ABSTRACT
The interleukin-23 (IL-23) pathway, T helper 17 (Th17) cells and γδ T cells, which respond to IL-23, have major pro-inflammatory roles. We have used unique IL-23 receptor (IL-23R) subunit-specific monoclonal antibodies, X67 and X68, and IL-12 receptor beta-1 subunit (IL-12Rß1) expression levels to evaluate the IL-23R complex on CD4 αß TCR Th17 cells and on γδ T cells. Both IL-23R and IL-12Rß1 subunits constitute the functional IL-23R. Expression of the IL-23R subunit by cultured Th17 cells was heterogeneous. Th17 cells expressed consistent high levels of the IL-12Rß1 subunit, which appeared a better predictor of responsiveness to IL-23 than the expression of the IL-23R subunit. Moreover, sorting memory CD4 T cells by high IL-12Rß1 expression selectively enriched cells committed to IL-17 production from the blood. IL-23R expression was also observed on freshly isolated and cultured γδ T cells and the cultured γδ T cells were not responsive to IL-23.
Subject(s)
Interleukin-12 Receptor beta 1 Subunit/metabolism , Protein Subunits/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Receptors, Interleukin/metabolism , Th17 Cells/metabolism , Animals , Antibodies, Monoclonal/metabolism , Cells, Cultured , Humans , Immunologic Memory , MiceABSTRACT
The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.
Subject(s)
Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/immunology , Immunoglobulin G/immunology , Receptors, IgG/chemistry , Receptors, IgG/immunology , Animals , Antigen-Antibody Complex/genetics , Crystallography, X-Ray , Epitope Mapping , Humans , Immunoglobulin G/chemistry , Mice , Models, Molecular , Polymorphism, Single Nucleotide , Protein Structure, Quaternary , Receptors, IgG/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Signal Transduction/immunology , Surface Plasmon ResonanceABSTRACT
The aggregation of cell surface Fc receptors by immune complexes induces a number of important antibody-dependent effector functions. It is becoming increasingly evident that the organization of key immune proteins has a significant impact on the function of these proteins. Comparatively little is known, however, about the nature of Fc receptor spatiotemporal organization. This review outlines the current literature concerning human Fc receptor spatial organization and physiological function.
Subject(s)
Receptors, Fc/immunology , Animals , Humans , Immunoglobulins/immunology , Receptors, Fc/chemistryABSTRACT
The effector memory CD4+ Th17 cells play critical roles in bacterial immunity and pathological inflammation in autoimmune conditions. ZSWIM1 is a gene encoding a protein of unknown function in leukocytes-but containing a zinc finger SWIM motif. In peripheral blood mononuclear cells, the expression of ZSWIM1 is highest in lymphocytes, and in particular shows greatest abundance in naive CD4+ T cells. Upon polarisation of naïve CD4+ T cells, ZSWIM1 expression is retained in Th17 cells but is selectively down regulated in Th1 cells. Similarly in in vitro expanded effector memory CD4+ T cells, ZSWIM1 was more abundant in Th17 cells compared to Th1 or Th17 polyfunctional (Th17pf) cells, which produce IL-17A and IFNγ. Although stimulation of cytokine production by PMA and ionomycin reduced ZSWIM1 expression, the relative differences in abundance between the cell types were maintained. The activation sensitive nature of ZSWIM1 expression suggests that it may play a novel role in the development or function of T helper cells.
Subject(s)
DNA-Binding Proteins/genetics , Th1 Cells/immunology , Th17 Cells/immunology , Zinc Fingers/genetics , Amino Acid Sequence , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Separation , Cells, Cultured , DNA-Binding Proteins/immunology , Gene Expression Regulation , Humans , Immunologic Memory , Interferon-gamma/biosynthesis , Interleukin-17/biosynthesis , Ionomycin/pharmacology , Lymphocyte Activation/drug effects , Molecular Sequence Data , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology , Th1 Cells/cytology , Th1 Cells/drug effects , Th17 Cells/cytology , Th17 Cells/drug effectsABSTRACT
Interleukin 17 producing T helper cells (Th17) and IFNγ producing Th1 cells are distinct subsets of effector memory CD4(+) T cells that are crucial to host immunity and have been linked to the pathology of certain inflammatory autoimmune diseases. We have developed a method for the isolation and long term culture of human Th17 and Th1 cells. Using allogeneic stimulation we have cultured homogeneous populations of Th17 and Th1 cells to large cell numbers. These alloreactive cell lines were established from CD4(+)CD45RO(+) memory T cells expressing, or lacking, CCR6 and CCR4. The Th17 cells were derived only from cells expressing both CCR6 and CCR4 whereas the Th1 cells, secreting IFNγ, were derived from cells lacking CCR6 and CCR4. The CCR6(+) and CCR4(+) memory T cells also gave rise to a third population of polyfunctional cells expressing both IL-17 and IFNγ. All cell populations expressed the TCR αß and the Th17 cells characteristically expressed CCR6, CCR4 and CD161. The use of this protocol will ultimately allow for the comparative analysis of the Th17 and Th1 cells.
