Modification of sulfhydryl groups of interleukin-8 (IL-8) receptor impairs binding of IL-8 and IL-8-mediated chemotactic response of human polymorphonuclear neutrophils.
Interleukin-8 (IL-8), a monocyte-derived neutrophil chemotactic agent, has a potential role in the regulation of inflammatory responses. The specific receptor for IL-8 has been identified and characterized on the surface of human neutrophils (Samanta, A. K., Oppenheim, J. J., and Matsushima, K. (1989) J. Exp. Med. 169, 1185-1189). The present study demonstrates that at least two sulfhydryl groups of this receptor from human neutrophils participate in the binding of IL-8. Incubation of neutrophils with sulfhydryl group-modifying reagents, N-ethylmaleimide and diazene dicarboxylic acid bis-N,N-dimethylamide (diamide), severely impaired the binding of 125I-IL-8 to neutrophils. Treatment with 0.8 mM N-ethylmaleimide and 0.4 mM diamide inhibit binding of 125I-IL-8 to the neutrophils by 62 and 60%, respectively. These inhibitory effects could be reversed by 84-87% by treatment with 2-4 mM dithiothreitol. The saturable amount of the ligand, IL-8, provided partial protection against the modifying reagents. N-Ethylmaleimide and diamide at a concentration of 0.4 mM reduced chemotactic migration of neutrophils in a Boyden chamber by 95 and 60%, respectively. At a concentration of 0.4 mM, N-ethylmaleimide reduced the IL-8-induced (10 micrograms/ml) release of myeloperoxidase by 50%. Under identical conditions, 0.4 mM diamide could reduce release of myeloperoxidase by 63%. Finally, N-ethylmaleimide severely affected the overall binding and total uptake of 125I-IL-8 to the neutrophils at 37 degrees C, a condition required for receptor-mediated internalization of the ligand and recycling of the receptor to the surface of neutrophils. Nitro blue tetrazolium reduction test of the lipopolysaccharide-stimulated neutrophils indicates that compared to control general metabolic functions of thiol-modified cells were markedly retained. These data suggest that at least two conformationally vicinal free reactive sulfhydryl groups are located in the binding domain of the receptor in neutrophils which are essential for IL-8-mediated biological responses.
Samanta
A K
AK
Division of Immunobiology, Indian Institute of Chemical Biology, Calcutta.
Dutta
S
S
Ali
E
E
D001665:Binding Sites; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte; D003958:Diamide / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D008024:Ligands; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000737:chemistry / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013696:Temperature
D016209:Interleukin-8; D008024:Ligands; D011971:Receptors, Immunologic; D023062:Receptors, Interleukin-8A; D013438:Sulfhydryl Compounds; D003958:Diamide; D005033:Ethylmaleimide