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Query: UNIPROT:P10145 (IL-8)
 23,849 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

GRO alpha and neutrophil-activating peptide 2 (NAP-2), like their analog interleukin 8 (IL-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, GRO alpha 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 GRO alpha and NAP-2, respectively, and 55-70% of low affinity (Kd = 30 nM). Two proteins of approximately 70 kDa and 44 kDa (p70 and p44) were specifically cross-linked with labeled GRO alpha, NAP-2, and IL-8. Unlabeled IL-8 fully inhibited this cross-linking and the binding of labeled GRO alpha or NAP-2 to the high-affinity sites on neutrophils or neutrophil membranes. Treatment of membranes with digitonin resulted in the preferential solubilization of a single receptor species, corresponding to p44, that bound GRO alpha and NAP-2 with low affinity (Kd = 30 nM) and IL-8 with high affinity (Kd = 0.4 nM). Exposure of neutrophil membranes to 100 microM guanosine 5'-[gamma-thio]triphosphate led to a 75-fold increase of the Kd in approximately 60% of the IL-8 receptors. High-affinity receptors for GRO alpha and NAP-2 were similarly affected. In contrast, guanosine 5'-[gamma-thio]triphosphate had no effect on the binding of IL-8 to p44 solubilized by digitonin. These results demonstrate that human neutrophils bear two classes of receptors for GRO alpha, NAP-2, and IL-8 (p70 and p44) that may differ in their mode of interaction with GTP regulatory proteins.
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PMID:High- and low-affinity binding of GRO alpha and neutrophil-activating peptide 2 to interleukin 8 receptors on human neutrophils. 143 44

Essentially pure preparations of normal density eosinophils obtained from patients with hypereosinophilic syndrome (HES) were stimulated with complement factor 5a (C5a), platelet-activating factor (PAF), FMLP and neutrophil-activating peptide (NAP-1/IL-8). Three responses were studied, the transient rise in cytosolic free calcium concentration ([Ca2+]i) (derived from indo-1 fluorescence), shape changes (measured by laser turbidimetry), and exocytosis of eosinophil peroxidase (EPO) (assessed by H2O2/luminol-dependent chemiluminescence). Responses were obtained with all four agonists, but C5a and PAF were by far more potent than FMLP and NAP-1/IL-8, which induced only minor effects. Pretreatment of the cells with pertussis toxin attenuated [Ca2+]i changes, EPO release and, to a lesser extent, shape changes, indicating that GTP-binding proteins of Gi-type are involved in receptor-dependent signal transduction processes leading to these responses. A clear dissociation was observed in the control of the shape change response and EPO exocytosis. The shape change was not affected by Ca2+ depletion or treatment with the protein kinase inhibitor staurosporine, but exocytosis was prevented by Ca2+ depletion and markedly enhanced by staurosporine. The activation of the contractile system, leading to shape changes and motility, thus appears to be independent of the classical signal transduction pathway involving phospholipase C, a [Ca2+]i rise and protein kinase C activation. Exocytosis is, as expected, Ca2+ dependent and appears to be under a negative control involving protein phosphorylations.
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PMID:Shape changes, exocytosis, and cytosolic free calcium changes in stimulated human eosinophils. 204 Jun 92

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.
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PMID:IL-8-induced signal transduction in T lymphocytes involves receptor-mediated activation of phospholipases C and D. 770 9

The formylpeptide (fMLP) and C5a chemoattractants were previously shown to cross-desensitize each other's ability to mobilize Ca2+ in leukocytes but not to affect nonchemoattractant Ca(2+)-mobilizing receptors, and vice versa. Our data show that all receptors studied underwent homologous desensitization. Interestingly, peptide chemoattractants (fMLP, C5a, and IL-8) desensitized each other's Ca(2+)-mobilizing responses, but had no effect on a Ca(2+)-mobilizing purinergic receptor. Lipid chemoattractant receptors (PAF and leukotriene B4) were also desensitized by peptide chemoattractants but not vice versa. In the presence of cytochalasin B, only fMLP and C5a caused the activation of phospholipase D in intact leukocytes and enhanced desensitization of IL-8 and C5a but not fMLP receptors. To measure receptor/G protein interactions, agonist-stimulated GTP gamma S binding to leukocyte membranes was measured. Whereas all peptide receptors underwent homologous desensitization, C5a and IL-8, but not fMLP, receptors were cross-desensitized by other peptide chemoattractants. Furthermore, PMA caused inhibition of C5a- and IL-8- but not fMLP-stimulated GTP gamma S binding. These data suggest that in addition to homologous desensitization, peptide chemoattractant receptors cross-desensitize one another by at least two processes. One can be detected at the level of receptor/G-protein interaction and possibly involves receptor phosphorylation by protein kinase C. The fMLP receptor is resistant to this process. The second process is distal to receptor/G-protein interaction and utilizes an undefined pathway to cross-desensitize the Ca2+ mobilization response to all peptide chemoattractants. We propose that receptor cross-desensitization in leukocytes is orchestrated at several levels by mechanisms with selectivity for types of chemoattractant receptors.
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PMID:Cross-desensitization of receptors for peptide chemoattractants. Characterization of a new form of leukocyte regulation. 808 98

