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  1. Targeting the Fc receptor in autoimmune disease

    PubMed Central

    Li, Xinrui; Kimberly, Robert P

    2014-01-01

    Introduction The Fc receptors and their interaction with immunoglobulin and innate immune opsonins such as CRP are key players in humoral and cellular immune responses. As the effector mechanism for some therapeutic monoclonal antibodies and often a contributor to the pathogenesis and progression of autoimmunity, FcRs are promising targets for treating autoimmune diseases. Areas covered This review discusses the nature of different Fc receptors and the various mechanisms of their involvement in initiating and modulating immunocyte functions and their biological consequences. It describes a range of current strategies in targeting Fc receptors and manipulating their interaction with specific ligands while presenting the pros and cons of these approaches. This review also discusses potential new strategies including regulation of FcR expression and receptor cross-talk. Expert opinion Fc receptors are appealing targets in the treatment of inflammatory autoimmune diseases. However, there are still knowledge limitations and technical challenges, the most important being a better understanding of the individual roles of each of the Fc receptors and enhancement of the specificity in targeting particular cell types and specific Fc receptors. PMID:24521454

  2. Fc receptors and immunity to parasites.

    PubMed

    Pleass, R J; Woof, J M

    2001-11-01

    Fc receptors (FcRs) are crucial in the immune system; they mediate a plethora of biological functions as diverse as antigen presentation, phagocytosis, cytotoxicity, induction of inflammatory cascades and modulation of immune responses. Parasites, in order to survive in the immunocompetent host, have devised ingenious methods to subvert this important aspect of the immune response. This article discusses the current thinking on FcRs, their role in immunity to parasites, and immune evasion strategies employed by parasites in their attempt to neutralize the important immune defense mechanisms mediated by these molecules. PMID:11872400

  3. Neonatal Fc Receptor Mediates Internalization of Fc in Transfected Human Endothelial Cells

    PubMed Central

    Goebl, Nancy A.; Babbey, Clifford M.; Datta-Mannan, Amita; Witcher, Derrick R.; Wroblewski, Victor J.

    2008-01-01

    The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels. PMID:18843053

  4. Tissue expression profile of human neonatal Fc receptor (FcRn) in Tg32 transgenic mice.

    PubMed

    Fan, Yao-Yun; Avery, Lindsay B; Wang, Mengmeng; O'Hara, Denise M; Leung, Sheldon; Neubert, Hendrik

    2016-07-01

    The neonatal Fc receptor (FcRn) is a homeostatic receptor responsible for prolonging immunoglobulin G (IgG) half-life by protecting it from lysosomal degradation and recycling it to systemic circulation. Tissue-specific FcRn expression is a critical parameter in physiologically-based pharmacokinetic (PBPK) modeling for translational pharmacokinetics of Fc-containing biotherapeutics. Using online peptide immuno-affinity chromatography coupled with high resolution mass spectrometry, we established a quantitative FcRn tissue protein expression profile in human FcRn (hFcRn) transgenic mice, Tg32 homozygous and hemizygous strains. The concentration of hFcRn across 14 tissues ranged from 3.5 to 111.2 pmole per gram of tissue. Our hFcRn quantification data from Tg32 mice will enable a more refined PBPK model to improve the accuracy of human PK predictions for Fc-containing biotherapeutics. PMID:27104806

  5. Identity of the elusive IgM Fc receptor (FcμR) in humans

    PubMed Central

    Oka, Satoshi; Kubagawa, Yoshiki; Torii, Ikuko; Takayama, Eiji; Kang, Dong-Won; Gartland, G. Larry; Bertoli, Luigi F.; Mori, Hiromi; Takatsu, Hiroyuki; Kitamura, Toshio; Ohno, Hiroshi; Wang, Ji-Yang

    2009-01-01

    Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcμR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcμR in human B-lineage cDNA libraries. FcμR is defined as a transmembrane sialoglycoprotein of ∼60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcα/μR) but exhibits an exclusive Fcμ-binding specificity. The cytoplasmic tail of FcμR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcμR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcμR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcμR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcμR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis. PMID:19858324

  6. Harnessing Fc receptor biology in the design of therapeutic antibodies.

    PubMed

    Sondermann, Peter; Szymkowski, David E

    2016-06-01

    The antibody Fc domain engages the small family of Fc receptors, expressed on cells of the immune system and beyond, to stimulate a rich diversity of positive and negative cell-mediated effector functions. The emergence of monoclonal antibodies for the treatment of various pathologic conditions has provided additional insights into Fc receptor biology, and has suggested new strategies to exploit Fc receptor interactions to create improved therapeutics. While most therapeutic IgGs approved to date have retained a native IgG Fc domain, the knowledge gained over the last decades has provided the opportunity to design tailored and more efficacious immunotherapies exhibiting fewer side effects and longer half-life. This review summarizes recent advances made in the design of biologics that modulate or exploit Fc receptor-IgG interactions, and describes innovative drugs currently under investigation in clinical trials that have been precisely tuned to achieve a desired therapeutic effect. PMID:27038127

  7. The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy

    PubMed Central

    Sockolosky, Jonathan T.; Szoka, Francis C.

    2015-01-01

    Immunoglobulin G (IgG)-based drugs are arguably the most successful class of protein therapeutics due in part to their remarkably long blood circulation. This arises from IgG interaction with the neonatal Fc receptor, FcRn. FcRn is the central regulator of IgG and albumin homeostasis throughout life and is increasingly being recognized as an important player in autoimmune disease, mucosal immunity, and tumor immune surveillance. Various engineering approaches that hijack or disrupt the FcRn-mediated transport pathway have been devised to develop long-lasting and non-invasive protein therapeutics, protein subunit vaccines, and therapeutics for treatment of autoimmune and infectious disease. In this review, we highlight the diverse biological functions of FcRn, emerging therapeutic opportunities, as well as the associated challenges of targeting FcRn for drug delivery and disease therapy. PMID:25703189

  8. The Long Elusive IgM Fc Receptor, FcμR

    PubMed Central

    Kubagawa, Hiromi; Oka, Satoshi; Kubagawa, Yoshiki; Torii, Ikuko; Takayama, Eiji; Kang, Dong-Won; Jones, Dewitt; Nishida, Naonori; Miyawaki, Toshio; Bertoli, Luigi F.; Sanders, Sheila K.; Honjo, Kazuhito

    2014-01-01

    IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both preimmune “natural” and antigen-induced “immune” IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcμR). We have recently identified a bona fide FcμR in both humans and mice. In this article we briefly review what we have learned so far about FcμR. PMID:24793544

  9. Human antibody-Fc receptor interactions illuminated by crystal structures.

    PubMed

    Woof, Jenny M; Burton, Dennis R

    2004-02-01

    Immunoglobulins couple the recognition of invading pathogens with the triggering of potent effector mechanisms for pathogen elimination. Different immunoglobulin classes trigger different effector mechanisms through interaction of immunoglobulin Fc regions with specific Fc receptors (FcRs) on immune cells. Here, we review the structural information that is emerging on three human immunoglobulin classes and their FcRs. New insights are provided, including an understanding of the antibody conformational adjustments that are required to bring effector cell and target cell membranes sufficiently close for efficient killing and signal transduction to occur. The results might also open up new possibilities for the design of therapeutic antibodies. PMID:15040582

  10. The old but new IgM Fc receptor (FcμR).

    PubMed

    Kubagawa, Hiromi; Kubagawa, Yoshiki; Jones, Dewitt; Nasti, Tahseen H; Walter, Mark R; Honjo, Kazuhito

    2014-01-01

    IgM is the first Ig isotype to appear during phylogeny, ontogeny and the immune response. The importance of both pre-immune "natural" and antigen-induced "immune" IgM antibodies in immune responses to pathogens and self-antigens has been established by studies of mutant mice deficient in IgM secretion. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed, but fail to fully account for the IgM-mediated immune protection and regulation of immune responses. Particularly, the role of the Fc receptor for IgM (FcμR) in such effector functions has not been explored until recently. We have identified an authentic FcμR in humans using a functional cloning strategy and subsequently in mice by RT-PCR and describe here its salient features and the immunological consequences of FcμR deficiency in mice. Since the FcμR we cloned was identical to Toso or Fas inhibitory molecule 3 (FAIM3), there have been spirited debates regarding the real function of FcμR/Toso/FAIM3 and we will also comment on this topic. PMID:25116093

  11. Fc receptors for immunoglobulins and their appearance during vertebrate evolution.

    PubMed

    Akula, Srinivas; Mohammadamin, Sayran; Hellman, Lars

    2014-01-01

    Receptors interacting with the constant domain of immunoglobulins (Igs) have a number of important functions in vertebrates. They facilitate phagocytosis by opsonization, are key components in antibody-dependent cellular cytotoxicity as well as activating cells to release granules. In mammals, four major types of classical Fc receptors (FcRs) for IgG have been identified, one high-affinity receptor for IgE, one for both IgM and IgA, one for IgM and one for IgA. All of these receptors are related in structure and all of them, except the IgA receptor, are found in primates on chromosome 1, indicating that they originate from a common ancestor by successive gene duplications. The number of Ig isotypes has increased gradually during vertebrate evolution and this increase has likely been accompanied by a similar increase in isotype-specific receptors. To test this hypothesis we have performed a detailed bioinformatics analysis of a panel of vertebrate genomes. The first components to appear are the poly-Ig receptors (PIGRs), receptors similar to the classic FcRs in mammals, so called FcRL receptors, and the FcR γ chain. These molecules are not found in cartilagous fish and may first appear within bony fishes, indicating a major step in Fc receptor evolution at the appearance of bony fish. In contrast, the receptor for IgA is only found in placental mammals, indicating a relatively late appearance. The IgM and IgA/M receptors are first observed in the monotremes, exemplified by the platypus, indicating an appearance during early mammalian evolution. Clearly identifiable classical receptors for IgG and IgE are found only in marsupials and placental mammals, but closely related receptors are found in the platypus, indicating a second major step in Fc receptor evolution during early mammalian evolution, involving the appearance of classical IgG and IgE receptors from FcRL molecules and IgM and IgA/M receptors from PIGR. PMID:24816777

  12. Chemoenzymatic Synthesis and Fcγ Receptor Binding of Homogeneous Glycoforms of Antibody Fc Domain. Presence of a Bisecting Sugar Moiety Enhances the Affinity of Fc to FcγIIIa Receptor

    PubMed Central

    Zou, Guozhang; Ochiai, Hirofumi; Huang, Wei; Yang, Qiang; Li, Cishan; Wang, Lai-Xi

    2011-01-01

    Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on antibody’s effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substratess, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter protophormiae (Endo-A) was found to be remarkably efficient to take various modified N-glycan core oxazolines, including the bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding homogeneous Fc glycoforms. Nevertheless, neither Endo-A, nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A and EndoM-N175Q), was able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. SPR binding studies with the synthetic IgG-Fc glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could significantly enhance the binding of Fc to FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to FcγRIIIa. On the orther hand, the presence of a bisecting GlcNAc or core fucosylation had little effect on the affinity of Fc to the inhibitory Fcγ receptor, FcγRIIb. Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high-affinity of Fc to both FcγRIIIa and FcγRIIb. The synthetic homogeneous Fc

  13. Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.

    PubMed

    Isoda, Yuya; Yagi, Hirokazu; Satoh, Tadashi; Shibata-Koyama, Mami; Masuda, Kazuhiro; Satoh, Mitsuo; Kato, Koichi; Iida, Shigeru

    2015-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296 in interactions

  14. Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors

    PubMed Central

    Isoda, Yuya; Yagi, Hirokazu; Satoh, Tadashi; Shibata-Koyama, Mami; Masuda, Kazuhiro; Satoh, Mitsuo; Kato, Koichi; Iida, Shigeru

    2015-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr–296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr–296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr–296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr–296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr–296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr–296 in

  15. Characterization of the human platelet Fc sub. gamma. receptor

    SciTech Connect

    King, M.

    1988-01-01

    Thrombocytopenia is often associated with immune complex disease and may in part be due to the interaction of circulating (IgG) immune complexes with an Fc{sub {gamma}} receptor on the platelet surface. Characterization of the immune complex-platelet interaction should provide for a better understanding of the pathophysiology of immune thrombocytpenia. To this end, a ligand binding assay, employing {sup 125}I-IgG trimer, was established. Receptor expression was determined by measuring the saturable binding of radiolabeled trimer to platelets at equilibrium. Normal human platelets were observed to express 8559 {plus minus} 852 binding sites for IgG trimer with a Kd of 12.5 {plus minus} 1.7 {times} 10{sup {minus}8} M. Binding of IgG trimer to human platelets was blocked following preincubation of the cells with an anti-Fc{sub {gamma}}RII monoclonal antibody. Furthermore, this binding was ionic-strength dependent but was unaffected by the presence of Mg{sup ++} or cytochalasin B. Platelet Fc{sub {gamma}} receptor modulation was examined by assessing the effects of various physiologic and pharmacologic on the ability of platelets to bind IgG trimer. Platelet Fc{sub {gamma}} receptor expression was not affected by thrombin, ADP, or {gamma}-interferon. However, in 7/12 normal donors, treatment of platelets with dexamethasone resulted in a decrease in the number of Fc{sub {gamma}} receptors expressed.

  16. Ligand Valency Affects Transcytosis, Recycling and Intracellular Trafficking Mediated by the Neonatal Fc Receptor

    PubMed Central

    Tesar, Devin B; Tiangco, Noreen E; Bjorkman, Pamela J

    2006-01-01

    The neonatal Fc receptor (FcRn) transports IgG across epithelial cell barriers to provide maternal antibodies to offspring and serves as a protection receptor by rescuing endocytosed IgG and albumin from lysosomal degradation. Here we describe the generation of polarized Madin–Darby canine kidney (MDCK) cells expressing rat FcRn (rFcRn) to investigate the potential requirement for ligand bivalency in FcRn-mediated transport. The rFcRn-MDCK cells bind, internalize and bidirectionally transcytose the bivalent ligands IgG and Fc across polarized cell monolayers. However, they cannot be used to study FcRn-mediated transport of the monovalent ligand albumin, as we observe no specific binding, internalization or transcytosis of rat albumin. To address whether ligand bivalency is required for transport, the ability of rFcRn to transcytose and recycle wild-type Fc homodimers (wtFc; two FcRn-binding sites) and a heterodimeric Fc (hdFc; one FcRn-binding site) was compared. We show that ligand bivalency is not required for transcytosis or recycling, but that wtFc is transported more efficiently than hdFc, particularly at lower concentrations. We also demonstrate that hdFc and wtFc have different intracellular fates, with more hdFc than wtFc being trafficked to lysosomes and degraded, suggesting a role for avidity effects in FcRn-mediated IgG transport. PMID:17004319

  17. Molecular recognition of antibody (IgG) by cellular Fc receptor (FcRI).

    PubMed

    Burton, D R; Jefferis, R; Partridge, L J; Woof, J M

    1988-11-01

    Earlier studies from this and other laboratories have provided indirect evidence for the involvement of the C gamma 2 domain of human IgG in the binding of IgG to the high affinity monocyte Fc receptor (FcRI). Two approaches have been used to extend these studies and to further localize the site of interaction on human IgG. Firstly, monoclonal antibodies (MAbs) directed against different epitopes on IgG were assayed for their capacity to inhibit the binding of radiolabelled IgG to human monocytes or U937 cells. The capacity of the MAbs to interact with their respective epitopes on FcR-bound IgG was also studied using indirect radiobinding and immunofluorescence assays. Secondly, a number of IgGs from several different species and fragments of human IgGs were assayed for their ability to inhibit the binding of radiolabelled IgG to human monocytes. The amino acid sequences of those IgGs exhibiting relatively tight, intermediate or weak binding to monocyte FcRs were compared. On the basis of these studies a possible monocyte FcR-binding site on human IgG is postulated, involving the lower hinge region of IgG (residues Leu 234-Ser 239) with possible involvement of the nearby N-proximal bend and two beta-strands (Gly 316-Lys 338). PMID:2975762

  18. Platelets deficient in glycoprotein I have normal Fc receptor expression.

    PubMed

    Pfueller, S L; de Rosbo, N K; Bilston, R A

    1984-04-01

    Platelet glycoprotein I (GPI) is known to be required for the interaction of platelets with ristocetin and factor VIII:von Willebrand factor (VIII:vWf). However, its role as Fc receptor is not clear. Some studies have shown that enzymatic removal of GPI destroys the ability of platelets to react with VIII:vWf but not their ability to bind Ig G (IgG). Others have shown that IgG immune complexes which block the Fc receptor also inhibit VIII:vWf interaction with platelets. This subject has been re-examined by testing the ability of platelets with reduced amounts of GPI to aggregate and undergo the release reaction in response to stimuli which act at the platelet Fc receptor. Platelets from two patients with Bernard-Soulier syndrome, congenitally deficient in GPI, both aggregated and released 14C-serotonin normally when exposed to latex particles coated with IgG. Levels of GPI were decreased experimentally in normal platelets by treating them with chymotrypsin. Platelets treated in this manner did not aggregate or release [14C]serotonin in response to ristocetin-VIII:vWf. They did, however, both aggregate and release when incubated with heat-aggregated IgG, antigen-antibody complexes or latex particles coated with IgG. Thus the presence of GPI is not a prerequisite for platelet stimulation via the Fc receptor. PMID:6231945

  19. Neonatal Fc receptor promotes immune complex-mediated glomerular disease.

    PubMed

    Olaru, Florina; Luo, Wentian; Suleiman, Hani; St John, Patricia L; Ge, Linna; Mezo, Adam R; Shaw, Andrey S; Abrahamson, Dale R; Miner, Jeffrey H; Borza, Dorin-Bogdan

    2014-05-01

    The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG. PMID:24357670

  20. Neonatal Fc Receptor Promotes Immune Complex–Mediated Glomerular Disease

    PubMed Central

    Olaru, Florina; Luo, Wentian; Suleiman, Hani; St. John, Patricia L.; Ge, Linna; Mezo, Adam R.; Shaw, Andrey S.; Abrahamson, Dale R.; Miner, Jeffrey H.

    2014-01-01

    The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex–mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex–mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG. PMID:24357670

  1. The role of Fc receptors in HIV prevention and therapy.

    PubMed

    Boesch, Austin W; Brown, Eric P; Ackerman, Margaret E

    2015-11-01

    Over the past decade, a wealth of experimental evidence has accumulated supporting the importance of Fc receptor (FcR) ligation in antibody-mediated pathology and protection in many disease states. Here we present the diverse evidence base that has accumulated as to the importance of antibody effector functions in the setting of HIV prevention and therapy, including clinical correlates, genetic associations, viral evasion strategies, and a rapidly growing number of compelling animal model experiments. Collectively, this work identifies antibody interactions with FcR as important to both therapeutic and prophylactic strategies involving both passive and active immunity. These findings mirror those in other fields as investigators continue to work toward identifying the right antibodies and the right effectors to be present at the right sites at the right time. PMID:26497529

  2. Inhibition by synthetic peptides from human IgG Fc of OKT4 binding and Fc receptor binding

    SciTech Connect

    Gaarde, W.A.; McClurg, M.R.; Hahn, G.S.; Plummer, J.M.

    1986-03-05

    Synthetic peptides from the Fc region of human IgG were tested for the ability to inhibit IgG rosette formation, /sup 125/I-IgG binding to Fc receptors on lymphocytes, and expression of the T4 cell determinant. The Fc/sub ..gamma../ peptides inhibited IgG rosette formation and competitively inhibited both /sup 125/I-IgG binding to Fc receptors and OKT4 binding to the T4 cell determinant. Peptides containing D-amino acids did not inhibit IgG rosettes, OKT4 binding or /sup 125/I-IgG binding. OKT4 binding to T4 on MNC was inhibited by heat aggregated IgG but not by monomeric IgG. OKT3, OKT8 and OKT11 binding to their determinants was not altered by Fc/sub ..gamma../ peptides, aggregated IgG or monomeric IgG. These results suggest that T4 antigen, Fc receptor for IgG and the Fc/sub ..gamma../ peptide binding site are in close proximity on the cell surface and when occupied by their respective ligands may sterically hinder binding to other sites. Alternatively, Fc/sub ..gamma../ peptides may indirectly regulate cell surface expression of T4 and/or Fc receptors for IgG. These Fc/sub ..gamma../ peptides inhibit the MLR, inhibit antigen induced T cell proliferation and reverse animal models of autoimmune disease. The immunoregulatory activities of these peptides may be related to their selective action on T4 helper/inducer lymphocytes expressing Fc receptors.

  3. Characterization of the Rabbit Neonatal Fc Receptor (FcRn) and Analyzing the Immunophenotype of the Transgenic Rabbits That Overexpresses FcRn

    PubMed Central

    Catunda Lemos, Ana Paula; Cervenak, Judit; Bender, Balázs; Hoffmann, Orsolya Ivett; Baranyi, Mária; Kerekes, Andrea; Farkas, Anita; Bősze, Zsuzsanna; Hiripi, László; Kacskovics, Imre

    2012-01-01

    The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies. PMID:22247762

  4. Characterization of the ligand binding site of the bovine IgA Fc receptor (bFc alpha R).

    PubMed

    Morton, H Craig; Pleass, Richard J; Woof, Jenny M; Brandtzaeg, Per

    2004-12-24

    Recently, we identified a bovine IgA Fc receptor (bFc alpha R), which shows high homology to the human myeloid Fc alpha R, CD89. IgA binding has previously been shown to depend on several specific residues located in the B-C and F-G loops of the membrane-distal extracellular domain 1 of CD89. To compare the ligand binding properties of these two Fc alpha Rs, we have mapped the IgA binding site of bFc alpha R. We show that, in common with CD89, Tyr-35 in the B-C loop is essential for IgA binding. However, in contrast to earlier observations on CD89, mutation of residues in the F-G loop did not significantly inhibit IgA binding. PMID:15485844

  5. Toll-like receptor 2 ligands regulate monocyte Fcγ receptor expression and function.

    PubMed

    Shah, Prexy; Fatehchand, Kavin; Patel, Hemal; Fang, Huiqing; Justiniano, Steven E; Mo, Xiaokui; Jarjoura, David; Tridandapani, Susheela; Butchar, Jonathan P

    2013-04-26

    Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy. PMID:23504312

  6. Fc Gamma Receptor IIIB (FcγRIIIB) Polymorphisms Are Associated with Clinical Malaria in Ghanaian Children

    PubMed Central

    Adu, Bright; Dodoo, Daniel; Adukpo, Selorme; Hedley, Paula L.; Arthur, Fareed K. N.; Gerds, Thomas A.; Larsen, Severin O.; Christiansen, Michael; Theisen, Michael

    2012-01-01

    Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-FcγRIIA and FcγRIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding FcγRIIA and FcγRIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding FcγRIIA p.H166R and FcγRIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. FcγRIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between FcγRIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: p = 0.0061; recessive: p = 0.097; dominant: p = 0.0076) of inheritance. The FcγRIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p = 0.049). The FcγRIIIB-NA2*03 allotype (CTGCGA), a variant of the classical FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p = 0.0092). The present study is the first to report an association between a variant of FcγRIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical FcγRIIIB allotypes. This

  7. Divergent roles for Fc receptors and complement in vivo.

    PubMed

    Ravetch, J V; Clynes, R A

    1998-01-01

    Recent results obtained in mice deficient in either FcRs or complement have revealed distinct functions for these two classes of molecules. While each is capable of interacting with antibodies or immune complexes, the two systems mediate distinct biological effector responses. Complement-deficient mice are unable to mediate innate immune responses to several bacterial pathogens and bacterial toxins, yet respond normally to the presence of cytotoxic antibodies and pathogenic immune complexes. In contrast, FcR-deficient mice display no defects in innate immunity or susceptibility to a variety of pathogens, yet they are unable to mediate inflammatory responses to cytotoxic IgG antibodies or IgG immune complexes, despite the presence of a normal complement system. These results lead to the surprising conclusion that these two systems have evolved distinct functions in host immunity, with complement and its receptors mediating the interaction of natural antibodies (IgM) with pathogens to effect protection, while FcRs couple the interaction of IgG antibodies to effector cells to trigger inflammatory sequelae. These results necessitate a fundamental revision of the role of these antibody-binding systems in the immune response. PMID:9597136

  8. Fc receptor like 3 in Chinese patients of Han nationality with Guillain-Barré syndrome.

    PubMed

    Sang, Daoqian; Chen, Qiming; Liu, Xiaolin; Qu, Hongdang; Wei, Daoxiang; Yin, Liang; Zhang, Lina

    2012-05-15

    Fc receptor like 3 gene (FcRL3) has been associated with some autoimmune diseases. Here, its role in Guillain-Barré syndrome (GBS) was evaluated by studying nine FcRL3 gene SNPs in a Chinese cohort of GBS patients. The frequencies of FcRL3-3-169C, FcRL3-6 intron3A, and FcRL3-8 exon15G alleles were significantly increased in GBS patients compared with healthy controls. The frequency of FcRL3-1→9 CCTGGAGAA haplotype was significantly increased, and the frequencies of FcRL3-1→9 CCTACAAAA,CCCACGAAA, and CCTGCGGAA haplotypes were significantly decreased compared with healthy controls. These results suggest that FcRL3 is associated with GBS incidence. PMID:22458979

  9. Human CD4+ T-Cells: A Role for Low-Affinity Fc Receptors

    PubMed Central

    Chauhan, Anil K.

    2016-01-01

    Both lymphoid and myeloid cells express Fc receptors (FcRs). Low-affinity FcRs engage circulating immune complexes, which results in the cellular activation and pro-inflammatory cytokine production. FcRs participate in the internalization, transport, and/or recycling of antibodies and antigens. Cytosolic FcRs also route these proteins to proteasomes and antigen-presentation pathways. Non-activated CD4+ T-cells do not express FcRs. Once activated, naive CD4+ T-cells express FcγRIIIa, which, upon IC ligation, provide a costimulatory signal for the differentiation of these cells into effector cell population. FcγRIIIa present on CD4+ T-cell membrane could internalize nucleic acid-containing ICs and elicit a cross-talk with toll-like receptors. FcγRIIIa common γ-chain forms a heterodimer with the ζ-chain of T-cell receptor complex, suggesting a synergistic role for these receptors. This review first summarizes our current understanding of FcRs on CD4+ T-cells. Thereafter, I will attempt to correlate the findings from the recent literature on FcRs and propose a role for these receptors in modulating adaptive immune responses via TLR signaling, nucleic acid sensing, and epigenetic changes in CD4+ T-cells. PMID:27313579

  10. Expression of murine Fc receptors for IgG.

    PubMed

    Schreiber, R E; Buku, A; Unkeless, J C

    1990-06-15

    There are two distinct genes that encode murine low affinity Fc gamma RII, murine Fc gamma RII alpha, and murine Fc gamma RII beta, which are transcribed in specific cell lineages. Fc gamma RII alpha transcripts are present in macrophages, NK cells, and mesangial cells; Fc gamma RII beta transcripts are expressed in Fc gamma R-bearing B cells, T cells, and macrophages. We have devised a sandwich ELISA to quantify the expression of Fc gamma RII alpha protein. The ELISA is specific for Fc gamma RII alpha, and does not detect the closely related Fc gamma RII beta protein. Upon stimulation with IFN-gamma the Fc gamma RII beta- macrophage cell line J774a expressed a twelvefold enhanced level of Fc gamma RII alpha protein. Peritoneal macrophages synthesized varying amounts of Fc gamma RII alpha. High levels of Fc gamma RII alpha were observed in resident and thioglycollate-elicited peritoneal macrophages, but no Fc gamma RII alpha was detected in Bacillus Calmette Guérin-elicited macrophages. J774a cells stimulated with rIL-6 bound approximately twice as much anti-Fc gamma RII mAb 2.4G2 IgG as did unstimulated controls. However, the Fc gamma RII alpha-specific ELISA showed no change in the amount of Fc gamma RII alpha expressed. A probe encompassing the extracellular coding sequence of Fc gamma RII beta hybridized to two distinct transcripts that were elevated in rIL-6-stimulated J774a cells. One of these transcripts had the same mobility in electrophoresis as Fc gamma RII alpha mRNA and hybridized to an Fc gamma RII alpha-specific probe, whereas the other transcript was larger and did not hybridize to probes specific for either Fc gamma RII alpha or Fc gamma RII beta. Moreover, we confirmed, with an Fc gamma RII beta-specific probe, that J774a cells do not make Fc gamma RII beta mRNA. Thus, the larger transcript appears to encode a novel Fc gamma RII. We suggest that the increased level of binding of the anti-Fc gamma RII mAb 2.4G2 to rIL-6-induced cells represents

  11. Crystal Structure of the HSV-1 Fc Receptor Bound to Fc Reveals a Mechanism for Antibody Bipolar Bridging

    SciTech Connect

    Sprague, E.R.; Wang, C.; Baker, D.; Bjorkman, P.J.; /Caltech /Howard Hughes Med. Inst.

    2007-08-08

    Herpes simplex virus type-1 expresses a heterodimeric Fc receptor, gE-gI, on the surfaces of virions and infected cells that binds the Fc region of host immunoglobulin G and is implicated in the cell-to-cell spread of virus. gE-gI binds immunoglobulin G at the basic pH of the cell surface and releases it at the acidic pH of lysosomes, consistent with a role in facilitating the degradation of antiviral antibodies. Here we identify the C-terminal domain of the gE ectodomain (CgE) as the minimal Fc-binding domain and present a 1.78-{angstrom} CgE structure. A 5-{angstrom} gE-gI/Fc crystal structure, which was independently verified by a theoretical prediction method, reveals that CgE binds Fc at the C{sub H}2-C{sub H}3 interface, the binding site for several mammalian and bacterial Fc-binding proteins. The structure identifies interface histidines that may confer pH-dependent binding and regions of CgE implicated in cell-to-cell spread of virus. The ternary organization of the gE-gI/Fc complex is compatible with antibody bipolar bridging, which can interfere with the antiviral immune response.

  12. Identification of a family of Fc receptor homologs with preferential B cell expression

    PubMed Central

    Davis, Randall S.; Wang, Yui-Hsi; Kubagawa, Hiromi; Cooper, Max D.

    2001-01-01

    Investigation of human genome sequences with a consensus sequence derived from receptors for the Fc region of Igs (FcR) led to the identification of a subfamily of five Ig superfamily members that we term the Fc receptor homologs (FcRHs). The closely linked FcRH genes are located in a chromosome 1q21 region in the midst of previously recognized FcR genes. This report focuses on the FcRH1, FcRH2, and FcRH3 members of this gene family. Their cDNAs encode type I transmembrane glycoproteins with 3–6 Ig-like extracellular domains and cytoplasmic domains containing consensus immunoreceptor tyrosine-based activating and/or inhibitory signaling motifs. The five FcRH genes are structurally related, and their protein products share 28–60% extracellular identity with each other. They also share 15–31% identity with their closest FcR relatives. The FcRH genes are expressed primarily, although not exclusively, by mature B lineage cells. Their conserved structural features, patterns of cellular expression, and the inhibitory and activating signaling potential of their transmembrane protein products suggest that the members of this FcRH multigene family may serve important regulatory roles in normal and neoplastic B cell development. PMID:11493702

  13. Computational modeling of the Fc αRI receptor binding in the Fc α domain of the human antibody IgA: Normal Modes Analysis (NMA) study

    NASA Astrophysics Data System (ADS)

    Jayasinghe, Manori; Posgai, Monica; Tonddast-Navaei, Sam; Ibrahim, George; Stan, George; Herr, Andrew; George Stan Group Collaboration; Herr's Group Team

    2014-03-01

    Fc αRI receptor binding in the Fc α domain of the antibody IgA triggers immune effector responses such as phagocytosis and antibody-dependent cell-mediated cytotoxicity in eukaryotic cells. Fc α is a dimer of heavy chains of the IgA antibody and each Fc α heavy chain which consisted of two immunoglobulin constant domains, CH2 and CH3, can bind one Fc αRI molecule at the CH2-CH3 interface forming a 2:1 stoichiometry. Experimental evidences confirmed that Fc αRI binding to the Fc α CH2-CH3 junction altered the kinetics of HAA lectin binding at the distant IgA1 hinge. Our focus in this research was to understand the conformational changes and the network of residues which co-ordinate the receptor binding dynamics of the Fc α dimer complex. Structure-based elastic network modeling was used to compute normal modes of distinct Fc α configurations. Asymmetric and un-liganded Fc α configurations were obtained from the high resolution crystal structure of Fc α-Fc αRI 2:1 symmetric complex of PDB ID 1OW0. Our findings confirmed that Fc αRI binding, either in asymmetric or symmetric complex with Fc α, propagated long-range conformational changes across the Fc domains, potentially also impacting the distant IgA1 hinge.

  14. Modulation of tumor growth by inhibitory Fcγ receptor expressed by human melanoma cells

    PubMed Central

    Cassard, Lydie; Cohen-Solal, Joël F.G.; Galinha, Annie; Sastre-Garau, Xavier; Mathiot, Claire; Galon, Jérôme; Dorval, Thierry; Bernheim, Alain; Fridman, Wolf H.; Sautès-Fridman, Catherine

    2002-01-01

    The efficacy of anti-tumor IgG reflects the balance between opposing signals mediated by activating and inhibitory Fcγ receptors (FcγRs) expressed by effector cells. Here, we show that human malignant melanoma cells express the inhibitory low-affinity Fcγ receptor FcγRIIB1 in 40% of tested metastases. When melanoma cells were grafted in nude mice, a profound inhibition of FcγRIIB1 tumor growth that required the intracytoplasmic region of the receptor was observed. IgG immune complexes (ICs) may be required for this inhibition, since sera from nude mice bearing tumors contained IgG that decreased the proliferation of FcγRIIB1-positive cells in vitro, and tumor development of FcγRIIB1-positive melanoma lines was not inhibited in antibody-defective severe combined immunodeficiency (SCID) mice. Passive immunization of SCID mice with anti–ganglioside GD2 antibody resulted in significant inhibition of growth of FcγRIIB1-positive tumors in an intracytoplasmic-dependent manner. Altogether, these data suggest that human melanoma cells express biologically active inhibitory FcγRIIB1, which regulates their development upon direct interaction with anti-tumor antibodies. Therefore, FcγR expression on human tumors may be one component of the efficacy of antibody-mediated therapies, and FcγR-positive tumors could be the most sensitive candidates for such treatments. PMID:12438452

  15. Expression of soluble human Neonatal Fc-receptor (FcRn) in Escherichia coli through modification of growth environment.

