Experimental Autoimmune Hemolytic Anemia RIII ...

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1998 92: 3997-4002

FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia Dirk Meyer, Carsten Schiller, Jürgen Westermann, Shozo Izui, Wouter L. W. Hazenbos, J. Sjef Verbeek, Reinhold E. Schmidt and J. Engelbert Gessner

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FcgRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia By Dirk Meyer, Carsten Schiller, Ju¨rgen Westermann, Shozo Izui, Wouter L. W. Hazenbos, J. Sjef Verbeek, Reinhold E. Schmidt, and J. Engelbert Gessner In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcgR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcgRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcgRIII, and comparing the clinical outcome to those in wild-type mice. FcgRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in

vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcgRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcgRIII reflects an activation of FcgRI that is normally coexpressed with FcgRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcgRIII and further suggest that FcgRI, in addition to FcgRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved. r 1998 by The American Society of Hematology.

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events.12-15 For instance, both FcR g-chain–deficient mice (which are unable to function through both FcgRI and FcgRIII) and FcgRIII KO mice exhibit an impaired Arthus reaction indicating for FcgRIII as the essential Fcg receptor in the initiation of IgG immune complex–triggered inflammation and autoimmune disease.14,16,17 FcR g-chain–deficient mice also argue for an important role of FcgRs expressed on macrophages in the pathogenesis of AIHA.18 But the specific FcgR classes involved could not be identified yet. Furthermore, until this study, the relationship between specific FcgRs such as FcgRIII and IgG isotypes for disease development of AIHA has not been investigated. In the present study, we tested the cytotoxic activities of IgG1 (105-2H) and IgG2a (34-3C) a murine RBC (MRBC) monoclonal antibodies (MoAbs) both reacting with the same autoantigen epitope identified as the erythrocyte anion channel band 3 on MRBCs19,20 in FcgRIII KO mice and their wild-type littermates. We show that mice lacking FcgRIII are protected to experimental AIHA determined through IgG1-dependent erythrophagocytosis. FcgRIII KO mice are not completely resistant to IgG2a-induced anemia indicating, in addition to macrophage

UTOIMMUNE hemolytic anemia (AIHA) is the oldest recognized autoimmune disease in humans. It is characterized by the production of pathogenic self-reactive antibodies causing anemia as a result of immune destruction of red blood cells (RBCs). The antibodies involved are classified as warm autoantibodies, cold agglutinins, and biphasic hemolysins which are cleared by distinct effector mechanisms.1 The predominant forms of AIHA involve warm IgG-autoantibodies accompanied with extravascular hemolysis. It is presumed that hemolysis of IgG opsonized RBCs in warm AIHA is largely mediated through either Fc and/or complement receptors expressed by phagocytic effector cells. Two recent studies in murine models of AIHA have suggested a more prominent role of Fc receptors than of complement activation. First, the treatment with recombinant granulocyte-macrophage colony-stimulating factor (GMCSF), which normally enhances FcgR-dependent phagocytosis, also accelerates the progression of spontaneous AIHA in New Zealand Black (NZB) mice.2 A similar effect of GM-CSF in accelerating the clearance of IgG-coated RBCs has been noted in humans.3 Second, experimentally induced AIHA occurs even in the absence of the complement components C3, C4, and C5, but requires the presence of Fc receptors in association with the common FcR g-chain.4 There are three classes of murine receptors for IgG, FcgR on leukocytes: the high-affinity receptor FcgRI, and the two low-affinity receptors, FcgRII and FcgRIII.5,6 Although FcgRI is capable of binding monomeric IgG2a, both FcgRII and FcgRIII have been proposed to interact preferentially with murine IgG1 and IgG2b immune complexes.7,8 These receptors are structurally related consisting of similar ligand-binding domains, but differ in their transmembrane and intracellular domains. The FcgRII isoforms, termed b1 and b2, are single subunit receptors with inhibitory functions. FcgRI and FcgRIII are multimeric receptors in association with the common FcR g-chain9-11 required for assembly and the triggering of various effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and the release of inflammatory mediators.6 Knock-out (KO) mice deficient in FcgRI, FcgRII, FcgRIII, and FcR g-chain allow dissecting the contribution of FcgRs to various normal and pathological immunological Blood, Vol 92, No 11 (December 1), 1998: pp 3997-4002

