Abstract
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.
Highlights
Ethylmaleimide for 30 min a t 37 "C resulted in inhibition of nitrobenzoic acid), N-succinimidyl 3(4-hydroxyphenyl)propionate, 1251-IL-8binding to neutrophils ina dose-dependent manner dithiothreitol, cysteine, N-formyl-methionyl-leucyl-phenylalanine, (Fig. 1).Treatment with 0.4 mM N-ethylmaleimide showed bovine serumalbumin,gelatin,lipopolysaccharide, and nitro blue tetrazoliumwere purchased from Sigma ChemicaClo., St
As the modified thiol groups wereindicated to be involved in ligand binding, we examined the effects of these modifications on the receptor-mediated release of myeloperoxidase.From Table 11,it canbe seen that compared to control (IL-8 concentration 10 Fg/ml) addition of 0.2, 0.4, and 0.8 mM N-ethylmaleimide blocked the release of myeloperoxidase by 22, 50, and 62%,respectively)
Any chemoattractant-induced migration of atarget cell requires stimulationand activation of the cells,which is mediated through the interaction of the chemoattractant with its specific receptors expressed on the surface of the responding cell
Summary
Intraperitoneal or intravenousinjections of IL-8 caused rapid PMN accumulation or neutrophilia [17, 18].Intradermal injection of IL-8 caused a dose-dependent accumulation of PMNs and lymphocytes at the site of injection (4, 5 , 10) Another neutrophil-activating peptide-2which is the truncated form of platelet connective tissue-activating recycling of the receptor to the surfacoef neutrophils. Nitro blue tetrazolium reduction test of the lipopolysaccharide-stimulated neutrophils indicatesthat compared to control general metabolic functions of thiolmodified cells weremarkedlyretained. These data suggest that atleast twoconformationally vicinal free reactive sulfhydryl groups are located in the binding domain of the receptorin neutrophils which are essential forIL-8-mediated biological responses.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
