Primary role of growth-related oncogene-α and granulocyte chemotactic protein-2 as neutrophil chemoattractants in chronic rhinosinusitis

BACKGROUND: The aetiology of chronic rhinosinusitis (CRS) remains unclear. The purpose of this study was to investigate neutrophil-attracting chemokine patterns in CRS without nasal polyposis. METHODS: The biological activity of the chemokines was identified using a two-step high-performance liquid chromatography (HPLC) technique combined with a bioassay in extracts from 55 CRS patients, and in the turbinate mucosa (TM) of patients (N=51) undergoing septumplasty. The biologic activity of each chemokine was assessed using blocking antibodies to chemokines. Immunolocalization of detected neutrophil chemokines was performed by quantitative evaluation of immunohistochemistry. Besides, PCR analysis was performed to quantify neutrophil chemokine mRNA. RESULTS: In CRS, the chemokines primarily detected by two-step HPLC were growth-related oncogene-alpha (GRO-alpha) and the granulocyte chemotactic protein-2 (GCP-2). Blocking of GCP-2 and GRO-alphad each resulted in chemotaxis inhibition rates of 43.3% and 35.9%, respectively, whereas anti-IL-8 and anti-ENA-78 had no effect. Both GCP-2 and GRO-alphad were generally synthesized by the surface epithelium and mucosal glands while GRO-alpha in particular was synthesized by endothelial cells, as shown by immunohistochemistry. The concentrations of the chemokines IL-8 and epithelial cell-derived neutrophil attractant-78 (ENA-78) were low in CRS and TM. CONCLUSION: It appears that both GRO-alpha and GCP-2 contribute to neutrophil chemotaxis in CRS, whereas IL-8 and ENA-78 appear to be of secondary importance for the chemotaxis of neutrophils in this condition. The expression of chemokines in mucosal gland cells is the main phenomenon involved in constitutive neutrophil chemotaxis in the TM.     

The CXC chemokine GCP-2/CXCL6 is predominantly induced in mesenchymal cells by interleukin-1beta and is down-regulated by interferon-gamma: comparison with interleukin-8/CXCL8

Human granulocyte chemotactic protein-2 (GCP-2)/CXCL6 is a CXC chemokine that functionally uses both of the IL-8/CXCL8 receptors to chemoattract neutrophils but that is structurally most related to epithelial cell-derived neutrophil attractant-78 (ENA-78)/CXCL5. This study provides the first evidence that GCP-2 protein is, compared with IL-8, weakly produced by some sarcoma, but less by carcinoma cells, and is tightly regulated in normal mesenchymal cells. IL-1beta was the predominant GCP-2 inducer in fibroblasts, chondrocytes, and endothelial cells, whereas IL-8 was equally well up-regulated in these cells by TNF-alpha, measles virus, or double-stranded RNA (dsRNA). In contrast, lipopolysaccharide (LPS) was a relatively better stimulus for GCP-2 versus IL-8 in fibroblasts. IFN-gamma down-regulated the GCP-2 production in fibroblasts induced by IL-1beta, TNF-alpha, LPS, or dsRNA. The kinetics of GCP-2 induction by IL-1beta, LPS, or dsRNA in fibroblasts differed from those of IL-8. Freshly isolated peripheral blood mononuclear leukocytes, which are a good source of IL-8 and ENA-78, failed to produce GCP-2. However, lung macrophages and blood monocyte-derived macrophages produced GCP-2 in response to LPS. Quantitatively, secretion of GCP-2 always remained inferior to that of IL-8, despite the fact that the ELISA recognized all posttranslationally modified GCP-2 isoforms. The expression of GCP-2 was confirmed in vivo by immunohistochemistry. The patterns of producer cell types, inducers and kinetics and the quantities of GCP-2 produced, suggest a unique role for GCP-2 in physiologic and pathologic processes.     

