J. Engelbert Gessner

Impaired IgG-dependent anaphylaxis and Arthus reaction in FcγRIII (CD16) deficient mice.

Abstract The family of receptors for IgG (FcγR) plays an essential role in antibody-mediated effector functions of the immune system. However, the specific contribution of each of the FcγR classes to in vivo immune reactions is still unclear. Here, we demonstrate that mice deficient for the ligand-binding α chain of FcγRIII lack NK cell–mediated antibody-dependent cytotoxicity and phagocytosis of IgG1-coated particles by macrophages. Strikingly, these mice lack IgG-mediated mast cell degranulation, are resistant to IgG-dependent passive cutaneous anaphylaxis, and exhibit an impaired Arthus reaction. These results indicate a prominent role for FcγRIII in inflammatory and anaphylactic responses, making this receptor a potential target in immunotherapy.

Expression and Function of the Mouse Collagen Receptor Glycoprotein VI Is Strictly Dependent on Its Association with the FcRγ Chain

Platelet glycoprotein (GP) VI has been proposed as the major collagen receptor for activation of human platelets. Human GPVI belongs to the immunoglobulin superfamily and is noncovalently associated with the FcRγ chain that is involved in signaling through the receptor. In mice, similar mechanisms seem to exist as platelets from FcRγ chain-deficient mice do not aggregate in response to collagen. However, the activating collagen receptor on mouse platelets has not been definitively identified. In the current study we examined the function and in vivo expression of GPVI in control and FcRγ chain-deficient mice with the first monoclonal antibody against GPVI (JAQ1). On wild type platelets, JAQ1 inhibited platelet aggregation induced by collagen but not PMA or thrombin. Cross-linking of bound JAQ1, on the other hand, induced aggregation of wild type but not FcRγ chain-deficient platelets. JAQ1 stained platelets and megakaryocytes from wild type but not FcRγ chain-deficient mice. Furthermore, JAQ1 recognized GPVI (approximately 60 kDa) in immunoprecipitation and Western blot experiments with wild type but not FcRγ chain-deficient platelets. These results strongly suggest that GPVI is the collagen receptor responsible for platelet activation in mice and demonstrate that the association with the FcRγ chain is critical for its expression and function.

A Codominant Role of FcγRI/III and C5aR in the Reverse Arthus Reaction

Recent attempts to specify the relative contribution of FcR and complement in various experimental systems of immune complex disease have led to opposing conclusions. As concluded in IgG FcRγ−/− mice, manifestation of disease is almost exclusively determined by FcγR on effector cells, arguing for a minor role of complement. In contrast, data obtained with C5aR−/− mice suggested that, dependent on the tissue site, complement is more important than FcγR. In this paper, we demonstrate that, in response to IgG immune complex formation, FcγRI/III- and C5aR-mediated pathways are both necessary and only together are they sufficient to trigger the full expression of inflammation in skin and lung. Moreover, both effector systems are not entirely independent, suggesting an interaction between FcγR and C5aR. Therefore, FcγR-mediated responses can be integrated through C5aR activation, which may explain why these two receptor pathways have previously been considered to dominate each other.

C5a Initiates the Inflammatory Cascade in Immune Complex Peritonitis

Immune complex (IC)-induced inflammation is integral to the pathogenesis of several autoimmune diseases. ICs activate the complement system and interact with IgG FcγR. In this study, we demonstrate that activation of the complement system, specifically generation of C5a, initiates the neutrophilic inflammation in IC peritonitis. We show that ablation of C5a receptor signaling abrogates neutrophil recruitment in wild-type mice and prevents the enhancement of neutrophil migration seen in FcγRIIB−/− mice, suggesting that C5aR signaling is the crucial initial event upstream of FcγR signaling. We also provide evidence that C5a initiates the inflammatory cascade both directly, through C5aR-mediated effector functions on infiltrating and resident peritoneal cells, and indirectly, through shifting the balance between activating and inhibitory FcγRs on resident cells toward an inflammatory phenotype. We conclude that complement activation and C5a generation are prerequisites for IC-induced inflammation through activating FcγR, which amplifies complement-induced inflammation in autoimmunity.

