (PS) is directly involved in the inhibition of phagocytosis by HMGB1, as blockade of HMGB1 by PS eliminates the effects of HMGB1 on efferocytosis. Confocal and FRET demonstrate that HMGB1 interacts with PS on the neutrophil surface. However, HMGB1 does not inhibit PS-independent phagocytosis of viable neutrophils. Bronchoalveolar lavage (BAL) fluid from Scnn+ mice, a murine model of cystic fibrosis lung disease, which contains elevated concentrations of HMGB1 inhibits neutrophil efferocytosis. Anti-HMGB1 antibodies reverse the inhibitory effect of Scnn+ BAL on efferocytosis, showing that this effect is due to HMGB1. These findings demonstrate that HMGB1 can modulate phagocytosis of apoptotic neutrophils and suggest an alternative mechanism by which HMGB1 is involved in enhancing inflammatory responses. PMID:18768881

  6. Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells.

    PubMed

    Liu, W; Ernst, J D; Broaddus, V C

    2000-09-01

    Phagocytosis of asbestos fibers may be a necessary step for asbestos-induced injury to mesothelial cells, but this has not been established because quantification of fiber uptake is difficult and ways to increase fiber phagocytosis without also increasing total dose were not available. We quantified phagocytosis by counting intracellular fibers after removing adherent fibers with trypsin; we selectively increased fiber phagocytosis by coating crocidolite asbestos fibers with the adhesive serum protein vitronectin (VN), which we have shown increases fiber uptake via integrins. We measured various aspects of asbestos-induced cytotoxicity: intracellular oxidation by the shift of fluorescence of cells loaded with an oxidative probe, DNA strand breakage by the alkaline unwinding ethidium bromide fluorometric assay, apoptosis by annexin V binding and by nuclear morphology, and cell-cycle progression. We found that, compared with control fibers or particles, asbestos increased intracellular oxidation, DNA strand breakage, and apoptosis. Selective increases in fiber uptake by VN-coating of the fibers further increased the oxidation, DNA strand breakage, and apoptosis, and induced a cell-cycle arrest in G2/M. Selective decreases in fiber uptake by cytochalasin or by integrin blockade with RGD peptides inhibited several of these measures of injury. We conclude that phagocytosis is important and perhaps necessary for asbestos-induced injury to mesothelial cells.

  7. Role of caveolae in Leishmania chagasi phagocytosis and intracellular survival in macrophages.

    PubMed

    Rodríguez, Nilda E; Gaur, Upasna; Wilson, Mary E

    2006-07-01

    Caveolae are membrane microdomains enriched in cholesterol, ganglioside M1 (GM1) and caveolin-1. We explored whether caveolae facilitate the entry of Leishmania chagasi into murine macrophages. Transient depletion of macrophage membrane cholesterol by 1 h exposure to methyl-beta-cyclodextrin (MbetaCD) impaired the phagocytosis of non-opsonized and serum-opsonized virulent L. chagasi. In contrast, MbetaCD did not affect the phagocytosis of opsonized attenuated L. chagasi. As early as 5 min after phagocytosis, virulent L. chagasi colocalized with the caveolae markers GM1 and caveolin-1, and colocalization continued for over 48 h. We explored the kinetics of lysosome fusion. Whereas fluorescent-labelled dextran entered macrophage lysosomes by 30 min after addition, localization of L. chagasi in lysosomes was delayed for 24-48 h after phagocytosis. However, after transient depletion of cholesterol from macrophage membrane with MbetaCD, the proportion of L. chagasi-containing phagosomes that fused with lysosomes increased significantly. Furthermore, intracellular replication was impaired in parasites entering after transient cholesterol depletion, even though lipid microdomains were restored by 4 h after treatment. These observations suggest that virulent L. chagasi localize in caveolae during phagocytosis by host macrophages, and that cholesterol-containing macrophage membrane domains, such as caveolae, target parasites to a pathway that promotes delay of lysosome fusion and intracellular survival.

  8. Upregulation of phagocytosis and candidicidal activity of macrophages exposed to the immunostimulant acemannan.

    PubMed

    Stuart, R W; Lefkowitz, D L; Lincoln, J A; Howard, K; Gelderman, M P; Lefkowitz, S S

    1997-02-01

    Previous studies by these investigators have shown that mannosylated bovine serum albumin (m-BSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans by resident murine peritoneal macrophages (MO). Upregulation of the above MO functions was associated with binding of m-BSA to the MO-mannose receptor. The present study was done to determine if the immunostimulant, acemannan prepared from aloe vera, could stimulate MO in a similar manner. Resident peritoneal MO collected from C57BL/6 mice were exposed to acemannan for 10 min. The RB was measured using chemiluminescence and demonstrated approximately a two-fold increase above the media controls. In studies involving phagocytosis, MO were exposed to acemannan, washed and exposed to Candida at a ratio of 1:5. The percent phagocytosis and Candida killing were determined using fluorescence microscopy. There was a marked increase in phagocytosis in the treated cultures (45%) compared to controls (25%). Macrophages exposed to acemannan for 10 min resulted in ca 38% killing of Candida albicans compared with 0-5% killing in controls. If MO were incubated with acemannan for 60 min, 98% of the yeast were killed compared to 0-5% in the controls. The results of the present study indicate that short term exposure of MO to acemannan upregulates the RB, phagocytosis and candidicidal activity. Further studies are needed to clarify the potential use of this immunostimulant as an anti-fungal agent.

  9. The adaptor protein GULP promotes Jedi-1-mediated phagocytosis through a clathrin-dependent mechanism.

    PubMed

    Sullivan, Chelsea S; Scheib, Jami L; Ma, Zhong; Dang, Rajan P; Schafer, Johanna M; Hickman, Francis E; Brodsky, Frances M; Ravichandran, Kodi S; Carter, Bruce D

    2014-06-15

    During the development of the peripheral nervous system, the large number of apoptotic neurons generated are phagocytosed by glial precursor cells. This clearance is mediated, in part, through the mammalian engulfment receptor Jedi-1. However, the mechanisms by which Jedi-1 mediates phagocytosis are poorly understood. Here we demonstrate that Jedi-1 associates with GULP, the mammalian homologue of CED-6, an adaptor protein required for phagocytosis mediated by the nematode engulfment receptor CED-1. Silencing GULP or mutating the NPXY motif in Jedi-1, which is required for GULP binding, prevents Jedi-1-mediated phagocytosis. How GULP promotes engulfment is not known. Of interest, we find that Jedi-1-induced phagocytosis requires GULP binding to clathrin heavy chain (CHC). During engulfment, CHC is tyrosine phosphorylated, which is required for Jedi-mediated engulfment. Both phosphoclathrin and actin accumulate around engulfed microspheres. Furthermore, knockdown of CHC in HeLa cells prevents Jedi-1-mediated engulfment of microspheres, and knockdown in glial precursors prevents the engulfment of apoptotic neurons. Taken together, these results reveal that Jedi-1 signals through recruitment of GULP, which promotes phagocytosis through a noncanonical phosphoclathrin-dependent mechanism. © 2014 Sullivan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  10. Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

    PubMed Central

    Lefkir, Yaya; Malbouyres, Marilyne; Gotthardt, Daniel; Ozinsky, Adrian; Cornillon, Sophie; Bruckert, Franz; Aderem, Alan A.; Soldati, Thierry; Cosson, Pierre; Letourneur, François

    2004-01-01

    The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1- cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1- cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1- cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation. PMID:14617812

  11. Influence of Glucose Levels on the In Vitro Phagocytosis of Bacteria by Human Neutrophils

    PubMed Central

    van Oss, Carel J.

    1971-01-01

    A previously developed in vitro method for studying the phagocytosis of bacteria and particles by human neutrophils was used to investigate the influence of different glucose levels on phagocytosis. It was found that high glucose levels (200, 400, and 800 mg of glucose/100 ml) significantly depressed the phagocytosis of Staphylococcus epidermidis, S. aureus, and Escherichia coli. At very low glucose levels, a somewhat decreased phagocytic activity was noted. The strongest phagocytic activity occurred at glucose concentrations of 50 and 100 mg/100 ml. A second effect noted at the higher (200, 400 and 800 mg/100 ml) glucose concentrations was a decreased adhesiveness of the neutrophils to solid surfaces. The mechanism of the decrease in phagocytosis and in neutrophil adhesiveness at higher glucose levels is unknown, but it is not linked to increased osmotic pressures due to the presence of glucose, as ethanol, at the same and even higher osmolal concentrations, had no effect on the phagocytosis. These results show that not only the phagocytic activity of those neutrophils that do adhere to a solid surface is diminished at higher glucose concentrations but also that fewer neutrophils adhere to solid surfaces at higher glucose levels. These two phenomena combined may provide at least part of the explanation for the well-known decrease in resistance to bacterial infections of diabetics. PMID:4117287

  12. MicroRNA-9 regulates non-small cell lung cancer cell invasion and migration by targeting eukaryotic translation initiation factor 5A2

    PubMed Central

    Xu, Guodong; Shao, Guofeng; Pan, Qiaoling; Sun, Lebo; Zheng, Dawei; Li, Minghui; Li, Ni; Shi, Huoshun; Ni, Yiming

    2017-01-01

    MicroRNAs (miRNAs) play a critical role in cancer development and progression. Bioinformatics analyses has identified eukaryotic translation initiation factor 5A2 (eIF5A2) as a target of miR-9. In this study, we attempted to determine whether miR-9 regulates non-small cell lung cancer (NSCLC) cell invasion and migration by targeting eIF5A2 We examined eIF5A2 expression using reverse transcription-quantitative PCR (RT-qPCR) and subsequently transfected A549 and NCI-H1299 NSCLC cells with a miR-9 mimic or miR-9 inhibitor to determine the migration and invasive capability of the cells via wound healing assay and Transwell invasion assay, respectively. E-cadherin and vimentin expression was detected with western blotting. The miR-9 mimic significantly reduced NSCLC cell invasive and metastatic ability, and the miR-9 inhibitor enhanced NSCLC cell migration activity, increasing the number of migrated cells. There was no significant difference between the negative control siRNA and miR-9 mimic groups after knockdown of eIF5A2; western blotting showed that miR-9 regulated E-cadherin and vimentin expression. These data show that miR-9 regulates NSCLC cell invasion and migration through regulating eIF5A2 expression. Taken together, our findings suggest that the mechanism of miR-9-regulated NSCLC cell invasion and migration may be related to epithelial-mesenchymal transition. PMID:28337276

  13. Regulation of HtrA2 on WT1 gene expression under imatinib stimulation and its effects on the cell biology of K562 cells.

