Bailey N C, Kelly C J
University of California, San Diego, La Jolla, USA.
Eur J Immunol. 1997 Sep;27(9):2302-9. doi: 10.1002/eji.1830270926.
Cytoplasmic granules of cytotoxic T lymphocytes contain several proteins that may be involved in cell-mediated cytotoxicity. We have previously described nephritogenic T cell clones that are cytotoxic to cultured renal proximal tubular epithelial cells (MCT). One of these clones, M52.34.1, expresses perforin, a cytotoxic mediator. We investigated the expression of other granule-associated proteases of M52.34.1. Granzymes A and B have been extensively studied in T cell-mediated cytotoxicity, and associated with tissue destruction in models of transplantation. However, the activity of other granzymes has not been as extensively investigated. We focused our studies on granzyme C. Northern blots showed very high levels of granzymes B and C mRNA expression in M52.34.1 cells 3 days following T cell activation. There was no expression of granzyme A mRNA. An antisense oligonucleotide made from the 5'-upstream region of the murine granzyme C exon 1 inhibited granzyme C mRNA expression in M52.34.1 when added at a concentration of 50 microM to the culture medium for 2 days. There was no inhibition of granzyme C mRNA expression with the sense oligonucleotide. The granzyme C antisense oligonucleotide inhibited M52.34.1 cytotoxicity to MCT at effector:target ratios of 20:1 and 40:1. M52.34.1 cells mediate inflammatory interstitial nephritis following adoptive transfer. If T cells were resuspended in 200 microM of the antisense oligonucleotide prior to subcapsular transfer, the recipient kidneys showed markedly diminished tubular cell destruction, suggesting that granzyme C can also be an important mediator of cytotoxicity in vivo.
细胞毒性T淋巴细胞的细胞质颗粒含有几种可能参与细胞介导的细胞毒性的蛋白质。我们之前描述过对培养的肾近端小管上皮细胞（MCT）具有细胞毒性的致肾炎性T细胞克隆。其中一个克隆M52.34.1表达穿孔素，一种细胞毒性介质。我们研究了M52.34.1中其他颗粒相关蛋白酶的表达。颗粒酶A和B在T细胞介导的细胞毒性中已得到广泛研究，并与移植模型中的组织破坏有关。然而，其他颗粒酶的活性尚未得到如此广泛的研究。我们将研究重点放在颗粒酶C上。Northern印迹显示，T细胞活化3天后，M52.34.1细胞中颗粒酶B和C的mRNA表达水平非常高。颗粒酶A的mRNA没有表达。由小鼠颗粒酶C外显子1的5'上游区域制成的反义寡核苷酸，当以50 microM的浓度添加到培养基中2天时，可抑制M52.34.1中颗粒酶C的mRNA表达。正义寡核苷酸对颗粒酶C的mRNA表达没有抑制作用。颗粒酶C反义寡核苷酸在效应细胞：靶细胞比例为20:1和40:1时，抑制M52.34.1对MCT的细胞毒性。M52.34.1细胞在过继转移后介导炎症性间质性肾炎。如果在被膜下转移前将T细胞重悬于200 microM的反义寡核苷酸中，受体肾脏的肾小管细胞破坏明显减少，这表明颗粒酶C在体内也可能是细胞毒性的重要介质。
