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.     

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.     

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.     

Stimulation of angiostatic platelet factor-4 variant (CXCL4L1/PF-4var) versus inhibition of angiogenic granulocyte chemotactic protein-2 (CXCL6/GCP-2) in normal and tumoral mesenchymal cells

Chemokines affect inflammation and cancer through leukocyte attraction and angiogenesis. Here, we demonstrate that CXCL4L1/platelet factor-4 variant (PF-4var), a highly angiostatic chemokine, is poorly chemotactic for phagocytes and is inducible in monocytes by inflammatory mediators but remained undetectable in macrophages and neutrophils. In addition, CXCL4L1/PF-4var production by mesenchymal tumor cells was evidenced in vitro and in vivo by specific ELISA and immunohistochemistry. CXCL4L1/PF-4var, but not CXCL4/PF-4, was coinduced with the angiogenic chemokine CXCL6/granulocyte chemotactic protein-2 (GCP-2) by cytokines, e.g., IL-1beta and IL-17, in sarcoma cells, but not in diploid fibroblasts. Furthermore, the induction of CXCL6/GCP-2 in endothelial cells by IL-1beta was enhanced synergistically by TNF-alpha but inhibited by IFN-gamma, which synergized with IL-1beta to produce the angiostatic CXCL10/IFN-gamma-induced protein-10. These findings indicate that the equilibrium between angiostatic and angiogenic factors during inflammation and tumor progression is rather complex and differs depending on the chemokine, cell type, and stimulus. Selective intervention in the chemokine network may drastically disturb this delicate balance of angiogenesis and tissue repair. Application of angiostatic CXCL4L1/PF-4var without attraction of protumoral phagocytes may be beneficial in cancer therapy.     

Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis

The CXC chemokine IFN-gamma-inducible protein-10 (IP-10/CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T cells and natural killer cells. Peripheral blood mononuclear cells (PBMC) produce low but significant amounts of IP-10/CXCL10 protein upon stimulation with double-stranded (ds) RNA, the Toll-like receptor 3 (TLR3) ligand. IFN-gamma is a superior IP-10/CXCL10inducer. The bacterial TLR4 and TLR2 ligands, LPS and peptidoglycan (PGN), inhibit IFN-gamma- or dsRNA-dependent IP-10/CXCL10 production in PBMC, whereas IL-8/CXCL8 production was enhanced. In fibroblasts a different picture emerges with IFN-gamma inducing moderate and dsRNA provoking strong IP-10/CXCL10 production. Furthermore, treatment of fibroblasts with IFN-gamma in combination with bacterial LPS or PGN results in a synergistic production of IP-10/CXCL10 and IL-8/CXCL8. The synergistic induction of IP-10/CXCL10 in fibroblasts is reflected by significantly enhanced IP-10/CXCL10 concentrations in synovial fluids of septic compared to osteoarthritis patients to reach on average higher levels than those of IL-8/CXCL8. These high amounts of IP-10/CXCL10 produced by connective tissue fibroblasts not only attract CXCR3 expressing activated Th1 cells and natural killer cells to sites of infection but may also antagonize the CCR3 dependent attraction of Th2 lymphocytes and exert CXCR3-independent, defensin-like antibacterial activity.     

Treatment of monocytes with interleukin (IL)-12 plus IL-18 stimulates survival, differentiation and the production of CXC chemokine ligands (CXCL)8, CXCL9 and CXCL10

During inflammation, interleukin (IL)-12 and IL-18 are produced by macrophages and other cell types such as neutrophils (IL-12), keratinocytes and damaged endothelial cells (IL-18). To explore the role of IL-12 and IL-18 in inflammatory innate immune responses we investigated their impact on human peripheral blood monocytes and mature bronchoalveolar lavage (BAL) macrophages. IL-12 and IL-18 together, but not alone, prevented spontaneous apoptosis of cultured monocytes, promoted monocyte clustering and subsequent differentiation into macrophages. These morphological changes were accompanied by increased secretion of CXC chemokine ligands (CXCL)9, CXCL10 (up to 100-fold, P < 0.001) and CXCL8 (up to 10-fold, P < 0.001) but not CCL3, CCL4 or CCL5. Mature macrophages (from BALs) expressed high basal levels of CXCL8, that were no modified upon stimulation with IL-12 and IL-18. In contrast, the basal production of CXCL9 and CXCL10 by BALs was increased by 10-fold (P < 0.001) in the presence of either IL-12 or IL-18 alone and by 50-fold in the presence of both cytokines. In conclusion, our results indicate a relevant role for IL-12 and IL-18 in the activation and resolution of inflammatory immune responses, by increasing the survival of monocytes and by inducing the production of chemokines. In particular, those that may regulate angiogenesis and promote the recruitment of monocytes, activated T cells (CXCL9 and CXCL10) and granulocytes (CXCL8).     

