Identification of a Receptor Required for the Anti-Inflammatory Activity of IVIG
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Identification of a receptor required for the INAUGURAL ARTICLE anti-inflammatory activity of IVIG Robert M. Anthonya, Fredrik Wermelinga,b, Mikael C. I. Karlssonb, and Jeffrey V. Ravetcha,1 aThe Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, NY 10065; and bDepartment of Medicine, Karolinska Institutet, 171 76 Stockholm, Sweden This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 25, 2006. Contributed by Jeffrey V. Ravetch, October 11, 2008 (sent for review October 1, 2008) The anti-inflammatory activity of intravenous Ig (IVIG) results from patients suffering from autoimmune diseases (3). Monomeric IgG, a minor population of the pooled IgG molecules that contains purified from the serum of thousands of healthy donors (IVIG) is terminal ␣2,6-sialic acid linkages on their Fc-linked glycans. These a commonly administered at high doses (1–2 g/kg) for the treatment anti-inflammatory properties can be recapitulated with a fully of a number of autoimmune diseases, including immune-mediated recombinant preparation of appropriately sialylated IgG Fc frag- thrombocytopenia, chronic inflammatory demyelinating polyneu- ments. We now demonstrate that these sialylated Fcs require a ropathy, Kawasaki Disease and Guillain-Barre syndrome, and is specific C-type lectin, SIGN-R1, (specific ICAM-3 grabbing non- widely used in other autoimmune disorders (4–6). integrin-related 1) expressed on macrophages in the splenic mar- A number of hypotheses have been advanced to explain the ginal zone. Splenectomy, loss of SIGN-R1؉ cells in the splenic paradoxical activity of high dose IgG, and include models that marginal zone, blockade of the carbohydrate recognition domain attribute the activity to the polyclonal binding specificities, encoded (CRD) of SIGN-R1, or genetic deletion of SIGN-R1 abrogated the in the variable domains of the administered antibodies that may anti-inflammatory activity of IVIG or sialylated Fc fragments. Al- counteract the activity of autoantibodies or inflammatory media- though SIGN-R1 has not previously been shown to bind to sialy- tors (6). Others have focused on the IgG Fc portion as the lated glycans, we demonstrate that it preferentially binds to anti-inflammatory component, and are supported by early clinical 2,6-sialylated Fc compared with similarly sialylated, biantennary studies in which preparations of Fc fragments were active in glycoproteins, thus suggesting that a specific binding site is cre- restoring platelet levels in autoimmune thrombocytopenia compa- ated by the sialylation of IgG Fc. A human orthologue of SIGN-R1, rable to that of intact IgG (7). Numerous mechanisms have been DC-SIGN, displays a similar binding specificity to SIGN-R1 but proposed to account for this activity of high dose IgG Fc fragments, differs in its cellular distribution, potentially accounting for some including competition for cellular Fc␥Rs, saturation of FcRn, and of the species differences observed in IVIG protection. These modulation of inhibitory pathways (6). Attempts to distinguish studies thus identify an antibody receptor specific for sialylated Fc, among these models have been hampered by a lack of in vivo models and present the initial step that is triggered by IVIG to suppress that recapitulate the anti-inflammatory activity of high dose IgG, inflammation. and an incomplete understanding of the biochemical composition of the active components within the polyclonal therapeutic required autoimmune disease ͉ DC-SIGN ͉ rheumatoid arthritis ͉ sialylated IgG Fc ͉ for in vivo activity. SIGN-R1 To address these shortcomings, we have developed murine inflammatory disease models that are attenuated by the anti- ince their initial discovery by von Behring and Kitasato over a inflammatory activity of high-dose IVIG or its Fc fragments, Scentury ago, immunoglobulins have served as prototypical including immune thrombocytopenia (8), serum-induced arthritis effector molecules of immune defense. Antigen binding, a property (9) and nephrotoxic nephritis (10). Thus, mice deficient in the IMMUNOLOGY of the variable domains of the molecule, confers on immunoglobu- macrophage growth/differentiation factor CSF-1 or mice lacking ␥ lins their remarkable diversity, while the constant region of the the inhibitory Fc RIIB receptor fail to respond to IVIG treatment molecule functions to couple antigenic recognition to cellular to attenuate thrombocytopenia, arthritis, or nephritis (8–10). Other effector pathways. It is through these Fc domains that immuno- pathways, such as the classical pathway of complement activation, globulins engage specific cellular receptors, translating the speci- for