Granulocyte chemotactic protein 2 acts via both IL- 8 receptors,CXCR1 and CXCR2

Interleukin-8 (IL-γ) acts on human neutrophils via two receptors, CXCR1 and CXCR2. It shares CXCR2 with all neutrophil-activating chemokines, which like IL-8 have a conserved Glu-Leu-Arg (ELR) N-terminal motif, but is generally considered to be the only relevant agonist for CXCR1. IL-8 has a basic residue at the sixth position after the second cysteine, which was suggested to contribute to CXCR1 specificity. Among the other ELR chemokines, only granulocyte chemotactic protein 2 (GCP-2) has such a basic determinant. Using Jurkat cells that stably express either CXCR1 or CXCR2, we studied receptor activation by IL-8, GCP-2 epithelial neutrophil-activating protein 2 (ENA-78) (which shares 77 % identical amino acids with GCP-2) and growth-regulated oncogene α (GROα). At 10 nM and higher concentrations, GCP-2 and IL-8 induced significant activation of CXCR1-expressing cells, but no activity was found with GROα and ENA-78. As expected, however, all four chemokines had similar activities on CXCR2-expressing cells. A variant of GCP-2 in which the basic residue, Arg20, was replaced by a glycine was synthesized. This derivative was ineffective on CXCR1, but was as active as wild-type GCP-2 in CXCR2-expressing cells. GCP-2 displaced radiolabeled IL-8 from both receptors with low affinity, and in this respect resembled ENA-78 and GROα. Our data show that GCP-2 acts via both IL-8 receptors and thus appears to be functionally more similar to IL-8 than to the other ELR chemokines. Activation of CXCR1 appears to depend significantly on the presence of a basic binding determinant close to the second cysteine.     

The Detection of a Novel Neutrophil-Activating Peptide (ENA-78) Using a Sensitive Elisa

Neutrophil elicitation into tissue is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. This event has gained renewed interest with the discovery of a family of small polypeptides (<10 kD). The salient features of these cytokines are the presence of four cysteine amino acids (first two separated by one amino acid; C-X-C) and their ability to induce neutrophil chemotaxis and activation. Recently, our laboratories have discovered a new member of this C-X-C chemotactic cytokine supergene family, neutrophil-activating peptide, ENA-78. ENA-78 shares significant amino acid sequence homology with neutrophil activating peptide-2 (NAP-2; 53%), growth regulated oncogene/melanoma growth stimulatory activity (GROα; 52%), and IL-8 (22%). In addition, ENA-78 appears to activate neutrophils through the IL-8 receptor. Since both in vitro and in vivo biological fluids may contain an array of chemotactic cytokines that may be relevant to the activation and chemotaxis of neutrophils, we have developed a highly specific and sensitive sandwich ELISA for the detection of ENA-78.     

The detection of a novel neutrophil-activating peptide (ENA-78) using a sensitive ELISA.

Neutrophil elicitation into tissue is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. This event has gained renewed interest with the discovery of a family of small polypeptides (less than 10 kD). The salient features of these cytokines are the presence of four cysteine amino acids (first two separated by one amino acid; C-X-C) and their ability to induce neutrophil chemotaxis and activation. Recently, our laboratories have discovered a new member of this C-X-C chemotactic cytokine supergene family, neutrophil-activating peptide, ENA-78. ENA-78 shares significant amino acid sequence homology with neutrophil activating peptide-2 (NAP-2; 53%), growth regulated oncogene/melanoma growth stimulatory activity (GRO alpha; 52%), and IL-8 (22%). In addition, ENA-78 appears to activate neutrophils through the IL-8 receptor. Since both in vitro and in vivo biological fluids may contain an array of chemotactic cytokines that may be relevant to the activation and chemotaxis of neutrophils, we have developed a highly specific and sensitive sandwich ELISA for the detection of ENA-78.     

Recombinant mouse granulocyte chemotactic protein-2: production in bacteria, characterization, and systemic effects on leukocytes.