    PubMed

    Zhang, Lixia; Li, Yan; Li, Xiaoyan; Zhang, Qing; Qiu, Shaowei; Zhang, Qi; Wang, Min; Xing, Haiyan; Rao, Qing; Tian, Zheng; Tang, Kejing; Wang, Jianxiang; Mi, Yingchang

    2017-09-01

    The aim of the present study was to investigate the regulation of Wilms Tumor 1 (WT1) by serine protease high-temperature requirement protein A2 (HtrA2), a member of the Htr family, in K562 cells. In addition, the study aimed to observe the effect of this regulation on cell biological functions and its associated mechanisms. Expression of WT1 and HtrA2 mRNA, and proteins following imatinib and the HtrA2 inhibitor 5-[5-(2-nitrophenyl) furfuryl iodine]-1, 3-diphenyl-2-thiobarbituric acid (UCF-101) treatment was detected with reverse transcription-quantitative polymerase chain reaction and western blot analysis. Subsequent to treatment with drugs and UCF-101, the proliferative function of K562 cells was detected using MTT assays, and the rate of apoptosis was detected using Annexin V with propidium iodide flow cytometry in K562 cells. The protein levels in the signaling pathway were analyzed using western blotting following treatment with imatinib and UCF-101. In K562 cells, imatinib treatment activated HtrA2 gene at a transcription level, while the WT1 gene was simultaneously downregulated. Following HtrA2 inhibitor (UCF-101) treatment, the downregulation of WT1 increased gradually. At the protein level, imatinib induced the increase in HtrA2 protein level and concomitantly downregulated WT1 protein level. Subsequent to HtrA2 inhibition by UCF-101, the WT1 protein level decreased temporarily, but eventually increased. Imatinib induced apoptosis in K562 cells, but this effect was attenuated by the HtrA2 inhibitor UCF-101, resulting in the upregulation of the WT1 protein level. However; UCF-101 did not markedly change the proliferation inhibition caused by imatinib. Imatinib activated the p38 mitogen activated protein kinase (p38 MAPK) signaling pathway in K562 cells, and UCF-101 affected the activation of imatinib in the p38 MAPK signaling pathway. Imatinib inhibited the extracellular signal-related kinase (ERK1/2) pathway markedly and persistently, but UCF-101

  14. NR5A2 Regulates Lhb and Fshb Transcription in Gonadotrope-Like Cells In Vitro, but Is Dispensable for Gonadotropin Synthesis and Fertility In Vivo

    PubMed Central

    Fortin, Jérôme; Kumar, Vikas; Zhou, Xiang; Wang, Ying; Auwerx, Johan; Schoonjans, Kristina; Boehm, Ulrich; Boerboom, Derek; Bernard, Daniel J.

    2013-01-01

    Successful mammalian reproduction depends on proper synthesis of the pituitary-derived glycoprotein hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several transcription factors cooperate to activate cell-specific and hormone-regulated expression of the gonadotropin beta subunits (Lhb and Fshb). Among these, NR5A1 (steroidogenic factor 1; SF1) has been shown to directly bind to the Lhb promoter, mediate basal and gonadotropin-releasing hormone (GnRH)-stimulated Lhb transcription, and possibly directly regulate Fshb expression. Recently, the closely-related NR5A2 was shown to activate the rat Lhb promoter in vitro. Here, we further characterized the role of NR5A2 in regulating gonadotropin synthesis. Ectopically expressed NR5A2 directly activated the murine Lhb promoter in a manner identical to that of NR5A1, whereas neither factor activated the murine Fshb promoter. In LβT2 gonadotrope-like cells, depletion of endogenous NR5A1 or NR5A2 impaired basal and GnRH-stimulated Lhb and Fshb transcription. To analyze the physiological role of NR5A2 in gonadotropes in vivo, we generated mice with a gonadotrope-specific deletion of Nr5a2. In contrast with our in vitro data, these mice had normal pituitary Lhb and Fshb expression and intact fertility. Together, our data establish that NR5A2 can act in a non-redundant manner to regulate Lhb and Fshb transcription in vitro, but is dispensable in vivo. PMID:23536856

  15. PacCYP707A2 negatively regulates cherry fruit ripening while PacCYP707A1 mediates drought tolerance.

    PubMed

    Li, Qian; Chen, Pei; Dai, Shengjie; Sun, Yufei; Yuan, Bing; Kai, Wenbin; Pei, Yuelin; He, Suihuan; Liang, Bin; Zhang, Yushu; Leng, Ping

    2015-07-01

    Sweet cherry is a non-climacteric fruit and its ripening is regulated by abscisic acid (ABA) during fruit development. In this study, four cDNAs (PacCYP707A1-4) encoding 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA, were identified in sweet cherry fruits using tobacco rattle virus-induced gene silencing (VIGS) and particle bombardment approaches. Quantitative real-time PCR confirmed significant down-regulation of target gene transcripts in VIGS-treated cherry fruits. In PacCYP707A2-RNAi-treated fruits, ripening and fruit colouring were promoted relative to control fruits, and both ABA accumulation and PacNCED1 transcript levels were up-regulated by 140%. Silencing of PacCYP707A2 by VIGS significantly altered the transcripts of both ABA-responsive and ripening-related genes, including the ABA metabolism-associated genes NCED and CYP707A, the anthocyanin synthesis genes PacCHS, PacCHI, PacF3H, PacDFR, PacANS, and PacUFGT, the ethylene biosynthesis gene PacACO1, and the transcription factor PacMYBA. The promoter of PacMYBA responded more strongly to PacCYP707A2-RNAi-treated fruits than to PacCYP707A1-RNAi-treated fruits. By contrast, silencing of PacCYP707A1 stimulated a slight increase in fruit colouring and enhanced resistance to dehydration stress compared with control fruits. These results suggest that PacCYP707A2 is a key regulator of ABA catabolism that functions as a negative regulator of fruit ripening, while PacCYP707A1 regulates ABA content in response to dehydration during fruit development. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  16. Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus.

    PubMed

    Sobczak, Magdalena; Wasik, Anna; Kłopocka, Wanda; Redowicz, Maria Jolanta

    2008-12-01

    Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.).

  17. Earthworm coelomocyte phagocytosis: An in vitro assay for terrestrial toxicity identification evaluation

    SciTech Connect

    Burch, S.W.; Goven, A.J.; Fitzpatrick, L.C.; Venables, B.J.; Callahan, C.A.

    1995-12-31

    An in vitro assay has been developed for rapid (48 h) evaluation of cytotoxic effects of exposure (24 h) of earthworm coelomocytes. The assay, inhibition of phagocytosis (24 h) of stained yeast cells and cell viability, links a traditional soil bioassay organism (Lumbricus terrestris) with a laboratory protocol for use in evaluating physical/chemical fractions resulting from terrestrial TIE manipulations. The assay was developed using copper sulfate as a reference toxicant. Copper exposures as low as 2--4 pg/ml. resulted in 20--60% inhibition of phagocytosis without significant decrease in cell viability. Exposures above 10 pg/ml resulted in reduced cell viability and inhibition of phagocytosis. The assay was successfully applied to terrestrial TIE fractions derived from extractions of soil from a PCP contaminated wood treatment site.

  18. Enhanced phagocytosis of group A streptococci M type 6 by oleic acid

    SciTech Connect

    Speert, D.P.; Quie, P.G.; Wannamaker, L.W.

    1981-04-01

    M protein, located on the surface fimbriae of group A streptococci, is antiphagocytic by unknown means. It is known that oleic acid kills group A streptococci and distorts the fimbriae. The effect of oleic acid on phagocytosis of group A streptococci was examined. Phagocytosis of a strain possessing M protein (M+) and its M- variant was assessed by uptake of radiolabeled bacteria and by chemiluminescence. The M- but not the M+ streptococci were well phagocytized and induced chemiluminescence. Oleic acid-killed and heat-killed streptococci (both M+ and M-) were readily phagocytized and induced sustained chemiluminescence. M+ streptococci killed by ultraviolet irradiation were inefficiently phagocytized and did not induce chemiluminescence. Oleic acid-killed M+ streptococci absorbed type-specific antibody. An extract of M protein reduced the bactericidal capacity of oleic acid. It is proposed that oleic acid may bind to and alter the M protein of group A streptococci and thereby enhance phagocytosis.

  19. Effects of gatifloxacin on phagocytosis, intracellular killing and oxidant radical production by human polymorphonuclear neutrophils.

    PubMed

    Braga, P C; Dal Sasso, M; Bovio, C; Zavaroni, E; Fonti, E

    2002-03-01

    The ingestion and killing of bacteria by phagocytic cells is an important step in the sequence of interactions between invading microorganisms and host defense systems and may be affected by antibiotics. We investigated the effects of gatifloxacin on the phagocytosis, killing and oxidative bursts of human polymorphonuclear neutrophils (PMNs). The percentage phagocytosis and the phagocytosis index were unaffected by exposure of Escherichia coli strains to sub-MICs of gatifloxacin to a 1/64 dilution. However a significant increase in percentage intraphagocytic killing and the killing index occurred in one E. coli strain at 1/32 MIC and in two strains at 1/16 MIC. The incubation of PMNs with sub-MICs and supra-MICs of gatifloxacin (to 32 MIC) did not affect the oxidative bursts.