example, appear to be dispensible for IVIG protection (8–10). ficity of antigen recognition into in vivo responses. Thus, IgG These results have lead us to propose a model in which Fc fragments molecules are able to trigger proinflammatory responses, such as of IgG interact with a regulatory macrophage population in the phagocytosis and tumor cell killing through the engagement and spleen, which, in turn, mediates the stimulation of an anti- cross-linking of cognate Fc receptors for IgG (Fc␥Rs) (1). Similarly, inflammatory pathway, ultimately increasing surface expression of IgG immune complexes, when deposited in end organs such as the the inhibitory Fc receptor on effector macrophages found at sites kidney, synovium or lung, can induce an inflammatory response of immune complex deposition (3, 6). initiated by the activation of Fc␥Rs on inflammatory cells, such as The high-dose requirement for the anti-inflammatory activity of macrophages and neutrophils, and trigger the tissue pathology IVIG (1–2 g/kg), suggested that the preparations of purified IgG observed in diseases such as systemic lupus erythematosis and from normal human donors contain a small fraction of active arthritis. Systematic analysis of the Fc domain interactions with therapeutic. We recently reported on the biochemical character- Fc␥Rs and the resulting in vivo biological properties of IgG ization of IVIG preparations that display anti-inflammatory activity in antibodies has highlighted the critical role of these interactions to the efficacy of antibodies in diverse settings, including therapeutic Author contributions: R.M.A., F.W., M.C.I.K., and J.V.R. designed research; R.M.A. and F.W. IgGs developed for the treatment of neoplastic diseases, developed performed research; R.M.A., M.C.I.K., and J.V.R. analyzed data; and R.M.A. and J.V.R. wrote in defense against microbial pathogens, and in understanding the the paper. mechanisms of tissue pathology in autoantibody mediated autoim- Conflict of interest statement: J.V.R is a founder and shareholder of Virdante Pharmaceu- mune diseases (2). tical, Inc. However, IgG has also been demonstrated to mediate anti- 1To whom correspondence should be addressed. E-mail: [email protected]. inflammatory activity when administered as very high doses to © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810163105 PNAS ͉ December 16, 2008 ͉ vol. 105 ͉ no. 50 ͉ 19571–19578 Downloaded by guest on October 3, 2021 the animal models described above, and demonstrated an absolute requirement for the Fc fragment and its unique, N-linked complex, biantennary glycan attached at Asn 297 (11). Further character- ization demonstrated that the anti-inflammatory activity in IVIG preparations depended on a minor fraction of IgG Fc that con- tained the fully processed N-linked glycan terminated in sialic acid, linked in an ␣2,6 linkage to the penultimate galactose (12). This fully processed glycan is found in 1–3% of IgG in IVIG, thus explaining the high dose requirement of IVIG. As expected, enrichment of this fraction by lectin affinity chromatography or in vitro sialylation, resulted in a concomitant reduced dose require- ment for in vivo activity. Glycoproteins displaying similar complex, biantennary glycans terminating in sialic acid failed to demonstrate anti-inflammatory activity, indicating that both the amino acid backbone of the IgG Fc and the associated glycan are required. The biantennary glycan found at Asn 297 in the Fc fragment is unusual in that it is structurally constrained within the cavity formed by the Fig. 1. A splenic macrophage population is required for IVIG anti- A and B chains of the IgG Fc, adopting a highly ordered confor- inflammatory activity. Wild-type C57BL/6 mice, mice deficient in B cells mation that contrasts to similar glycans exposed on the solvent face (JHDϪ/Ϫ), mice deficient in CD4ϩ T cells (CD4Ϫ/Ϫ), mice deficient in both B and of proteins (13). This biochemical analysis of the active fraction of T cells (Rag1Ϫ/Ϫ), and splenectomized mice were treated with IVIG and K/BxN IVIG was confirmed by the preparation of a fully recombinant, sera, and footpad swelling was monitored. Means and standard deviation of human IgG1 Fc with a biantennary glycan terminating in ␣2,6-sialic day 6 clinical scores from 4–5 mice per group are plotted, and are represen- tative of three separate experiments; *, P Ͻ 0.05 as determined by a Student acid-galactose linkages that recapitulated the in vivo anti- t test. inflammatory activity of intact IVIG, albeit at a much reduced dose (12). The in vivo activity of this recombinant preparation was enhanced 35-fold compared with the activity of IVIG. C-type lectin involved in the binding of S. pneumonia and dextran In the present study, we sought to define the