Metalloproteinases are involved in lipopolysaccharide- and tumor necrosis factor-alpha-mediated regulation of CXCR1 and CXCR2 chemokine receptor expression

The neutrophil-specific G-protein-coupled chemokine receptors, CXCR1 and CX CR2, bind with high affinity to the potent chemoattractant interleukin-8 (I L-8). The mechanisms of IL-8 receptor regulation are not well defined, alth ough previous studies have suggested a process of ligand-promoted internali zation as a putative regulatory pathway. Herein, we provide evidence for tw o distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy dat a showed a redistribution of CXCR1 expression from the cell surface of neut rophils to internal compartments after stimulation with IL-8, whereas stimu lation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alp ha (TNF-alpha) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investig ate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-alpha-induced downmodulation of IL-8 receptors, we tested a panel of p roteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF- alpha was most dramatically inhibited by metalloproteinase inhibitors; 1,10 -phenanthroline and EDTA significantly attenuated LPS- and TNF-alpha-induce d loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibi tors also blocked the release of CXCR1 cleavage fragments into the cell sup ernatants of LPS- and TNF-alpha-stimulated neutrophils, In addition, while treatment of neutrophils with LPS and TNF-alpha inhibited IL-8 receptor-med iated calcium mobilization and IL-8-directed neutrophil chemotaxis, both 1, 10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8-mediated downmodulation o f CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, thes e findings may provide further insight into the mechanisms of leukocyte reg ulation during immunologic and inflammatory responses. (C) 1999 by The Amer ican Society of Hematology.