B-CELL DIFFERENTIATION FACTOR-INDUCED HUMAN B-CELL MATURATION - STIMULATION OF INTRACELLULAR CALCIUM-RELEASE

We have recently identified a novel human B cell differentiation facto r, 446-BCDF, derived from anti-CD3-stimulated peripheral blood (PB) T cells, This novel cytokine, which may act through a pertussis toxin-se nsitive G(i)-linked receptor, induces a 5- to 100-fold increase in imm unoglobulin (Ig) secretion by SAC (0.001%, v/v)-activated PB B cells, Coculture of B cells with 446-BCDF induces a decrease in intracellular cAMP which is necessary but not sufficient to drive terminal B cell d ifferentiation. A second signal appears to be required, We therefore m easured Ca2+ flux in indo-1 AM-loaded PB B cells, Stimulation with 446 -BCDF resulted in an immediate rise in intracellular Ca2+ comparable t o that seen with the anti-IgM mAb HB57, Ca2+ appeared to be mobilized from internal stores as pretreatment with BAPTA but not EG;TA inhibite d the response, Ca2+ mobilization was critical for the induction of di fferentiation as BAPTA pretreatment of PB B cells completely inhibited Ig secretion without affecting cell viability. In contrast, neither S AG, rIL6, IL2, IFN-gamma, nor IL4 could mobilize Ca2+, Pertussis toxin , a G(i) and G(0) protein inhibitor, was able to inhibit 446-BCDF-indu ced Ca2+ flux as well as Ig secretion. To determine whether the Ca2+ f lux was generated in the course of inositol phosphate turnover, we mea sured IP3 turnover and the translocation of PKC from cytosol to membra ne. An increase in IP3 comparable to that seen with a monoclonal anti- human IgM antibody was noted and was specifically inhibited by the 446 -BCDF-specific mAb 929, Interestingly, no membrane PKC was demonstrabl e in either SAC- or BCDF-stimulated B cells, although PMA (50 ng/ml) c ould directly activate PKC. To confirm these findings functionally, B cells were stimulated with SAC and 446-BCDF in the presence of two kno wn PKC inhibitors, staurosporin and calphostin. No inhibition of Ig se cretion was detected at any concentration tested (0.39-100 nM staurosp orin and 0.0625-1 mu M calphostin C). These data suggest that inductio n of B cell differentiation is a Ca2+-dependent and PTX-sensitive even t, (C) 1995 Academic Press, Inc.