FcR -chain deficient (Fcer1g/) mice express no functional activating FcRs but do express the inhibitory FcRIIB. impact on activating FcRs that may control allergy, autoimmunity and cancer. The inflammatory responses in experimental autoimmune nephritis7,8, arthritis9, IC alveolitis10and peritonitis11are mediated by activating FcRs and C5aR. The emerging paradigm is usually that C5a decreases the cellular activation threshold by upregulation of the ratio between activating and inhibitory FcRs (known as the A/I ratio)10,11, whereas activating FcRs eventually drive the pro-inflammatory effector functions12. However, C5a recruits and activates inflammatory cells such as neutrophils, macrophages and mast cells impartial of FcRs2. FcR -chain deficient (Fcer1g/) mice express no functional activating FcRs but do express the inhibitory FcRIIB. They are guarded from IC-mediated inflammation despite the presence of C5a. We hypothesized that one mechanism underlying such protection could Mouse monoclonal to SMN1 be an anti-inflammatory property of IgG IC suppressing C5a-mediated effector functions. To test our hypothesis, we injected C5a into the peritoneal cavity of BALB/c (WT) orFcer1g/mice. As expected, we observed strong neutrophil recruitment in both mouse strains (Fig. NNC 55-0396 1a). IgG1 is the most abundant isotype of serum antibody (Ab) in mice. The preferential binding of IgG1 to inhibitory FcRIIB is considered a protective mechanism NNC 55-0396 to prevent incidental activation of circulating myeloid cells by binding of IC to activating FcRs12. Intravenous injection of IC comprising the anti-TNP-IgG1 clone 107.3 and TNP-OVA (107.3 IC)1330 min prior to C5a administration reduced the C5a-mediated neutrophil migration into the peritoneum in wt andFcer1g/mice but not inFcgr2b/mice(Fig. 1aandSupplementary Fig.1). We found no anti-inflammatory effect for monomeric 107.3-Ab (data not shown). Further, 107.3-IC blocked IC-peritonitis11in WT but not inFcgr2b/mice, demonstrating a critical role of NNC 55-0396 FcRIIB NNC 55-0396 for 107.3 IC-mediated inhibition of C5a-dependent inflammation(Fig. 1b). == Physique 1. IgG1 immune complexes (IC) inhibit C5a-mediated inflammatory responses in vivo and in vitro by an FcRIIB-dependent mechanism. == Peritoneal migration of neutrophils in response to C5a (a) or OVA/anti-OVA-IC (b) 107.3-IC in WT,Fcer1g/orFcgr2b/mice. Impact of 107.3-IC on(c)CD11b expression and (d) iC3b-dependent adhesion in neutrophils fromFcer1g/andFcgr2b/mice. C5a-mediated migration of BM-derived neutrophils (e) and increase in [Ca2+]iin BM-derived cells (f) in WT,Fcer1g/orFcgr2b/mice 107.3-IC. (g) Gating of BM neutrophils according to their FSC/SSC pattern (left dot-plot) and the expression of the Gr-1 marker (1sthistogram around the left). Dose-dependent impact of 107.3-IC on C5a-mediated ERK1/2 phosphorylation in Gr-1+WT BM neutrophils. (h) Effect of a C5a receptor antagonist (C5aRA) on C5a-mediated ERK1/2 phosphorylation. White histogram: ERK phosphorylation in the absence of C5a (background); grey histogram: treatment with C5a NNC 55-0396 (5 107M; 1 min); magenta histogram: C5a (5 107M; 1 min) 107.3 IC (g) or C5aRA (h) at the indicated concentrations. Results in (c) and (g) are representative of at least three impartial experiments. Values in (a) and (b), and (d-f) are means s.e.m. (n=5-10/group). *P<0.05, **P<0.01, ***P<0.001. The mechanisms leading to C5aR-mediated extravasation and tissue recruitment of neutrophils involve the 2 2 integrin CD11b/CD18 (CR3)14. C5a dose-dependently upregulated CD11b expression on neutrophils from WT (data not shown) andFcer1g/mice(Supplementary Fig. 2), which was significantly reduced upon 107.3-IC treatment. This reduction was absent with cells fromFcgr2b/mice(Fig. 1c). Further, 107.3-IC administration reduced C5a-dependent neutrophil adhesion with cells fromFcer1/but not fromFcgr2b/mice(Fig. 1d). Next, we assessed the impact of 107.3 IC on C5a-mediated chemotaxis(Supplementary Figs. 3a, d)and found a dose-dependent inhibition of C5a-mediated chemotaxis with BM neutrophils and peritoneal macrophages(Fig. 1eandSupplementary Fig. 3c,d)from WT andFcer1g/but not fromFcgr2b/mice. Further, pharmacological targeting of FcRIIB abrogated.