Huang R, Cioffi J, Kimberly R, Edberg J, Mayer L
Division of Clinical Immunology, Mount Sinai Medical Center, New York, New York 10029, USA.
Cell Immunol. 1995 Sep;164(2):227-33. doi: 10.1006/cimm.1995.1165.
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 Gi-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 Ca2+ flux in indo-1 AM-loaded PB B cells. Stimulation with 446-BCDF resulted in an immediate rise in intracellular Ca2+ comparable to that seen with the anti-IgM mAb HB57. Ca2+ appeared to be mobilized from internal stores as pretreatment with BAPTA but not EGTA inhibited the response. Ca2+ 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-gamma, nor IL4 could mobilize Ca2+. Pertussis toxin, a Gi and G0 protein inhibitor, was able to inhibit 446-BCDF-induced Ca2+ flux as well as Ig secretion. To determine whether the Ca2+ flux was generated in the course of inositol phosphate turnover, we measured IP3 turnover and the translocation of PKC from cytosol to membrane. 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 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 nM staurosporin and 0.0625-1 microM calphostin C). These data suggest that induction of B cell differentiation is a Ca(2+)-dependent and PTX-sensitive event.
我们最近鉴定出一种新型人类B细胞分化因子，即446-BCDF，它源自抗CD3刺激的外周血（PB）T细胞。这种新型细胞因子可能通过一种对百日咳毒素敏感的Gi偶联受体发挥作用，可使经SAC（0.001%，v/v）激活的PB B细胞的免疫球蛋白（Ig）分泌增加5至100倍。B细胞与446-BCDF共培养会导致细胞内cAMP减少，这是驱动B细胞终末分化所必需的，但并不充分。似乎还需要第二个信号。因此，我们检测了用indo-1 AM负载的PB B细胞中的Ca2+通量。用446-BCDF刺激导致细胞内Ca2+立即升高，与抗IgM单克隆抗体HB57刺激时所见相当。Ca2+似乎是从内部储存库中动员出来的，因为用BAPTA预处理可抑制该反应，而用EGTA预处理则不能。Ca2+动员对于诱导分化至关重要，因为用BAPTA预处理PB B细胞可完全抑制Ig分泌，而不影响细胞活力。相比之下，SAC、rIL6、IL2、IFN-γ或IL4均不能动员Ca2+。百日咳毒素是一种Gi和G0蛋白抑制剂，能够抑制446-BCDF诱导的Ca2+通量以及Ig分泌。为了确定Ca2+通量是否在肌醇磷酸周转过程中产生，我们检测了IP3周转以及PKC从胞质溶胶向细胞膜的转位。观察到IP3增加，与用单克隆抗人IgM抗体刺激时所见相当，并且被446-BCDF特异性单克隆抗体929特异性抑制。有趣的是，在SAC或BCDF刺激的B细胞中均未检测到膜PKC，尽管PMA（50 ng/ml）可直接激活PKC。为了从功能上证实这些发现，在存在两种已知的PKC抑制剂星形孢菌素和钙磷蛋白的情况下，用SAC和446-BCDF刺激B细胞。在任何测试浓度（0.39 - 100 nM星形孢菌素和0.0625 - 1 μM钙磷蛋白C）下均未检测到对Ig分泌的抑制作用。这些数据表明，B细胞分化的诱导是一个Ca(2+)依赖性且对PTX敏感的事件。
