These data suggest that MIP8a Fab treatment of FcαRI on macrophages affects the expression of FcγRIIb and DC-sIGn. After injection of FcαRIR209L/FcRγ Tg macrophages into non-transgenic mice, all mice were injected with HAF prior to CpG. At day 14, mice treated with an unrelated control IgG developed elevated proteinuria, deposition of IgG and IgM, glomerular expansion and hypercellular changes and infiltration of CD11b+/F4/80+ macrophage in glomeruli and interstitial PI3K inhibitor tissue (Fig. 7a,b). However, all these signs of renal disease were attenuated
significantly in mice treated with MIP8a Fab (Fig. 7a,b). These data suggest that MIP8a Fab treatment of FcαRI on macrophages is sufficient to protect against development of HAF-CpG-GN. We next analysed the effect of anti-FcαRI (MIP8a Fab) pretreatment on TLR-9 signal transduction in response to CpG-ODN in the FcαRR209L/FcRγ RAW 264·7 macrophage cell line (clone I3D). Key events in CpG-ODN-mediated signals, such as p38 and p42–p44 ERK MAPKs phosphorylation [20], are shown in Fig. 8a. However, these phosphorylations were RG 7204 inhibited strongly in I3D after preincubation with MIP8a Fab but not with control Fab (Fig. 8a). This inhibition was concentration- and time-dependent and showed the maximum effect after 12 h of preincubation
(Fig. 8b,c). This treatment, although unlikely, does not kill the target cells (Fig. S2). We also tested the effect of the physiological ligand IgA. Incubation with human monomeric Histone demethylase IgA, but not IgG, resulted in an inhibitory response in I3D (Fig. 9). Figure 10 shows the effect of MIP8a Fab on CpG-ODN-induced transcriptional activation of the NF-κB/AP-1 cascade using a NF-κB/AP-1-Lux reporter
construct. FcαRI/FcRγ transfected RAW 264·7 (I3D) cells transiently transfected with a NF-κB/AP-1-Lux reporter construct showed increased-NF-κB/AP-1 activity after CpG-ODN treatment. CpG DNA-activated NF-κB/AP-1-Lux was inhibited significantly after preincubation with MIP8a Fab but not with control Fab (Fig. 10). These results were dose- and time-dependent (data not shown). MIP8a Fab itself had no effect on basal NF-kB/AP-1-Lux activity (data not shown). Taken together, MIP8a Fab inhibits CpG-induced activation of the NF-κB/AP-1-Lux activity, providing a molecular basis for its inhibition of HAF-CpG-GN. I3D cells produced significantly greater amounts of TNF-α/MCP-1 after exposure to CpG (100 ng/ml) for 4 h (Fig. 11c), as described previously [19]. However, CpG-triggered TNF-α/MCP-1 production was inhibited significantly by pretreatment with MIP8a Fab but not control IgG (Fig. 11a). The inhibitory effect of MIP8a Fab was concentration-dependent with maximal inhibition at a Fab concentration of 10 µg/ml (Fig. 11b). MIP8a Fab at 10 µg/ml effectively inhibited CpG-induced TNF-α/MCP-1 production in I3D cells over a wide range of CpG concentrations (25–500 ng/ml).