Increased expression of fractalkine (CX3CL1) and its receptor, CX3CR1, in Wegener's granulomatosis--possible role in vascular inflammation.

OBJECTIVE: Based on the function of fractalkine (CX3CL1), the unique member of the CX3C chemokine subfamily, in endothelial-related inflammation, we hypothesized a role for CX3CL1 and its receptor (CX3CR) in Wegener's granulomatosis (WG). In the present study, this hypothesis was tested by different experimental approaches. METHODS: We examined plasma levels of CX3CL1 (enzyme immunoassay) and CX3CR1 expression in peripheral blood mononuclear cells (PBMCs) (real-time quantitative RT-PCR and flow cytometry) in 18 WG patients and 15 healthy controls. In eight of these individuals, we also examined CX3CR1-mediated chemotaxis and adhesion in T cells and monocytes as well as the effects of CX3CL1 on monocyte chemoattractant protein (MCP) 1 levels in PBMC supernatants. RESULTS: Our main findings were: (i) WG patients had markedly increased plasma levels of CX3CL1, with particularly high levels in those with active disease, (ii) These increased CX3CL1 levels were accompanied by enhanced expression of its corresponding receptor, CX3XR1, in PBMC, primarily reflecting an increased proportion of CX3CR1(+)CD3(+)CD4(+) T cells and (iii) The up-regulation of CX3CR1 in PBMC from WG patients affected their functional potential as shown by CX3CL1-induced enhancement of chemotaxis, adhesion, responses as well as MCP-1 stimulation. CONCLUSION: Based on the ability of CX3CL1 to promote leucocyte infiltration into the vessel wall of inflammatory levels, it is tempting to hypothesize that increased CX3CL1/CX3CR1 interaction could be involved in the pathogenesis of the granulomatous vasculitis characterizing WG.

Recruitment of CD16+ monocytes into synovial tissues is mediated by fractalkine and CX3CR1 in rheumatoid arthritis patients.

CD16+ monocytes, identified as a minor population of monocytes in human peripheral blood, have been implicated in several inflammatory diseases, including rheumatoid arthritis (RA). Fractalkine (FKN, CX3CL1), a member of the CX3 C subfamily, is induced by pro-inflammatory cytokines, while a receptor for FKN, CX3CR1, is capable of mediating both leukocyte migration and firm adhesion. Here, we investigated the role of FKN and CX3CR1 in activation of CD16+ monocytes and their recruitment into synovial tissues in RA patients. High levels of soluble FKN were detected in the synovial fluid and sera of RA patients. Circulating CD16+ monocytes showed a higher level of CX3CR1 expression than CD16- monocytes in both RA patients and healthy subjects. High level expression of CX3CR1 was also seen in CD16+ monocytes localized to the lining layer in RA synovial tissue. In the in vitro culture experiments, IL-10 induced CX3CR1 expression on the surface of monocytes, and TNFalpha induced membrane-bound FKN as well as soluble FKN expression in synovial fibroblasts. Moreover, soluble FKN was capable of inducing IL-1beta and IL-6 by activated monocytes. These results suggest that FKN might preferentially mediate migration and recruitment of CD16+ monocytes, and might contribute to synovial tissue inflammation.

Increased serum levels of soluble fractalkine (CX3CL1) correlate with disease activity in rheumatoid vasculitis.

OBJECTIVE: To determine levels of soluble fractalkine (sFkn) in rheumatoid arthritis (RA) patients with and without rheumatoid vasculitis (RV), and to assess the relationship of sFkn levels to disease activity. METHODS: Serum was obtained from 98 RA patients (54 without vasculitis, 36 with extraarticular manifestations but without histologically proven vasculitis, and 8 with histologically proven vasculitis) and from 38 healthy individuals. Levels of sFkn were measured by enzyme-linked immunosorbent assay. Expression of Fkn and CX(3)CR1 was quantified by real-time polymerase chain reaction. Vasculitis disease activity was assessed using the Birmingham Vasculitis Activity Score and the Vasculitis Activity Index. RESULTS: Serum sFkn levels were significantly higher in patients with RA than in controls and were significantly higher in RA patients with RV than in those without vasculitic complications. Statistically significant correlations were observed between serum sFkn levels in RA patients and levels of C-reactive protein, rheumatoid factor, immune complex, and complement. In the RV group, sFkn levels also correlated with disease activity. Immunohistochemical analysis indicated that Fkn levels were associated mainly with endothelial cells in vasculitic arteries. In addition, expression of CX(3)CR1 messenger RNA was significantly greater in peripheral blood mononuclear cells from patients with active RV than in those from other RA patients or controls. Notably, serum sFkn levels were significantly diminished following successful treatment and clinical improvement. CONCLUSION: These findings suggest that Fkn and CX(3)CR1 play crucial roles in the pathogenesis of RV and that sFkn may serve as a serologic inflammatory marker of disease activity in RA patients with vasculitis.

Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, is elevated in coronary artery disease and is reduced during statin therapy.

OBJECTIVE: Recent data derived primarily from studies in animal models suggest that fractalkine (CX3CL1) and its cognate receptor, CX3CR1, play a role in atherogenesis. We, therefore, hypothesized that enhanced CX3CL1/CX3CR1 expression may promote atherogenesis in patients with coronary artery disease (CAD). METHODS AND RESULTS: We examined the plasma levels of CX3CL1 and CX3CR1 expression in peripheral blood mononuclear cells (PBMC) in various CAD populations (30 patients with previous myocardial infarction, 40 patients with stable angina, 40 patients with unstable angina, and a total of 35 controls) and used various experimental approaches to characterize CX3CL1-mediated leukocyte responses. We found that the plasma levels of CX3CL1 are greatly increased in CAD, particularly in unstable disease. The parallel increase of CX3CR1 expression in PBMC was predominantly attributable to an expansion of the (CX3CR1+)(CD3+)(CD8+) T cell subset and was associated with enhanced chemotactic, adhesive, and inflammatory responses to CX3CL1. Statin therapy for 6 months reduced the expression of CX3CL1 and CX3CR1, reaching statistical significance for both parameters only during aggressive (atorvastatin, 80 mg qd) but not conventional (simvastatin, 20 mg qd) therapy. Consequently, the functional responses of the PBMC to CX3CL1 including migration, adhesion, and secretion of interleukin-8 were attenuated by the treatments. CONCLUSIONS: Our results suggest that the CX3CL1/CX3CR1 dyad may contribute to atherogenesis and plaque destabilization in human CAD.

Fractalkine in the peritoneal fluid of women with endometriosis.

OBJECTIVE: The objective of this study was to evaluate the presence of fractalkine in the ascites and the association between fractalkine levels in the ascites and endometriosis. METHODS: Peritoneal fluids and peripheral blood samples were obtained from patients undergoing laparoscopy for infertility work-up or laparoscopic cystectomy. Three samples of peritoneum were obtained from patients undergoing hysterectomy. Western blotting, RT-PCR and immunohistochemistry were performed. RESULTS: Fractalkine protein was detected in the ascites. Positive staining was confirmed in peritoneal surface cells and perivascular cells of the peritoneum. CX3CR1 positive cells were present in the cells in the peritoneal fluid. The fractalkine concentrations in the ascites of patients with endometriosis were lower than those without endometriosis. There was no significant difference between serum fractalkine levels in patients with and without endometriosis. CONCLUSION: The decreased level of fractalkine found in the peritoneal fluid of patients with endometriosis may contribute to the pathogenesis of endometriosis.

Elevated levels of soluble fractalkine in active systemic lupus erythematosus: potential involvement in neuropsychiatric manifestations.

OBJECTIVE: To determine levels of the soluble form of the chemokine fractalkine (sFkn) and its receptor, CX(3)CR1, in patients with systemic lupus erythematosus (SLE) with neuropsychiatric involvement (NPSLE) and in SLE patients without neuropsychiatric involvement, and to assess their relationship with disease activity and organ damage. METHODS: Levels of sFkn in serum and cerebrospinal fluid (CSF) were measured by enzyme-linked immunosorbent assay. Expression of Fkn and CX(3)CR1 was quantified using real-time polymerase chain reaction. Surface expression of CX(3)CR1 on peripheral blood mononuclear cells (PBMCs) was determined by flow cytometry. Disease activity and organ damage were assessed using the SLE Disease Activity Index (SLEDAI) and the Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index, respectively. RESULTS: Serum sFkn levels were significantly higher in patients with SLE than in patients with rheumatoid arthritis (RA) or healthy controls. In addition, significant correlations between serum sFkn levels and the SLEDAI, the SLICC/ACR Damage Index, anti-double-stranded DNA and anti-Sm antibody titers, immune complex levels (C1q), and serum complement levels (CH50) were observed. Expression of CX(3)CR1 was significantly greater in PBMCs from patients with active SLE than in those from RA patients or healthy controls. Levels of sFkn were also significantly higher in CSF from untreated patients with newly diagnosed NPSLE than in SLE patients without neuropsychiatric involvement; treatment reduced both serum and CSF levels of sFkn in patients with SLE. CONCLUSION: Soluble Fkn and CX(3)CR1 may play key roles in the pathogenesis of SLE, including the neuropsychiatric involvement. Soluble Fkn is also a serologic marker of disease activity and organ damage in patients with SLE, and its measurement in CSF may be useful for the diagnosis of NPSLE and followup of patients with NPSLE.