Distinct Tissue Site-Specific Requirements of Mast Cells and Complement Components C3/C5a Receptor in IgG Immune Complex-Induced Injury of Skin and Lung

We induced the passive reverse Arthus reaction to IgG immune complexes (IC) at different tissue sites in mice lacking C3 treated or not with a C5aR-specific antagonist, or in mice lacking mast cells (KitW/KitW-v mice), and compared the inflammatory responses with those in the corresponding wild-type mice. We confirmed that IC inflammation of skin can be mediated largely by mast cells expressing C5aR and FcγRIII. In addition, we provided evidence for C3-independent C5aR triggering, which may explain why the cutaneous Arthus reaction develops normally in C3−/− mice. Furthermore, some, but not all, of the acute changes associated with the Arthus response in the lung were significantly more intense in normal mice than in C3−/− or KitW/KitW-v mice, indicating for C3- and mast cell-dependent and -independent components. Finally, we demonstrated that C3 contributed to the elicitation of neutrophils to alveoli, which corresponded to an increased synthesis of TNF-α, macrophage-inflammatory protein-2, and cytokine-induced neutrophil chemoattractant. While mast cells similarly influenced alveolar polymorphonuclear leukocyte influx, the levels of these cytokines remained largely unaffected in mast cell deficiency. Together, the phenotypes of C3−/− mice and KitW/KitW-v mice suggest that complement and mast cells have distinct tissue site-specific requirements acting by apparently distinct mechanisms in the initiation of IC inflammation.

STIM1 is essential for Fcγ receptor activation and autoimmune inflammation

Fcgamma receptors (FcgammaRs) on mononuclear phagocytes trigger autoantibody and immune complex-induced diseases through coupling the self-reactive immunoglobulin G (IgG) response to innate effector pathways, such as phagocytosis, and the recruitment of inflammatory cells. FcRgamma-based activation is critical in the pathogenesis of these diseases, although the contribution of FcgammaR-mediated calcium signaling in autoimmune injury is unclear. Here we show that macrophages lacking the endoplasmic reticulum-resident calcium sensor, STIM1, cannot activate FcgammaR-induced Ca(2+) entry and phagocytosis. As a direct consequence, STIM1 deficiency results in resistance to experimental immune thrombocytopenia and anaphylaxis, autoimmune hemolytic anemia, and acute pneumonitis. These results establish STIM1 as a novel and essential component of FcgammaR activation and also indicate that inhibition of STIM1-dependent signaling might become a new strategy to prevent or treat IgG-dependent immunologic diseases.

Mouse FcγRII is a negative regulator of FcγRIII in IgG immune complex-triggered inflammation but not in autoantibody-induced hemolysis

Murine low‐affinity receptors for IgG, FcγRII and FcγRIII, differ by their distinct capacities in mediating down‐regulation or activation of cellular effector functions, respectively. In this study, antibodies detecting the mouse Ly‐17.1 / 2 alloantigen system are demonstrated to be specific for FcγRII with no cross‐reactivities to other FcγR, including FcγRIII. Using these FcγRII‐specific monoclonal antibodies (mAb), the significance of FcγRII inhibition of FcγRIII was examined in two models of autoantibody [autoimmune hemolytic anemia (AIHA)]‐ and IgG immune complex‐induced (Arthus reaction) inflammation in C57BL / 6 mice in comparison with FcγRII– / – and FcγRIII– / – mice. Our results demonstrate that both FcγRIII and FcγRII contributed to the binding of erythrocytes opsonized with the pathogenic IgG1 autoreactive anti‐murine red blood cell antibody 105‐2H. However, the functional blocking with anti‐FcγRII mAb in C57BL / 6 mice and the lack of FcγRII expression in FcγRII– / – mice, which both lowered the threshold level of FcγRIII‐triggered phagocytosis in vitro, did not results in enhanced disease development of 105‐2H mAb‐induced AIHA in vivo. This was in sharp contrast to cutaneous Arthus reaction, where FcγRIII‐mediated activation was inhibited by FcγRII. Together these results show that murine AIHA is markedly different from other FcγR‐dependent inflammatory diseases where FcγRIII is normally counterregulated by FcγRII.

