FUNCTIONAL LINKAGE OF THE GRO-BETA AND IL-8 RECEPTORS ON THE SURFACE OF HUMAN NEUTROPHILS

Grobeta and IL-8 are two pro-inflammatory cytokines with chemotactic a ctivities on neutrophils. Binding studies were performed to ascertain whether their similar biological activities are mediated through the s ame receptor. Since Grobeta lacks tyrosine residues, recombinant Grobe ta containing an additional carboxy-terminal tyrosine residue (Grobeta -Tyr) was produced in transfected COS cells, purified to homogeneity a nd radiolabelled with (INa)-I-125. Saturation experiments using I-125 !-Grobeta-Tyr allowed us to identify high affinity receptors on human neutrophils (Kd: 2 +/- 0.5 nM and Bmax: 4760 +/- 761 sites/cell). Expe riments using I-125!-IL-8 as ligand, showed no significative differen ces in affinity (Kd: 4 +/- 0.9 nM) but about two times the number of s ites (11316 +/- 1810 sites/cell). In competition experiments using I- 125!-Grobeta-Tyr, unlabelled IL-8 and Grobeta-Tyr generated superposab le displacement curves (IC50: 0.69 +/- 0.15 nM and 0.42 +/- 0.11 nM, r espectively). Interesting, IL-8 binding sites could be down-regulated by Grobeta and IL-8, indicating that the two binding sites may be asso ciated. Cross-linking experiments using I-125!-IL-8 revealed two majo r bands at 70 and 140 kDa, whereas experiments with I-125!-Grobeta-Ty r showed only the 70 kDa band. Taken together, these results suggest t hat the human neutrophil IL-8/Grobeta receptor is a dimeric complex wi th two high affinity binding sites for IL-8 and of those two, only one is shared by Grobeta.