TNF-alpha and IL-4 regulate expression of IL-13 receptor alpha2 on human fibroblasts

Two interleukin 13 receptors (IL-13Rs) have been identified as IL-13Ralpha1 and IL-13Ralpha2. IL-13Ralpha1 is composed of a heterodimer consisting of IL-13Ralpha1 and IL-4 receptor alpha (IL-4Ralpha) as a signaling subunit. In contrast, IL-13Ralpha2 is known as a decoy receptor for IL-13. In this study, we investigated the expression of IL-13Rs on human fibroblasts. IL-13Ralpha2 was significantly up-regulated after stimulation with tumor necrosis factor-alpha (TNF-alpha) and/or IL-4. In contrast, IL-13Ralpha1 was constitutively detectable and was not up-regulated. After the induction of IL-13alpha2 by IL-4, STAT6 phosphorylation through IL-13Ralpha1 by IL-13 was inhibited. We also detected large intracellular pools of IL-13Ralpha2 in fibroblasts quantitatively. Furthermore, mobilization of the IL-13Ralpha2 protein stores from the cytoplasm to the cell surface was prevented by an inhibitor of protein transport, brefeldin-A. These results indicate that TNF-alpha and IL-4 synergistically up-regulate the expression of IL-13Ralpha2 decoy receptor on human fibroblasts by inducing gene expression and mobilizing intracellular receptors, and thus may down-regulate the IL-13 signaling.     

MicroRNA-424 regulates the expression of CX3CL1 (fractalkine) in human microvascular endothelial cells during Rickettsia rickettsii infection

Cytokines and chemokines trigger complex intracellular signaling through specific receptors to mediate immune cell recruitment and activation at the sites of infection. CX3CL1 (Fractalkine), a membrane-bound chemokine also capable of facilitating intercellular interactions as an adhesion molecule, contributes to host immune responses by virtue of its chemoattractant functions. Published studies have documented increased CX3CL1 expression in target tissues in a murine model of spotted fever rickettsiosis temporally corresponding to infiltration of macrophages and recovery from infection. Because pathogenic rickettsiae primarily target vascular endothelium in the mammalian hosts, we have now determined CX3CL1 mRNA and protein expression in cultured human microvascular endothelial cells (HMECs) infected in vitro with Rickettsia rickettsii. Our findings reveal 15.5 ± 4.0-fold and 12.3 ± 2.3-fold increase in Cx3cl1 mRNA expression at 3 h and 24 h post-infection, coinciding with higher steady-state levels of the corresponding protein in comparison to uninfected HMECs. Since CX3CL1 is a validated target of microRNA (miR)-424-5p (miR-424) and our earlier findings demonstrated robust down-regulation of miR-424 in R. rickettsii-infected HMECs, we further explored the possibility of regulation of CX3CL1 expression during rickettsial infection by miR-424. As expected, R. rickettsii infection resulted in 87 ± 5% reduction in miR-424 expression in host HMECs. Interestingly, a miR-424 mimic downregulated R. rickettsii-induced expression of CX3CL1, whereas an inhibitor of miR-424 yielded a converse up-regulatory effect, suggesting miR-424-mediated regulation of CX3CL1 during infection. Together, these findings provide the first evidence for the roles of a host microRNA in the regulation of an important bifunctional chemokine governing innate immune responses to pathogenic rickettsiae.     

15-Deoxy-D12,14-prostaglandin J2 inhibits CX3CL1/fractalkine expression in human endothelial cells

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma)is a member of nuclear hormone receptor superfamily, and is knownto play a role in various biological processes including inflammatoryresponses and adipocyte differentiation. CX3CL1/fractalkineis a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes.Endothelial cells produce fractalkine when stimulated with cytokinessuch as interleukin-1 (IL-1), tumour necrosis factor-alpha andinterferon-gamma (IFN-gamma). We herein report that 15-deoxy-n12,14 -prostaglandinJ2 (15d-PGJ2), a PPAR-gamma agonist,inhibits the expression of fractalkine induced by IFN-gamma orIL-1beta in human endothelial cells. Agonist for PPAR-alpha (WY14643)or PPAR-gamma (ciglitazone) did not inhibit the cytokine-inducedfractalkine expression, and the effect of 15d-PGJ2 maybe independent of PPAR. 15-Deoxy-D12,14 prostaglandinJ2 also inhibited the adhesion of blood mononuclear cellsto endothelial monolayers treated with IFN-gamma or IL-1beta.The data suggest that 15d-PGJ2 regulates inflammatoryreactions, at least in part, through the inhibition of fractalkineexpression and leucocyte traffic through the endothelium.     

Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1)

Fractalkine (CX3CL1) is an unusual member of the chemokine family that is synthesized with its chemokine domain at the end of a mucin-rich, transmembrane stalk. This membrane-bound localization allows fractalkine to function as an adhesion molecule for cells bearing its receptor, CX3CR1. In addition, fractalkine can be proteolytically released from the cell surface, generating a soluble molecule that functions as a chemoattractant similar to the other members of the chemokine family. In this study, we have examined the mechanisms that regulate the conversion between these two functionally distinct forms of fractalkine. We demonstrate that under normal conditions fractalkine is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it becomes a target for metalloproteinase-dependent cleavage that causes the release of a fragment containing the majority of the fractalkine extracellular domain. We show that the cleavage of fractalkine can be markedly enhanced by stimulating cells with phorbol 12-myristate 13-acetate (PMA), and we identify tumor necrosis factor-alpha converting enzyme (TACE; ADAM17) as the protease responsible for this PMA-induced fractalkine release. In addition, we provide data showing that TACE-mediated fractalkine cleavage occurs at a site distinct from the dibasic juxtamembrane motif that had been suggested previously based on protein sequence homologies. The identification of TACE as a major protease responsible for the conversion of fractalkine from a membrane-bound adhesion molecule to a soluble chemoattractant will provide new information for understanding the physiological function of this chemokine.     

Inhibition of CX3CL1 (fractalkine) improves experimental autoimmune myositis in SJL/J mice

Idiopathic inflammatory myopathy is a chronic inflammatory muscle disease characterized by mononuclear cell infiltration in the skeletal muscle. The infiltrated inflammatory cells express various cytokines and cytotoxic molecules. Chemokines are thought to contribute to the inflammatory cell migration into the muscle. We induced experimental autoimmune myositis (EAM) in SJL/J mice by immunization with rabbit myosin and CFA. In the affected muscles of EAM mice, CX3CL1 (fractalkine) was expressed on the infiltrated mononuclear cells and endothelial cells, and its corresponding receptor, CX3CR1, was expressed on the infiltrated CD4 and CD8 T cells and macrophages. Treatment of EAM mice with anti-CX3CL1 mAb significantly reduced the histopathological myositis score, the number of necrotic muscle fibers, and infiltration of CD4 and CD8 T cells and macrophages. Furthermore, treatment with anti-CX3CL1 mAb down-regulated the mRNA expression of TNF-alpha, IFN-gamma, and perforin in the muscles. Our results suggest that CX3CL1-CX3CR1 interaction plays an important role in inflammatory cell migration into the muscle tissue of EAM mice. The results also point to the potential therapeutic usefulness of CX3CL1 inhibition and/or blockade of CX3CL1-CX3CR1 interaction in idiopathic inflammatory myopathy.     

Soluble interleukin-6 receptor alpha inhibits the cytokine-Induced fractalkine/CX3CL1 expression in human vascular endothelial cells in culture

Soluble form of IL-6 receptor alpha (sIL-6R) is known to serve as an agonist, without exogenous IL-6, on endothelial cells which do not express IL-6R but have only IL-6 receptor beta chain, gp130. We investigated the effect of sIL-6R on fractalkine expression in human umbilical vein endothelial cells (HUVECs) in culture. sIL-6R markedly inhibited HUVEC fractalkine/CX3CL1 expression induced by interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma. IL-1alpha-induced fractalkine expression was inhibited by sIL-6R in time- and concentration-dependent manners. The experiment using actinomycin D indicated that sIL-6R lowered the stability of fractalkine mRNA. The inhibitory effect of sIL-6R was reversed by anti-gp130 neutralizing antibody. sIL-6R inhibited adhesion of mononuclear cells (MNCs) to HUVEC monolayers stimulated with IFN-gamma, but it did not inhibit the adhesion to monolayers stimulated with IL-1alpha. MNC chemotactic activity of conditioned medium of HUVEC stimulated with IL-1alpha or IFN-gamma was inhibited by co-treatment with sIL-6R. sIL-6R may play a regulatory role in immune responses by modulating the interaction between leukocytes and the vascular endothelium.     