Subject(s)
Autoimmunity , Cell Culture Techniques/methods , Cell Differentiation , Cell Separation/methods , Th1 Cells/immunology , Th17 Cells/immunology , Cells, Cultured , Cytokines/biosynthesis , Cytokines/immunology , Humans , Phenotype , Th1 Cells/cytology , Th17 Cells/cytologyABSTRACT
The major human Fc receptor, huFcgammaRIIa, is implicated in the development of autoimmune arthritis in humans but until recently has not been studied in mouse models. We evaluated potential roles of FcgammaRIIa by using transgenic mice expressing the receptor. We examined two models of induced autoimmune arthritis pristane-induced arthritis (PIA) and collagen-induced arthritis (CIA) as well as the anti-collagen-II antibody-induced arthritis (CAIA) model. In the induced arthritis models PIA and CIA, the transgenic mice developed a more severe arthritis than the other arthritis-prone SJL or DBA1 mice. Interestingly, anti-collagen-II antibodies were elevated in PIA in the susceptible mice. In the CIA model, the highly susceptible transgenic mouse had IgG subclass levels equivalent to the unaffected and disease resistant C57BL/6 mouse strain implying that the FcgammaRIIa lowers the threshold of IgG dependent leukocyte activation. This is consistent with the greatly enhanced sensitivity of the FcgammaRIIa transgenic mice to CAIA which clearly indicates a role for the receptor at least at the inflammatory effector cell level. Other roles for huFcgammaRIIa or other gene products in the development of autoimmunity cannot be ruled out however, especially as the mice exhibited elevated Th1 or Th17 CD4 T cells in the draining lymph nodes.
Subject(s)
Arthritis/immunology , Autoimmune Diseases/immunology , Interleukin-17/immunology , Receptors, IgG/immunology , Animals , Arthritis/genetics , Autoimmune Diseases/genetics , Disease Models, Animal , Flow Cytometry , Genetic Predisposition to Disease , Humans , Interferon-gamma/immunology , Mice , Mice, Transgenic , Receptors, IgG/genetics , T-Lymphocytes/immunology , Up-RegulationABSTRACT
The majority of human subjects who receive subcutaneous allergen immunotherapy (IT) develop decreased sensitivity to their allergens. Multiple factors may explain the efficacy of IT, some evidence support a role for allergen specific IgG antibodies. There is controversy whether such antibodies act by blocking allergen binding to IgE or initiation of active inhibitory signaling through low affinity IgG receptors (FcgammaRIIB) on mast cells and basophils. In this study, we addressed this question using peripheral blood from cat non-allergic, cat allergic, and immunotherapy-treated cat allergic subjects. Blood from subjects who received IT contain IgG antibodies that mediate inhibition of basophil activation by a mechanism that is blocked by antibodies specific for the inhibitory IgG receptor FcgammaRIIB. Surprisingly, inhibition was also blocked by aglycosylated, putatively non-FcR binding, antibodies that are specific for the FcgammaRIIA, suggesting a contribution of this receptor to the observed effect. Consistent with a cooperative effect, ex vivo basophils were found to express both IgG receptors. In other studies we found that basophils from subjects who were both chronically exposed to allergen and were producing both cat allergen specific IgE and IgG, are hyporesponsive to allergen. These studies confirm that IgG antibodies produced during IT act primarily by stimulation of inhibitory signaling, and suggest that FcgammaRIIA and FcgammaRIIB function cooperatively in activation of inhibitory signaling circuit. We suggest that under normal physiologic conditions in which only a small proportion of FcepsilonRI are occupied by IgE of a single allergen specificity, FcgammaRIIA co-aggregation may, by providing activated Lyn, be required to fuel activation of inhibitory FcgammaRIIB function.