    PubMed

    Ng, Woei Kean; Lim, Theam Soon; Lai, Ngit Shin

    2016-11-01

    Neonatal Fc-receptor (FcRn) with its affinity to immunoglobulin G (IgG) has been the subject of many pharmacokinetic studies in the past century. This protein is well known for its unique feature in maintaining the circulating IgG from degradation in blood plasma. FcRn is formed by non-covalent association between the α-chain with the β-2-microglobulin (β2m). Many studies have been conducted to produce FcRn in the laboratory, mainly using mammalian tissue culture as host for recombinant protein expression. In this study, we demonstrate a novel strategy to express the α-chain of FcRn using Escherichia coli as the expression host. The expression vector that carries the cDNA of the α-chain was transformed into expression host, Rosetta-gami 2 strain for inducible expression. The bacterial culture was grown in a modified growth medium which constitutes of terrific broth, sodium chloride (NaCl), glucose and betaine. A brief heat shock at 45 °C was carried out after induction, before the temperature for expression was reduced to 22 °C and grown for 16 h. The soluble form of the α-chain of FcRn expressed was tested in the ELISA and dot blot immunoassay to confirm its native functionality. The results implied that the α-chain of FcRn expressed using this method is functional and retains its pH-dependent affinity to IgG. Our study significantly suggests that the activity of human FcRn remain active and functional in the absence of β2m. PMID:27412717

  16. Spontaneous expression of a low affinity Fc receptor for IgA (Fc alpha R) on human B cell lines.

    PubMed Central

    Millet, I; Briere, F; Vincent, C; Rousset, F; Andreoni, C; De Vries, J E; Revillard, J P

    1989-01-01

    Expression of receptors for IgA (Fc alpha Rs) was investigated on a panel of 35 human B cell lines by labelling with human secretory IgA (0.5 mg/ml) and flow cytometry analysis after staining with fluoresceinated goat anti-human secretory component and/or anti-alpha chain F(ab')2 fragments. Receptors for IgA could be demonstrated on one out of nine Burkitt's lymphoma cell lines, three out of five myeloma cell lines and five out of 21 lymphoblastoid cell lines. The percentage of Fc alpha R-positive cells within the same B cell line varied upon repeated examination. Human dimeric IgA1 lambda myeloma protein revealed the same number of IgA receptor positive cells as did secretory IgA, whereas monomeric IgA did not bind to Fc alpha R. Detection of Fc alpha R was not inhibited when the tests were carried out in the presence of human dimeric IgG, IgM, asialo-orosomucoid, and secretory component but it was abrogated by pre-treatment of the cells with trypsin. The binding characteristics of Fc alpha Rs were studied on the myeloma cell line Esteve, using 125I-labelled human dimeric IgA and secretory IgA. The binding was dose-dependent with rapid kinetics and specific inhibition by unlabelled secretory IgA. Scatchard plot analysis resulted in an equilibrium constant K ranging from 3.2 to 4.7 x 10(6) M/l. No correlation was observed between Fc alpha R expression and differentiation stage, monoclonality, polyclonality of the cell lines, or Ig class produced by the B cells. PMID:2788048

  17. Dimeric FcγR Ectodomains as Probes of the Fc Receptor Function of Anti-Influenza Virus IgG.

    PubMed

    Wines, Bruce D; Vanderven, Hillary A; Esparon, Sandra E; Kristensen, Anne B; Kent, Stephen J; Hogarth, P Mark

    2016-08-15

    Ab-dependent cellular cytotoxicity, phagocytosis, and Ag presentation are key mechanisms of action of Abs arising in vaccine or naturally acquired immunity, as well of therapeutic mAbs. Cells expressing the low-affinity FcγRs (FcγRII or CD32 and FcγRIII or CD16) are activated for these functions when receptors are aggregated following the binding of IgG-opsonized targets. Despite the diversity of the Fc receptor proteins, IgG ligands, and potential responding cell types, the induction of all FcγR-mediated responses by opsonized targets requires the presentation of multiple Fc regions in close proximity to each other. We demonstrated that such "near-neighbor" Fc regions can be detected using defined recombinant soluble (rs) dimeric low-affinity ectodomains (rsFcγR) that have an absolute binding requirement for the simultaneous engagement of two IgG Fc regions. Like cell surface-expressed FcγRs, the binding of dimeric rsFcγR ectodomains to Ab immune complexes was affected by Ab subclass, presentation, opsonization density, Fc fucosylation, or mutation. The activation of an NK cell line and primary NK cells by human IgG-opsonized influenza A hemagglutinin correlated with dimeric rsFcγRIIIa binding activity but not with Ab titer. Furthermore, the dimeric rsFcγR binding assay sensitively detected greater Fc receptor activity to pandemic H1N1 hemagglutinin after the swine influenza pandemic of 2009 in pooled human polyclonal IgG. Thus these dimeric rsFcγR ectodomains are validated, defined probes that should prove valuable in measuring the immune-activating capacity of IgG Abs elicited by infection or vaccination or experimentally derived IgG and its variants. PMID:27385782

  18. The roles of complement receptor 3 and Fcγ receptors during Leishmania phagosome maturation

    PubMed Central

    Polando, Rachel; Dixit, Upasna Gaur; Carter, Cristina R.; Jones, Blake; Whitcomb, James P.; Ballhorn, Wibke; Harintho, Melissa; Jerde, Christopher L.; Wilson, Mary E.; McDowell, Mary Ann

    2013-01-01

    Leishmania are intracellular parasites adapted to surviving in macrophages, whose primary function is elimination of invading pathogens. Leishmania entry into host cells is receptor-mediated. These parasites are able to engage multiple host cell-surface receptors, including MR, TLRs, CR3, and FcγRs. Here, we investigated the role of CR3 and FcγR engagement on the maturation of Leishmania-containing phagosomes using CD11b−/− and FcγR−/− macrophages, and assessing EEA1 and lysosome-associated proteins is necessary for the phagosome maturation delay, characteristic of Leishmania infection. Leishmania-containing phagosomes do not fuse with lyosomes until 5 h postinfection in WT mice. Phagolysosome fusion occurs by 1 h in CD11b and FcγR common chain KO macrophages, although receptor deficiency does not influence Leishmania entry or viability. We also investigated the influence of serum components and their effects on phagosome maturation progression. Opsonization with normal mouse serum, complement-deficient serum, or serum from Leishmania-infected mice all influenced phagosome maturation progression. Our results indicate that opsonophagocytosis influences phagosomal trafficking of Leishmania without altering the intracellular fate. PMID:23543768

  19. Micropatterned ligand arrays to study spatial regulation in Fc receptor signaling

    PubMed Central

    Torres, Alexis J.; Holowka, David

    2013-01-01

    Summary Fc receptor signaling plays a fundamental role in the adaptive immune response. A plethora of Fc receptors (e.g. Fc gamma, Fc-alpha and Fc-epsilon) are expressed on different immune cells, including natural killer cells, macrophages, mast cells and neutrophils. Receptor clustering and activation by multivalent ligands or opsonized particles induces a signaling cascade that leads to targeted secretion of chemical mediators (i.e. histamine, cytokines and chemokines) and phagocytosis, among other responses. Spatial targeting and compartmentalization are common mechanisms of regulation in Fc receptor signaling. However, the tools for studying these dynamic interactions have been limited. To overcome these limitations in our model system, microfabricated surfaces containing spatially defined ligands are used to cluster and activate IgE receptors (FcεRI), involved in allergic responses by mast cells. Micron-scale control of cell activation allows investigation of spatially regulated mechanisms for intracellular signaling with fluorescence microscopy. This approach in conjunction with biochemical techniques has proven to be valuable for investigating immune receptor signaling. PMID:21701976

  20. Functional and clinical consequences of Fc receptor polymorphic and copy number variants

    PubMed Central

    Bournazos, S; Woof, J M; Hart, S P; Dransfield, I

    2009-01-01

    Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leucocytes, thereby linking the humoral and cellular components of immunity. Indeed, engagement of Fc receptors by immunoglobulins initiates a range of immunoregulatory processes that might also play a role in disease pathogenesis. In the circulation, five main types of immunoglobulins (Ig) exist – namely IgG, IgA, IgE, IgM and IgD and receptors with the ability to recognize and bind to IgG (Fcγ receptor family), IgE (FcεRI and CD23), IgA (CD89; Fcα/µR) and IgM (Fcα/µR) have been identified and characterized. However, it is astonishing that nearly all the known human Fc receptors display extensive genetic variation with clear implications for their function, thus representing a substantial genetic risk factor for the pathogenesis of a range of chronic inflammatory disorders. PMID:19604264

  1. Functional and clinical consequences of Fc receptor polymorphic and copy number variants.

    PubMed

    Bournazos, S; Woof, J M; Hart, S P; Dransfield, I

    2009-08-01

    Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leucocytes, thereby linking the humoral and cellular components of immunity. Indeed, engagement of Fc receptors by immunoglobulins initiates a range of immunoregulatory processes that might also play a role in disease pathogenesis. In the circulation, five main types of immunoglobulins (Ig) exist - namely IgG, IgA, IgE, IgM and IgD and receptors with the ability to recognize and bind to IgG (Fc gamma receptor family), IgE (Fc epsilon RI and CD23), IgA (CD89; Fc alpha/microR) and IgM (Fc alpha/microR) have been identified and characterized. However, it is astonishing that nearly all the known human Fc receptors display extensive genetic variation with clear implications for their function, thus representing a substantial genetic risk factor for the pathogenesis of a range of chronic inflammatory disorders. PMID:19604264

  2. Control of Cytokine Production by Human Fc Gamma Receptors: Implications for Pathogen Defense and Autoimmunity

    PubMed Central

    Vogelpoel, Lisa T. C.; Baeten, Dominique L. P.; de Jong, Esther C.; den Dunnen, Jeroen

    2015-01-01

    Control of cytokine production by immune cells is pivotal for counteracting infections via orchestration of local and systemic inflammation. Although their contribution has long been underexposed, it has recently become clear that human Fc gamma receptors (FcγRs), which are receptors for the Fc region of immunoglobulin G (IgG) antibodies, play a critical role in this process by controlling tissue- and pathogen-specific cytokine production. Whereas individual stimulation of FcγRs does not evoke cytokine production, FcγRs cell-type specifically interact with various other receptors for selective amplification or inhibition of particular cytokines, thereby tailoring cytokine responses to the immunological context. The physiological function of FcγR-mediated control of cytokine production is to counteract infections with various classes of pathogens. Upon IgG opsonization, pathogens are simultaneously recognized by FcγRs as well as by various pathogen-sensing receptors, leading to the induction of pathogen class-specific immune responses. However, when erroneously activated, the same mechanism also contributes to the development of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. In this review, we discuss control of cytokine production as a novel function of FcγRs in human innate immune cells in the context of homeostasis, infection, and autoimmunity and address the possibilities for future therapeutic exploitation. PMID:25759693

  3. INDUCTION OF ACTIVATION ANTIGENS ON HUMAN NATURAL KILLER CELLS MODIFIED THROUGH THE FC-Y RECEPTOR

    EPA Science Inventory

    NK cells, defined here as lymphocytes bearing the CD16 Ag found on the NK cell Fc-y receptor (FcR), are known to enter a proliferative and activated state in response to stimulation with IL-2 as assessed by clonal expansion, short-term DNA synthesis, and de novo expression of lym...

  4. Certolizumab pegol does not bind the neonatal Fc receptor (FcRn): Consequences for FcRn-mediated in vitro transcytosis and ex vivo human placental transfer.

    PubMed

    Porter, Charlene; Armstrong-Fisher, Sylvia; Kopotsha, Tim; Smith, Bryan; Baker, Terry; Kevorkian, Lara; Nesbitt, Andrew

    2016-08-01

    Antibodies to tumor necrosis factor (anti-TNF) are used to treat inflammatory diseases, which often affect women of childbearing age. The active transfer of these antibodies across the placenta by binding of the Fc-region to the neonatal Fc receptor (FcRn) may result in adverse fetal or neonatal effects. In contrast to other anti-TNFs, certolizumab pegol lacks an Fc-region. The objective of this study was to determine whether the structure of certolizumab pegol limits active placental transfer. Binding affinities of certolizumab pegol, infliximab, adalimumab and etanercept to human FcRn and FcRn-mediated transcytosis were determined using in vitro assays. Human placentas were perfused ex vivo to measure transfer of certolizumab pegol and positive control anti-D IgG from the maternal to fetal circulation. FcRn binding affinity (KD) was 132nM, 225nM and 1500nM for infliximab, adalimumab and etanercept, respectively. There was no measurable certolizumab pegol binding affinity, similar to that of the negative control. FcRn-mediated transcytosis across a cell layer (mean±SD; n=3) was 249.6±25.0 (infliximab), 159.0±20.2 (adalimumab) and 81.3±13.1ng/mL (etanercept). Certolizumab pegol transcytosis (3.2±3.4ng/mL) was less than the negative control antibody (5.9±4.6ng/mL). No measurable transfer of certolizumab pegol from the maternal to the fetal circulation was observed in 5 out of 6 placentas that demonstrated positive-control IgG transport in the ex vivo perfusion model. Together these results support the hypothesis that the unique structure of certolizumab pegol limits its transfer through the placenta to the fetus and may be responsible for previously reported differences in transfer of other anti-TNFs from mother to fetus. PMID:27123565

  5. Polymorphisms of Immunoglobulin G Fc Receptors in Pediatric Guillain-Barré Syndrome.

    PubMed

    Mansour, Lobna A; Girgis, Marian Y; Abdulhay, Mohamed; ElEinein, Ehab I Abou; ElHawary, Rabab; Hanna, Mariam Onsy F

    2016-06-01

    Introduction Guillain-Barré syndrome (GBS) is an autoimmune peripheral neuropathy characterized by demyelination and axonal damage. Biallelic functional polymorphisms in the immunoglobulin G Fc receptors (FcγR)-FcγRIIA: H131/R131, FcγRIIIA: V158/F158, and FcγRIIIB: NA1/NA2 affect the affinity of the IgG-FcγR interaction, therefore, diseases such as GBS in which this interaction plays a critical role might be influenced by the polymorphisms. Methods We evaluated the role of FcγR polymorphisms in susceptibility to GBS in Egyptian pediatric patients and the association of the variant alleles with neurophysiological types, severity, and outcome of the disease. A total of 50 patients with GBS and 50 controls were examined for FcγR polymorphisms by allele-specific polymerase chain reaction. Results FcγRIIA H131 allele (p = < 0.0001; odds ratio [OR] = 4.78; 95% confidence interval [CI], 2.62-8.70) and FcγRIIA H/H131 genotype (p = < 0.0001 ; OR = 10.56; 95% CI, 3.59-31.06) were significantly increased in GBS patients while FcγRIIIA and FcγRIIIB allelic distributions were similar among patients and controls. The FcγR genotypes showed no association with neurophysiological types of GBS, severity or outcome of the disease. Conclusions These findings reflect that FcγRIIA H131 allele may represent a risk marker for susceptibility to GBS. PMID:27064330

  6. Mouse model recapitulating human Fcγ receptor structural and functional diversity

    PubMed Central

    Smith, Patrick; DiLillo, David J.; Bournazos, Stylianos; Li, Fubin; Ravetch, Jeffrey V.

    2012-01-01

    The in vivo biological activities of IgG antibodies result from their bifunctional nature, in which antigen recognition by the Fab is coupled to the effector and immunomodulatory diversity found in the Fc domain. This diversity, resulting from both amino acid and glycan heterogeneity, is translated into cellular responses through Fcγ receptors (FcγRs), a structurally and functionally diverse family of cell surface receptors found throughout the immune system. Although many of the overall features of this system are maintained throughout mammalian evolution, species diversity has precluded direct analysis of human antibodies in animal species, and, thus, detailed investigations into the unique features of the human IgG antibodies and their FcγRs have been limited. We now report the development of a mouse model in which all murine FcγRs have been deleted and human FcγRs, encoded as transgenes, have been inserted into the mouse genome resulting in recapitulation of the unique profile of human FcγR expression. These human FcγRs are shown to function to mediate the immunomodulatory, inflammatory, and cytotoxic activities of human IgG antibodies and Fc engineered variants and provide a platform for the detailed mechanistic analysis of therapeutic and pathogenic IgG antibodies. PMID:22474370

  7. Impact of IgG2 high molecular weight species on neonatal Fc receptor binding assays.

    PubMed

    Zhang, Yuling; Mathur, Abhishek; Maher, Gwen; Arroll, Thomas; Bailey, Robert

    2015-11-15

    A cell-based assay and a solution neonatal Fc receptor (FcRn) binding assay were implemented for the characterization of an IgG2 antibody after observation that different product lots exhibited unexpected differences in FcRn binding in the cell-based format with membrane-bound FcRn. The experiments described here suggest that the apparent differences observed in the FcRn binding across different product lots in the cell-based format can be attributed to the different levels of the higher order high molecular weight species (HMWs) in them. A strong correlation between FcRn binding in the cell-based format and the percentage (%) higher order HMWs suggests that small amounts (∼0.1%) of the latter could cause the enhanced apparent FcRn binding (% relative binding ranging from 50 to 100%) in the format. However, when the binding was assessed with recombinant FcRn in soluble form, avidity effects were minimal and the assay format exhibited less sensitivity toward the differences in higher order HMWs levels across product lots. In conclusion, a solution-based assay may be a more appropriate assay to assess FcRn binding of the dominant species of an Fc-fusion protein or monoclonal antibody if minor differences in product variants such as higher order HMWs are shown to affect the binding significantly. PMID:26255698

  8. Chimeric Antigen Receptors With Mutated IgG4 Fc Spacer Avoid Fc Receptor Binding and Improve T Cell Persistence and Antitumor Efficacy

    PubMed Central

    Jonnalagadda, Mahesh; Mardiros, Armen; Urak, Ryan; Wang, Xiuli; Hoffman, Lauren J; Bernanke, Alyssa; Chang, Wen-Chung; Bretzlaff, William; Starr, Renate; Priceman, Saul; Ostberg, Julie R; Forman, Stephen J; Brown, Christine E

    2015-01-01

    The success of adoptive therapy using chimeric antigen receptor (CAR)–expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here, we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (FcγRs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble FcγRs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid FcγR binding would improve CAR+ T cell engraftment and antitumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)) or incorporated a CH2 deletion (CD19Rch2Δ). These mutations reduced binding to soluble FcγRs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2Δ T cells exhibited improved persistence and more potent CD19-specific antilymphoma efficacy in NSG mice. Together, these studies suggest that optimal CAR function may require the elimination of cellular FcγR interactions to improve T cell persistence and antitumor responses. PMID:25366031

  9. Insights from Fc receptor biology: a route to improved antibody reagents.

    PubMed

    Woof, Jenny M

    2012-01-01

    Fc receptors and their interaction with antibodies will be a major theme at the forthcoming FASEB Science Research Conference on Immunoreceptors to be held in Snowmass this July (details available at www.faseb.org/src/home.aspx, follow the tabs for Immunoreceptors). Since its inception in the mid 1980s, this meeting series has maintained a focus on Fc receptors, and this year's meeting will be no exception. PMID:22531437

  10. Insights from Fc receptor biology: A route to improved antibody reagents

    PubMed Central

    2012-01-01

    Fc receptors and their interaction with antibodies will be a major theme at the forthcoming FASEB Science Research Conference on Immunoreceptors to be held in Snowmass this July (details available at www.faseb.org/src/home.aspx, follow the tabs for Immunoreceptors). Since its inception in the mid 1980s, this meeting series has maintained a focus on Fc receptors, and this year’s meeting will be no exception. PMID:22531437

  11. Quantitative Analysis of Human Neonatal Fc Receptor (FcRn) Tissue Expression in Transgenic Mice by Online Peptide Immuno-Affinity LC-HRMS.

    PubMed

    Fan, Yao-Yun; Neubert, Hendrik

    2016-04-19

    Neonatal Fc receptor (FcRn) is the homeostatic receptor responsible for the long half-life of endogenous IgG by protecting it from lysosomal degradation. Understanding systemic FcRn tissue expression is important to predict and design the half-life of therapeutic antibodies and Fc-coupled biotherapeutics. To this end, we measured human FcRn (hFcRn) tissue expression in Tg32, a human FcRn knock-in transgenic mouse model, for which a strong correlation of drug clearance to humans has been demonstrated. Building an hFcRn tissue expression profile in Tg32 was enabled by the development of a tissue preparation procedure composed of bead-based protein extraction and protein precipitation using acetone followed by pellet digestion with trypsin. Digests were then loaded onto an online peptide immuno-affinity flow configuration hyphenated with reversed phase nanoflow chromatography and coupled with high resolution mass spectrometry to quantify hFcRn derived peptides. The workflow allowed bypassing some of the challenges typically associated with membrane protein analysis. We demonstrated acceptable precision and bias for measuring hFcRn in tissue matrices, typically within 20% coefficient of variation and relative error. We also report hFcRn expression in several Tg32 tissues. We anticipate that establishing a quantitative approach for hFcRn in tissues will enable the systematic measurement of hFcRn concentrations to further increase the accuracy of physiologically based pharmacokinetic (PBPK) models for PK prediction of Fc-containing biotherapeutics. This is anticipated to improve the translation of pharmacokinetic data from preclinical model systems to humans. PMID:27012525

  12. X-ray Crystal Structures of Monomeric and Dimeric Peptide Inhibitors in Complex with the Human Neonatal Fc Receptor, FcRn

    SciTech Connect

    Mezo, Adam R.; Sridhar, Vandana; Badger, John; Sakorafas, Paul; Nienaber, Vicki

    2010-10-28

    The neonatal Fc receptor, FcRn, is responsible for the long half-life of IgG molecules in vivo and is a potential therapeutic target for the treatment of autoimmune diseases. A family of peptides comprising the consensus motif GHFGGXY, where X is preferably a hydrophobic amino acid, was shown previously to inhibit the human IgG:human FcRn protein-protein interaction (Mezo, A. R., McDonnell, K. A., Tan Hehir, C. A., Low, S. C., Palombella, V. J., Stattel, J. M., Kamphaus, G. D., Fraley, C., Zhang, Y., Dumont, J. A., and Bitonti, A. J. (2008) Proc. Natl. Acad. Sci. U.S.A., 105, 2337-2342). Herein, the x-ray crystal structure of a representative monomeric peptide in complex with human FcRn was solved to 2.6 {angstrom} resolution. The structure shows that the peptide binds to human FcRn at the same general binding site as does the Fc domain of IgG. The data correlate well with structure-activity relationship data relating to how the peptide family binds to human FcRn. In addition, the x-ray crystal structure of a representative dimeric peptide in complex with human FcRn shows how the bivalent ligand can bridge two FcRn molecules, which may be relevant to the mechanism by which the dimeric peptides inhibit FcRn and increase IgG catabolism in vivo. Modeling of the peptide:FcRn structure as compared with available structural data on Fc and FcRn suggest that the His-6 and Phe-7 (peptide) partially mimic the interaction of His-310 and Ile-253 (Fc) in binding to FcRn, but using a different backbone topology.

  13. Development and Characterization of Monoclonal Antibodies Specific for Mouse and Human Fcγ Receptors.

    PubMed

    Tutt, Alison L; James, Sonya; Laversin, Stéphanie A; Tipton, Thomas R W; Ashton-Key, Margaret; French, Ruth R; Hussain, Khiyam; Vaughan, Andrew T; Dou, Lang; Earley, Alexander; Dahal, Lekh N; Lu, Chen; Dunscombe, Melanie; Chan, H T Claude; Penfold, Christine A; Kim, Jinny H; Potter, Elizabeth A; Mockridge, C Ian; Roghanian, Ali; Oldham, Robert J; Cox, Kerry L; Lim, Sean H; Teige, Ingrid; Frendéus, Bjorn; Glennie, Martin J; Beers, Stephen A; Cragg, Mark S

    2015-12-01

    FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic. PMID:26512139

  14. Materno-Fetal Transfer of Preproinsulin Through the Neonatal Fc Receptor Prevents Autoimmune Diabetes.

    PubMed

    Culina, Slobodan; Gupta, Nimesh; Boisgard, Raphael; Afonso, Georgia; Gagnerault, Marie-Claude; Dimitrov, Jordan; Østerbye, Thomas; Justesen, Sune; Luce, Sandrine; Attias, Mikhaël; Kyewski, Bruno; Buus, Søren; Wong, F Susan; Lacroix-Desmazes, Sebastien; Mallone, Roberto

    2015-10-01

    The first signs of autoimmune activation leading to β-cell destruction in type 1 diabetes (T1D) appear during the first months of life. Thus, the perinatal period offers a suitable time window for disease prevention. Moreover, thymic selection of autoreactive T cells is most active during this period, providing a therapeutic opportunity not exploited to date. We therefore devised a strategy by which the T1D-triggering antigen preproinsulin fused with the immunoglobulin (Ig)G Fc fragment (PPI-Fc) is delivered to fetuses through the neonatal Fc receptor (FcRn) pathway, which physiologically transfers maternal IgGs through the placenta. PPI-Fc administered to pregnant PPIB15-23 T-cell receptor-transgenic mice efficiently accumulated in fetuses through the placental FcRn and protected them from subsequent diabetes development. Protection relied on ferrying of PPI-Fc to the thymus by migratory dendritic cells and resulted in a rise in thymic-derived CD4(+) regulatory T cells expressing transforming growth factor-β and in increased effector CD8(+) T cells displaying impaired cytotoxicity. Moreover, polyclonal splenocytes from nonobese diabetic (NOD) mice transplacentally treated with PPI-Fc were less diabetogenic upon transfer into NOD.scid recipients. Transplacental antigen vaccination provides a novel strategy for early T1D prevention and, further, is applicable to other immune-mediated conditions. PMID:25918233

  15. Characterization and screening of IgG binding to the neonatal Fc receptor.

    PubMed

    Neuber, Tobias; Frese, Katrin; Jaehrling, Jan; Jäger, Sebastian; Daubert, Daniela; Felderer, Karin; Linnemann, Mechthild; Höhne, Anne; Kaden, Stefan; Kölln, Johanna; Tiller, Thomas; Brocks, Bodo; Ostendorp, Ralf; Pabst, Stefan

    2014-01-01

    The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding. PMID:24802048

  16. Fc receptors and their influence on efficacy of therapeutic antibodies for treatment of viral diseases.

    PubMed

    Chan, Kuan Rong; Ong, Eugenia Z; Mok, Darren Z L; Ooi, Eng Eong

    2015-01-01

    The lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. In recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, Ebola virus and Hendra virus. The binding affinity of these antibodies can directly impact their therapeutic efficacy. However, we and others have also demonstrated that the subtype of Fc-gamma receptors (FcγRs) engaged influences the stoichiometric requirement for virus neutralization. Hence, the development of therapeutic antibodies against infectious diseases should consider the FcγRs engaged and Fc-effector functions involved. This review highlights the current state of knowledge about FcγRs and FcγR effector functions involved in virus neutralization, with emphasis on factors that can affect FcγR engagement. A better understanding of Fc-FcγR interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects. PMID:26466016

  17. Involvement of Fc Receptors in Disorders of the Central Nervous System

    PubMed Central

    Okun, Eitan; Mattson, Mark P.; Arumugam, Thiruma V.

    2009-01-01

    Immunoglobulins (Igs) are proteins with a highly variable antigen-binding domain and a constant region (Fc domain) that binds to a cell surface receptor (FcR). Activation of FcRs in immune cells (lymphocytes, macrophages and mast cells) triggers effector responses including cytokine production, phagocytosis and degranulation). In addition to their roles in normal responses to infection or tissue injury, and in immune-related diseases, FcRs are increasingly recognized for their involvement in neurological disorders. One or more FcRs are expressed in microglia, astrocytes, oligodendrocytes and neurons. Aberrant activation of FcRs in such neural cells may contribute to the pathogenesis of major neurodegenerative conditions including Alzheimer's disease, Parkinson's disease, ischemic stroke and multiple sclerosis. On the other hand, FcRs may play beneficial roles in counteracting pathological processes; for example, FcRs may facilitate removal of amyloid peptides from the brain and so protect against Alzheimer's disease. Knowledge of the functions of FcRs in the nervous system in health and disease is leading to novel preventative and therapeutic strategies for stroke, Alzheimer's disease and other neurological disorders. PMID:19844812

  18. Fc receptors and their influence on efficacy of therapeutic antibodies for treatment of viral diseases

    PubMed Central

    Chan, Kuan Rong; Ong, Eugenia Z; Mok, Darren ZL; Ooi, Eng Eong

    2015-01-01

    The lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. In recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, Ebola virus and Hendra virus. The binding affinity of these antibodies can directly impact their therapeutic efficacy. However, we and others have also demonstrated that the subtype of Fc-gamma receptors (FcγRs) engaged influences the stoichiometric requirement for virus neutralization. Hence, the development of therapeutic antibodies against infectious diseases should consider the FcγRs engaged and Fc-effector functions involved. This review highlights the current state of knowledge about FcγRs and FcγR effector functions involved in virus neutralization, with emphasis on factors that can affect FcγR engagement. A better understanding of Fc-FcγR interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects. PMID:26466016

  19. Fc receptors do not mediate African swine fever virus replication in macrophages

    SciTech Connect

    Alcami, A.; Vinuela, E. )

    1991-04-01

    Titration experiments in swine macrophages have shown that African swine fever virus infectivity was not enhanced in the presence of antiviral antibodies. The early viral protein synthesis and the viral DNA replication in swine macrophages infected with virus-antibody complexes were inhibited in the presence of high doses of uv-inactivated virus, which saturated specific virus receptors, but not when Fc receptors were saturated with antibodies. These results indicate that African swine fever virus does not infect swine macrophages through Fc receptors and that the normal entry pathway through virus receptors is not bypassed by the virus-antibody complexes.

  20. Effects of mononuclear phagocyte system modulating agents on Fc and C3 receptors of adherent cells.

    PubMed Central

    Hitomi, M.; Shimizu, F.

    1985-01-01

    Agents which modulate the mononuclear phagocyte system (MPS) were examined for their effects on Fc and C3 receptors of adherent cells (A-cells) as judged by rosette formation. Dextran sulphate, carrageenan, and immune complexes, known as MPS suppressants, reduced the percentage of receptor-positive A-cells, while levamisole, known as a MPS-activator, increased the percentage in vitro. The changes in the percentage of Fc receptor were parallel to those of the C3 receptor in vitro. The effects of these agents were also examined in vivo. PMID:2408651

  1. The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

    PubMed Central

    Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

    2015-01-01

    The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates. PMID:25658443

  2. Defect of Fc receptors and phenotypical changes in sinusoidal endothelial cells in human liver cirrhosis.

    PubMed Central

    Muro, H.; Shirasawa, H.; Kosugi, I.; Nakamura, S.

    1993-01-01

    To analyze the pathological changes occurring in Fc receptors (FcRs) in sinusoidal endothelial cells (SECs) in chronic liver diseases, we first characterized immunohistochemically the SEC FcRs by using monoclonal antibodies (MAbs) to FcRs and then investigated the distribution of the SEC FcRs by using peroxidase-antiperoxidase IgG complexes as a ligand on frozen sections. MAb 2E1 to FcRII reacted with SECs in a similar manner to peroxidase-antiperoxidase IgG and blocked the peroxidase-antiperoxidase IgG binding to SECs, whereas MAbs 3G8 and Leu-11b to FcRIII did not. FcRs in normal liver were found along the sinusoidal walls, except for those in the outer periportal zones, but FcRs in chronic active hepatitis and cirrhosis were intermittently or focally absent. The lengths of the FcR-positive portion of sinusoids in unit areas were respectively about 54% and 76% of the normal values in active and inactive cirrhosis. Where FcRs were absent, the MAbs CD36, CD31, and EN4 revealed the presence of sinusoids and, in active cirrhosis, frequently the thickening of liver cell plates. The FcR-negative SECs in the outer periportal zones of normal livers were different from the SECs of other sites in the presence of PAL-E antigen and a rich amount of EN4 antigen, though these sinusoids possessed Kupffer cells and no perisinusoidal deposition of laminin. The FcR-negative SECs in liver diseases occasionally presented the character of ordinary blood vessels, viz., PAL-E antigen, CD34 antigen, and a deficiency of Kupffer cells, regardless of perisinusoidal laminin deposition. However, they preserved the character of normally FcR-possessing SECs, viz., CD36 antigen, and a small amount of EN4 and CD31 antigens. These findings indicate that the outer-periportal SECs in normal livers are phenotypically different from other SECs and that the SECs in diseased livers frequently undergo phenotypical changes, including loss of FcRs, regardless of perisinusoidal laminin deposition, i

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  5. Human cytomegalovirus Fcγ binding proteins gp34 and gp68 antagonize Fcγ receptors I, II and III.

    PubMed

    Corrales-Aguilar, Eugenia; Trilling, Mirko; Hunold, Katja; Fiedler, Manuela; Le, Vu Thuy Khanh; Reinhard, Henrike; Ehrhardt, Katrin; Mercé-Maldonado, Eva; Aliyev, Enver; Zimmermann, Albert; Johnson, David C; Hengel, Hartmut

    2014-05-01

    Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity. PMID:24830376

  6. Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection.

    PubMed

    Chan, Kuan Rong; Zhang, Summer Li-Xin; Tan, Hwee Cheng; Chan, Ying Kai; Chow, Angelia; Lim, Angeline Pei Chiew; Vasudevan, Subhash G; Hanson, Brendon J; Ooi, Eng Eong

    2011-07-26

    The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes. PMID:21746897

  7. The human IgA-Fc alpha receptor interaction and its blockade by streptococcal IgA-binding proteins.

    PubMed

    Woof, J M

    2002-08-01

    IgA plays a key role in immune defence of the mucosal surfaces. IgA can trigger elimination mechanisms against pathogens through the interaction of its Fc region with Fc alpha Rs (receptors specific for the Fc region of IgA) present on neutrophils, macrophages, monocytes and eosinophils. The human Fc alpha R (CD89) shares homology with receptors specific for the Fc region of IgG (Fc gamma Rs) and IgE (Fc epsilon RIs), but is a more distantly related member of the receptor family. CD89 interacts with residues lying at the interface of the two domains of IgA Fc, a site quite distinct from the homologous regions at the top of IgG and IgE Fc recognized by Fc gamma R and Fc epsilon RI respectively. Certain pathogenic bacteria express surface proteins that bind to human IgA Fc. Experiments with domain-swap antibodies and mutant IgAs indicate that binding of three such proteins (Sir22 and Arp4 of Streptococcus pyogenes and beta protein of group B streptococci) depend on sites in the Fc interdomain region of IgA, the binding region also used by CD89. Further, we have found that the streptococcal proteins can inhibit interaction of IgA with CD89, and have thereby identified a mechanism by which a bacterial IgA-binding protein may modulate IgA effector function. PMID:12196121

  8. Monoclonal Antibodies Specific for Human IgM Fc Receptor Inhibit Ligand-binding Activity

    PubMed Central

    Kubagawa, Yoshiki; Honjo, Kazuhito; Kang, Dong-Won

    2014-01-01

    A panel of six different murine hybridoma clones secreting IgG monoclonal antibodies (MAbs) specific for the human IgM Fc receptor (FcμR) was generated. All MAbs specifically precipitated a major protein of ∼60 kDa from membrane lysates of FcμR-bearing, but not FcμR-negative, cells as did IgM-ligands. Pre-incubation of membrane lysate of FcμR-bearing cells with these MAbs completely removed the ∼60 kDa IgM-reactive protein. By using recombinant human/mouse chimeric FcμR proteins, the epitope recognized by HM7 and HM10 MAbs was mapped to the Ig-like domain of human FcμR, whereas the other MAbs recognized the stalk region. Pre-incubation of FcμR+ cells with the Ig-like domain-specific MAbs, but not with others, markedly inhibited subsequent IgM-ligand binding. A similar, but much weaker, inhibition was also observed when the incubation order was reversed. When FcμR+ cells were simultaneously incubated with both IgM-ligands and MAbs, HM7 MAb efficiently competed with IgM for FcμR binding. Unlike control Jurkat cells, FcμR-bearing cells were resistant to apoptosis induced by agonistic IgM anti-Fas MAb (CH11); however, addition of the HM7 MAb inhibited the interaction of the Fc portion of CH11 MAb with FcμR, thereby promoting apoptosis of FcμR-bearing Jurkat cells. The variable regions of the HM7 MAb were composed of Ighv14-3, Ighd1-2, and Ighj2 for the γ2b heavy chain and Igk3-4 and Igkj2 for the κ light chain. These findings suggest that HM7 MAb efficiently blocks the ligand-binding activity of FcμR. PMID:25545208

  9. No association of primary Sjögren's syndrome with Fcγ receptor gene variants.

    PubMed

    Haldorsen, K; Appel, S; Le Hellard, S; Bruland, O; Brun, J G; Omdal, R; Kristjansdottir, G; Theander, E; Fernandes, C P D; Kvarnström, M; Eriksson, P; Rönnblom, L; Herlenius, M W; Nordmark, G; Jonsson, R; Bolstad, A I

    2013-06-01

    The genetic background of primary Sjögren's syndrome (pSS) is partly shared with systemic lupus erythematosus (SLE). Immunoglobulin G Fc receptors are important for clearance of immune complexes. Fcγ receptor variants and gene deletion have been found to confer SLE risk. In this study, four Fcγ receptor single-nucleotide polymorphisms (SNPs) and one copy number variation (CNV) were studied. Swedish and Norwegian pSS patients (N=527) and controls (N=528) were genotyped for the Fcγ receptor gene variant FCGR2A H131R (rs1801274) by the Illumina GoldenGate assay. FCGR3A F158V (rs396991) was analysed in 488 patients and 485 controls, FCGR3B rs447536 was analysed in 471 patients and 467 controls, and FCGR3B rs448740 was analysed in 478 cases and 455 controls, using TaqMan SNP genotyping assays. FCGR3B CNV was analysed in 124 patients and 139 controls using a TaqMan copy number assay. None of the SNPs showed any association with pSS. Also, no FCGR3B CNV association was detected. The lack of association of pSS with Fcγ receptor gene variants indicates that defective immune complex clearance may not be as important in pSS pathogenesis as in SLE, and may point to important differences between SLE and pSS. PMID:23552400

  10. Human neutrophil Fcγ receptors initiate and play specialized nonredundant roles in antibody-mediated inflammatory diseases

    PubMed Central

    Tsuboi, Naotake; Asano, Kenichi; Lauterbach, Michael; Mayadas, Tanya N.

