The ability of CD40 signaling to regulate B cell growth, survival, differentiation, and Ig class switching involves many changes in gene expression. Using cDNA expression arrays and Northern blotting, we found that CD40 signaling increased the mRNA levels for pim-1, a protooncogene that encodes a serine/threonine protein kinase. Subsequent experiments showed that CD40 engagement also increased both Pim-1 protein levels and Pim-1 kinase activity in B cells. We then investigated the signaling pathways by which CD40 regulates Pim-1 expression and found that CD40 up-regulates Pim-1 primarily via the activation of NF-κB. Inhibiting the activation of NF-κB, either by treating cells with a chemical inhibitor, BAY11-7082, or by inducibly expressing a superrepressor form of IκBα, significantly impaired the ability of CD40 to increase Pim-1 protein levels. Because Pim-1 expression is associated with cell proliferation and survival, we asked whether this correlated with the ability of CD40 signaling to prevent anti-IgM-induced growth arrest in the WEHI-231 murine B cell line, a model for Ag-induced clonal deletion. We found that the anti-IgM-induced growth arrest in WEHI-231 cells correlated with a substantial decrease in Pim-1 levels. In contrast, culturing WEHI-231 cells with either anti-CD40 Abs or with the B cell mitogen LPS, both of which prevent the anti-IgM-induced growth arrest, also prevented the rapid decline in Pim-1 levels. This suggests that Pim-1 could regulate the survival and proliferation of B cells.
The imidazoquinoline R-848, originally identified as a highly effective antiviral agent, has recently been shown to be capable of potent B lymphocyte activation. The B cell-activating properties of R-848 are strikingly similar to the effects of the CD40 ligand CD154. The present study demonstrates that this similarity extends to the intracellular signaling pathways triggered by the compound, although both overlapping and distinct mechanisms of signaling were seen. Like CD40 ligation, R-848 stimulated activation of the stress-activated protein kinases c-Jun kinase and p38 and activated the NF-κB family of transcription factors. Both R-848- and CD40-mediated B cell differentiation were dependent upon NF-κB activation, although the relative importance of individual NF-κB family members appeared to differ between R-848- and CD40-mediated signals. Both signals were partially dependent upon induction of TNF-α and IL-6, and the cytoplasmic adaptor molecule TNF receptor-associated factor 2 is involved in both R-848- and CD40-mediated differentiation.
B cells of mouse and human can be divided into distinct subpopulations, differing in distribution and phenotype. It is not known, however, whether B cell subsets respond similarly to signals mediated by cognate interactions with T cells, such as ligation of the B cell class II MHC molecule. Mouse splenic B cells proliferate in response to a combination of non-mitogenic anti-mu mAb, IL-4 and class II MHC-specific mAbs. In order to assess the response of B cell subpopulations to these signals, B cells were separated on the basis of CD23 expression. Previous studies have shown that CD23 expression is useful in distinguishing marginal zone from follicular B cells and peritoneal B1 from B2 B cells. B cells of both subsets responded to the combination of signals, but CD23- B cells showed a higher response. Splenic B cells were also purified from several strains of autoimmune mice, of interest because their disease features expansion of CD23- B cells and autoantibody production. While normal B cells showed no response to non-mitogenic anti-mu mAb alone, B cells from autoimmune mice showed a marked decrease in proliferation. Addition of IL-4 plus class II-specific mAbs restored increased proliferation. Again, responses were higher in CD23- than in CD23+ B cells. These findings suggest that CD23- B cells are especially responsive to signals delivered through class II and the IL-4 receptor. These signals may allow preferential rescue of CD23-B cells from antigen-mediated tolerance and result in their hyperexpansion in response to autoreactive T cells.
Expression of the Thy-1 membrane antigen is generally confined to thymocytes and T lymphocytes, but its expression on B lymphocytes can be induced by culture with the lymphokine IL-4. IL-4 was first reported as a soluble factor capable of participating in the activation of B cells. However, it has been shown that the proliferative response of B cells to IL-4 is dependent upon both their stage of differentiation and their prior exposure to other activating signals--under some conditions, IL-4 can inhibit B cell functions. The present study was designed to determine whether IL-4 signaling induces Thy-1 expression on all B lymphocytes, or whether this induction is dependent upon IL-4-mediated activation. We examined the role of IL-4 in regulating both mRNA and protein levels of Thy-1 in three mouse B cell lines with distinct growth responses to IL-4. IL-4 was required for Thy-1 expression in cells which were dependent upon IL-4 for continuous growth in culture but markedly decreased Thy-1 expression in cells which are growth-inhibited by IL-4. In a mutant subclone of the latter cells in which IL-4 signaling does not cause growth inhibition, IL-4 did not affect Thy-1 expression. The regulation of Thy-1 expression by IL-4 is manifest at the level of Thy-1 mRNA. Thus, IL-4 can both positively and negatively influence B cell expression of Thy-1, depending on the growth response of the cells to IL-4.
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