Differences in IgG1 production between WT and Camp−/− B cells could be explained if there was a change in CSR to IgG1 and a linear relationship has been shown
between the amount of B-cell sterile Iγ1 transcript and CSR 36. Alternatively, the amount of IgG1 mRNA production could be increased in the WT cells compared with Camp−/− cells. Therefore, to determine the amount of Iγ1 and IgG1 mRNA in WT and Camp−/− cells, B cells were sort-purified and activated as described earlier and total RNA was isolated on days 2–4. Semi-quantitative RT-PCR showed no significant difference in the levels of Iγ1 transcript over the time course analyzed (Fig. 4E), suggesting no change in CSR. However, the level of IgG1 mRNA was significantly www.selleckchem.com/products/PD-0325901.html higher in the WT compared
with Camp−/− B cells (Fig. 4F), suggesting that mCRAMP was increasing either the rate or stability of the IgG1 mRNA. To determine the stability of the IgG1 mRNA, actinomycin D was added to the B-cell cultures on day 5 and total RNA was collected every 2 h for a total of 12 h. The stability of the IgG1 mRNA did not differ significantly between the WT and Camp−/− B cells (Fig. 4G). Thus, it appears that mCRAMP production by B cells increases the amount of IgG1 produced per cell by increasing the rate of IgG1 mRNA trancription, without affecting CSR or the stability of the IgG1 mRNA. Our data presented in Fig. 2 show that mCRAMP negatively regulates the level of T-cell IL-4 production in vitro, while our data presented in Fig. 3 show that mCRAMP positively regulates the level of B-cell IgG1 PD 332991 production in vitro. However, the antibody responses to TI-1, TI-2, and TD antigens have not been investigated extensively in Camp−/− mice to date. To investigate the antibody response in vivo to these three groups of antigens, WT and Camp−/− mice were immunized with either TNP-LPS (TI-1), S. pneumoniae (TI-2), or TNP-OVA absorbed to Alum (TD). The levels of IgM and IgG3 antibodies against TNP and
phosphorylcholine(PC) were determined by ELISA and showed no significant difference between WT and Camp−/− mice (Fig. 5A–D), Selleckchem Paclitaxel similar to our findings with LPS-activated B cells in vitro. Mice were also immunized i.p. and s.c. with TNP-OVA absorbed in alum on days 0 and 21 and the level of serum IgG1 antibody was measured. TNP-specific IgG1 was significantly higher in the Camp−/− mice following the second i.p. immunization (Fig. 5E) and first s.c. immunization (Fig. 5F). TNP-specific IgG2b and IgG2c were also determined and no differences were detected between WT and Camp−/− mice (data not shown). Overall, these results suggest that mCRAMP negatively regulates the TD antibody response in vivo, although the specific cell type responding to and affected by mCRAMP remains unknown.