Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons.     

Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts

Although fibroblasts are important in providing a structural framework for most tissues, they also appear to be active participants in the inflammatory process via the production of specific mediators. The production of inflammatory mediators by fibroblasts is especially important in relation to their strategic location within connective tissue as they may act as a cellular communication bridge between the interstitium and vasculature. In this paper, we demonstrate that fibroblasts may participate in these inflammatory reactions by the production of a neutrophil chemotactic factor (NCF) with characteristics similar to a recently isolated and cloned monocyte-derived NCF. Either tumor necrosis factor-alpha-, interleukin-1 alpha-, or interleukin-1 beta-stimulated fibroblasts showed both a time- and dose-dependent increase in steady-state levels of NCF mRNA and secretion of chemotactic activity. In contrast, lipopolysaccharide and interleukin-6 failed to induce fibroblast-derived NCF. The expression of fibroblast-derived NCF mRNA was first detectable by 30 min poststimulation, whereas chemotactic activity was significantly observed 3-4 h postchallenge. Heat-inactivated monokine (100 degrees C) failed to induce NCF mRNA expression, suggesting that only the active proteins are capable of inducing NCF. Gel filtration analysis using high pressure liquid chromatography indicated peak chemotactic activity with an approximate molecular mass of 8000 daltons. This peak of NCF activity was found to be relatively stable to both heat and trypsin inactivation. Specificity of the fibroblast-derived neutrophil chemotactic activity was demonstrated with inhibition of chemotaxis by the addition of neutralizing antibody directed against recombinant human neutrophil chemotactic factor. These data provide evidence that monokine-treated fibroblasts can synthesize a potent chemotactic agent with molecular and physicochemical characteristics similar to monocyte-derived NCF and that this factor may contribute to neutrophil-mediated disease processes.     

Cytokine-induced gene expression of a neutrophil chemotactic factor/IL-8 in human hepatocytes

The liver participates in inflammation via the elaboration of acute phase proteins from hepatocytes in response to IL-1, TNF-alpha, and IL-6/INF-beta 2/hepatocyte-stimulating factor. In addition, some inflammatory states of the liver are characterized by leukocyte infiltrates. Here we demonstrate that human hepatocyte lines are capable of expressing mRNA and biologic activity for a neutrophil chemotactic factor (NCF)/IL-8 in response to the inflammatory mediators IL-1 alpha, IL-1 beta, and TNF. Two human hepatoma cell lines (SK-Hep and Hep-G2) displayed a time- and dose-dependent increase in steady state levels of NCF/IL-8 mRNA and secretion of chemotactic activity in response to TNF and IL-1. Neutralizing antibody to NCF/IL-8 inhibited hepatocyte-derived chemotactic activity by 88%. In contrast to IL-1 and TNF, hepatocytes did not respond to LPS or IL-6 within the time and dose parameters used above. Although the expression of NCF/IL-8 mRNA (1.8 kb) was first detectable between 1 and 2 h poststimulation, significant chemotactic bioactivity was not observed until about 4 h. Heat-inactivated (100 degrees C, 30 min) cytokine failed to induced NCF/IL-8 mRNA synthesis, and cotreatment of cells with cytokine and cycloheximide super-induced NCF/IL-8 mRNA while inhibiting production of bioactivity. Thus, NCF/IL-8 expression is a primary induction phenomenon. Our data demonstrate the stimulus specific induction of NCF/IL-8 in hepatocytes and suggest that cytokine cell-to-cell communication circuits may be important in neutrophil-mediated inflammatory processes in the liver.     

Monokine-induced gene expression of a human endothelial cell-derived neutrophil chemotactic factor

Monokines have been increasingly recognized as communication signals that interact with both immune and non-immune cells during inflammation. Specifically, interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) possess potent effector activities on various cell types. We present novel data demonstrating that human endothelial cells are a major source of a neutrophil chemotactic factor (NCF) synthesized upon stimulation with either IL-1 alpha, IL-1 beta, or TNF-alpha; but not with interleukin-6 (IL-6). Northern blot analysis demonstrated that 20 ng/ml of either IL-1 or TNF-alpha could induce endothelial cells to express significant levels of NCF mRNA, while IL-6 was not active in this system. These data demonstrate that monokines play an important role in mediating acute inflammation via induction of an endothelial cell-derived NCF.     

Generation of interleukin-8 by plasmin from AVLPR-interleukin-8, the human fibroblast-derived neutrophil chemotactic factor

Plasmin mainly cleaved the Arg5-Ser6 bond of Arg-Val-Leu-Pro-Arg-interleukin-8 (AVLPR-IL-8) produced by human dermal fibroblasts, which resulted in the conversion of AVLPR-IL-8 to IL-8 and the inactive pentapeptide, though a minor cleavage of AVLPR-IL-8 by plasmin at Lys8-Glu9 bond occurred.     