From the Departments of Clinical Immunology and Functional Anatomy, Hannover Medical School, Hannover, Germany; the Department of Pathology, CMU, University of Geneva, Geneva, Switzerland; and the Department of Immunology, University Hospital Utrecht, Utrecht, The Netherlands. Submitted April 7, 1998; accepted August 24, 1998. Supported by the Deutsche Forschungsgemeinschaft Grants No. Ge892/2-2 and SFB 265/A09 to J.E.G. and R.E.S., and by a grant from the Swiss National Foundation for Scientific Research to S.I. Address reprint requests to J. Engelbert Gessner, PhD, Department of Clinical Immunology, Hannover Medical School, Carl-Neuberg Str 1, 30625 Hannover, Germany; e-mail: [email protected]. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate this fact. r 1998 by The American Society of Hematology. 0006-4971/98/9211-0062$3.00/0 3997


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FcgRIII, the contribution of FcgRI. These findings suggest that differences in IgG isotype specificities of individual FcgRs are critical in the disease process of AIHA. MATERIALS AND METHODS Mice. FcgRIII-deficient and wild-type littermates were developed in collaboration with the group of Dr J.S. Verbeek (Utrecht, The Netherlands), as described previously.14 All mice were bred and maintained under dry barrier conditions in the animal facilities at the Hannover Medical School (Hannover, Germany). Mice were studied at 2 to 4 months of age. All experiments received institutional approval. aMRBC and other antibodies. 105-2H (IgG1), and 34-3C (IgG2a) monoclonal aMRBC autoantibodies were obtained by fusion of spleen cells from unmanipulated NZB mice as described.19 Hybridoma cells were maintained in RPMI/10% fetal calf serum (FCS). Culture supernatants were concentrated by precipitation in 50% saturated ammonium sulfate followed by purification with protein A affinity chromatography (Pharmacia, Uppsala, Sweden) and dialysis against phosphate-buffered saline (PBS). Purity was confirmed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Concentrations of aMRBC MoAb were determined by Ig class-specific enzyme-linked immunosorbent assay (ELISA; DAKO, Hamburg, Germany). Other antibodies in use were: 2.4G2,21 which is directed against FcgRII/III (Pharmingen, San Diego, CA), M1/70 against Mac-1 (Pharmingen), the isotype-matched control antibodies 5E5 (murine IgG1), and W6/32 (murine IgG2a). Polyclonal rabbit-anti–rat-IgG (Z0494) and monoclonal APAAP (alkaline phophatase anti-alkaline phosphatase) rat IgG (D0488) were obtained from DAKO. Phagocytosis of IgG-opsonized MRBCs by peritoneal macrophages. Peritoneal macrophages elicited by intraperitoneal injection with 1 mL 3% thioglycolate (DIFCO Laboratories, Detroit, MI) were flushed out the peritoneal cavity on day 3 postinjection and suspended in PBS. Freshly isolated MRBCs were washed two times with ice-cold PBS by centrifugation at 1,600 rpm and processed for opsonization. Hereby, 10 µL of pelleted MRBCs were incubated at 4°C for 60 minutes with 10 µL of aMRBC MoAbs at saturating (determined by fluorescence-activated cell sorting [FACS] analysis) concentrations. Aliquots of 50 µL of 1% opsonized MRBC suspension were added to 50 µL peritoneal macrophages preparation and incubated at 37°C for 60 minutes. For some experiments, peritoneal macrophages were first incubated for 30 minutes at 4°C with the anti-FcgRII/III blocking antibody 2.4G2. Noningested extracellular MRBC were lysed by hypotonic shock, immediately followed by two washes with PBS. Peritoneal macrophages were conventionally stained with Giemsa/hematoxylin-eosin, and phagocytosis was determined by light microscopy. Peritoneal macrophages containing more than two MRBCs were considered as phagocytic. Experimental AIHA. Hemolytic anemia was induced by a single intraperitoneal (IP) injection of the pathogenic aMRBC autoantibodies

