Involvement of Virus-Induced Interferon Production in IgG Autoantibody-Mediated Anemia.

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.

High FcγR Expression on Intratumoral Macrophages Enhances Tumor-Targeting Antibody Therapy.

Therapy with tumor-specific Abs is common in the clinic but has limited success against solid malignancies. We aimed at improving the efficacy of this therapy by combining a tumor-specific Ab with immune-activating compounds. In this study, we demonstrate in the aggressive B16F10 mouse melanoma model that concomitant application of the anti-TRP1 Ab (clone TA99) with TLR3-7/8 or -9 ligands, and IL-2 strongly enhanced tumor control in a therapeutic setting. Depletion of NK cells, macrophages, or CD8+ T cells all mitigated the therapeutic response, showing a coordinated immune rejection by innate and adaptive immune cells. FcγRs were essential for the therapeutic effect, with a dominant role for FcγRI and a minor role for FcγRIII and FcγRIV. FcγR expression on NK cells and granulocytes was dispensable, indicating that other tumoricidal functions of NK cells were involved and implicating that FcγRI, -III, and -IV exerted their activity on macrophages. Indeed, F4/80+Ly-6C+ inflammatory macrophages in the tumor microenvironment displayed high levels of these receptors. Whereas administration of the anti-TRP1 Ab alone reduced the frequency of these macrophages, the combination with a TLR agonist retained these cells in the tumor microenvironment. Thus, the addition of innate stimulatory compounds, such as TLR ligands, to tumor-specific Ab therapy could greatly enhance its efficacy in solid cancers via optimal exploitation of FcγRs.

A Restricted Role for FcγR in the Regulation of Adaptive Immunity.

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity.

The essential role of complement in antibody-mediated resistance to Salmonella.

Vaccines can serve as essential tools to prevent bacterial diseases via the induction of long-lasting IgG responses. The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc-gamma receptors (FcγRs) can potentially play a crucial role in IgG-mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene-targeted mice lacking either FcγRI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody-mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O-antigen in gene-targeted mice, we demonstrate that the complement system is essential for the IgG-mediated reduction of bacterial numbers in the tissues.

FcγR interaction is not required for effective anti-PD-L1 immunotherapy but can add additional benefit depending on the tumor model.

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.

Anti-Tumor Necrosis Factor With a Glyco-Engineered Fc-Region Has Increased Efficacy in Mice With Colitis.

BACKGROUND & AIMS: Although tumor necrosis factor (TNF) antagonists reduce many clinical features of inflammatory bowel disease, complete mucosal healing occurs in fewer than 50% of patients. The Fc-region of monoclonal antibodies against TNF has immunosuppressive properties via effects on macrophage polarization. We examined the interaction between the anti-TNF Fc-region and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) increases the efficacy of anti-TNF in mice with colitis. METHODS: We generated Rag1-/- mice that lack all activating FcγRs (FcγRI, FcγRIII, and FcγRIV; called FcγR-/-Rag1-/- mice). We produced hypo-fucosylated antibodies against mouse and human TNF (adalimumab). Colitis was induced in mice by transfer of CD4+CD45RBhi to FcγR-/-Rag1-/- or Rag1-/- littermates; mice were given different antibodies against TNF or isotype (control) antibodies and disease activity index scores were determined. Colon tissues were collected and analyzed by histology. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors. T-cell proliferation and proportions of CD206+ (immune regulatory) macrophages were measured in mixed lymphocyte reactions. Human PBMCs were genotyped for FCGR3A158 (the FcγRIIIa-158F allotype displays a lower Fc binding affinity) using the TaqMan single nucleotide polymorphism genotype assay. RESULTS: Rag1-/- mice with colitis given anti-TNF had near complete mucosal healing and Rag1-/- mice given an isotype control antibody developed severe colitis. In contrast, FcγR-/-Rag1-/- mice were refractory to the effects of anti-TNF: their histological colitis scores were as severe as those from FcγR-/-Rag1-/- mice given a control antibody. Colons from Rag1-/- mice that received anti-TNF had an increased number of CD206+ macrophages compared with Rag1-/- mice given control antibody; in FcγR-/-Rag1-/- mice given anti-TNF these numbers were as low as FcγR-/-Rag1-/- given the control antibody. In human PBMCs, anti-TNF increased the number of CD206+ macrophages: this required expression of FcγRIIIa; numbers of these cells were reduced in PBMCs with the low-affinity FcγRIIIa-158F genotype. A hypo-fucosylated form of adalimumab bound human FcγRIIIa with a higher affinity than control adalimumab. When hypo-fucosylated adalimumab was added to PBMCs, a larger number of CD206+ macrophages formed and T-cell proliferation was reduced, compared with addition of a control adalimumab. Hypo-fucosylated adalimumab increased the number of CD206+ macrophages in PMBCs that expressed the low-affinity FcγRIIIa. In mice with colitis, hypo-fucosylated anti-TNF significantly increased the number of CD206+ macrophages in the colon compared with control anti-TNF and was more effective in reducing colitis severity as measured by histology. CONCLUSIONS: In a study of the in vitro and in vivo mechanisms of anti-TNF, we found FcγR engagement by anti-TNF to be required for reduction of colitis in mice and development of CD206+ macrophages. A hypo-fucosylated form of anti-TNF binds FcγRIIIa with higher affinity and induces development of CD206+ macrophages in human PBMCs, especially PBMCs that express low-affinity FcγRIIIa. Hypo-fucosylated anti-TNF might be more effective in patients with inflammatory bowel disease.