Up regulated expression of fractalkine/CX3CL1 and CX3CR1 in patients with systemic sclerosis.

BACKGROUND: Fractalkine expressed on endothelial cells mediates activation and adhesion of leucocytes expressing its receptor, CX(3)CR1. Soluble fractalkine exhibits chemotactic activity for leucocytes expressing CX(3)CR1. OBJECTIVE: To determine the role of fractalkine and its receptor in systemic sclerosis (SSc) by assessing their expression levels in patients with this disease. METHODS: The expression of fractalkine and CX(3)CR1 in the skin and lung tissues was immunohistochemically examined. Circulating soluble fractalkine levels were examined by enzyme linked immunosorbent assay (ELISA). Blood samples from patients with SSc were stained for CX(3)CR1 with flow cytometric analysis. RESULTS: CX(3)CR1 levels on peripheral monocytes/macrophages and T cells were found to be raised in patients with diffuse cutaneous SSc. The numbers of cells expressing CX(3)CR1, including monocytes/macrophages, were increased in the lesional skin and lung tissues from patients with diffuse cutaneous SSc. Fractalkine was strongly expressed on endothelial cells in the affected skin and lung tissues. Soluble fractalkine levels were significantly raised in sera and were associated with raised erythrocyte sedimentation rates, digital ischaemia, and severity of pulmonary fibrosis. CONCLUSIONS: Up regulated expression of fractalkine and CX(3)CR1 cooperatively augments the recruitment of mononuclear cells expressing CX(3)CR1 into the affected tissue of SSc, leading to inflammation and vascular injury.

The chemokine fractalkine in patients with severe traumatic brain injury and a mouse model of closed head injury.

The potential role of the chemokine Fractalkine (CX3CL1) in the pathophysiology of traumatic brain injury (TBI) was investigated in patients with head trauma and in mice after experimental cortical contusion. In control individuals, soluble (s)Fractalkine was present at low concentrations in cerebrospinal fluid (CSF) (12.6 to 57.3 pg/mL) but at much higher levels in serum (21,288 to 74,548 pg/mL). Elevation of sFractalkine in CSF of TBI patients was observed during the whole study period (means: 29.92 to 535.33 pg/mL), whereas serum levels remained within normal ranges (means: 3,100 to 59,159 pg/mL). Based on these differences, a possible passage of sFractalkine from blood to CSF was supported by the strong correlation between blood-brain barrier dysfunction (according to the CSF-/serum-albumin quotient) and sFractalkine concentrations in CSF (R = 0.706; P < 0.01). In the brain of mice subjected to closed head injury, neither Fractalkine protein nor mRNA were found to be augmented; however, Fractalkine receptor (CX3CR1) mRNA steadily increased peaking at 1 week postinjury (P < 0.05, one-way analysis of variance). This possibly implies the receptor to be the key factor determining the action of constitutively expressed Fractalkine. Altogether, these data suggest that the Fractalkine-CX3CR1 protein system may be involved in the inflammatory response to TBI, particularly for the accumulation of leukocytes in the injured parenchyma.

Involvement of fractalkine and macrophage inflammatory protein-1 alpha in moderate-severe depression.

Moderate-severe depression (MSD) is linked to overexpression of proinflammatory cytokines and chemokines. Fractalkine (FKN) and macrophage inflammatory protein-1 alpha (MIP-1alpha) are, respectively, members of CX3C and C-C chemokines, and both are involved in recruiting and activating mononuclear phagocytes in the central nervous system. We analysed the presence of FKN and MIP-1alpha in sera of untreated MSD patients and healthy donors. High FKN levels were observed in all MSD patients as compared with values only detectable in 26% of healthy donors. MIP-1alpha was measurable in 20% of patients, while no healthy donors showed detectable chemokine levels. In conclusion, we describe a previously unknown involvement of FKN in the pathogenesis of MSD, suggesting that FKN may represent a target for a specific immune therapy of this disease.

Expression of fractalkine and its receptor, CX3CR1, in atopic dermatitis: possible contribution to skin inflammation.