; Fig 1. Phagocytosis of IgG-opsonized MRBCs. Thioglycolateelicited peritoneal macrophages from FcgRIII wild-type (h) and FcgRIII KO (j) mice were incubated with MRBCs opsonized with (A) pathogenic aMRBC MoAbs of the IgG1 (105-2H) and IgG2a (34-3C) isotypes or with medium alone. In addition, macrophages from FcgRIII wild-type (B) and FcgRIII KO (C) mice were first incubated with (j) or without (h) the 2.4G2 antibody, which is directed against FcgRII and FcgRIII, and subsequently opsonized with the aMRBC 105-2H and 34-3C. After 1 hour of incubation at 37°C, extracellular erythrocytes were lysed by hypotonic shock and the percentage of positive peritoneal macrophages that had ingested more than two erythrocytes was assessed microscopically. Results are expressed as the mean values 6 SEM of five individual experiments. Significances are determined by Student’s t-test (*P F .05; **P F .001).


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Ingested erythrocytes were shown by their endogeneous peroxidase using H2O2 and diaminobenzidine as substrates. Thus, Mac-11 macrophages phagocytosing erythrocytes were seen as blue cells with brown content. The slides were counterstained with hematoxylin and mounted in glycergel (DAKO). RESULTS AND DISCUSSION

Fig 2. Experimental AIHA induced by aMRBC antibodies in FcgRIII wild-type and FcgRIII KO mice. (A and B) Daily hematocrits of FcgRIII wild-type (h) and FcgRIII KO (j) mice injected with the pathogenic aMRBC MoAbs 105-2H (A) and 34-3C (B). No decrease of Ht level was observed for mice injected with nonpathogenic isotype control MoAbs (s). Ht values lower than 40% were considered as anemic. Shown are the mean values obtained from 5 to 10 mice in each group (SD F3%).

105-2H (450 µg) or 34-3C (120 µg) or the same amounts of isotypematched control antibodies 5E5 (mIgG1) or W6/32 (mIgG2a). In some experiments mice received IP 10 µg naja naja cobra venom factor (Calbiochem, La Jolla, CA) 1 day before and 2 days after the injection of 34-3C. This treatment depletes serum levels of complement C3 as determined by CH50-measurements. For FcgRII and FcgRIII blockade 250 µg 2.4G2 MoAb was injected IP 24 hours before and 24 and 72 hours after administration of aMRBC 34-3C.22 Blood samples obtained from the retroorbital plexus were collected into heparinized microhematocrit capillary tubes and centrifuged for 5 minutes at 12,000 rpm in a microfuge. Hematocrits measured by the percentage of packed RBCs were directly determined after centrifugation. Histopathology. Mice were killed at day 2 after injection of pathogenic aMRBC, and major organs including spleens and livers were processed for histological examination. Tissues were fixed in 10% buffered formaline, embedded in paraffin, and stained with hematoxylin and eosin (H 1 E) according to conventional procedures. In further experiments tissues were prepared for immunocytochemical techniques. Mac-11 cells were revealed by incubating cryostat sections for 30 minutes with the rat antibody M1/70 and then incubated with the bridging anitbody Z0494 and the rat-APAAP antibody complex (D0488) for 30 minutes. The last two steps were repeated for 15 minutes followed by visualization using naphthyl phosphate and Fast blue.23

