Proteolysis of monocyte CD14 by human leukocyte elastase inhibits lipopolysaccharide-mediated cell activation

Human leukocyte elastase (HLE), a polymorphonuclear neutrophil (PMN) serine proteinase, is proteolytically active on some membrane receptors at the su rface of immune cells. The present study focused on the effect of HLE on th e expression of CD14, the main bacterial lipopolysaccharide (LPS) receptor at the surface of monocytes. HLE exhibited a time- and concentration-depend ent downregulatory effect on CD14 surface expression. A 30-minute incubatio n of 3 mu M 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 H LE, this enzyme was incubated with recombinant human CD14 (M-r similar to 5 7,000), and proteolysis was further analyzed by immunoblot analysis. Cleava ge of the CD14 molecule was directly evidenced by the generation of short-l ived fragments (M-r similar to 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., whe n only the CD14-dependent pathway was involved. At a higher LPS concentrati on, such as 10 mu g/ml, when CD14-independent pathways were operative, this inhibition was overcome. The direct proteolysis by HLE of the membrane CD1 4 expressed on monocytes illustrates a potential anti-inflammatory effect o f HLE through inhibition of LPS-mediated cell activation.