Signal transduction initiated by B cell Ag receptor (BCR) cross-linking plays an important role in the development and activation of B cells. Therefore, considerable effort has gone into determining the biochemical signaling events initiated by the BCR and delineating which events participate in specific biological responses to Ag. We used two inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 and MEK2, PD98059, and U0126, to assess the role the Ras-mitogen-activated protein kinase pathway plays in several BCR-induced responses. PD98059 or U0126 treatment substantially inhibited the BCR-induced activation of the extracellular signal-regulated kinase (ERK) forms of mitogen-activated protein kinase in the immature B cell line WEHI-231, in immature splenic B cells, and in mature splenic B cells. However, MEK-ERK inhibition did not block BCR-induced growth arrest or apoptosis of WEHI-231 cells or apoptosis of immature splenic B cells, indicating that the MEK-ERK pathway is not required for these events. In contrast, PD98059 and U0126 treatment did inhibit the up-regulation of specific BCR-induced proteins, including the transcription factor Egr-1 in WEHI-231 and mature splenic B cells, and the CD44 adhesion molecule and CD69 activation marker in mature splenic B cells. Moreover, both inhibitors suppressed BCR-induced proliferation of mature splenic B cells, in the absence and in the presence of IL-4. Therefore, activation of the MEK-ERK pathway is necessary for a subset of B cell responses to Ag.

Signaling by the BCR causes proliferation and resistance to Fas-induced apoptosis in mature B cells, but growth arrest and apoptosis in immature B cells. We have identified a variant of the immature B cell line WEHI 231 that retains the apoptotic response to the BCR but has acquired susceptibility to Fas-induced apoptosis. The Fas susceptibility was associated with increased Fas expression on the cell surface and down-regulated IgD expression. These cells exhibited a distinctive functional relationship in response to signals from the BCR, Fas and CD40: BCR stimulation markedly promoted Fas-mediated apoptosis (and vice versa) and Fas-induced apoptosis was not subject to modulation by CD40 signaling. While BCR-induced apoptosis was effectively rescued by CD40, it was not affected by the expression of a dominant-negative FADD. The mechanistic distinctions between BCR- and Fas-induced apoptosis were further characterized by the differential effects of different caspase inhibitors on these two processes which imply the involvement of different subsets of caspases. For BCR-induced apoptosis, we provide evidence that the final apoptotic destruction phase can be inhibited by the pan-caspase inhibitor BOC-Asp-FMK (BD) and that, in the presence of BD, the BCR only induces growth arrest which is reversible. The striking enhancing effects of Fas on BCR-induced apoptosis seen in the variant cells prompted us to examine if a similar cooperation in induction of apoptosis occurs in the highly tolerizable immature B cells of the spleen. We found that the splenic immature B population contains a significant number of Fas-expressing cells, but neither Fas-induced apoptosis nor an enhancing effect of Fas on BCR-induced apoptosis of these cells was detected in vitro.