BACKGROUND: Fractalkine (FKN) induces activation and adhesion of leukocytes expressing its receptor, CX(3)CR1. FKN is released from the cell surface through proteolytic cleavage as soluble FKN (sFKN). OBJECTIVE: We sought to assess FKN and CX(3)CR1 expression in the skin, serum sFKN levels, and CX(3)CR1 expression on blood leukocytes in patients with atopic dermatitis (AD). METHODS: FKN and CX(3)CR1 expression in the skin was examined immunohistochemically. mRNA expression of FKN, thymus and activation-regulated chemokine, and macrophage-derived chemokine in the skin was assessed by means of real-time RT-PCR. Serum sFKN levels were assessed by using ELISA. Blood leukocytes were stained for CX(3)CR1 by means of flow cytometric analysis. RESULTS: FKN was strongly expressed on endothelial cells in skin lesions of patients with AD and psoriasis but not in normal skin. FKN mRNA levels in AD lesional skin increased to a similar extent to thymus and activation-regulated chemokine and macrophage-derived chemokine mRNA levels. CX(3)CR1-expressing cells in the affected skin of patients with AD or psoriasis increased compared with those in normal skin. Serum sFKN levels were increased in patients with AD but not in patients with psoriasis relative to levels in healthy control subjects. Serum sFKN levels were associated with the disease severity and decreased with the improvement of skin lesions in patients with AD. CX(3)CR1(+) cell frequencies and CX(3)CR1 expression levels were decreased in CD8(+) T cells, monocytes, and natural killer cells from patients with AD, but this was not observed in patients with psoriasis. CONCLUSIONS: These results suggest that through functions in both membrane-bound and soluble forms, FKN plays an important role in the trafficking of CX(3)CR1(+) leukocytes during the inflammation caused by AD.

Novel role of the membrane-bound chemokine fractalkine in platelet activation and adhesion.

Chemokines released by the endothelium have proaggregatory properties on platelets. Fractalkine, a recently discovered membrane-bound chemokine with a transmembrane domain, is expressed in vascular injury; however, the effects of fractalkine on platelets have not yet been investigated. Blood was taken from healthy Wistar-Kyoto rats and the expression of the fractalkine receptor on platelets was demonstrated. The modulation of surface expression of P-selectin was assessed by flow cytometry. P-selectin expression was significantly enhanced by in vitro stimulation with recombinant rat fractalkine compared with baseline levels. Selectively inhibiting the function of recombinant fractalkine by an antagonizing antibody or the disruption of the G-protein-coupled intracellular signaling cascade of the fractalkine receptor by pertussis toxin (PTX) completely prevented fractalkine-mediated platelet activation. Preincubation with apyrase significantly attenuated the fractalkine-induced degranulation. In a flow chamber model of platelet adhesion, stimulation with fractalkine significantly enhanced platelet adhesion to collagen and fibrinogen. Similar to P-selectin expression, enhanced adhesion could be prevented by the antagonizing antibody or preincubation of platelets with PTX. Fractalkine, which is overexpressed in atherosclerosis and vascular injury, contributes to platelet activation and adhesion and hence is likely to play a pathophysiologically important role for increased thrombogenesis in vascular diseases.

The CX3C chemokine fractalkine in allergic asthma and rhinitis.

BACKGROUND: Unlike other chemokines, fractalkine is expressed as a membrane-bound form, mainly on endothelial and epithelial cells, and can be shed as a soluble chemotactic form. Fractalkine can capture leukocytes expressing its receptor (CX(3)CR(1)), including T lymphocytes, rapidly and firmly in an integrin-independent manner. Because of its dual activity, fractalkine plays a major role in the transendothelial and transepithelial migration of leukocytes during inflammation. OBJECTIVE: We sought to study the fractalkine-CX(3)CR(1) axis in patients with allergic airways diseases. METHODS: Plasma fractalkine levels were measured by means of ELISA in 19 control subjects and 55 patients with symptomatic allergic rhinitis, asthma, or both, and CX(3)CR(1) function was studied by using triple-color flow cytometry in circulating T-lymphocyte subpopulations. Segmental allergen challenge was performed in 16 allergic asthmatic patients to analyze fractalkine expression and inflammatory cell recruitment in bronchoalveolar lavage fluid and bronchial biopsy specimens. RESULTS: Compared with control subjects, patients with symptomatic allergic rhinitis and asthmatic patients had increased circulating fractalkine levels, and CX(3)CR(1) function was upregulated in circulating CD4(+) T lymphocytes. Twenty-four hours after segmental allergen challenge, bronchoalveolar lavage fluid soluble fractalkine concentrations increased and correlated with the total number of recruited cells. Bronchial epithelial and endothelial cells expressed high levels of the membrane-bound form of fractalkine before and after challenge. CONCLUSION: Allergic asthma and rhinitis are associated with systemic and bronchial upregulation of the chemotactic axis fractalkine-CX(3)CR(1). This might contribute to the rapid recruitment of circulating CD4(+) T lymphocytes in the airways after allergen stimulation.