    2008-01-01

    Summary Antibody-antigen complex mediated inflammation is integral to the pathogenesis of many autoimmune diseases. Mice deficient in the γ-chain of Fc-receptors are protected in IgG-mediated glomerulonephritis and the Arthus reaction and FcR-bearing mast cells and macrophages have been assigned primary roles in these processes. Here we demonstrate that neutrophil selective transgenic expression of the two uniquely human activating FcγRs, FcγRIIA and FcγRIIIB was sufficient to restore susceptibility to progressive anti-glomerular basement membrane (GBM) nephritis and the cutaneous Reverse Passive Arthus (RPA) reaction in γ-chain deficient mice. Both FcγRIIA and FcγRIIIB mediated robust neutrophil accumulation in tissues suggesting direct roles for these human receptors in IC-induced neutrophil recruitment, while FcγRIIA alone mediated organ injury. In an acute model of anti-GBM nephritis, both FcγRIIIB and FcγRIIA promoted initial neutrophil recruitment to glomerular immune-complexes (ICs) accessible to circulating cells, while FcγRIIA further sustained accumulation. In a model of soluble ICs deposited strictly within the post-capillary venules of the cremaster muscle, FcγRIIIB was solely responsible for converting initial selectin-dependent tethers to slow rolling and adhesion. However, in the cremaster RPA reaction, dependent on vascular and tissue accumulation of soluble ICs, FcγRIIA predominated in neutrophil recruitment that was dependent on G-protein coupled receptor activation. Thus, human FcγRs on neutrophils serve as the primary molecular links between ICs and immunological disease with FcγRIIA promoting tissue injury, and FcγRIIIB and FcγRIIA displaying specialized context-dependent functions in IC-induced neutrophil recruitment. PMID:18538590

  11. Differential Expression of Functional Fc-Receptors and Additional Immune Complex Receptors on Mouse Kidney Cells

    PubMed Central

    Suwanichkul, Adisak; Wenderfer, Scott E.

    2013-01-01

    The precise mechanisms by which circulating immune complexes accumulate in the kidney to form deposits in glomerulonephritis are not well understood. In particular, the role of resident cells within glomeruli of the kidney has been widely debated. Immune complexes have been shown to bind one glomerular cell type (mesangial cells) leading to functional responses such as pro-inflammatory cytokine production. To further assess the presence of functional immunoreceptors on resident glomerular cells, cultured mouse renal epithelial, endothelial, and mesangial cells were treated with heat-aggregated mouse IgG or preformed murine immune complexes. Mesangial and renal endothelial cells were found to bind IgG complexes, whereas glomerular epithelial cell binding was minimal. A blocking antibody for Fc-gamma receptors reduced binding to mesangial cells but not renal endothelial cells, suggesting differential immunoreceptor utilization. RT-PCR and immunostaining based screening of cultured renal endothelial cells showed limited low-level expression of known Fc-receptors and Igbinding proteins. The interaction between mesangial cells and renal endothelial cells and immune complexes resulted in distinct, cell-specific patterns of chemokine and cytokine production. This novel pathway involving renal endothelial cells likely contributes to the predilection of circulating immune complex accumulation within the kidney and to the inflammatory responses that drive kidney injury. PMID:23911392

  12. Limited role of charge matching in the interaction of human immunoglobulin A with the immunoglobulin A Fc receptor (Fc alpha RI) CD89.

    PubMed

    Pleass, Richard J; Dehal, Prabhjyot K; Lewis, Melanie J; Woof, Jenny M

    2003-07-01

    Human immunoglobulin A (IgA) mediates protective effector mechanisms through interaction with specific cellular Fc receptors (Fc alpha RI). Two IgA Fc interdomain loops (Leu257-Leu258 in the CH2 domain and Pro440-Phe443 in the CH3 domain) have previously been identified as critical for binding to Fc alpha RI. On the receptor, the interaction site for IgA has been localized to the EC1 domain. The essential Fc alpha RI residues involved are Tyr35, Tyr81 and Arg82, with contributions also from Arg52 and to a lesser extent from His85 and Tyr86. The basic nature of the side chains of some of the receptor residues implicated in ligand binding suggested that charge matching might play some role in the interaction. To address this possibility, we have generated five IgA1 mutants with point substitutions in acidic residues lying close to the putative interaction site and assessed their abilities to bind Fc alpha RI on human neutrophils. Mutants E254A, E254L and E437A displayed affinities for Fc alpha RI comparable to that of wild-type IgA1, while mutants D255A and D255V had only slightly reduced affinities for the receptor. Therefore, electrostatic interactions appear unlikely to play a significant role in the IgA-Fc alpha RI interaction. Moreover, the lack of effect of mutations in residues adjacent to those previously implicated in binding, reaffirms the importance of the interdomain loops in Fc alpha RI binding. PMID:12807477

  13. Structural Heterogeneity and Functional Domains of Murine Immunoglobulin G Fc Receptors

    NASA Astrophysics Data System (ADS)

    Ravetch, Jeffrey V.; Luster, Andrew D.; Weinshank, Richard; Kochan, Jarema; Pavlovec, Amalia; Portnoy, Daniel A.; Hulmes, Jeffrey; Pan, Yu-Ching E.; Unkeless, Jay C.

    1986-11-01

    Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule Eβ. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.

  14. Targeting Sindbis virus-based vectors to Fc receptor-positive cell types

    SciTech Connect

    Klimstra, William B.; Williams, Jacqueline C.; Ryman, Kate D.; Heidner, Hans W. . E-mail: hans.heidner@utsa.edu

    2005-07-20

    Some viruses display enhanced infection for Fc receptor (FcR)-positive cell types when complexed with virus-specific immunoglobulin (Ig). This process has been termed antibody-dependent enhancement of viral infection (ADE). We reasoned that the mechanism of ADE could be exploited and adapted to target alphavirus-based vectors to FcR-positive cell types. Towards this goal, recombinant Sindbis viruses were constructed that express 1 to 4 immunoglobulin-binding domains of protein L (PpL) as N-terminal extensions of the E2 glycoprotein. PpL is a bacterial protein that binds the variable region of antibody kappa light chains from a range of mammalian species. The recombinant viruses incorporated PpL/E2 fusion proteins into the virion structure and recapitulated the species-specific Ig-binding phenotypes of native PpL. Virions reacted with non-immune serum or purified IgG displayed enhanced binding and ADE for several species-matched FcR-positive murine and human cell lines. ADE required virus expression of a functional PpL Ig-binding domain, and appeared to be Fc{gamma}R-mediated. Specifically, ADE did not occur with Fc{gamma}R-negative cells, did not require active complement proteins, and did not occur on Fc{gamma}R-positive murine cell lines when virions were bound by murine IgG-derived F(ab'){sub 2} fragments.

  15. Fc gamma receptor IIa (CD32) polymorphism in fulminant meningococcal septic shock in children.

    PubMed

    Bredius, R G; Derkx, B H; Fijen, C A; de Wit, T P; de Haas, M; Weening, R S; van de Winkel, J G; Out, T A

    1994-10-01

    Antibodies are essential in host defense against Neisseria meningitidis. Therefore, interactions among IgG and Fc receptors (Fc gamma R) on phagocytes may be crucial. Genetic polymorphic forms of Fc gamma RIIa (CD32) express different functional activities. In a retrospective study, Fc gamma R polymorphisms were determined in 25 children who survived fulminant meningococcal septic shock: 11 had Fc gamma RIIa-R/R131, the poor IgG2-binding allotype, which is a significantly more frequent rate than found in a healthy white population (44% vs. 23%; P = .028; odds ratio = 2.67; 95% confidence interval, 1.09-6.53). The relevance of this finding was further supported by the fact that neutrophils with the Fc gamma RIIa-R/R131 allotype phagocytized N. meningitidis opsonized with polyclonal IgG2 antibodies less effectively than did IIa-H/H131 neutrophils. Our findings suggest an important role for anti-N. meningitidis IgG2 and the Fc gamma RIIa polymorphism in host defense against systemic meningococcal infections. PMID:7930726

  16. Alternative splicing of the human IgA Fc receptor CD89 in neutrophils and eosinophils.

    PubMed

    Pleass, R J; Andrews, P D; Kerr, M A; Woof, J M

    1996-09-15

    Receptors for the Fc portion of IgA (Fc alpha R) trigger important immunological elimination processes against IgA-coated targets. Investigation of human Fc alpha R (CD89) transcripts in neutrophils, eosinophils and a monocyte-like cell line, THP-1, with the use of reverse transcriptase PCR, Northern blotting and RNase protection analysis, has provided evidence in these cell types for at least two distinct transcripts generated by alternative splicing. The cDNAs derived from the two major transcripts of both neutrophils and eosinophils have been cloned and sequenced. For both cell types, the larger clone represents the previously described full-length receptor, whereas the second, shorter, splice variant lacks the entire second, membrane-proximal, Ig-like domain. Stable CHO-K1 transfectants have been obtained for both full-length and truncated variant neutrophil receptors. Whereas the full-length receptor is recognized by a panel of five anti-Fc alpha R monoclonal antibodies (mAbs), the shorter variant is bound weakly by only two of the antibodies, suggesting that the epitopes recognized by the majority of the mAbs lie at least in part in the second Ig-like domain of Fc alpha R. Both full-length and splice variant forms of the receptor bind secretory IgA, but the weak binding to serum IgA seen with the full-length receptor is not evident with the shorter variant. Alternative splicing might therefore serve as a means of diversifying Fc alpha R structure and function. PMID:8836118

  17. Expression and immunohistochemical localization of the neonatal Fc receptor (FcRn) in the mammary glands of the Egyptian water buffalo.

    PubMed

    Sayed-Ahmed, Ahmed; Kassab, Mohamed; Abd-Elmaksoud, Ahmed; Elnasharty, Mohamed; El-Kirdasy, Ahmed

    2010-07-01

    Although a marginal placental transfer of maternal immunoglobulin (Ig) has been demonstrated in buffalo, the colostrum still provides the main source of immune components and nutrients to neonate buffalo calves. The neonatal Fc receptor (FcRn) transports maternal Ig across the gut wall and is involved in the transport of IgG in the mammary gland. In this study we used RT-PCR to examine the gene expression of FcRn in the mammary gland during several physiological states of the Egyptian water buffalo. The buffalo FcRn showed a high sequence homology to that of other mammalian species and especially the cow. Immunohistochemistry demonstrated positive immunolabelling of FcRn in the epithelial cells of the acini and ducts of the examined mammary gland tissue. Remarkable differences in both the cellular localization and in the intensity of FcRn immunopositivity were observed depending on the functional state of the mammary gland tissues. In late pregnancy, the FcRn immunolabelling was homogeneously distributed in the cytoplasm of the epithelial cells. In recently parturient animals, positive FcRn immunolabelling was mainly located at the luminal surface and apical cytoplasm of the mammary gland epithelium, while in dry and lactating animals, the FcRn immunolabelling was in the apical cytoplasm of the cells. The strongest FcRn immunolabelling was observed in late pregnancy and in recently parturient animals. In conclusion, the present data support the notion that FcRn might be involved in the transfer of maternal immunoglobulins and in the local defense mechanism of the mammary gland. PMID:19481783

  18. n-3 Polyunsaturated fatty acids inhibit Fc ε receptor I-mediated mast cell activation.

    PubMed

    Wang, Xiaofeng; Ma, David W L; Kang, Jing X; Kulka, Marianna

    2015-12-01

    In vivo models show that n-3 polyunsaturated fatty acids (PUFA) inhibit some of the processes associated with allergic inflammation but the direct effect of n-3 PUFA on mast cells, the major effector cells in allergy, is poorly understood. We sought to determine the effect and mechanism of n-3 PUFA on Fc ε receptor I (FcεRI)-mediated signal transduction and mast cell activation. Bone marrow-derived mast cells (BMMC) were differentiated from bone marrow obtained from C57BL/6 wild-type (WT) and fat-1 transgenic mice. The fat-1 mice express fatty acid n-3 desaturase and produce endogenous n-3 PUFA. For comparison, exogenous n-3 PUFA were supplemented to WT BMMC and human mast cell (LAD2) cultures. Fat-1 BMMC released less β-hexosaminidase (β-hex) and cysteinyl leukotrienes and produced less tumor necrosis factor and chemokine (C-C motif) ligand 2. n-3 PUFA supplementation reduced LAD2 and BMMC degranulation (β-hex release) following FcεRI activation. Fat-1 BMMC expressed less constitutive Lyn and linker of activated T cells (LAT), and FcεRI-mediated phosphorylation of Lyn, spleen tyrosine kinase and LAT were reduced in fat-1 BMMC. Although the expression of surface and whole cell FcεRI was similar in WT and fat-1 BMMC, unstimulated fat-1 BMMC showed reduced FcεRI localization to lipid rafts, and stimulation with antigen resulted in aberrant FcεRI shuttling to the rafts. Our results show that n-3 PUFA suppress FcεRI-mediated activation of mast cells, which results in reduced mediator release. This effect is associated with a decrease in LAT and Lyn expression as well as abnormal shuttling of FcεRI to lipid rafts. PMID:26363927

  19. Staphylococci-induced Human Platelet Injury Mediated by Protein A and Immunoglobulin G Fc Fragment Receptor

    PubMed Central

    Hawiger, Jack; Steckley, Sylvia; Hammond, Dianne; Cheng, Charles; Timmons, Sheila; Glick, Alan D.; Des Prez, Roger M.

    1979-01-01

    Bloodstream infections with staphylococci are accompanied by thromboembolic complications. We have studied the mechanism of the interaction of staphylococci with human blood platelets. Staphylococci that possess protein A, a bacterial receptor for the Fc fragment of immunoglobulin G (IgG), caused aggregation of human platelets in whole plasma accompanied by release of [3H]serotonin. These reactions were time and concentration dependent, requiring two or more staphylococci per platelet to give maximal response within 5 min. The interaction between staphylococci and platelets required the presence of cell wall-bound protein A and of IgG with an intact Fc fragment. It did not require an intact complement system. Cell wall-bound protein A (solid phase) was capable of aggregating human platelets in whole plasma. In contrast, free, solubilized protein A (fluid phase) did not cause measurable aggregation, and release of [3H]serotonin was reduced. An excess of free, solubilized protein A blocked aggregation of human platelets induced by staphylococci in whole plasma. The role of the Fc fragment of IgG in the staphylococci-human platelet interaction was demonstrated by an experiment in which free, isolated Fc fragment blocked aggregation of platelets in whole plasma induced by staphylococci. Furthermore, binding of 125I-protein A to human platelets was demonstrated in the presence of complete IgG with intact Fc fragment but not in the presence of the F(ab)2 fragment. Binding of the protein A-IgG complex to the human platelet Fc receptor was paralleled by the release of [3H]serotonin. These results represent a novel example of the interaction of two phylogenetically different Fc receptors, one on prokaryotic staphylococci and the other on human platelets. Their common ligand, IgG, is amplified by one Fc receptor (protein A) to react with another Fc receptor present on human platelets, which results in membrane-mediated aggregation and release reaction occurring in whole

  20. Mincle, the receptor for mycobacterial cord factor, forms a functional receptor complex with MCL and FcεRI-γ.

    PubMed

    Lobato-Pascual, Ana; Saether, Per Christian; Fossum, Sigbjørn; Dissen, Erik; Daws, Michael R

    2013-12-01

    Upon receptor activation, the myeloid C-type lectin receptor Mincle signals via the Syk-CARD9-Bcl10-MALT1 pathway. It does so by recruiting the ITAM-bearing FcεRI-γ. The related receptor macrophage C-type Lectin (MCL) has also been shown to be associated with Syk and to be dependent upon this signaling axis. We have previously shown that MCL co-precipitates with FcεRI-γ, but were unable to show a direct association, suggesting that MCL associates with FcεRI-γ via another molecule. Here, we have used rat primary cells and cell lines to investigate this missing link. A combination of flow cytometric and biochemical analysis showed that Mincle and MCL form heteromers on the cell surface. Furthermore, association with MCL and FcεRI-γ increased Mincle expression and enhanced phagocytosis of Ab-coated beads. The results presented in this paper suggest that the Mincle/MCL/FcεRI-γ complex is the functionally optimal form for these C-type lectin receptors on the surface of myeloid cells. PMID:23921530

  1. Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor

    PubMed Central

    Andersen, Jan Terje; Dalhus, Bjørn; Cameron, Jason; Daba, Muluneh Bekele; Plumridge, Andrew; Evans, Leslie; Brennan, Stephan O.; Gunnarsen, Kristin Støen; Bjørås, Magnar; Sleep, Darrell; Sandlie, Inger

    2012-01-01

    Albumin is the most abundant protein in blood where it has a pivotal role as a transporter of fatty acids and drugs. Like IgG, albumin has long serum half-life, protected from degradation by pH-dependent recycling mediated by interaction with the neonatal Fc receptor, FcRn. Although the FcRn interaction with IgG is well characterized at the atomic level, its interaction with albumin is not. Here we present structure-based modelling of the FcRn–albumin complex, supported by binding analysis of site-specific mutants, providing mechanistic evidence for the presence of pH-sensitive ionic networks at the interaction interface. These networks involve conserved histidines in both FcRn and albumin domain III. Histidines also contribute to intramolecular interactions that stabilize the otherwise flexible loops at both the interacting surfaces. Molecular details of the FcRn–albumin complex may guide the development of novel albumin variants with altered serum half-life as carriers of drugs. PMID:22215085

  2. Human neutrophil Fc receptor-mediated adhesion under flow: a hollow fibre model of intravascular arrest.

    PubMed

    D'Arrigo, C; Candal-Couto, J J; Greer, M; Veale, D J; Woof, J M

    1995-04-01

    Human polymorphonuclear cells (PMN) were found to adhere to a novel model of blood vessel wall-associated IgG. The internal surfaces of cellulose acetate hollow fibres, of comparable internal diameter to small blood vessels, were coated with normal serum human IgG, heat-aggregated IgG (HAIgG), laminin or fibrinogen. Under conditions of flow mimicking those in a small vessel, PMN were found to adhere markedly only to immunoglobulin-coated fibres. Arrest on HAIgG was inhibited by excess soluble IgG but not by bovine serum albumin (BSA), demonstrating that the adhesion was IgG-specific and presumably mediated by Fc gamma R on the PMN surface. Pre-adsorption of serum components onto HAIgG-coated fibres enhanced PMN arrest, due most probably to fixation of complement components by immobilized HAIgG, resulting in additional potential to entrap PMN via complement receptors such as CR3. Treatment of PMN with the regulatory neuropeptide substance P also enhanced adhesion to HAIgG-coated fibres and caused increased surface expression of Fc gamma RI, Fc gamma RII and Fc gamma RIII. A mouse cell line derived from L cells, hR4C6, stably transfected with human Fc gamma RII, was found to adhere under flow to HAIgG-coated fibres, whilst untransfected parent L cells did not. This adhesion was similarly inhibited by excess soluble IgG, confirming the capability of Fc gamma R to mediate cell arrest. The study strongly suggests that Fc gamma R may play an important role in intravascular PMN arrest and we speculate that in inflammatory diseases PMN may adhere via Fc gamma R to immobilized immunoglobulin on the vascular endothelium, with subsequent degranulation and tissue damage. PMID:7535210

  3. Altering Antibody-Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability.

    PubMed

    Hamblett, Kevin J; Le, Tiep; Rock, Brooke M; Rock, Dan A; Siu, Sophia; Huard, Justin N; Conner, Kip P; Milburn, Robert R; O'Neill, Jason W; Tometsko, Mark E; Fanslow, William C

    2016-07-01

    Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index. PMID:27248573

  4. Fc Receptor-Like Proteins in Pathophysiology of B-cell Disorder

    PubMed Central

    Capone, Mollie; Bryant, John Matthew; Sutkowski, Natalie; Haque, Azizul

    2016-01-01

    Members of the family of Fc receptor-like (FcRL) proteins, homologous to FcγRI, have been identified by multiple research groups. Consequently, they have been described using multiple nomenclatures including Fc receptor homologs (FcRH), immunoglobulin superfamily receptor translocation-associated genes (IRTA), immunoglobulin-Fc-gp42-related genes (IFGP), Src homology 2 domain-containing phosphatase anchor proteins (SPAP), and B cell cross-linked by anti-immunoglobulin M-activating sequences (BXMAS). They are now referred to under a unified nomenclature as FCRL. Eight different human FCRL genes have been identified, all of which appear to be related to the genes of the immunoglobulin superfamily (IgSF) of cellular adhesion molecules. These type 1 transmembrane glycoproteins are composed of different combinations of 5 types of immunoglobulin-like domains, with each protein consisting of 3 to 9 domains, and no individual domain type conserved throughout all of the FCRL proteins. Ligands for the majority of the FCRLs remain unknown. In general, FCRL expression is restricted to lymphocytes and is primarily expressed in B-lymphocytes, supporting FCRL’s involvement in a variety of immune disorders. Most FCRLs functionally repress B-cell activation; however, they might have dual roles in lymphocyte functions as these proteins often possess immunoreceptor tyrosine activation (ITAM) and inhibitory (ITIM) motif elements. The biological functions of these newly recognized FCRL proteins are just beginning to emerge, and might provide the insight necessary for understanding pathophysiology of lymphocyte disorders and treating different immune diseases. PMID:27446638

  5. Prognostic significance of the heterogenous expression of IgG Fc receptors in B-cell chronic lymphocytic leukemia.

    PubMed

    Schranz, V; Gráf, F

    1992-03-01

    Receptors for the Fc part of IgG (Fc gamma R) are expressed in three forms on peripheral blood lymphocytes. The presence of the releasable form (Fc gamma R(REL.)) as well as of the two nonreleasable forms with lower (Fc gamma R(LOW)) and higher (Fc gamma R(HIGH)) cellular avidity was correlated with survival in 63 patients with B-cell chronic lymphocytic leukemia (B-CLL). High percentage of cells with Fc gamma R(LOW) as well as high "absolute" number of cells carrying the two nonreleasable forms of Fc gamma R were connected to unfavorable prognosis. Combining these three parameters, an Fc gamma R constellation was defined which pointed to a favorable prognosis (in 24 patients) when all three parameters were low, but detected short survivors when all three data were high (in 14 patients). The Fc gamma R constellation was capable of identifying patients with better or worse prognosis within groups that were homogeneous regarding some other known prognostic factors. Fc gamma R constellation as a prognostic factor was shown to be independent of age, sex, and Rai and Binet stages, but it was found to be connected with the total tumor mass score (TTM). The three forms of Fc gamma R on B cells might reflect stages of B-cell activation. Differences in Fc gamma R constellations between patients with B-CLL would thus correspond to differently activated B-cell clones with variable prognosis. PMID:1571409

  6. Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function.

    PubMed

    Ren, Li; Campbell, Amanda; Fang, Huiqing; Gautam, Shalini; Elavazhagan, Saranya; Fatehchand, Kavin; Mehta, Payal; Stiff, Andrew; Reader, Brenda F; Mo, Xiaokui; Byrd, John C; Carson, William E; Butchar, Jonathan P; Tridandapani, Susheela

    2016-02-01

    The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function. PMID:26627823

  7. Subclass specificity of the Fc receptor for human IgG on K562.

    PubMed

    Chiofalo, M S; Teti, G; Goust, J M; Trifiletti, R; La Via, M F

    1988-07-01

    The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2). PMID:2968843

  8. In Vitro Glycoengineering of IgG1 and Its Effect on Fc Receptor Binding and ADCC Activity

    PubMed Central

    Thomann, Marco; Schlothauer, Tilman; Dashivets, Tetyana; Malik, Sebastian; Avenal, Cecile; Bulau, Patrick; Rüger, Petra; Reusch, Dietmar

    2015-01-01

    The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity. PMID:26266936

  9. Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus

    PubMed Central

    Hargreaves, Chantal E.; Iriyama, Chisako; Rose-Zerilli, Matthew J. J.; Nagelkerke, Sietse Q.; Hussain, Khiyam; Ganderton, Rosalind; Lee, Charlotte; Machado, Lee R.; Hollox, Edward J.; Parker, Helen; Latham, Kate V.; Kuijpers, Taco W.; Potter, Kathleen N.; Coupland, Sarah E.; Davies, Andrew; Stackpole, Michael; Oates, Melanie; Pettitt, Andrew R.; Glennie, Martin J.; Cragg, Mark S.; Strefford, Jonathan C.

    2015-01-01

    Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics. PMID:26545243

  10. Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus.

    PubMed

    Hargreaves, Chantal E; Iriyama, Chisako; Rose-Zerilli, Matthew J J; Nagelkerke, Sietse Q; Hussain, Khiyam; Ganderton, Rosalind; Lee, Charlotte; Machado, Lee R; Hollox, Edward J; Parker, Helen; Latham, Kate V; Kuijpers, Taco W; Potter, Kathleen N; Coupland, Sarah E; Davies, Andrew; Stackpole, Michael; Oates, Melanie; Pettitt, Andrew R; Glennie, Martin J; Cragg, Mark S; Strefford, Jonathan C

    2015-01-01

    Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics. PMID:26545243

  11. Identification and characterization of a Fc receptor activity on the Toxoplasma gondii tachyzoite.

    PubMed

    Vercammen, M; el Bouhdidi, A; Ben Messaoud, A; de Meuter, F; Bazin, H; Dubremetz, J F; Carlier, Y

    1998-01-01

    The Immunoglobulin (Ig) binding capacity of Toxoplasma gondii tachyzoites was investigated using fluorescence flow-cytometry analysis. Polyclonal mouse, human and rat immunoglobulins without specific anti-Toxoplasma activity bound to parasites in a concentration-dependent manner, saturating them at circulating serum concentrations. The immunoglobulin class and subclass specificity of binding was investigated using irrelevant monoclonal antibodies. IgM, IgA and IgG reacted with the parasite membrane. The attachment of mouse IgM to the parasite surface was hampered by mouse IgG1, IgG2a, IgG2b and IgG3. The binding of mouse IgG was proportionally reduced with increasing concentrations of mouse monoclonal IgM. The binding of murine immunoglobulin was diminished when in presence of human IgG. Purified Fc- but not Fab portions of immunoglobulins, fixed to parasites. Using labelled calibrated beads, the Ig binding capacity of parasites was estimated to be 6900 +/- 500 sites per tachyzoite. The Kd of the T. gondii Fc Receptor (FcR) activity was determined at 1.4 +/- 0.1 microM (mean +/- SEM). Such FcR activity was reduced by phospholipase C, trypsin and pronase treatment of the parasites. These data show a low affinity FcR activity on T. gondii tachyzoites which recognizes Ig of different species and isotypes and is likely supported by a glycosyl-phosphatidylinositol (GPI)-anchored surface protein of the parasite. PMID:9491416

  12. Suppression of Fcγ-receptor-mediated antibody effector function during persistent viral infection.

    PubMed

    Yamada, Douglas H; Elsaesser, Heidi; Lux, Anja; Timmerman, John M; Morrison, Sherie L; de la Torre, Juan Carlos; Nimmerjahn, Falk; Brooks, David G

    2015-02-17

    Understanding how viruses subvert host immunity and persist is essential for developing strategies to eliminate infection. T cell exhaustion during chronic viral infection is well described, but effects on antibody-mediated effector activity are unclear. Herein, we show that increased amounts of immune complexes generated in mice persistently infected with lymphocytic choriomeningitis virus (LCMV) suppressed multiple Fcγ-receptor (FcγR) functions. The high amounts of immune complexes suppressed antibody-mediated cell depletion, therapeutic antibody-killing of LCMV infected cells and human CD20-expressing tumors, as well as reduced immune complex-mediated cross-presentation to T cells. Suppression of FcγR activity was not due to inhibitory FcγRs or high concentrations of free antibody, and proper FcγR functions were restored when persistently infected mice specifically lacked immune complexes. Thus, we identify a mechanism of immunosuppression during viral persistence with implications for understanding effective antibody activity aimed at pathogen control. PMID:25680277

  13. CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

    PubMed

    Amash, Alaa; Wang, Lin; Wang, Yawen; Bhakta, Varsha; Fairn, Gregory D; Hou, Ming; Peng, Jun; Sheffield, William P; Lazarus, Alan H

    2016-04-15

    Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions. PMID:26944929

  14. Imaging and measuring the biophysical properties of Fc gamma receptors on single macrophages using atomic force microscopy

    SciTech Connect

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

    2013-09-06

    Highlights: •Nanoscale cellular ultra-structures of macrophages were observed. •The binding affinities of FcγRs were measured directly on macrophages. •The nanoscale distributions of FcγRs were mapped on macrophages. -- Abstract: Fc gamma receptors (FcγR), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of FcγRs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measure the biophysical properties (affinity and distribution) of FcγRs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of FcγRs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the FcγRs, revealing the nanoscale distribution of FcγRs on local areas of macrophages. The experimental results can improve our understanding of FcγRs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy.

  15. High-Affinity Fc Receptor Expression Indicates Relative Immaturity in Human Monocytes.

    PubMed

    Clanchy, Felix I L

    2016-05-01

    Within monocyte heterogeneity, subsets represent discrete, well-characterized phenotypes. Although many studies have highlighted differences between subsets, there is evidence that subpopulations represent contiguous stages in a maturational series. As CD14(hi)CD64(hi) monocytes have higher proliferative potential than CD14(hi)CD64(lo) monocytes, the surface marker profile on 4 subsets defined by CD14 and CD64 was measured. The profiles were compared to that of subsets defined by the high-affinity IgE receptor (FcɛRIα), CD16, and CD14; further differences in size, granularity, and buoyancy were measured in subsets delineated by these markers. There was a positive correlation between proliferative monocyte (PM) prevalence and CD64 expression on the classical monocyte subset, and also between PM prevalence and circulating FcɛRIα(+) monocytes. The expression of CD64, the high-affinity IgG receptor, on canonical human monocyte subsets was determined before and after short-term culture, and in response to interleukin (IL)-6, IL-10, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and interferon-γ; the influence of these cytokines on monocyte subset transition was also measured. The loss of FcɛRIα expression preceded an increase in CD16 expression in whole blood cultures. These data indicate that high-affinity Fc receptors are expressed on less mature monocytes and that FcɛRIα(+) monocytes are developmentally antecedent to the canonical classical and intermediate monocyte subsets. PMID:26714112

  16. Altered Ig levels and antibody responses in mice deficient for the Fc receptor for IgM (FcμR).

    PubMed

    Honjo, Kazuhito; Kubagawa, Yoshiki; Jones, Dewitt M; Dizon, Brian; Zhu, Zilu; Ohno, Hiroshi; Izui, Shozo; Kearney, John F; Kubagawa, Hiromi

    2012-09-25

    Cell surface Fc receptor for IgM antibody (FcμR) is the most recently identified member among FcRs. We determined the cellular distribution of mouse FcμR and the functional consequences of Fcmr disruption. Surface FcμR expression was restricted to B-lineage cells, from immature B to plasma cells, except for a transient down-modulation during germinal center reactions. Fcmr ablation had no significant effect on overall B- and T-cell development, but led to a reduction of marginal zone B cells and an increase in splenic B1 B cells. Preimmune serum IgM in mutant mice was significantly elevated as were natural autoantibodies. When immunized with live attenuated pneumococci, mutant mice mounted robust antibody responses against phosphorylcholine, but not protein, determinants compared with wild-type mice. By contrast, upon immunization with a hapten-carrier conjugate, nitrophenyl-coupled chicken γ-globulin (NP-CGG), the mutant mice had a diminished primary IgG1 response to both NP and CGG. These findings suggest that FcμR has an important role in IgM homeostasis and regulation of humoral immune responses. PMID:22984178

  17. Functional characteristics of enhanced Fc receptor expression of beta 2 integrin-deficient bovine mononuclear phagocytes.

    PubMed

    Nagahata, H; Higuchi, H; Goji, N; Noda, H; Kuwabara, M

    1996-01-01

    Fc receptor expression, cytoplasmic Ca2+ signaling, chemiluminescent (CL) response, and electron spin resonance (ESR) combined with spin trapping of blood mononuclear phagocytes from control heifers and a heifer with leukocyte adhesion deficiency (LAD) were evaluated to elucidate the relationships between complement receptor type 3 (CR3) and Fc receptor expression and their functional responses. The mean fluorescence intensity of fluorescein isothiocyanate (FITC)-conjugated anti-bovine IgG bound to mononuclear phagocytes from the heifer with LAD was 1.8-fold higher than that of control heifers. The mean increments of cytoplasmic Ca2+ concentrations of mononuclear phagocytes from the heifer with LAD stimulated with OPZ, Agg-IgG, and PMA were 39.4 (P < 0.05), 118, and 71.6% compared with those of control heifers. A 1.27-fold increase in the CL response relative to control heifers was detected when mononuclear phagocytes from the heifer with LAD were stimulated with Agg-IgG. The OPZ-induced CL response of mononuclear phagocytes from the heifer with LAD was significantly (P < 0.05) decreased, whereas the PMA-induced CL response was similar to that of control heifers. The ESR spectrum of mononuclear phagocytes from the heifer with LAD was increased when stimulated with Agg-IgG, and was impaired when stimulated by OPZ compared with that of control heifers. The ESR spectrum of mononuclear phagocytes stimulated with PMA was similar in control heifers and the heifer with LAD. Fc receptors on mononuclear phagocytes from the heifer with LAD were enhanced, and their cytoplasmic Ca2+ signaling, CL response, and ESR-spin trapping when stimulated with Agg-IgG and OPZ appeared to be associated with enhanced Fc receptors. PMID:8805104

  18. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

    PubMed Central

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  19. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome.

