APOPTOSIS IN SPLENIC B-LYMPHOCYTES - REGULATION BY PROTEIN-KINASE-C AND IL-4

Small dense splenic B lymphocytes from adult specific pathogen-free mi ce were shown to undergo apoptosis in vitro as indicated by internucle osomal DNA fragmentation, hypodiploid DNA content of isolated nuclei, and morphologic features by electron microscopy. Unstimulated cultures showed spontaneous apoptosis increasing gradually and monotonically f rom <2 to 32% of B cells by 16 h. The rate of accumulation of apoptoti c cells was reduced by the addition of IL-4 or PMA, but not by the ina ctive phorbol ester, 4alphaPDD. In contrast, inhibitors of protein kin ase C (H7 and staurosporine) increased the percentage of cells undergo ing apoptosis to >70% by 12 h; HA 1004, genistein, and herbimycin A al l had no effect on apoptosis. Thus, protein kinase C activity regulate s apoptosis, but there is no evidence that protein kinases A and G and tyrosine kinases are involved. Cycloheximide increased apoptosis, ind icating that apoptosis may be restrained in B cells by the presence of one or more labile protective proteins. The percentage of apoptotic c ells measured by flow cytometry and the percentage of fragmented DNA m easured by the diphenylamine method were nearly equal, regardless of t he method of apoptotic regulation. Together with the absence of nuclei with flow cytometric properties intermediate between normal and apopt otic, these results suggest that in individual B cells apoptosis progr esses rapidly to completion. These data suggest a fundamental change i n our concept of the life-style of the ''resting'' B cell: instead of a dormant cell remaining unchanged until it receives activation signal s, the mature spleen B cell appears programmed to die by apoptosis unl ess rescued by specific agents, such protein kinase C activators or IL -4.