Goepel Friederike, Weinmann Pamela, Schymeinsky Jürgen, Walzog Barbara
Department of Physiology, Freie Universität, Berlin, Germany.
J Leukoc Biol. 2004 May;75(5):836-43. doi: 10.1189/jlb.0703317. Epub 2004 Feb 3.
In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis of human polymorphonuclear neutrophils (PMN). Whereas TNF-alpha-mediated apoptosis was almost absent in the presence of the caspase-8 inhibitor Z-Ac-Ala-Glu-Val-Asp-7-fluoromethyl ketone (Z-AEVD-FMK), the inhibitor had no effect on spontaneous apoptosis, suggesting that spontaneous apoptosis was independent of caspase-8. Subsequently, we identified different isoforms of caspase-10 in human PMN and found high expression of caspase-10/b and/or -10/d and low expression of caspase-10/a and -10/c at the mRNA level. At the protein level, freshly isolated PMN showed high expression of caspase-10/b and -10/d as well as moderate expression of caspase-10/a and -10/c. Upon spontaneous apoptosis, caspase-10/b was down-regulated, which was accompanied by the appearance of a specific 47-kDa caspase-10/b cleavage product and an increased caspase-10 activity. In contrast, no down-regulation of caspase-10/a, -10/c, or -10/d was observed, suggesting that spontaneous apoptosis was associated with a differential activation of caspase-10/b. This was confirmed by the finding that spontaneous apoptosis was inhibited in the presence of Z-Ile-Glu-Thr-Asp (Z-IETD)-FMK, which blocks caspase-10. However, no down-regulation of caspase-10 isoforms was observed in the presence of TNF-alpha, suggesting that caspase-10 was not involved in TNF-alpha-induced apoptosis. Taken together, our study demonstrates that spontaneous and TNF-alpha-mediated apoptosis of PMN have different molecular requirements. Whereas TNF-alpha-mediated apoptosis depends on the activation of caspase-8, spontaneous apoptosis requires the activation of caspase-10/b. This finding may reveal that PMN apoptosis in different (patho-) physiological settings results from distinct molecular mechanisms.
在本研究中，我们探究了人多形核中性粒细胞（PMN）自发凋亡以及肿瘤坏死因子α（TNF-α）介导的凋亡的分子机制。在存在半胱天冬酶-8抑制剂Z-乙酰丙氨酰-谷氨酰-缬氨酰-天冬氨酸-7-氟甲基酮（Z-AEVD-FMK）的情况下，几乎不存在TNF-α介导的凋亡，而该抑制剂对自发凋亡没有影响，这表明自发凋亡不依赖于半胱天冬酶-8。随后，我们在人PMN中鉴定出半胱天冬酶-10的不同同工型，并发现其在mRNA水平上半胱天冬酶-10/b和/或-10/d高表达，而半胱天冬酶-10/a和-10/c低表达。在蛋白质水平上，新鲜分离的PMN显示半胱天冬酶-10/b和-10/d高表达，以及半胱天冬酶-10/a和-10/c中度表达。在自发凋亡过程中，半胱天冬酶-10/b被下调，同时出现一种特定的47 kDa半胱天冬酶-10/b裂解产物，并且半胱天冬酶-10活性增加。相比之下，未观察到半胱天冬酶-10/a、-10/c或-10/d的下调，这表明自发凋亡与半胱天冬酶-10/b的差异性激活有关。这一发现通过以下结果得到证实：在存在阻断半胱天冬酶-10的Z-异亮氨酰-谷氨酰-苏氨酰-天冬氨酸（Z-IETD）-FMK的情况下，自发凋亡受到抑制。然而，在存在TNF-α的情况下，未观察到半胱天冬酶-10同工型的下调，这表明半胱天冬酶-10不参与TNF-α诱导的凋亡。综上所述，我们的研究表明PMN的自发凋亡和TNF-α介导的凋亡具有不同的分子需求。TNF-α介导的凋亡依赖于半胱天冬酶-8的激活，而自发凋亡需要半胱天冬酶-10/b的激活。这一发现可能揭示了在不同（病理-）生理环境下PMN凋亡是由不同的分子机制导致的。
