B Cell Differentiation Factor-Induced Human B Cell Maturation: Stimulation of Intracellular Calcium Release

Abstract

We have recently identified a novel human B cell differentiation factor, 446-BCDF, derived from anti-CD3-stimulated peripheral blood (PB) T cells. This novel cytokine, which may act through a pertussis toxin-sensitive G i-linked receptor, induces a 5- to 100-fold increase in immunoglobulin (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 differentiation. A second signal appears to be required. We therefore measured Ca 2+ flux in indo-1 AM-loaded PB B cells. Stimulation with 446-BCDF resulted in an immediate rise in intracellular Ca 2+ comparable to that seen with the anti-IgM mAb HB57. Ca 2+ appeared to be mobilized from internal stores as pretreatment with BAPTA but not EGTA inhibited the response. Ca 2+ mobilization was critical for the induction of differentiation as BAPTA pretreatment of PB B cells completely inhibited Ig secretion without affecting cell viability. In contrast, neither SAC, rIL6, IL2, IFN-γ, nor IL4 could mobilize Ca 2+. Pertussis toxin, a G i and G o protein inhibitor, was able to inhibit 446-BCDF-induced Ca 2+ flux as well as Ig secretion. To determine whether the Ca 2+ flux was generated in the course of inositol phosphate turnover, we measured IP 3 turnover and the translocation of PKC from cytosol to membrane. An increase in IP 3 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 demonstrable in either SAC- or BCDF-stimulated B cells, although PMA (50 ng/ml) could directly activate PKC. To confirm these findings functionally, B cells were stimulated with SAC and 446-BCDF in the presence of two known PKC inhibitors, staurosporin and calphostin. No inhibition of Ig secretion was detected at any concentration tested (0.39-100 n M staurosporin and 0.0625-1 μ M calphostin C). These data suggest that induction of B cell differentiation is a Ca 2+-dependent and PTX-sensitive event.