The CC chemokine MCP-1 stimulates surface expression of CX3CR1 and enhances the adhesion of monocytes to fractalkine/CX3CL1 via p38 MAPK

The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.     

Protective roles of the fractalkine/CX3CL1-CX3CR1 interactions in alkali-induced corneal neovascularization through enhanced antiangiogenic factor expression

Macrophages accumulate during the course of corneal neovascularization, but its mechanisms and roles still remain elusive. To address these points, we herein examined corneal neovascularization after alkali injury in mice deficient in fractalkine receptor/CX3CR1, which is normally expressed by macrophages. After alkali injury, the mRNA expression of CX3CR1 was augmented along with accumulation of F4/80-positive macrophages and Gr-1-positive neutrophils in the corneas. Compared with wild-type mice, CX3CR1-deficient mice exhibited enhanced corneal neovascularization 2 wk after injury, as evidenced by enlarged CD31-positive areas. Concomitantly, the accumulation of F4/80-positive macrophages, but not Gr-1-positive neutrophils, was markedly attenuated in CX3CR1-deficient mice compared with wild-type mice. The intraocular mRNA expression of vascular endothelial growth factor (VEGF) was enhanced to similar extents in wild-type and CX3CR1-deifient mice after the injury. However, the mRNA expression of antiangiogenic factors, thrombospondin (TSP) 1, TSP-2, and a disintegrin and metalloprotease with thrombospondin (ADAMTS) 1, was enhanced to a greater extent in wild-type than CX3CR1-deificient mice. A double-color immunofluorescence analysis demonstrated that F4/80-positive cells also expressed CX3CR1 and ADAMTS-1 and that TSP-1 and ADAMTS-1 were detected in CX3CR1-positive cells. CX3CL1 enhanced TSP-1 and ADAMTS-1, but not VEGF, expression by peritoneal macrophages. Moreover, topical application of CX3CL1 inhibited corneal neovascularization at 2 wk, along with enhanced intraocular expression of TSP-1 and ADAMTS-1 but not VEGF. Thus, these observations indicate that accumulation of CX3CR1-positive macrophages intraocularly can dampen alkali-induced corneal neovascularization by producing antiangiogenic factors such as TSP-1 and ADAMTS-1 and suggest the potential therapeutic efficacy of using CX3CL1 against alkali-induced corneal neovascularization.     

Hydrogen sulfide inhibits the development of atherosclerosis with suppressing CX3CR1 and CX3CL1 expression

Hydrogen sulfide, as a novel gaseous mediator, has been suggested to play a key role in atherogenesis. However, the precise mechanisms by which H(2)S affects atherosclerosis remain unclear. Therefore, the present study aimed to investigate the potential role of H(2)S in atherosclerosis and the underlying mechanism with respect to chemokines (CCL2, CCL5 and CX3CL1) and chemokine receptors (CCR2, CCR5, and CX3CR1) in macrophages. Mouse macrophage cell line RAW 264.7 or mouse peritoneal macrophages were pre-incubated with saline or NaHS (50 μM, 100 μM, 200 μM), an H(2)S donor, and then stimulated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS). It was found that NaHS dose-dependently inhibited IFN-γ or LPS-induced CX3CR1 and CX3CL1 expression, as well as CX3CR1-mediated chemotaxis in macrophages. Overexpression of cystathionine γ-lyase (CSE), an enzyme that catalyzes H(2)S biosynthesis resulted in a significant reduction in CX3CR1 and CX3CL1 expression as well as CX3CR1-mediated chemotaxis in stimulated macrophages. The inhibitory effect of H(2)S on CX3CR1 and CX3CL1 expression was mediated by modulation of proliferators-activated receptor-γ (PPAR-γ) and NF-κB pathway. Furthermore, male apoE(-/-) mice were fed a high-fat diet and then randomly given NaHS (1 mg/kg, i.p., daily) or DL-propargylglycine (PAG, 10 mg/kg, i.p., daily). NaHS significantly inhibited aortic CX3CR1 and CX3CL1 expression and impeded aortic plaque development. NaHS had a better anti-atherogenic benefit when it was applied at the early stage of atherosclerosis. However, inhibition of H(2)S formation by PAG increased aortic CX3CR1 and CX3CL1 expression and exacerbated the extent of atherosclerosis. In addition, H(2)S had minimal effect on the expression of CCL2, CCL5, CCR2 and CCR5 in vitro and in vivo. In conclusion, these data indicate that H(2)S hampers the progression of atherosclerosis in fat-fed apoE(-/-) mice and downregulates CX3CR1 and CX3CL1 expression on macrophages and in lesion plaques.     