Granulocyte chemotactic protein-2 (GCP-2) is an important neutrophil chemotactic factor in the mouse that belongs to the CXC chemokine family. Although the local tissular effects of chemokines are well known, only recently has the systemic regulation of leukocytes become accepted. To study the pharmacokinetics of mouse GCP-2 and the systemic effects on leukocytes, we expressed a potent natural isoform of mouse GCP-2, GCP-2(9-78), in Escherichia coli and produced electrophoretically pure material. GCP-2(9-78) was 10-fold more potent to chemoattract neutrophils than recombinant GCP-2(5-78). After intravenous (i.v.) injection in mice, GCP-2(9-78) persisted in the circulation with an average half-life of 42 min. When a bolus of 1 mg/kg recombinant mouse GCP-2(9-78) was injected systemically, a significant effect on circulating leukocytes was observed. After a neutropenic phase, at its height at 1 h after injection, neutrophil numbers increased to a maximum at 4 h postinjection, and a concomitant decrease in lymphocyte numbers was observed. In control mice injected with isotonic saline, changes in leukocyte numbers were less pronounced and followed a different kinetic. Whereas tissular neutrophil chemotaxis to GCP-2 is influenced by gelatinase B, the systemic effects on neutrophilia and lymphopenia were not different in gelatinase B-deficient and wild-type mice. These data reinforce the idea that chemokines, including GCP-2, influence the homeostasis of circulating leukocyte numbers.     

CXCR1-binding chemokines in inflammatory bowel diseases: down-regulated IL-8/CXCL8 production by leukocytes in Crohn's disease and selective GCP-2/CXCL6 expression in inflamed intestinal tissue

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) that are characterized by chronic intestinal inflammation and a constant influx of leukocytes mediated by pro-inflammatory cytokines and chemokines. The intestinal expression of the CXCR1-binding chemokines IL-8/CXCL8 and GCP-2/CXCL6 and the participation of immunocompetent cells in IBD were evaluated. IL-8 production by peripheral blood mononuclear cells (PBMC) from IBD patients, stimulated with endotoxin, plant lectin or double-stranded RNA, was significantly lowered in patients with CD, but not in UC patients or healthy subjects. The reduced chemokine production by PBMC from IBD patients was both IL-8 and CD specific, but not inducer dependent. In serum, most chemokines remained undetectable, while the levels of those that were measurable remained unaltered in IBD patients. GCP-2, but not ENA-78/CXCL5, nor IL-8, were highly expressed by endothelial cells in inflamed intestinal tissue of IBD patients. In contrast, stimulated endothelial cell cultures produced more IL-8 than GCP-2. The selective GCP-2 staining of endothelial cells at sites of ulcerations suggests that GCP-2, despite its low production capacity in vitro, plays a role in IBD that is different from that of structurally (ENA-78) and functionally (IL-8) related ELR(+) CXC chemokines. Thus, the chemokine network shows complementarity rather than redundancy.     

Interleukin-8 and neutrophil leucocytes: adhesion, spreading, polarisation, random motility, chemotaxis and deactivation in assays using 'sparse-pore' polycarbonate (nuclepore) membranes in the Boyden chamber.

Adhesion, spreading, chemotaxis and deactivation of chemotactic responses of separated human peripheral blood neutrophil leucocytes under the influence of interleukin-8 (IL-8) were tested using a previously described Boyden chamber-type method involving 'sparse-pore' polycarbonate (Nuclepore) filtration membrane. The random motilities of neutrophils in similar concentrations of IL-8 were tested using corresponding chambers with polycarbonate membranes of standard pore densities. In addition, polarisation of neutrophils in suspension in various concentrations of IL-8, and the possibility of deactivation of this polarisation response by IL-8 itself or by N-formyl-methionyl-leucyl-phenylalanine (FMLP) were examined. Neutrophils exhibited maximum chemotaxis to IL-8 in a concentration of 100 ng/ml, and to a degree which was similar to the maximum response to FMLP (10(-7) M). This chemotactic response to IL-8 was markedly reduced by pretreatment of the cells with either 100 ng/ml IL-8 (deactivation) or 10(-7) M FMLP (cross-deactivation). On the other hand, the chemotactic response of the neutrophils to FMLP was reduced by pretreatment with FMLP but was not deactivated by pretreatment with IL-8 (i.e. deactivation but not cross-deactivation). Neutrophils in suspension were maximally polarised by 100 ng/ml IL-8, and to lesser degrees by 1, 10 and 1,000 ng/ml IL-8. The detailed morphology of the polar cells was not distinguishable from that induced by 10(-8) M FMLP. Pretreatment of the cells with either IL-8 or FMLP resulted in no reduction of polarisation in response to subsequent exposure to either agent (i.e. neither deactivation nor cross-deactivation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Synergy between proinflammatory ligands of G protein-coupled receptors in neutrophil activation and migration