Phagocytosis of IgG1 versus IgG2a-coated MRBCs in FcgRIII-deficient mice. We first investigated the in vitrophagocytosis of MRBCs opsonized with 105-2H (IgG1) and 34-3C (IgG2a) using thioglycolate-elicited peritoneal macrophages that normally express FcgRI, FcgRII, and FcgRIII. High levels of phagocytosis were evident with both 105-2H and 34-3C in wild-type mice (Fig 1A). The phagocytosis was either slightly (34-3C) or substantially (105-2H) diminished in the presence of the anti-FcgRII/III antibody 2.4G2 (Fig 1B). This is consistent with our observation that FcgRIII KO mice lack phagocytosis of 105-2H opsonized MRBCs (Fig 1A), indicating an apparent specificity of IgG1 for FcgRIII. In case of the IgG2a aMRBC 34-3C the reduced phagocytosis in FcgRIII KO mice was not further decreased by 2.4G2 (Fig 1A and C). These data show that the specificity of complexed IgG2a for FcgRI15 is not absolute. It appears that FcgRIII contributes to some extent to the binding and phagocytosis of IgG2a-coated MRBCs. FcgRIII-deficient mice are resistant to experimental AIHA induced by pathogenic IgG1 but not IgG2a aMRBC MoAbs. We next examined the pathogenicity of aMRBC by a single IP injection of purified MoAb in FcgRIII KO mice or their wild-type controls. As shown in Fig 2A and B, 450 µg of the IgG1 MoAb 105-2H or 120 µg of the IgG2a MoAb 34-3C was required to develop a strong but transient AIHA with an average hematocrit (Ht) of 23% and 21% at day 4 after injection in wild-type mice, respectively. Under these conditions the decrease in Ht induced by 105-2H and 34-3C recovered to normal levels of about 40% to 50% around day 7. In contrast, FcgRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H with mean Ht levels remaining at $40% (Fig 2A). 34-3C induced a less severe but persistent

Fig 3. Experimental AIHA in FcgRIII wild-type and FcgRIII KO mice treated with CVF and 2.4G2. Mean hematocrits of FcgRIII wild-type (h) and FcgRIII KO (j) mice at day 4 after injection induced by the aMRBC antibody 34-3C. Hatched areas indicate the differences in AIHA induction obtained by treatments with cobra venom factor to deplete complement C3 (CVF) or with the anti-FcgR MoAb 2.4G2 to block specifically the two low-affinity receptors FcgRII and FcgRIII (2.4G2). Results are expressed as the percentage of mean hematocrit 6 SEM obtained from 5 to 10 mice in each group.


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Fig 4. Histopathology from anemic FcgRIII wild-type and FcgRIII KO mice. Representative histological appearance of liver (A) and spleen (B) from FcgRIII wild-type and FcgRIII KO mice on day 2 after AIHA-induction by the injection of the pathogenic IgG1 105-2H and IgG2a 34-3C MoAbs (hematoxylin and eosin stained; inset: higher magnification).


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anemia with a decrease in Ht levels to 28% in FcgRIII KO mice (Fig 2B), indicating that, in addition to FcgRIII, either FcgRI and FcgRII, or both, may have a contributory role. However, two observations argue against a significant contribution of FcgRII but rather indicate that FcgRI is responsible for this residual anemic phenotype. First, the prior administration of the FcgRII and FcgRIII blocking antibody 2.4G2 resulted in partial protection from 34-3C-induced AIHA in wild-type controls at similar levels to those in FcgRIII KO mice not receiving 2.4G2 (Fig 3). Second, blocking FcgRII in the absence of FcgRIII through 2.4G2 did not further improve the protective effect in FcgRIII KO mice (Fig 3). These results are consistent with previous evidence obtained with FcR g-chain knock-out mice deficient for both FcgRI and FcgRIII but not FcgRII, in which the profound anemia normally induced by 34-3C is completely absent.18 Histopathological examination of mice treated with IgG1 aMRBC 105-2H showed a marked degree of erythrophagocytosis in the liver accompanied by splenic engorgement in wildtype mice but not in FcgRIII-deficient mice, as assessed by conventional eosin/hematoxylin staining (Fig 4). Studies in op/op mice have indicated that in addition to the splenic macrophage, the hepatic Kupffer cell may be an important effector cell to the development of AIHA.18 Thus, we also performed immunohistochemistry with the M1/70 MoAb specific for the Mac-1 antigen on macrophages residing in the spleen and the liver. This allows a more quantitative estimation of FcgR-dependent erythrophagocytosis, especially in the liver. From a total number of 196 6 23 Mac-11 cells detected per mm2 liver section at day 2 postinjection, 27 6 5 (n 5 6) were Benzidin-positive containing ingested MRBCs (Fig 5). This pathology, equivalent to 13.8% erythrophagocytosis observed