  20. Study of histamine effects on phagocytosis and enzyme secretion of Tetrahymena pyriformis.

    PubMed

    Darvas, Z; Madarász, B; László, V

    1999-01-01

    1. The biogenic amine histamine develops effects not only in mammalian cells and tissues but in ciliated unicellular Tetrahymena as well. In addition to binding and internalization of labelled histamine, low concentrations can stimulate the phagocytosis of cells in inorganic salt solution. 2. In inorganic solution Tetrahymena cells secrete acid hydrolases to the medium. High concentration of histamine (10 mM) decreases the secretion of three investigated acid hydrolases in a different manner. We think that in this process the primary determinant is the alkaline character of histamine. 3. The effect of histamine on phagocytosis differs from the effect on secretion since the low, physiological concentration of histamine stimulates phagocytosis, the higher concentrations inhibit it. In the background of these effects possibly the hormone character is dominant. It is supported by the fact that histamine antagonists influence the process differently.

  1. Regulation of the Low Dose Radiation Paracrine-Specific Anchorage-Independent Growth Response by Annexin A2

    SciTech Connect

    Weber, Thomas J.; Opresko, Lee K.; Waisman, David M.; Newton, Gregory J.; Quesenberry, Ryan D.; Bollinger, Nikki; Moore, Ronald J.; Smith, Richard D.

    2009-07-13

    ABSTRACT-Here we identify release of annexin A2 into the culture medium in response to low dose X-ray radiation exposure and establish functional linkages to an established paracrine factor-mediated anchorage-independent growth response. Using a standard bicameral coculture model, we observe that annexin A2 levels associated with non-irradiated neighboring cells seeded in the lower chamber (annexin A2 silenced [shRNA] JB6 cells) are increased upon coculture with irradiated (10-50 cGy) JB6 cells seeded in the upper chamber, relative to coculture with sham exposed JB6 cells seeded in the upper chamber, suggesting that annexin A2 released into the medium is capable of communicating in a paracrine fashion. Using a previously established coculture model, we observed that the paracrine factor-mediated anchorage-independent growth response to low dose X-ray radiation is markedly reduced when irradiated annexin A2 silenced (shRNA) JB6 cells are used, relative to coculture with irradiated annexin A2 competent vector control counterparts. These observations suggest that annexin A2 is functionally linked to the radiation paracrine factor-specific anchorage-independent growth response in JB6 cells.

  2. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells.

    PubMed

    He, Gen-Lin; Luo, Zhen; Yang, Ju; Shen, Ting-Ting; Chen, Yi; Yang, Xue-Sen

    2016-01-01

    Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer's disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1-42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases.

  3. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells

    PubMed Central

    Yang, Ju; Shen, Ting-ting; Chen, Yi; Yang, Xue-Sen

    2016-01-01

    Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer’s disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1–42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases. PMID:26824354

  4. A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages.

    PubMed

    Crowley, M T; Costello, P S; Fitzer-Attas, C J; Turner, M; Meng, F; Lowell, C; Tybulewicz, V L; DeFranco, A L

    1997-10-06

    Receptors on macrophages for the Fc region of IgG (FcgammaR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcgammaR cross-linking. Macrophages derived from Syk-deficient (Syk-) mice were defective in phagocytosis of particles bound by FcgammaRs, as well as in many FcgammaR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk- macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk- macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcgammaRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcgammaR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcgammaR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcgammaR engagement, accompanied by a delay in FcgammaR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcgammaR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcgammaR's analogous to models of signaling by the B and T cell antigen receptors.

  5. Role of Yops and adhesins in resistance of Yersinia enterocolitica to phagocytosis.

    PubMed

    Grosdent, Nadine; Maridonneau-Parini, Isabelle; Sory, Marie-Paule; Cornelis, Guy R

    2002-08-01

    Yersinia enterocolitica is a pathogen endowed with two adhesins, Inv and YadA, and with the Ysc type III secretion system, which allows extracellular adherent bacteria to inject Yop effectors into the cytosol of animal target cells. We tested the influence of all of these virulence determinants on opsonic and nonopsonic phagocytosis by PU5-1.8 and J774 mouse macrophages, as well as by human polymorphonuclear leukocytes (PMNs). The adhesins contributed to phagocytosis in the absence of opsonins but not in the presence of opsonins. In agreement with previous results, YadA counteracted opsonization. In every instance, the Ysc-Yop system conferred a significant level of resistance to phagocytosis. Nonopsonized single-mutant bacteria lacking either YopE, -H, -T, or -O were phagocytosed significantly more by J774 cells and by PMNs. Opsonized bacteria were phagocytosed more than nonopsonized bacteria, and mutant bacteria lacking either YopH, -T, or -O were phagocytosed significantly more by J774 cells and by PMNs than were wild-type (WT) bacteria. Opsonized mutants lacking only YopE were phagocytosed significantly more than were WT bacteria by PMNs but not by J774 cells. Thus, YopH, -T, and -O were involved in all of the phagocytic processes studied here but YopE did not play a clear role in guarding against opsonic phagocytosis by J774. Mutants lacking YopP and YopM were, in every instance, as resistant as WT bacteria. Overexpression of YopE, -H, -T, or -O alone did not confer resistance to phagocytosis, although it affected the cytoskeleton. These results show that YopH, YopT, YopO, and, in some instances, YopE act synergistically to increase the resistance of Y. enterocolitica to phagocytosis by macrophages and PMNs.

  6. Impact of resolvin E1 on murine neutrophil phagocytosis in type 2 diabetes.

    PubMed

    Herrera, Bruno S; Hasturk, Hatice; Kantarci, Alpdogan; Freire, Marcelo O; Nguyen, Olivia; Kansal, Shevali; Van Dyke, Thomas E

    2015-02-01

    Diabetic complications involve inflammation-mediated microvascular and macrovascular damage, disruption of lipid metabolism, glycosylation of proteins, and abnormalities of neutrophil-mediated events. Resolution of inflamed tissues to health and homeostasis is an active process mediated by endogenous lipid agonists, including lipoxins and resolvins. This proresolution system appears to be compromised in type 2 diabetes (T2D). The goal of this study was to investigate unresolved inflammation in T2D. Wild-type (WT) and genetically engineered mice, including T2D mice (db/db), transgenic mice overexpressing the human resolvin E1 (RvE1) receptor (ERV1), and a newly bred strain of db/ERV1 mice, were used to determine the impact of RvE1 on the phagocytosis of Porphyromonas gingivalis in T2D. Neutrophils were isolated and incubated with fluorescein isothiocyanate-labeled P. gingivalis, and phagocytosis was measured in a fluorochrome-based assay by flow cytometry. Mitogen-activated protein kinase (MAPK) (p42 and p44) and Akt (Thr308 and Ser473) phosphorylation was analyzed by Western blotting. The mouse dorsal air pouch model was used to evaluate the in vivo impact of RvE1. Results revealed that RvE1 increased the neutrophil phagocytosis of P. gingivalis in WT animals but had no impact in db/db animals. In ERV1-transgenic and ERV1-transgenic diabetic mice, phagocytosis was significantly increased. RvE1 decreased Akt and MAPK phosphorylation in the transgenic animals. In vivo dorsal air pouch studies revealed that RvE1 decreases neutrophil influx into the pouch and increases neutrophil phagocytosis of P. gingivalis in the transgenic animals; cutaneous fat deposition was reduced, as was macrophage infiltration. The results suggest that RvE1 rescues impaired neutrophil phagocytosis in obese T2D mice overexpressing ERV1.

  7. An invertebrate-specific and immune-responsive microRNA augments oyster haemocyte phagocytosis by targeting CgIκB2

    PubMed Central

    Chen, Hao; Zhou, Zhi; Wang, Hao; Wang, Lingling; Wang, Weilin; Liu, Rui; Qiu, Limei; Song, Linsheng

    2016-01-01

    Nuclear factor (NF)-κB pathway is an evolutionally conserved pathway in activating immune response, in which IκBs can repress the activation. In the present study, cgi-miR-2d, an invertebrate-specific microRNA, was proved to regulate CgIκB2 expression and haemocyte phagocytosis during bacterial infection in oyster Crassostrea gigas. The expression of cgi-miR-2d was significantly up-regulated after Vibrio splendidus challenge, while CgIκB2 transcripts decreased. Significant decreases in both luminescence and CgIκB2 3′UTR level was observed after transfection of cgi-miR-2d in CgIκB2 3′UTR luciferase reporter assay. CgIκB2 mRNA level decreased significantly (0.51-fold of control group, p < 0.05) in gain-of-function assay of cgi-miR-2d in vivo while it increased markedly (1.27-fold, p < 0.05) when cgi-miR-2d was repressed (0.10-fold, p < 0.01). A significant increase of haemocyte phagocytosis rate was observed in cgi-miR-2d overexpression group (p < 0.01), consistent with results in CgIκB2 knock-down group (p < 0.01). Moreover, the apoptosis rate of haemocytes was found significantly declined (28.57%, p < 0.01) in gain-of-function assay of cgi-miR-2d. Together, those results not only depicted the functional conservation of miR-2d family in anti-apoptosis of oysters but also highlighted its interaction with phagocytosis by modulating NF-κB pathway, which might dedicate critically to the well-balance of host immune response. PMID:27404434

  8. Using Image-Based Flow Cytometry to Assess Monocyte Oxidized LDL Phagocytosis Capacity.

    PubMed

    Henning, Andrea L; Venable, Adam S; Prado, Eric A; McFarlin, Brian K

    2016-01-01

    The examination of monocyte phagocytosis of modified lipoproteins is important to the understanding of plaque deposition and the development of atherosclerosis. Current methods lack the high-throughput image-based analysis capabilities, which may yield novel information concerning monocyte activity in disease processes. Specifically, this method identifies monocyte phagocytosis of oxidized LDL along with a change in adhesion molecules and scavenger receptors. Our laboratory is currently implementing this method as a means to study how acute dietary modifications alter risk of developing atherosclerosis.