    PubMed

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  20. Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection

    PubMed Central

    Palaniyandi, Senthilkumar; Zeng, Rongyu; Tuo, Wenbin; Roopenian, Derry C.; Zhu, Xiaoping

    2011-01-01

    IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about where and how IgG enters the lumen of the genital tract and the exact role local IgG plays in preventing sexually transmitted diseases. We demonstrate here that the neonatal Fc receptor, FcRn, is expressed in female genital tract epithelial cells of humans and mice and binds IgG in a pH-dependent manner. In vitro we show that FcRn mediates bidirectional IgG transport across polarized human endometrial HEC-1-A monolayers and primary human genital epithelial cells. Furthermore, endosomal acidification appears to be a prerequisite for FcRn-mediated IgG transcytosis; IgG transcytosis was demonstrated in vivo by translocation of systemically administered IgG into the genital lumen in WT but not FcRn-KO mice. The biological relevance of FcRn-transported IgG was demonstrated by passive immunization using herpes simplex virus-2 (HSV-2)–specific polyclonal serum, which conferred significantly higher protection against intravaginal challenge infection by the HSV-2 186 strain in WT mice than in FcRn-KO mice. These studies demonstrate that FcRn-mediated transport is a mechanism by which IgG can act locally in the female genital tract in immune surveillance and in host defense against sexually transmitted diseases. PMID:21368166

  1. Targeted IgA Fc receptor I (FcαRI) therapy in the early intervention and treatment of pristane-induced lupus nephritis in mice.

    PubMed

    Liu, C; Kanamaru, Y; Watanabe, T; Tada, N; Horikoshi, S; Suzuki, Y; Liu, Z; Tomino, Y

    2015-09-01

    The Fc receptor I for IgA (FcαRI) down-regulates humoral immune responses and modulates the risk of autoimmunity. This study aimed to investigate whether FcαRI targeting can affect progression of pristine-induced lupus nephritis. In the first experiment (early intervention), four groups of animals were evaluated: untreated FcαRI/FcRγ transgenic (Tg) mice and Tg mice administered control antibody (Ctr Fab), saline and anti-FcαRI Fab [macrophage inflammatory protein (MIP)-8a], respectively, three times a week for 29 weeks, after being injected once intraperitoneally with 0·5 ml pristane. In the second experiment, antibody injection started after the onset of nephritis and was carried out for 2 months, with similar groups as described above. MIP-8a improved proteinuria, decreased the amounts of glomerular injury markers, serum interleukin (IL)-6, IL-1 and monocyte chemoattractant protein (MCP)-1, and F4/80 macrophages in the interstitium and glomeruli, in both experiments. When MIP-8a was used as early intervention, a decrease in mouse serum anti-nuclear antibody (ANA) titres and reduced deposition of immunoglobulins in glomeruli were observed. This effect was associated with reduced serum titres of immunoglobulin (Ig)G2a but not IgG1, IgG2b and IgG3. Furthermore, pathological analysis showed lower glomerular activity index and less fibronectin in MIP-8a treated mice. This study suggests that FcαRI targeting could halt disease progression and lupus activation by selective inhibition of cytokine production, leucocyte recruitment and renal inflammation. Our findings provide a basis for the use of FcαRI as a molecular target for the treatment of lupus. PMID:25907714

  2. Enhanced expression of Fc receptors on neutrophils from calves with leukocyte adhesion deficiency.

    PubMed

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

    1995-01-01

    The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P < 0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 micrograms/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18). PMID:8577284

  3. Stimulatory effects of ingenols from Euphorbia kansui on the expression of macrophage Fc receptor.

    PubMed

    Matsumoto, T; Cyong, J C; Yamada, H

    1992-06-01

    Immune complex binding to macrophages was enhanced by treatment with an E. kansui extract. Systematic fractionation of the extract led to the characterization of 3-O-(2'E,4'Z-decadienoyl)- and 3-O-(2,3-dimethylbutyryl)-13- O-n-dodecanoyl-13-hydroxyingenol as the active principles. Immune complex binding to macrophages by the action of these compounds increased in a dose-dependent manner. When each ingenol (10 nM) was added to the separated culture medium, the immune complex binding ability of macrophages increased up to 2-fold, respectively. Scatchard analysis showed the enhanced expression of the Fc-receptor for gamma-globulin by the action of each ingenol to macrophages. This Fc-receptor upregulation was dependent on RNA synthesis, suggesting a possible de novo synthesis. PMID:1409980

  4. Interactions of phagocytes with the Lyme disease spirochete: role of the Fc receptor

    SciTech Connect

    Benach, J.L.; Fleit, H.B.; Habicht, G.S.; Coleman, J.L.; Bosler, E.M.; Lane, B.P.

    1984-10-01

    The phagocytic capacity of murine and human mononuclear and polymorphonuclear phagocytes (including peripheral blood monocytes and neutrophils), rabbit and murine peritoneal exudate cells, and the murine macrophage cell line P388D1 against the Lyme disease spirochete was studied. All of these cells were capable of phagocytosing the spirochete; phagocytosis was measured by the uptake of radiolabeled spirochetes, the appearance of immunofluorescent bodies in phagocytic cells, and electron microscopy. Both opsonized and nonopsonized organisms were phagocytosed. The uptake of opsonized organisms by neutrophils was blocked by a monoclonal antibody specific for the Fc receptor and by immune complexes; these findings suggested that most phagocytosis is mediated by the Fc receptor. Similarly, the uptake of opsonized organisms by human monocytes was inhibited by human monomeric IgG1 and by immune complexes. These results illustrate the role of immune phagocytosis of spirochetes in host defense against Lyme disease.

  5. Fully human monoclonal antibody inhibitors of the neonatal fc receptor reduce circulating IgG in non-human primates.

    PubMed

    Nixon, Andrew E; Chen, Jie; Sexton, Daniel J; Muruganandam, Arumugam; Bitonti, Alan J; Dumont, Jennifer; Viswanathan, Malini; Martik, Diana; Wassaf, Dina; Mezo, Adam; Wood, Clive R; Biedenkapp, Joseph C; TenHoor, Chris

    2015-01-01

    The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach - depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases. PMID:25954273

  6. Structural and phylogenetic analysis of the MHC class I-like Fc receptor gene

    SciTech Connect

    Kandil, Eman; Ishibashi, Teruo; Kasahara, Masanori

    1995-06-01

    The intestinal epithelium of neonatal mice and rats expresses an Fc receptor that mediates selective uptake of IgG in mothers`milk. This receptor (FcRn), which helps newborn animals to acquire passive immunity, is an MHC class I-like heterodimer made up of a heavy chain and {beta}{sub 2}-microglobulin. In the present study, we determined the genomic structure of a mouse gene (FcRn) encoding the heavy of FcRn. The overall exon-intron organization of the Fcrn gene was similar to that of the Fcrn gene, thus providing structural evidence that Fcrn os a bona fide class I gene. The 5{prime}-flanking region of the Fcrn gene contained the binding motifs for two cytokine-inducible transcription factors, NF-IL6 and NF1. However, regulatory elements found in MHC class I genes (enhancer A, enhancer B, and the IFN response element) were absent. Phylogenetic tree analysis suggested that, like the MICA, AZGP1, and CD1 genes, the Fcrn gene diverged form MHC class I genes after the emergence of amphibians but before the split of placental and marsupial mammals. Consistent with this result, Southern blot analysis with a mouse Fcrn cDNA probe detected cross-hybridizing bands in various mammalian species and chickens. Sequence analysis of the Fcrn gene isolated from eight mouse strains showed that the membrane-distal domain of FcRn has at least three amino acid variants. The fact that Fcrn is a single copy gene indicates that it is expressed in both the neonatal intestine and the fetal yolk sac. 74 refs., 7 figs., 2 tabs.

  7. Emerging Functions of Natural IgM and Its Fc Receptor FCMR in Immune Homeostasis

    PubMed Central

    Wang, Hongsheng; Coligan, John E.; Morse, Herbert C.

    2016-01-01

    Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions. PMID:27014278

  8. Cloning and characterization of an immunoglobulin A Fc receptor from cattle.

    PubMed

    Morton, H Craig; Pleass, Richard J; Storset, Anne K; Dissen, Erik; Williams, John L; Brandtzaeg, Per; Woof, Jenny M

    2004-02-01

    Here, we describe the cloning, sequencing and characterization of an immunoglobulin A (IgA) Fc receptor from cattle (bFcalphaR). By screening a translated EST database with the protein sequence of the human IgA Fc receptor (CD89) we identified a putative bovine homologue. Subsequent polymerase chain reaction (PCR) amplification confirmed that the identified full-length cDNA was expressed in bovine cells. COS-1 cells transfected with a plasmid containing the cloned cDNA bound to beads coated with either bovine or human IgA, but not to beads coated with bovine IgG2 or human IgG. The bFcalphaR cDNA is 873 nucleotides long and is predicted to encode a 269 amino-acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a short cytoplasmic tail devoid of known signalling motifs. Genetically, bFcalphaR is more closely related to CD89, bFcgamma2R, NKp46, and the KIR and LILR gene families than to other FcRs. Moreover, the bFcalphaR gene maps to the bovine leucocyte receptor complex on chromosome 18. Identification of the bFcalphaR will aid in the understanding of IgA-FcalphaR interactions, and may facilitate the isolation of FcalphaR from other species. PMID:15027906

  9. Emerging Functions of Natural IgM and Its Fc Receptor FCMR in Immune Homeostasis.

    PubMed

    Wang, Hongsheng; Coligan, John E; Morse, Herbert C

    2016-01-01

    Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions. PMID:27014278

  10. Crosslinking of surface antibodies and Fc sub. gamma. receptors: Theory and application

    SciTech Connect

    Wofsy, C.; Goldstein, B. Los Alamos National Lab., NM )

    1991-03-15

    In an immune response, the crosslinking of surface immunoglobulin (sIg) on B cells by multiply-bound ligand activates a range of cell responses, culminating in the production of antibody-secreting cells. However, when the crosslinking agent is itself an antibody, B cell activation is inhibited. Solution antibody (IgG) can bind simultaneously to sIg and to another cell surface receptor, Fc{sub {gamma}}R, co-crosslinking' the distinct receptors. Experiments point to co-crosslinking as the inhibitory signal. It is not clear how co-crosslinking inhibits B cell stimulation. The authors construct and analyze a mathematical model aimed at clarifying the nature and mechanisms of action of the separate cell signals controlling B cell responses to antibodies. Basophils and mast cells respond to the crosslinking of cell surface antibody by releasing histamine. Like B cells, basophils also express FC{sub {gamma}}R. They use their model to analyze new data on the effect of antibody-induced co-crosslinking of the two types of receptor on this family of cells. Predictions of the model indicate that an observed difference between the response patterns induced by antibodies and by antibody fragments that cannot bind to FC{sub {gamma}}R can be explained if co-crosslinking is neither inhibitory nor stimulatory in this system.

  11. The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fcα receptor interactions.

    PubMed

    Mattu, T S; Pleass, R J; Willis, A C; Kilian, M; Wormald, M R; Lellouch, A C; Rudd, P M; Woof, J M; Dwek, R A

    1998-01-23

    The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction with various Fc receptors (Fc Rs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab and Fc as well as three recombinant IgA1 molecules, including two N-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated that O-glycans are located at Thr228, Ser230, and Ser232, while O-glycan sites at Thr225 and Thr236 are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where < 10% contain sialic acid. This paper contains the first report of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα 2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary structures. Further analysis of these data suggested that processing at one Fc N-glycosylation site affects the other. Neutrophil Fcα R binding studies, using recombinant IgA1, indicated that neither the tailpiece region nor the N-glycans in the C alpha 2 domain contribute to IgA1-neutrophil Fcα R binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the Fcγ R. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence of both the primary sequence and S-S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα 2 N-glycans provides an explanation for the

  12. Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

    PubMed Central

    Zheng, L; Nibbering, P H; Zomerdijk, T P; van Furth, R

    1994-01-01

    Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes. Images PMID:7927687

  13. RSV neutralization by palivizumab, but not by monoclonal antibodies targeting other epitopes, is augmented by Fc gamma receptors.

    PubMed

    van Mechelen, Lenny; Luytjes, Willem; de Haan, Cornelis A M; Wicht, Oliver

    2016-08-01

    Palivizumab efficiently blocks respiratory syncytial virus (RSV) infection in vitro. However, virus neutralization assays generally omit Fc region-mediated effects. We investigated the neutralization activity of RSV-specific monoclonal antibodies on cells with Fc receptors. Subneutralizing concentrations of antibodies resulted in antibody-dependent enhancement of RSV infection in monocytic cells. Contrary to antibodies targeting other epitopes, the neutralization by palivizumab was augmented in cells with Fc receptors. This unrecognized characteristic of palivizumab may be relevant for its performance in vivo. PMID:27185625

  14. Structural basis for binding of human IgG1 to its high-affinity human receptor FcγRI

    PubMed Central

    Kiyoshi, Masato; Caaveiro, Jose M.M.; Kawai, Takeaki; Tashiro, Shinya; Ide, Teruhiko; Asaoka, Yoshiharu; Hatayama, Kouta; Tsumoto, Kouhei

    2015-01-01

    Cell-surface Fcγ receptors mediate innate and adaptive immune responses. Human Fcγ receptor I (hFcγRI) binds IgGs with high affinity and is the only Fcγ receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcγRI's interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFcγRI and human Fc, at 1.80 Å resolution, revealing an unique hydrophobic pocket at the surface of hFcγRI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFcγRI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFcγRI. PMID:25925696

  15. Variation in the expression of macrophage Fc epsilon receptors in relation to experimental rat schistosome infection.

    PubMed

    Pestel, J; Joseph, M; Dessaint, J P; Capron, A

    1988-01-01

    The relationship between IgE and rat peritoneal macrophages during the course of Schistosoma mansoni infection was examined. Immune macrophages exhibited an IgE-dependent schistosomulicidal activity mainly between the 5th and the 7th weeks. At this period the percentages of anti-IgE rosettes whose variations appeared consecutive to the variations in the serum IgE level were higher. Following incubation in a serum-free medium immune macrophages could release membrane-associated IgE, to a certain extent, and consequently formed rosettes with IgE-coated erythrocytes, indicating the existence of Fc epsilon receptors. Thus, in terms of rosettes, their number varied in the course of the disease. In fact IgE-rich serum (i.e. at day 42) as IgE molecules could induce this macrophage Fc epsilon receptor. Moreover when transferred to syngeneic rats in association with parasite-specific IgE Fc epsilon R2+ macrophages led to some protection against challenge infection. All the data reported here emphasize the determining role of macrophages and IgE in immunity to schistosomiasis. PMID:2962948

  16. Neonatal Fc Receptor-Mediated IgG Transport Across Porcine Intestinal Epithelial Cells: Potentially Provide the Mucosal Protection.

    PubMed

    Guo, Jinyue; Li, Fei; He, Qigai; Jin, Hui; Liu, Mei; Li, Shaowen; Hu, Sishun; Xiao, Yuncai; Bi, Dingren; Li, Zili

    2016-06-01

    It has been well characterized that piglets can absorb colostrum IgG across the intestine to neonatal bloodstream and a certain level of IgG has been found in the mucosal secretions of the porcine intestinal tract. However, little is known about how the maternal IgG transport across the intestinal barrier and how IgG enter the lumen of intestinal tract. In this study, we demonstrated that the porcine neonatal Fc receptor (pFcRn) was expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2) as well as in kidney cells (PK-15), and pFcRn was mainly distributed in the apical side of the polarized IPEC-J2 cells. Analyzing the phylogenetic relatedness of this gene we found that swine and human neonatal Fc receptor (FcRn) amino acid sequence are closer than rodents. We also showed that pFcRn mediated bidirectional IgG transport across polarized IPEC-J2 cells and bound to IgG in a pH-dependent manner. Furthermore, pFcRn-transcytosed viral-specific IgG reduced the transmissible gastroenteritis virus (TGEV) yield from the luminal direction by a 50% tissue culture infective dose (TCID50) assay. Our results indicate that pFcRn-dependent bidirectional IgG transport across the intestinal epithelium plays critical role in the acquisition of humoral immunity in early life and in host defense at mucosal surfaces. PMID:26982157

  17. The anorexic effect of Ex4/Fc through GLP-1 receptor activation in high-fat diet fed mice.

    PubMed

    Liu, Rui; Ma, Duan; Li, Yiming; Hu, Renming; Peng, Yongde; Wang, Qinghua

    2014-08-01

    Exendin-4 (Ex4), a peptide initially found in the saliva of the Gila monster, can activate the signaling pathway of the incretin hormone glucagon-like peptide-1 (GLP-1) through the GLP-1 receptor (GLP-1R). We previously reported that a chimera protein consisting of Ex4 and mouse IgG heavy chain constant regions (Ex4/Fc) can exert biological effects of GLP-1, such as improving glycemic control and ameliorating manifestations in diabetic mice. The aim of this study was to determine whether Ex4/Fc is effective in modulating energy homeostasis in mice. Our results showed that in vivo expression of Ex4/Fc by intramuscular injection of the plasmid encoding Ex4/Fc followed by local electroporation effectively decreased food intake in the mice on high-fat diet (HFD) feeding. In addition, the reduced energy intake was associated with the decreased excrements from the Ex4/Fc-treated HFD mice but not the Fc control mice. Remarkably, the Ex4/Fctreated HFD mice displayed significantly lower triglyceride (TG) levels when compared with the control mice. Interestingly, while the leptin levels were not changed, the circulating ghrelin levels were higher in Ex4/Fc mice than those in the Fc control mice. These results suggested that Ex4/Fc can improve energy metabolism and lipid metabolism through GLP-1R in mice under excessive nutrition conditions. PMID:24951724

  18. Fc Receptor-Mediated Phagocytosis in Tissues as a Potent Mechanism for Preventive and Therapeutic HIV Vaccine Strategies

    PubMed Central

    Sips, Magdalena; Krykbaeva, Marina; Diefenbach, Thomas J.; Ghebremichael, Musie; Bowman, Brittany A.; Dugast, Anne-Sophie; Boesch, Austin W.; Streeck, Hendrik; Kwon, Douglas S.; Ackerman, Margaret E.; Suscovich, Todd J.; Brouckaert, Peter; Schacker, Timothy W.; Alter, Galit

    2016-01-01

    While the development of a protective HIV vaccine is the ultimate goal of HIV research, to date only one HIV vaccine trial, the RV144, has successfully induced a protective response. The 31% protection from infection achieved in the RV144 trial was linked to the induction of non-neutralizing antibodies, able to mediate ADCC, suggestive of an important role of Fc-mediated functions in protection. Likewise, Fc-mediated antiviral activity was recently shown to play a critical role in actively suppressing the viral reservoir, however, the Fc-effector mechanisms within tissues that provide protection from or after infection are largely unknown. Here we aimed to define the landscape of effector cells and Fc-receptors present within vulnerable tissues. We found negligible Fc-receptor expressing NK cells in the female reproductive and gastrointestinal mucosa. Conversely, Fc-receptor expressing macrophages were highly enriched in most tissues, but neutrophils mediated superior antibody-mediated phagocytosis. Modifications in Fc domain of VRC01 antibody increased phagocytic responses in both phagocytes. These data suggest that non-ADCC mediated mechanisms, such as phagocytosis and neutrophil activation, are more likely to play a role in preventative vaccine or reservoir-eliminating therapeutic approaches. PMID:26883728

  19. Neonatal Fc Receptor Binding Tolerance toward the Covalent Conjugation of Payloads to Cysteine 34 of Human Albumin Variants.

    PubMed

    Petersen, Steffan S; Kläning, Eva; Ebbesen, Morten F; Andersen, Birgitte; Cameron, Jason; Sørensen, Esben S; Howard, Kenneth A

    2016-02-01

    The long circulatory half-life of albumin facilitated by the interaction with the cellular recycling neonatal Fc receptor (FcRn) is utilized for drug half-life extension. FcRn engagement effects following covalent attachment of cargo to cysteine 34, however, have not been investigated. Poly(ethylene glycol) polymers were used to study the influence of cargo molecular weight on human FcRn engagement of recombinant wild type (WT) albumin and an albumin variant engineered for increased FcRn binding. Decreased affinity was observed for all conjugates; however, the engineered albumin maintained an affinity above that of unmodified wild type albumin that promotes it as an attractive drug delivery platform. PMID:26654692

  20. Expression of Fc gamma and complement receptors in monocytes of X-linked agammaglobulinaemia and common variable immunodeficiency patients.

    PubMed

    Amoras, A L B; da Silva, M T N; Zollner, R L; Kanegane, H; Miyawaki, T; Vilela, M M S

    2007-12-01

    Recently we reported that monocyte phagocytosis and chemotaxis are impaired in X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVI) patients. Few data exist on the in vivo expression of receptors for the constant region of immunoglobulin (IgG) (Fc gammaR) and complement receptors (CR) in these patients. The objective of this study was to investigate the expression of Fc gammaR and CR on monocytes from XLA and CVI patients and compare it to that of healthy controls. Whole blood samples were obtained from 10 patients with XLA, 12 with CVI and 18 healthy controls. Monocyte phenotype was determined by flow cytometry with gating on CD14+ cells. Surface expression of Fc gammaRI (CD64), Fc gammaRII (CD32) and Fc gammaRIII (CD16), CR1 (CD35) and CR3 (CD11b and CD18) was measured by determination of the proportion of CD14+ cells positive for each receptor and by receptor density. Compared to controls, a significantly higher percentage of CD16 and CD35+ monocytes from XLA (P = 0.002 and P = 0.007, respectively) were observed. The relative fluorescence intensity (RFI) expression of Fc gammaRII (CD32) and Fc gammaRIII (CD16) were significantly lower on CVI monocytes compared to controls (P = 0.001 and P = 0.035, respectively). XLA patients, who have a reduction of Bruton's tyrosine kinase (Btk), showed normal or increased percentages of monocytes expressing Fc gamma and complement receptors. CVI patients, who have normal expression of Btk, showed reduced expression of CD16 and CD32 on monocytes. Inefficient chemotaxis and phagocytosis, reported previously in XLA patients, could be due to defects of cytoplasmatic transduction mechanisms. PMID:17900300

  1. Sequential Engagement of FcεRI on Mast Cells and Basophil Histamine H4 Receptor and FcεRI in Allergic Rhinitis

    PubMed Central

    Shiraishi, Yoshiki; Jia, Yi; Domenico, Joanne; Joetham, Anthony; Karasuyama, Hajime; Takeda, Katsuyuki; Gelfand, Erwin W.

    2012-01-01

    Histamine H4 receptor (H4R)-deficient mice (H4R−/−), H4R antagonist-treated WT mice, and WT mice depleted of basophils failed to develop early (EPR) or late phase (LPR) nasal responses following allergen sensitization and challenge. Basophil transfer from WT but not H4R−/− mice restored the EPR and LPR in H4R−/− mice. Following passive sensitization with OVA-specific IgE, FcεRI−/− recipients of WT basophils plus OVA and histamine developed an EPR and LPR. OVA-IgE passively sensitized FcεRI−/− recipients of H4R−/− basophils and OVA and histamine challenge failed to develop an EPR or LPR, and basophils were not detected in nasal tissue. In contrast, recipients of basophils from IL-13−/− and IL-4−/−/IL-13−/− mice developed an EPR but not LPR. These results demonstrate the development of allergic rhinitis proceeded in two distinct stages: histamine release from FcεRI-activated mast cells, followed by histamine-mediated recruitment of H4R-expressing basophils to the nasal cavity and activation through FcεRI. PMID:23241885

  2. Biochemical signal transmitted by Fc gamma receptors: phospholipase A2 activity of Fc gamma 2b receptor of murine macrophage cell line P388D1.

    PubMed Central

    Suzuki, T; Saito-Taki, T; Sadasivan, R; Nitta, T

    1982-01-01

    The detergent lysate of the P388D1 macrophage cell line was subjected to affinity chromatography on two different media, Sepharose coupled to heat-aggregated human IgG (IgG-Sepharose) and Sepharose coupled to the phosphatidylcholine analog rac-1-(9-carboxyl)nonyl-2-hexadecylglycero-3-phosphocholine (PC-Sepharose). Both IgG- and phosphatidylcholine-binding proteins were further purified by Sephadex G-100 gel filtration and isoelectric focusing in the presence of 6 M urea. The isolated IgG-binding proteins specifically bound to IgG2a, but not to IgG2b, whereas the isolated phosphatidylcholine-binding proteins specifically bound to IgG2b but not to IgG2a. Phosphatidylcholine-binding proteins possessed a typical phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4), which was maximal (10 mumol/min per mg of protein) at pH 9.5, depended on Ca2+, and was specific for cleavage of fatty acid from the C-2 position of the glycerol backbone of phosphatidylcholine. The noted enzymatic activity was augmented 4-fold by preincubating phosphatidylcholine-binding proteins with heat-aggregated murine IgG2b but not with IgG2a. IgG-binding proteins, on the other hand, are devoid of any detectable phospholipase A2 activity. Thus, the functional significance of Fc gamma 2b receptor of P388D1 macrophage cell line would be the generation of phospholipase A2 activity at the cell surface upon specific binding to Fc gamma 2b fragment. PMID:6804944

  3. A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life

    PubMed Central

    Souders, Colby A; Nelson, Stuart C; Wang, Yang; Crowley, Andrew R; Klempner, Mark S; Thomas, William

    2015-01-01

    Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is well-known that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties. PMID:26018774

  4. A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life.

    PubMed

    Souders, Colby A; Nelson, Stuart C; Wang, Yang; Crowley, Andrew R; Klempner, Mark S; Thomas, William

    2015-01-01

    Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is well-known that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties. PMID:26018774

  5. Macrophage Membrane Potential Changes Associated with γ 2b/γ 1 Fc Receptor-Ligand Binding

    NASA Astrophysics Data System (ADS)

    Young, John Ding-E; Unkeless, Jay C.; Kaback, H. Ronald; Cohn, Zanvil A.

    1983-03-01

    We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (Δ Psi ) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in Δ Psi , but a sustained depolarization lasting ≈ 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+, K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.

  6. Localization of the binding site for the human high-affinity Fc receptor on IgG.

    PubMed

    Duncan, A R; Woof, J M; Partridge, L J; Burton, D R; Winter, G

    1988-04-01

    A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen. PMID:2965792

  7. Human Fc(gamma) receptors for differentiation in throat cultures of group C "Streptococcus equisimilis" and group C "Streptococcus milleri".

    PubMed

    Lebrun, L; Guibert, M; Wallet, P; de Maneville, M M; Pillot, J

    1986-11-01

    The biochemical characteristics and the presence of human Fc(gamma) receptors of 52 throat isolates of group C beta-hemolytic streptococci were examined. Among these isolates, 38 were identified as "Streptococcus milleri" and 14 were identified as "Streptococcus equisimilis." The differentiation of group C "S. equisimilis" from "S. milleri" with identical group antigens was easy to perform by the measurement of the size of the hemolytic zone on a sheep blood agar plate in an anaerobic atmosphere and by biochemical tests (Voges-Proskauer test). A clear-cut criterion for differentiation was noted among these isolates, i.e., the presence of Fc(gamma) receptors. "S. equisimilis," which are generally associated with pharyngitis, possess human Fc(gamma) receptors, while "S. milleri", which are generally isolated from healthy persons, have no such receptors. PMID:3771760

  8. Distribution of type I Fc epsilon-receptors on the surface of mast cells probed by fluorescence resonance energy transfer.

    PubMed Central

    Kubitscheck, U; Schweitzer-Stenner, R; Arndt-Jovin, D J; Jovin, T M; Pecht, I

    1993-01-01

    The aggregation state of type I Fc epsilon-receptors (Fc epsilon RI) on the surface of single living mast cells was investigated by resonance fluorescence energy transfer. Derivatization of Fc epsilon RI specific ligands, i.e., immunoglobulin E or Fab fragments of a Fc epsilon RI specific monoclonal antibody, with donor and acceptor fluorophores provided a means for measuring receptor clustering through energy transfer between the receptor probes. The efficiency of energy transfer between the ligands carrying distinct fluorophores was determined on single cells in a microscope by analyzing the photobleaching kinetics of the donor fluorophore in the presence and absence of receptor ligands labeled with acceptor fluorophores. To rationalize the energy transfer data, we developed a theoretical model describing the dependence of the energy transfer efficiency on the geometry of the fluorescently labeled macromolecular ligands and their aggregation state on the cell surface. To this end, the transfer process was numerically calculated first for one pair and then for an ensemble of Fc epsilon RI bound ligands on the cell surface. The model stipulates that the aggregation state of the Fc epsilon RI is governed by an attractive lipid-protein mediated interaction potential. The corresponding pair-distribution function characterizes the spatial distribution of the ensemble. Using this approach, the energy transfer efficiency of the ensemble was calculated for different degrees of receptor aggregation. Comparison of the theoretical modeling results with the experimental energy transfer data clearly suggests that the Fc epsilon RI are monovalent, randomly distributed plasma membrane proteins. The method provides a novel approach for determining the aggregation state of cell surface components. PMID:8431535

  9. TRIM21: a cytosolic Fc receptor with broad antibody isotype specificity

    PubMed Central

    Foss, Stian; Watkinson, Ruth; Sandlie, Inger; James, Leo C; Andersen, Jan Terje

    2015-01-01

    Antibodies are key molecules in the fight against infections. Although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. This postentry viral defense mechanism requires binding of the antibody to a cytosolic Fc receptor named tripartite motif containing 21 (TRIM21). In contrast to other Fc receptors, TRIM21 shows remarkably broad isotype specificity as it does not only bind IgG but also IgM and IgA. When viral pathogens coated with these antibody isotypes enter the cytosol, TRIM21 is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. In addition, inflammatory signaling is induced. As such, TRIM21 acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. Here, we summarize our current understanding of how TRIM21 orchestrates humoral immunity in the cytosolic environment. PMID:26497531

  10. The IgG Fc receptor, FcγRIIB, is a target for deregulation by chromosomal translocation in malignant lymphoma

    PubMed Central

    Callanan, Mary B.; Le Baccon, Patricia; Mossuz, Pascal; Duley, Samuel; Bastard, Christian; Hamoudi, Rifat; Dyer, Martin J.; Klobeck, Gustav; Rimokh, Ruth; Sotto, Jean Jacques; Leroux, Dominique

    2000-01-01

    Rearrangement of chromosomal bands 1q21–23 is one of the most frequent chromosomal aberrations observed in hematological malignancy. The genes affected by these rearrangements remain poorly characterized. Typically, 1q21–23 rearrangements arise during tumor evolution and accompany disease-specific chromosomal rearrangements such as t(14;18) (BCL2) and t(8;14) (MYC), where they are thus thought to play an important role in tumor progression. The pathogenetic basis of this 1q21–23-associated disease progression is currently unknown. In this setting, we surveyed our series of follicular lymphoma for evidence of recurring 1q21–23 breaks and identified three cases in which a t(14;18)(q32;q21) was accompanied by a novel balanced t(1;22)(q22;q11). Molecular cloning of the t(1;22) in a cell line (B593) derived from one of these cases and detailed fluorescent in situ hybridization mapping in the two remaining cases identified the FCGR2B gene, which encodes the immunoreceptor tyrosine-based inhibition motif-bearing IgG Fc receptor, FcγRIIB, as the target gene of the t(1;22)(q22;q11). We demonstrate deregulation of FCGR2B leading to hyperexpression of FcγRIIb2 as the principal consequence of the t(1;22). This is evidence that IgG Fc receptors can be targets for deregulation through chromosomal translocation in lymphoma. It suggests that dysregulation of FCGR2B may play a role in tumor progression in follicular lymphoma. PMID:10618414

  11. Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor.

    PubMed

    Burvenich, Ingrid J G; Farrugia, William; Lee, Fook T; Catimel, Bruno; Liu, Zhanqi; Makris, Dahna; Cao, Diana; O'Keefe, Graeme J; Brechbiel, Martin W; King, Dylan; Spirkoska, Violeta; Allan, Laura C; Ramsland, Paul A; Scott, Andrew M

    2016-01-01

    IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic. PMID:27030023

  12. Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

    PubMed Central

    McConnell, I; Hurd, C M

    1976-01-01

    Immunoglobulin (Ig) bearing, antigen-binding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibody-secreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibody-secreting cells. These receptors are either lost from the B cell surface as they differentiate into antibody-secreting cells or are blocked in vivo by immune complexes or C3b. PMID:800392

  13. IgG transport across mucosal barriers by neonatal Fc receptor for IgG and mucosal immunity.

    PubMed

    Yoshida, Masaru; Masuda, Atsuhiro; Kuo, Timothy T; Kobayashi, Kanna; Claypool, Steven M; Takagawa, Tetsuya; Kutsumi, Hiromu; Azuma, Takeshi; Lencer, Wayne I; Blumberg, Richard S

    2006-12-01

    Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain significant quantities of IgG. The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. The FcRn can then recycle the IgG/antigen complex back across the intestinal barrier into the lamina propria for processing by dendritic cells and presentation to CD4(+) T cells in regional organized lymphoid structures. FcRn, through its ability to secrete and absorb IgG, thus integrates luminal antigen encounters with systemic immune compartments and, as such, provides essential host defense and immunoregulatory functions at the mucosal surfaces. PMID:17051393

  14. Bruton’s Tyrosine Kinase Mediates FcγRIIa/Toll-Like Receptor–4 Receptor Crosstalk in Human Neutrophils

    PubMed Central

    Krupa, Agnieszka; Fudala, Rafal; Florence, Jon M.; Tucker, Torry; Allen, Timothy C.; Standiford, Theodore J.; Luchowski, Rafal; Fol, Marek; Rahman, Moshiur; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2013-01-01

    Previous observations by our laboratory indicate that the presence of anti–IL-8 autoantibody:IL-8 immune complexes in lung fluids from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) comprises an important prognostic indicator in the development and ultimate outcome of ALI/ARDS. We also showed that these complexes display proinflammatory activity toward neutrophils through the engagement of FcγRIIa receptors. Because sepsis is one of the most common risk factors for ALI/ARDS, the initial goal of our present study involved investigating the effects of LPS on the expression of FcγRIIa receptors in neutrophils. Our results indicate that LPS triggers an increase in the expression of FcγRIIa on the neutrophil surface, which leads to shortening of the molecular distance between FcγRIIa and Toll-like receptor–4 (TLR4). When such neutrophils are stimulated with anti–IL-8:IL-8 complexes, the TLR4 cascade becomes activated via the engagement of FcγRIIa. The underlying molecular mechanism has been subsequently examined and involves Bruton’s tyrosine kinase (Btk). In conclusion, our study reveals the existence of Btk-dependent molecular cooperation between FcγRIIa and TLR4 signaling cascades in LPS-“primed” human neutrophils. Furthermore, we used fluorescence lifetime imaging to study the interactions between TLR4 and FcγRIIa in human alveolar neutrophils from patients with ALI/ARDS. The results from these experiments confirm the existence of the molecular cooperation between TLR4 and FcγRIIa. PMID:23239500

  15. Dynamics of mononuclear phagocyte system Fc receptor function in systemic lupus erythematosus. Relation to disease activity and circulating immune complexes.