Involvement of Fcα/µ Receptor in IgM Anti-Platelet, but Not Anti-Red Blood Cell Autoantibody Pathogenicity in Mice.

IgM anti-mouse platelet autoantibodies cause thrombocytopenia by mediating uptake of opsonized thrombocytes, whereas IgM anti-erythrocyte autoantibodies induce anemia through a phagocytosis-independent cell destruction. In this article, we show that infection with lactate dehydrogenase-elevating virus, a benign mouse arterivirus, exacerbates the pathogenicity of IgM anti-platelet, but not anti-erythrocyte autoantibodies. To define the role of Fcα/µ receptor (Fcα/µR) in IgM-mediated thrombocytopenia and anemia, we generated mice deficient for this receptor. These animals were resistant to IgM autoantibody-mediated thrombocytopenia, but not anemia. However, the lactate dehydrogenase-elevating virus-induced exacerbation of thrombocytopenia was not associated with enhanced Fcα/µR expression on macrophages. These results indicate that Fcα/µR is required for the pathogenicity of IgM anti-platelet autoantibodies but is not sufficient to explain the full extent of the disease in virally infected animals.

FcγRIIb on myeloid cells rather than on B cells protects from collagen-induced arthritis.

Extensive analysis of a variety of arthritis models in germline KO mice has revealed that all four receptors for the Fc part of IgG (FcγR) play a role in the disease process. However, their precise cell type-specific contribution is still unclear. In this study, we analyzed the specific role of the inhibiting FcγRIIb on B lymphocytes (using CD19Cre mice) and in the myeloid cell compartment (using C/EBPαCre mice) in the development of arthritis induced by immunization with either bovine or chicken collagen type II. Despite their comparable anti-mouse collagen autoantibody titers, full FcγRIIb knockout (KO), but not B cell-specific FcγRIIb KO, mice showed a significantly increased incidence and severity of disease compared with wild-type control mice when immunized with bovine collagen. When immunized with chicken collagen, disease incidence was significantly increased in pan-myeloid and full FcγRIIb KO mice, but not in B cell-specific KO mice, whereas disease severity was only significantly increased in full FcγRIIb KO mice compared with incidence and severity in wild-type control mice. We conclude that, although anti-mouse collagen autoantibodies are a prerequisite for the development of collagen-induced arthritis, their presence is insufficient for disease development. FcγRIIb on myeloid effector cells, as a modulator of the threshold for downstream Ab effector pathways, plays a dominant role in the susceptibility to collagen-induced arthritis, whereas FcγRIIb on B cells, as a regulator of Ab production, has a minor effect on disease susceptibility.

The inhibiting Fc receptor for IgG, FcγRIIB, is a modifier of autoimmune susceptibility.

FcγRIIB-deficient mice generated in 129 background (FcγRIIB(129)(-/-)) if back-crossed into C57BL/6 background exhibit a hyperactive phenotype and develop lethal lupus. Both in mice and humans, the Fcγr2b gene is located within a genomic interval on chromosome 1 associated with lupus susceptibility. In mice, the 129-derived haplotype of this interval, named Sle16, causes loss of self-tolerance in the context of the B6 genome, hampering the analysis of the specific contribution of FcγRIIB deficiency to the development of lupus in FcγRIIB(129)(-/-) mice. Moreover, in humans genetic linkage studies revealed contradictory results regarding the association of "loss of function" mutations in the Fcγr2b gene and susceptibility to systemic lupus erythematosis. In this study, we demonstrate that FcγRIIB(-/-) mice generated by gene targeting in B6-derived ES cells (FcγRIIB(B6)(-/-)), lacking the 129-derived flanking Sle16 region, exhibit a hyperactive phenotype but fail to develop lupus indicating that in FcγRIIB(129)(-/-) mice, not FcγRIIB deficiency but epistatic interactions between the C57BL/6 genome and the 129-derived Fcγr2b flanking region cause loss of tolerance. The contribution to the development of autoimmune disease by the resulting autoreactive B cells is amplified by the absence of FcγRIIB, culminating in lethal lupus. In the presence of the Yaa lupus-susceptibility locus, FcγRIIB(B6)(-/-) mice do develop lethal lupus, confirming that FcγRIIB deficiency only amplifies spontaneous autoimmunity determined by other loci.

Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.

To elucidate the mechanisms of glomerulonephritis, including Goodpasture's syndrome, mouse models are used that use heterologous Abs against the glomerular basement membrane (GBM) with or without preimmunization with foreign IgG from the same species. These studies have revealed the requirement of either FcgammaR or complement, depending on the experimental model used. In this study, we provide evidence that both FcgammaR and complement are obligatory for a full-blown inflammation in a novel attenuated passive model of anti-GBM disease. We demonstrate that administration of subnephritogenic doses of rabbit anti-GBM Abs followed by a fixed dose of mouse mAbs to rabbit IgG, allowing timing and dosing for the induction of glomerulonephritis, resulted in reproducible complement activation via the classical pathway of complement and albuminuria in wild-type mice. Because albuminuria was absent in FcR-gamma-chain(-/-) mice and reduced in C3(-/-) mice, a role for both FcgammaR and complement is postulated. Because C1q(-/-) and C4(-/-) mice lacking a functional classical and lectin pathway did develop albuminuria, we suggest involvement of the alternative pathway of complement. Anti-GBM glomerulonephritis occurs acutely following the administration of mouse anti-rabbit IgG, and proceeds in a chronic fashion dependent on both FcgammaR and complement. This novel attenuated model allows elucidating the relative contribution of different mediator systems of the immune system to the development of renal injury, and also provides a platform for the assessment of different treatment protocols and evaluation of drugs that ultimately may be beneficial for the treatment of anti-GBM mediated glomerulonephritides.

Immunoglobulin-free light chains elicit immediate hypersensitivity-like responses.

Immunoglobulin (Ig)-free light chains IgLC are present in serum and their production is augmented under pathological conditions such as multiple sclerosis, rheumatoid arthritis and neurological disorders. Until now, no (patho)physiological function has been ascribed to circulating Ig light chains. Here we show that IgLCs can confer mast cell dependent hypersensitivity in mice. Antigenic stimulation results in plasma extravasation, cutaneous swelling and mast-cell degranulation. We show that IgLCs have a crucial role in development of contact sensitivity, which could be completely prevented by a novel IgLC antagonist. Although IgE and IgG(1) are central to the induction of immediate hypersensitivity reactions, our results show that IgLCs have similar activity. IgLCs may therefore be a novel factor in the humoral immune response to antigen exposure. Our findings open new avenues in investigating the pathogenesis of autoimmune diseases and their treatments.

Immunogenicity of rat-neu+ mouse mammary tumours determines the T cell-dependent therapeutic efficacy of anti-neu monoclonal antibody treatment.

The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2+ breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu+ tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours.

Highly B lymphocyte-specific tamoxifen inducible transgene expression of CreER T2 by using the LC-1 locus BAC vector.

The generation of cell type specific inducible Cre transgenic mice is the most challenging and limiting part in the development of spatio-temporally controlled knockout mouse models. Here we report the generation and characterization of a B lymphocyte-specific tamoxifen-inducible Cre transgenic mouse strain, LC-1-hCD19-CreER(T2). We utilized the human CD19 promoter for expression of the tamoxifen-inducible Cre recombinase (CreER(T2)) gene, embedded in genomic sequences previously reported to give minimal position effects after transgenesis. Cre recombinase activity was evaluated by cross-breeding the LC-1-hCD19-CreER(T2) strain with a strain containing a floxed gene widely expressed in the hematopoietic system. Cre activity was only detected in the presence of tamoxifen and was restricted to B lymphocytes. The efficacy of recombination ranged from 27 to 61% in the hemizygous and homozygous mice, respectively. In conclusion, the LC-1-hCD19-CreER(T2) strain is a powerful tool to study gene function specifically in B lymphocytes at any chosen time point in the lifecycle of the mouse.

FcγRI expression on macrophages is required for antibody-mediated tumor protection by cytomegalovirus-based vaccines.

Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.

A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

Generation of embryonic stem cells and mice for duchenne research.

