Le-Barillec K, Si-Tahar M, Balloy V, Chignard M
Unité de Pharmacologie Cellulaire, Unité Associée IP/Institut National de la Santé et de la Recherche Médicale 485, Institut Pasteur, 75015 Paris, France.
J Clin Invest. 1999 Apr;103(7):1039-46. doi: 10.1172/JCI5779.
Human leukocyte elastase (HLE), a polymorphonuclear neutrophil (PMN) serine proteinase, is proteolytically active on some membrane receptors at the surface of immune cells. The present study focused on the effect of HLE on the expression of CD14, the main bacterial lipopolysaccharide (LPS) receptor at the surface of monocytes. HLE exhibited a time- and concentration-dependent downregulatory effect on CD14 surface expression. A 30-minute incubation of 3 microM HLE was required to display 95% disappearance of the receptor. This downregulation resulted from a direct proteolytic process, not from a shedding consecutive to monocyte activation as observed upon challenge with phorbol myristate acetate (PMA). To confirm that CD14 is a substrate for HLE, this enzyme was incubated with recombinant human CD14 (Mr approximately 57,000), and proteolysis was further analyzed by immunoblot analysis. Cleavage of the CD14 molecule was directly evidenced by the generation of short-lived fragments (Mr approximately 47,000 and 30,000). As a consequence of the CD14 proteolysis, a decrease in the responsiveness of monocytes to LPS was observed, as assessed by measuring tumor necrosis factor-alpha (TNF-alpha) formation. This inhibition was only observed with 1 ng/ml of LPS, i.e., when only the CD14-dependent pathway was involved. At a higher LPS concentration, such as 10 microgram/ml, when CD14-independent pathways were operative, this inhibition was overcome. The direct proteolysis by HLE of the membrane CD14 expressed on monocytes illustrates a potential anti-inflammatory effect of HLE through inhibition of LPS-mediated cell activation.
人白细胞弹性蛋白酶（HLE）是一种多形核中性粒细胞（PMN）丝氨酸蛋白酶，对免疫细胞表面的某些膜受体具有蛋白水解活性。本研究聚焦于HLE对单核细胞表面主要细菌脂多糖（LPS）受体CD14表达的影响。HLE对CD14表面表达呈现出时间和浓度依赖性的下调作用。需要用3微摩尔HLE孵育30分钟才能使该受体消失95%。这种下调是由直接的蛋白水解过程导致的，而非像在用佛波酯（PMA）刺激时观察到的那样，是单核细胞激活后的脱落所致。为证实CD14是HLE的底物，将该酶与重组人CD14（分子量约57,000）一起孵育，然后通过免疫印迹分析进一步分析蛋白水解情况。CD14分子的切割通过产生短寿命片段（分子量约47,000和30,000）直接得到证实。作为CD14蛋白水解的结果，通过测量肿瘤坏死因子-α（TNF-α）的形成评估发现，单核细胞对LPS的反应性降低。仅在1纳克/毫升的LPS时观察到这种抑制作用，即仅涉及CD14依赖性途径时。在更高的LPS浓度，如10微克/毫升，当CD14非依赖性途径起作用时，这种抑制作用被克服。HLE对单核细胞表面表达的膜CD14进行直接蛋白水解，说明了HLE通过抑制LPS介导的细胞激活可能具有的抗炎作用。