  9. Endomorphins 1 and 2 modulate chemotaxis, phagocytosis and superoxide anion production by microglia.

    PubMed

    Azuma, Y; Ohura, K; Wang, P L; Shinohara, M

    2001-09-03

    We evaluate the role of endomorphins 1 and 2 on microglial functions. Endomorphins 1 and 2 blocked phagocytosis of Escherichia coli. In addition, both markedly inhibited chemotaxis toward zymosan-activated serum. In contrast, when microglia was preincubated with these endomorphins, followed by incubation with LPS before stimulation with phorbol 12-myristate 13-acetate (PMA) at 200 nM, they potentiated superoxide anion production. Furthermore, when microglia was preincubated with these endomorphins together with PMA at 20 nM, followed by stimulation with PMA at 200 nM, superoxide anion production was potentiated. These results suggest that endomorphins 1 and 2 modulate phagocytosis, chemotaxis and superoxide anion production by microglia.

  10. Methods: implementation of in vitro and ex vivo phagocytosis and respiratory burst function assessments in safety testing.

    PubMed

    Freebern, Wendy J; Bigwarfe, Tammy J; Price, Karen D; Haggerty, Helen G

    2013-01-01

    Functional innate immune assessments, including phagocytosis and respiratory burst, are at the forefront of immunotoxicology evaluation in pre-clinical animal species. Although in the clinic and in academic science, phagocytosis, and respiratory burst assessments have been reported for over two decades, the implementation of phagocytosis and respiratory burst analyses in toxicology safety programs is just recently gaining publicity. Discussed herein are general methods, both microtiter plate-based and flow cytometric-based, for assessing phagocytosis and respiratory burst in pre-clinical species including mouse, rat, dog, and monkey. This methods-centric discussion includes a review of technologies and descriptions of method applications, with examples of results from analyses testing reported inhibitors (rottlerin, wortmannin, and SB203580) of phagocytosis and respiratory burst. Justification of implementation, strategic experimental design planning, and feasibility aspects of evaluating test article effects on phagocytosis and respiratory burst function are described within the context of a case study. The case study involves investigation of the effects of a small molecule p38 kinase inhibitor, BMS-582949, on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro, as well as ex vivo in these innate immune cells from monkeys administered BMS-582949 during a 1-week repeat dose investigative study. The results of the in vitro and ex vivo assessments demonstrated that BMS-582949 inhibited phagocytosis and respiratory burst. These findings correlated with incidences of opportunistic infections observed in rat and monkey toxicity studies.

  11. Vitamin D-mediated regulation of CYP21A2 transcription - a novel mechanism for vitamin D action.

    PubMed

    Lundqvist, Johan; Wikvall, Kjell; Norlin, Maria

    2012-10-01

    1α,25-Dihydroxyvitamin D(3) has recently been reported to decrease expression and activity of CYP21A2. In this paper, we have studied the mechanisms for the 1α,25-dihydroxyvitamin D(3)-mediated effect on CYP21A2 transcriptional rate. We have studied the effects of 1α,25-dihydroxyvitamin D(3) using luciferase reporter constructs containing different lengths of the CYP21A2 promoter. These constructs were transfected into cell lines derived from human and mouse adrenal cortex. The mechanism for the effects of vitamin D on the CYP21A2 promoter was studied using chromatin immunoprecipitation assay, mutagenesis and gene silencing by siRNA. 1α,25-Dihydroxyvitamin D(3) was found to alter the promoter activity via a VDR-mediated mechanism, including the comodulators VDR interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF). The involvement of comodulator VDIR was confirmed by gene silencing. We identified a vitamin D response element in the CYP21A2 promoter. Interaction between this novel response element and VDR, WSTF and VDIR was shown by chromatin immunoprecipitation assay. When this sequence was deleted, the effect of 1α,25-dihydroxyvitamin D(3) was abolished, indicating that this sequence in the CYP21A2 promoter functions as a vitamin D response element. Interestingly, an altered balance between nuclear receptors and comodulators reversed the suppressing effect of vitamin D to a stimulatory effect. This paper reports data important for the understanding of the mechanisms for vitamin D-mediated suppression of gene expression as well as for the vitamin D-mediated effects on CYP21A2. We report a novel mechanism for effects of 1α,25-dihydroxyvitamin D(3). Copyright © 2012 Elsevier B.V. All rights reserved.

  12. CD147 regulates cancer migration via direct interaction with Annexin A2 and DOCK3-β-catenin-WAVE2 signaling

    PubMed Central

    Feng, Fei; Wu, Jiao; Yang, Xiang-Min; Chen, Zhi-Nan; Jiang, Jian-Li

    2016-01-01

    The acquisition of inappropriate migratory feature is crucial for tumor metastasis. It has been suggested that CD147 and Annexin A2 are involved in regulating tumor cell movement, while the regulatory mechanisms are far from clear. In this study, we demonstrated that CD147 physically interacted with the N-terminal domain of Annexin A2 and decreased Annexin A2 phosphorylation on tyrosine 23. In vitro kinase assay showed that the I domain of CD147 was indispensable for CD147-mediated downregulation of Annexin A2 phosphorylation by Src. Furthermore, we determined that p-Annexin A2 promoted the expression of dedicator of cytokinesis 3 (DOCK3) and DOCK3 blocked β-catenin nuclear translocation, resulting in inhibition of β-catenin signaling. In addition, DOCK3 inhibited lamellipodium dynamics and tumor cell movement. Also, we found that β-catenin signaling increased WAVE2 expression. Therefore, DOCK3 was characterized as a negative regulator of WAVE2 expression via inhibiting β-catenin signaling. Our study provides the first evidence that CD147 promotes tumor cell movement and metastasis via direct interaction with Annexin A2 and DOCK3-β-catenin-WAVE2 signaling axis. PMID:26716413

  13. CD147 regulates cancer migration via direct interaction with Annexin A2 and DOCK3-β-catenin-WAVE2 signaling.

    PubMed

    Cui, Hong-Yong; Wang, Shi-Jie; Miao, Ji-Yu; Fu, Zhi-Guang; Feng, Fei; Wu, Jiao; Yang, Xiang-Min; Chen, Zhi-Nan; Jiang, Jian-Li

    2016-02-02

    The acquisition of inappropriate migratory feature is crucial for tumor metastasis. It has been suggested that CD147 and Annexin A2 are involved in regulating tumor cell movement, while the regulatory mechanisms are far from clear. In this study, we demonstrated that CD147 physically interacted with the N-terminal domain of Annexin A2 and decreased Annexin A2 phosphorylation on tyrosine 23. In vitro kinase assay showed that the I domain of CD147 was indispensable for CD147-mediated downregulation of Annexin A2 phosphorylation by Src. Furthermore, we determined that p-Annexin A2 promoted the expression of dedicator of cytokinesis 3 (DOCK3) and DOCK3 blocked β-catenin nuclear translocation, resulting in inhibition of β-catenin signaling. In addition, DOCK3 inhibited lamellipodium dynamics and tumor cell movement. Also, we found that β-catenin signaling increased WAVE2 expression. Therefore, DOCK3 was characterized as a negative regulator of WAVE2 expression via inhibiting β-catenin signaling. Our study provides the first evidence that CD147 promotes tumor cell movement and metastasis via direct interaction with Annexin A2 and DOCK3-β-catenin-WAVE2 signaling axis.

  14. FGF acts as a co-transmitter through Adenosine A2A receptor to regulate morphological and physiological synaptic plasticity

    PubMed Central

    Flajolet, Marc; Wang, Zhongfeng; Futter, Marie; Shen, Weixing; Nuangchamnong, Nina; Bendor, Jacob; Palaszewski, Iwona; Nairn, Angus C.; Surmeier, D. James; Greengard, Paul

    2009-01-01

    Summary Abnormalities of striatal function have been implicated in several major neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, and depression. Adenosine, by activation of A2A receptors, antagonizes dopamine signaling at D2 receptors and A2A receptor antagonists have been tested as therapeutic agents for Parkinson's disease. We report here a direct physical interaction between the G protein-coupled A2A receptor and the receptor tyrosine kinase FGF receptor. Concomitant activation of these two classes of receptors, but not individual activation of either one alone, causes a robust activation of the MAPK/ERK pathway, differentiation and neurite extension of PC12 cells, spine morphogenesis in primary neuronal cultures, and cortico-striatal plasticity induced by a novel A2AR/FGFR-dependent mechanism. The discovery of a direct physical interaction between the A2A and FGF receptors and the robust physiological consequences of this association shed light on the mechanism underlying FGF functions as a co-transmitter and open new avenues for therapeutic interventions. PMID:18953346

  15. Synergistic Up-Regulation of Vascular Endothelial Growth Factor Expression in Murine Macrophages by Adenosine A2A Receptor Agonists and Endotoxin

    PubMed Central

    Leibovich, Samuel Joseph; Chen, Jiang-Fan; Pinhal-Enfield, Grace; Belem, Paula C.; Elson, Genie; Rosania, Anthony; Ramanathan, Madhuri; Montesinos, Carmen; Jacobson, Marlene; Schwarzschild, Michael A.; Fink, J. Stephen; Cronstein, Bruce

    2002-01-01

    Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by ∼500%; interferon-γ (IFN-γ) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by ∼50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5′-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A2 receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5′-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A2A receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N6-cyclopentyl adenosine (CCPA), an adenosine A1 agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-γ (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC50 < 10 ng/ml), but not of IFN-γ, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS−/− mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A2 or A1 receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A3−/− and A2A−/− mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A3−/− but not A2A−/− mice, confirming the role of adenosine A2A receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by

  16. HsfA1d and HsfA1e involved in the transcriptional regulation of HsfA2 function as key regulators for the Hsf signaling network in response to environmental stress.

