Immunity, Vol. 3, 21-26, July, 1995, Copyright 0 1995 by Cell Press Cytotoxic Antibodies Trigger Inflammation Through Fc Receptors

Raphael Clynes and Jeffrey V. Ravetch a wide array of effector functions, including phagocytosis, Program in Molecular Biology antibody-dependent cellular cytotoxicity, and release of Memorial Sloan-Kettering Center inflammatory mediators, which ultimatelycanleadtocellu- 1275 York Avenue lar destruction and the amplification of the inflammatory New York, New York 10021 response (Gallin, 1993). Either or both of these latter two mechanisms have been presumed responsible for the ex- travascular hemolysis seen in warm AIHA and ITP. Summary In the mouse, there are three classesof Fc receptors for (IgG): the high affinity FqRI, Pathogenic self-reactive antibodies are a significant capable of binding monomeric IgG, and the two low aff inky cause of morbidlty and mortality and contribute to both receptors, FcyRll and FcyRIII, which bind polymeric IgG. cytotoxic and -trlggered inflamma- While FcyRll is a single subunit receptor, both FcyRl and tory dlsorders, typified by rheumatic diseases, autoim- FcyRlll are hetero-oligomeric complexes and require an mune hemolytic anemia, and thrombocytopenla. Roles additional chain for their assembly and signaling: the ho- have been proposed for Fc receptors, complement, modimeric y subunit (Ra et al., 1989; Lanier et al., 1989; and complement receptors in the pathogenesis of Kurosaki and Ravetch, 1989; Ernst et al., 1993). All three these disorders, although the contribution of each to classes of Fc receptors are expressed on myeloid lineage autoimmune injury is unclear. y chain-deficient mice cells, including macrophages and neutrophils. In contrast, lacking FcyRl and FcyRlll are resistant to the develop FcyRlll is the sole FcR on natural killer cells (reviewed by ment of experimental immune hemolytic anemla in- Ravetch, 1994). duced by polyclonal rabbit anti-mouse red blood cell Previous work using FcRy chain-deficient mice that are IgG antibodies. Thls resistance Is primarily a conse- unable to express FcyRl and FcrRlll (Takai et al., 1994) quence of ineffective erythrophagocytosls, resulting revealed an unexpected role for engagement from the lack of FcyRs on mononuclear phagocytes. in the initiation of IgG immune complex-triggered type Similarly, y chain-deficient mice are completely resis- Ill inflammation, as demonstrated by the absence of an tant to the development of experimental Immune in these mice (Sylvestre and Ravetch, thrombocytopenia induced by mouse anti-platelet an- 1994). In the present study, we provide evidence that Fc tibodies. These data suggest that Fc receptors play receptor engagement is pivotal to the development of dis- an integral role in the pathogenesis of type II hypersen- ease in two models of type II ; namely, sitlvity and suggest potentlal therapeutic beneflts of experimental immune thrombocytopenia and hemolytic Fc receptor blockade. anemia. Genetic susceptibility to AIHA has been demonstrated Introduction in a number of inbred mouse lines and studied most exten- sively in NZB mice. Nearly all NZB mice develop AIHA Pathogenic self-reactive antibody either in the form of solu- by the age of 9 months, highlighted by the serological ble immune complexes or as cellular bound cytotoxic anti- appearance of autoreactive anti-red blood cell antibodies body produces autoimmune injury as a consequence of (Howie and Helyer, 1988). A number of NZBderived patho activation of the inflammatory response (Ravetch, 1994). genie anti-mouse red blood cell (aMRBC) hybridomas Type II inflammation, which is characterized by patho- have been described (Reininger et al., 1990), and two of genic cytotoxic antibody, contributes to the pathogenesis these monoclonal antibodies (MAbs), 34-3C and 31-90, of human and is specifically causal have revealed that two disparate pathogenic mechanisms in the development of autoimmune hemolytic anemia are responsible for the anemia observed (Shibata et al., (AIHA), immune thrombocytopenic purpura (ITP), and 1990). Goodpasture’s Disease. Three discrete pathways may re- Autoimmune thrombooytopenia and the appearance of sult in the destruction of the antibody-coated target cell autoreactive anti-platelet antibodies occurs in genetically (Roitt et al., 1993). In one pathway, the direct activation susceptible inbred mouse strains (Mizutani et al., 1990). Re- of both the early and late components of the complement cently, a pathogenic IgGl MAb @AS) was derived from BSX cascade can result in the formation of the membrane at- mice, which induces a rapid transient thrombocytopenia in tack complex producing pores in the cell membrane and intravenously injected animals (Mizutani et ‘al., 1993). The directly lyse the target cell. This mechanism is presumed pathogenic mechanism of this experimental immune throm- to predominate in causing intravascular hemolysis in bocytopenia has not yet been described. In this study, we acute hemolytic transfusion reactions after the administra- examined the development of experimental autoimmune tion of ABO-incompatible blood (Snyder, 1995). Recruit- thrombocytopenia and hemolytic anemia in FcyRI- and ment and activation of cellular effecters may be accom- Fc-rRllldeficient mice, as well as in osteopetrotic (op/op) plished by the two other pathways: engagement of C3b mice, which have abnormal mononuclear cell development or FcyR receptors, or both. Ligand cross-linking of these as a consequence of the functional loss of CSF-1 (Yoshida FoyRs on effector cells in vitro initiates the activation of et al., 1990; WiktorJedrzejczak et al., 1990; Felix et al., Immunity 22