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease in which muscle is continuously damaged, resulting in loss of muscle tissue and function. Antisense-mediated exon skipping is a promising therapeutic approach for DMD. This method uses sequence specific antisense oligonucleotides (AONs) to reframe disrupted dystrophin transcripts. As AONs function in a sequence specific manner, human specific AONs cannot be tested in the mdx mouse, which carries a mutation in the murine Dmd gene. We have previously generated a mouse model carrying the complete human DMD gene (hDMD mouse) integrated in the mouse genome to overcome this problem. However, as this is not a disease model, it cannot be used to study the effect of AON treatment on protein level and muscle function. Therefore, our long term goal is to generate deletions in the human DMD gene in a mouse carrying the hDMD gene in an mdx background. Towards this aim, we generated a male ES cell line carrying the hDMD gene while having the mdx point mutation. Inheritance of the hDMD gene by the ES cell was confirmed both on DNA and mRNA level. Quality control of the ES cells revealed that the pluripotency marker genes Oct-4 and Nanog are well expressed and that 85% of cells have 40 chromosomes. Germ line competence of this cell line has been confirmed, and 2 mice strains were derived from this cell line and crossed back on a C57BL6 background: hDMD/mdx and mdx(BL6).

Destructive arthritis in the absence of both FcgammaRI and FcgammaRIII.

Fc receptors for IgG (FcgammaR) have been implicated in the development of arthritis. However, the precise contribution of the individual FcgammaR to joint pathology is unclear. In this study, the role of the different FcgammaR was assessed both in an active and in a passive mouse model of arthritis by analyzing disease development in double and triple knockout (KO) offspring from crosses of FcgammaRI KO, FcgammaRIII KO, FcgammaRI/III double KO, or FcR gamma-chain KO with the FcgammaRII KO on C57BL6 background, which is susceptible for collagen-induced arthritis (CIA). In the active CIA model, onset was significantly delayed in the absence of FcgammaRIII, whereas incidence and maximum severity were significantly decreased in FcgammaRI/II/III triple KO but not in FcgammaRII/III double KO and FcgammaRI/II double KO mice as compared with FcgammaRII KO animals. Remarkably, fully destructive CIA developed in FcgammaRI/II/III triple KO mice. In contrast, FcR gamma/FcgammaRII double KO mice were resistant to CIA. These findings were confirmed with the passive KRN serum-induced arthritis model. These results indicate that all activating FcgammaR play a role in the development of arthritis, mainly in the downstream effector phase. FcgammaRIII is critically required for early arthritis onset, and FcgammaRI can substantially contribute to arthritis pathology. Importantly, FcgammaRI and FcgammaRIII were together dispensable for the development of destructive arthritis but the FcR gamma-chain was not, suggesting a role for another FcR gamma-chain associated receptor, most likely FcgammaRIV. In addition, FcgammaRII plays a negative regulatory role in both the central and effector phase of arthritis.

Macrophages induce the inflammatory response in the pulmonary Arthus reaction through G alpha i2 activation that controls C5aR and Fc receptor cooperation.

Complement and FcgammaR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcgammaRIIB, increased levels of activating FcgammaRIII, and highly induced FcgammaR-mediated TNF-alpha and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of G(i)-type G protein signaling in C5aR-mediated control of the regulatory FcgammaR system in vitro, and analysis of the various C5aR-, FcgammaR-, and G(i)-deficient mice verifies the importance of Galpha(i2)-associated C5aR and the FcgammaRIII-FcgammaRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcgammaRIII-positive cells into C5aR- and FcgammaRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcgammaRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcgammaRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Galpha(i2)-dependent signal for modulating the two opposing FcgammaR, FcgammaRIIB and FcgammaRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.

Both Fcgamma receptor I and Fcgamma receptor III mediate disease in accelerated nephrotoxic nephritis.

Recognition of immune complexes in glomeruli by activator Fcgamma receptors (FcgammaRI and FcgammaRIII) is an important step in the development of glomerulonephritis. The low-affinity receptor (FcgammaRIII) has previously been shown to be important in passive heterologous immune complex glomerulonephritis. However, most forms of human glomerulonephritis involve an active immune response, and the relative importance of FcgammaRI (high-affinity receptor) and FcgammaRIII in an active model of glomerulonephritis is not known. We have now studied accelerated nephrotoxic nephritis in FcgammaRIII-/- mice and FcgammaRI/III double-deficient mice, and compared them with matched wild-type controls and FcRgamma chain-deficient (FcRgamma-/-) mice. Mice were immunized against sheep IgG and injected with sheep anti-mouse glomerular basement membrane antibody 5 days later. Both FcgammaRI/III double-deficient mice and FcRgamma-/- mice were strongly protected from renal injury. In contrast, FcgammaRIII-/- mice developed substantial nephritis, although there was a dose-dependent partial protection from glomerular crescents and thrombosis. Despite this histological protection from injury, the macrophage infiltrate was not reduced, implying a dissociation of macrophage accumulation from activation in the absence of activatory FcgammaRIII. Therefore, both FcgammaRI and FcgammaRIII play a role in this active model of glomerulonephritis, because both had to be deficient to protect markedly from disease.