The macrophage-derived neutrophil chemotactic factor, MNCF: a lectin with TNF-alpha-like activities on neutrophils

The macrophage-derived neutrophil chemotactic factor (MNCF) is an α-galactoside-binding lectin, known to induce dexamethasone-insensitive neutrophil recruitment. We further characterized MNCF effects on neutrophils and showed that it shares with TNF-α the ability to delay apoptosis and to trigger degranulation. MNCF and TNF-α effects show similar kinetics and involve Src kinases and MAPKinases dependent pathways. They were, however, clearly distinguished, since the soluble TNF-receptor etanercept prevented TNF but not MNCF effects, while melibiose disaccharide inhibited MNCF but not TNF effects. Absorption of MNCF on detoxi-gel did not alter its properties, precluding an LPS contamination effect. By contrast, galectin-3 required LPS to activate neutrophils. Specific antibodies allowed to further demonstrate that MNCF and galectin-3 are two distinct molecules. Finally, MNCF- and IL-8-induced neutrophil activation differed by their kinetic and sensitivity to pertussis toxin. In conclusion, MNCF is a distinct neutrophil agonist, with pro-inflammatory activities involving its carbohydrate recognition domain.     

Interleukin-1 induced gene expression of neutrophil activating protein (interleukin-8) and monocyte chemotactic peptide in human synovial cells

We report here that human synovial cells stimulated by interleukin-1 alpha and interleukin-1 beta express mRNA for both IL-8 (neutrophil chemotactic peptide) and monocyte chemotactic protein. IL-1 stimulated synovial cells from both osteoarthritis and rheumatoid arthritis patients exhibited similar mRNA expression of interleukin-8 and monocyte chemotactic protein. A capacity to produce factors selectively chemotactic for neutrophils, lymphocytes and monocytes provides a mechanism whereby synovial cells can facilitate inflammatory arthritis.     

Granulocyte colony-stimulating factor negatively regulates Toll-like receptor agonist-induced cytokine production in human neutrophils

We studied the effect of G-CSF on TLR agonist-induced cytokine production in human neutrophils. Human neutrophils produced IL-8 and TNF-α in response to stimulation with TLR agonists such as LPS and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine. This response was dependent on activation of ERK, p38, and PI3K, but not JNK. TLR agonist-induced cytokine production in neutrophils was inhibited by G-CSF, whereas it was enhanced by GM-CSF, and GM-CSF-mediated enhancement was attenuated by G-CSF. G-CSF and GM-CSF did not affect TLR agonist-induced phosphorylation of ERK, p38, JNK, Akt, and IκBα. STAT3 activation was much greater in G-CSF-stimulated neutrophils than that in GM-CSF-stimulated cells. G-CSF-mediated STAT3 phosphorylation and inhibition of TLR agonist-induced cytokine production were prevented by pretreatment of cells with AG-490 (JAK2 inhibitor). These findings suggest that G-CSF and GM-CSF exert the opposite effects on TLR agonist-induced cytokine production, and G-CSF negatively regulates TLR agonist-induced cytokine production in neutrophils via activation of STAT3.     

Interleukin 2 (IL 2) up-regulates its own receptor on a subset of human unprimed peripheral blood lymphocytes and triggers their proliferation

Several reports indicate that human peripheral blood lymphocytes (PBL) seeded in culture with purified or recombinant interleukin 2 (IL 2) immediately after separation from the blood display a substantial level of proliferation at day 5 or 6, even in the absence of any activating signal. The spontaneously IL 2 proliferating cells are large lymphocytes, and they co-purify on a Percoll gradient in the large granular lymphocytes (third (LGL) fraction) together with the natural killer (NK) activity. When LGL were separated into NKH1 (an NK-specific surface marker)-positive and NKH1-negative cells by fluorescence-activated cell sorting (FACS), proliferating cells were mainly found in the NKH1-negative fraction. On the contrary, when cells from Percoll fraction 3 were separated into OKT3-negative and positive cells, the majority of the proliferating cell was found in the OKT3-positive cells. These results indicate that spontaneously IL 2 proliferating (SIP) cells most probably belong to the T cell lineage, but are distinct from NK cells. Surprisingly, cells from this Percoll fraction examined immediately after separation from the blood do not express detectable amounts of IL 2 receptors as assessed by three different techniques: binding of [3H]IL 2, binding of [125I]anti-Tac antibodies, and FACS analysis with the use of anti-Tac antibodies. However, after 18 hr of culture in IL 2-supplemented medium, 5 to 7% of these cells became Tac-positive by FACS analysis. Additional analysis of IL 2 receptor induced in culture with IL 2 was performed by [125I]anti-TAC binding and by [3H]IL 2 binding. Scatchard analysis of [3H]IL 2 binding, in the range of concentrations leading to the detection of high-affinity binding sites, showed an affinity constant similar to that of conventional phytohemagglutinin blasts. The role of IL 2/IL 2 receptor interaction in the proliferation process was confirmed by the fact that proliferation, in contrast with NK activation, was clearly inhibited by anti-Tac antibodies. When LGL activated with IL 2 for 60 hr were sorted into Tac+ and Tac- cells, equal levels of NK activity was found in the two fractions. Proliferation, however, was only observed in the Tac+ population. We interpret these results to indicate that SIP cells are preactivated cells circulating in the blood. They are large cells and represent a very small proportion of circulating lymphocytes (0.3%). They express a subliminal amount of IL 2 receptor. Cultivated in the presence of IL 2, IL 2 receptor expression is enhanced to a detectable level, and the SIP cells begin to proliferate. These SIP cells could be activated T cells present in every normal individual.     

