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 l igand or, more effectively, by treatment with soluble monoclonal antib ody (mAb) directed against CD40. The present study examines the intrac ellular signal transduction pathways through which rescue from spontan eous apoptosis is engendered in GC B cells following ligation of surfa ce CD40. Cross-linking the surface CD40 of GC B cells with mAb consist ently resulted in enhanced tyrosine phosphorylation on a number of dis tinct substrates: this process could be blocked, in a dose-dependent f ashion, by pre-treating GC B cells with the selective protein tyrosine kinase(s) (PTK) inhibitor, herbimycin A. Moreover, the pattern of pho sphorylation on tyrosine observed following treatment with anti-CD40 w as remarkably similar to that triggered by polyvalent anti-Ig. By cont rast, anti-CD40 failed to stimulate the increase in inositol 1,4,5-tri sphosphate and cytosolic free calcium observed in both GC B cells and resting B lymphocytes following ligation of surface Ig. The involvemen t of the signaling pathways generated in the rescue of GC B cells from apoptosis was studied by using selective inhibitors of PTK and of ext racellular and intracellular Ca2+. Pre-incubation with the PTK inhibit or herbimycin A (5 muM) abrogated anti-CD40-mediated rescue of GC B ce lls from apoptosis, while genistein (40 muM) and the tyrphostins AG490 (10 muM) and AG814 (25 muM) significantly inhibited this process. Con sistent with these results, herbimycin A (5 muM) abolished the express ion of the 26 kDa bcl-2 protooncogene product, which confers resistanc e to apoptosis, normally observed following culture with anti-CD40. Th e Ca2+ chelators BAPTA and EGTA did not significantly affect CD40-prom oted 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.