    PubMed

    Kimberly, R P; Parris, T M; Inman, R D; McDougal, J S

    1983-02-01

    Seventeen pairs of longitudinal studies of mononuclear phagocyte system (MPS) Fc receptor function in 15 patients with systemic lupus were performed to explore the dynamic range of Fc receptor dysfunction in lupus and to establish the relationships between MPS function, clinical disease activity and circulating immune complexes (CIC). Fc receptor function was measured by the clearance of IgG sensitized autologous erythrocytes. At the time of first study the degree of MPS dysfunction was correlated with both clinical activity (P less than 0.05) and CIC (P less than 0.05). At follow-up patients with a change in clinical status show significantly larger changes in clearance function compared to clinically stable patients (206 min vs 7 min; P less than 0.001). MPS function changed concordantly with a change in clinical status in all cases (P = 0.002). Longitudinal assessments did not demonstrate concordance of changes in MPS function and CIC, measured by three different assays. The MPS Fc receptor defect in systemic lupus is dynamic and closely associated with disease activity. The lack of concordance of the defect with changes in CIC suggests that either CIC does not adequately reflect receptor site saturation or that other factors may also contribute to the magnitude of MPS dysfunction. PMID:6839542

  16. Fyn kinase controls Fc{epsilon}RI receptor-operated calcium entry necessary for full degranulation in mast cells

    SciTech Connect

    Sanchez-Miranda, Elizabeth; Ibarra-Sanchez, Alfredo; Gonzalez-Espinosa, Claudia

    2010-01-22

    IgE-antigen-dependent crosslinking of the high affinity IgE receptor (Fc{epsilon}RI) on mast cells leads to degranulation, leukotriene synthesis and cytokine production. Calcium (Ca{sup 2+}) mobilization is a sine qua non requisite for degranulation, allowing the rapid secretion of stored pro-inflammatory mediators responsible for allergy symptoms. Fyn is a Src-family kinase that positively controls Fc{epsilon}RI-induced mast cell degranulation. However, our understanding of the mechanism connecting Fyn activation to secretion of pre-synthesized mediators is very limited. We analyzed Fc{epsilon}RI-dependent Ca{sup 2+} mobilization in bone marrow-derived mast cells (BMMCs) differentiated from WT and Fyn -/- knock out mice. Fyn -/- BMMCs showed a marked defect in extracellular Ca{sup 2+} influx after Fc{epsilon}RI crosslinking but not after thapsigargin addition. High concentrations of Gadolinium (Gd{sup 3+}) partially blocked Fc{epsilon}RI-induced Ca{sup 2+} influx in WT cells but, in contrast, completely inhibited Ca{sup 2+} mobilization in Fyn -/- cells. Low concentrations of an inhibitor of the canonical transient receptor potential (TRPC) Ca{sup 2+} channels (2-aminoethoxyphenyl-borane, 2-APB) blocked Fc{epsilon}RI-induced maximal Ca{sup 2+} rise in WT but not in Fyn -/- cells. Ca{sup 2+} entry through Fyn-controlled, 2-APB sensitive channels was found to be important for full degranulation and IL-2 mRNA accumulation in WT cells. Immunoprecipitation assays showed that Fyn kinase interacts with TRPC 3/6/7 channels after IgE-antigen stimulation, but its association is not related to protein tyrosine phosphorylation. Results indicate Fyn kinase mediates the receptor-dependent activation of TRPC channels that contribute to degranulation in Fc{epsilon}RI-stimulated mast cells.

  17. MHC Class I-Related Neonatal Fc Receptor for IgG Is Functionally Expressed in Monocytes, Intestinal Macrophages, and Dendritic Cells1

    PubMed Central

    Zhu, Xiaoping; Meng, Gang; Dickinson, Bonny L.; Li, Xiaotong; Mizoguchi, Emiko; Miao, Lili; Wang, Yuansheng; Robert, Caroline; Wu, Benyan; Smith, Phillip D.; Lencer, Wayne I.; Blumberg, Richard S.

    2010-01-01

    The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the β2-microglobulin (β2m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical FcγRs: FcγRI, FcγRII, and FcγRIII. PMID:11207281

  18. Characterization and comparison of commercially available TNF receptor 2-Fc fusion protein products

    PubMed Central

    Tan, Qingqiao; Guo, Qingcheng; Fang, Chen; Wang, Chong; Li, Bohua; Wang, Hao; Li, Jing; Guo, Yajun

    2012-01-01

    Because of rapidly increasing market demand and rising cost pressure, the innovator of etanercept (Enbrel®) will inevitably face competition from biosimilar versions of the product. In this study, to elucidate the differences between the reference etanercept and its biosimilars, we characterized and compared the quality attributes of two commercially available, biosimilar TNF receptor 2-Fc fusion protein products. Biosimilar 1 showed high similarity to Enbrel® in critical quality attributes including peptide mapping, intact mass, charge variant, purity, glycosylation and bioactivity. In contrast, the intact mass and MS/MS analysis of biosimilar 2 revealed a mass difference indicative of a two amino acid residue variance in the heavy chain (Fc) sequences. Comprehensive glycosylation profiling confirmed that biosimilar 2 has significantly low sialylated N-oligosaccharides. Biosimilar 2 also displayed significant differences in charge attributes compared with the reference product. Interestingly, biosimilar 2 exhibited similar affinity and bioactivity levels compared with the reference product despite the obvious difference in primary structure and partial physiochemical properties. For a biosimilar development program, comparative analytical data can influence decisions about the type and amount of animal and clinical data needed to demonstrate biosimilarity. Because of the limited clinical experience with biosimilars at the time of their approval, a thorough knowledge surrounding biosimilars and a case-by-case approach are needed to ensure the appropriate use of these products. PMID:23032066

  19. Defect in the membrane expression of high affinity 72-kD Fc gamma receptors on phagocytic cells in four healthy subjects.

    PubMed Central

    Ceuppens, J L; Baroja, M L; Van Vaeck, F; Anderson, C L

    1988-01-01

    Three different receptors for the Fc portion of IgG (FcR) have been characterized on human leukocytes. We have identified four healthy members of one family, whose blood phagocytic cells lack functional 72 kD high-affinity FcRI. Their monocytes were unable to bind the Fc portion of mouse (m)-IgG2a and of monomeric human IgG, and they were unreactive with two anti-FcRI monoclonal antibodies. Thus, FcRI is either absent, expressed at very low density, or is so structurally altered as to be unable to bind both its ligand and the anti-FcRI antibodies. The failure to bind the Fc portion of mIgG2a underlies the previously reported inability of these monocytes to support T cell mitogenesis on OKT3 stimulation. FcRI was not inducible upon incubation of their monocytes or neutrophils in gamma interferon. However, their monocytes were able to bind aggregated human IgG, and to phagocytose IgG-coated particles in vitro. Both functions could be blocked with a monoclonal antibody to the 40-kD low-affinity FcRII and therefore apparently were mediated exclusively through FcRII. This also demonstrates that FcRII can mediate phagocytosis independently. Despite the FcRI defect, these subjects had no circulating immune complexes, no evidence of autoimmune pathology and no increased susceptibility to infections. PMID:2969920

  20. The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

    PubMed Central

    Starkl, Philipp; Bevins, Charles L.; Scheiner, Otto; Boltz-Nitulescu, George; Wrba, Fritz; Jensen-Jarolim, Erika

    2010-01-01

    Background IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium. Methodology/Principal Findings FcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains. Conclusions/Significance Our data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested. PMID:20126404

  1. Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.

    PubMed Central

    Davidson-Moncada, Jan K; Lopez-Lluch, Guillermo; Segal, Anthony W; Dekker, Lodewijk V

    2002-01-01

    Protein kinases involved in the activation of the NADPH oxidase by Fc gamma receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70-80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fc gamma receptors. Down-regulation of PKC isotypes resulted in inhibition of Fc gamma-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-mu (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fc gamma-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40(phox) and p47(phox). Taken together, these data indicate that Fc gamma receptors engage PKD in the regulation of the NADPH oxidase. PMID:11903052

  2. Fc receptor-mediated phagocytosis, superoxide production and calcium signaling of beta 2 integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Sawada, C; Higuchi, H; Teraoka, H; Yamaguchi, M

    1997-01-01

    Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ([Ca2+]i) signaling of complement receptor type 3 (CR3)-deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers. The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca2+]i signals in CR3-deficient neutrophils stimulated with Agg-IgG or concanavalin A were different with mean peak [Ca2+]i concentrations of 78% and 41.9%, respectively, of those of control neutrophils. These findings suggest that Fc receptor-mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces. PMID:9343828

  3. Relationship between Fc receptors, antigen-binding sites on T and B cells, and H-2 complex-associated determinants.

    PubMed

    Basten, A; Miller, J F; Abraham, R

    1975-03-01

    The relationship between H-2 complex-associated determinants, Fc receptors, and specific antigen-recognition sites on T and B cells was examined by binding and functional assays. The Fc receptor was detected by radiolabeled immune complexes or aggregated human IgG. Both these reagents selectively bound to B cells, not to T cells. When spleen cells, from mice primed to several antigens, were exposed to highly substituted radioactive aggregates, their capacity to transfer both a direct and indirect plaque-forming cell response to these antigens was abrogated. Addition of B cells, but not of T cells, restored responsiveness. Complexed Ig binding to Fc receptors was prevented by pretreatment of mixed lymphoid cell populations with antisera directed against membrane components on the same cell (e.g., H-2) and on other cells (e.g., theta). The lack of specificity of inhibition was thought to be due to the formation on cell surfaces of antigen-antibody complexes which would then attach to the Fc receptor during the incubation precedure. Specific blockade of the Fc receptor during the incubation procedure. Specific blockade of the Fc receptor however occurred when B cells were pretreated with the Fab fragments of anti-H-2 antibody. This was demonstrated autoradiographically and by inhibition of aggregate-induced suicide. The blocking activity of ante-H-2 Fab was removed by absorption with spleen cells from thymectomized irradiated mice but not with thymus cells of appropriate specificity. This suggested that the antibodies involved had specificity for determinants on the B-cell membrane distinct from those coded by the K or D end of the H-2 complex, and either absent from, or poorly represented on, thymus cells. Specific antigen-induced suicide of B cells was achieved simply by incubating the cells with radioactive antigen in the cold. T-cell suicide on the other hand required that the 125I-labeled antigen be presented to the T cells at 37 degrees-C on the surface of

  4. IgG Suppresses Antibody Responses in Mice Lacking C1q, C3, Complement Receptors 1 and 2, or IgG Fc-Receptors

    PubMed Central

    Bergström, Joakim J. E.; Heyman, Birgitta

    2015-01-01

    Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes. PMID:26619292

  5. The MHC Class II-Associated Invariant Chain Interacts with the Neonatal Fcγ Receptor and Modulates Its Trafficking to Endosomal/Lysosomal Compartments1

    PubMed Central

    Ye, Lilin; Liu, Xindong; Rout, Subrat N.; Li, Zili; Yan, Yongqi; Lu, Li; Kamala, Tirumalai; Nanda, Navreet K.; Song, Wenxia; Samal, Siba K.; Zhu, Xiaoping

    2009-01-01

    The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-β2-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-γ-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-γ. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain. PMID:18684948

  6. Fc Gamma Receptor IIA (CD32A) R131 Polymorphism as a Marker of Genetic Susceptibility to Sepsis.

    PubMed

    Beppler, Jaqueline; Koehler-Santos, Patrícia; Pasqualim, Gabriela; Matte, Ursula; Alho, Clarice Sampaio; Dias, Fernando Suparregui; Kowalski, Thayne Woycinck; Velasco, Irineu Tadeu; Monteiro, Renato C; Pinheiro da Silva, Fabiano

    2016-04-01

    Sepsis is a devastating disease that can affect humans at any time between neonates and the elderly and is associated with mortality rates that range from 30 to 80 %. Despite intensive efforts, its treatment has remained the same over the last few decades. Fc receptors regulate multiple immune responses and have been investigated in diverse complex diseases. FcγRIIA (CD32A) is an immunoreceptor, tyrosine-based activation motif-bearing receptor that binds immunoglobulin G and C-reactive protein, important opsonins in host defense. We conducted a study of 702 patients (184 healthy individuals, 171 non-infected critically ill patients, and 347 sepsis patients) to investigate if genetic polymorphisms in the CD32A coding region affect the risk of septic shock. All individuals were genotyped for a variant at position 131 of the FcγRIIA gene. We found that allele G, associated with the R131 genotype, was significantly more frequent in septic patients than in the other groups (p = 0.05). Our data indicate that FcγRIIA genotyping can be used as a marker of genetic susceptibility to sepsis. PMID:26490967

  7. Functional co-localization of monocytic aminopeptidase N/CD13 with the Fc{gamma} receptors CD32 and CD64

    SciTech Connect

    Riemann, Dagmar; Wulfaenger, Jens

    2005-06-17

    Information about the function of aminopeptidase N/CD13 on monocytes is limited. In order to gain more insight into its interaction with other proteins, we have identified molecules that co-localize with the membrane ectoenzyme at the cell surface of monocytes. Using laser scanning and electron microscopy as well as fluorescence resonance energy transfer (FRET) measured by flow cytometry we show that monocytic CD13 co-localized with the Fc{gamma} receptor II/CD32 after Fc receptor ligation by a CD32-specific antibody. FRET was also observed between CD13 and the Fc{gamma} receptor I/CD64, but not with the myeloid marker CD33 representing a member of the sialoadhesin family. Our results imply a novel functional role of CD13 and Fc{gamma} receptors as members of a multimeric receptor complex. Further studies have to be done to elucidate common signaling pathways of these molecules.

  8. Identification and characterization of a novel splicing variant of the MHC classI-related Neonatal Fc receptor for IgG

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The neonatal Fc receptor for IgG (FcRn), a MHC class I-related molecule, functions to transport maternal IgG into the fetus or newborn via placenta and/or intestine and protects IgG from catabolism. The mRNAs of several MHC class I-related molecules have multiple splicing variants. In the course of ...

  9. Linkage on chromosome 3 of autoimmune diabetes and defective Fc receptor for lgG in NOD mice

    SciTech Connect

    Prins, J.B.; Todd, J.A.; Rodrigues, N.R.; Ghosh, S. ); Hogarth, P.M. ); Wicker, L.S.; Podolin, P.L.; Gaffney, E.; Peterson, L.B.; Fischer, P.A.; Sirotina, A. )

    1993-04-30

    A congenic, non-obese diabetic (NOD) mouse strain that contains a segment of chromosome 3 from the diabetes-resistant mouse strain B6.PL-Thy-1[sup a] was less susceptible to diabetes than NOD mice. A fully penetrant immunological defect also mapped to this segment, which encodes the high-affinity Fc receptor for immunoglobulin G (lgG), Fc[gamma]Rl. The NOD Fcgr1 allele, which results in a deletion of the cytoplasmic tail, caused a 73 percent reduction in the turnover of cell surface receptor-antibody complexes. The development of congenic strains and the characterization of Mendelian traits that are specific to the disease phenotype demonstrate the feasibility of dissecting the pathophysiology of complex, non-Mendelian diseases.

  10. Neonatal Fc receptor promoter gene polymorphism does not predict pharmacokinetics of IVIg or the clinical course of GBS.

    PubMed

    Fokkink, Willem-Jan R; Haarman, Annechien E G; Tio-Gillen, Anne P; van Rijs, Wouter; Huizinga, Ruth; van Doorn, Pieter A; Jacobs, Bart C

    2016-07-01

    Treatment of Guillain-Barré syndrome with a standard course of high-dose intravenous immunoglobulin (IVIg) results in a variable clinical recovery which is associated with changes in serum IgG levels after treatment. The neonatal Fc-receptor protects IgG from degradation, and a genetic polymorphism in its promoter region that influences the expression of Fc-receptor, may in part explain the variation in IgG levels and outcome. This polymorphism was determined by polymerase chain reaction in a cohort of 257 patients with Guillain-Barré syndrome treated with IVIg. We could not demonstrate a relation between this polymorphism, the pharmacokinetics of IVIg, or the clinical course and outcome. PMID:27386503

  11. Differential Fc-receptor engagement drives an anti-tumor vaccinal effect

    PubMed Central

    DiLillo, David J.; Ravetch, Jeffrey V.

    2015-01-01

    Summary Passively-administered anti-tumor mAbs rapidly kill tumor targets via FcγR-mediated cytotoxicity (ADCC), a short-term process. However, anti-tumor mAb treatment can also induce a vaccinal effect, in which mAb-mediated tumor death induces a long-term anti-tumor cellular immune response. To determine how such responses are generated, we utilized a murine model of an anti-tumor vaccinal effect against a model neoantigen. We demonstrate that FcγR expression by CD11c+ antigen-presenting cells is required to generate anti-tumor T cell responses upon ADCC-mediated tumor clearance. Using FcγR-humanized mice, we demonstrate that anti-tumor huIgG1 must engage hFcγRIIIA on macrophages to mediate ADCC, but also engage hFcγRIIA, the sole hFcγR expressed by human DCs, to generate a potent vaccinal effect. Thus, while next-generation anti-tumor antibodies with enhanced binding to only hFcγRIIIA are now in clinical use, ideal anti-tumor antibodies must be optimized for both cytotoxic effects as well as hFcγRIIA engagement on DCs to stimulate long-term anti-tumor cellular immunity. PMID:25976835

  12. Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

    PubMed Central

    2004-01-01

    We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP-76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cγ2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (∼10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRγ chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti-(α2 integrin) antibodies Ha1/29 and HMα2, but not by blockade of αIIbβ3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI–FcRγ chain complex, and is facilitated by binding of collagen to integrin α2β1. PMID:15283702

  13. [The changes of expression and Fc-gamma-receptor's polypeptide composition of fetal small intestine enterocytes in Bos primigenius taurus L].

    PubMed

    Masiuk, D M; Nedzvets'kyĭ, V S; Tsvilikhovs'kyĭ, M I; Nerush, P O

    2008-01-01

    The expression and polypeptide composition of Fc-gamma-receptors of enterocytes from Bos primigenius taurus L. intestine at 3-, 5-, 7-, 9- month of fetal development was investigated. The results of immunobloting show similar composition of Fc-gamma-receptors extracted from apical and basolateral membranes. The proteins that bind IgG after PAAG electrophoresis and transferring on nitrocellulose were observed as 120, 87, 72 and 43 kDa polypeptide line. The changes of each polypeptide contents were related to the changes of total content of Fc-gamma-receptor proteins. The rise in concentration of Fc-gamma-receptors on apical membrane was observed from 3-rd to 7-th month of fetal development. Maximal concentration of these proteins was detected on enterocytes at 7-th month of fetal development. Fc-gamma-receptors content on basolateral side was higher than on apical side. The presence of Fc-gamma-receptors on enterocyte's membrane indicates on active recycling of these receptors on plasma membrane and reflects early development of immune system in Bos primigenius taurus L. fetus. PMID:18416181

  14. α2β1 Integrin, GPVI Receptor, and Common FcRγ Chain on Mouse Platelets Mediate Distinct Responses to Collagen in Models of Thrombosis

    PubMed Central

    Marjoram, Robin J.; Li, Zhengzhi; He, Li; Tollefsen, Douglas M.; Kunicki, Thomas J.; Dickeson, S. Kent; Santoro, Samuel A.; Zutter, Mary M.

    2014-01-01

    Objective Platelets express the α2β1 integrin and the glycoprotein VI (GPVI)/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ−/−) or the complex was depleted. The development of α2β1−/− and GPVI−/− mice permits side-by-side comparison to address contributions of these collagen receptors in vivo and in vitro. Approach and Results To understand the different roles played by the α2β1 integrin, the GPVI receptor or FcRγ subunit in collagen-stimulated hemostasis and thrombosis, we compared α2β1−/−, FcRγ−/−, and GPVI−/− mice in models of endothelial injury and intravascular thrombosis in vivo and their platelets in collagen-stimulated activation in vitro. We demonstrate that both the α2β1 integrin and the GPVI receptor, but not the FcRγ subunit influence carotid artery occlusion in vivo. In contrast, the GPVI receptor and the FcRγ chain, but not the α2β1 integrin, play similar roles in intravascular thrombosis in response to soluble Type I collagen. FcRγ−/− platelets showed less attenuation of tyrosine phosphorylation of several proteins including RhoGDI when compared to GPVI−/− and wild type platelets. The difference between FcRγ−/− and GPVI−/− platelet phosphotyrosine levels correlated with the in vivo thrombosis findings. Conclusion Our data demonstrate that genetic deletion of GPVI receptor, FcRγ chain, or the α2β1 integrin changes the thrombotic potentials of these platelets to collagen dependent on the stimulus mechanism. The data suggest that the FcRγ chain may provide a dominant negative effect through modulating signaling pathways in platelets involving several tyrosine phosphorylated proteins such as RhoGDI. In addition, these findings

  15. Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. VIII. Expression and shedding of Fc gamma receptors on metastatic tumor cell variants.

    PubMed

    Schirrmacher, V; Jacobs, W

    1979-01-01

    The expression of receptors for the Fc portion of IgG immunoglobin molecules was studied on tumor cell lines with high and low metastatic capacity. Two tumor cell lines from DBA/2 mice that had high metastatic activity, ESb and MDAY-D2, contained a high percentage of Fc receptor positive cells, as detected in a rosette assay with IgG antibody-coated erythrocytes (EA). In contrast, the low metastatic parental line Eb, from which ESb was derived, contained only a low percentage of EA-rosette-forming cells. ESb ascites tumor cells adapted to tissue culture in the presence of 2-mercaptoethanol (2ME) had a high expression of Fc receptors, whereas a cell line adapted to tissue culture in the absence of 2ME had a low expression of Fc receptors. "Soluble" Fc receptors were detectable by their ability to bind to EA and to cause blocking of rosette formation. They were found to be present in fluids from tumor-bearing animals, such as serum and cell-free ascites. Even animals with an ascites tumor of the low-metastatic line Eb contained "soluble" Fc receptors. The results are discussed with regard to their possible significance for tumor metastasis. PMID:522481

  16. Ligation of Macrophage Fcγ Receptors Recapitulates the Gene Expression Pattern of Vulnerable Human Carotid Plaques

    PubMed Central

    Lennartz, Michelle R.; Aggarwal, Ankur; Michaud, Tanya M.; Feustel, Paul J.; Jones, David M.; Brosnan, M. Julia; Keller, Rebecca S.; Loegering, Daniel J.; Kreienberg, Paul B.

    2011-01-01

    Stroke is a leading cause of death in the United States. As ∼60% of strokes result from carotid plaque rupture, elucidating the mechanisms that underlie vulnerability is critical for therapeutic intervention. We tested the hypothesis that stable and vulnerable human plaques differentially express genes associated with matrix degradation. Examination established that femoral, and the distal region of carotid, plaques were histologically stable while the proximal carotid plaque regions were vulnerable. Quantitative RT-PCR was used to compare expression of 22 genes among these tissues. Distal carotid and femoral gene expression was not significantly different, permitting the distal carotid segments to be used as a paired control for their corresponding proximal regions. Analysis of the paired plaques revealed differences in 16 genes that impact plaque stability: matrix metalloproteinases (MMP, higher in vulnerable), MMP modulators (inhibitors: lower, activators: higher in vulnerable), activating Fc receptors (FcγR, higher in vulnerable) and FcγR signaling molecules (higher in vulnerable). Surprisingly, the relative expression of smooth muscle cell and macrophage markers in the three plaque types was not significantly different, suggesting that macrophage distribution and/or activation state correlates with (in)stability. Immunohistochemistry revealed that macrophages and smooth muscle cells localize to distinct and non-overlapping regions in all plaques. MMP protein localized to macrophage-rich regions. In vitro, treatment of macrophages with immune complexes, but not oxidized low density lipoprotein, C-reactive protein, or TNF-α, induced a gene expression profile similar to that of the vulnerable plaques. That ligation of FcγR recapitulates the pattern of gene expression in vulnerable plaques suggests that the FcγR → macrophage activation pathway may play a greater role in human plaque vulnerability than previously appreciated. PMID:21814555

  17. Downmodulation of vaccine-induced immunity and protection against the intracellular bacterium Francisella tularensis by the inhibitory receptor FcγRIIB.

    PubMed

    Franz, Brian J; Li, Ying; Bitsaktsis, Constantine; Iglesias, Bibiana V; Pham, Giang; Sunagar, Raju; Kumar, Sudeep; Gosselin, Edmund J

    2015-01-01

    Fc gamma receptor IIB (FcγRIIB) is the only Fc gamma receptor (FcγR) which negatively regulates the immune response, when engaged by antigen- (Ag-) antibody (Ab) complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft), a Category A biothreat agent. We utilized inactivated Ft (iFt) as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO) or wildtype (WT) mice were challenged with Ft-live vaccine strain (LVS). While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed. PMID:25961064

  18. Downmodulation of Vaccine-Induced Immunity and Protection against the Intracellular Bacterium Francisella tularensis by the Inhibitory Receptor FcγRIIB

    PubMed Central

    Franz, Brian J.; Li, Ying; Bitsaktsis, Constantine; Iglesias, Bibiana V.; Pham, Giang; Sunagar, Raju; Kumar, Sudeep; Gosselin, Edmund J.

    2015-01-01

    Fc gamma receptor IIB (FcγRIIB) is the only Fc gamma receptor (FcγR) which negatively regulates the immune response, when engaged by antigen- (Ag-) antibody (Ab) complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft), a Category A biothreat agent. We utilized inactivated Ft (iFt) as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO) or wildtype (WT) mice were challenged with Ft-live vaccine strain (LVS). While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed. PMID:25961064

  19. Human Eosinophils Express the High Affinity IgE Receptor, FcεRI, in Bullous Pemphigoid

    PubMed Central

    Messingham, Kelly N.; Holahan, Heather M.; Frydman, Alexandra S.; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A.

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  20. Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

    PubMed

    Messingham, Kelly N; Holahan, Heather M; Frydman, Alexandra S; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  1. Murine B-cell stimulatory factor 1 (interleukin 4) increases expression of the Fc receptor for IgE on mouse B cells.

    PubMed Central

    Hudak, S A; Gollnick, S O; Conrad, D H; Kehry, M R

    1987-01-01

    We have studied the activity of mouse B-cell stimulatory factor 1 (interleukin 4, IL-4) on resting splenic B cells and on a B-cell hybridoma. Purified T-cell-derived as well as recombinant IL-4 was shown to increase the expression of the low-affinity Fc receptor for IgE (Fc epsilon R) on a majority of B lymphocytes in a 24-hr culture period. Levels of Fc epsilon R expression increased 2- to 3-fold on splenic B cells and up to 6-fold on a B-cell hybridoma. The effect was inhibited by an anti-IL-4 monoclonal antibody and by mouse gamma-interferon. Other recombinant lymphokines exhibited no effect on either Fc epsilon R expression or the induction by IL-4. The presence of IgE during the stimulation with IL-4 resulted in an additional increase in Fc epsilon R expression. These data and results showing that IgE prevents Fc epsilon R turnover while IL-4 increases the rate of Fc epsilon R synthesis suggest that the mechanisms by which IgE and IL-4 increase Fc epsilon R expression are likely to be different. The starting population of splenic B cells expressed low levels of Fc epsilon R and was relatively uniform in size (small). After greater than 48 hr of culture with IL-4, viable B cells had not undergone DNA synthesis and consisted mainly of larger highly Fc epsilon R-positive cells (23%) and medium-sized Fc epsilon R-positive cells (60%). A possible role for Fc epsilon R in certain B-cell maturation pathways is discussed. Images PMID:2955412

  2. Interaction of human IgG chimeric antibodies with the human FcRI and FcRII receptors: requirements for antibody-mediated host cell-target cell interaction.

    PubMed

    Walker, M R; Woof, J M; Brüggemann, M; Jefferis, R; Burton, D R

    1989-04-01

    Chimeric monoclonal antibodies (McAb), specific for the hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP), expressing human IgG1, IgG2, IgG3 and IgG4 subclass constant domains, have been examined for their ability to interact with the human FcRII receptor. Human red blood cells (RBC) sensitized by each of these McAbs have been assayed for their ability to form rosettes with the human histiocytic lymphoma U937 cell line, human B cell line Daudi and erythroblastoid K562 cell line. IgG1 and IgG3 sensitized RBC formed significant rosettes with the FcR- and FcRII+ Daudi and K562 cell lines, the percentage of cells forming rosettes being directly proportional to the degree of sensitization of the RBC. Bromelin treating Daudi cells did not alter this pattern of reactivity, whereas bromelin treated FcRI+ and FcRII+ U937 cells formed significant resettes with IgG1, IgG3 and IgG4 sensitized RBC, demonstrating a difference in the IgG subclass specificity between human FcRI and FcRII. Murine IgG2b anti-NIP sensitized RBC did not form rosettes with any cell line tested; however, RBC sensitized by some members of a panel of murine IgG1 McAb, specific for the glycophorin A molecule, were able to form rosettes with Daudi, U937 and K562 cells. This interaction was enhanced by bromelin treating the Daudi or U937 cells and can be correlated to the disposition of the epitopes recognized, relative to the target cell membrane, those McAbs recognizing epitopes furthest from the RBC surface being most effective in interacting with FcRII. The data are interpreted in terms of a simple model for antibody-mediated cell--cell interaction. PMID:2716734

  3. Fc Gamma Receptor 3A Polymorphism and Risk for HIV-Associated Cryptococcal Disease

    PubMed Central

    Rohatgi, Soma; Gohil, Shruti; Kuniholm, Mark H.; Schultz, Hannah; Dufaud, Chad; Armour, Kathryn L.; Badri, Sheila; Mailliard, Robbie B.; Pirofski, Liise-anne

    2013-01-01

    ABSTRACT Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4+ T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4+ T cell decline, and nadir CD4+ T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation. PMID:23982074

  4. Genotypes of NK cell KIR Receptors, Their Ligands, and Fcγ Receptors in the Response of Neuroblastoma Patients to Hu14.18-IL2 Immunotherapy

    PubMed Central

    Delgado, DC; Hank, JA; Kolesar, J; Lorentzen, D; Gan, J; Seo, S; Kim, KM; Shusterman, S; Gillies, SD; Reisfeld, RA; Yang, R; Gadbaw, B; DeSantes, KD; London, WB; Seeger, RC; Maris, JM; Sondel, PM

    2010-01-01

    Response to immunocytokine (IC) therapy is dependent on natural killer (NK) cells in murine neuroblastoma (NBL) models. Furthermore, killer immunoglobulin-like receptor (KIR) / KIR-ligand mismatch is associated with improved outcome to autologous stem cell transplant for NBL. Additionally, clinical anti-tumor response to monoclonal antibodies has been associated with specific polymorphic-FcγR alleles. Relapsed/refractory NBL patients received the hu14.18-IL2 IC (humanized anti-GD2 monoclonal antibody linked to human IL2) in a Children’s Oncology Group (COG) Phase II trial. In this report, these patients were genotyped for KIR, HLA and FcR alleles to determine if KIR receptor-ligand mismatch or specific FcγR alleles were associated with anti-tumor response. Thirty-eight of 39 patients enrolled had DNA available for analysis; 24 were found to have autologous KIR/KIR -ligand mismatch; 14 were matched. Seven of 24 mismatched patients experienced either complete response or improvement of their disease after IC therapy. There was no response or comparable improvement of disease in patients who were matched. Thus KIR/KIR-ligand mismatch was associated with response/improvement to IC (p = 0.03). There was a trend towards patients with the FcγR2A 131-H/H genotype showing a higher response rate compared to other FcγR2A genotypes (p = 0.06). These analyses indicate that response or improvement of relapsed/refractory neuroblastoma patients after IC treatment is associated with autologous KIR/KIR-ligand mismatch, consistent with a role for NK cells in this clinical response. PMID:20935224

  5. Evidence for immunoglobulin Fc receptor-mediated prostaglandin2 and platelet-activating factor formation by cultured rat mesangial cells

    SciTech Connect

    Neuwirth, R.; Singhal, P.; Diamond, B.; Hays, R.M.; Lobmeyer, L.; Clay, K.; Schlondorff, D.

    1988-09-01

    The possibility of Fc-dependent uptake of IgG immune complexes was examined in subcultured rat mesangial cells free of monocytes. 195Au-labeled colloidal gold particles were coated either with BSA only or with BSA followed by rabbit anti-BSA-IgG or the F(ab')2 fragment of the IgG. Mesangial cells preferentially took up 195Au particles covered with BSA-anti-BSA-IgG over those covered with BSA or the F(ab')2 fragment. This uptake was a time-dependent and saturable process inhibitable by sodium azide or cytochalasin B. Using phase-contrast microscopy in the light reflectance mode, it was established that essentially all mesangial cells took up IgG-coated gold particles. By electron microscopy the process was shown to consist of vesicular uptake with delivery to endosomes. Mesangial binding-uptake of the IgG-covered particles was associated with stimulation of PGE2 synthesis and production of platelet-activating factor, a lipid mediator of inflammation. To characterize the potential Fc receptor for IgG we used the rosetting technique with sheep red blood cells coated with IgG subclass-specific mouse monoclonal antibodies. 50% of mesangial cells exhibited rosetting with red cells coated with mouse IgG2a, whereas negligible rosetting was observed with IgG2b or IgG1. Competition experiments confirmed the specificity of IgG2a binding. We conclude that cultured rat mesangial cells exhibit specific receptors for IgG and that occupancy of Fc receptors results in endocytosis and is associated with generation of PGE2 and platelet-activating factor. These observations may be of significance for immune-mediated glomerular diseases.

  6. A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand*

    PubMed Central

    Sun, Yi; Vandenbriele, Christophe; Kauskot, Alexandre; Verhamme, Peter; Hoylaerts, Marc F.; Wright, Gavin J.

    2015-01-01

    Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (KD ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation. PMID:25713122

  7. Neutrophils and their Fcγ receptors are essential in a mouse model of transfusion-related acute lung injury

    PubMed Central

    Looney, Mark R.; Su, Xiao; Van Ziffle, Jessica A.; Lowell, Clifford A.; Matthay, Michael A.

    2006-01-01

    Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion-related mortality. To explore the pathogenesis of TRALI, we developed an in vivo mouse model based on the passive transfusion of an MHC class I (MHC I) mAb (H2Kd) to mice with the cognate antigen. Transfusion of the MHC I mAb to BALB/c mice produced acute lung injury with increased excess lung water, increased lung vascular and lung epithelial permeability to protein, and decreased alveolar fluid clearance. There was 50% mortality at a 2-hour time point after Ab administration. Pulmonary histology and immunohistochemistry revealed prominent neutrophil sequestration in the lung microvasculature that occurred concomitantly with acute peripheral blood neutropenia, all within 2 hours of administration of the mAb. Depletion of neutrophils by injection of anti-granulocyte mAb Gr-1 protected mice from lung injury following MHC I mAb challenge. FcRγ–/– mice were resistant to MHC I mAb–induced lung injury, while adoptive transfer of wild-type neutrophils into the FcRγ–/– animals restored lung injury following MHC I mAb challenge. In conclusion, in a clinically relevant in vivo mouse model of TRALI using an MHC I mAb, the mechanism of lung injury was dependent on neutrophils and their Fcγ receptors. PMID:16710475

  8. Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons

    NASA Technical Reports Server (NTRS)

    Mohamed, Habib A.; Mosier, Dennis R.; Zou, Ling L.; Siklos, Laszlo; Alexianu, Maria E.; Engelhardt, Jozsef I.; Beers, David R.; Le, Wei-dong; Appel, Stanley H.

    2002-01-01

    Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals. Copyright 2002 Wiley-Liss, Inc.