    PubMed

    Nishizawa-Yokoi, Ayako; Nosaka, Ryota; Hayashi, Hideki; Tainaka, Hitoshi; Maruta, Takanori; Tamoi, Masahiro; Ikeda, Miho; Ohme-Takagi, Masaru; Yoshimura, Kazuya; Yabuta, Yukinori; Shigeoka, Shigeru

    2011-05-01

    Heat shock transcription factor A2 (HsfA2) acts as a key component of the Hsf signaling network involved in cellular responses to various types of environmental stress. However, the mechanism governing the regulation of HsfA2 expression is still largely unknown. We demonstrated here that a heat shock element (HSE) cluster in the 5'-flanking region of the HsfA2 gene is involved in high light (HL)-inducible HsfA2 expression. Accordingly, to identify the Hsf regulating the expression of HsfA2, we analyzed the effect of loss-of-function mutations of class A Hsfs on the expression of HsfA2 in response to HL stress. Overexpression of an HsfA1d or HsfA1e chimeric repressor and double knockout of HsfA1d and HsfA1e Arabidopsis mutants (KO-HsfA1d/A1e) significantly suppressed the induction of HsfA2 expression in response to HL and heat shock (HS) stress. Transient reporter assays showed that HsfA1d and HsfA1e activate HsfA2 transcription through the HSEs in the 5'-flanking region of HsfA2. In the KO-HsfA1d/A1e mutants, 560 genes, including a number of stress-related genes and several Hsf genes, HsfA7a, HsfA7b, HsfB1 and HsfB2a, were down-regulated compared with those in the wild-type plants under HL stress. The PSII activity of KO-HsfA1d/A1e mutants decreased under HL stress, while the activity of wild-type plants remained high. Furthermore, double knockout of HsfA1d and HsfA1e impaired tolerance to HS stress. These findings indicated that HsfA1d and HsfA1e not only regulate HsfA2 expression but also function as key regulators of the Hsf signaling network in response to environmental stress.

  17. IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fcα/μ Receptor.

    PubMed

    Weinstein, Jonathan R; Quan, Yi; Hanson, Josiah F; Colonna, Lucrezia; Iorga, Michael; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira; Elkon, Keith B; Möller, Thomas

    2015-12-01

    Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia.

  18. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

    PubMed

    Takeuchi, Yusuke; Morise, Jyoji; Morita, Ippei; Takematsu, Hiromu; Oka, Shogo

    2015-01-01

    The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

  19. Secretory and cytosolic phospholipase A2 activities and expression are regulated by oxytocin and estradiol during labor.

    PubMed

    Farina, Mariana Gabriela; Billi, Silvia; Leguizamón, Gustavo; Weissmann, Carina; Guadagnoli, Tamara; Ribeiro, Maria Laura; Franchi, Ana Maria

    2007-08-01

    The release of arachidonic acid from membrane glycerophospholipids through the action of phospholipases (PLs) is the first step in the biosynthesis of prostaglandins (PGs). In reproductive tissues, the most important PLs are cytosolic PLA(2) (cPLA(2)) and types IIA and V of the secretory isoform (sPLA(2)). The aim of this work was to investigate the role of ovarian steroid hormones and oxytocin (OT) in the regulation of rat uterine PLA(2) activity and expression during pregnancy and labor. The activity of sPLA(2) increased near labor, whereas cPLA(2) activity augmented towards the end of gestation. The levels of sPLA(2) IIA and cPLA(2) mRNA showed an increase before labor (P<0.05, day 21), whereas sPLA(2) V mRNA was not regulated during pregnancy. The administration of atosiban (synthetic OT antagonist) together with tamoxifen (antagonist of estrogen receptors) was able to decrease cytosolic and secretory PLA(2) activities, diminish the expression of sPLA(2) IIA and cPLA(2), as well as decrease PGF(2 alpha) production before the onset of labor (P<0.01). The ovarian steroid did not affect PLA(2) during pregnancy. Collectively, these findings indicate that in the rat uterus, both 17beta-estradiol and OT could be regulating the activity and the expression of the secretory and the cytosolic isoforms of PLA(2), thus controlling PGF(2 alpha) synthesis prior to the onset of labor.

  20. Transient up-regulation of the rostrocaudal gradient of ephrin A2 in the tectum coincides with reestablishment of orderly projections during optic nerve regeneration in goldfish.

    PubMed

    Rodger, J; Bartlett, C A; Beazley, L D; Dunlop, S A

    2000-11-01

    During development, a graded expression of ephrin A2 has been implicated in retinotectal map formation. Here we have examined ephrin A2 expression during optic nerve regeneration in the mature goldfish. In the tecta of normal animals, a gradient of ephrin A2 expression is detected in cell bodies within the stratum fibrosum et griseum superficiale with more immunopositive cells caudally than rostrally. The gradient in the mature animal presumably reflects the plasticity associated with continued retinal and tectal neurogenesis. During optic nerve regeneration, expression throughout the tectum is increased by 1 month as a strong rostrocaudal gradient. The gradient declines to normal by 3 months. The up-regulation of ephrin A2 during optic nerve regeneration is likely to be instrumental in reestablishing the retinotectal map.

  1. Plexin-A4-dependent retrograde semaphorin 3A signalling regulates the dendritic localization of GluA2-containing AMPA receptors.

    PubMed

    Yamashita, Naoya; Usui, Hiroshi; Nakamura, Fumio; Chen, Sandy; Sasaki, Yukio; Hida, Tomonobu; Suto, Fumikazu; Taniguchi, Masahiko; Takei, Kohtaro; Goshima, Yoshio

    2014-03-06

    The dendritic targeting of neurotransmitter receptors is vital for dendritic development and function. However, how such localization is established remains unclear. Here we show that semaphorin 3A (Sema3A) signalling at the axonal growth cone is propagated towards the cell body by retrograde axonal transport and drives AMPA receptor GluA2 to the distal dendrites, which regulates dendritic development. Sema3A enhances glutamate receptor interacting protein 1-dependent localization of GluA2 in dendrites, which is blocked by knockdown of cytoplasmic dynein heavy chain. PlexinA (PlexA), a receptor component for Sema3A, interacts with GluA2 at the immunoglobulin-like Plexin-transcription-factor domain (PlexA-IPT) in somatodendritic regions. Overexpression of PlexA-IPT suppresses dendritic localization of GluA2 and induces aproximal bifurcation phenotype in the apical dendrites of CA1 hippocampal neurons. Thus, we propose a control mechanism by which retrograde Sema3A signalling regulates the glutamate receptor localization through trafficking of cis-interacting PlexA with GluA2 along dendrites.

  2. RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional level.

    PubMed

    Lai, Yi-Chyi; Peng, Hwei-Ling; Chang, Hwan-You

    2003-02-01

    The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2. The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2. The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease.

  3. RmpA2, an Activator of Capsule Biosynthesis in Klebsiella pneumoniae CG43, Regulates K2 cps Gene Expression at the Transcriptional Level

    PubMed Central

    Lai, Yi-Chyi; Peng, Hwei-Ling; Chang, Hwan-You

    2003-01-01

    The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2. The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2. The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease. PMID:12533454

  4. Optical tracking of phagocytosis with an activatable profluorophore metabolically incorporated into bacterial peptidoglycan.

    PubMed

    Tian, Yunpeng; Yu, Mingzu; Li, Zhu; Han, Jiahuai; Yang, Liu; Han, Shoufa

    2015-08-18

    Phagocytosis is critical for immunity against pathogens. Prior imaging using dye-labeled synthetic beads or green fluorescent protein-expressing bacteria is limited by "always-on" signals which compromise discerning phagocytosed particles from adherent particles. Targeting cellular internalization of pathogens into acidic phagolysosomes, we herein report "turn-on" fluorescence imaging of phagocytosis with viable bacteria featuring peptidoglycans covalently modified with rhodamine-lactam responsive to acidic pH. Culturing of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) with d-lysine conjugated rhodamine-lactam and fluorescein isocyanate (FITC) leads to efficient metabolic incorporation of FITC and rhodamine-lactam into bacterial peptidoglycan. E. coli and S. aureus become red-emissive upon phagocytosis into Raw 264.7 macrophages. With FITC as the reference signal, the mono- and dual-color emission allow efficient in situ distinction of ingested bacteria from extracellular bacteria. Given the ease of optical peptidoglycan labeling, the prevalence of microbial peptidoglycan and preservation of microbial surface landscape, this approach would be of use for investigation on microbial pathogenesis and high-throughput screening of immunomodulators of phagocytosis.

  5. Dynamin 2 is required for actin assembly in phagocytosis in Sertoli cells

    SciTech Connect

    Otsuka, Atsushi; Abe, Tadashi; Watanabe, Masami; Yagisawa, Hitoshi; Takei, Kohji; Yamada, Hiroshi

    2009-01-16

    Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P{sub 2}]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P{sub 2} either by expression of PH domain of PLC{delta} or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.

  6. Rediae of echinostomatid and heterophyid trematodes suppress phagocytosis of haemocytes in Littorina littorea (Gastropoda: Prosobranchia).

    PubMed

    Iakovleva, Nadya V; Shaposhnikova, Tania G; Gorbushin, Alexander M

    2006-05-01

    A modulation of the phagocytic activity of hemocytes from the common periwinkle Littorina littorea by secretory-excretory products (SEP) released by trematode rediae during axenic in vitro cultivation was studied. The SEP released by the parasites Himasthla elongata (Echinostomatidae) and Cryptocotyle lingua (Heterophyidae) were found to inhibit the phagocytosis of zymozan particles by periwinkle hemocytes. The specificity of SEP effects was assessed: SEP of Himasthla militaris and Cryptocotyle concavum, two trematodes belonging to the same genera but infecting another closely related prosobranch snail Hydrobia ulvae, were also shown to be able to suppress L. littorea hemocytes phagocytic activity. However, no decrease in phagocytosis rate was observed when SEP of H. elongata and C. lingua were applied to monolayers of hemocytes from the bivalve mollusc Mytilus edulis. SEP from H. elongata was fractionated; only those fractions containing proteins of molecular weight more than 50 kDa were shown to possess inhibitory activity. Different H. elongata SEP concentrations were tested in for their ability to suppress phagocytosis by L. littorea hemocytes. Even very low SEP concentrations were shown to retain their ability to decrease phagocytosis rate, the inhibitory effect being dose-dependent. Hemocytes derived from snails naturally infected with H. elongata were also found to have lower phagocytic ability as compared to healthy individuals.

