IM directly inhibits CSR in activated B cells through down-regulation of AID. (A) IgG1 expression levels in spleen cells cultured in conditioning medium containing 12.5 μg/mL LPS and 7.5 ng/mL IL-4 with 0, 1, 2.5, 5, 7.5, and 10μM IM for 72 hours were 15.8%, 12.5%, 9.8%, 7.6%, 5.8%, and 2.9% of untreated controls, respectively. Reduction of IgG1 expression was induced by IM dose-dependently. (B) Real-time RT-PCR in spleen cells cultured in conditioning medium containing IL-4 and LPS for 72 hours indicated that expression of AID was decreased by IM dose-dependently. Significant differences were found between 0 and 1μM or 10μM IM. *P < .05. The y-axis represents AID mRNA levels relative to the no-IM control. The levels of AID mRNA at each IM concentration were calculated relative to the internal control (GAPDH); n = 6. (C) The level of the germline transcript of IgG1 in spleen cells cultured in conditioning medium containing IL-4 and LPS for 72 hours was not decreased in contrast to AID mRNA expression levels, which were decreased by IM in a dose-dependent manner. Significant differences were found between 0, 1, and 10μM IM. *P < .05. The y-axis represents expression levels of the IgG1 germline transcript relative to the no-IM control in the same manner as that in panel B; n = 4. (D) IgA expression levels in CH12F3-2A cells cultured in conditioning medium containing 7.5 μg/mL IL-4, 0.3 ng/mL TGF-β1, and 40% CD40 ligand with 0, 5, 10, and 20μM IM for 72 hours were reduced in an IM dose-dependent manner. (E) Cell proliferation, division, and apoptosis in spleen cells cultured in conditioning medium containing IL-4 and LPS for 72 hours were investigated using BrdU, annexin V, and CFSE assays. The BrdU incorporation rate of 10μM IM was 45.9%, whereas that of 0μM IM was 58.1%. Cell fluorescence of 10μM IM using the CFSE assay was shifted, but that of 1μM IM was not shifted to the right compared with that of 0μM IM. Annexin V analysis of 10μM IM was 39.9%, whereas that of 0μM IM was 35.1%. These results indicate that IM affects cell proliferation but not apoptosis. (F) Immunohistochemical analysis of spleens from mice that were administered SRBC with or without IM. Serial sections of spleens were prepared from nonimmunized (i,iv,vii,x), SRBC-immunized (ii,v,viii,xi), or SRBC-immunized + IM (50 mg/kg; iii,vi,ix,xii) animals. (i-vi) H&E staining. (vii-xii) Immunohistochemical analysis of AID. Low-power fields are shown in panels i to iii and vii to ix. High-power fields are shown in subpanels iv to vi and x to xii. Individual germinal centers from SRBC-immunized IM (+) mice were significantly smaller than those from SRBC-immunized IM (−) mice and were comparable with those from nonimmunized mice. AID expression, which was induced in germinal center-activated B cells, was barely detectable in spleens of IM-treated mice but was strongly positive in those of nontreated mice. Moreover, IM significantly suppressed AID expression, even in the residual germinal centers. (G) Real-time RT-PCR analysis of AID and FACS analysis of IgG1 expression of spleen cells harvested from SRBC-immunized mice with or without 50 mg/kg IM. The top panel shows relative normalized AID mRNA expression, and the bottom panel shows surface IgG1 expression of total splenocytes. A significant difference was found between SRBC (+) and SRBC (+) IM 50 mg/kg (+) for both AID and IgG1. *P < .05. The y-axis represents the relative ratio of the relative expression level of AID mRNA (top panel) and the percentage of surface IgG1 expression of total splenocytes (bottom panel). Normalized values obtained for SRBC (+) IM 50 mg/kg (+) were derived from SRBC (+); n = 2.