Subject(s)
Basophils/immunology , Desensitization, Immunologic , Immunoglobulin G/immunology , Receptors, IgG/immunology , Adult , Animals , Cats , Female , Humans , Immunoglobulin G/biosynthesis , Injections, Subcutaneous , Male , Middle Aged , Up-RegulationABSTRACT
The interaction of immune complexes with the human Fc receptor, FcgammaRIIa, initiates the release of inflammatory mediators and is implicated in the pathogenesis of human autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, so this FcR is a potential target for therapy. We have used the three-dimensional structure of an FcgammaRIIa dimer to design small molecule inhibitors, modeled on a distinct groove and pocket created by receptor dimerization, adjacent to the ligand-binding sites. These small chemical entities (SCEs) blocked immune complex-induced platelet activation and aggregation and tumor necrosis factor secretion from macrophages in a human cell line and transgenic mouse macrophages. The SCE appeared specific for FcgammaRIIa, as they inhibited only immune complex-induced responses and had no effect on responses to stimuli unrelated to FcR, for example platelet stimulation with arachidonic acid. In vivo testing of the SCE in FcgammaRIIa transgenic mice showed that they inhibited the development and stopped the progression of collagen-induced arthritis (CIA). The SCEs were more potent than methotrexate and anti-CD3 in sustained suppression of CIA. Thus, in vitro and in vivo activity of these SCE FcgammaRIIa receptor antagonists demonstrated their potential as anti-inflammatory agents for autoimmune diseases involving immune complexes.
Subject(s)
Antirheumatic Agents/chemistry , Antirheumatic Agents/therapeutic use , Arthritis, Experimental/drug therapy , Drug Design , Receptors, IgG/antagonists & inhibitors , Animals , Antirheumatic Agents/chemical synthesis , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Blood Platelets/drug effects , Blood Platelets/immunology , Blood Platelets/metabolism , Disease Models, Animal , Humans , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Transgenic , Platelet Activation/drug effects , Platelet Activation/immunology , Protein Conformation , Receptors, IgG/chemistry , Receptors, IgG/genetics , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunology , U937 CellsABSTRACT
Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor gamma-chain (FcRgamma) immunoreceptor tyrosine-activation motif (ITAM) and the calmodulin inhibitor, W7, dissociate calmodulin from GPVI and induce metalloproteinase-mediated GPVI ectodomain shedding. We investigated whether signaling by another ITAM-bearing receptor on platelets, FcgammaRIIa, also down-regulates GPVI expression. Agonists that signal through FcgammaRIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, FcgammaRIIa also underwent rapid proteolysis in platelets treated with agonists for FcgammaRIIa (VM58/14A2) or GPVI/FcRgamma (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that FcgammaRIIa also bound calmodulin and W7 induced FcgammaRIIa cleavage. However, unlike GPVI, the approximate 30-kDa FcgammaRIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, micro-calpain cleaved an FcgammaRIIa tail-fusion protein at (222)Lys/(223)Ala and (230)Gly/(231)Arg, upstream of the ITAM domain. These findings suggest simultaneous activation of distinct extracellular (metalloproteinase-mediated) and intracellular (calpain-mediated) proteolytic pathways irreversibly inactivating platelet GPVI/FcRgamma and FcgammaRIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by FcgammaRIIa after immunologic insult.
Subject(s)
Antigens, CD/metabolism , Blood Platelets/metabolism , Platelet Activation/physiology , Platelet Membrane Glycoproteins/metabolism , Receptors, IgG/metabolism , Amino Acid Sequence , Antigens, CD/chemistry , Calmodulin/antagonists & inhibitors , Calmodulin/metabolism , Calpain/metabolism , Enzyme Inhibitors/pharmacology , Humans , Immunoglobulins/blood , Ligands , Metalloproteases/metabolism , Molecular Sequence Data , Platelet Activation/drug effects , Platelet Membrane Glycoproteins/chemistry , Protein Structure, Tertiary , Receptors, IgG/chemistry , Signal Transduction/physiology , Sulfonamides/pharmacology , Thrombocytopenia/bloodABSTRACT
The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcgammaRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcgammaRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcgammaRIIa may be an essential feature of the FcgammaRIIa signaling cascade.
