High- and low-affinity binding of GRO alpha and neutrophil-activating peptide 2 to interleukin 8 receptors on human neutrophils.

Abstract

GROa and neutrophil-activating peptide 2 (NAP-2), like their analog interleukin 8 , are considered to be inflammatory mediators since they recruit and activate neutrophil leukocytes. After introduction of tyrosines by substitution for other residues at the C terminus, GROa and NAP-2 were labeled with 125I and used for binding studies. A total of 60,000-90,000 receptors per neutrophil were found for either ligand. Of these 30-45% were of high affinity with a mean Kd value of 0.3 and 0.7 nM for GROa and NAP-2, respectively, and 55-70% of low affinity (Kd = 30 nM). Two proteins of "70 kDa and 44 kDa (p70 and p44) were specifially cross-linked with labeled GROa, NAP-2, and IL-8. Unlabeled IL-8 fully inhibited this crossing and the binding oflabeled Among the growing number of interleukin 8 (IL-8)-related chemotactic cytokines, neutrophil-activating peptide 2 (NAP-2) and GROa were studied extensively because oftheir possible involvement in the pathophysiology of inflammation (1-4) and tumor growth (5, 6). Responses with neutrophils, the principal target cells for all three cytokines, include chemotaxis, shape change, mobilization of cytosolic free calcium, release of granule components, upregulation of adhesion receptors, and the respiratory burst (7-12). Several recent reports have described receptors for IL-8 on human neutrophils (13)(14)(15)(16)(17). With one exception (13), these studies show that IL-8 binds to a single class of high-affinity receptors (Kd = 0.2-4 nM) with densities reported to be between 20,000 and 75,000 sites per cell. Cross-linking experiments revealed either one (14) or two (15, 17) receptor proteins with molecular masses ranging from 44 to 78 kDa. Recently, cDNAs for two IL-8 receptors with seven putative transmembrane domains typical of guanine nucleotide binding protein (G protein)-coupled receptors have been described (18, 19).Studies of the biochemical and binding properties of receptors for GROa and NAP-2 were hampered by the absence of tyrosines suitable for radioiodination. By substitution of residues at or close to the C terminus with tyrosines, we have obtained analogs with similar biological activities as the natural peptides that could be labeled to high-specific activities with 1251. Using these analogs, we were able to identify GROa and NAP-2 receptors on human neutrophils by direct binding assays and to compare them with the receptors for IL-8. The results of the present paper demonstrate the existence of two distinct receptors on human neutrophils that recognize GROa and NAP-2 as well as IL-8. MATERIALS AND METHODSMaterials. Aprotinin, chymostatin, leupeptin, bovine serum albumin, EDTA, PEG 6000, diisopropyl fluorophosphate, deoxycholate, Nonidet P40, polyethylenimine, and D-(+)-sucrose were obtained from Fluka; gelatin and phenylmethylsulfonyl fluoride were from Sigma; disuccinimidyl suberate was from Pierce; DNase I was from Boehringer Mannheim; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and dodecyl f3-maltoside were from Calbiochem-Behring; d...

Both interleukin‐8 receptors independently mediate chemotaxis

“…The competition for binding of iz51-IL-8 by unlabelled IL-8 and GROcz is shown in [4][5][6]. The present findings are in agreement with previous observations in transiently transfected E293 cells [13].…”

“…press two distinct IL-8 receptors [4,5] which are now termed IL-8Rl and IL-8R2 [2]. IL-8 binds to both receptors with high affinity, whereas GROa, GRO/3, GROr and NAB-2 bind with high affinity to IL-8R2 (Kd 0.2-2.5 nM) and low affinity to IL-8Rl (& 20&500 nM) [4-71. The cDNAs for IL-8Rl and IL-8R2 were cloned and shown to code for homologous proteins of 350 and 360 amino acids with seven putative transmembrane domains, which are typical for G-protein coupled receptors [8,9].…”

Identification of Distinct Surface-Expressed and Intracellular CXC-Chemokine Receptor 2 Glycoforms in Neutrophils:N-Glycosylation Is Essential for Maintenance of Receptor Surface Expression

“…There have been several attempts to determine the molecular characteristics of IL-8R by cross-linking studies using radiolabeled ligands. These resulted in the detection of either a single surfaceexpressed molecule (27,28) or multiple variants diverging in molecular size (29,30). The only report providing some evidence for glycosylation of IL-8R (27) did not, however, discriminate between the different receptor types CXCR-1 and CXCR-2, which were only later shown to be coexpressed on neutrophils (31).…”

Selective induction of monocyte and not neutrophil-attracting chemokines after influenza A virus infection.

“…of Transfusion Medicine, University of Marburg, Germany, for buffy coat blood. Received for publication 13 May 1996 and in revised form 15July I996.…”

“…Members of the CC-chemokine subfamily, such as MIP-lcl 1 (macrophage inflammatory protein-lc~), MCP-1 (monocyte chemotactic protein-I), and RANTES (regulated upon activation, normal T cell expressed and secreted) preferentially attract monocytes and lymphocytes (12). The CXC-chemokines which contain an ELR-motifpreceding the first cysteine, such as IL-8 (interleukin-8) or GRO-ci (melanoma growth stimulatory activity) are major neutrophil chemoattractants (13).…”