Differential and additive effects of platelet-derived chemokines on monocyte arrest on inflamed endothelium under flow conditions

Platelet-derived chemokines, such as regulated on activation, normal T expressed and secreted (RANTES; CC chemokine ligand 5), platelet factor 4 [PF4; CXC chemokine ligand 4 (CXCL4)], and epithelial neutrophil-activating protein 78 (ENA-78; CXCL5), or precursors, such as beta-thromboglobulin, which can be processed to neutrophil-activating protein-2 (NAP-2; CXCL7), may play an important role in monocyte recruitment during atherogenesis. Platelets can deposit chemokines on inflamed endothelium; however, little is known about differential or additive effects of platelet chemokines on monocyte arrest. Here, we demonstrate that preincubation of activated human microvascular endothelial cells (HMVECs) with RANTES, PF4, or NAP-2 but not ENA-78 dose-dependently increased surface immobilization and subsequent monocyte arrest in flow. RANTES was the most potent and efficient arrest chemokine. Pretreatment of HMVECs with beta-thromboglobulin enhanced monocyte arrest in the presence of cathepsin G generating NAP-2. Combined pretreatment of HMVECs with RANTES and PF4 at suboptimal concentrations synergistically increased arrest, and preincubation with chondroitinase ABC abrogated RANTES- and PF4-induced monocyte arrest. This was associated with reduced expression of chondroitin sulfate, RANTES, and PF4 on the HMVEC surface. Perfusion of HMVECs with platelets known to deposit RANTES and PF4 on the endothelial surface enhanced monocyte arrest, which was inhibited by Met-RANTES, chondroitinase, or a blocking antibody to PF4 but not to ENA-78. The relevance of platelet-derived chemokines was confirmed in adhesion assays with activated whole blood, where Met-RANTES and to a lesser extent, antibodies to PF4 and NAP-2 inhibited arrest of CD14-positive monocytes. Thus, multiple platelet-derived chemokines and processable precursors, which can be presented by specific endothelial proteoglycans, may contribute and cooperate differentially to induce monocyte recruitment.     

Chemokines in the Inflammatory Bowel Diseases

Ulcerative colitis and Crohn's disease are characterized by chronic intestinal inflammation. Intestinal bacteria initiate the activation of intestinal inflammatory processes, which are mediated by proinflammatory cytokines and chemokines. In inflammatory bowel disease, intestinal inflammation is not downregulated, in part due to defective or absent inhibitory processes. Studies to date have demonstrated that IL-8, MCP-1, and ENA-78 are highly expressed in the intestinal mucosa in areas of active Crohn's disease and ulcerative colitis. Neutrophils and macrophages in the inflamed intestine synthesize and secrete large amounts of chemokines in patients with inflammatory bowel disease. Increased chemokine expression has also been observed in epithelial cells, endothelial cells, and smooth muscle cells. Future trials of specific agents capable of inhibiting chemokine synthesis and secretion or blocking chemokine–chemokine receptor interaction will be important to study in patients with ulcerative colitis and Crohn's disease.     

Expression of the neutrophil-activating CXC chemokine ENA-78/CXCL5 by human eosinophils

BACKGROUND: Eosinophils are seen at sites of inflammation in diseases such as helminthic infestation, asthma, ulcerative colitis and some neoplastic diseases. They are also associated with connective tissue remodelling, for example in longstanding asthma. In the present study, we investigated whether eosinophils express the CXC chemokine epithelial cell-derived neutrophil activating peptide (ENA-78/CXCL5), a chemokine that can activate neutrophils and in addition possesses angiogenic properties. Immunocytochemistry detected CXCL5 in eosinophils and the peptide was localized in the specific granules by immunoelectron microscopy. METHODS AND RESULTS: In eosinophil lysates, 12 +/- 2 pg (mean +/- SEM) of CXCL5 was detected per 106 cells by enzyme-linked immunosorbent assay (ELISA). Weak constitutive expression of CXCL5, as well as the related CXC chemokine IL-8/CXCL8, could be detected in freshly isolated eosinophils by RT-PCR. However, during prolonged incubation of eosinophils, a strong increase in both CXCL5 and IL-8/CXCL8 expression was seen, as detected by RT-PCR, and increasing amounts of CXCL5 peptide with time were detected in the incubation medium by ELISA. Addition of TNF-alpha neutralizing antibodies during prolonged incubation significantly inhibited CXCL5 production, demonstrating involvement of auto- and paracrine effects from TNF-alpha produced by eosinophils themselves. Addition of IFN-gamma showed a strong inhibitory effect on CXCL5 synthesis. CONCLUSION: These findings suggest that, through expression of CXCL5, eosinophils can recruit and activate CXC receptor 2 (CXCR2)-bearing cells such as neutrophils at sites of inflammation. Eosinophils may also promote connective tissue remodelling through release of this peptide.     