The chemokine dose and the time period during which the chemotactic gradient is established determine the number of leukocytes that infiltrate inflamed tissues. At suboptimal chemokine concentrations, neutrophils may require a priming agent or a second stimulus for full activation. An interesting mode of cooperative action to reach maximal migration is synergy between chemokines. This was first observed between the plasma CC chemokine regakine-1 and the tissue CXC chemokine ligand interleukin-8 (IL-8/CXCL8) in neutrophil chemotaxis. Addition of antibodies against IL-8 or regakine-1 in the Boyden microchamber assay abrogated this synergy. Other CC chemokines, such as CC chemokine ligand-2 monocyte chemotactic protein-1 (MCP-1/CCL2), MCP-2 (CCL8), and MCP-3 (CCL7) as well as the CXC chemokine receptor-4 (CXCR4) agonist stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12), also dose-dependently enhanced neutrophil chemotaxis toward a suboptimal concentration of IL-8. These chemokines synergized equally well with the anaphylatoxin C5a in neutrophil chemotaxis. Alternatively, IL-8 and C5a did not synergize with an inactive precursor form of CXCL7, connective tissue-activating peptide-III/CXCL7, or the chemoattractant neutrophil-activating peptide-2/CXCL7. In the chemotaxis assay under agarose, MCP-3 dose-dependently increased the migration distance of neutrophils toward IL-8. In addition, the combination of IL-8 and MCP-3 resulted in enhanced neutrophil shape change. AMD3100, a specific CXCR4 inhibitor, reduced the synergistic effect between SDF-1alpha and IL-8 significantly. SDF-1alpha, but not MCP-1, synergized with IL-8 in chemotaxis with CXCR1-transfected, CXCR4-positive Jurkat cells. Thus, proinflammatory chemokines (IL-8, MCP-1), coinduced during infection in the tissue, synergize with each other or with constitutive chemokines (regakine-1, SDF-1alpha) to enhance the inflammatory response.     

In vivo neutrophil recruitment by granulocyte chemotactic protein-2 is assisted by gelatinase B/MMP-9 in the mouse.

Granulocyte chemotactic protein-2 (GCP-2) of the mouse is a potent neutrophil chemotactic and activating factor in vitro and in vivo. Gelatinase B/matrix metalloproteinase-9 is released from neutrophils within 1 h after stimulation with GCP-2. In vitro neutrophil chemotaxis by GCP-2 was not impaired by specific inhibitory monoclonal antibodies (mAb) against gelatinase B, indicating that gelatinase B is not involved in chemotaxis of neutrophils through polycarbonate filters. To investigate if gelatinase B degranulation is involved in in vivo cell migration toward GCP-2, experiments were performed with gelatinase B knockout mice. When mouse GCP-2 was injected intradermally in mice, a dose-dependent neutrophil chemotactic response was observed, and this cell migration was significantly impaired in young mice by genetic gelatinase B knockout. In adult vs. young gelatinase B-deficient mice, such compensatory mechanisms as higher basal neutrophil counts and less impairment of chemotaxis toward local GCP-2 injection were observed. These experiments prove the concept that gelatinase B release under pressure of GCP-2 is a relevant, but not exclusive, effector mechanism of neutrophil chemotaxis in vivo and that known mechanisms, other than the release of gelatinase B, allow for a full-blown chemotactic response and compensate for gelatinase B deficiency in adult life in the mouse.     

