Two monoclonal antibodies, anti-IL8Rl and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GROa, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 1251-labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8Rl in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GROa. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GROa was inhibited by anti-IL8R2 but was not affected by anti-IL8Rl. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GROca or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8Rl, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GROa or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL8Rl but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-SRi.Chemokines constitute a large family of structurally related proteins which activate and attract leukocytes. Interleukin 8 (IL-8) and its analogs, GROa, GRO,3, GROT, NAP-2, and ENA-78, act predominantly on neutrophils and are called CXC chemokines because their first two conserved cysteines are separated by one amino acid (1). CXC chemokines share two distinct receptors, IL-8 receptors 1 (IL-8R1) and IL-8 receptor 2 (IL-8R2), which are both expressed in high numbers on neutrophils (1). IL-8 binds with high affinity (Kd values between 0.5 to 3 nM) to both receptors, whereas all the other CXC chemokines have high affinity for IL-8R2 only (2, 3). The two receptors have considerable amino acid sequence identity within the seven transmembrane domains and connecting loops, but differ almost totally in their NH2-and COOHterminal regions. This suggests that they not only possess distinct ligand-binding properties but also signal differently. Because the receptors are expressed together on neutrophils, their individual properties were studied in Jurkat cells after transfection of the respective cDNA. IL-8R1 and IL-8R2 were found to function ind...
The activation of protein kinase C by diacylglycerol and by tumour promoters has implicated this enzyme in transmembrane signalling and in the regulation of the cell cycle. In vitro studies revealed that catalytic activity requires the presence of calcium and phospholipids with a preference for phosphatidylserine. Diacylglycerol and tumour promoters such as phorbol esters bind to the enzyme, leading to its activation while sharply increasing its affinity for Ca2+ and phospholipid. Addition of diacylglycerol analogues or phorbol esters to intact cells results in the phosphorylation of specific polypeptides. Several cellular processes, including hormone and neurotransmitter release and receptor down-regulation, are modulated by the activation of protein kinase C, while phorbol ester-induced stimulation of the enzyme in whole cells has been associated with its translocation from the cytoplasm to the plasma membrane. Moreover, the use of Ca2+ ionophores has revealed an apparent synergism between Ca2+ mobilization and protein kinase C activation. This synergism has recently also been found to apply to receptor down-regulation (ref. 23 and accompanying paper). Here we describe a reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied. The results suggest a rationale for concomitant Ca2+ mobilization and diacylglycerol formation in response to some hormones, neurotransmitters and growth factors.
Activated neutrophils secrete two forms of IL-8 with 77 and 72 amino acids, and , along with proteinases that could process these cytokines. Significant conversion of IL-8(77) to more potent, N-terminally truncated forms was observed upon incubation with neutrophil granule lysates and purified proteinase-3. IL-8(72) was considerably more resistant to proteolytic processing than . The present observations indicate that neutrophil proteinases released in inflamed tissues convert IL-8 to more active forms and therefore tend to conserve or enhance, rather than decrease IL-8 activity.
The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous Gram-positive bacteria and Candida albicans as well as the Gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human β-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.