Fig 5. Quantitative analysis of in vivo-erythrophagocytosis in the liver from anemic FcgRIII wild-type and FcgRIII KO mice. Liver sections from FcgRIII wild-type (h) and FcgRIII KO (j) mice on day 2 after injection induced by either 105-2H or 34-3C aMRBCs were processed for Mac-1 immunostaining of macrophages counterstained with benzidin for the detection of erythrocytes. The amount of Mac-11 liver macrophages containing ingested erythrocytes per mm2 was assessed microscopically. Results are expressed as the mean values 6 SEM obtained from three to five mice in each group. Significance is determined by Student’s t-test (*P F .05; **P F .001).

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for wild-type controls, was completely absent in FcgRIII KO mice, supporting the notion that AIHA induced by 105-2H is exclusively mediated through FcgRIII. In the case of the IgG2a aMRBC 34-3C, a reduced pathology was seen in FcgRIIIdeficient mice (38 6 4 Mac-1/Benzidin-positive cells, n 5 5) compared with wild-type mice (62 6 2 Mac-1/Benzidinpositive cells, n 5 5). The reduction in erythrophagocytosis by around 40% in FcgRIII KO mice is consistent with the idea that FcgRIII contributes significantly but not exclusively to the histopathological manifestations typical for AIHA induced by 34-3C. The contribution of both FcgRI and FcgRIII may also account for the stronger pathogenicity observed in general with 34-3C (IgG2a) compared with 105-2H (IgG1).19 The role of complement in AIHA induced by 105-2H and 34-3C has been analyzed by depleting complement C3 with cobra venom factor (CVF). Similar to several other reports,24 CVF-treated wild-type and FcgRIII KO mice were not significantly protected from anemia (Fig 3 and data not shown), indicating only a minor role, if any, of complement receptormediated erythrophagocytosis in this model of AIHA. Concluding remarks. In the present study we identified FcgRIII to be involved in the disease process of murine AIHA. Loss of FcgRIII in mice resulted in complete protection from disease development induced by the cytotoxic IgG1 autoantibody 105-2H. The lack of IgG1-mediated erythrophagocytosis in vitro and in vivo in FcgRIII KO mice coincides with a strong reduction in anemia. This result provides direct in vivo evidence that the interaction between IgG1 and FcgRIII contributes significantly to the development of experimental AIHA. In case of 34-3C, partial protection from anemia occurred in FcgRIII-deficient mice, indicating that FcgRIII is normally involved in experimental AIHA caused by this IgG2a autoantibody. It further suggests that IgG2a may also act via FcgR other than FcgRIII, the most likely being the high-affinity receptor FcgRI. This was supported by functional blocking studies using the anti-FcgRII/III 2.4G2 antibody indicating a minor, if any, role of FcgRII on macrophages. In accordance, the predominance of FcgRI and not FcgRII has been recently shown in the phagocytosis of IgG2a-coated MRBCs.25 Because the 105-2H and 34-3C MoAbs reacted with the same autoantigenic epitope on MRBCs their relative affinities for FcgRIII (IgG1 5 IgG2a) and FcgRI (IgG2a . . . IgG1)5,6 are thus of prime importance for the differences in induction of anemia. Previous studies on the pathogenesis of murine idiopathic thrombocytopenic purpura (ITP) suggested a role for FcgR,26 supported by findings that disease development induced by the cytotoxic anti-platelet 6A6 MoAb is abolished in FcR g-chain– deficient mice.18 Because the 6A6 autoantibody was of the IgG1 subclass, we suggest that the pathology of ITP induced by this antibody may be predominantly mediated by FcgRIII. Confirmatory studies in FcgRIII-deficient mice would further strengthen this hypothesis. Our observation, at least, that FcgRIII-deficient mice have higher platelet counts (1,640 6 220 3 103/µL, n 5 6) than normal mice (1,160 6 120 3 103/µL, n 5 9) suggests a role for FcgRIII also in the clearance of IgG-coated platelets. The findings that individual FcgR interact differently with IgG isotypes in mediating autoimmune injury might be relevant for the potential use of these receptors as therapeutic targets in the treatment of AIHA in humans. Clinical studies in which AIHA