Downmodulation of CXCL8/IL-8 receptors on neutrophils after recruitment in the airways

BACKGROUND: CXCL8/IL-8 is the most significant chemokine for neutrophils, and CXC chemokine receptor (CXCR) 1 and 2 are its 2 receptors, which are downmodulated by CXCL8/IL-8 and endotoxin on activated neutrophils. OBJECTIVE: We sought to evaluate the expression of the CXCL8/IL-8 receptors and the activation marker CD11b on neutrophils in peripheral blood and in the site of airway inflammation. METHODS: The flow cytometric expression of CXCR1, CXCR2, and CD11b was evaluated on peripheral blood and induced sputum neutrophils in patients with nonsevere asthma with greater than 60% sputum neutrophils, in patients with chronic obstructive pulmonary disease (COPD), and in healthy control subjects. RESULTS: Asthmatic patients and patients with COPD had comparable expressions of CXCR1, CXCR2, and CD11b on peripheral blood and sputum neutrophils. Compared with control subjects, the peripheral neutrophil expression of CXCR2 was lower in patients with COPD ( P = .03) and that of CD11b was higher in asthmatic patients and patients with COPD ( P < .02 and P < .002). The expression of the CXCL8/IL-8 receptors on sputum neutrophils was markedly lower than on peripheral blood neutrophils ( P < .0001). The downmodulation of CXCL8/IL-8 receptors was also present in healthy control subjects but less than that seen in asthmatic patients. The difference between peripheral blood and sputum expression of CXCL8/IL-8 receptors correlated with serum CXCL8/IL-8 levels. In asthmatic patients the expression of CXCR1 and CXCR2 on sputum neutrophils negatively correlated with sputum neutrophils. CONCLUSION: In neutrophilic asthma the expression of CXCL8/IL-8 receptors on peripheral and sputum neutrophils is similar to COPD and negatively correlated with the inflammatory infiltrate in the airways. The downmodulation of CXCL8/IL-8 receptors detected in the airways should be taken into account for an eventual therapeutic inhibition of these receptors.     

Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-gamma) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll-Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-gamma and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-gamma-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-gamma+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial-lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD-even in an inactive state of disease-exert an increased capacity for IFN-gamma induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-gamma-producing CD8+ lymphocytes.     

Chemokine CXCL11 links microbial stimuli to intestinal inflammation

Interleukin (IL)-17 plays an important role in the pathogenesis in a number of immune inflammatory disorders. This study aims to investigate the mechanism by which microbial product flagellin is involved in the development of T helper type (Th)17 cells. Serum levels of IL-17 and CXCL9-11 in patients with ulcerative colitis (UC) were evaluated. The source and mechanism of CXC11 release in intestinal mucosa were examined with colonic biopsies from UC patients and a colitis mouse model. The role of flagellin in the development of Th17 cells was studied with a cell co-culture system. High serum levels of CXCL11 and IL-17 were observed in UC. Flagellin could induce the production of CXCL11 in CD14(+) cells that facilitated the development of Th17 cells. In a skewed Th1 response environment flagellin induces intestinal inflammation, with IL-17 expression predominant. CXCR3/CXCL11 pathway is involved in microbial product flagellin-induced intestinal inflammation in which the Th17 response plays an important role.     

Granulocyte chemotactic protein-2 mediates adaptive immunity in part through IL-8Rbeta interactions