Urokinase receptor (uPAR) regulates complement receptor 3 (CR3)-mediated neutrophil phagocytosis

Urokinase receptor (uPAR) associates in cis with complement receptor 3 (CR3). In the present study, we addressed whether this coupling regulates CR3-mediated phagocytosis. CR3-mediated attachment of iC3b-opsonized sheep red blood cells to human neutrophils and internalization of these cells were reduced by removal of cell-bound uPAR by phosphatidylinositol-specific phospholipase C and reconstituted in the presence of soluble uPAR. The attachment and internalization were suppressed in the presence of anti-uPAR polyclonal antibody, proteolytically inactive urokinase and saccharides that disrupt interaction of uPAR with CR3. Thus, uPAR acts as a cofactor for iC3b binding to CR3 and regulates CR3-mediated phagocytosis.     

Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages.

Synthesis of the low-density-lipoprotein (LDL) receptor protein by cultured human monocyte-derived macrophages was demonstrated by immunoprecipitation of [35S]methionine-labelled cell extracts with a monoclonal antibody to the bovine adrenal LDL receptor. Although the antibody does not bind to or inhibit binding of 125I-LDL to the LDL receptor on intact fibroblasts, it specifically binds to a protein in extracts of human skin fibroblasts, of Mr approx. 130,000 under non-reducing conditions, that is able to bind LDL. In monocyte-derived macrophages, as in fibroblasts, the receptor is synthesized as a low-Mr precursor that is converted into the mature protein. The half-life of the precursor in human macrophages is approx. 44 min. In cells from two homozygous familial-hypercholesterolaemic subjects, only the precursor form of the receptor is synthesized. Detection of abnormalities of LDL-receptor synthesis in human mononuclear cells may be a useful aid in diagnosis of familial hypercholesterolaemia that is simpler and quicker than methods requiring growth of cultured skin fibroblasts.     

Identification of a monocyte-derived factor which regulates synthesis of insulin receptors on activated T-lymphocytes (MIRRF)

The regulation of the insulin receptor on the activated T-lymphocyte was studied. It has been previously shown that the monocyte with its constitutive insulin receptor can signal the quiescent T-lymphocyte with respect to ambient insulin concentration which regulates the copies of insulin receptors synthesized during the lymphocyte activation event. In this communication it is shown that the vehicle by which the monocyte signals the T-lymphocyte is a soluble, small molecular weight protein. Initially a bioassay was established to test the putative monocyte-derived factor in which freshly prepared purified populations of monocytes were incubated with insulin, extensively washed, and replated with lymphocytes in microwells or across a 3 microns filter from lymphocytes using the appearance of insulin receptors on T lymphocytes responding to lectin as measured by a radioligand binding assay as the outcome variable. Dose response and time course relationships were established to develop the ideal conditions for the bioassay. It was shown that the monocyte-derived insulin receptor regulatory factor (MIRRF) could be readily detected in conditioned medium of insulin-incubated and then washed monocytes as a starting point for attempts at later purification. Using rats fed an essential fatty acid deficient diet (EFAD), incapable of generating standard prostanoids, it was demonstrated that the MIRRF was readily detectable in our standard bioassay revealing that the factor was not a member of the arachidonic acid family. Lastly, it was shown that MIRRF is cycloheximide sensitive and either is a protein or requires protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of receptor for granulocyte colony-stimulating factor on human circulating neutrophils

We have made a mutein of human G-CSF with more stable, and potent biological activity. Using 125 I-labeled mutein human G-CSF, high affinity binding sites were identified on human circulating neutrophils. Receptor number per cell was 560 with a Kd of 250 pM. The human G-CSF receptor was identified as a single subunit protein of Mr approximately 150,000.     