  7. Mycobacterium llatzerense, a waterborne Mycobacterium, that resists phagocytosis by Acanthamoeba castellanii

    PubMed Central

    Delafont, Vincent; Samba-Louaka, Ascel; Cambau, Emmanuelle; Bouchon, Didier; Moulin, Laurent; Héchard, Yann

    2017-01-01

    Nontuberculous mycobacteria (NTM) are environmental bacteria increasingly associated to public health problems. In water systems, free-living amoebae (FLA) feed on bacteria by phagocytosis, but several bacteria, including many NTM, are resistant to this predation. Thus, FLA can be seen as a training ground for pathogenic bacteria. Mycobacterium llatzerense was previously described as frequently associated with FLA in a drinking water network. The present study aimed to characterize the interactions between M. llatzerense and FLA. M. llatzerense was internalised by phagocytosis and featured lipid inclusions, suggesting a subversion of host resources. Moreover, M. llatzerense survived and even multiplied in presence of A. castellanii. Using a genomic-based comparative approach, twelve genes involved in phagocytosis interference, described in M. tuberculosis, were identified in the M. llatzerense genome sequenced in this study. Transcriptomic analyses showed that ten genes were significantly upregulated during the first hours of the infection, which could partly explain M. llatzerense resistance. Additionally, M. llatzerense was shown to actively inhibit phagosome acidification. In conclusion, M. llatzerense presents a high degree of resistance to phagocytosis, likely explaining its frequent occurrence within FLA in drinking water networks. It underscores that NTM should be carefully monitored in water networks to prevent human health concerns. PMID:28393860

  8. The High PMNs Phagocytosis Resistance of Enterococcal Isolates from RTx Patients

    PubMed Central

    Daca, Agnieszka; Witkowski, Jacek M.; Bryl, Ewa; Rutkowski, Bolesław

    2015-01-01

    Infections caused by opportunistic pathogens such as enterococci remain difficult to manage, especially in immunocompromised patients. Because of infections' limited symptoms in such patients the additional problems are to find proper diagnostic criteria and the management of infection. Here we aimed to compare the resistance of commensal enterococcal strains and RTx patients' isolates, to PMNs phagocytosis. Thirty-six enterococcal urine and faecal isolates from RTx patients and 17 faecal isolates from healthy volunteers were cultured in planktonic and biofilm forms in 37°C or 42°C. Another tested variable was the addition of immunosuppressant to the culture media. Bacterial cells were stained with fluorescent reporter (CFDA, PI) and incubated with PMNs. Results of phagocytosis were estimated as a mean fluorescence intensity (MFI) of PMNs using flow cytometry. Commensal enterococci cultured in all abovementioned (37°C and 42°C/the addition of immunosuppressant) conditions were less resistant to phagocytosis compared to RTx isolates. Observed significant difference in phagocytosis resistance suggests that patients in immunosuppression are colonized with high risk strains which may lead to the development of infection. PMID:25861625

  9. The high PMNs phagocytosis resistance of enterococcal isolates from RTx patients.

    PubMed

    Jarzembowski, Tomasz; Daca, Agnieszka; Witkowski, Jacek M; Bryl, Ewa; Rutkowski, Bolesław

    2015-01-01

    Infections caused by opportunistic pathogens such as enterococci remain difficult to manage, especially in immunocompromised patients. Because of infections' limited symptoms in such patients the additional problems are to find proper diagnostic criteria and the management of infection. Here we aimed to compare the resistance of commensal enterococcal strains and RTx patients' isolates, to PMNs phagocytosis. Thirty-six enterococcal urine and faecal isolates from RTx patients and 17 faecal isolates from healthy volunteers were cultured in planktonic and biofilm forms in 37°C or 42°C. Another tested variable was the addition of immunosuppressant to the culture media. Bacterial cells were stained with fluorescent reporter (CFDA, PI) and incubated with PMNs. Results of phagocytosis were estimated as a mean fluorescence intensity (MFI) of PMNs using flow cytometry. Commensal enterococci cultured in all abovementioned (37°C and 42°C/the addition of immunosuppressant) conditions were less resistant to phagocytosis compared to RTx isolates. Observed significant difference in phagocytosis resistance suggests that patients in immunosuppression are colonized with high risk strains which may lead to the development of infection.

  10. DEVELOPMENTAL EXPOSURE TO A THYROID DISRUPTING CHEMICAL STIMULATES PHAGOCYTOSIS IN JUVENILE SPRAGUE-DAWLEY RATS

    EPA Science Inventory

    Developmental Exposure to a Thyroid Disrupting Chemical Stimulates Phagocytosis in Juvenile Sprague-Dawley Rats.
    AA Rooney1, R Matulka2, and R Luebke3. 1NCSU/US EPA CVM, Department of Anatomy, Physiological Sciences and Radiology, Raleigh, NC;2UNC Department of Toxicology, Cha...

  11. Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells.

    PubMed

    Wu, J; Liu, W; Koenig, K; Idell, S; Broaddus, V C

    2000-11-01

    Biological modification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nevertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, confirming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is important in fiber-induced toxicity for mesothelial cells.

  12. Mechanism involved in phagocytosis and killing of Listeria monocytogenes by Acanthamoeba polyphaga.

    PubMed

    Akya, Alisha; Pointon, Andrew; Thomas, Connor

    2009-10-01

    Intra-cellular pathogen, Listeria monocytogenes, is capable of invasion and survival within mammalian cells. However, Acanthamoeba polyphaga trophozoites phagocytose and rapidly degrade Listeria cells. In order to provide more information on amoeba phagocytosis and killing mechanisms, this study used several inhibitor agents known to affect the phagocytosis and killing of bacteria by eukaryotes. Amoebae were pre-treated with mannose, cytochalasin D, wortmannin, suramin, ammonium chloride, bafilomycin A and monensin followed by co-culture with bacteria. Phagocytosis and killing of bacterial cells by amoeba trophozoites was assessed using plate counting methods and microscopy. The data presented indicates that actin polymerisation and cytoskeletal rearrangement are involved in phagocytosis of L. monocytogenes cells by A. polyphaga trophozoites. Further, both phagosomal acidification and phagosome-lysosome fusion are involved in killing and degradation of L. monocytogenes cells by A. polyphaga. However, the mannose-binding protein receptor does not play an important role in uptake of bacteria by amoeba trophozoites. In conclusion, this data reveals the similar principles of molecular mechanisms used by different types of eukaryotes in uptake and killing of bacteria.

  13. Kaurane diterpenes protect against apoptosis and inhibition of phagocytosis in activated macrophages.

    PubMed

    de las Heras, B; Hortelano, S; Girón, N; Bermejo, P; Rodríguez, B; Boscá, L

    2007-09-01

    The kaurane diterpenes foliol and linearol are inhibitors of the activation of nuclear factor kappaB, a transcription factor involved in the inflammatory response. Effects of these diterpenes on apoptosis and phagocytosis have been analysed in cultured peritoneal macrophages and in the mouse macrophage cell line, RAW 264.7. Macrophages were maintained in culture and activated with pro-inflammatory stimuli in the absence or presence of diterpenes. Apoptosis and the phagocytosis in these cells under these conditions were determined. Incubation of macrophages with a mixture of bacterial lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) induced apoptosis through a NO-dependent pathway, an effect significantly inhibited by foliol and linearol in the low muM range, without cytotoxic effects. Apoptosis in macrophages induced by NO donors was also inhibited. The diterpenes prevented apoptosis through a mechanism compatible with the inhibition of caspase-3 activation, release of cytochrome c to the cytosol and p53 overexpression, as well as an alteration in the levels of proteins of the Bcl-2 family, in particular, the levels of Bax. Cleavage of poly(ADP-ribose) polymerase, a well-established caspase substrate, was reduced by these diterpenes. Treatment of cells with foliol and linearol decreased phagocytosis of zymosan bioparticles by RAW 264.7 cells and to a greater extent by peritoneal macrophages. Both diterpenes protected macrophages from apoptosis and inhibited phagocytosis, resulting in a paradoxical control of macrophage function, as viability was prolonged but inflammatory and phagocytic functions were impaired.

  14. A Simple Microscopy Assay to Teach the Processes of Phagocytosis and Exocytosis

    ERIC Educational Resources Information Center

    Gray, Ross; Gray, Andrew; Fite, Jessica L.; Jordan, Renee; Stark, Sarah; Naylor, Kari

    2012-01-01

    Phagocytosis and exocytosis are two cellular processes involving membrane dynamics. While it is easy to understand the purpose of these processes, it can be extremely difficult for students to comprehend the actual mechanisms. As membrane dynamics play a significant role in many cellular processes ranging from cell signaling to cell division to…

  15. DEVELOPMENTAL EXPOSURE TO A THYROID DISRUPTING CHEMICAL STIMULATES PHAGOCYTOSIS IN JUVENILE SPRAGUE-DAWLEY RATS

    EPA Science Inventory

    Developmental Exposure to a Thyroid Disrupting Chemical Stimulates Phagocytosis in Juvenile Sprague-Dawley Rats.
    AA Rooney1, R Matulka2, and R Luebke3. 1NCSU/US EPA CVM, Department of Anatomy, Physiological Sciences and Radiology, Raleigh, NC;2UNC Department of Toxicology, Cha...