Chemokines constitute a large family of structurally related proteins that play a role in leukocyte migration and differentiation. Indeed, the early expression of human CXC chemokine receptor 1 (hCXCR1) and hCXCR2 [homologous to mouse interleukin (IL)-8Rbeta] ligands by the epithelium is a hallmark of the mucosal host defense. Mice lack IL-8; however, granulocyte chemotactic protein-2 (GCP-2)/lipopolysaccharide-induced CXC chemokine, a murine homologue of human GCP-2, has 32% and 61% sequence identity to human IL-8 and GCP-2, respectively, and binds hCXCR1, hCXCR2, and mouse IL-8Rbeta. To better understand the role of GCP-2 in adaptive immunity and as a nasal adjuvant, we characterized the exogenous effects of this CXC chemokine on cellular and humoral mucosal immune responses. GCP-2 significantly enhanced serum immunoglobulin G (IgG) and mucosal IgA antibodies through increased cytokine secretion by CD4+ T cells. These alterations in humoral and cellular responses were preceded by an increase in the number of B cells in the nasal tract, a decrease in the number of CD4+ T cells in the nasal tract as well as cervical lymph nodes, and an increase in the number of neutrophils in the nasal tract 12 h after GCP-2 immunization. This chemokine also modulated CD28 expression by CD4+ T cells during CD3epsilon stimulation of wild-type mice. GCP-2 increased CD80 and CD86 expression on B cells during in vitro stimulation in a dose-dependent manner. In contrast, cytokine and costimulatory molecule enhancement by GCP-2 was not induced by lymphocytes from IL-8Rbeta-/- mice, suggesting that GCP-2 modulates cellular immunity in part through IL-8Rbeta interactions.     

TLR ligands and cytokines induce CXCR3 ligands in endothelial cells: enhanced CXCL9 in autoimmune arthritis

CXC chemokines are potent attractants of neutrophil granulocytes, T cells or natural killer cells. Toll-like receptors (TLR) recognize microbial components and are also activated by endogenous molecules possibly implicated in autoimmune arthritis. In contrast to CXC chemokine ligand 8 (CXCL8), no CXC chemokine receptor 3 (CXCR3) ligand (ie CXCL9, CXCL10 and CXCL11) was induced by bacterial TLR ligands in human microvascular endothelial cells (HMVEC). However, peptidoglycan (PGN), double-stranded (ds) RNA or lipopolysaccharide (LPS) (TLR2, TLR3 or TLR4 ligands, respectively) synergized with interferon-gamma (IFN-gamma) at inducing CXCL9 and CXCL10. In contrast, enhanced CXCL11 secretion was only obtained when IFN-gamma was combined with TLR3 ligand. Furthermore, flagellin, loxoribine and unmethylated CpG oligonucleotide (TLR5, TLR7 and TLR9 ligands, respectively) did not enhance IFN-gamma-dependent CXCR3 ligand production in HMVEC. In analogy with TLR ligands, tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), in combination with IFN-gamma, synergistically induced CXCL9 and CXCL11 in HMVEC and human fibroblasts, two fundamental cell types delineating the joint cavity. Etanercept, a humanized soluble recombinant p75 TNF-receptor/IgG(1)Fc fusionprotein, neutralized synergistic CXCL9 production induced by TNF-alpha plus IFN-gamma, but not synergy between IFN-gamma and the TLR ligands PGN or LPS. Synovial chemokine concentrations exemplify the physiopathological relevance of the observed in vitro chemokine production patterns. In synovial fluids of patients with spondylarthropathies (ie ankylosing spondylitis or psoriatic arthritis) or rheumatoid arthritis, significantly enhanced CXCL9, but not CXCL11 levels, were detected compared to concentrations in synovial fluids of patients with metabolic crystal-induced arthritis. Thus, CXCL9 is an important chemokine in autoimmune arthritis.     

Modulation of chemokine production in lung microvascular endothelial cells by dopamine is mediated via an oxidative mechanism

Serum concentrations of catecholamines are high in patients with sepsis or acute respiratory distress syndrome (ARDS). Because chemokines mediate the recruitment of neutrophils into inflammatory sites, we addressed the question of whether dopamine (DA) is able to influence chemokine production in endothelial cells under basal and proinflammatory conditions. To this end, lung microvascular endothelial cells (LMVEC) were stimulated or not for 24 h with the bacterial toxins lipopolysaccharide (LPS) (1 microg/ml) or lipoteichonic acid (LTA) (10 microg/ml) in the presence or absence of various concentrations of DA (1-100 microg/ml). Whereas under basal and stimulatory conditions, the addition of DA to endothelial cells dose-dependently increased IL-8 production, the production of ENA-78 and Gro-alpha was significantly inhibited (P < 0.01). This effect could still be demonstrated when the cells were stimulated for up to 3 h with LPS before DA administration. Similar findings were detected for the mRNA expression of these chemokines. The influence of DA on chemokine production was not receptor mediated and could be prevented by antioxidants or radical scavengers. Moreover, addition of H(2)O(2) to endothelial cells gave results similar to those observed with DA stimulation, suggesting a pivotal role for reactive oxygen species in DA-mediated modulation of chemokine production in endothelial cells. Our data thus demonstrate that DA administration results in the induction of oxidative stress, with profound effects on endothelial chemokine production.