The class II IL-8 receptor (IL-8R) binds both melanoma growth stimulatory activity (MGSA) and IL-8 with high affinity. Reverse transcriptase polymerase chain reaction demonstrates that the class II IL-8R mRNA, which has previously been detected only in cells of hematopoietic lineage, is also expressed in non-hematopoietic cell types shown to respond to MGSA or IL-8. To study the signaling mechanism by MGSA through the class II IL-8R in non-hematopoietic cells, this receptor was overexpressed in the 3ASubE human placental and the 293 human kidney cell lines. Membrane preparations of the class II IL-8R expressing 3ASubE transfectants exhibited a 2.3 +/- 0.2-fold increase in GTP gamma 35S binding, which was sensitive to pertussis toxin, in response to MGSA treatment (0.2 microM). This MGSA response was not observed in cells transfected with the parental expression vector. In vivo phosphorylation studies demonstrated that the class II IL-8R was basally phosphorylated in the untreated transfectants, and MGSA (5 nM) treatment markedly enhanced the phosphorylation of this receptor. The MGSA-induced receptor phosphorylation was both time and concentration dependent and could be mimicked by treatment with the calcium ionophore A23187. Phosphoamino acid analysis indicated that the MGSA-induced receptor phosphorylation was on serine residue(s), suggesting that a serine kinase is activated in response to MGSA binding to the class II IL-8R in non-hematopoietic cells.
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PMID:Melanoma growth stimulatory activity enhances the phosphorylation of the class II interleukin-8 receptor in non-hematopoietic cells. 829 49

Interleukin-8 (IL-8) is a polymorphonuclear leukocyte (PMN) chemoattractant and activator which mediates its effects through specific cell-surface receptors. Indirect evidence indicates that guanine nucleotide regulatory proteins (G proteins) are necessary for transmembrane signaling. The present study characterizes IL-8 receptors in isolated PMN membrane fractions and shows direct regulation of these receptors by guanine nucleotides. The binding of [125I]IL-8 to subcellular fractions of PMNs showed specific binding in a low-density membrane fraction containing alkaline phosphatase, but not in primary or secondary granules. The binding of [125I]IL-8 was rapid and reversible. The equilibrium dissociation constant (Kd) of the receptor ranged from 5.0-12.4 nM and there were 1.58-5.90 . 10(10) receptors/mg protein. The dose-response curves for the competitive binding of three different forms of IL-8 to the receptor labeled by [125I]IL-8 corresponded with their ability to produce chemotaxis and granule exocytosis in PMNs. Treatment of membranes with the nonhydrolyzable analogs of GTP, GMP-PNP and GTP gamma S, inhibited the binding of [125I]IL-8. GMP-PNP decreased the affinity of the IL-8 receptor by approx. 2-fold without altering the total receptor number. These findings demonstrate that IL-8 receptors in PMN membranes are of high affinity and are convertible to a low-affinity state in the presence of guanine nucleotides, suggesting a direct role for G proteins in transmembrane signaling by this cytokine.
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PMID:Characterization of interleukin-8 receptors in human neutrophil membranes: regulation by guanine nucleotides. 832 78

The effect of IL-8 on the in vitro locomotion of human IL-2-activated natural killer (IANK) cells was studied. It was observed that IL-8 induces chemokinesis in these cells, as determined by their migration in modified Boyden chambers. Bacterial toxins such as cholera toxin or pertussis toxin inhibited IL-8-induced chemokinetic activity, suggesting the involvement of guanine nucleotide-binding (G) proteins in IL-8 signal transduction in these cells. Pertussis toxin ADP-ribosylates a 39-kDa protein, whereas cholera toxin ADP-ribosylates a 43- to 45-kDa protein. Pretreatment of IANK cell membranes with 0.01 or 0.1 ng/ml of IL-8 and/or 5 microM GTP-gamma S did not affect pertussis toxin- or cholera toxin-dependent ADP-ribosylation. Western blot analysis showed that IANK cell membranes possess one Gi (39 kDa), two Gs (43 kDa and 45 kDa), and one Go (39 kDa). Pretreatment of IANK cell membranes with concentrations between 0.001 to 1.0 ng/ml of IL-8 resulted in the disappearance of the 39 kDa Go, but not Gi or Gs protein(s), suggesting that IL-8 receptors expressed on IANK cells are coupled to Go. Various concentrations of IL-8 enhanced the binding of GTP-gamma 35 S to IANK cell membranes, which further indicates the coupling of G proteins to IL-8 receptors in IANK cells.
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PMID:IL-8 induces the locomotion of human IL-2-activated natural killer cells. Involvement of a guanine nucleotide binding (Go) protein. 838 37