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have been treated in similar ways are rather limited. Thus, current approaches on targeting FcgR binding sites7 in combination with humanized FcgR mouse models27-29 will be useful to establish the significance of either FcgRIII or FcgRI blockade as therapeutic modalities for human AIHA. ACKNOWLEDGMENT We thank Margot Zielinska for the isotype-matched control antibodies (5E5, W6/32) and Frank Heusohn for support in histology data processing. REFERENCES 1. Engelfriet CP, Overbeeke MAM, von dem Borne AEGKr: Autoimmune hemolytic anemia. Semin Hematol 29:3, 1992 2. Berney T, Shibata T, Merino R, Chicheportiche Y, Kindler V, Vassalli P, Izui S: Murine autoimmune hemolytic anemia resulting from Fcg receptor-mediated erythrophagocytosis: Protection by erythropoietin but not by interleukin-3, and aggravation by granulocytemacrophage colony-stimulating factor. Blood 79:2960, 1992 3. Rossmann MD, Ruiz P, Comber P, Gomez F, Rottem M, Schreiber AD: Modulation of macrophage Fcg receptors by rGM-CSF. Exp Hematol 21:177, 1993 4. Sylvestre DL, Clynes R, Ma M, Warren H, Caroll MC, Ravetch JV: Immunoglobulin G-mediated inflammatory responses develop normally in complement deficient mice. J Exp Med 184:2385, 1996 5. Ravetch JV, Kinet J-P: Fc receptors. Annu Rev Immunol 9:457, 1991 6. Gessner JE, Heiken H, Tamm A, Schmidt RE: The IgG Fc receptor family (FcgR). Ann Hematol 76:231, 1998 7. Tamm A, Kister A, Nolte KU, Gessner JE, Schmidt RE: The IgG binding site of human FcgRIIIB receptor involves CC‘ and FG loops of the membrane-proximal domain. J Biol Chem 271:3659, 1996 8. Tamm A, Schmidt RE: IgG binding sites on human Fcg receptors. Int Rev Immunol 16:57, 1997 9. Ra C, Jouvin M-H, Blank U, Kinet J-P: A macrophage Fcg receptor and the mast cell receptor for immunoglobulin E share an identical subunit. Nature 341:752, 1989 10. Ernst LK, Duchemin AM, Anderson CL: Association of the high-affinity receptor for IgG (FcgRI) with the g subunit of the IgE receptor. Proc Natl Acad Sci USA 90:6023, 1993 11. Radeke HH, Gessner JE, Uciechowski P, Ma¨gert H-J, Schmidt RE, Resch K: Intrinsic human glomerular mesangial cells can express receptors for IgG complexes (hFcgRIII-A) and the associated FceRI g-chain. J Immunol 153:1281, 1994 12. Gavin AL, Barnes N, Dijstelbloem HM, Hogarth PM: Identification of the mouse IgG3 receptor. Implications for antibody effector function at the interface between innate and adaptive immunity. J Immunol 160:20, 1998 13. Takai T, Ono M, Hikida M, Ohmori H, Ravetch JV: Augmented humoral and anaphylactic responses in FcgRII-deficient mice. Nature 379:346, 1996 14. Hazenbos WLW, Gessner JE, Hofhuis FMA, Kuipers H, Meyer D, Heijnen IAFM, Schmidt RE, Sandor M, Capel PJA, Dae¨ron M, van

