EFFECT OF BACTERIAL TOXINS ON HUMAN B-CELL ACTIVATION .2. MITOGENIC ACTIVITY OF THE B-SUBUNIT OF CHOLERA-TOXIN

The B subunit of cholera toxin (CT) but not the entire CT was found to induce the proliferation of resting human B lymphocytes. A significant mitogenic effect was observed for B subunit concentrations > 1-mu-g/ml and reached a maximum of stimulation at 10-mu-g/ml. As already described for B lymphocytes preactivated with Staphylococcus aureus Cowan Strain I (SAC), B lymphocytes preactivated with the B subunit of CT, but not with the entire CT, were able to proliferate in response to exogenous interleukin 2 (IL 2) and to the low-molecular weight B cell growth factor (BCGF). To determine the transmembrane signaling system used by the B subunit of CT to mediate its biological effects, we compared the transmembrane signals used by the entire CT, its B subunit and SAC. In comparison to the entire CT, which directly activates adenylate cyclase and increases intracellular cAMP levels, neither the B subunit nor SAC modified the cAMP content. In contrast, although SAC induced inositol phosphate generation neither CT nor the separate subunits were able to induce such a production. Moreover, changes in the fluorescence of indo-1-loaded B lymphocytes revealed that mitogenic doses of either the B subunit or SAC induced a rapid and sustained increase in cytoplasmic free Ca2+ concentration ([Ca2+]i). The effect of the B subunit appeared to be largely dependent on the presence of extracellular Ca2+, because in Ca2+-free medium no [Ca2+]i uptake was observed. In contrast, the SAC-induced [Ca2+]i uptake is substantially, but not totally, inhibited in Ca2+-free medium, suggesting that part of the rise in [Ca2+]i was due to the release from internal stores. Moreover, fluorimetric measurements on loaded cells with 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein revealed that SAC induced a rapid cytoplasmic alkalinization via activation of Na+/H+ exchange, whereas the entire CT and its B subunit had no effect on intracellular pH. Taken together, these data suggest that, in comparison to SAC, the mitogenic effect of the B subunit of CT was mediated through different intracellular biochemical pathways.