Alkan S S, Rutschmann S, Grogg D, Erb P
Department of Allergy/Immunology, Ciba-Geigy Ltd., Basel, Switzerland.
Cell Immunol. 1993 Aug;150(1):137-48. doi: 10.1006/cimm.1993.1185.
The effects of CGP 41251, a specific inhibitor of protein kinase C (PKC), its inactive derivative CGP 42700, and of staurosporine have been analyzed in vitro on T lymphocyte functions. The proliferation of fresh human peripheral blood lymphocytes stimulated with antigen (PPD) or anti-CD3 mAb was strongly inhibited by both staurosporine (IC50 < 0.01 microM) and CGP 41251 (IC50 = 0.092 microM) but not by the PKC inactive compound CGP 42700 (IC50 > 10 microM). Antigen-specific activation and proliferation of mouse lymph node T cells was inhibited by staurosporine and CGP 41251 with IC50 values of 0.008 and 0.05 microM, respectively. The inactive derivative caused 50% inhibition in this mouse T cell assay only at concentrations of 25 microM. In order to evaluate possible differential effects of PKC inhibitors on CD4+ T cell subsets, murine T helper cell type 1 (Th1) and type 2 (Th2) clones were used. The KLH-specific clone 9/6 secretes IL-2 and IFN-gamma (Th1), whereas clone 9A/B does not secrete these lymphokines but secretes IL-4 and IL-5 (Th2). It was found that CGP 41251 inhibited antigen-induced proliferation of both Th1 and Th2 equally well with an IC50 of 0.02 microM. Furthermore, CGP 41251 inhibited the IL-2 or IL-2 and IL-4-mediated growth of Th1 and Th2 cells (IC50, 0.08 and 0.02 microM, respectively). Moreover, CGP 41251, but not CGP 42700, inhibited antigen-specific killing of target cells by Th1 clones (IC50 = 0.2 microM), a phenomenon which does not require cell proliferation. When Th1 cells were preincubated with the compound, washed, and rested for 24 hr, they killed the target cells, whereas killing by similarly preincubated, washed, but not rested Th1 cells was inhibited. Thus, the inhibitory effect of CGP 41251 is reversible and excludes the possibility of inhibition due to toxicity at the IC50 dose given. The comparison of CGP 41251 and staurosporine showed that although CGP 41251 has lower activity, it is more specific and much less toxic than staurosporine. Thus, CGP 41251 is more suitable for PKC studies.
蛋白激酶C（PKC）的特异性抑制剂CGP 41251、其无活性衍生物CGP 42700以及星形孢菌素对T淋巴细胞功能的影响已在体外进行了分析。用抗原（PPD）或抗CD3单克隆抗体刺激的新鲜人外周血淋巴细胞的增殖受到星形孢菌素（IC50 < 0.01 microM）和CGP 41251（IC50 = 0.092 microM）的强烈抑制，但不受PKC无活性化合物CGP 42700（IC50 > 10 microM）的抑制。星形孢菌素和CGP 41251抑制小鼠淋巴结T细胞的抗原特异性激活和增殖，IC50值分别为0.008和0.05 microM。在该小鼠T细胞试验中，无活性衍生物仅在浓度为25 microM时引起50%的抑制。为了评估PKC抑制剂对CD4 + T细胞亚群可能的差异作用，使用了小鼠1型辅助性T细胞（Th1）和2型辅助性T细胞（Th2）克隆。KLH特异性克隆9/6分泌IL - 2和IFN - γ（Th1），而克隆9A/B不分泌这些细胞因子，但分泌IL - 4和IL - 5（Th2）。发现CGP 41251以0.02 microM的IC50同等程度地抑制Th1和Th2的抗原诱导增殖。此外，CGP 41251抑制Th1和Th2细胞由IL - 2或IL - 2和IL - 4介导的生长（IC50分别为0.08和0.02 microM）。而且，CGP 41251而非CGP 42700抑制Th1克隆对靶细胞的抗原特异性杀伤（IC50 = 0.2 microM），这一现象不需要细胞增殖。当Th1细胞与该化合物预孵育、洗涤并静置24小时后，它们能够杀伤靶细胞，而同样预孵育、洗涤但未静置的Th1细胞的杀伤作用受到抑制。因此，CGP 41251的抑制作用是可逆的，排除了在给定IC50剂量下因毒性导致抑制的可能性。CGP 41251与星形孢菌素的比较表明，尽管CGP 41251活性较低，但它比星形孢菌素更具特异性且毒性小得多。因此，CGP 41251更适合用于PKC研究。
