Anti-inflammatory Activity of IVIG Mediated Through the Inhibitory Fc Receptor

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Science  19 Jan 2001:
Vol. 291, Issue 5503, pp. 484-486
DOI: 10.1126/science.291.5503.484


The molecular basis for the anti-inflammatory property of intravenous gamma globulin (IVIG) was investigated in a murine model of immune thrombocytopenia. Administration of clinically protective doses of intact antibody or monomeric Fc fragments to wild-type or Fcγ receptor–humanized mice prevented platelet consumption triggered by a pathogenic autoantibody. The inhibitory Fc receptor, FcγRIIB, was required for protection, because disruption either by genetic deletion or with a blocking monoclonal antibody reversed the therapeutic effect of IVIG. Protection was associated with the ability of IVIG administration to induce surface expression of FcγRIIB on splenic macrophages. Modulation of inhibitory signaling is thus a potent therapeutic strategy for attenuating autoantibody-triggered inflammatory diseases.

Although first introduced for the treatment of hypogammaglobulinemia, IVIG has since been shown to have broad therapeutic applications in the treatment of infectious and inflammatory diseases (1). The polyclonal specificities found in these preparations have been demonstrated to be responsible for some of the biological effects of IVIG. For example, IVIG has been used as prophylaxis against infectious agents and in the treatment of necrotizing dermatitis (2). Independent of these antigen-specific effects, IVIG has well-recognized anti-inflammatory activities, generally attributed to the immunoglobulin G (IgG) Fc domains. These activities, first applied for the treatment of immune thrombocytopenia (ITP) (3, 4), have been extended to the treatment of a variety of immune mediated inflammatory disorders including autoimmune cytopenias, Guillain-Barrésyndrome, myasthenia gravis, anti–Factor VIII autoimmune disease, dermatomyositis, vasculitis, and uveitis (5–10). A variety of explanations have been put forward to account for these activities, including Fc receptor blockade, attenuation of complement-mediated tissue damage, neutralization of autoantibodies by antibodies to idiotype, neutralization of superantigens, modulation of cytokine production, and down-regulation of B cell responses (11–14). However, the importance of any of these mechanisms to the in vivo activity of IVIG has not been established.

To investigate the mechanism by which IVIG mediated its anti-inflammatory effects, we used a murine model of ITP. As described previously, the murine IgG2a anti-platelet monoclonal antibody (mAb) 6A6 triggers a rapid consumption of platelets when injected intravenously into wild-type mice (15,16), mimicking the effect of a pathogenic autoantibody. As in human ITP, IVIG, administered at 1.0 g/kg, was protective (Fig. 1A). This protective effect was dose-dependent, with 50% protection observed at 0.5 g/kg, decreasing to a negligible effect at 0.1 g/kg (17). Administration of equimolar concentrations of Fab or Fc fragments, followed by 6A6, revealed that only the Fc fragment conferred the protective effect of IVIG (Fig. 1B). The pathogenic effect of 6A6 was dependent on the presence of the low-affinity activating IgG Fc receptor, FcγRIII. In its absence (Fig. 1C), mice were also protected from the ITP induced by 6A6. In contrast, deleting the high-affinity activating FcγR, FcγRI, was not protective (17). To further define the mechanisms of IVIG protection, we developed a transgenic model in which the endogenous murine FcγRIII gene was replaced by a human transgene encoding the FcγIIIA protein, expressed on natural killer (NK) cells, macrophages, and mast cells (18). These FcγR-humanized mice were fully susceptible to the pathogenic effect of this murine IgG2a antibody (Fig. 1D), despite the lack of FcγRIII expression on neutrophils. Thus, the requirement for neutrophils in the pathogenicity of this form of ITP was minimal. In contrast, splenectomy was partially protective (17), implicating splenic effector cells, such as macrophages, in the FcR-dependent pathogenicity of ITP. Protection in these FcR-humanized mice was observed either upon IVIG administration or by blocking the human IIIA with the monoclonal antibody to human FcγRIII, 3G8 (Fig. 1D). However as defined below, the mechanisms underlying these two treatments are distinct.

Figure 1

(A) IVIG protects against experimental immune thrombocytopenia. Wild-type BALB/c mice were injected with IVIG (1.0 g/kg) before administration of anti-platelet mAb 6A6 (15, 16). Mean platelet counts from untreated (blue circles) and IVIG-treated (red circles) mice are presented. (B) The Fc portion of IVIG protects against ITP. Equimolar concentrations of IVIG (open triangles), Fab (yellow triangles), or Fc fragments (red triangles) were administered 1 hour before 6A6 and compared with 6A6 alone (black triangles). (C) ITP is mediated by activation Fc receptors. ITP was induced in wild-type (wt; blue circles) and FcR γ-chain–deficient mice (green circles). (D) ITP in hRIIIATg/RIII−/− mice. Nadir platelet counts after 6A6 injection are depicted after administration of mAb 3G8 or IVIG (26–28).

