Conversely, blocking IL-6R did not alter the level of STAT3 phosphorylation in B cells incubated with IL-10, indicating that it did not rely on IL-6 production, as also indicated by measuring IgA level by ELISA (Fig. 4b). IL-6 increased IgA production by approximately twofold compared to untreated cells and IL-10 increased IgA production by more than 10-fold. Addition of the IL-10R blocking PKC inhibitor antibody to IL-10-treated B cells significantly decreased IgA production to nearly baseline levels, whereas the addition of the IL-6R blocking antibody did not affect IgA production. Moreover, when B cells were incubated for 120 min with blocking peptides against pNF-κB p65 and/or pSTAT3 and then stimulated with sCD40L

and IL-10, the additional IgA production following stimulation was unaffected by blocking IL-6R (data not shown). B cells

were also incubated with an IL-6R blocking antibody to rule out instantaneous binding (recapture) of released IL-6 to IL-6R. B cells were stimulated with sCD40L alone, IL-10 alone or sCD40L + IL-10 for 0–60 min and then IL-6 production by stimulated B cells was assayed by ELISA. IL-6 was not detected in any of the B cell cultures after 1–2 days (data not shown). We therefore conclude that IL-10 has a direct role in IgA production without an IL-6 shift and that IL-6 does not play an essential role in CD40L–IL-10-driven IgA production. PBMC were stimulated in the presence or absence of blocking peptides against pNF-κB p65 and/or pSTAT3 at various concentrations (0–10 µg/ml; Fig. 5a) before initiation of the 12-day culture experiments. IgA Urocanase ELISAs were performed to identify the optimal concentration for each selleck kinase inhibitor peptide. IgA synthesis decreased in parallel with increased concentrations of blocking peptide against pNF-κB p65 and/or pSTAT3, with the lowest IgA level being observed at a concentration of 5 µg/ml. Next, PBMC were stimulated in the presence or absence of the same blocking peptides against pNF-κB p65 and/or pSTAT3 (5 µg/ml) at various time-points (0–240 min; Fig. 5b) before initiation of the 12-day culture experiments. IgA synthesis decreased in parallel with longer incubation times of blocking peptide

against pNF-κB p65 and/or pSTAT3, with the lowest IgA level being observed at an exposure time of 120 min. The pNF-κB p50 blocking peptide was tested under similar conditions and was not shown to be associated with a significant decrease in IgA synthesis at any of the blocking peptide concentrations tested (data not shown). Inhibition of IgA production was not due to in vitro toxicity of the blocking peptides against pNF-κB p50 or pNF-κB p65 or pSTAT3, as determined by counting the viable cells after 120 min of exposure to XTT during the 12 days of culture (Materials and methods, data not shown). In this set of experiments, we used PBMC in order to determine the optimal concentration and incubation time for the inhibitory peptides.