tion. In contrast, the administration of a polyclonal rabbit anti-MRBC sera would be expected to activate both com- plement-dependent and independent pathways, resulting in both intravascular and extravascular hemolysis (Vitale et al., 1967). Injection of MAb 34-3C resulted in an average hemato- crit in wild-type mice of 23% at day 4 postinjection, while in knockout mice hematocrit levels remained at 45% (Figure 3A) and had no evidence of hepatic erythrophagocytosis (see Figure 2A). In contrast, MAb 31-9D resulted in hema- 20- tocrits of 25% in both wild-type and knockout mice, while 0 1 2 3 4 5 6 7 4C6 reduced the hematocrits of wild-type and knockout TIME (days) animals to 35% (Figures 38,3C). Histopathological exami- Figure 1. Experimental Murine Immune Hemolytic Anemia Induced nation of knockout animals injected with 31-9D revealed by Rabbit Polyclonal Anti-Mouse Red Blood Cell IgG similar degrees of splenomegaly and microscopic aggluti- Daily hematocrits of wild-type (+/+, closed circles and squares) and nation in the sinusoids of the liver and spleen (data not homozygous y chain-deficient littermates (-I-. open circles and shown). Additionally, knockout and wild-type animals squares). Circles denote uninjected control animals and squares de- note animals injected with rabbit anti-mouse RBC IgG. showed similar rates of recovery with stabilization of hema- tocrits at 40%-500/b by day 7. This recovery process was accompanied by similar histological evidence of extra- 1990). We show that FcyR-bearing cells are critical to the medullary hematopoiesis in the liver and spleen and equiv- development of these models of type II , and alent levels of reticulocytosis in the peripheral blood (data that the hepatic macrophage is of greater importance in im- not shown). The role of complement in the residual anemia mune hemolytic anemia than in immune thrombocytopenia. induced in knockout mice by RaMRBC was determined by depleting complement C3 with cobra venom factor. Cobra Reeulte and Discussion venom factor-treated mice were not significantly pro- tected from anemia (Figure 3C), indicating that comple- Experlmental Immune Hemolytic Anemia ment-mediated pathways do not significantly contribute in y-j- Mice to the residual pathology seen in FcR-deficient animals. To determine whether cytotoxic antibodies require FcRs to Thus, despite the fact that complement and complement mediate their effects, FcyRI- and FcyRllldeficient mice or receptors are normal in these mice and can be activated their wild-type littermates were injected intraperiioneally with by appropriate stimuli (Sylvestre and Ravetch, 1994) and 200 ug of a polyclonal rabbit anti-mouse red blood cell IgG incubation of RaMRBC IgG with mouse RBCs and serum, fraction. As shown in Figure 1, wild-type littermates became in vitro, results in hemolysis, the in vivo role of complement profoundly anemic with an average hematocrit of 23.7% on in this model of immune hemolytic anemia appears to be day 4 postinjection, whereas knockout mice were relatively minimal. resistant to the pathogenic effects of this antibody, resulting in an average hematocrit of 36.5%. Experimental Immune Hemolytic Anemia We next investigated the mechanisms of the FcRdepen- In op/op Mice dent and FcR-independent anemia induced by RaMRBC The FcrR-expressing cell responsibleforthe experimental IgG. Histopathological examination of the livers and spleens AIHA we observe is likely to be the splenic macrophage of treated animals revealed a greater degree of hepato- and hepatic Kupffer cell. We have found that the adult op/ splenomegaly in wild-type mice injected with RaMRBC, with op mouse, which carries a mutation in the CSF-1 , is prominent evidence of hepatic erythrophagocytosis (Figure less susceptible to the induction of anemia by MAb 34-3C 2A) and splenic engorgement (Figure 28) as compared (Table 1). These mice have greatly reduced numbers of with their knockout littermates. These data suggest that hepatic and splenic marginal macrophages (Cecchini et polyclonal RaMRBC IgG is likely to be inducing anemia al., 1994) implicating these CSF-l-dependent mononu- predominately through erythrophagocytosis, mediated clear phagocytes in the AIHA mediated by erythrophago- through FcR engagement. The persistent though dimin- cytosis of antibody-coated RBCs through FcyRIII. ished pathology observed in the knockout animals is likely to be the result of FcR-independent processes, such as Experimental Immune Thrombocytopenia in y-l- agglutination (Figure 28). Additional evidence for these and oplop Mice conclusions was obtained by the use of three mouse MAbs Similar results were obtained in another model of type II directed against mouse RBCs, which elicit autoimmune hypersensitivity, experimentally induced immune throm- anemia through different mechanisms. MAb 34-3C has bocytopenia. The capacity of knockout mice to develop been shown to induce erythrophagocytosis by peritoneal thrombocytopenia after challenge with MAb 6A6 (Mizutani macrophages in vitro and by splenic and hepatic mononu- et al., 1993) an IgGl specific for mouse platelets, was clear cells in vivo. The anemia induced by MAb 31-9D dramatically different from their wild-type littermates (Fig- is mediated instead by red blood cell agglutination and ure 4). Wild-type animals demonstrated a rapid induction subsequent hepatic and splenic mechanical sequestra- of thrombocytopenia following antibody challenge and de- Etotoxlc Antibodies Trigger Inflammation Via FqR