Previous studies have demonstrated that the responses triggered by FcRγIII are counterbalanced by the inhibitory receptor FcγRIIB, under conditions when the effector cell coexpresses both molecules. This coupling has been seen in murine models of glomerulonephritis (19), in syngenic and xenograft tumor protection studies with anti-tumor antibodies, and in immune complex–triggered inflammatory responses in the skin and lung (20–22). However, in those models, deletion of RIIB enhanced sensitivity to cytotoxic IgGs and immune complexes. In the model of ITP described here, RIIB-deficient mice were as sensitive to the pathogenic effects of 6A6 as their wild-type counterparts (Fig. 2). This was the case even at limiting concentrations of autoantibody, indicating that the effector cells mediating ITP are not substantially modulated by RIIB. However, in contrast to wild-type mice, RIIB-deficient mice were defective in their protective responses through IVIG administration (Fig. 2A). Similarly, blocking RIIB with a specific mAb (anti-Ly17.2) in wild-type mice treated with IVIG negated its protective effect (Fig. 2B). These data demonstrated a clear requirement for FcγRIIB in the mechanism of IVIG protection, suggesting that IVIG might modulate RIIB expression or function, in a manner that counteracts the activation responses triggered by FcRIII engagement. Staining of splenic macrophages before and after treatment with IVIG revealed a 60% increase in expressing high levels of FcγRIIB after IVIG treatment (Fig. 3), supporting a model in which enhanced surface expression of RIIB is responsible for mediating the protective effect of IVIG. Because FcγRIIB mediates its inhibitory effect by the recruitment of the SH2-containing inositol phosphatase SHIP, an intracellular signaling molecule with pleiotropic effects on phosphatidylinositol (3,4,5)triphosphate (PIP3)–dependent signaling cascades (23,24), modulating RIIB expression could result in greatly amplified inhibitory effects. However, additional effects of IVIG on SHIP-dependent signaling cannot be excluded.

Figure 2

The protective effect of IVIG is mediated through the inhibitory receptor RIIB. (A) Mean platelet counts after 6A6 injection into RIIB−/− mice are shown for IVIG-treated (1.0 g/kg; yellow circles) and untreated (blue circles) groups (29). Red circles indicate IVIG + 6A6 in wild-type controls. The panel is representative of four different experiments. (B) RIIB blocking was performed in wild-type mice with anti-Ly17.2 (30). Mean platelet counts are presented for mice pretreated with IVIG (1.0 g/kg; red circles) or IVIG + anti-Ly17.2 mAb (6 μg/g; yellow circles) before 6A6 injection.

Figure 3

(A) In vivo induction of spleen RIIB+ cells by IVIG. Wild-type mice were injected with IVIG (1.0 g/kg). After 4 hours, splenocytes were analyzed by FACS for mac-1 and RIIB expression. (B) Representative FACS dot plots on mac-1– and Ly17.2-stained B220-negative splenocytes from one control and one IVIG-treated mouse.

Protection is seen with the Fc fragment of IVIG alone (Fig. 1B), and we therefore investigated whether the inductive response was dependent on the high-affinity Fc receptor, FcRI, which is capable of binding this molecule. FcγRI-deficient mice were susceptible to the pathogenic effects of 6A6 and were fully protected by IVIG administration (Fig. 4). Similar results were observed with complement-deficient mice (Fig. 4), indicating that the mechanism by which Fc fragments of IVIG induce protection through RIIB does not require these classical antibody-binding components. These data support the conclusion that IVIG mediates its protective effect by its ability to induce the expression of the inhibitory FcγRIIB receptor on effector cells that would otherwise trigger clearance of the opsonized platelets. A pronounced effect of even a modest induction of RIIB can be predicted in cells where activation through FcγRIII is not previously balanced by an inhibitory receptor. ITP can also be treated by blocking the activation receptor, FcRIII, as shown in Fig. 1D. This blocking effect on activation receptors may explain the therapeutic effect of anti-D in the treatment of ITP (25), where complexes of anti-D–opsonized red blood cells are likely to compete for FcRIII occupancy with complexes of autoantibody-opsonized platelets.

Figure 4

Effects of IVIG on 6A6-induced thrombocytopenia in wild-type, FcγRI-, C3-, complement receptor 1/2 (CRI/II)–, and RIIB-deficient mice. The panel shows mean nadir platelet counts reached after injection with IVIG (1.0 g/kg) followed by mAb 6A6 (0.3 μg/g; red) or 6A6 alone (blue).

The present report demonstrates that manipulation of the inhibitory FcR pathway is a practical therapeutic means for controlling autoantibody-mediated inflammation. IVIG is thus a demonstrated therapeutic whose mechanism of action is targeted to this inhibitory pathway. The emergence of additional molecules of this class can be anticipated on the basis of the insights provided by this mechanism of IVIG action.

  • * To whom correspondence should be addressed. E-mail: ravetch{at}


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