Subject(s)
Antigens, CD/genetics , Antigens, CD/metabolism , Receptors, IgG/genetics , Receptors, IgG/metabolism , Signal Transduction/genetics , Signal Transduction/immunology , Animals , Antigens, CD/physiology , Binding Sites/genetics , Binding Sites/immunology , CHO Cells , Cricetinae , Cricetulus , Dimerization , Down-Regulation/genetics , Humans , Immunoglobulin G/metabolism , Ligands , Methotrexate/metabolism , Mutagenesis, Site-Directed , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation , Proline/genetics , Receptors, IgG/physiology , Serine/genetics , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
OBJECTIVE: The major human Fc receptor, FcgammaRIIa, is the most widespread activating FcR. Our aim was to determine the role of FcgammaRIIa in a transgenic mouse model of immune complex-mediated autoimmunity and to characterize the development of spontaneous autoimmune disease. METHODS: Arthritis was induced in normal and FcgammaRIIa-transgenic mice by immunization with type II collagen (CII) or by transfer of arthritogenic anti-CII antibodies. Also, mice that spontaneously developed autoimmune disease were assessed by clinical scoring of affected limbs, histology and serology, and measurement of autoantibody titers and cytokine production. RESULTS: FcgammaRIIa-transgenic mice developed collagen-induced arthritis (CIA) more rapidly than did archetypal CIA-sensitive DBA/1 (H-2q) mice, while nontransgenic C57BL/6 (H-2b) mice did not develop CIA when similarly immunized. Passive transfer of a single dose of anti-CII antibody induced a more rapid, severe arthritis in FcgammaRIIa-transgenic mice than in nontransgenic animals. In addition, most immune complex-induced production of tumor necrosis factor alpha by activated macrophages occurred via FcgammaRIIa, not the endogenous mouse FcR. A spontaneous, multisystem autoimmune disease developed in aging (>20 weeks) transgenic mice (n = 25), with a 32% incidence of arthritis, and by 45 weeks, all mice had developed glomerulonephritis and pneumonitis, and most had antihistone antibodies. Elevated IgG2a levels were seen in mice with CIA and in those with spontaneous disease. CONCLUSION: The presence of enhanced passive and induced autoimmunity, as well as the emergence of spontaneous autoimmune disease at 20-45 weeks of age, suggest that FcgammaRIIa is a very important factor in the pathogenesis of autoimmune inflammation and a possible target for therapeutic intervention.
Subject(s)
Antigens, CD/genetics , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Hypersensitivity/genetics , Hypersensitivity/immunology , Receptors, IgG/genetics , Animals , Antibodies, Antinuclear/blood , Arthritis, Experimental/diagnostic imaging , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Disease Models, Animal , Disease Susceptibility , Female , Glomerulonephritis/genetics , Glomerulonephritis/immunology , Histones/immunology , Humans , Immunoglobulin G/blood , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Pneumonia/genetics , Pneumonia/immunology , Pregnancy , Radiography , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Soluble low-affinity receptors for IgG are known to inhibit immune complex (IC)-mediated inflammation, and expression by leukocytes is elevated in several inflammatory diseases. Immunoglobulin M (IgM) rheumatoid factors (RF), anti-Fc autoantibodies, are found in autoimmune diseases, such as rheumatoid arthritis (RA), as well as in normal immune responses. This study demonstrated that soluble FcgammaRIIa inhibits the interaction of rheumatoid factors with ICs. The recombinant soluble low-affinity FcgammaR, rsFcgammaRIIa, partially inhibited (30-70%) the rate of precipitation of soluble ICs by RF-positive RA sera. This required the normal interaction of FcgammaRIIa with Fc as the effect could be abrogated with the Fab fragment of the blocking mAb IV-3. Furthermore, rsFcgammaRIIa partially inhibited (40%) the binding of a monoclonal IgM RF (RF-AN) to an IC formed by IgG2 antibody binding to an antigen-coated biosensor chip. Since RF-AN has been characterized by crystallography to bind to the CH2/CH3 interface of the IgG-Fc, and leukocyte FcgammaRIIa binds to a distinct site centred on the lower hinge, this inhibition is uncompetitive. Some inhibition (15%) of staphylococcal protein A binding to IC was also observed. As soluble FcgammaRIIa disrupts Fc:Fc interactions in IgG-ICs, we propose that this alteration of the IC also reduces the accessibility of Fc portions in the IC, resulting in the partial inhibition of ligands, particularly IgM RF, which bind Fc. We propose that the high concentrations of soluble FcgammaR found during inflammation can affect the properties of ICs and their interaction with the immune system.