On the role of complement and Fc γ-receptors in the Arthus reaction

Abstract The contribution of either the complement system or the activation of Fc receptors for IgG (FcγRs) to the inflammatory response in immune complex (IC) disease is puzzling. A series of studies has been performed in mice with engineered deficiencies of either FcγRs, the complement components C3, C4 or the C5a receptor. In addition, different C5-deficient mice strains have been evaluated. Mice with gene targeted disruption of the γ-subunit, which mediates surface expression and signal transduction of the high affinity Fc receptor type I for IgG (FcγRI), the low affinity receptor Fc receptor type III for IgG (FcγRIII) and the high affinity receptor type I for IgE (IgϵRI), showed an impaired inflammatory response in the reverse passive Arthus reaction in skin, peritoneum and lung. These data suggest, that the activation of FγRs is the initial event triggering the inflammatory cascade in IC disease. On the other hand, C5aR deficient mice are either protected from tissue injury induced by ICs, as in the lung, or the degree of the inflammatory response is markedly attenuated, as in peritoneum and skin. A detailed analysis of data obtained with the different knock-out strains revealed that both the activation of the complement system as well as the activation of different effector cells via FcγRs contribute to the inflammatory sequelae leading to tissue destruction in IC disease. The relative contributions of FcγRI or FcγRIII and the main effector cells through which these receptors mediate their effector functions are tissue dependent. The activation of the C5a receptor pathway appears to be the prominent contribution of the complement system.

Both FcγRIV and FcγRIII are essential receptors mediating type II and type III autoimmune responses via FcRγ-LAT-dependent generation of C5a

FcγRIV is a relatively new IgG Fc receptor (FcγR) that is reported to contribute to the pathogenesis of autoimmune diseases, although its specific role in relation to FcγRIII, complement and IgG2 subclasses remains uncertain. Here we define FcγRIV on macrophages as a receptor for soluble IgG2a/b complexes but not for cellular bound IgG2a and show that simultaneous activation of FcγRIV and FcγRIII is critical to mediate certain type II/III autoimmune responses. FcγRIII‐deficient mice display compensatory enhanced FcγRIV expression, are protected from lung inflammation after deposition of IgG complexes, and show reduced sensitivity to IgG2a/b‐mediated hemolytic anemia, indicating that increased FcγRIV alone is not sufficient to trigger these diseases in the absence of FcγRIII. Importantly, however, blockade of FcγRIV is also effective in inhibiting phagocytosis and cytokine production in IgG2b‐induced anemia and acute lung injury, processes that display a further dependence on C5a anaphylatoxin receptor. Using gene deletion and functional inhibition studies, we found that FcγRIII and FcγRIV are each essential to trigger an FcRγ‐linker for activation of T‐cell‐dependent signal that drives C5a production in the Arthus reaction. Together, the results demonstrate a combined requirement for FcγRIII and FcγRIV in autoimmune injury, and identify the linker for activation of T cells adaptor as an integral component of linked FcγR and C5a anaphylatoxin receptor activation to generate inflammation.

C5a Receptor Targeting in Neointima Formation After Arterial Injury in Atherosclerosis-Prone Mice

Background— Receptor binding of complement C5a leads to proinflammatory activation of many cell types, but the role of receptor-mediated action during arterial remodeling after injury has not been studied. In the present study, we examined the contribution of the C5a receptor (C5aR) to neointima formation in apolipoprotein E–deficient mice employing a C5aR antagonist (C5aRA) and a C5aR-blocking monoclonal antibody. Methods and Results— Mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C5aRA and anti-C5aR-blocking monoclonal antibody or vehicle control. Compared with controls, neointima formation was significantly reduced in mice receiving C5aRA or anti-C5aR-blocking monoclonal antibody for 1 week but not for 3 weeks, attributable to an increased content of vascular smooth muscle cells, whereas a marked decrease in monocyte and neutrophil content was associated with reduced vascular cell adhesion molecule-1. As assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry, C5aR was expressed in lesional and cultured vascular smooth muscle cells, upregulated by injury or tumor necrosis factor-&agr;, and reduced by C5aRA. Plasma levels and neointimal plasminogen activator inhibitor-1 peaked 1 week after injury and were downregulated in C5aRA-treated mice. In vitro, C5a induced plasminogen activator inhibitor-1 expression in endothelial cells and vascular smooth muscle cells in a C5aRA-dependent manner, possibly accounting for higher vascular smooth muscle cell immigration. Conclusions— One-week treatment with C5aRA or anti-C5aR-blocking monoclonal antibody limited neointimal hyperplasia and inflammatory cell content and was associated with reduced vascular cell adhesion molecule-1 expression. However, treatment for 3 weeks failed to reduce but rather stabilized plaques, likely by reducing vascular plasminogen activator inhibitor-1 and increasing vascular smooth muscle cell migration.