  16. Involvement of lipoprotein PpiA of Streptococcus gordonii in evasion of phagocytosis by macrophages.

    PubMed

    Cho, K; Arimoto, T; Igarashi, T; Yamamoto, M

    2013-10-01

    Streptococcus gordonii is a commensal gram-positive bacterium that resides in the human oral cavity, and is one of the most common causes of infective endocarditis (IE). Bacterial surface molecules play an important role in establishing IE, and several S. gordonii proteins have been implicated in binding to host cells during the establishment of IE. In this study, we identified a putative lipoprotein, peptidyl-prolyl cis/trans isomerase (PpiA), and clarified its role in evasion of phagocytosis by macrophages. Attenuation of the gene encoding prolipoprotein diacylglyceryl transferase (Lgt) altered the localization of PpiA from the cell surface to the culture supernatant, indicating that PpiA is lipid-anchored in the cell membrane by Lgt. Both human and murine macrophages showed higher phagocytic activity towards ppiA and lgt mutants than the wild-type, indicating that the presence of PpiA suppresses phagocytosis of S. gordonii. Human macrophages treated with dextran sulfate had significantly impaired phagocytosis of S. gordonii, suggesting that class A scavenger receptors in human macrophages are involved in the phagocytosis of S. gordonii. These results provide evidence that S. gordonii lipoprotein PpiA plays an important role in inhibiting phagocytic engulfment and in evasion of the host immune response.

  17. Nuclear receptors license phagocytosis by trem2+ myeloid cells in mouse models of Alzheimer's disease.

    PubMed

    Savage, Julie C; Jay, Taylor; Goduni, Elanda; Quigley, Caitlin; Mariani, Monica M; Malm, Tarja; Ransohoff, Richard M; Lamb, Bruce T; Landreth, Gary E

    2015-04-22

    Alzheimer's disease (AD) is characterized by a robust inflammatory response elicited by the accumulation and subsequent deposition of amyloid (Aβ) within the brain. The brain's immune cells migrate to and invest their processes within Aβ plaques but are unable to efficiently phagocytose and clear plaques from the brain. Previous studies have shown that treatment of myeloid cells with nuclear receptor agonists increases expression of phagocytosis-related genes. In this study, we elucidate a novel mechanism by which nuclear receptors act to enhance phagocytosis in the AD brain. Treatment of murine models of AD with agonists of the nuclear receptors PPARγ, PPARδ, LXR, and RXR stimulated microglial phagocytosis in vitro and rapidly induced the expression of the phagocytic receptors Axl and MerTK. In murine models of AD, we found that plaque-associated macrophages expressed Axl and MerTK and treatment of the cells with an RXR agonist further induced their expression, coincident with the rapid reduction in plaque burden. Further characterization of MerTK(+)/Axl(+) macrophages revealed that they also expressed the phagocytic receptor TREM2 and high levels of CD45, consistent with a peripheral origin of these cells. Importantly, in an ex vivo slice assay, nuclear receptor agonist treatment reversed the AD-related suppression of phagocytosis through a MerTK-dependent mechanism. Thus, nuclear receptor agonists increase MerTK and Axl expression on plaque-associated immune cells, consequently licensing their phagocytic activity and promoting plaque clearance.

  18. A Simple Microscopy Assay to Teach the Processes of Phagocytosis and Exocytosis

    ERIC Educational Resources Information Center

    Gray, Ross; Gray, Andrew; Fite, Jessica L.; Jordan, Renee; Stark, Sarah; Naylor, Kari

    2012-01-01

    Phagocytosis and exocytosis are two cellular processes involving membrane dynamics. While it is easy to understand the purpose of these processes, it can be extremely difficult for students to comprehend the actual mechanisms. As membrane dynamics play a significant role in many cellular processes ranging from cell signaling to cell division to…

  19. Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera) worker castes.

    PubMed

    Hystad, Eva Marit; Salmela, Heli; Amdam, Gro Vang; Münch, Daniel

    2017-01-01

    Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells) that clear pathogens from hemolymph (blood) by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees), and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which-together with confocal microscopy and flow cytometry-allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity.

  20. Phagocytosis and Inflammation: Exploring the effects of the components of E-cigarette vapor on macrophages.

    PubMed

    Ween, Miranda P; Whittall, Jonathan J; Hamon, Rhys; Reynolds, Paul N; Hodge, Sandra J

    2017-08-01

    E-cigarettes are perceived as harmless; however, evidence of their safety is lacking. New data suggests E-cigarettes discharge a range of compounds capable of physiological damage to users. We previously established that cigarette smoke caused defective alveolar macrophage phagocytosis. The present study compared the effect E-cigarette of components; E-liquid flavors, nicotine, vegetable glycerine, and propylene glycol on phagocytosis, proinflammatory cytokine secretion, and phagocytic recognition molecule expression using differentiated THP-1 macrophages. Similar to CSE, phagocytosis of NTHi bacteria was significantly decreased by E-liquid flavoring (11.65-15.75%) versus control (27.01%). Nicotine also decreased phagocytosis (15.26%). E-liquid, nicotine, and E-liquid+ nicotine reduced phagocytic recognition molecules; SR-A1 and TLR-2. IL-8 secretion increased with flavor and nicotine, while TNFα, IL-1β, IL-6, MIP-1α, MIP-1β, and MCP-1 decreased after exposure to most flavors and nicotine. PG, VG, or PG:VG mix also induced a decrease in MIP-1α and MIP-1β We conclude that E-cigarettes can cause macrophage phagocytic dysfunction, expression of phagocytic recognition receptors and cytokine secretion pathways. As such, E-cigarettes should be treated with caution by users, especially those who are nonsmokers. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  1. The Mannose Receptor Is Involved in the Phagocytosis of Mycobacteria-Induced Apoptotic Cells

    PubMed Central

    2016-01-01

    Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-β and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB) infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect. PMID:27413759

  2. A simple microscopy assay to teach the processes of phagocytosis and exocytosis.

    PubMed

    Gray, Ross; Gray, Andrew; Fite, Jessica L; Jordan, Renée; Stark, Sarah; Naylor, Kari

    2012-01-01

    Phagocytosis and exocytosis are two cellular processes involving membrane dynamics. While it is easy to understand the purpose of these processes, it can be extremely difficult for students to comprehend the actual mechanisms. As membrane dynamics play a significant role in many cellular processes ranging from cell signaling to cell division to organelle renewal and maintenance, we felt that we needed to do a better job of teaching these types of processes. Thus, we developed a classroom-based protocol to simultaneously study phagocytosis and exocytosis in Tetrahymena pyriformis. In this paper, we present our results demonstrating that our undergraduate classroom experiment delivers results comparable with those acquired in a professional research laboratory. In addition, students performing the experiment do learn the mechanisms of phagocytosis and exocytosis. Finally, we demonstrate a mathematical exercise to help the students apply their data to the cell. Ultimately, this assay sets the stage for future inquiry-based experiments, in which the students develop their own experimental questions and delve deeper into the mechanisms of phagocytosis and exocytosis.

  3. Indirect role for COPI in the completion of FCgamma receptor-mediated phagocytosis.

    PubMed

    Hackam, D J; Botelho, R J; Sjolin, C; Rotstein, O D; Robinson, J M; Schreiber, A D; Grinstein, S

    2001-05-25

    Recent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of epsilonCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcgammaRIIA). In the presence of functional COPI, FcgammaRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcgammaRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus.

  4. The effects of macrophage source on the mechanism of phagocytosis and intracellular survival of Leishmania

    PubMed Central

    Hsiao, Chia-Hung Christine; Ueno, Norikiyo; Shao, Jian Q.; Schroeder, Kristin R.; Moore, Kenneth C.; Donelson, John E.; Wilson, Mary E.

    2011-01-01

    Leishmania spp. protozoa are obligate intracellular parasites that replicate in macrophages during mammalian infection. Efficient phagocytosis and survival in macrophages are important determinants of parasite virulence. Macrophage lines differ dramatically in their ability to sustain intracellular Leishmania infantum chagasi (Lic). We report that the U937 monocytic cell line supported the intracellular replication and cell-to-cell spread of Lic during 72 hours after parasite addition, whereas primary human monocyte-derived macrophages (MDMs) did not. Electron microscopy and live cell imaging illustrated that Lic promastigotes anchored to MDMs via their anterior ends and were engulfed through symmetrical pseudopods. In contrast, U937 cells bound Lic in diverse orientations, and extended membrane lamellae to reorient and internalize parasites through coiling phagocytosis. Lic associated tightly with the parasitophorous vacuole (PV) membrane in both cell types. PVs fused with LAMP-1-expressing compartments 24 hours after phagocytosis by MDMs, whereas U937 cell PVs remained LAMP-1 negative. The expression of one phagocytic receptor (CR3) was higher in MDMs than U937 cells, leading us to speculate that parasite uptake proceeds through dissimilar pathways between these cells. We hypothesize that the mechanism of phagocytosis differs between primary versus immortalized human macrophage cells, with corresponding differences in the subsequent intracellular fate of the parasite. PMID:21723411

  5. Concentration-dependent effect of fibrinogen on IgG-specific antigen binding and phagocytosis.

    PubMed

    Boehm, Tobias Konrad; Sojar, Hakimuddin; Denardin, Ernesto

    2010-01-01

    In this paper, we aim to characterize fibrinogen-IgG interactions, and explore how fibrinogen alters IgG-mediated phagocytosis. Using enzyme-linked binding assays, we found that fibrinogen binding to IgG is optimized for surfaces coated with high levels of IgG. Using a similar method, we have shown that for an antigen unable to specifically bind fibrinogen, fibrinogen enhances binding of antibodies towards that antigen. For binding of IgG antibodies to cells expressing Fc receptors, we found a bimodal binding response, where low levels of fibrinogen enhance binding of antibody to Fc receptors and high levels reduce it. This corresponds to a bimodal effect on phagocytosis of IgG-coated particles, which is inhibited in the presence of excess IgG during coating of the particles with antibodies and fibrinogen. We conclude that fibrinogen can modulate phagocytosis of IgG-coated particles in vitro by changing IgG binding behavior, and that high fibrinogen levels could negatively affect phagocytosis.