  9. Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

    PubMed

    Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi

    2016-07-01

    The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β. PMID:26940508

  10. Separate Fc-receptors for immunoglogulins IgG2a and IgG2b on an established cell line of mouse macrophages.

    PubMed

    Walker, W S

    1976-04-01

    The specificity of Fe-receptors on IC-21 cells, an established line of mouse peritoneal macrophages with antibody-dependent effector cell activity has been examined. Only IgG2a and IgG2b myeloma proteins bound readily to IC-21 Fc-receptors, the former in nonaggregated as well as aggregated form, the latter only as aggregated complexes. Thus, IgG2a bound in a manner characteristic of classically defined cytophilic antibody, whereas the binding of IgG2b appeared to be mediated by Fc-receptors for antigen-antibody complexes. Evidence is presented in support of the view that IC-21 macrophages possess separate and distinct Fc-receptor sites for these two immunoglobulins. PMID:1254971

  11. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcɛRI.

    PubMed

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2013-05-15

    The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcɛRI) is the central protein interaction in IgE-mediated allergic reactions. The cross-linking of the IgE/FcɛRI complex, through cognate allergens, on the surface of mast cells and basophil cells results in mediator release, and thus leads to the symptoms of type I hypersensitivity responses in mammals. To develop a baseline value for subsequent equine anti-allergy drug and vaccine research, the interaction of equine IgE with its high-affinity FcɛRI receptor was investigated following the cloning and expression of equine IgE with specificity for NIP-HSA (4-hydroxy-5-iodo-3-nitrophenylacetic acid conjugated to human serum albumin). Receptor recognition and effector functions were assessed in Rat Basophil Leukemia (RBL-2H3.1) cells transfected with the α chain of equine and canine FcɛRI. Results obtained showed that the equine FcɛRI receptor recognizes both equine and canine IgE and supports similar β-hexosaminidase release levels from RBL cells transfected with equine FcɛRI, peaking at 36.68% at 100ngml(-1) antigen and 32.00% at 100ngml(-1) antigen respectively. Furthermore, the binding kinetics of the equine IgE to the equine FcɛRI receptor and the canine IgE to the same receptor was measured to be KA=6.33×10(9)M(-1) and KA=1.84×10(9)M(-1) respectively. This research established basic reagents and vitro assay systems to underpin the development of rational therapeutic intervention strategies to combat equine allergic manifestations. PMID:23485176

  12. Complement component C3b and immunoglobulin Fc receptors on neutrophils from calves with leukocyte adhesion deficiency.

    PubMed

    Worku, M; Paape, M J; Di Carlo, A; Kehrli, M E; Marquardt, W W

    1995-04-01

    Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence-related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (LAD) on ligand binding and receptor expression. We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from LAD, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (CL) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of CL was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1. A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from LAD-affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and LAD-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between LAD-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from LAD-affected calves.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7785817

  13. Complex Formation Facilitates Endocytosis of the Varicella-Zoster Virus gE:gI Fc Receptor

    PubMed Central

    Olson, Julie K.; Grose, Charles

    1998-01-01

    Open reading frames within the unique short segment of alphaherpesvirus genomes participate in egress and cell-to-cell spread. The case of varicella-zoster virus (VZV) is of particular interest not only because the virus is highly cell associated but also because its most prominent cell surface protein, gE, bears semblance to the mammalian Fc receptor FcγRII. A previous study demonstrated that when expressed alone in cells, VZV gE was endocytosed from the cell surface through a tyrosine localization motif in its cytoplasmic tail (J. K. Olson and C. Grose, J. Virol. 71:4042–4054, 1997). Since VZV gE is normally found in association with gI in the infected cell, the present study was directed at defining the trafficking of the VZV gE:gI protein complex. First, VZV gI underwent endocytosis and recycling when it was expressed alone in cells, and interestingly, VZV gI contained a methionine-leucine internalization motif in its cytoplasmic tail. Second, VZV gI was found by confocal microscopy to colocalize with VZV gE during endocytosis and recycling in cells. Third, by a quantitative internalization assay, VZV gE:gI was shown to undergo endocytosis more efficiently (steady state, 55 to 60%) than either gE alone (steady state, ∼32%) or gI alone (steady state, ∼45%). Further, examination of endocytosis-deficient mutant proteins demonstrated that VZV gI exerted a more pronounced effect than gE on internalization of the complex. Most importantly, therefore, these studies suggest that VZV gI behaves as an accessory component by facilitating the endocytosis of the major constituent gE and thereby modulating the trafficking of the entire cell surface gE:gI Fc receptor complex. PMID:9445058

  14. Expression Profile of Human Fc Receptors in Mucosal Tissue: Implications for Antibody-Dependent Cellular Effector Functions Targeting HIV-1 Transmission

    PubMed Central

    Cheeseman, Hannah M.; Carias, Ann M.; Evans, Abbey B.; Olejniczak, Natalia J.; Ziprin, Paul; King, Deborah F. L.; Hope, Thomas J.; Shattock, Robin J.

    2016-01-01

    The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the initial events in

  15. ANTIBODY-MEDIATED PROTECTION AGAINST GENITAL HERPES SIMPLEX VIRUS TYPE 2 DISEASE IN MICE BY FC GAMMA RECEPTOR -DEPENDENT AND -INDEPENDENT MECHANISMS

    PubMed Central

    Chu, Chin-Fun; Meador, Michael G.; Young, Christal G.; Strasser, Jane E.; Bourne, Nigel; Milligan, Gregg N.

    2008-01-01

    The ability of antibody (Ab) to modulate HSV pathogenesis is well recognized but the mechanisms by which HSV-specific IgG antibodies protect against genital HSV-2 disease are not well understood. The requirement for Ab interactions with Fcγ receptors (FcγR) in protection was examined using a murine model of genital HSV-2 infection. IgG antibodies isolated from the serum of HSV-immune mice protected normal mice against HSV-2 disease when administered prior to genital HSV-2 inoculation. However, protection was significantly diminished in recipient mice lacking the gamma chain subunit utilized in FcγRI, FcγRIII, FcγRIV and FcepsilonRI receptors and in normal mice depleted of Gr-1+ immune cell populations known to express FcγR, suggesting protection was largely mediated by an FcγR-dependent mechanism. To test whether neutralizing Ab might provide superior protection, a highly neutralizing HSV glycoprotein D (gD)- specific monoclonal antibody (mAb) was utilized. Similar to results with HSV-specific polyclonal IgG, administration of the gD-specific mAb did not prevent initial infection of the genital tract but resulted in lower virus loads in the vaginal epithelium and provided significant protection against disease and acute infection of the sensory ganglia; however, this protection was independent of host FcγR expression and was manifest in mice depleted of Gr-1+ immune cells. Together, these data demonstrate that substantial Ab-mediated protection against genital HSV-2 disease could be achieved by either FcγR-dependent or -independent mechanisms. These studies suggest that HSV vaccines might need to elicit multiple, diverse antibody effector mechanisms to achieve optimal protection. PMID:17950908

  16. Isotype-specific immunoregulation; characterization and function of Fc receptors on T-T hybridomas which produce murine IgA-binding factor.

    PubMed

    Kurita, T; Kiyono, H; Komiyama, K; Grossi, C E; Mestecky, J; McGhee, J R

    1986-06-01

    Several methods have been used in the present study to characterize Fc receptors (FcR) expressed on T-T hybridomas derived from mouse Peyer's patch T helper (Th) cell clones that preferentially support IgA responses. These T hybridomas (designated Th HA cells) produce IgA-binding factor (IBF alpha) which regulates antigen-dependent IgA responses. The ultrastructure of Th HA cells and the distribution of Fc alpha R on these cell lines were determined by colloidal gold (CG) immunoelectron microscopy (IEM). When Th HA cells were incubated with purified mouse IgA followed by CG-labeled anti-IgA, an even pattern of CG was distributed on the cell membrane. To ensure that binding occurred through Fc alpha R, Th HA cells were mixed with MOPC 315 IgA anti-DNP, followed by staining with CG-labeled TNP-human serum albumin. This resulted in an identical pattern of gold particle distribution, confirming expression of Fc alpha R on Th HA cells. No Fc mu R or Fc gamma 1R were detectable on Th HA cells by IEM. Immunocytoadherence with TNP-conjugated erythrocytes confirmed that Th HA cells were Fc alpha R+; however, no IgM or IgG rosettes were seen. When these cell lines were analyzed by flow cytometry (FACS) using IgA, IgM, or IgG1 and FITC-labeled anti-H chain-specific antibodies, 55 to 65% of cultured Th HA cells expressed Fc alpha R, and 11 to 18% expressed Fc mu R; however, no Fc gamma 1R was detectable on Th HA cells. The use of ELISA with Th HA cells as antigen confirmed the expression of Fc alpha R and the presence of less Fc mu R on these two cell lines. Solubilized membrane fractions derived from Th HA cells were tested for the presence of FcR by ELISA and for biologic function for support of IgA responses in Peyer's patch B cell cultures. Both Fc alpha R and Fc mu R were detected in fractions derived from Th HA cells. Furthermore, these fractions supported in vitro IgA anti-sheep erythrocyte responses, comparable to those obtained with Th HA cell culture supernatants

  17. Virus Attenuation after Deletion of the Cytomegalovirus Fc Receptor Gene Is Not due to Antibody Control

    PubMed Central

    Crnković-Mertens, Irena; Messerle, Martin; Milotić, Irena; Szepan, Uwe; Kučić, Natalija; Krmpotić, Astrid; Jonjić, Stipan; Koszinowski, Ulrich H.

    1998-01-01

    The murine cytomegalovirus (MCMV) fcr-1 gene codes for a glycoprotein located at the surface of infected cells which strongly binds the Fc fragment of murine immunoglobulin G. To determine the biological significance of the fcr-1 gene during viral infection, we constructed MCMV fcr-1 deletion mutants and revertants. The fcr-1 gene was disrupted by insertion of the Escherichia coli lacZ gene. In another mutant, the marker gene was also deleted, by recombinase cre. As expected for its hypothetical role in immunoevasion, the infection of mice with fcr-1 deletion mutants resulted in significantly restricted replication in comparison with wild-type MCMV and revertant virus. In mutant mice lacking antibodies, however, the fcr-1 deletion mutants also replicated poorly. This demonstrated that the cell surface-expressed viral glycoprotein with FcR activity strongly modulates the virus-host interaction but that this biological function is not caused by the immunoglobulin binding property. PMID:9445038

  18. The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus-infected rhesus macaques.

    PubMed

    O'Connell, Karyn E; Guo, Wen; Serra, Carlo; Beck, Matthew; Wachtman, Lynn; Hoggatt, Amber; Xia, Dongling; Pearson, Chris; Knight, Heather; O'Connell, Micheal; Miller, Andrew D; Westmoreland, Susan V; Bhasin, Shalender

    2015-04-01

    There are no approved therapies for muscle wasting in children infected with human immunodeficiency virus (HIV), which portends poor disease outcomes. To determine whether a soluble ActRIIb receptor Fc fusion protein (ActRIIB.Fc), a ligand trap for TGF-β/activin family members including myostatin, can prevent or restore loss of lean body mass and body weight in simian immunodeficiency virus (SIV)-infected juvenile rhesus macaques (Macaca mulatta). Fourteen pair-housed, juvenile male rhesus macaques were inoculated with SIVmac239 and, 4 wk postinoculation (WPI) treated with intramuscular injections of 10 mg ⋅ kg(-1) ⋅ wk(-1) ActRIIB.Fc or saline placebo. Body weight, lean body mass, SIV titers, and somatometric measurements were assessed monthly for 16 wk. Age-matched SIV-infected rhesus macaques were injected with saline. Intervention groups did not differ at baseline. Gains in lean mass were significantly greater in the ActRIIB.Fc group than in the placebo group (P < 0.001). Administration of ActRIIB.Fc was associated with greater gains in body weight (P = 0.01) and upper arm circumference than placebo. Serum CD4(+) T-lymphocyte counts and SIV copy numbers did not differ between groups. Administration of ActRIIB.Fc was associated with higher muscle expression of myostatin than placebo. ActRIIB.Fc effectively blocked and reversed loss of body weight, lean mass, and fat mass in juvenile SIV-infected rhesus macaques. PMID:25466897

  19. The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus-infected rhesus macaques

    PubMed Central

    O’Connell, Karyn E.; Guo, Wen; Serra, Carlo; Beck, Matthew; Wachtman, Lynn; Hoggatt, Amber; Xia, Dongling; Pearson, Chris; Knight, Heather; O’Connell, Micheal; Miller, Andrew D.; Westmoreland, Susan V.; Bhasin, Shalender

    2015-01-01

    There are no approved therapies for muscle wasting in children infected with human immunodeficiency virus (HIV), which portends poor disease outcomes. To determine whether a soluble ActRIIb receptor Fc fusion protein (ActRIIB.Fc), a ligand trap for TGF-β/activin family members including myostatin, can prevent or restore loss of lean body mass and body weight in simian immunodeficiency virus (SIV)-infected juvenile rhesus macaques (Macaca mulatta). Fourteen pair-housed, juvenile male rhesus macaques were inoculated with SIVmac239 and, 4 wk postinoculation (WPI) treated with intramuscular injections of 10 mg ⋅ kg−1 ⋅ wk−1 ActRIIB.Fc or saline placebo. Body weight, lean body mass, SIV titers, and somatometric measurements were assessed monthly for 16 wk. Age-matched SIV-infected rhesus macaques were injected with saline. Intervention groups did not differ at baseline. Gains in lean mass were significantly greater in the ActRIIB.Fc group than in the placebo group (P < 0.001). Administration of ActRIIB.Fc was associated with greater gains in body weight (P = 0.01) and upper arm circumference than placebo. Serum CD4+ T-lymphocyte counts and SIV copy numbers did not differ between groups. Administration of ActRIIB.Fc was associated with higher muscle expression of myostatin than placebo. ActRIIB.Fc effectively blocked and reversed loss of body weight, lean mass, and fat mass in juvenile SIV-infected rhesus macaques.—O’Connell, K. E., Guo, W., Serra, C., Beck, M., Wachtman, L., Hoggatt, A., Xia, D., Pearson, C., Knight, H., O’Connell, M., Miller, A. D., Westmoreland, S. V., Bhasin, S. The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus-infected rhesus macaques. PMID:25466897

  20. Fcγ Receptor I Alpha Chain (CD64) Expression in Macrophages Is Critical for the Onset of Meningitis by Escherichia coli K1

    PubMed Central

    Selvaraj, Suresh K.; Wooster, David G.; Babu, M. Madan; Schreiber, Alan D.; Verbeek, J. Sjef; Prasadarao, Nemani V.

    2010-01-01

    Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa−/− mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa−/− macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa−/− mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1. PMID:21124939

  1. Distribution and Dynamics of Rat Basophilic Leukemia Immunoglobulin E Receptors (FcɛRI) on Planar Ligand-Presenting Surfaces

    PubMed Central

    Spendier, Kathrin; Carroll-Portillo, Amanda; Lidke, Keith A.; Wilson, Bridget S.; Timlin, Jerilyn A.; Thomas, James L.

    2010-01-01

    Abstract There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. We have quantified the spatiotemporal dynamics of the redistribution of immunoglobulin E-loaded receptors (IgE-FcɛRI) on rat basophilic leukemia-2H3 mast cells in contact with fluid and gel-phase membranes displaying ligands for immunoglobulin E, using total internal reflection fluorescence microscopy. To clearly separate the kinetics of receptor redistribution from cell spreading, and to precisely define the initial contact time (±50 ms), micropipette cell manipulation was used to bring individual cells into contact with surfaces. On ligand-free surfaces, there are micron-scale heterogeneities in fluorescence that likely reflect regions of the cell that are more closely apposed to the substrate. When ligands are present, receptor clusters form with this same size scale. The initial rate of accumulation of receptors into the clusters is consistent with diffusion-limited trapping with D ∼10−1μm2/s. These results support the hypothesis that clusters form by diffusion to cell-surface contact regions. Over longer timescales (>10 s), individual clusters moved with both diffusive and directed motion components. The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes. PMID:20643056

  2. Interleukin 4 reduces expression of inhibitory receptors on B cells and abolishes CD22 and Fc gamma RII-mediated B cell suppression.

    PubMed

    Rudge, Elizabeth U; Cutler, Antony J; Pritchard, Nicholas R; Smith, Kenneth G C

    2002-04-15

    Inhibitory receptors CD22, Fc gamma RII (CD32), CD72, and paired immunoglobulin-like receptor (PIR)-B are critically involved in negatively regulating the B cell immune response and in preventing autoimmunity. Here we show that interleukin 4 (IL-4) reduces expression of all four on activated B cells at the level of messenger RNA and protein. This reduced expression is dependent on continuous exposure to IL-4 and is mediated through Stat6. Coligation of Fc gamma RII to the B cell receptor (BCR) via intact IgG increases the B cell activation threshold and suppresses antigen presentation. IL-4 completely abolishes these negative regulatory effects of Fc gamma RII. CD22 coligation with the BCR also suppresses activation -- this suppression too is abolished by IL-4. Thus, IL-4 is likely to enhance the B cell immune response by releasing B cells from inhibitory receptor suppression. By this coordinate reduction in expression of inhibitory receptors, and release from CD22 and Fc gamma RII-mediated inhibition, IL-4 is likely to play a role in T cell help of B cells and the development of T helper cell type 2 responses. Conversely, B cell activation in the absence of IL-4 would be more difficult to achieve, contributing to the maintenance of B cell tolerance in the absence of T cell help. PMID:11956299

  3. Mediation of macrophage cytolytic and phagocytic activities by antibodies of different classes and class-specific Fc-receptors.

    PubMed

    Walker, W S

    1977-08-01

    The classes of antibodies that mediate the phagocytosis and cytolysis of 51Cr-labeled chicken erythrocytes by IC-21 macrophages, an established line of mouse peritoneal macrophages, were identified. The phagocytic activity of IC-21 macrophages, as determined by a functional inhibition assay with mouse myeloma proteins, depended mainly on IgM and IgG2a antibodies and to a lesser extent on IgG2b antibodies. Extracellular cytolysis of target cells was mediated solely by IgG2b antibodies. These results correlate with the previously documented specificities of discrete Fc-receptors for IgG2a and IgG2b immunoglobulins on IC-21 cells. Thus, phagocytosis and cytolysis appear to be mediated by antibodies of different classes operating through separate and distinct sites on the surface of IC-21 macrophages. PMID:886183

  4. Fc Gamma Receptor Signaling in Mast Cells Links Microbial Stimulation to Mucosal Immune Inflammation in the Intestine

    PubMed Central

    Chen, Xiao; Feng, Bai-Sui; Zheng, Peng-Yuan; Liao, Xue-Qing; Chong, Jasmine; Tang, Shang-Guo; Yang, Ping-Chang

    2008-01-01

    Microbes and microbial products are closely associated with the pathogenesis of inflammatory bowel disease (IBD); however, the mechanisms behind this connection remain unclear. It has been previously reported that flagellin-specific antibodies are increased in IBD patient sera. As mastocytosis is one of the pathological features of IBD, we hypothesized that flagellin-specific immune responses might activate mast cells that then contribute to the initiation and maintenance of intestinal inflammation. Thirty-two colonic biopsy samples were collected from IBD patients. A flagellin/flagellin-specific IgG/Fc gamma receptor I complex was identified on biopsied mast cells using both immunohistochemistry and co-immunoprecipitation experiments; this complex was shown to co-localize on the surfaces of mast cells in the colonic mucosa of patients with IBD. In addition, an ex vivo study showed flagellin-IgG was able to bind to human mast cells. These cells were found to be sensitized to flagellin-specific IgG; re-exposure to flagellin induced the mast cells to release inflammatory mediators. An animal model of IBD was then used to examine flagellin-specific immune responses in the intestine. Mice could be sensitized to flagellin, and repeated challenges with flagellin induced an IBD-like T helper 1 pattern of intestinal inflammation that could be inhibited by pretreatment with anti-Fc gamma receptor I antibodies. Therefore, flagellin-specific immune responses activate mast cells in the intestine and play important roles in the pathogenesis of intestinal immune inflammation. PMID:18974296

  5. Immune Complex-Induced, Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcγReceptors

    PubMed Central

    Mula, Ramanjaneya V. R.; Machiah, Deepa; Holland, Lauren; Wang, Xinyu; Parihar, Harish; Sharma, Avadhesh C.; Selvaraj, Periasamy; Shashidharamurthy, Rangaiah

    2016-01-01

    Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcγRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcγRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcγR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an in vitro vasculitis model. Dimeric FcγR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcγR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcγRs. PMID:27101012

  6. Dengue vascular leakage is augmented by mast cell degranulation mediated by immunoglobulin Fcγ receptors

    PubMed Central

    Syenina, Ayesa; Jagaraj, Cyril J; Aman, Siti AB; Sridharan, Aishwarya; St John, Ashley L

    2015-01-01

    Dengue virus (DENV) is the most significant human arboviral pathogen and causes ∼400 million infections in humans each year. In previous work, we observed that mast cells (MC) mediate vascular leakage during DENV infection in mice and that levels of MC activation are correlated with disease severity in human DENV patients (St John et al., 2013b). A major risk factor for developing severe dengue is secondary infection with a heterologous serotype. The dominant theory explaining increased severity during secondary DENV infection is that cross-reactive but non-neutralizing antibodies promote uptake of virus and allow enhanced replication. Here, we define another mechanism, dependent on FcγR-mediated enhanced degranulation responses by MCs. Antibody-dependent mast cell activation constitutes a novel mechanism to explain enhanced vascular leakage during secondary DENV infection. DOI: http://dx.doi.org/10.7554/eLife.05291.001 PMID:25783751

  7. Impact of Fc gamma-receptor polymorphisms on the response to rituximab treatment in children and adolescents with mature B cell lymphoma/leukemia.

    PubMed

    Burkhardt, Birgit; Yavuz, Deniz; Zimmermann, Martin; Schieferstein, Jutta; Kabickova, Edita; Attarbaschi, Andishe; Lisfeld, Jasmin; Reiter, Alfred; Makarova, Olga; Worch, Jennifer; Bonn, Bettina R; Damm-Welk, Christine

    2016-09-01

    Recent studies in adult lymphoma patients have indicated a correlation between polymorphisms of Fc gamma-receptors (FcγRs, encoded by the respective FCGR genes) and the response to rituximab treatment. In vitro, cells expressing FcγRIIIa-158V mediate antibody-dependent cellular cytotoxicity (ADCC) more efficiently than cells expressing FcγRIIIa-158F. The impact of the FCGR2A-131HR polymorphism is unclear. In this study, the FCGR polymorphisms FCGR3A-158VF and FCGR2A-131HR were analyzed in pediatric patients with mature aggressive B cell non-Hodgkin lymphoma/leukemia (B-NHL). Pediatric patients received a single dose of rituximab monotherapy. Response was evaluated on day 5 followed by standard chemotherapy for B-NHL. Among 105 evaluable patients, a response to rituximab was observed in 21 % of those homozygous for FcγRIIa-131RR (5/24) compared to 48 % of patients who were HH and HR FcγRIIa-131 allele carriers (18/34 and 21/47, respectively; p = 0.044). Among patients with the FCGR3A-158 polymorphism, those homozygous for the FF genotype had a significantly favorable rituximab response rate of 59 % (22/37) compared to 32 % in patients who were FcγRIIIa-158VV and FcγRIIIa-VF allele carriers (2/9 and 20/59, respectively; p = 0.022). A stringent phase II response evaluation of children and adolescents with B-NHL after one dose of rituximab monotherapy showed a significant association between the rituximab response rate and FCGR polymorphisms. These findings support the hypothesis that FCGR polymorphisms represent patient-specific parameters that influence the response to rituximab. PMID:27376362

  8. FCGR2C Polymorphisms Associated with HIV-1 Vaccine Protection Are Linked to Altered Gene Expression of FcReceptors in Human B Cells

    PubMed Central

    Peng, Xinxia; Li, Shuying S.; Gilbert, Peter B.; Geraghty, Daniel E.; Katze, Michael G.

    2016-01-01

    The phase III Thai RV144 vaccine trial showed an estimated vaccine efficacy (VE) to prevent HIV-1 infection of 31.2%, which has motivated the search for immune correlates of vaccine protection. In a recent report, several single nucleotide polymorphisms (SNPs) in FCGR2C were identified to associate with the level of VE in the RV144 trial. To investigate the functional significance of these SNPs, we utilized a large scale B cell RNA sequencing database of 462 individuals from the 1000 Genomes Project to examine associations between FCGR2C SNPs and gene expression. We found that the FCGR2C SNPs that associated with vaccine efficacy in RV144 also strongly associated with the expression of FCGR2A/C and one of them also associated with the expression of Fc receptor-like A (FCRLA), another Fcreceptor (FcγR) gene that was not examined in the previous report. These results suggest that the expression of FcγR genes is influenced by these SNPs either directly or in linkage with other causal variants. More importantly, these results motivate further investigations into the potential for a causal association of expression and alternative splicing of FCGR2C and other FcγR genes with the HIV-1 vaccine protection in the RV144 trial and other similar studies. PMID:27015273

  9. FCGR2C Polymorphisms Associated with HIV-1 Vaccine Protection Are Linked to Altered Gene Expression of FcReceptors in Human B Cells.

    PubMed

    Peng, Xinxia; Li, Shuying S; Gilbert, Peter B; Geraghty, Daniel E; Katze, Michael G

    2016-01-01

    The phase III Thai RV144 vaccine trial showed an estimated vaccine efficacy (VE) to prevent HIV-1 infection of 31.2%, which has motivated the search for immune correlates of vaccine protection. In a recent report, several single nucleotide polymorphisms (SNPs) in FCGR2C were identified to associate with the level of VE in the RV144 trial. To investigate the functional significance of these SNPs, we utilized a large scale B cell RNA sequencing database of 462 individuals from the 1000 Genomes Project to examine associations between FCGR2C SNPs and gene expression. We found that the FCGR2C SNPs that associated with vaccine efficacy in RV144 also strongly associated with the expression of FCGR2A/C and one of them also associated with the expression of Fc receptor-like A (FCRLA), another Fcreceptor (FcγR) gene that was not examined in the previous report. These results suggest that the expression of FcγR genes is influenced by these SNPs either directly or in linkage with other causal variants. More importantly, these results motivate further investigations into the potential for a causal association of expression and alternative splicing of FCGR2C and other FcγR genes with the HIV-1 vaccine protection in the RV144 trial and other similar studies. PMID:27015273

  10. Fc Receptor-like 5 Expression Distinguishes Two Distinct Subsets of Human Circulating Tissue-like Memory B Cells.

    PubMed

    Li, Huifang; Borrego, Francisco; Nagata, Satoshi; Tolnay, Mate

    2016-05-15

    Fc receptor-like (FCRL) 5 is a novel IgG binding protein expressed on B cells, with the capacity to regulate Ag receptor signaling. We assessed FCRL5 expression on circulating B cells from healthy donors and found that FCRL5(+) cells are most enriched among atypical CD21(-/lo)/CD27(-) tissue-like memory (TLM) B cells, which are abnormally expanded in several autoimmune and infectious diseases. Using multicolor flow cytometry, FCRL5(+) TLM cells were found to express more CD11c and several inhibitory receptors than did the FCRL5(-) TLM subset. The homing receptor profiles of the two TLM subsets shared features consistent with migration away from lymphoid tissues, but they also displayed distinct differences. Analysis of IgH V regions in single cells indicated that although both subsets are diverse, the FCRL5(+) subset accumulated significantly more somatic mutations. Furthermore, the FCRL5(+) subset had more switched isotype expression and more extensive proliferative history. Microarray analysis and quantitative RT-PCR demonstrated that the two TLM subsets possess distinct gene expression profiles, characterized by markedly different CD11c, SOX5, T-bet, and RTN4R expression, as well as differences in expression of inhibitory receptors. Functional analysis revealed that the FCRL5(+) TLM subset responds poorly to multiple stimuli compared with the FCRL5(-) subset, as reflected by reduced calcium mobilization and blunted cell proliferation. We propose that the FCRL5(+) TLM subset, but not the FCRL5(-) TLM subset, underwent Ag-driven development and is severely dysfunctional. The present study elucidates the heterogeneity of TLM B cells and provides the basis to dissect their roles in the pathogenesis of inflammatory and infectious diseases. PMID:27076679

  11. Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy.

    PubMed

    Valerius, T; Repp, R; de Wit, T P; Berthold, S; Platzer, E; Kalden, J R; Gramatzki, M; van de Winkel, J G

    1993-08-01

    Three different classes of Fc receptors for IgG (Fc gamma R) are currently distinguished in humans, of which polymorphonuclear phagocytes (PMN) normally express both low-affinity receptor classes--Fc gamma RII (CD32) and Fc gamma RIII (CD16). During therapy with granulocyte colony-stimulating factor (G-CSF), neutrophils from patients with various malignancies and different hematologic disorders were found to additionally express high levels of the receptor with high affinity for IgG (Fc gamma RI; CD64). For these patients, the relative fluorescence intensity (rFI) for Fc gamma RI was 5.3 (range, 1.7 to 10.3; n = 19), compared with 1.0 (range, 1.0 to 1.1; n = 8) for healthy donors. The expression of Fc gamma RI during G-CSF therapy could be confirmed by using a panel of six CD64-specific antibodies, and by showing mRNA for Fc gamma RI. So far, three genes for Fc gamma RI have been identified, encoding four distinct transcription products. By reverse transcriptase-polymerase chain reaction technology, transcripts for both membrane-associated isoforms (hFc gamma RIa and hFc gamma RIb2) could be detected. The functional activity of Fc gamma RI on PMN during G-CSF therapy was shown by measuring binding of monomeric human IgG and antibody-dependent cellular cytotoxicity (ADCC). Thus, Fc gamma RI-positive neutrophils displayed enhanced ADCC activity to glioma (A1207), squamous cell (A431), and ovarian (SK-ov3) carcinoma cell lines. The involvement of Fc gamma RI in this increased cytotoxic activity was shown by blocking Fc gamma receptors with monoclonal antibodies, and by using F(ab')2 x F(ab')2-bispecific antibodies with specificities against tumor-related antigens and Fc gamma RI, resulting in solely Fc gamma RI-mediated cytotoxicity. Therapeutically, this additional Fc receptor on PMN may increase the efficacy of experimental antibody therapy. PMID:7687898

  12. Protein kinase activity associated with Fc. gamma. /sub 2a/ receptor of a murine macrophage like cell line, P388D/sub 1/

    SciTech Connect

    Hirata, Y.; Suzuki, T.

    1987-12-15

    The properties of protein kinase activity associated with Fc receptor specific for IgG/sub 2a/(Fc..gamma../sub 2a/R) of a murine macrophage like cell line, P388D/sub 1/, were investigated. IgG/sub 2a/-binding protein isolated from the detergent lysate of P388D/sub 1/ cells by affinity chromatography of IgG-Sepharose was found to contain four distinct proteins of M/sub r/ 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with (..gamma..-/sup 32/P)ATP. The autophosphorylation of Fc..gamma../sub 2a/ receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Phosphorylation of casein catalyzed by Fc..gamma../sub 2a/ receptor complex was dependent on casein concentration, increased with time or temperature, was dependent on the concentration of ATP and Mg/sup 2 +/, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn/sup 2 +/ or KCl or by a small amount of heparin and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc..gamma../sub 2a/ receptor used ATP as substrate with an apparent K/sub m/ of 2 ..mu..M as well as GTP with an apparent K/sub m/ of 10 ..mu..M. Prior heating (60/sup 0/C for 15 min) or treatment with protease (trypsin or Pronase) of Fc..gamma../sub 2a/ receptor complex almost totally abolished casein kinase activity. Thin-layer chromatography of a partial acid hydrolysate of the phosphorylated casein showed that the site of phosphorylation is at a seryl residue. These results suggest that Fc..gamma../sub 2//sub a/ receptor forms a molecule complex with protein kinase, whose characteristics resemble those of type II casein kinase but are different from those of cyclic nucleotide dependent protein kinase or from those of C protein kinase.

  13. [Preparation and the biological effect of fusion protein GLP-1-exendin-4/ IgG4(Fc) fusion protein as long acting GLP-1 receptor agonist].

    PubMed

    Zheng, Yun-cheng

    2015-12-01

    GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for treatment of diabetes due to its short half-life (t½, 2-5 min). Exendin-4 is a polypeptide isolated from lizard saliva, which can bind to GLP-1 receptor, produce physiological effects similar to GLP-1, t½ up to 2.5 h, therefore, we developed a long-lasting GLP-1 receptor agonists and GLP-1-exendin-4 fusion IgG4 Fc [GLP-1-exendin-4/ IgG4(Fc)]. We constructed the eukaryotic expression vector of human GLP-1-exendin-4/IgG4(Fc)-pOptiVEC- TOPO by gene recombination technique and expressed the fusion protein human GLP-1-IgG4 (Fc) in CHO/DG44 cells. The fusion protein stimulated the INS-1 cells secretion of insulin, GLP-1, exendin-4 and fusion protein in CD1 mice pharmacokinetic experiments, as well as GLP-1, exendin-4 and fusion protein did anti-diabetic effect on streptozotocin induced mice. Results demonstrated that the GLP-1-exendin-4/IgG4(Fc) positive CHO/DG44 clones were chosen and the media from these positive clones. Western blotting showed that one protein band was found to match well with the predicted relative molecular mass of human GLP-1-exendin-4/IgG4(Fc). Insulin RIA showed that GLP-1-exendin-4/IgG4(Fc) dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (ip), the fusion protein peaked at 30 min in circulation and maintained a plateau for 200 h. Natural biological half-life of exendin-4 was (1.39 ± 0.28) h, GLP-1 in vivo t½ 4 min, indicating that fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1-exendin-4/IgG4(Fc) was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced diabetes in mice, longer duration of the biological activity of the fusion protein. The biological activity was significantly higher than that of GLP-1 and exendin-4. GLP-1-exendin-4/IgG4(Fc) has good anti-diabetic activity

  14. Expression and Characterization of a Potent Long-Acting GLP-1 Receptor Agonist, GLP-1-IgG2σ-Fc.

    PubMed

    Yang, Yi; Chen, Fang; Wan, Deyou; Liu, Yunhui; Yang, Li; Feng, Hongru; Cui, Xinling; Gao, Xin; Song, Haifeng

    2016-01-01

    Human GLP-1 (glucagon-like peptide-1) can produce a remarkable improvement in glycemic control in patients with type 2 diabetes. However, its clinical benefits are limited by its short half-life, which is less than 2 min because of its small size and rapid enzymatic inactivation by dipeptidyl peptidase IV. We engineered GLP-1-IgG2σ-Fc, a 68-kDa fusion protein linking a variant human GLP-1 (A8G/G26E/R36G) to a human IgG2σ constant heavy-chain. A stably transfected Chinese hamster ovary cell line was obtained using electroporation. Western blotting showed that the expressed protein was immunoreactive to both GLP-1 and IgG antibodies. GLP-1-IgG2σ-Fc stimulated insulin secretion from INS-1 cells in a dose- and glucose-dependent manner and increased insulin mRNA expression. The half-life of GLP-1-IgG2σ-Fc in cynomolgus monkeys was approximately 57.1 ± 4.5 h. In the KKAy mouse model of diabetes, one intraperitoneal injection of GLP-1-IgG2σ-Fc (1 mg/kg) reduced blood glucose levels for 5 days. A 4-week repeat-administration study identified sustained effects on blood glucose levels. Oral glucose tolerance tests conducted at the beginning and end of this 4-week period showed that GLP-1-IgG2σ-Fc produced a stable glucose lowering effect. In addition, KKAy mice treated with GLP-1-IgG2σ-Fc showed statistically significant weight loss from day 23. In conclusion, these properties of GLP-1-IgG2σ-Fc demonstrated that it represented a potential long-acting GLP-1 receptor agonist for the treatment of type 2 diabetes. PMID:27232339

  15. Expression and Characterization of a Potent Long-Acting GLP-1 Receptor Agonist, GLP-1-IgG2σ-Fc

    PubMed Central

    Yang, Yi; Chen, Fang; Wan, Deyou; Liu, Yunhui; Yang, Li; Feng, Hongru; Cui, Xinling; Gao, Xin; Song, Haifeng

    2016-01-01

    Human GLP-1 (glucagon-like peptide-1) can produce a remarkable improvement in glycemic control in patients with type 2 diabetes. However, its clinical benefits are limited by its short half-life, which is less than 2 min because of its small size and rapid enzymatic inactivation by dipeptidyl peptidase IV. We engineered GLP-1-IgG2σ-Fc, a 68-kDa fusion protein linking a variant human GLP-1 (A8G/G26E/R36G) to a human IgG2σ constant heavy-chain. A stably transfected Chinese hamster ovary cell line was obtained using electroporation. Western blotting showed that the expressed protein was immunoreactive to both GLP-1 and IgG antibodies. GLP-1-IgG2σ-Fc stimulated insulin secretion from INS-1 cells in a dose- and glucose-dependent manner and increased insulin mRNA expression. The half-life of GLP-1-IgG2σ-Fc in cynomolgus monkeys was approximately 57.1 ± 4.5 h. In the KKAy mouse model of diabetes, one intraperitoneal injection of GLP-1-IgG2σ-Fc (1 mg/kg) reduced blood glucose levels for 5 days. A 4-week repeat-administration study identified sustained effects on blood glucose levels. Oral glucose tolerance tests conducted at the beginning and end of this 4-week period showed that GLP-1-IgG2σ-Fc produced a stable glucose lowering effect. In addition, KKAy mice treated with GLP-1-IgG2σ-Fc showed statistically significant weight loss from day 23. In conclusion, these properties of GLP-1-IgG2σ-Fc demonstrated that it represented a potential long-acting GLP-1 receptor agonist for the treatment of type 2 diabetes. PMID:27232339

  16. Analysis of Response Elements Involved in the Regulation of the Human Neonatal Fc Receptor Gene (FCGRT)

    PubMed Central

    Mikulska, Joanna E.

