Granzye B activity in target cells detects attack by cytotoxic lymphocytes
Granzye B activity in target cells detects attack by cytotoxic lymphocytes
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DOI：
10.4049/jimmunol.179.6.3812
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发表时间：
2007-09-15
影响因子：
4.4
通讯作者：
Henkart, Pierre A.
中科院分区：
文献类型：
--
作者：
Packard, Beverly Z.;Telford, William G.;Henkart, Pierre A.
淋巴细胞介导的细胞毒性通过颗粒胞吐作用，通过穿孔素介导的颗粒酶从 CTL 和 NK 细胞转移到靶细胞中，在靶细胞中触发 caspase 激活和其他死亡途径。颗粒酶 B (GzB) 是该途径中的主要细胞毒性效应物，多个小组已使用免疫检测研究了其在靶细胞中的命运。在这项研究中，我们使用了新开发的细胞可渗透的荧光 GzB 底物来测量与细胞毒性效应物接触后三个不同活靶标中的蛋白酶活性。尽管在 CTL 或 NK92 效应细胞中无法测量到 GzB 活性，但在效应子-靶标接合后，该活性很快在整个靶细胞质中变得可检测到。我们将 GzB 底物与对 caspase 3 具有选择性的第二种荧光底物结合起来，以便可以进行细胞毒性的流式细胞术和荧光共聚焦显微镜研究。对于这两个效应子，Caspase 3 活性出现在所有三个靶标内的 GzB 活性之后。靶细胞中 Bcl-2 的过度表达对裂解、NK 或 CTL 传递的 GzB 活性或靶标 caspase 3 的激活影响极小。检测靶标 GzB 活性，随后检测 caspase 3 激活，可提供细胞毒性淋巴细胞造成的潜在致命损伤的独特读数。
Lymphocyte-mediated cytotoxicity via granule exocytosis operates by the perforin-mediated transfer of granzymes from CTLs and NK cells into target cells where caspase activation and other death pathways are triggered. Granzyme B (GzB) is a major cytotoxic effector in this pathway, and its fate in target cells has been studied by several groups using immunodetection. In this study, we have used a newly developed cell-permeable fluorogenic GzB substrate to measure this protease activity in three different living targets following contact with cytotoxic effectors. Although no GzB activity is measurable in CTL or NK92 effector cells, this activity rapidly becomes detectable throughout the target cytoplasm after effector-target engagement. We have combined the GzB substrate with a second fluorogenic substrate selective for caspase 3 to allow both flow cytometry and fluorescence confocal microscopy studies of cytotoxicity. With both effectors, caspase 3 activity appears subsequent to that of GzB inside all three targets. Overexpression of Bcl-2 in target cells has minimal effects on lysis, NK- or CTL-delivered GzB activity, or activation of target caspase 3. Detection of target GzB activity followed by caspase 3 activation provides a unique readout of a potentially lethal injury delivered by cytotoxic lymphocytes.