The chemokine CXCL3 is responsible for the constitutive chemotactic activity of bovine milk for neutrophils

Bovine milk is known to exert a potent chemotactic activity on neutrophils, but the responsible agent has not been identified. The objective of the study was to characterize the main biochemical component responsible for this chemotactic activity. A neutrophil shape change assay was used to locate active milk fractions separated by chromatography. A single protein was isolated and identified by amino acid sequencing and mass spectrometry as CXCL3. Recombinant bovine chemokines and specific antibodies were used to show that normal milk contains active concentrations of CXCL1 (1-5ng/ml) and CXCL3 (100-500ng/ml), whereas CXCL2 and CXCL8/IL-8 were not detected. Depletion experiments with antibodies showed that CXCL3 was the main chemotaxin for neutrophils in normal (non-mastitic) milk. The chemokine CXCL3 was located by immunohistochemistry in mammary epithelial cells, and abundant mRNA was found in uninflamed mammary tissue, suggesting constitutive secretion by the lactating mammary epithelium. These results indicate that CXCL3/GRO-gamma is the major chemotactic factor for neutrophils in bovine milk in the absence of inflammation, and that it is secreted constitutively in milk by mammary epithelial cells. This finding prompts the question of the biological significance of permanent high concentrations of a CXC chemokine in milk.     

Shiga Toxins Induce, Superinduce, and Stabilize a Variety of C-X-C Chemokine mRNAs in Intestinal Epithelial Cells, Resulting in Increased Chemokine Expression

Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and ENA-78. Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-alpha protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection.     

Chemokines synergize in the recruitment of circulating neutrophils into inflamed tissue

The innate immune response against micro-organisms is mediated by phagocytes, attracted by chemokines and other G protein-coupled receptor (GPCR) ligands. Originally, we observed increased neutrophil migration by the interaction of inflammatory CXC chemokines such as IL-8/CXCL8 and granulocyte chemotactic protein (GCP)-2/CXCL6 with regakine-1, a CC chemokine constitutively present in plasma. We here demonstrate statistically significant synergy between regakine-1 and the neutrophil attractants C5a or IL-8/CXCL8 in inducing neutrophil shape change and migration under agarose. In addition, regakine-1 attracted human bone marrow granulocytes and enhanced their chemotactic response to IL-8/CXCL8 in a dose-dependent manner. Thus, plasma chemokines may regulate the number of circulating leukocytes under homeostatic conditions and may facilitate extra recruitment of bone marrow neutrophils during inflammation. Indeed, in vivo, regakine-1 provoked a mild neutrophilia in rabbits upon intravenous injection. We also observed that the CC chemokines regakine-1 and monocyte chemotactic protein-3/CCL7 as well as the CXC chemokine stromal cell-derived factor-1alpha/CXCL12 co-operated with murine GCP-2 after intraperitoneal co-administration to increase neutrophil influx in mice. These data demonstrate that inducible and constitutive GPCR ligands synergize to enhance inflammation and facilitate a more effective immune response.     

Ozone-induced bronchial epithelial cytokine expression differs between healthy and asthmatic subjects

BACKGROUND: Ozone (O3) is a common air pollutant associated with adverse health effects. Asthmatics have been suggested to be a particularly sensitive group. OBJECTIVE: This study evaluated whether bronchial epithelial cytokine expression would differ between healthy and allergic asthmatics after ozone exposure, representing an explanatory model for differences in susceptibility. METHODS: Healthy and mild allergic asthmatic subjects (using only inhaled beta2-agonists prn) were exposed for 2 h in blinded and randomized sequence to 0.2 ppm of O3 and filtered air. Bronchoscopy with bronchial mucosal biopsies was performed 6 h after exposure. Biopsies were embedded in GMA and stained with mAbs for epithelial expression of IL-4, IL-5, IL-6, IL-8, IL-10, TNF-alpha, GRO-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), fractalkine and ENA-78. RESULTS: When comparing the two groups at baseline, the asthmatic subjects showed a significantly higher expression of IL-4 and IL-5. After O3 exposure the epithelial expression of IL-5, GM-CSF, ENA-78 and IL-8 increased significantly in asthmatics, as compared to healthy subjects. CONCLUSION: The present study confirms a difference in epithelial cytokine expression between mild atopic asthmatics and healthy controls, as well as a differential epithelial cytokine response to O3. This O3-induced upregulation of T helper type 2 (Th2)-related cytokines and neutrophil chemoattractants shown in the asthmatic group may contribute to a subsequent worsening of the airway inflammation, and help to explain their differential sensitivity to O3 pollution episodes.