ENDOGENOUS ACTIVATION OF APOPTOSIS IN BURSAL LYMPHOCYTES - INHIBITIONBY PHORBOL ESTERS AND PROTEIN-SYNTHESIS INHIBITORS

The bursa of Fabricius represents the primary immune organ where immat ure B cells undergo maturational changes in avian species. Isolation o f bursal lymphocytes for analysis in cell culture results in the rapid endogenous activation of apoptosis, After 2 h of incubation, over 45% of the lymphocytes were shown to be undergoing apoptosis and by 6 h 8 0% were undergoing apoptosis as demonstrated by a terminal deoxynucleo tidyltransferase-mediated dUTP-fluorescein isothiocynate nick end-labe ling Bow-cytometric analysis. These results were corroborated by a pro pidium iodide-staining flow-cytometric assay and by an agarose gel ele ctrophoresis DNA fragmentation assay that demonstrated internucleosoma l DNA cleavage of genomic DNA in apoptotic bursal lymphocytes, Endogen ous activation of apoptosis in bursal lymphocytes could be inhibited i n a dose-dependent fashion with the phorbol ester, phorbol la-myristat e 13-acetate, but not the phorbol ester antagonist 4 alpha-phorbol 12, 13-didecanoate. In addition, apoptosis could be inhibited in a dose-de pendent fashion with inhibitors of protein translation, cycloheximide, and puromycin, as well as the transcriptional inhibitor actinomycin D . These results suggest that endogenous activation of bursal lymphocyt e apoptosis may be mediated by the protein kinase C signal transductio n pathway and activation of this process appears to be dependent upon denovo protein biosynthesis. (C) 1998 Academic Press.