Serum level of soluble CX3CL1/fractalkine is elevated in patients with polymyositis and dermatomyositis, which is correlated with disease activity

Introduction

Polymyositis (PM) and dermatomyositis (DM) are chronic inflammatory muscle diseases, in which chemokines are thought to contribute to inflammatory cell migration into muscle. In this study, we retrospectively analyzed the expressions of CX3CL1/fractalkine and its corresponding receptor, CX3CR1, in muscle and lung with interstitial lung disease (ILD) of PM patients and DM patients, and determined the correlation between serum soluble CX3CL1 level and disease activity.

Methods

Expressions of CX3CL1 and CX3CR1 in muscle and lung tissue were analyzed by immunohistochemistry. Serum CX3CL1 concentrations were measured by ELISA. For evaluation of patients' disease activity, serum creatinine kinase, manual muscle testing, and the alveolar-arterial oxygen pressure difference were used independently.

Results

CX3CL1 was expressed on infiltrated mononuclear cells and endothelial cells in muscle affected by PM and DM and in lung with ILD, whereas CX3CR1 was expressed on some CD4⁺ T cells, a majority of CD8⁺ T cells, and most macrophages in muscle, and on infiltrated mononuclear cells in the lung. Serum soluble CX3CL1 was significantly higher in PM patients and DM patients than in healthy controls. The CX3CL1 level was correlated with serum creatinine kinase and manual muscle testing score. In patients with PM and DM with ILD, serum CX3CL1 was also correlated with alveolar-arterial oxygen pressure difference. Furthermore, CX3CL1 was significantly decreased after conventional treatment.

Conclusions

The interaction between CX3CL1 and CX3CR1 might contribute to the inflammatory cell infiltration into affected muscle and lung with ILD in PM patients and DM patients. Serum CX3CL1 level could be a surrogate marker of disease activity.     

Decreased expression of the fractalkine receptor CX3CR1 on circulating monocytes as new feature of sepsis-induced immunosuppression

Although it is known that septic shock rapidly induces immune dysfunctions, which contribute to the impaired clearance of microorganisms observed in patients, the mechanisms for this phenomenon remain incompletely understood. We recently observed, in a microarray study, an altered circulating leukocyte CX3CR1 mRNA expression associated with patients' mortality. As monocytes play a central role in septic shock pathophysiology and express high levels of CX3CR1, we therefore further investigated the alteration of CX3CR1 expression and of its ligand fractalkine (CX3CL1) on those cells in this clinical condition. We observed that CX3CR1 expression (both mRNA and protein) was severely down-regulated in monocytes and consequently associated with a lack of functionality upon fractalkine challenge. Importantly, nonsurvivors presented with significantly sustained lower expression in comparison with survivors. This down-regulation was reproduced by incubation of cells from healthy individuals with LPS, whole bacteria (Escherichia coli and Staphylococcus aureus), and, to a lower extent, with corticosteroids-in accordance with the concept of LPS-induced monocyte deactivation. In addition, CX3CL1 serum concentrations were elevated in patients supporting the hypothesis of increased cleavage of the membrane-anchored form expressed by endothelial cells. As CX3CR1/CX3CL1 interaction preferentially mediates arrest and migration of proinflammatory cells, the present observations may contribute to patients' inability to kill invading microorganisms. This could represent an important new feature of sepsis-induced immunosuppression.     

Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion

The seven-transmembrane receptor CX(3)CR1 is a specific receptor for the novel CX(3)C chemokine fractalkine (FKN) (neurotactin). In vitro data suggest that membrane anchoring of FKN, and the existence of a shed, soluble FKN isoform allow for both adhesive and chemoattractive properties. Expression on activated endothelium and neurons defines FKN as a potential target for therapeutic intervention in inflammatory conditions, particularly central nervous system diseases. To investigate the physiological function of CX(3)CR1-FKN interactions, we generated a mouse strain in which the CX(3)CR1 gene was replaced by a green fluorescent protein (GFP) reporter gene. In addition to the creation of a mutant CX(3)CR1 locus, this approach enabled us to assign murine CX(3)CR1 expression to monocytes, subsets of NK and dendritic cells, and the brain microglia. Analysis of CX(3)CR1-deficient mice indicates that CX(3)CR1 is the only murine FKN receptor. Yet, defying anticipated FKN functions, absence of CX(3)CR1 interferes neither with monocyte extravasation in a peritonitis model nor with DC migration and differentiation in response to microbial antigens or contact sensitizers. Furthermore, a prominent response of CX(3)CR1-deficient microglia to peripheral nerve injury indicates unimpaired neuronal-glial cross talk in the absence of CX(3)CR1.     

A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX(3)CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-alpha-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX(3)CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX(3)CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX(3)CR1 Ab. Treatment with the anti-CX(3)CR1 Ab significantly prolonged allograft survival from 7 +/- 1 to 49 +/- 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.     

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.     

Fractalkine/CX3CL1 production by human airway smooth muscle cells: induction by IFN-gamma and TNF-alpha and regulation by TGF-beta and corticosteroids

Chemokine synthesis by airway smooth muscle cells (ASMC) may be an important process underlying inflammatory cell recruitment in airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Fractalkine (FKN) is a recently described CX(3)C chemokine that has dual functions, serving as both a cell adhesion molecule and a chemoattractant for monocytes and T cells, expressing its unique receptor, CX(3)CR1. We investigated FKN expression by human ASMC in response to the proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma, the T helper 2-type cytokines IL-4, IL-10, and IL-13, and the fibrogenic cytokine transforming growth factor (TGF)-beta. Neither of these cytokines alone had any significant effect on ASMC FKN production. Combined stimulation with IFN-gamma and TNF-alpha induced FKN mRNA and protein expression in a time- and concentration-dependent manner. TGF-beta had a significant inhibitory effect on cytokine-induced FKN mRNA and protein expression. Dexamethasone (10(-8)-10(-6) M) significantly upregulated cytokine-induced FKN mRNA and protein expression. Finally, we used selective inhibitors of the mitogen-activated protein kinases c-Jun NH(2)-terminal kinase (JNK) (SP-610025), p38 (SB-203580), and extracellular signal-regulated kinase (PD-98095) to investigate their role in FKN production. SP-610025 (25 microM) and SB-203580 (20 microM), but not PD-98095, significantly attenuated cytokine-induced FKN protein synthesis. IFN-gamma- and TNF-alpha-induced JNK phosphorylation remained unaltered in the presence of TGF-beta but was inhibited by dexamethasone, indicating that JNK is not involved in TGF-beta- or dexamethasone-mediated regulation of FKN production. In summary, FKN production by human ASMC in vitro is regulated by inflammatory and anti-inflammatory factors.     

IL-15 alters expression and function of the chemokine receptor CX3CR1 in human NK cells

The chemokine receptor CX3CR1 is thought to regulate inflammation in part by modulating NK cell adhesion, migration, and killing in response to its ligand CX3CL1 (fractalkine). Recent reports indicate that IL-15, which is essential for development and survival of NK cells, may negatively regulate CX3CR1 expression, however, the effects of the cytokine on human NK cell CX3CR1 expression and function have not been fully delineated. Here, we demonstrate that short term culture in IL-15 decreases surface expression of CX3CR1 on cultured CD56+ cells from human blood resulting in diminished chemotaxis and calcium flux in response to CX3CL1. Cells cultured long term in IL-15 (more than five days) completely lost surface expression as well as mRNA and protein for CX3CR1. The effect was specific since mRNA for CCR5 was increased and mRNA for CXCR4 was unchanged in these cells by IL-15. Thus, exogenous IL-15 is a negative regulator of CX3CR1 expression and function in human CD56+ NK cells. The data imply that the use of IL-15 alone to expand NK cells ex vivo for immunotherapy may produce cells impaired in their ability to traffic to sites of inflammation.