Deactivation of human neutrophil chemotaxis by chemoattractants: effect on receptors for the chemotactic factor f-Met-Leu-Phe

Normal human peripheral blood PMN were exposed to varying concentrations of partially purified chemotactic complement fragments (C5fr) and a chemotactic peptide N-formyl methionylleucylphenylalanine (f-Met-Leu-Phe). This exposure resulted in a decreased chemotactic response termed deactivation of chemotaxis. Deactivation was found to be nonpreferential for the deactivating stimulus when high concentrations of either f-Met-Leu-Phe (10(-6) M) or C5fr (20 micrograms/ml) were used. When PMN were incubated with lower concentrations of C5fr (10 micrograms/ml), there was preferential deactivation towards C5fr. Similarly, preferential deactivation of chemotaxis was observed when PMN were incubated with 10(-6) M f-Met-Leu-Phe, but this was transient and cells were nonpreferentially deactivated 60 min after the initial exposure to f-Met-Leu-Phe. The availability of receptors for tritiated f-Met-Leu-Phe was examined by Scatchard analyses and measurement of reversible f-Met-Leu-[3H]Phe binding to C5fr and f-Met-Leu-Phe-deactivated PMN. When PMN f-Met-Leu-Phe receptors were studied immediately after exposure to concentrations of C5fr causing either preferential or nonpreferential deactivation, there was increased receptor availability compared with control PMN. In contrast, PMN deactivated with high concentrations of f-Met-Leu-Phe 10(-6) M) had a transient decrease in the number of receptors followed 1 hr later by an increase in the number of receptors. This was similar to the functional correlate of preferential deactivation of chemotaxis immediately after incubation with f-Met-Leu-Phe followed by nonpreferential deactivation in these same PMN. The data indicate that preferential deactivation of chemotaxis may be associated with a preferential decrease (down-regulation) of chemoattractant receptors and that nonpreferential deactivation is associated with an increase in chemoattractant receptors.     

The chemoattractant des-Arg74-C5a regulates the expression of its own receptor on a monocyte-like cell line.

The interaction of the chemoattractant des-Arg74-C5a (C5a des Arg) with its receptor on a human monocyte-like cell line, U-937, was examined. The data obtained suggest that C5a des Arg receptor expression is regulated by the extracellular concentration of C5a des Arg itself.     

Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8) resides in a gene cluster along with several other members of the platelet factor 4 gene superfamily

Summary Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8, suggested gene symbol IL8) is a cytokine that chemoattracts and activates neutrophils. Using a panel of human-rodent cell hybrids that preferentially segregate human chromosomes and in situ hybridization, the MDNCF/IL-8 gene was placed on the human gene map at position 4q12-q21. This is the same location where at least three other members (platelet factor 4, melanoma growth stimulatory activity, and interferon- induced factor) of the platelet factor 4 gene superfamily reside. In addition, a restriction fragment length polymorphism was identified using MDNCF as a probe in screening genomic DNA digested with HindIII from unrelated individuals.On leave from Genetics Section, Instituto Nacional do Cancer (RJ)/ Department of Genetics, Universidade Federal do Rio de Janeiro, Brazil     

Liver X receptor (LXR) regulates human adipocyte lipolysis

The Liver X receptor (LXR) is an important regulator of carbohydrate and lipid metabolism in humans and mice. We have recently shown that activation of LXR regulates cellular fuel utilization in adipocytes. In contrast, the role of LXR in human adipocyte lipolysis, the major function of human white fat cells, is not clear. In the present study, we stimulated in vitro differentiated human and murine adipocytes with the LXR agonist GW3965 and observed an increase in basal lipolysis. Microarray analysis of human adipocyte mRNA following LXR activation revealed an altered gene expression of several lipolysis-regulating proteins, which was also confirmed by quantitative real-time PCR. We show that expression and intracellular localization of perilipin1 (PLIN1) and hormone-sensitive lipase (HSL) are affected by GW3965. Although LXR activation does not influence phosphorylation status of HSL, HSL activity is required for the lipolytic effect of GW3965. This effect is abolished by PLIN1 knockdown. In addition, we demonstrate that upon activation, LXR binds to the proximal regions of the PLIN1 and HSL promoters. By selective knock-down of either LXR isoform, we show that LXRα is the major isoform mediating the lipolysis-related effects of LXR. In conclusion, the present study demonstrates that activation of LXRα up-regulates basal human adipocyte lipolysis. This is at least partially mediated through LXR binding to the PLIN1 promoter and down-regulation of PLIN1 expression.