    2015-01-01

    Human epithelial, endothelial and PMA-differentiated THP-1 cell lines were used as model systems to study the transcriptional regulation of the human FCGRT gene encoding the alpha chain of hFcRn. The data obtained from site-directed mutagenesis in transient transfection experiments indicate that the Sp1 sites at positions -641, -635, and -313, CF1/YY1 elements at positions -586 and -357, and the AP-1 motif at -276 within the-660/-233 fragment of the human FCGRT promoter (hFCGRT) participate in the regulation of human FCGRT in all selected cell lines. However, their individual contribution to promoter activity is not equivalent. The Sp1 binding site at -313 and the AP-1 site at -276 are critical for the activity of the hFCGRT promoter in epithelial and endothelial cells. Moreover, the CF1/YY1 site at -586 in differentiated THP-1 cells, plays an essential role in the transcriptional activity of the promoter. In addition, the C/EBPbeta binding site at -497 of the hFCGRT promoter in epithelial and endothelial cells, and the C/EBPbeta motif located at -497 and -233 within the hFCGRT promoter in differentiated THP-1 cells may function as positive regulatory sequences in response to LPS or PMA stimulation. EMSA and supershift analyses showed that the functionally identified binding motifs in the hFCGRT promoter were able to specifically interact with their corresponding (Sp1, Sp2, Sp3, c-Fos, c-Jun, YY1, and C/EBPbeta or C/EBPdelta) transcription factors (TFs), suggesting their possible involvement in the regulation of the human FCGRT gene expression. PMID:26252948

  17. Preclinical safety evaluation of recombinant adeno-associated virus 2 vector encoding human tumor necrosis factor receptor-immunoglobulin Fc fusion gene.

    PubMed

    Zhou, Xiaobing; Shen, Lianzhong; Liu, Li; Wang, Chao; Qi, Weihong; Zhao, Aizhi; Wu, Xiaobing; Li, Bo

    2016-03-01

    Recombinant adeno-associated virus (rAAV) 2 vector gene therapy offers promise for the healing of Rheumatoid arthritis. To support the clinical development of the candidate gene therapeutic product in China, a comprehensive preclinical safety assessment of rAAV2 encoding human TNF receptor-immunoglobulin Fc fusion gene (rAAV2/human TNFR:Fc), were conducted in 3 species of experimental animals. No abnormal findings were observed in mice following single intravenous administration with test article. Compared with the control group, no differences in mean body weight, food consumption in rats and monkeys following the repeated intraarticular administration with rAAV2/human TNFR:Fc. There were also no significant adverse effects due to treatment noted by clinical chemistry, hematology and pathology assessments. After intraarticular administration with rAAV2/human TNFR:Fc, the vector DNA initially distributed to spleen, lymph nodes, and joint synovium. The vector DNA cleared rapidly as it could be detected mainly at the site of injection by 91 d post-administration (182 d for monkey). Taken together, localized delivery of rAAV2/human TNFR:Fc showed no significant toxicity in mice, rats, and monkeys, which support the planned clinical evaluation of this product. PMID:26837862

  18. Pretreatment with a soluble activin type IIB receptor/Fc fusion protein improves hypoxia-induced muscle dysfunction

    PubMed Central

    Pistilli, Emidio E.; Bogdanovich, Sasha; Mosqueira, Matias; Lachey, Jennifer; Seehra, Jasbir

    2010-01-01

    Hypoxia, or reduced oxygen, occurs in a variety of clinical and environmental situations. Hypoxic exposure is associated with decreased muscle mass and a concomitant reduction in exercise capacity, although the exact mechanisms are not completely understood. The activin type IIB receptor (ActRIIB) is a receptor for transforming growth factor-β (TGFβ) superfamily members that are involved in the negative regulation of lean tissue mass. Given that hypoxia has negative effects on muscle mass and function and that modulation of the ActRIIB has been shown to increase muscle mass, we tested the hypothesis that pharmacological targeting of the ActRIIB for 2 wk would attenuate the loss of muscle mass and function in mice after exposure to normobaric hypoxia. ActRIIB modulation was achieved using a soluble activin receptor/Fc fusion protein (sActRIIB) in mice housed in a hypoxic chamber for 1 or 2 wk. Hypoxia induced a reduction in body weight in PBS- and sActRIIB-treated mice, although sActRIIB-treated mice remained larger throughout the hypoxic exposure. The absolute forces generated by extensor digitorum longus muscles were also significantly greater in sActRIIB- than PBS-treated mice and were more resistant to eccentric contraction-induced force drop after eccentric lengthening contractions. In summary, sActRIIB pretreatment attenuated hypoxia-induced muscle dysfunction. These data suggest that targeting the ActRIIB is an effective strategy to counter hypoxia-induced muscle dysfunction and to preacclimatize to hypoxia in clinical or high-altitude settings. PMID:19864340

  19. Pretreatment with a soluble activin type IIB receptor/Fc fusion protein improves hypoxia-induced muscle dysfunction.

    PubMed

    Pistilli, Emidio E; Bogdanovich, Sasha; Mosqueira, Matias; Lachey, Jennifer; Seehra, Jasbir; Khurana, Tejvir S

    2010-01-01

    Hypoxia, or reduced oxygen, occurs in a variety of clinical and environmental situations. Hypoxic exposure is associated with decreased muscle mass and a concomitant reduction in exercise capacity, although the exact mechanisms are not completely understood. The activin type IIB receptor (ActRIIB) is a receptor for transforming growth factor-beta (TGFbeta) superfamily members that are involved in the negative regulation of lean tissue mass. Given that hypoxia has negative effects on muscle mass and function and that modulation of the ActRIIB has been shown to increase muscle mass, we tested the hypothesis that pharmacological targeting of the ActRIIB for 2 wk would attenuate the loss of muscle mass and function in mice after exposure to normobaric hypoxia. ActRIIB modulation was achieved using a soluble activin receptor/Fc fusion protein (sActRIIB) in mice housed in a hypoxic chamber for 1 or 2 wk. Hypoxia induced a reduction in body weight in PBS- and sActRIIB-treated mice, although sActRIIB-treated mice remained larger throughout the hypoxic exposure. The absolute forces generated by extensor digitorum longus muscles were also significantly greater in sActRIIB- than PBS-treated mice and were more resistant to eccentric contraction-induced force drop after eccentric lengthening contractions. In summary, sActRIIB pretreatment attenuated hypoxia-induced muscle dysfunction. These data suggest that targeting the ActRIIB is an effective strategy to counter hypoxia-induced muscle dysfunction and to preacclimatize to hypoxia in clinical or high-altitude settings. PMID:19864340

  20. Stimulatory function of paired immunoglobulin-like receptor-A in mast cell line by associating with subunits common to Fc receptors.

    PubMed

    Ono, M; Yuasa, T; Ra, C; Takai, T

    1999-10-15

    Paired Ig-like receptors (PIR) are polymorphic type I transmembrane proteins belonging to an Ig superfamily encoded by multiple isotypic genes. They are expressed on immune cells such as mast cells, macrophages, and B lymphocytes. Two subtypes of PIR have been classified according to the difference in the primary structure of the PIR transmembrane and cytoplasmic regions. These subtypes are designated as PIR-A and PIR-B. In this study, the transmembrane and cytoplasmic regions of the PIR-A subtype were shown to mediate activation signal events such as cytoplasmic calcium mobilization, protein tyrosine phosphorylations, and degranulation in rat mast cell line RBL-2H3. The association of the Fc receptor gamma and beta subunits with PIR-A was shown to be responsible for PIR-A function but not required for membrane expression of PIR-A on COS-7 cells. We further revealed the role of two charged amino acid residues in the transmembrane region, namely arginine and glutamic acid, in PIR-A function and its association with the above subunits. In contrast to the inhibitory nature of the PIR-B subtype, present findings reveal that PIR-A potentially acts as a stimulatory receptor in mast cells, suggesting a mechanism for regulation of mast cell functions by the PIR family. PMID:10514523

  1. Differential gene expression of activating Fcγ receptor classifies active tuberculosis regardless of human immunodeficiency virus status or ethnicity.

    PubMed

    Sutherland, J S; Loxton, A G; Haks, M C; Kassa, D; Ambrose, L; Lee, J-S; Ran, L; van Baarle, D; Maertzdorf, J; Howe, R; Mayanja-Kizza, H; Boom, W H; Thiel, B A; Crampin, A C; Hanekom, W; Ota, M O C; Dockrell, H; Walzl, G; Kaufmann, S H E; Ottenhoff, T H M

    2014-04-01

    New diagnostics and vaccines for tuberculosis (TB) are urgently needed, but require an understanding of the requirements for protection from/susceptibility to TB. Previous studies have used unbiased approaches to determine gene signatures in single-site populations. The present study utilized a targeted approach, reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), to validate these genes in a multisite study. We analysed ex vivo whole blood RNA from a total of 523 participants across four sub-Saharan countries (Ethiopia, Malawi, South Africa, and The Gambia) with differences in TB and human immunodeficiency virus (HIV) status. We found a number of genes that were expressed at significantly lower levels in participants with active disease than in those with latent TB infection (LTBI), with restoration following successful TB treatment. The most consistent classifier of active disease was FCGR1A (high-affinity IgG Fc receptor 1 (CD64)), which was the only marker expressed at significantly higher levels in participants with active TB than in those with LTBI before treatment regardless of HIV status or genetic background. This is the first study to identify a biomarker for TB that is not affected by HIV status or geo-genetic differences. These data provide valuable clues for understanding TB pathogenesis, and also provide a proof-of-concept for the use of RT-MLPA in rapid and inexpensive validation of unbiased gene expression findings. PMID:24205913

  2. The Split Virus Influenza Vaccine rapidly activates immune cells through Fcγ receptors.

    PubMed

    O'Gorman, William E; Huang, Huang; Wei, Yu-Ling; Davis, Kara L; Leipold, Michael D; Bendall, Sean C; Kidd, Brian A; Dekker, Cornelia L; Maecker, Holden T; Chien, Yueh-Hsiu; Davis, Mark M

    2014-10-14

    Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes. PMID:25203448

  3. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    PubMed

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs. PMID:26026665

  4. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI.

    PubMed

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2014-01-01

    The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml(-1) of antigen. This assay was modified from previous assays used to study human and canine allergic responses. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease. PMID:25406512

  5. 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.

  6. The Split Virus Influenza Vaccine rapidly activates immune cells through Fcγ Receptors

    PubMed Central

    O’Gorman, William E.; Huang, Huang; Wei, Yu-Ling; Davis, Kara L.; Leipold, Michael D.; Bendall, Sean C.; Kidd, Brian A.; Dekker, Cornelia L.; Maecker, Holden T.; Chien, Yueh-Hsiu; Davis, Mark M.

    2014-01-01

    Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or “split” viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors—specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus “splitting” inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes. PMID:25203448

  7. The dimeric platelet collagen receptor GPVI-Fc reduces platelet adhesion to activated endothelium and preserves myocardial function after transient ischemia in mice.

    PubMed

    Schönberger, Tanja; Ziegler, Melanie; Borst, Oliver; Konrad, Ildiko; Nieswandt, Bernhard; Massberg, Steffen; Ochmann, Carmen; Jürgens, Tobias; Seizer, Peter; Langer, Harald; Münch, Götz; Ungerer, Martin; Preissner, Klaus T; Elvers, Margitta; Gawaz, Meinrad

    2012-10-01

    Platelets play a critical role in the pathophysiology of reperfusion, sepsis, and cardiovascular diseases. In a multiple step process, they adhere to activated endothelium and release proinflammatory cytokines thereby promoting the inflammatory process. Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and triggers platelet activation and primary hemostasis. Activation of GPVI leads to stable platelet adhesion and degranulation of platelet granules. However, GPVI is critically involved in platelet adhesion to activated endothelium without exposure of subendothelial matrix. Earlier studies show that the soluble GPVI-Fc binds to collagen and protects mice from atherosclerosis and decreases neointima proliferation after arterial injury. Here, we show for the first time that recombinant GPVI-Fc binds to activated endothelium mainly via vitronectin and prevents platelet/endothelial interaction. Administration of GPVI-Fc reduced infarct size and preserved cardiac function in a mouse model of myocardial infarction. This process was associated with reduced GPVI-induced platelet degranulation and release of proinflammatory cytokines in vitro and in vivo. Taken together, administration of GPVI-Fc offers a novel strategy to control platelet-mediated inflammation and to preserve myocardial function following myocardial infarction. PMID:22814400

  8. Paired immunoglobulin-like receptor (PIR)-A is involved in activating mast cells through its association with Fc receptor gamma chain.

    PubMed

    Maeda, A; Kurosaki, M; Kurosaki, T

    1998-09-01

    Paired immunoglobulin-like receptor (PIR)-A and PIR-B possess similar ectodomains with six immunoglobulin-like loops, but have distinct transmembrane and cytoplasmic domains. PIR-B bears immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in its cytoplasmic domain that recruit Src homology (SH)2 domain-containing tyrosine phosphatases SHP-1 and SHP-2, leading to inhibition of B and mast cell activation. In contrast, the PIR-A protein has a charged Arg residue in its transmembrane region and a short cytoplasmic domain that lacks ITIM sequences. Here we show that Fc receptor gamma chain, containing an immunoreceptor tyrosine-based activation motif (ITAM), associates with PIR-A. Cross-linking of this PIR-A complex results in mast cell activation such as calcium mobilization in an ITAM-dependent manner. Thus, our data provide evidence for the existence of two opposite signaling pathways upon PIR aggregation. PIR-A induces the stimulatory signal by using ITAM in the associated gamma chain, whereas PIR-B mediates the inhibitory signal through its ITIMs. PMID:9730901

  9. Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus

    PubMed Central

    2010-01-01

    Introduction More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy. Methods Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR. Results Circulating CD14+ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy. Conclusions Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients. PMID:20478071

  10. Molecular and functional characteristics of the Fcalpha/muR, a novel Fc receptor for IgM and IgA.

    PubMed

    Shibuya, Akira; Honda, Shin-Ichiro

    2006-12-01

    IgM is the first antibody to be produced in a humoral immune response and is a major isotope of natural antibodies and may play an important role in innate immunity. On the other hand, IgA is a secreted antibody at the mucosal membrane such as the gastrointestinal and respiratory tracts and protects from initial invasion of microbes. However, how these antibodies are involved in immunity has been poorly elucidated. We previously identified a novel Fc receptor for IgA and IgM, designated Fcalpha/mu receptor (Fcalpha/muR), whose gene is closely located at the polymeric immunoglobulin receptor (poly-IgR), also a receptor for IgA and IgM, in the Fc receptor gene cluster on the chromosome 1. In contrast to the the poly-IgR that is expressed on epithelial, but not hematopoietic, cells, Fcalpha/muR is constitutively expressed on the majority of B lymphocytes and macrophages in the spleen and at the center of the secondary lymphoid follicles. The Fcalpha/muR mediates endocytosis Staphylococcus aureus /anti-S. aureus IgM antibody immune complexes by B lymphocytes, for which the dileucine motif in the cytoplasmic tail of the mouse Fcalpha/muR is responsible. These results reveal a new mechanism in the primary stage of immune defense against microbes. PMID:17061088

  11. Fc receptor-bearing peripheral blood mononuclear cells in breast cancer patients: a possible marker of tumour burden and prognosis.

    PubMed Central

    Bray, J; McPherson, T A

    1981-01-01

    Indirect immunofluorescence was used to identify and quantitate peripheral blood mononuclear (PBM) cells possessing high avidity Fc receptors in 105 patients upon referral to the breast cancer clinic at the Cross Cancer Institute. The cell detected was shown to be a non-adherent PBM, probably belonging to the T or null cell population. The mean percentage +/- 2 standard deviations of PBM-positive cells in 75 patients with no disease or benign breast disease was 5.3 +/- ;6.7, and this was significantly (P less than 0.001) less than the percentage found for 31 patients with breast cancer. The percentage of PBM-positive cells correlated directly with tumour burden in patient with small (less than or equal to 5 cm) tumours without regional node or extranodal metastases (5/13 had greater than or equal to 12% positive PBM) and in those with small tumours plus regional node metastases, but without extranodal metastases (8/10 had greater than or equal to 12% positive PBM). This correlation was less, however, in patients with large tumours (greater than 5 cm), and in those with extranodal metastases (4/8 had greater than or equal to 12% positive PBM), and in patients tested postoperatively (1/13 had greater than or equal to 12% positive PBM) even though 6!13 had regional node metastases at the time of surgery. Thus, this relatively simple assay, which can be done on peripheral blood samples, may turn out to be useful in patients with breast cancer as a prognostic marker insofar as it may be an indirect indicator of tumour burden preoperatively. If so, it may lead to a more aggressive postoperative adjuvant therapy approach to the subpopulation of node-negative PBM-positive breast cancer patients than is currently used for node-negative patients. PMID:7035033

  12. Insulin receptor binding motif tagged with IgG4 Fc (Yiminsu) works as an insulin sensitizer to activate Akt signaling in hepatocytes.

    PubMed

    Wang, J; Zou, T; Yang, H X; Gong, Y Z; Xie, X J; Liu, H Y; Liao, D F

    2015-01-01

    Insulin resistance is a key feature of obesity and type 2 diabetes mellitus (T2DM). Interaction of insulin with the insulin receptor (IR) leads to both its auto-phosphorylation and phosphorylation of tyrosine residues on the IR substrate (IRS) proteins, initiating the activation of intracellular signaling cascades. The metabolic effects of IRS are known to be mediated through pathways involving phosphatidyl-inositol 3-kinase (PI-3K), which result in the activation of Akt signaling. The C-terminal region of the IR ectodomain is required to facilitate the conformational changes that are required for high-affinity binding to insulin. Furthermore, the CH2 and CH3 domains in the Fc fragments of immunoglobulins are responsible for their binding to the Fc receptor, which triggers transcytosis. In this study, we created a fusion peptide of the C-terminal end of the human IR ectodomain with the IgG4 Fc fragment, including an intervening polyG fragment to ensure enough space for insulin binding. We named this new peptide "Yiminsu", meaning an insulin sensitizer. The results of our analyses show that Yiminsu significantly facilitates insulin signaling via the activation of Akt in hepatocytes in a dose- and time-dependent manner. Further studies are required to determine whether Yiminsu can act as an insulin sensitizer. PMID:26345813

  13. Fc receptors for IgG (Fc gamma Rs) on human monocytes and macrophages are not infectivity receptors for human immunodeficiency virus type 1 (HIV-1): studies using bispecific antibodies to target HIV-1 to various myeloid cell surface molecules, including the Fc gamma R.

    PubMed Central

    Connor, R I; Dinces, N B; Howell, A L; Romet-Lemonne, J L; Pasquali, J L; Fanger, M W

    1991-01-01

    Fc gamma Rs (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) are highly expressed on human mononuclear phagocytes and function in the clearance of immune complexes and opsonized pathogens. We have examined the role of Fc gamma R in mediating antibody-dependent clearance of HIV-1 by human monocytes and monocyte-derived macrophages by using bispecific antibodies (BsAbs) to independently target the virus to Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Virus production was markedly reduced in monocytes cultured with strain HIV-1IIIB opsonized with BsAbs that target the virus to either Fc gamma RI or Fc gamma RII compared to monocytes cultured with virus in the absence of BsAbs or in the presence of BsAbs that target the virus to non-Fc gamma R surface antigens (CD33 and HLA-A,B,C). These results were confirmed using the monotropic isolate HIV-1JRFL. Interaction of HIV-1JRFL with Fc gamma RI or Fc gamma RII on human monocytes and Fc gamma RI, Fc gamma RII, or Fc gamma RIII on monocyte-derived macrophages resulted in markedly reduced levels of virus production in these cultures. Moreover, HIV-1 infection of monocytes and monocyte-derived macrophages was completely blocked by anti-CD4 monoclonal antibodies, indicating that interaction with CD4 is required for infectivity even under conditions of antibody-mediated binding of HIV-1 to Fc gamma R. Thus, we propose that highly opsonized HIV-1 initiates high-affinity multivalent interactions with Fc gamma R that trigger endocytosis and intracellular degradation of the antibody-virus complex. At lower levels of antibody opsonization, there are two few interactions with Fc gamma R to initiate endocytosis and intracellular degradation of the antibody-virus complex, but there are enough interactions to stabilize the virus at the cell surface, allowing antibody-dependent enhancement of HIV-1 infection through high-affinity CD4 interactions. However, our results suggest that interaction of highly opsonized HIV-1 with Fc gamma Rs

  14. Soluble Monomeric IgG1 Fc*

    PubMed Central

    Ying, Tianlei; Chen, Weizao; Gong, Rui; Feng, Yang; Dimitrov, Dimiter S.

    2012-01-01

    Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives. PMID:22518843

  15. Adherence of Legionella pneumophila to guinea pig peritoneal macrophages, J774 mouse macrophages, and undifferentiated U937 human monocytes: role of Fc and complement receptors.

    PubMed Central

    Husmann, L K; Johnson, W

    1992-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen of alveolar macrophages. Although previous studies have demonstrated that specific antibody facilitates uptake of L. pneumophila by phagocytic cells, the role of complement has been unclear. Thus, we have examined the relative contributions of Fc gamma- and complement receptor-mediated adherence to guinea pig peritoneal macrophages, U937 human monocytic cells, and J774 mouse macrophage cells. Opsonization of L. pneumophila (Philadelphia 2) with polyclonal immunoglobulin G promoted maximum adherence to guinea pig macrophages. In contrast, incubation in the presence of 20% fresh nonimmune human serum from a single donor did not promote adherence. The results obtained with U937 and J774 cells paralleled those obtained with guinea pig macrophages. In the absence of specific antibody, opsonization with guinea pig complement did not enhance adherence of the Philadelphia 1, Philadelphia 2, or Knoxville strain. However, when complement was added to heat-inactivated, specific antiserum, a fourfold increase in the number of adherent organisms was observed. Blocking studies utilizing membrane receptor-specific monoclonal antibodies demonstrated that both Fc and complement receptors mediated adherence of organisms treated with complement in the presence of specific antibody. These results suggest that complement augments adherence of L. pneumophila only when acting in concert with specific antibody. PMID:1452353

  16. Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc 'spacer' domain in the extracellular moiety of chimeric antigen receptors avoids 'off-target' activation and unintended initiation of an innate immune response.

    PubMed

    Hombach, A; Hombach, A A; Abken, H

    2010-10-01

    Chimeric antigen receptors (CARs, immunoreceptors) are frequently used to redirect T cells with pre-defined specificity, in particular towards tumour cells for use in adoptive immunotherapy of malignant diseases. Specific targeting is mediated by an extracellularly located antibody-derived binding domain, which is joined to the transmembrane and intracellular CD3ζ moiety for T-cell activation. Stable CAR expression in T cells, however, requires a spacer domain interposed between the binding and the transmembrane domain and which is commonly the constant IgG1 Fc domain. We here revealed that CARs with Fc spacer domain bind to IgG Fc gamma receptors (FcγRs), thereby unintentionally activating innate immune cells, including monocytes and natural killer (NK) cells, which consequently secrete high amounts of pro-inflammatory cytokines. Engineered T cells, on the other hand, are likewise activated by FcγR binding resulting in cytokine secretion and lysis of monocytes and NK cells independently of the redirected specificity. To reduce FcγR binding, we modified the spacer domain without affecting CAR expression and antigen binding. Engineered with the modified CAR, T cells are not activated in presence of FcγR(+) cells, thereby minimizing the risk of off-target activation while preserving their redirected targeting specificity. PMID:20555360

  17. Forced expression of the Fc receptor gamma-chain renders human T cells hyperresponsive to TCR/CD3 stimulation.

    PubMed

    Nambiar, Madhusoodana P; Fisher, Carolyn U; Kumar, Anil; Tsokos, Christos G; Warke, Vishal G; Tsokos, George C

    2003-03-15

    High level expression of Fc epsilon RI gamma chain replaces the deficient TCR zeta-chain and contributes to altered TCR/CD3-mediated signaling abnormalities in T cells of patients with systemic lupus erythematosus. Increased responsiveness to Ag has been considered to lead to autoimmunity. To test this concept, we studied early signaling events and IL-2 production in fresh cells transfected with a eukaryotic expression vector encoding the Fc epsilon RI gamma gene. We found that the overexpressed Fc epsilon RI gamma chain colocalizes with the CD3 epsilon chain on the surface membrane of T cells and that cross-linking of the new TCR/CD3 complex leads to a dramatic increase of intracytoplasmic calcium concentration, protein tyrosine phosphorylation, and IL-2 production. We observed that overexpression of Fc epsilon RI gamma is associated with increased phosphorylation of Syk kinase, while the endogenous TCR zeta-chain is down-regulated. We propose that altered composition of the CD3 complex leads to increased T cell responsiveness to TCR/CD3 stimulation and sets the biochemical grounds for the development of autoimmunity. PMID:12626537

  18. CTLA4Fcε, a novel soluble fusion protein that binds B7 molecules and the IgE receptors, and reduces human in vitro soluble CD23 production and lymphocyte proliferation.

    PubMed

    Perez-Witzke, Daniel; Miranda-García, María Auxiliadora; Suárez, Nuris; Becerra, Raquel; Duque, Kharelys; Porras, Verónica; Fuenmayor, Jaheli; Montano, Ramon Fernando

    2016-05-01

    Immunoglobulin E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fcε, by fusing the ectodomain of the immunoregulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fcε appeared as a 70 000 MW polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fcε binds to IgE receptors FcεRI and FcεRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fcε to FcεRII/CD23 appeared stronger than that of IgE. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fcε to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fcε - but not IgE - induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, so blocking sCD23 generation. CTLA4Fcε caused a concentration-dependent reduction of lymphocyte proliferation in human peripheral blood mononuclear cell samples stimulated in vitro with concanavalin A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses. PMID:26801967

  19. Efficiency of immunoglobulin G replacement therapy in common variable immunodeficiency: correlations with clinical phenotype and polymorphism of the neonatal Fc receptor.

    PubMed

    Gouilleux-Gruart, V; Chapel, H; Chevret, S; Lucas, M; Malphettes, M; Fieschi, C; Patel, S; Boutboul, D; Marson, M-N; Gérard, L; Lee, M; Watier, H; Oksenhendler, E

    2013-02-01

    Treatment of common variable immunodeficiency disorders (CVID) is based on replacement therapy using intravenous (i.v.) or subcutaneous (s.c.) immunoglobulin (Ig)G. Interindividual variation of IgG dose is common. A total of 380 CVID patients on stable IgG replacement from two prospective cohorts were analysed. An 'efficiency' index was defined as the ratio of serum IgG trough level minus IgG residual to the average weekly dose of IgG infusion. A reduced efficiency of IgG was associated independently with the i.v. route (P < 0·001) and with the presence of at least one CVID disease-related phenotype (lymphoproliferation, autoimmune cytopenia or enteropathy) (P < 0·001). High IgG efficiency was noted in patients homozygotes for the variable number tandem repeat (VNTR) 3/3 polymorphism of the neonatal Fc receptor gene [IgG Fc fragment receptor transporter alpha chain (FCGRT)] promoter, and this was particularly significant in patients treated with IVIG (P < 0.01). In a multivariate analysis, FCGRT VNTR 3/3 genotype (P = 0·008) and high serum albumin (P < 0·001) were associated independently with increased efficiency of i.v. Ig. PMID:23286945

  20. Efficiency of immunoglobulin G replacement therapy in common variable immunodeficiency: correlations with clinical phenotype and polymorphism of the neonatal Fc receptor

    PubMed Central

    Gouilleux-Gruart, V; Chapel, H; Chevret, S; Lucas, M; Malphettes, M; Fieschi, C; Patel, S; Boutboul, D; Marson, M-N; Gérard, L; Lee, M; Watier, H; Oksenhendler, E

    2013-01-01

    Treatment of common variable immunodeficiency disorders (CVID) is based on replacement therapy using intravenous (i.v.) or subcutaneous (s.c.) immunoglobulin (Ig)G. Interindividual variation of IgG dose is common. A total of 380 CVID patients on stable IgG replacement from two prospective cohorts were analysed. An ‘efficiency’ index was defined as the ratio of serum IgG trough level minus IgG residual to the average weekly dose of IgG infusion. A reduced efficiency of IgG was associated independently with the i.v. route (P < 0·001) and with the presence of at least one CVID disease-related phenotype (lymphoproliferation, autoimmune cytopenia or enteropathy) (P < 0·001). High IgG efficiency was noted in patients homozygotes for the variable number tandem repeat (VNTR) 3/3 polymorphism of the neonatal Fc receptor gene [IgG Fc fragment receptor transporter alpha chain (FCGRT)] promoter, and this was particularly significant in patients treated with IVIG (P < 0.01). In a multivariate analysis, FCGRT VNTR 3/3 genotype (P = 0·008) and high serum albumin (P < 0·001) were associated independently with increased efficiency of i.v. Ig. PMID:23286945

  1. Mechanism-Based Competitive Binding Model to Investigate the Effect of Neonatal Fc Receptor Binding Affinity on the Pharmacokinetic of Humanized Anti-VEGF Monoclonal IgG1 Antibody in Cynomolgus Monkey.

    PubMed

    Ng, Chee M; Fielder, Paul J; Jin, Jin; Deng, Rong

    2016-07-01

    The quantitative relationship between neonatal Fc receptor (FcRn) binding affinity at both acidic and physiological pH and the pharmacokinetics of protein engineered FcRn IgG1 variants has not yet been reported. Our objective was to develop a quantitatively mechanism-based competitive binding model to describe the effects of FcRn binding affinity at acidic and physiological pH on the pharmacokinetics of anti-VEGF IgG1 antibodies when both endogenous and exogenous antibodies are competing for the same FcRn. Pharmacokinetic (PK) and FcRn binding data from five Fc variants of humanized anti-VEGF IgG1 monoclonal antibodies with wide range of FcRn binding affinity were used for the analysis. Sixty-seven anti-VEGF IgG1 antibody-treated animals and 25 control animals with simulated endogenous IgG levels were used to develop the final model. A hybrid iterative two stages and Monte Carlo parametric expectation-maximization method was used to obtain the final model parameters estimates. The final model well described the observed PK data. Quantitative FcRn binding affinity-pharmacokinetics relationships was constructed to provide important biological insights in better understanding of the FcRn binding effect on pharmacokinetics of anti-VEGF IgG1 antibodies in cynomolgus monkeys and served as an important model-based drug discovery platform to guide the design and development of the future generation of anti-VEGF or other therapeutic IgG1 antibodies. PMID:27075465

  2. Antibody-Mediated Fcγ Receptor-Based Mechanisms of HIV Inhibition: Recent Findings and New Vaccination Strategies

    PubMed Central

    Holl, Vincent; Peressin, Maryse; Moog, Christiane

    2009-01-01

    The HIV/AIDS pandemic is one of the most devastating pandemics worldwide. Today, the major route of infection by HIV is sexual transmission. One of the most promising strategies for vaccination against HIV sexual infection is the development of a mucosal vaccine, which should be able to induce strong local and systemic protective immunity. It is believed that both humoral and cellular immune responses are needed for inducing a sterilizing protection against HIV. Recently, passive administration of monoclonal neutralizing antibodies in macaques infected by vaginal challenge demonstrated a crucial role of FcγRs in the protection afforded by these antibodies. This questioned about the role of innate and adaptive immune functions, including ADCC, ADCVI, phagocytosis of opsonized HIV particles and the production of inflammatory cytokines and chemokines, in the mechanism of HIV inhibition in vivo. Other monoclonal antibodies - non-neutralizing inhibitory antibodies - which recognize immunogenic epitopes, have been shown to display potent FcγRs-dependent inhibition of HIV replication in vitro. The potential role of these antibodies in protection against sexual transmission of HIV and their biological relevance for the development of an HIV vaccine therefore need to be determined. This review highlights the potential role of FcγRs-mediated innate and adaptive immune functions in the mechanism of HIV protection. PMID:21994593

  3. PET/CT and MR imaging biomarker of lipid-rich plaques using [64Cu]-labeled scavenger receptor (CD68-Fc).

    PubMed

    Bigalke, Boris; Phinikaridou, Alkystis; Andia, Marcelo E; Cooper, Margaret S; Schuster, Andreas; Wurster, Thomas; Onthank, David; Münch, Götz; Blower, Philip; Gawaz, Meinrad; Nagel, Eike; Botnar, Rene M

    2014-11-15

    Continued uptake of modified low-density lipoproteins (LDL) by the scavenger receptor, CD68, of activated macrophages is a crucial process in the development of atherosclerotic plaques and leads to the formation of foam cells. Eight-weeks-old male Apolipoprotein E-deficient (ApoE(-/-)) mice (n = 6) were fed a high-fat diet for 12 weeks. C57BL/6J wildtype (WT) mice served as controls (n = 6). Positron emission tomography (PET) with an acquisition time of 1800 s (NanoPET/CT scanner; Mediso, Hungary & Bioscan, USA) was carried out 24h after intravenous tail vein administration of 50 µl (64)Cu-CD68-Fc (~20-30 µg labeled protein/mouse containing approximately 10-12 MBq (64)Cu-CD68-Fc per mouse). Three days after PET/CT, all mice received an intravenous administration of 0.2 mmol/kg body weight of a gadolinium-based elastin-binding contrast agent to assess plaque burden and vessel wall remodeling. Two hours after injection, mice were imaged in a 3T clinical MR scanner (Philips Healthcare, Best, NL) using a dedicated single loop surface coil (23 mm). Enhanced (64)Cu-CD68-Fc uptake was found in the aortic arches of ApoE(-/-) compared to WT mice (ApoE(-/-) mice:10.5 ± 1.5 Bq/cm(3) vs. WT mice: 2.1 ± 0.3 Bq/cm(3); P = 0.002). Higher gadolinium-based elastin-binding contrast agent uptake was also detected in the aortic arch of ApoE(-/-) compared to WT mice using R(1) maps (R(1) = 1.47 ± 0.06 s(-1) vs. 0.92 ± 0.05 s(-1); P <0.001). Radiolabeled scavenger receptor ((64)Cu-CD68-Fc) may help to target foam cell rich plaques with high content of oxidized LDL. This novel imaging biomarker tool may have potential to identify unstable plaques and for risk stratification. PMID:25499394

  4. Utility of Neutrophil Fcγ Receptor I (CD64) Index as a Biomarker for Mucosal Inflammation in Pediatric Crohn's Disease

    PubMed Central

    Minar, Phillip; Haberman, Yael; Jurickova, Ingrid; Wen, Ting; Rothenberg, Marc E.; Kim, Mi-Ok; Saeed, Shehzad A.; Baldassano, Robert N.; Stephens, Michael; Markowitz, James; Rosh, Joel; Crandall, Wallace V.; Heyman, Melvin B.; Mack, David R.; Griffiths, Anne M.; Baker, Susan S.; Hyams, Jeffrey S.; Kugathasan, Subra; Denson, Lee A.

