Intravenous immunoglobulin (IVIG) is used widely in the treatment of various autoimmune conditions. Despite its frequent use, the immunomodulatory mechanism of IVIG is still not fully clear. Recently, the ratio of activating immunoglobulin G Fc receptors (FcγRI, FcγRIII and FcγRIV) and the inhibitory FcγRIIB expression on macrophages was shown to be a key to the therapeutic action of IVIG, as IVIG can cause a high FcγRIIB:FcγRI/III/IV ratio in mouse models of disease (Bruhns et al, 2003; Kaneko et al, 2006). On the other hand, neutralisation of the C3a and C5a complement activation products is another pathway in the action of IVIG (Basta et al, 2003). Previously, it was demonstrated that C5a is a major regulator of the balanced FcR system in both FcR-mediated inflammation (Shushakova et al, 2002) and autoimmunity (Kumar et al, 2006) in mice. Anti-C5aR antibodies and FcγR blocking agents each mimic the anti-inflammatory action of IVIG in vivo (Skokowa et al, 2005), indicating that IVIG may produce its FcγR modulatory effects directly at the level of C5a, but molecular evidence for this possibility is lacking. The present study examined the effect of IVIG on the ability of C5a to differentially regulate FcγR gene expressions in RAW 264·7 macrophage cells transfected with luciferase reporter plasmids containing either the FcγRIIB (−727/+585) or FcγRIII (−1117/+18) gene promoter sequences. Optimal responses of enhanced versus suppressed FcγRIII and FcγRIIB promoter activities in transfected RAW 264·7 cells were obtained by stimulating them with 4·6 nmol/l C5a (50 ng of rhC5a; Sigma-Aldrich, Steinheim, Germany) for 2–4 h (data not shown). These data indicate that C5a activates inverse FcγR regulation in macrophages, confirming previous findings in FcγR mRNA and protein detection assays (Shushakova et al, 2002; Skokowa et al, 2005). We then stimulated macrophages for 4 h with rhC5a in the presence of increasing amounts of IVIG (Octagam®; Octapharma, Langenfeld, Germany). IVIG at 22·5 nmol/l fully blocked the upregulation of FcγRIII and suppression of FcγRIIB gene activities by C5a (Fig 1). We also performed controls to determine the specificity of the blocking effects of IVIG. The cytokine interferon-γ (IFN-γ) exhibits a similar capacity to C5a in triggering inverse FcγR modulation (Radeke et al, 2002). In contrast to C5a, however, FcγR gene regulation induced by IFNγ was not inhibited by IVIG (Fig 1A), indicating selective binding between IVIG and C5a, but not IFNγ, as recently suggested (Basta et al, 2003). Finally, we tested the efficacy of additional IVIG preparations, including a second batch of Octagam®, as well as Beriglobin® (Aventis, Marburg, Germany) and gamunex® (Bayer Vital, Leverkusen, Germany). As shown in Fig 1B, all these IVIG preparations were equally effective (at 22·5 nmol/l) in reversing the C5a-induced upregulation of FcγRIII and downregulation of FcγRIIB genes in macrophages. C5a-induced upregulation of FcγRIII and downregulation of FcγRIIB on macrophages is reversed in the presence of intravenous immunoglobulin (IVIG). (A, B) RAW 264·7 cells were transfected with the indicated FcγRIII (−1117/+18) (left panels) and FcγRIIB (−727/+585) (right panels) promoter constructs plus the renilla luciferase gene as control. After 48 h, an equal volume of complete medium or medium supplemented with 4·6 nmol/l rhC5a and interferon-γ (300 U) in the presence or absence of the indicated doses of IVIG (Octagam®) was added to the cells. (B) In further experiments, FcR-transfected RAW 264·7 cells were stimulated with 4·6 nmol/l rhC5a in the presence of a fivefold molar excess (22·5 nmol/l) of IVIG obtained from different manufacturers (Octagam®, gamunex®, Beriglobin®). (A, B) After an additional 4 h of culture, luciferase activity in the cell lysates was determined by luminescence spectroscopy. Results represent means ± SEM of firefly luciferase counts normalised to renilla luciferase of 6–8 (in A) or 2 (in B) independent transfections. Significant differences were determined by Student's t-test (*P < 0·02; **P < 0·001). The study by Bruhns et al (2003) discussed an indirect mechanism by which IVIG may cause a high FcγRIIB expression level. In that model, IVIG interacted with an as yet undefined receptor on sensor cells to result in the release of mediators capable of inducing expression of FcγRIIB on macrophages. In line with the observations by Basta et al (2003), however, our data now indicate that IVIG-mediated neutralisation of C5a is critical for a sustained high FcγRIIB:FcγRIII ratio expression on macrophages, supporting a more direct mechanism of IVIG in the FcγRIIB-based immune suppression of antibody-dependent autoimmune diseases.
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