Endogenous Activation of Apoptosis in Bursal Lymphocytes: Inhibition by Phorbol Esters and Protein Synthesis Inhibitors

The bursa of Fabricius represents the primary immune organ where immature B cells undergo maturational changes in avian species. Isolation of 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 80% were un-dergoing apoptosis as demonstrated by a terminal deoxynucleotidyltransferase-mediated dUTP-fluores-cein isothiocynate nick end-labeling flow-cytometric analysis. These results were corroborated by a propidium iodide-staining flow-cytometric assay and by an agarose gel electrophoresis DNA fragmentation assay that demonstrated internucleosomal DNA cleavage of genomic DNA in apoptotic bursal lymphocytes. Endogenous activation of apoptosis in bursal lymphocytes could be inhibited in a dose-dependent fashion with the phorbol ester, phorbol 12-myristate 13-acetate, but not the phorbol ester antagonist 4α-phorbol 12,13-didecanoate. In addition, apoptosis could be inhibited in a dose-dependent 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 lymphocyte apoptosis may be mediated by the protein kinase C signal transduction pathway and activation of this process appears to be dependent uponde novoprotein biosynthesis.