    2014-01-01

    Background Neutrophil expression of the Fcγ receptor I (CD64) is upregulated in adult patients with clinically active inflammatory bowel disease (IBD). We tested the relationship of CD64 with mucosal inflammation and clinical relapse in pediatric Crohn's disease (CD). Methods In a cohort of 208 newly diagnosed CD and 43 non-IBD controls, ileal expression of FcγRI/S100A9 was determined by RNA sequencing from biopsies obtained at ileocolonoscopy. In a second cohort, we tested for the peripheral blood polymorphonuclear neutrophil (PMN) CD64 index from 26 newly diagnosed CD, 30 non-IBD controls and 83 children with established CD. Results Ileal FcγRIA mRNA expression was significantly elevated in CD at diagnosis compared with non-IBD controls (p<0.001), and correlated with ileal S100A9 (calprotectin) expression (r=0.83, p<0.001). The median(range) PMN CD64 index for newly diagnosed CD was 2.3(0.74-9.3) compared with 0.76(0.39-1.2) for non-IBD controls (p<0.001) with 96% sensitivity and 90% specificity at the cut point of 1.0. The PMN CD64 index significantly correlated with mucosal injury as measured by the Simple Endoscopic Score-CD (SES-CD, r=0.62, p<0.001). CD patients in clinical remission receiving maintenance therapy with a PMN CD64 index <1.0 had a sustained remission rate of 95% over the following 12 months compared with 56% in those with a PMN CD64 index >1.0 (p<0.01). Conclusions An elevated PMN CD64 index is associated with both mucosal inflammation and an increased risk for clinical relapse in pediatric CD. The PMN CD64 index is a reliable marker for sustained remission in CD patients receiving maintenance therapy. PMID:24788216

  5. Induction of Fc receptors for IgA on murine T cell hybridoma by human monoclonal IgA and by high molecular weight IgA in IgA nephropathy.

    PubMed Central

    Chevailler, A; Monteiro, R C; Daëron, M; Lesavre, P

    1987-01-01

    A reproducible immunocyto-adherence assay has been developed to study the modulation of Fc receptors for IgA (Fc alpha R), using a murine T cell hybridoma (T2D4), which expresses Fc receptors for all known isotypes of secreted immunoglobulins. By using sheep red blood cells coated with the hapten 2-4-6 trinitrophenyl (TNP), as indicator cells, and a murine monoclonal IgA (MOPC 315) antibody with anti-TNP activity, we were able to study the Fc alpha R on T2D4 cells. We found that: (a) murine Fc alpha R can bind human monoclonal IgA, and this binding is isotype specific since it was inhibited by human monoclonal IgA but not by human monoclonal IgG or IgM; (b) the expression of murine Fc alpha R is unducible by human monoclonal IgA, and this effect is isotype specific since it is not observed with human monoclonal IgM or IgG (c) sera from patients with IgA nephropathy can also induce Fc alpha R expression; by contrast, no induction was observed with normal human sera, (d) in one serum from an IgA-nephropathy patient, the inducer factor was characterized by affinity chromatography on anti-IgA-Sepharose and by gel filtration: high molecular weight IgA, probably IgA aggregates or immune complexes were recognized to be responsible for the induction of murine Fc alpha R expression. PMID:3497739

  6. A Transmembrane Polymorphism of Fcγ Receptor IIb Is Associated with Kidney Deficiency Syndrome in Rheumatoid Arthritis

    PubMed Central

    Mo, Na; Lai, Ruogu; Luo, Shizi; Xie, Jianglin; Wang, Xizi; Liu, Lijuan; Liu, Xiaoling

    2016-01-01

    Objective. The purpose is to investigate the role of kidney deficiency and the association between kidney deficiency and a polymorphism FcγRIIb 695T>C coding for nonsynonymous substitution IIe232Thr (I232T) in rheumatoid arthritis (RA). Methods. Clinical parameters and autoantibodies were analyzed and genotyping was performed in 159 kidney deficiency and 161 non-kidney-deficiency RA patients. Results. The age of disease onset and disease duration exhibited significant differences between two groups (P < 0.01). Patients with kidney deficiency tend to have higher activity of disease (P < 0.05). Anti-cyclic citrullinated peptides antibodies (ACPA) levels of patients with kidney deficiency were higher than the controls (P = 0.039). 125 (78.6%) kidney deficiency and 114 (70.8%) non-kidney-deficiency patients had both ACPA-positive and RF-positive (P = 0.04, OR = 3.29). FcγRIIb I232TT homozygotes were identified in 10 of 159 (6.3%) kidney deficiency subjects and 1 of 161 (0.6%) controls (P = 0.000, OR = 16.45). Furthermore, in pooled genotype analysis, I232IT and I232TT homozygotes were significantly enriched in kidney deficiency individuals compared with the controls (P = 0.000, OR = 3.79). Frequency of T allele was associated with kidney deficiency RA population (P = 0.000, OR = 3.18). Conclusion. This study confirmed that kidney deficiency was closely associated with disease activity and autoimmune disorder in RA. Kidney deficiency in RA is first to reveal a strong genetic link to FcγRIIb variants. PMID:27051449

  7. Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics.

    PubMed

    Bajardi-Taccioli, Adriana; Blum, Andrew; Xu, Chongfeng; Sosic, Zoran; Bergelson, Svetlana; Feschenko, Marina

    2015-10-01

    Recycling of antibodies and Fc containing therapeutic proteins by the neonatal Fc receptor (FcRn) is known to prolong their persistence in the bloodstream. Fusion of Fc fragment of IgG1 to other proteins is one of the strategies to improve their pharmacokinetic properties. Accurate measurement of Fc-FcRn binding provides information about the strength of this interaction, which in most cases correlates with serum half-life of the protein. It can also offer insight into functional integrity of Fc region. We investigated FcRn binding activity of a large set of Fc-fusion samples after thermal stress by the method based on AlphaScreen technology. An unexpected significant increase in FcR binding was found to correlate with formation of aggregates in these samples. Monomer purified from a thermally-stressed sample had normal FcRn binding, confirming that its Fc portion was intact. Experiments with aggregates spiked into a sample with low initial aggregation level, demonstrated strong correlation between the level of aggregates and FcRn binding. This correlation varied significantly in different methods. By introducing modifications to the assay format we were able to minimize the effects of aggregated species on FcRn binding, which should prevent masking functional changes of Fc-fusion protein. Biolayer interferometry (BLI) was used as an alternative method to measure FcRn binding. Both optimized AlphaScreen- and BLI-based assays were sensitive to structural changes in Fc portion of the molecule, such as oxidation of methionines 252 and 428, and therefore suitable for characterization of FcRn binding. PMID:26254986

  8. Improved tumor imaging and therapy via i.v. IgG–mediated time-sequential modulation of neonatal Fc receptor

    PubMed Central

    Singh Jaggi, Jaspreet; Carrasquillo, Jorge A.; Seshan, Surya V.; Zanzonico, Pat; Henke, Erik; Nagel, Andrew; Schwartz, Jazmin; Beattie, Brad; Kappel, Barry J.; Chattopadhyay, Debjit; Xiao, Jing; Sgouros, George; Larson, Steven M.; Scheinberg, David A.

    2007-01-01

    The long plasma half-life of IgG, while allowing for enhanced tumor uptake of tumor-targeted IgG conjugates, also results in increased background activity and normal-tissue toxicity. Therefore, successful therapeutic uses of conjugated antibodies have been limited to the highly sensitive and readily accessible hematopoietic tumors. We report a therapeutic strategy to beneficially alter the pharmacokinetics of IgG antibodies via pharmacological inhibition of the neonatal Fc receptor (FcRn) using high-dose IgG therapy. IgG-treated mice displayed enhanced blood and whole-body clearance of radioactivity, resulting in better tumor-to-blood image contrast and protection of normal tissue from radiation. Tumor uptake and the resultant therapeutic response was unaltered. Furthermore, we demonstrated the use of this approach for imaging of tumors in humans and discuss its potential applications in cancer imaging and therapy. The ability to reduce the serum persistence of conjugated IgG antibodies after their infusion can enhance their therapeutic index, resulting in improved therapeutic and diagnostic efficacy. PMID:17717602

  9. Improved tumor imaging and therapy via i.v. IgG-mediated time-sequential modulation of neonatal Fc receptor.

    PubMed

    Jaggi, Jaspreet Singh; Carrasquillo, Jorge A; Seshan, Surya V; Zanzonico, Pat; Henke, Erik; Nagel, Andrew; Schwartz, Jazmin; Beattie, Brad; Kappel, Barry J; Chattopadhyay, Debjit; Xiao, Jing; Sgouros, George; Larson, Steven M; Scheinberg, David A

    2007-09-01

    The long plasma half-life of IgG, while allowing for enhanced tumor uptake of tumor-targeted IgG conjugates, also results in increased background activity and normal-tissue toxicity. Therefore, successful therapeutic uses of conjugated antibodies have been limited to the highly sensitive and readily accessible hematopoietic tumors. We report a therapeutic strategy to beneficially alter the pharmacokinetics of IgG antibodies via pharmacological inhibition of the neonatal Fc receptor (FcRn) using high-dose IgG therapy. IgG-treated mice displayed enhanced blood and whole-body clearance of radioactivity, resulting in better tumor-to-blood image contrast and protection of normal tissue from radiation. Tumor uptake and the resultant therapeutic response was unaltered. Furthermore, we demonstrated the use of this approach for imaging of tumors in humans and discuss its potential applications in cancer imaging and therapy. The ability to reduce the serum persistence of conjugated IgG antibodies after their infusion can enhance their therapeutic index, resulting in improved therapeutic and diagnostic efficacy. PMID:17717602

  10. Evidence for TLR4 and FcRγ-CARD9 activation by cholera toxin B subunit and its direct bindings to TREM2 and LMIR5 receptors.

    PubMed

    Phongsisay, Vongsavanh; Iizasa, Ei'ichi; Hara, Hiromitsu; Yoshida, Hiroki

    2015-08-01

    Cholera toxin (CTX) is a virulent factor of Vibrio cholerae that causes life-threatening diarrheal disease. Its non-toxic subunit CTB has been extensively studied for vaccine delivery. In immune cells, CTB induces a number of signaling molecules related to cellular activation and cytokine production. The mechanisms by which CTB exerts its immunological effects are not understood. We report here the immunological targets of CTB. The unexpected finding that GM1 ganglioside inhibited NF-κB activation in human monocytes stimulated with CTX and agonists of Toll-like receptors (TLR) suggests the possibility of CTX-TLR interaction. Indeed, CTX-induced IL-6 production was substantially reduced in MyD88(-/-) or TLR4(-/-) macrophages. Ectopic expression of TLR4 was required for CTX-induced NF-κB activation in HEK 293 cells. Furthermore, the inflammatory capacity of CTB was lost in the absence of TLR4, adaptor protein FcRγ, or its downstream signaling molecule CARD9. Attempts have been made to identify CTB-binding targets from various C-type lectin and immunoglobulin-like receptors. CTB targeted not only GM1 and TLR4 but also TREM2 and LMIR5/CD300b. CTB-TREM2 interaction initiated signal transduction through adaptor protein DAP12. The binding of CTB inhibited LMIR5 activation induced by its endogenous ligand 3-O-sulfo-β-d-galactosylceramide C24:1. In summary, CTB targets TLR4, FcRγ-CARD9, TREM2, and LMIR5. These findings provide new insights into the immunobiology of cholera toxin. PMID:26021803

  11. Inhibition of HIV Env binding to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120

    PubMed Central

    Binley, James M; Ngo-Abdalla, Stacie; Moore, Penny; Bobardt, Michael; Chatterji, Udayan; Gallay, Philippe; Burton, Dennis R; Wilson, Ian A; Elder, John H; de Parseval, Aymeric

    2006-01-01

    During natural HIV infection, an array of host receptors are thought to influence virus attachment and the kinetics of infection. In this study, to probe the interactions of HIV envelope (Env) with various receptors, we assessed the inhibitory properties of various anti-Env monoclonal antibodies (mAbs) in binding assays. To assist in detecting Env in attachment assays, we generated Fc fusions of full-length wild-type gp120 and several variable loop-deleted gp120s. Through investigation of the inhibition of Env binding to cell lines expressing CD4, CCR5, DC-SIGN, syndecans or combinations thereof, we found that the broadly neutralizing mAb, 2G12, directed to a unique carbohydrate epitope of gp120, inhibited Env-CCR5 binding, partially inhibited Env-DC-SIGN binding, but had no effect on Env-syndecan association. Furthermore, 2G12 inhibited Env attachment to primary monocyte-derived dendritic cells, that expressed CD4 and CCR5 primary HIV receptors, as well as DC-SIGN, and suggested that the dual activities of 2G12 could be valuable in vivo for inhibiting initial virus dissemination and propagation. PMID:16817962

  12. Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics

    PubMed Central

    Rath, Timo; Baker, Kristi; Dumont, Jennifer A.; Peters, Robert T.; Jiang, Haiyan; Qiao, Shuo-Wang; Lencer, Wayne I.; Pierce, Glenn F.; Blumberg, Richard S.

    2016-01-01

    Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn–Fc interaction can generate longer-lasting and more effective therapeutics. PMID:24156398

  13. N-terminal amphipathic helix of Amphiphysin can change the spatial distribution of immunoglobulin E receptors (FcεRI) in the RBL-2H3 mast cell synapse

    PubMed Central

    Spendier, Kathrin

    2015-01-01

    Biomembranes undergo extensive shape changes as they perform vital cellular functions or become diseased. To understand the mechanisms by which lipids and proteins control membrane curvature during various processes, researchers have identified and engineered many curvature-inducing and curvature-sensing proteins and peptides. In this paper, a simple experiment was performed to show qualitatively how membrane remodeling by N-terminal amphipathic helix of Amphiphysin affects the spatial distribution of the transmembrane Fc receptor protein (FcεRI) in mast cells. Results indicate that an elevated concentration of amphipathic helices can interfere with the formation of a typical mast cell synapse. PMID:26835247

  14. Antibody Fc: Linking Adaptive and Innate Immunity

    PubMed Central

    Reichert, Janice M.

    2014-01-01

    Antibody Fc: Linking Adaptive and Innate Immunity, edited by Margaret E. Ackerman and Falk Nimmerjahn and published by Academic Press, provides a highly detailed examination of the involvement of the antibody Fc in mechanisms critical to both innate and adaptive immune responses. Despite a recent increase in format diversity, most marketed antibodies are full-length IgG molecules and the majority of the commercial clinical pipeline of antibody therapeutics is composed of Fc-containing IgG molecules, which underscores the importance of understanding how the Fc domain affects biological responses. The book is divided into six sections that include a total of 20 chapters. In order of their appearance, the sections provide extensive coverage of effector mechanisms, effector cells, Fc receptors, variability of the Fc domain, genetic associations, and evolving areas.

  15. Analysis of Fcγ Receptor IIIa and IIa Polymorphisms: Lack of Correlation with Outcome in Trastuzumab-Treated Breast Cancer Patients

    PubMed Central

    Hurvitz, Sara A.; Betting, David J.; Stern, Howard M.; Quinaux, Emmanuel; Stinson, Jeremy; Seshagiri, Somasekar; Zhao, Ying; Buyse, Marc; Mackey, John; Driga, Adrian; Damaraju, Sambasivarao; Sliwkowski, Mark X.; Robert, Nicholas J.; Valero, Vicente; Crown, John; Falkson, Carla; Brufsky, Adam; Pienkowski, Tadeusz; Eiermann, Wolfgang; Martin, Miguel; Bee, Valerie; Marathe, Omkar; Slamon, Dennis J.; Timmerman, John M.

    2013-01-01

    Purpose The mechanisms by which trastuzumab imparts clinical benefit remain incompletely understood. Antibody-dependent cellular cytotoxicity via interactions with Fcγ receptors (FcγR) on leukocytes may contribute to its anti-tumor effects. Single nucleotide polymorphisms (SNPs) in FCGR3A and FCGR2A genes lead to amino acid substitutions at positions 158 and 131 respectively and affect binding of antibodies to FcγR such that 158V/V and 131H/H bind with highest affinity. This study aimed to determine whether high affinity SNPs are associated with disease free survival (DFS) among patients with HER2-positive non-metastatic breast cancer. Experimental Design Genomic DNA was isolated from 1,286 patients enrolled in a trial of adjuvant trastuzumab-based chemotherapy. Genotyping was performed using Sanger sequencing and Sequenom mass spectrometry. Results 1,189 patient samples were successfully genotyped for FCGR3A and 1,218 for FCGR2A. Compared to the overall results of the BCIRG006 study, in the subset of patients genotyped in this analysis, a less robust improvement in DFS was observed for the trastuzumab arms compared to control arm (HR=0.842, P=0.1925). When stratified for prognostic features, the HR in favor of trastuzumab was consistent with that of the overall study (HR=0.74, P=0.036). No correlation between DFS and FCGR3A/2A genotypes was seen for trastuzumab-treated patients (158V/V vs V/F vs F/F, P=0.98; 131H/H vs H/R vs R/R, P=0.76; 158V/V and/or 131H/H vs others, P=0.67). Conclusion This analysis evaluating the association between FCGR3A/2A genotypes and trastuzumab efficacy in HER2-positive breast cancer did not demonstrate a correlation between FCGR3A-V/F and FCGR2A-H/R SNPs and DFS in patients treated with trastuzumab. PMID:22504044

  16. Human Antibodies Can Cross Guinea Pig Placenta and Bind Its Neonatal Fc Receptor: Implications for Studying Immune Prophylaxis and Therapy during Pregnancy

    PubMed Central

    Struble, Evi Budo; Ma, Li; Zhong, Lilin; Lesher, A.; Beren, Joel; Zhang, Pei

    2012-01-01

    Despite increased use of monoclonal and polyclonal antibody therapies, including during pregnancy, there is little data on appropriate animal models that could humanely be used to understand determinants of protection and to evaluate safety of these biologics in the mother and the developing fetus. Here, we demonstrate that pregnant guinea pigs can transport human IgG transplacentally at the end of pregnancy. We also observe that human IgG binds to an engineered soluble variant of the guinea pig neonatal Fc receptor in vitro in a manner similar to that demonstrated for the human variant, suggesting that this transplacental transport mirrors the receptor-based mechanism seen in humans. Using an intravenous antihepatitis B-specific immune globulin preparation as an example, we show that this transport results in neutralizing activity in the mother and the newborn that would potentially be prophylactic against hepatitis B viral infection. These preliminary data lay the groundwork for introducing pregnant guinea pigs as an appropriate model for the evaluation of antibody therapies and advancing the health of women and neonates. PMID:22991567

  17. Human antibodies can cross guinea pig placenta and bind its neonatal Fc Receptor: implications for studying immune prophylaxis and therapy during pregnancy.

    PubMed

    Struble, Evi Budo; Ma, Li; Zhong, Lilin; Lesher, A; Beren, Joel; Zhang, Pei

    2012-01-01

    Despite increased use of monoclonal and polyclonal antibody therapies, including during pregnancy, there is little data on appropriate animal models that could humanely be used to understand determinants of protection and to evaluate safety of these biologics in the mother and the developing fetus. Here, we demonstrate that pregnant guinea pigs can transport human IgG transplacentally at the end of pregnancy. We also observe that human IgG binds to an engineered soluble variant of the guinea pig neonatal Fc receptor in vitro in a manner similar to that demonstrated for the human variant, suggesting that this transplacental transport mirrors the receptor-based mechanism seen in humans. Using an intravenous antihepatitis B-specific immune globulin preparation as an example, we show that this transport results in neutralizing activity in the mother and the newborn that would potentially be prophylactic against hepatitis B viral infection. These preliminary data lay the groundwork for introducing pregnant guinea pigs as an appropriate model for the evaluation of antibody therapies and advancing the health of women and neonates. PMID:22991567

  18. Activation of the JAK/STAT-1 Signaling Pathway by IFN-γ Can Down-Regulate Functional Expression of the MHC Class I-Related Neonatal Fc Receptor for IgG1

    PubMed Central

    Liu, Xindong; Ye, Lilin; Bai, Yu; Mojidi, Habi; Simister, Neil E.; Zhu, Xiaoping

    2009-01-01

    Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-γ. However, in this study we found that IFN-γ down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-γ resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-γ activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-γ, whereas expression of JAK1 or STAT-1 protein restored the IFN-γ inhibitory effect on luciferase activity. The repressive effect of IFN-γ on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-γ activation site sequence and by over-expression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-γ stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-γ. PMID:18566411

  19. Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

    PubMed

    Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

    2016-03-01

    Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women. PMID:26713784

  20. Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI.

    PubMed Central

    Yao, Z; Jackson, W; Grose, C

    1993-01-01

    Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane. Images PMID:8392591

  1. Aspergillus Cell Wall Chitin Induces Anti- and Proinflammatory Cytokines in Human PBMCs via the FcReceptor/Syk/PI3K Pathway

    PubMed Central

    Becker, K. L.; Aimanianda, V.; Wang, X.; Gresnigt, M. S.; Ammerdorffer, A.; Jacobs, C. W.; Gazendam, R. P.; Joosten, L. A. B.; Netea, M. G.

    2016-01-01

    ABSTRACT Chitin is an important cell wall component of Aspergillus fumigatus conidia, of which hundreds are inhaled on a daily basis. Previous studies have shown that chitin has both anti- and proinflammatory properties; however the exact mechanisms determining the inflammatory signature of chitin are poorly understood, especially in human immune cells. Human peripheral blood mononuclear cells were isolated from healthy volunteers and stimulated with chitin from Aspergillus fumigatus. Transcription and production of the proinflammatory cytokine interleukin-1β (IL-1β) and the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra) were measured from the cell culture supernatant by quantitative PCR (qPCR) or enzyme-linked immunosorbent assay (ELISA), respectively. Chitin induced an anti-inflammatory signature characterized by the production of IL-1Ra in the presence of human serum, which was abrogated in immunoglobulin-depleted serum. Fc-γ-receptor-dependent recognition and phagocytosis of IgG-opsonized chitin was identified as a novel IL-1Ra-inducing mechanism by chitin. IL-1Ra production induced by chitin was dependent on Syk kinase and phosphatidylinositol 3-kinase (PI3K) activation. In contrast, costimulation of chitin with the pattern recognition receptor (PRR) ligands lipopolysaccharide, Pam3Cys, or muramyl dipeptide, but not β-glucan, had synergistic effects on the induction of proinflammatory cytokines by human peripheral blood mononuclear cells (PBMCs). In conclusion, chitin can have both pro- and anti-inflammatory properties, depending on the presence of pathogen-associated molecular patterns and immunoglobulins, thus explaining the various inflammatory signatures reported for chitin. PMID:27247234

  2. Registration of 'FC1028', 'FC1037, 'FC1038' and, 'FC1036' multigerm sugarbeet germplasm with multiple disease resistances

    Technology Transfer Automated Retrieval System (TEKTRAN)

    FC1028’, ‘FC1036’, ‘FC1037’, and ‘FC1038’ (PI 665053, PI 665054, PI 665055, PI 665056) sugarbeet germplasms (Beta vulgaris L.) were released from 20111027, 09-FC1036, 20111025, and 04-FC1038 seed lots, respectively, and tested under the designations 04-FC1028; 05-, 06-, 07-, 08-, 09-FC1036; 04-FC10...

  3. Expression Profile of Human Fc Receptor-Like 1, 2, and 4 Molecules in Peripheral Blood Mononuclear Cells of Patients with Hashimoto's Thyroiditis and Graves' Disease.

    PubMed

    Rostamzadeh, D; Dabbaghmanesh, M H; Shabani, M; Hosseini, A; Amirghofran, Z

    2015-08-01

    Recently identified Fc receptor-like (FCRL) molecules are new members of the immunoglobulin superfamily dominantly expressed by B cells. Although FCRL expression patterns have been studied in normal and malignant cells, their biological functions and roles remain to be clearly identified in humans. Research has particularly focused on FCRL gene polymorphisms in autoimmune diseases, however, their involvement in the pathogenesis of autoimmune diseases is an interesting field for investigation. In the present study, we have investigated the gene expression profiles of FCRL1, 2, and 4 in 2 common thyroid diseases, Hashimoto's thyroiditis (HT) and Graves' disease (GD). FCRL1, 2, and 4 expressions were determined in peripheral blood samples of 55 HT patients, 40 GD patients and equal numbers of normal subjects by quantitative real-time PCR. Our results showed downregulation of FCRL1 and upregulation of FCRL2 transcripts in both HT and GD groups compared to healthy counterparts. Overexpression of FCRL4 was observed only in GD patients compared to controls. A significant correlation was observed between all FCRL gene expression levels in HT patients. Only FCRL2 and 4 had a correlation in GD patients. In addition, FCRL1, 2, and 4 gene expressions showed no correlations with the level of anti-thyroid peroxidase antibody (anti-TPO) or anti-thyroglobulin (anti-Tg) antibody from patients' sera. In conclusion, expressions of activating or inhibitory FCRL1, 2, and 4 showed significant alterations in HT and GD patients compared to healthy subjects. PMID:25738996

  4. A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B

    PubMed Central

    Haridan, Umi Shakina; Mokhtar, Umairah; Machado, Lee R.; Abdul Aziz, Abu Thalhah; Shueb, Rafidah Hanim; Zaid, Masliza; Sim, Benedict; Mustafa, Mahiran; Nik Yusof, Nik Khairudin; Lee, Christopher K. C.; Abu Bakar, Suhaili; AbuBakar, Sazaly; Hollox, Edward J.; Boon Peng, Hoh

    2015-01-01

    The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method’s performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs. PMID:25594501

  5. Fcγ and Complement Receptors and Complement Proteins in Neutrophil Activation in Rheumatoid Arthritis: Contribution to Pathogenesis and Progression and Modulation by Natural Products

    PubMed Central

    Paoliello-Paschoalato, Adriana Balbina; Marchi, Larissa Fávaro; de Andrade, Micássio Fernandes; Kabeya, Luciana Mariko; Donadi, Eduardo Antônio; Lucisano-Valim, Yara Maria

    2015-01-01

    Rheumatoid arthritis (RA) is a highly disabling disease that affects all structures of the joint and significantly impacts on morbidity and mortality in RA patients. RA is characterized by persistent inflammation of the synovial membrane lining the joint associated with infiltration of immune cells. Eighty to 90% of the leukocytes infiltrating the synovia are neutrophils. The specific role that neutrophils play in the onset of RA is not clear, but recent studies have evidenced that they have an important participation in joint damage and disease progression through the release of proteolytic enzymes, reactive oxygen species (ROS), cytokines, and neutrophil extracellular traps, in particular during frustrated phagocytosis of immune complexes (ICs). In addition, the local and systemic activation of the complement system contributes to the pathogenesis of RA and other IC-mediated diseases. This review discusses (i) the participation of Fcγ and complement receptors in mediating the effector functions of neutrophils in RA; (ii) the contribution of the complement system and ROS-dependent and ROS-independent mechanisms to joint damage in RA; and (iii) the use of plant extracts, dietary compounds, and isolated natural compounds in the treatment of RA, focusing on modulation of the effector functions of neutrophils and the complement system activity and/or activation. PMID:26346244

  6. Significant Association Between Fc Receptor-Like 3 Polymorphisms (-1901A>G and -658C>T) and Neuromyelitis Optica (NMO) Susceptibility in the Chinese Population.

    PubMed

    Wang, Xinling; Yu, Tao; Yan, Qichang; Wang, Wei; Meng, Nan; Li, Xuejiao; Luo, Yahong

    2016-01-01

    Neuromyelitis optica (NMO) is an autoimmune disorder. In pathogenesis, NMO-immunoglobulin G (NMO-IgG) selectively binds to aquaporin-4 (AQP4) and resulted in neuritis, myelitis, and brain lesion. Fc receptor-like 3 (FCRL3) gene encodes a member of the immunoglobulin receptor superfamily, which plays an important part in regulating immune activities. This study aimed at investigating the association between FCRL3 polymorphisms and NMO susceptibility and, hopefully, to contribute to the development of novel methods for diagnosis and treatment of NMO. We selected 150 NMO patients and 300 healthy controls from the Chinese population. Tag single nucleotide polymorphisms (SNPs) were identified with reference to CBI-dbSNP and HapMap databases. DNA were extracted and amplified. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was applied to determine the polymorphisms. χ (2), odds ratio (OR), and 95 % confidence interval (95 % CI) were presented to evaluate genotype distribution and association between SNPs and NMO susceptibility. Six out of 15 SNPs were selected according to the filter. No significant altered genotype distribution was observed concerning -11G>C, -166C>T, -219G>C, and -1629C>G polymorphisms. The G allele of -1901A>G variation was demonstrated to be more frequent in patients compared with controls (P < 0.001). The T allele of -658C>T polymorphism was significantly more prevalent in NMO patients than controls (P = 0.009). In summary, the study revealed that the G allele in -1901A>G polymorphism and T allele in -658C>T polymorphism are genetic risk factors for NMO in the Chinese population. Further research is needed to account for different ethnicities and clarify the mechanisms behind, which might contribute to the elucidation of novel diagnosis methods. PMID:25575677

  7. IgG-Mediated Immune Suppression to Erythrocytes by Polyclonal Antibodies Can Occur in the Absence of Activating or Inhibitory Fcγ Receptors in a Full Mouse Model.

    PubMed

    Bernardo, Lidice; Yu, Honghui; Amash, Alaa; Zimring, James C; Lazarus, Alan H

    2015-09-01

    Polyclonal anti-D has been used to prevent RhD-negative mothers from becoming immunized against RhD positive fetal erythrocytes, and this mechanism has been referred as Ab or IgG-mediated immune suppression (AMIS). Although anti-D has been highly successful, the inhibitory mechanisms remain poorly understood. Two major theories behind AMIS involve the binding of IgG to activating or inhibitory FcγR, which can induce either erythrocyte clearance or immune inhibition, respectively. In this work, we explored the absolute role of activating and inhibitory FcγR in the AMIS mechanism using the HOD mouse model of RBC immunization. HOD mice contain a RBC-specific recombinant protein composed of hen egg lysozyme (HEL), OVA and human transmembrane Duffy Ag, and erythrocytes from HOD mice can stimulate an immune response to HEL. To assess the contribution of activating and inhibitory FcγR to AMIS, C57BL/6 versus FcRγ-chain(-/-) or FcγRIIB(-/-) mice were used as recipients of HOD-RBC alone or together with anti-HEL Abs (i.e., AMIS) and the resulting immune response to HEL evaluated. We show that anti-HEL polyclonal Abs induce the same degree of AMIS effect in mice lacking these IgG binding receptors as compared with wild-type mice. In agreement with this, F(ab')2 fragments of the AMIS Ab also significantly reduced the Ab response to the HOD cells. In conclusion, successful inhibition of in vivo Ab responses to HOD-RBC by polyclonal IgG can occur independently of activating or inhibitory FcγR involvement. These results may have implications for the understanding of RhD prophylaxis. PMID:26188060

  8. Registration of FC1018, FC1019, FC1020, and FC1022, Sugarbeet Multigerm Pollinator Germplasms with Disease Resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    FC1018’, ‘FC1019’, ‘FC1020’, and ‘FC1022’ (PI 658059, PI 658060, PI 658061, PI 658062, respectively) sugarbeet germplasm (Beta vulgaris L.) were released in 2009 from 05-FC1018, 05-FC1019, 07-/08-FC1020 and 05-FC101022 seed lots, respectively, and tested under those designations. They were develo...

  9. Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor.

    PubMed

    Leatherbarrow, R J; Rademacher, T W; Dwek, R A; Woof, J M; Clark, A; Burton, D R; Richardson, N; Feinstein, A

    1985-04-01

    Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events. PMID:4033665

  10. Trastuzumab triggers phagocytic killing of high HER2 cancer cells in vitro and in vivo by interaction with Fcγ receptors on macrophages.

    PubMed

    Shi, Yun; Fan, Xuejun; Deng, Hui; Brezski, Randall J; Rycyzyn, Michael; Jordan, Robert E; Strohl, William R; Zou, Quanming; Zhang, Ningyan; An, Zhiqiang

    2015-05-01

    Trastuzumab has been used for the treatment of HER2-overexpressing breast cancer for more than a decade, but the mechanisms of action for the therapy are still being actively investigated. Ab-dependent cell-mediated cytotoxicity mediated by NK cells is well recognized as one of the key mechanisms of action for trastuzumab, but trastuzumab-mediated Ab-dependent cellular phagocytosis (ADCP) has not been established. In this study, we demonstrate that macrophages, by way of phagocytic engulfment, can mediate ADCP and cancer cell killing in the presence of trastuzumab. Increased infiltration of macrophages in the tumor tissue was associated with enhanced efficacy of trastuzumab whereas depletion of macrophages resulted in reduced antitumor efficacy in mouse xenograft tumor models. Among the four mouse FcγRs, FcγRIV exhibits the strongest binding affinity to trastuzumab. Knockdown of FcγRIV in mouse macrophages reduced cancer cell killing and ADCP activity triggered by trastuzumab. Consistently, an upregulation of FcγRIV expression by IFN-γ triggered an increased ADCP activity by trastuzumab. In an analogous fashion, IFN-γ priming of human macrophages increased the expression of FcγRIII, the ortholog of murine FcγRIV, and increased trastuzumab-mediated cancer cell killing. Thus, in two independent systems, the results indicated that activation of macrophages in combination with trastuzumab can serve as a therapeutic strategy for treating high HER2 breast cancer by boosting ADCP killing of cancer cells. PMID:25795760