Lomas D A, Stone S R, Llewellyn-Jones C, Keogan M T, Wang Z M, Rubin H, Carrell R W, Stockley R A
Department of Medicine, University of Cambridge, United Kingdom.
J Biol Chem. 1995 Oct 6;270(40):23437-43. doi: 10.1074/jbc.270.40.23437.
Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.
中性粒细胞趋化作用在炎症反应中起重要作用，过度或持续的中性粒细胞趋化作用可能会加剧组织损伤。抑制剂的作用表明，一种或多种丝氨酸蛋白酶参与了人类中性粒细胞对趋化因子的迁移和形态变化。使用单特异性抗体、氯甲基酮抑制剂以及α1-抗胰蛋白酶和α1-抗糜蛋白酶的反应位点突变体来探究参与趋化作用的蛋白酶的特异性。针对组织蛋白酶G的特异性抗体抑制了趋化作用。此外，组织蛋白酶G和α-糜蛋白酶的快速抑制剂在多次盲孔试验中抑制了中性粒细胞对趋化因子N-甲酰-L-蛋氨酰-L-亮氨酰-L-苯丙氨酸（fMLP）和酵母聚糖激活血清的趋化作用，在琼脂糖下的迁移试验中抑制了对fMLP的趋化作用。使趋化作用降低50%的抗糜蛋白酶突变体浓度可使游离组织蛋白酶G失活，半衰期为1.5 - 3秒，而产生类似趋化作用抑制所需的氯甲基酮浓度可使组织蛋白酶G失活，半衰期为345秒。这些数据表明这两类抑制剂的作用方式不同。实际上，组织蛋白酶G的氯甲基酮抑制剂（Z-甘氨酰-亮氨酰-苯丙氨酸氯甲基酮）以及程度较轻的糜蛋白酶抑制剂（苄氧羰基-甘氨酰-甘氨酰-苯丙氨酸氯甲基酮）通过阻止暴露于fMLP的纯化中性粒细胞的形态变化来介导其作用。抗糜蛋白酶即使在能够使中性粒细胞趋化作用降低50%的浓度下，也不会影响对fMLP的形态变化。这些结果支持细胞表面蛋白酶参与细胞迁移的调控，并表明抗糜蛋白酶和氯甲基酮具有不同的作用方式。这为合理设计针对中性粒细胞膜酶的抗炎药物开辟了可能性。
