Bonvini E, DeBell K E, Kolber M A, Hoffman T, Hodes R J, Taplits M S
Laboratory of Cell Biology, US Food and Drug Administration, Bethesda, MD 20892.
J Immunol. 1989 Jul 15;143(2):587-95.
Two murine, keyhole limpet hemocyanin-specific, Th cell clones were studied for their ability to respond to antibody-mediated stimulation of the TCR complex or to Ag-pulsed accessory cells by hydrolyzing inositol phospholipids. Both clones were positive for the determinant expressed on the epsilon chain of CD3 that is recognized by the mAb, 145-2C11 (2C11 mAb); one clone also expressed the V beta 8 epitope of the alpha/beta chains of the TCR recognized by the F23.1 mAb. Treatment of these cells with 2C11 or F23.1 mAb adsorbed onto polystyrene beads induced a time-dependent accumulation of inositol phosphates (IP). Keyhole limpet hemocyanin-pulsed accessory cells which expressed the appropriate MHC phenotype also induced IP accumulation, whereas no response was induced by medium-treated or MHC congenic accessory cells. The hydrolysis of inositol phospholipids induced by TCR perturbation depended upon the presence of exogenous Ca2+; Mg2+ did not substitute for Ca2+. Treatment of cells with ionomycin at concentrations up to 30 microM was unable to induce hydrolysis of inositol phospholipids, indicating that entrance of Ca2+ was itself insufficient to generate IP. Stimulated IP generation was rapidly blocked upon addition of EGTA to the incubation medium. Reducing the level of exogenous Ca2+ decreased the production of inositol mono-, bis-, and trisphosphate isomers similarly, suggesting that extracellular Ca2+ was required for the initiation of the hydrolysis rather than affecting phospholipase C affinity for its substrates. We concluded that activation of inositol phospholipid hydrolysis by perturbation of the TCR complex in the Th cell clones under investigation displays a Ca2+-dependent component which is likely to be proximal to IP generation.
研究了两个针对钥孔血蓝蛋白的鼠源Th细胞克隆，观察它们通过水解肌醇磷脂对抗体介导的TCR复合物刺激或对抗原脉冲刺激的辅助细胞作出反应的能力。两个克隆对CD3ε链上由单克隆抗体145 - 2C11（2C11 mAb）识别的决定簇呈阳性；一个克隆还表达了由F23.1 mAb识别的TCRα/β链的Vβ8表位。用吸附在聚苯乙烯珠上的2C11或F23.1 mAb处理这些细胞会诱导肌醇磷酸（IP）随时间积累。表达适当MHC表型的钥孔血蓝蛋白脉冲刺激的辅助细胞也会诱导IP积累，而经培养基处理的或MHC同基因辅助细胞则不会诱导反应。TCR扰动诱导的肌醇磷脂水解依赖于外源Ca2+的存在；Mg2+不能替代Ca2+。用浓度高达30μM的离子霉素处理细胞无法诱导肌醇磷脂水解，这表明Ca2+的进入本身不足以产生IP。向孵育培养基中添加EGTA后，刺激的IP生成迅速被阻断。降低外源Ca2+水平同样会降低肌醇单磷酸、双磷酸和三磷酸异构体的产生，这表明细胞外Ca2+是水解起始所必需的，而不是影响磷脂酶C对其底物的亲和力。我们得出结论，在所研究的Th细胞克隆中，TCR复合物扰动激活肌醇磷脂水解表现出一个依赖Ca2+的成分，该成分可能在IP生成的近端。
