ABSTRACT
KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was expressed in Escherichia coli by using a maltose binding protein vector and biochemically characterized as a ligand for both murine and human polymorphonuclear neutrophils (PMN). On murine PMN, KC is both a potent chemoattractant and up-regulator of Mac-1 cell surface expression. On human PMN, in contrast, KC exhibits dissociation of its chemoattractant and Mac-1 up-regulatory activities. Although KC strongly increases Mac-1 expression on human PMN, it does not induce chemotaxis in vitro. 125I-KC-Tyr binds to both mouse and human PMN with two classes of binding sites, including high affinity sites of 0.8 and 2 nM, with approximately 9,000 and 10,000 sites per cell, respectively. On mouse PMN, human macrophage inflammatory protein (MIP)-2 alpha and MIP-2 beta compete for 125I-KC-Tyr binding with high affinity, whereas the murine beta-chemokine TCA-3 does not compete. KC binds to human PMN by the IL-8 type B receptor and to murine PMN by a murine IL-8 type B receptor homologue. 125I-KC-Tyr also binds to human RBC with a single class of high affinity sites. KC mRNA is constitutively expressed in multiple murine tissues. With human IL-8 and KC cDNA as probes, a mouse neutrophil exudate library was screened: KC and MIP-2 were the dominant chemokine species found. Thus, KC appears to be intimately involved in murine inflammation and its constitutive expression may have a role in the basal trafficking of neutrophils.
Subject(s)
Cytokines/biosynthesis , Cytokines/physiology , Inflammation Mediators/immunology , Animals , Ascitic Fluid/cytology , Base Sequence , Cells, Cultured , Chemokine CXCL1 , Chemokine CXCL2 , Chemokines , Chemokines, CXC , Cytokines/metabolism , Humans , Inflammation Mediators/metabolism , Macrophage-1 Antigen/biosynthesis , Mice , Molecular Sequence Data , Monokines/physiology , Neutrophils/metabolism , Receptors, Interleukin/genetics , Receptors, Interleukin-8A , Recombinant Proteins/biosynthesis , Transfection/geneticsABSTRACT
KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was synthesized as a recombinant protein in Escherichia coli and binds with 0.8 nM affinity to mouse neutrophils. Human neutrophils also bind recombinant KC at a site competitive with human interleukin (IL8) and Gro-alpha/MGSA, consistent with binding at the IL8 type B receptor (IL8RB). The cDNA corresponding to human IL8RB hybridizes strongly with two restriction fragments in murine genomic DNA, representing candidate receptor genes for KC. Molecular cloning of both mouse genomic DNA and neutrophil exudate cell cDNA libraries yielded a receptor with approximately 68% sequence identity to both the human IL8 type A and B receptors. Transient expression of the murine receptor cDNA in COS cells conferred binding ability to KC and a related gene product, macrophage inflammatory protein-2 (MIP-2) with high affinity (approximately 5 nM). Human IL8 was a poor agonist for this expressed receptor (Kd = approximately 400 nM). The potent activity of human IL8 on mouse polymorphonuclear neutrophils is not consistent with binding on the cloned receptor and suggests that murine homologues of IL8 and an IL8 type A receptor remain to be identified. Our data indicate that KC is the murine homologue of human Gro-alpha, and the KC receptor is an IL8 type B receptor homologue capable of binding both KC and macrophage inflammatory protein-2 with high affinity.
