DISTRIBUTION OF CAMP IN SECONDARY FOLLICLES AND ITS EXPRESSION IN B-CELL APOPTOSIS AND CD40-MEDIATED SURVIVAL

Apoptosis occurs at a high rate among B cells in germinal centres (GCs). Isolated GC B cells undergo apoptosis spontaneously when cultured in vitro: such self-destruction can be arrested by protein kinase C-activating phorbol esters and by ligating surface CD40. This study sought to explore whether the cAMP-dependent second messenger system played a role in the regulation of GC cell apoptosis and in ligand-promoted survival. First, the distribution of cAMP in GCs was analysed by immunofluorescence staining and confocal laser scanning microscopy in situ: cytoplasmic cAMP was expressed by all cells but was most abundant in the constitutive cells of the light zones, where GC B cell apoptosis and rescue occur. Isolated GC B cells were found to express higher levels of cAMP than quiescent B lymphocytes as assessed both by a competitive binding assay and a newly developed flow cytometric method. Culturing GC B cells alone increased cAMP level, while those cells rescued with the phorbol ester phorbol myristate acetate (PMA) and anti-CD40 exhibited decreased cAMP levels. Resting B lymphocytes showed no change in cAMP level following culture alone, but a significant increase in cAMP levels when co-cultured with PMA and anti-CD40. These data suggest that given identical signals, resting B cells and GC B cells exhibit differential regulation of the cAMP-dependent second messenger system; moreover, this second messenger system appears to be involved in the regulation of apoptosis in GC B cells.