+/+ RaMRBC -/- RaMRBC

Figure 2. Histological Aappearance of the Liver and Spleen in Wild-Type (u*) and Homozygous y Chain-Deficient Littermates (y*-) Injected with Rabbit aMRBC IgG or MAb 34-3C Liver and spleen are shown in (A) and (B), respectively. Immunity 24

Table 1. Hemolytic Anemia and Thrombocytopenia in Wild-Type, FcR Knockout, and Osteopetrotic Mice

Antibody Wild type ?-lb op/op a-MRBC 34-S 23% 46% 34% a-platelet 6A6 16% 66% 16% Data presented as mean absolute hematccrtt values or as percent of baseline platelet counts from five animals in each group. Mice (2-4 months old) were used with antibody doses adjusted to body weight.

resulted in a partial protection from GAG-induced thrombo- cytopenia, with platelet counts reaching 46% of baseline I I 1 2 3 4 5 6 7 levels. These results are consistent with several clinical studies in which ITP has been treated with inhibitors of TIME WV) FcyRssuchas intravenousyglobulin, invivoimmunecom- plexes generated by anti-hRBC IgG, Fc fragments, and antibodies against F~Rlll, with varying degrees of suc- 318D B cess(Soubrane et al., 1993; Debre et al., 1993; Blanchette et al., 1994; Clarkson et al., 1986; Salama et al., 1983). In contrast with the situation with experimental AIHA, im- mune thrombocytopenia is identical in wild-type and oplop mice (Table l), indicating that different FcrR-expressing cells are responsible for the pathology of these two exam- ples of type II inflammation.

