Microinjection of macromolecules into normal murine lymphocytes by means of cell fusion. II. Enhancement and suppression of mitogenic responses by microinjection of monoclonal anti-cyclic AMP into B lymphocytes.

Reproducible methods are now available for introducing protein molecules such as antibodies into normal murine lymphocytes by fusion with protein molecule-containing erythrocyte ghosts. Monoclonal antibodies against cyclic AMP were raised by hybridoma technique and packed into erythrocyte ghosts. Then, monoclonal anti-cyclic AMP containing ghosts were fused with splenic B lymphocytes by polyethylene glycol-mediated fusion at various intervals after LPS stimulation. This method made it possible for us to quantitatively microinject antibodies into B lymphocytes. Microinjection of anti-cyclic AMP antibody molecules into lymphocytes at a very early stage of LPS stimulation resulted in a marked enhancement of DNA synthetic responses as well as increased numbers of plaque-forming cells. Intracellular cyclic AMP levels were found to be markedly decreased after microinjection of monoclonal anti-cyclic AMP, suggesting that lowering the intracellular cyclic-AMP level in the B lymphocytes at an early stage of stimulation might have induced the enhanced proliferative as well as differentiative responses to LPS. Similar enhancing effects on cell proliferation were obtained when antibodies were injected 18 hr after stimulation. Microinjection of anti-cyclic AMP at 12 hr after culture, however, inhibited the DNA synthetic responses, and induction of plaque-forming cells was suppressed when anti-cyclic AMP was injected 6 hr after LPS stimulation. The present data suggest the biphasic regulatory roles of cyclic AMP at the early stage of B lymphocyte activation. This approach may be useful in identifying regulatory molecules in B lymphocyte induced by mitogenic or antigenic stimulation.