Non-cytotoxic functions of lymphoycte granule exocytosis

淋巴细胞颗粒胞吐作用的非细胞毒性功能

• 批准号: 6758411
• 负责人: Pierre A Henkart
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6758411
• 关键词: beta N acetylhexosaminidase; cell population study; chemokine; cytotoxic T lymphocyte; exocytosis; flow cytometry; granule; helper T lymphocyte; interferons; lymphocyte; secretion

Effector T lymphocytes function by secretion of mediators which influence other cells. Secretion occurs via two pathways the "constitutive" pathway, in which newly synthesized proteins pass through the Golgi and are immediately released by exocytosis of small vesicles; and the "regulated" pathway in which mediators are stored in larger granules until TcR engagement signals their exocytosis. In lymphocytes the regulated pathway has been exclusively associated with cytotoxicity and the secretion of perforin and granzymes (granule exocytosis pathway). We investigated the T cell pathway used for chemokine secretion, particularly the major T cell product RANTES. Purified populations of both CD4+ and CD8+ human T cell blasts were found to secrete RANTES within two hours after TcR stimulation by plate-bound anti-CD3. The regulated pathway was implicated by the lack of inhibition by cycloheximide (which blocks protein synthesis) and Brefeldin A (which blocks Golgi export). At times greater than 3 hours after TcR triggering these drugs block secretion, indicating a switch to the constitutive pathway. Flow cytometry of permeabilized cells shows that RANTES is present in both CD8+ and CD4+ T cell blasts and that cytoplasmic RANTES is lost rapidly after TcR crosslinking (~ 6x faster than granule markers). By confocal microscopy intracellular RANTES appears as vesicles that do not colocalize with proteins such as perforin and granzymes in the lysosomal granules. This lack of colocalization was confirmed by the higher resolution technique of deconvolution microscopy, both visually and by statistical analysis of digital pixel intensities. Immunogold staining of ultrathin EM sections confirmed that RANTES is present in vesicles that are morphologically distinct from the granules. Thus we have described a novel regulated secretory pathway in T lymphocytes, capable of rapid delivery of preformed chemokines after antigen recognition.

效应T淋巴细胞通过分泌影响其他细胞的介质发挥功能。分泌通过两条途径发生：“组成性”途径，其中新合成的蛋白质穿过高尔基体并通过小囊泡的胞吐作用立即释放;和“调节性”途径，其中介质储存在较大颗粒中，直到TcR接合发出胞吐作用信号。在淋巴细胞中，调节途径仅与细胞毒性以及穿孔素和颗粒酶的分泌（颗粒胞吐途径）相关。我们研究了用于趋化因子分泌的T细胞途径，特别是主要的T细胞产物RANTES。发现纯化的CD 4+和CD 8+人T细胞母细胞群体在平板结合的抗CD 3的TcR刺激后2小时内分泌RANTES。受调节的途径与放线菌酮（其阻断蛋白质合成）和布雷菲德菌素A（其阻断高尔基体输出）缺乏抑制有关。在TcR触发后超过3小时的时间，这些药物阻断分泌，表明向组成性途径的转换。透化细胞的流式细胞术显示RANTES存在于CD 8+和CD 4 + T细胞母细胞中，并且在TcR交联后细胞质RANTES迅速丢失（比颗粒标记物快约6倍）。通过共聚焦显微镜，细胞内RANTES表现为不与溶酶体颗粒中的蛋白质如穿孔素和颗粒酶共定位的囊泡。这种缺乏共定位证实了更高的分辨率技术的去卷积显微镜，无论是视觉和数字像素强度的统计分析。免疫胶体金染色法证实RANTES存在于囊泡中，囊泡在形态上与颗粒不同。因此，我们已经描述了一种新的调节分泌途径，在T淋巴细胞，能够快速交付预先形成的趋化因子抗原识别后。