CSF and serum levels of soluble fractalkine (CX3CL1) in inflammatory diseases of the nervous system.

The new CX(3)C-chemokine fractalkine (CX(3)CL1) was measured by Western blot in the cerebrospinal fluid (CSF) and serum of patients with inflammatory diseases of the peripheral and central nervous system (Bell's palsy, BP; Guillain-Barré Syndrome, GBS; multiple sclerosis, MS; viral meningitis, VM; bacterial meningitis, BM) and patients with noninflammatory neurological diseases (controls). In controls, fractalkine was detectable at low concentrations in the CSF and, at much higher levels, in serum. In all inflammatory neurological diseases under study, CSF fractalkine levels were significantly (p<0.01) increased vs. controls (BM>>GBS>VM>MS>BP>controls). In serum, fractalkine levels were significantly increased only in MS patients. The fractalkine CSF/serum ratios (a measure of the chemotactic gradient) were significantly elevated in BM, VM and GBS; furthermore, they tended to be increased in BP and to be decreased in MS. The elevated fractalkine CSF/serum ratios in diseases without CSF pleocytosis (GBS, BP) and a lack of correlation between fractalkine levels and CSF leukocyte counts suggested that soluble fractalkine is not a major chemokine in the CSF. There was no evidence of significant intrathecal production of fractalkine as the mean fractalkine indices (fractalkine CSF/serum ratio:albumin CSF/serum ratio) were <1 in all inflammatory diseases and not significantly elevated vs. controls.

Increased intrathecal release of soluble fractalkine in HIV-infected patients.

The CX(3)C chemokine fractalkine is suggested to play an important role in inflammatory brain diseases, for example, because of its chemotactic properties. To investigate the release of soluble fractalkine in HIV-induced brain diseases fractalkine levels were determined in cerebrospinal fluid (CSF) and serum samples of HIV-infected patients with (n = 10) and without (n = 23) HIV-induced CNS complications, using semiquantitative Western blot analysis. Fractalkine CSF levels were significantly elevated (p < 0.05) in HIV-infected patients with CNS diseases compared with those without, and compared with HIV-negative controls (n = 23). Fractalkine serum concentrations did not differ between the two groups of HIV-infected patients, but were significantly elevated (p < 0.05) in HIV-infected patients with CNS complications compared with HIV-negative controls. Levels of fractalkine did not correlate with the CSF and serum HIV load and other CSF parameters. In one patient with HIV-associated dementia and myelopathy CSF fractalkine levels decreased on initiation of antiretroviral therapy and subsequent clinical improvement. In conclusion, intrathecal fractalkine release was observed in the majority of patients with HIV infection. The highest levels of soluble fractalkine were detected in CSF (and serum) samples of patients with HIV-induced CNS disorders. These results suggest a dysregulation of brain soluble fractalkine release during HIV infection.

Migration of CX3CR1-positive T cells producing type 1 cytokines and cytotoxic molecules into the synovium of patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by chronic inflammation of multiple joints. Large numbers of T cells, which produce type 1 cytokines, infiltrate into RA synovium. Chemokines and chemokine receptors are considered to contribute to the T cell infiltration. In this study, we examined the role of CX3CL1/fractalkine and its receptor CX3C chemokine receptor 1 (CX3CR1) in the T cell migration into RA synovium. METHODS: Using flow cytometry, immunohistochemistry, and reverse transcription-polymerase chain reaction, we analyzed CX3CR1 expression by peripheral blood and synovial T cells, and CX3CL1 expression in synovium from patients with RA. Cytokine and cytotoxic molecule expression by CX3CR1-positive T cells was analyzed by flow cytometry. RESULTS: CX3CR1 expression by peripheral CD4+ and CD8+ T cells was up-regulated in RA patients. The peripheral CD4+ and CD8+ T cells expressing CX3CR1 predominantly produced interferon-gamma and tumor necrosis factor alpha, and expressed cytotoxic molecules such as granzyme A and perforin. Furthermore, CX3CR1+,CD3+ T cells infiltrated into RA synovium. CX3CL1, the unique ligand of CX3CR1, was expressed by endothelial cells and synoviocytes in RA synovium, but not in osteoarthritis synovium. CONCLUSION: Our findings suggest that the interactions of CX3CL1 and CX3CR1 might contribute to the accumulation of CX3CR1+ T cells expressing type 1 cytokines and possessing cytotoxic granules in RA synovium.