Here we show that short-term injection of IL-33 in mice elicits a transient IgM autoantibody response that is dependent on IL-33-mediated induction of BAFF
Here we show that short-term injection of IL-33 in mice elicits a transient IgM autoantibody response that is dependent on IL-33-mediated induction of BAFF. also released during necrosis (53, 54), we evaluated the possibility that repeated exposure to IL-33 could lead to the formation of autoantibodies. Injection of C57BL/6 mice with 500 ng of IL-33 daily for four consecutive days resulted in an increase in the number of lymphocytes (data not shown) similar to what was reported previously with seven consecutive daily doses of 400 ng IL-33 (16). We quantified anti-DNA titers RGS10 by measuring total Ig, IgM, and IgG at 1, 2, and 3 weeks after the first IL-33 dose. We GOAT-IN-1 observed that IL-33 injections produced significantly higher total Ig titers at week 1, compared with mice injected with PBS (Physique ?(Figure1).1). This increase was driven by IgM as no increase in IgG anti-DNA titers were observed (Physique ?(Figure1).1). The autoantibody response appeared to be transient as the total Ig and IgM titers decreased at week 2 with a further reduction observed at week 3 (Physique ?(Figure1).1). The results show that repeated IL-33 injections induce a transient anti-DNA response. Open in a separate window Physique 1 IL-33 induced autoantibody formation. C57BL/6 mice (= 10 mice/group) were injected i.p. with PBS or 500 ng IL-33 daily for four consecutive days. Serum was collected at 1, 2, and 3 weeks after the first IL-33 injection then total Ig, IgM, and IgG anti-DNA titers in PBS (open symbols, dashed line) and IL-33 (closed symbols, solid line) injected mice were decided. Data are representative of two impartial experiments. Area under curve (AUC) was decided, comparing PBS to IL-33 injection by two-tailed unpaired 0.05, ns, not significant. BAFF is usually Induced by IL-33 and Mediates B Cell Expansion and Autoantibody Formation Excessive BAFF-mediated survival of B cells can potentially lead to the generation of autoreactive B cells (39, 42). It has been reported that multiple injections of IL-33 in mice increase the B cell population (16, 55). Therefore, we hypothesized a role for BAFF in the observed IL-33-induced autoantibody response. Indeed, serum BAFF levels were significantly increased following four consecutive IL-33 injections compared with PBS injected mice (Physique ?(Figure2A).2A). A corresponding increase in the number of splenic B cells was also observed (Physique ?(Figure2B).2B). To confirm that BAFF was directly responsible for the observed increases in B cell numbers and anti-DNA antibody titers, we repeated the experiment in the presence of a neutralizing BAFF antibody. Mice were injected 1 day prior to the start of the IL-33 or PBS injection series with either a BAFF neutralizing antibody or an isotype control antibody (100 g/mouse). As expected, treatment of PBS-injected mice with a BAFF neutralizing antibody caused a significant decrease in the numbers of B cells in the spleen when compared with control antibody (Physique ?(Figure2C).2C). Consistent with previous results (Physique ?(Figure2B)2B) we observed that, in mice that were pre-treated with the control antibody, IL-33 injections induced a significant increase in B cell numbers (Figure ?(Figure2C).2C). In the presence of the BAFF antibody, injection of IL-33 did not result in elevation of the number of B cells GOAT-IN-1 beyond what was observed in PBS-injected mice that received control antibody (Physique ?(Figure2C).2C). In addition, BAFF neutralization prevented the IL-33-induced increase in anti-DNA titers compared with the control antibody treated mice (Physique ?(Figure2D).2D). These results indicate that an increase in BAFF is responsible for both the increase in splenic B cell numbers and anti-DNA titers following repeated injections with IL-33. Open in a separate window Physique 2 BAFF is necessary for B cell expansion and autoantibody formation. C57BL/6 mice (= 5 mice/group) were injected i.p. with PBS or 500 ng IL-33 daily for four consecutive days. Serum was collected GOAT-IN-1 and spleens were harvested 24 h after the last injection then processed for flow cytometry. (A) serum BAFF concentrations in PBS (white bar) and IL-33 (black bar) injected mice were decided and (B) total (CD19+) B cell numbers (* GOAT-IN-1 0.05 by two-tailed unpaired = 5.