de Winkel JGJ, Verbeek JS: Impaired IgG-dependent anaphylaxis and arthus reaction in FcgRIII (CD16) deficient mice. Immunity 5:181, 1996 15. Takai T, Li M, Sylvestre DL, Clynes R, Ravetch JV: FcR g chain deletion results in pleiotropic effector cell defects. Cell 76:519, 1994 16. Sylvestre DL, Ravetch JV: Fc receptors initiate the Arthus reaction: Redefining the inflammatory cascade. Science 265:1095, 1994 17. Clynes R, Dumitru C, Ravetch JV: Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis. Science 279:1052, 1998 18. Clynes R, Ravetch JV: Cytotoxic antibodies trigger inflammation through Fc receptors. Immunity 3:21, 1995 19. Shibata T, Berney T, Reininger L, Chicheportiche Y, Ozaki S, Shirai T, Izui S: Monoclonal anti-erythrocyte autoantibodies derived from NZB mice cause autoimmune hemolytic anemia by two distinct pathogenic mechanisms. Int Immunol 2:1133, 1990 20. Oliveira GGS, Izui S, Ravirajan CT, Mageed RAK, Lydyard PM, Elson CJ, Barker RN: Diverse antigen specificity of erythrocytereactive monoclonal autoantibodies from NZB mice. Clin Exp Immunol 105:313, 1996 21. Unkeless JC: Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J Exp Med 142:580, 1979 22. Kurlander RJ, Ellison DM, Hall J: The blockade of Fc receptormediated clearance of immune complexes in vivo by a monoclonal antibody (2.4G2) directed against Fc receptors on murine leukocytes. J Immunol 133:855, 1984 23. Westermann J, Smith T, Peters U, Tschernig T, Pabst R, Steinhoff G, Sparshott SM, Bell EB: Both activated and nonactivated leukocytes from the periphery continuously enter the thymic medulla of adult rats: Phenotypes, sources and magnitude of traffic. Eur J Immunol 26:1866, 1996 24. Izui S, Reininger L, Shibata T, Berney T: Pathogenesis of autoimmune hemolytic anemia in New Zealand Black mice. Crit Rev Oncol Hematol 17:53, 1994 25. Schiller C, Meyer D, Schmidt RE, Gessner JE: Murine low affinity Fcg receptors can be distinguished by anti-Ly-17 antibodies. Immunol Lett 56:401, 1997 (abstr) 26. Mizutani H, Engelman RW, Kurata Y, Ikehara S, Good RA: Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW 3 BXSB) F1 mice. Blood 82:837, 1993 27. Gessner JE, Grussenmeyer T, Kolanus W, Schmidt RE: The human low affinity immunoglobulin G Fc receptor III-A and III-B genes: Molecular characterization of the promoter regions. J Biol Chem 270:1350, 1995 28. Gessner JE, Grussenmeyer T, Dumbsky M, Schmidt RE: Separate promoters from proximal and medial control regions contribute to the natural killer cell specific transcription of the human FcgRIII-A (CD16-A) receptor gene. J Biol Chem 271:30755, 1996 29. Li M, Wirthmueller U, Ravetch JV: Reconstitution of human FcgRIII cell type specificity in transgenic mice. J Exp Med 183:1259, 1996