PROTEIN-TYROSINE PHOSPHORYLATION IS MANDATORY FOR CD40-MEDIATED RESCUE OF GERMINAL CENTER B-CELLS FROM APOPTOSIS

Spontaneous apoptosis in germinal center (GC) B cells can be arrested either by engaging cell surface immunoglobulin (Ig) with immobilized ligand or, more effectively, by treatment with soluble monoclonal antibody (mAb) directed against CD40. The present study examines the intracellular signal transduction pathways through which rescue from spontaneous apoptosis is engendered in GC B cells following ligation of surface CD40. Cross-linking the surface CD40 of GC B cells with mAb consistently resulted in enhanced tyrosine phosphorylation on a number of distinct substrates: this process could be blocked, in a dose-dependent fashion, by pre-treating GC B cells with the selective protein tyrosine kinase(s) (PTK) inhibitor, herbimycin A. Moreover, the pattern of phosphorylation on tyrosine observed following treatment with anti-CD40 was remarkably similar to that triggered by polyvalent anti-Ig. By contrast, anti-CD40 failed to stimulate the increase in inositol 1,4,5-trisphosphate and cytosolic free calcium observed in both GC B cells and resting B lymphocytes following ligation of surface Ig. The involvement of the signaling pathways generated in the rescue of GC B cells from apoptosis was studied by using selective inhibitors of PTK and of extracellular and intracellular Ca2+. Pre-incubation with the PTK inhibitor herbimycin A (5 muM) abrogated anti-CD40-mediated rescue of GC B cells from apoptosis, while genistein (40 muM) and the tyrphostins AG490 (10 muM) and AG814 (25 muM) significantly inhibited this process. Consistent with these results, herbimycin A (5 muM) abolished the expression of the 26 kDa bcl-2 protooncogene product, which confers resistance to apoptosis, normally observed following culture with anti-CD40. The Ca2+ chelators BAPTA and EGTA did not significantly affect CD40-promoted rescue. Taken together, these results indicate that CD40 of GC B cells is coupled to functional PTK but not to the phosphatidylinositol signaling pathway and that tyrosine phosphorylation is mandatory for CD40-mediated rescue of GC B cells from apoptosis.