  6. Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes.

    PubMed

    Kampen, Annette H; Tollersrud, Tore; Larsen, Stig; Roth, James A; Frank, Dagmar E; Lund, Arve

    2004-01-01

    Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.

  7. High Fc Density Particles Result in Binary Complement Activation but Tunable Macrophage Phagocytosis

    NASA Astrophysics Data System (ADS)

    Sulchek, Todd; Pacheco, Patricia; White, David

    2014-03-01

    Macrophage phagocytosis and complement system activation represent two key components of the immune system and both can be activated through the presentation of multiple Fc domains of IgG antibodies. We have created functionalized micro- and nanoparticles with various densities of Fc domains to understand the modulation of the immune system for eventual use as a novel immunomodulation platform. Phagocytosis assays were carried out by adding functionalized particles to macrophage cells and quantitatively determined using fluorescent microscopy and flow cytometry. Complement system activation by the functionalized particles in human serum was quantified with an enzyme immunoassay. Our phagocytosis assay revealed a strong dependence on particle size and Fc density. For small particles, as the Fc density increased, the number of particles phagocytosed also increased. Large particles were phagocytosed at significantly lower levels and showed no dependency on Fc density. Complement was successfully activated at levels comparable to positive controls for small particles at high Fc densities. However at low Fc densities, there is a significant decrease in complement activation. This result suggests a binary response for complement system activation with a threshold density for successful activation. Therefore, varying the Fc density on micro/nanoparticles resulted in a tunable response in macrophage phagocytosis while a more binary response for complement activation.

  8. Circadian regulation of intestinal lipid absorption by apolipoprotein AIV involves forkhead transcription factors A2 and O1 and microsomal triglyceride transfer protein.

    PubMed

    Pan, Xiaoyue; Munshi, Mohamed Khalid; Iqbal, Jahangir; Queiroz, Joyce; Sirwi, Alaa Ahmed; Shah, Shrenik; Younus, Abdullah; Hussain, M Mahmood

    2013-07-12

    We have shown previously that Clock, microsomal triglyceride transfer protein (MTP), and nocturnin are involved in the circadian regulation of intestinal lipid absorption. Here, we clarified the role of apolipoprotein AIV (apoAIV) in the diurnal regulation of plasma lipids and intestinal lipid absorption in mice. Plasma triglyceride in apoAIV(-/-) mice showed diurnal variations similar to apoAIV(+/+) mice; however, the increases in plasma triglyceride at night were significantly lower in these mice. ApoAIV(-/-) mice absorbed fewer lipids at night and showed blunted response to daytime feeding. To explain reasons for these lower responses, we measured MTP expression; intestinal MTP was low at night, and its induction after food entrainment was less in apoAIV(-/-) mice. Conversely, apoAIV overexpression increased MTP mRNA in hepatoma cells, indicating transcriptional regulation. Mechanistic studies revealed that sequences between -204/-775 bp in the MTP promoter respond to apoAIV and that apoAIV enhances expression of FoxA2 and FoxO1 transcription factors and their binding to the identified cis elements in the MTP promoter at night. Knockdown of FoxA2 and FoxO1 abolished apoAIV-mediated MTP induction. Similarly, knockdown of apoAIV in differentiated Caco-2 cells reduced MTP, FoxA2, and FoxO1 mRNA levels, cellular MTP activity, and media apoB. Moreover, FoxA2 and FoxO1 expression showed diurnal variations, and their expression was significantly lower in apoAIV(-/-) mice. These data indicate that apoAIV modulates diurnal changes in lipid absorption by regulating forkhead transcription factors and MTP and that inhibition of apoAIV expression might reduce plasma lipids.

  9. Up-regulation of ribosome biogenesis by MIR196A2 genetic variation promotes endometriosis development and progression

    PubMed Central

    Yang, Ching-Wen; Chang, Hui-Wen; Lu, Cheng-Chan; Chen, Chih-Mei; Chan, Carmen; Chung, Ching; Tseng, Chun-Cheng; Hwang, Tritium; Sheu, Jim Jinn-Chyuan; Tsai, Fuu-Jen

    2016-01-01

    Aberrant miRNA expression has been reported in endometriosis and miRNA gene polymorphisms have been linked to cancer. Because certain ovarian cancers arise from endometriosis, we genotyped seven cancer-related miRNA single nucleotide polymorphisms (MiRSNPs) to investigate their possible roles in endometriosis. Genetic variants in MIR196A2 (rs11614913) and MIR100 (rs1834306) were found to be associated with endometriosis development and related clinical phenotypes, such as infertility and pain. Downstream analysis of the MIR196A2 risk allele revealed upregulation of rRNA editing and protein synthesis genes, suggesting hyper-activation of ribosome biogenesis as a driving force for endometriosis progression. Clinical studies confirmed higher levels of small nucleolar RNAs and ribosomal proteins in atypical endometriosis lesions, and this was more pronounced in the associated ovarian clear cell carcinomas. Treating ovarian clear cells with CX5461, an RNA polymerase I inhibitor, suppressed cell growth and mobility followed by cell cycle arrest at G2/M stage and apoptosis. Our study thus uncovered a novel tumorigenesis pathway triggered by the cancer-related MIR196A2 risk allele during endometriosis development and progression. We suggest that anti-RNA polymerase I therapy may be efficacious for treating endometriosis and associated malignancies. PMID:27741504

  10. Overload training inhibits phagocytosis and ROS generation of peritoneal macrophages: role of IGF-1 and MGF.

    PubMed

    Xiao, Weihua; Chen, Peijie; Wang, Ru; Dong, Jingmei

    2013-01-01

    We tested the hypothesis that overload training inhibits the phagocytosis and the reactive oxygen species (ROS) generation of peritoneal macrophages (Mϕs), and that insulin-like growth factor-1(IGF-1) and mechano-growth factor (MGF) produced by macrophages may contribute to this process. Rats were randomized to two groups, sedentary control group (n = 10) and overload training group (n = 10). The rats of overload training group were subjected to 11 weeks of experimental training protocol. Blood sample was used to determine the content of hemoglobin, testosterone, and corticosterone. The phagocytosis and the ROS generation of Mϕs were measured by the uptake of neutral red and the flow cytometry, respectively. IGF-1 and MGF mRNA levels in Mϕs were determined by real-time PCR. In addition, we evaluated the effects of IGF-1 and MGF peptide on phagocytosis and ROS generation of Mϕs in vitro. The data showed that overload training significantly decreased the body weight (19.3 %, P < 0.01), the hemoglobin (13.5 %, P < 0.01), the testosterone (55.3 %, P < 0.01) and the corticosterone (40.6 %, P < 0.01) in blood. Moreover, overload training significantly decreased the phagocytosis (27 %, P < 0.05) and the ROS generation (35 %, P < 0.01) of Mϕs. IGF-1 and MGF mRNA levels in Mϕs from overload training group increased significantly compared with the control group (21-fold and 92-fold, respectively; P < 0.01). In vitro experiments showed that IGF-1 had no significant effect on the phagocytosis and the ROS generation of Mϕs. Unlike IGF-1, MGF peptide impaired the phagocytosis of Mϕs in dose-independent manner. In addition, MGF peptide of some concentrations (i.e., 1, 10, 50, 100 ng/ml) significantly inhibited the ROS generation of Mϕs. These results suggest that overload training inhibits the phagocytosis and the ROS generation of peritoneal macrophages, and that MGF produced by macrophages may play a key role in this process. This may represent a novel mechanism of

  11. Photobiomodulation with 670 nm light increased phagocytosis in human retinal pigment epithelial cells

    PubMed Central

    Fuma, Shinichiro; Murase, Hiromi; Kuse, Yoshiki; Tsuruma, Kazuhiro; Shimazawa, Masamitsu

    2015-01-01

    Purpose Photobiomodulation is the treatment with light in the far-red to near-infrared region of the spectrum and has been reported to have beneficial effects in various animal models of disease, including an age-related macular degeneration (AMD) mouse model. Previous reports have suggested that phagocytosis is reduced by age-related increased oxidative stress in AMD. Therefore, we investigated whether photobiomodulation improves phagocytosis caused by oxidative stress in the human retinal pigment epithelial (ARPE-19) cell line. Methods ARPE-19 cells and human primary retinal pigment epithelium (hRPE) cells were incubated and irradiated with near-infrared light (670 nm LED light, 2,500 lx, twice a day, 250 s/per time) for 4 d. Next, hydrogen peroxide (H2O2) and photoreceptor outer segments (POS) labeled using a pH-sensitive fluorescent dye were added to the cell culture, and phagocytosis was evaluated by measuring the fluorescence intensity. Furthermore, cell death was observed by double staining with Hoechst33342 and propidium iodide after photobiomodulation. CM-H2DCFDA, JC-1 dye, and CCK-8 were added to the cell culture to investigate the reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell viability, respectively. We also investigated the expression of phagocytosis-related proteins, such as focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK). Results Oxidative stress inhibited phagocytosis, and photobiomodulation increased the oxidative stress-induced hypoactivity of phagocytosis in ARPE-19 cells and hRPE cells. Furthermore, H2O2 and photobiomodulation did not affect cell death in this experimental condition. Photobiomodulation reduced ROS production but did not affect cell viability or mitochondrial membrane potential. The expression of phosphorylated MerTK increased, but phosphorylated FAK was not affected by photobiomodulation. Conclusions These findings indicate that near-infrared light photobiomodulation (670 nm) may

  12. Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis

    PubMed Central

    Ludwig, Leif S.; Cho, Hyunjii; Wakabayashi, Aoi; Eng, Jennifer C.; Ulirsch, Jacob C.; Fleming, Mark D.; Lodish, Harvey F.; Sankaran, Vijay G.

    2015-01-01

    Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume, MCV). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into h