Concluding Remarks These studies argue for a dominant role for Fc receptor engagement at an early step in the cascade of inflamma- tion initiated by cytotoxicantibodies in thetype II hypersen- sitivity class of inflammation. Despite the ability of these antibodies to fix complement and mediate target cell lysis in vitro, the situation in vivo appears to be quite different. As was found for immune complex-initiated inflammation (Sylvestre and Ravetch, 1994), Fc receptor engagement is also an early and critical step in the cascade of type II inflammation, supported by these data and in vitro studies on defects in y-l- mice in antibody-dependent cellular cyto- toxicity and phagocytosis in natural killer cells and mono- nuclear phagocytes (Takai et al., 1994; data not shown). The activation of effector cells by antibody-coated RBCs has been thought to be mediated by the synergistic coop eration of FcyR and C3b receptors (Schreiber and Frank,

Figure 3. Experimental Murine Immune Hemolytic Anemia Induced by Pathogenic Anti-Mouse RBC antibodies in Wild-Type and Homozy- gous y Chain-Deficient Littermates (A and B) Daily hematocrits of wild-type (+I+, closed squares) and homozygous y chain-deficient littermates (-I-, open squares) are shown for mice injected with MAb 34-X (A) and 31-9D (B). (C) Nadir hematocrits achieved by mice injected with pathogenic mouse aMRBC MAbs and rabbit aMRBC IgG. Mean hematocrits ob- tained from five mice in each group is presented. Hatched areas indi- cate the differences in anemia induction attributable to cobra venom factor treatment (CVF).

veloped platelet counts 18% of baseline levels within 2 Figure 4. Experimental Immune Murine Thrombocytopenia Is Medi- hr postinjection. FcR-deficient mice are resistant to the ated by Fc Receptors pathogenic effects of this antibody, retaining platelet counts Platelet counts (x lol/ul) from y-‘- mice (open squares) and y+‘+ mice 86% of baseline levels. Prior administration of an FcyRll (closed squares) and y+‘+ mice treated with Fc blocking antibody 2.462 and FcyRlll blocking antibody, 2.4G2 (Unkeless, 1979), (triangles) are shown. Mean data from groups of four mice are shown. zptoxic Antibodies Trigger Inflammation Via FcyR

1972) on the splenic macrophage and hepatic Kuppfer was diluted in buffer containing ammonium oxalate (Unopette Kits, ceil. Our data using a polyclonal RaMRBC IgG fraction Becton-Dickinson). Platelets were counted using a hematocytometer under a phase-contrast microscope. Fc-blocked r+‘+ mice received a shows that in a system capable of both complement- maximal inhibitory dose (Kurlander et al., 1964) of 6 pg of MAb 2.4G2 dependent and independent activation pathways, Fc-rR (Pharmingen) per gram of body weight intravenously 1 hr prior to injec- receptor loss rather than complement depletion prevents tion of MAb 6A6. immune hemolysis and argues for a minimal contributory role for complement activation. Confirmatory studies in complement-deficient animals would further strengthen We are grateful to Dr. S. lzui for providing anti-RBC MAbs; to Dr. R. this argument. These data further suggest that the hepatic Good for the anti-platelet MAb; and to Drs. D. Sylvestre, R. Pearse, Kuppfer cell or splenic marginal macrophage, which are and M. Sheffery for their helpful comments on the manuscript. C. reduced in adult op/op mice, are dominant in clearance of Chiang provided expert assistance in the preparation of the figures; the secretarial assistance of C. Ritter is gratefully acknowledged. These IgG-opsonized RBCs by erythrophagocytosis. In contrast, studiesweresupported bygrantsfrom the National Institutesof Health, this same cell population is not required for clearance of the Dewitt Wallace Research Laboratory, and the Memorial Sloan- IgGcoated platelets. Kettering Cancer Center Clinical Scholars Program (National Cancer These studies are especially relevant to the treatment Institute grant CA-09512). of ITP and AIHA in humans. 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