In this investigation we studied the modulation of human NK- and CTL-mediated cytotoxicity in response to extracellular nucleotides. NK cell-mediated cytotoxicity (CMC) was inhibited in a dose-dependent manner by ATP/ADP, GTP/GDP, and by pentasodium triphosphate (PST), whereas MHC-restricted CTL were inhibited by GTP/GDP and PST, but not by ATP/ADP. Triphosphates were the most potent inhibitors, followed by diphosphates and monophosphates which were the least effective, suggesting that the inhibition was not due to the sugars nor adenosine and guanosine nucleotides, but rather to the increasing negative charges. Cultured CTL, fresh NK cells that had been incubated with IL-2 for 18 hr and IL-2-dependent NK 3.3 cells were all inhibited by GTP, but not by ATP. This differential regulation of fresh NK cells and CTL by exogenous nucleotides is dependent upon the presence of IL-2, but IL-4, IL-6, and IL-8 did not have any effect. Mouse CTL are resistant to ATP presumably because they contain high levels of ecto-ATPases. Different levels of ecto-ATPase activity in human CTL and NK cells may therefore explain the difference in the responses of these effector cells to extracellular nucleotides. To test this possibility we determined the levels of ecto-ATPases in human CTL and NK cells and showed that CTL contained five times more ecto-ATPases than NK cells. Incubation of NK cells with IL-2 or IL-4 did not significantly change the level of ecto-ATPase activity on NK cells. Treatment of NK cells with IL-2 also did not significantly change the substrate specificity of NK-ecto-ATPases toward the extracellular ATP and GTP. Furthermore, treatment of CTL and NK cells with a potent ecto-ATPase inhibitor, 5'-fluorosulfonylbenzoyladenosine (FSBA), did not significantly alter the effect of exogenous nucleotides on the lytic potential of CTL and NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of resting and IL-2-activated human cytotoxic lymphocytes by exogenous nucleotides: role of IL-2 and ecto-ATPases. 849 83

Interleukin-8 (IL-8), the prototypic member of the CXC subfamily of chemokines, induces in neutrophils chemotaxis, the respiratory burst, granule release, and increased cell adhesion. The IL-8 receptor is a seven-transmembrane spanning receptor coupled to specific heterotrimeric G proteins including Gi and G16. IL-8 stimulation of its receptor on neutrophils activates Ras GTP loading and the mitogen-activated protein kinase (MAPK) pathway including Raf-1 and B-Raf. The properties of IL-8 stimulation of the MAPK pathway differ from those observed for chemoattractants such as C5a. Even though Ras GTP loading is similar for IL-8 and C5a, the maximal activation of Raf-1 and B-Raf is approximately 2-fold and 3-7-fold, respectively, less for IL-8 than that observed for C5a. Raf-1 activation is rapid but transient, returning to near basal levels by 10 min. B-Raf activation is slower in onset and does not return to basal levels for nearly 30 min. IL-8 activation of MAPK follows a time course suggesting an involvement of both Raf-1 and B-Raf. Surprisingly, wortmannin, at low concentrations, inhibits Raf-1, B-Raf, and MAPK activation in response to IL-8 and C5a demonstrating a role for phosphatidylinositol 3-kinase in the activation of Raf kinases in G protein-coupled receptor systems in human neutrophils. Furthermore, wortmannin inhibits IL-8 stimulated granule release and neutrophil adherence. These findings demonstrate the control of Raf kinases, the MAPK pathway and specific neutrophil functions by phosphatidylinositol 3-kinase enzymes.
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PMID:Interleukin-8 regulation of the Ras/Raf/mitogen-activated protein kinase pathway in human neutrophils. 857 62

CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines IL-8, MGSA/GRO, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we have mutated two putative G protein-coupling regions of CXCR2 and characterized the effects of these mutations on ligand-activated signal transductions: aspartic acid 89 in the second transmembrane domain and the HRAMR sequence (BBXXB motif, found in the third intracellular loop where B indicates a basic amino acid and X represents any amino acid). The Asp89 was replaced by either asparagine (D89N) or glutamic acid (D89E). For the BBXXB motif, the first two basic amino acids were mutated to two neutral isoleucines (HR-II), or alternatively, two isoleucines were inserted between alanine and methionine (II-insert). When expressed in human embryonic kidney 293 cells, the D89E mutant was localized intracellularly with no detectable cell surface expression. In contrast, D89N, HR-II, and II-insert mutants displayed cell surface expression, with Kd values and expression levels similar to that of the wild-type transfectant. The ability of the mutants to transduce signal was assessed by ligand-stimulated GTPgamma35S binding, mobilization of intracellular free Ca2+, and chemotaxis assays. Both D89N and HR-II mutants signaled similarly to a wild-type receptor in all three assays. However, the II-insert mutant exhibited a loss of ligand-stimulated GTPgamma35S binding, calcium mobilization, and chemotaxis. Unexpectedly, this receptor underwent ligand-induced sequestration comparable to wild-type CXCR2. These data indicate that Asp89 and the basic amino acids in the third intracellular domain do not play essential roles in ligand-induced signal transduction through CXCR2. However, proper secondary structure and orientation of the third intracellular loop of CXCR2 are essential for ligand-mediated signal transduction but not for receptor sequestration.
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PMID:Interruption of G protein-coupling in CXCR2 does not alter ligand binding, but eliminates ligand-activation of GTPgamma35S binding, calcium mobilization, and chemotaxis. 939 46


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