Differential CXC and CX3C Chemokine Expression Profiles in Aqueous Humor of Patients With Specific Endogenous Uveitic Entities.

Purpose: To determine the levels of the neutrophil chemoattractants CXCL1, CXCL2, CXCL5, CXCL6, and CXCL8, the T helper 1 chemoattractants CXCL9, CXCL10 and CXCL11, the lymphoid chemokines CXCL12 and CXCL13 and the soluble form of the transmembrane chemokines CXCL16 and CX3CL1, in aqueous humor samples from patients with specific uveitic entities. Methods: Aqueous humor samples from patients with active uveitis associated with Behçet's disease (n = 13), sarcoidosis (n = 8), HLA-B27-related inflammation (n = 12), Vogt-Koyanagi-Harada (VKH) disease (n = 12), and healthy controls (n = 9) were assayed with the use of a multiplex assay. Results: All chemoattractant levels were significantly higher in all patients than in the controls. The levels of all neutrophil chemoattractants and CXCL10, CXCL16, and CX3CL1 were significantly higher in nongranulomatous uveitis (Behçet's disease and HLA-B27-associated uveitis) than in granulomatous uveitis (sarcoidosis and VKH disease), whereas the levels of the B cell chemoattractant CXCL13 were significantly higher in granulomatous uveitis than in nongranulomatous uveitis. CXCL13 levels were highest in the patients with VKH disease. CXCL9, CXCL11, and CXCL12 levels did not differ significantly. Conclusions: Inflammation in nongranulomatous uveitis appears to be driven by neutrophils and T helper 1 lymphocytes, whereas B lymphocytes may contribute to the inflammatory process in granulomatous uveitis, particularly in VKH disease.

The spreading of HIV-1 infection in the human organism is caused by fractalkine trafficking of the infected lymphocytes--a review, hypothesis and implications for treatment.

The reviews on HIV-1/AIDS [1-8] highlighted the mechanism by which HIV-1 virions utilize dendritic cells (DCs) for transport from the genitals, the portal of virus infection, to the draining lymph nodes where DCs carry HIV-1 virions and present viral antigens by HLA class I and II to CD4(+) T cells. Interaction of the T cells with viral antigens presented by HLA class II molecules polarizes them to become Th2 cells, the targets of HIV-1 infection and producers of HIV-1 progeny virions. The T cells which interact with viral antigen presented by HLA class I polarize to become Th1 cells, which stimulate the CD8(+) T cell precursors to develop into antiviral cytotoxic T cells. In addition, HIV-1 virions shed gp120 glycoprotein molecules which bind to IgE immunoglobulin molecules bound to FCepsilonRI+ innate system cells (basophils, mast cells and monocytes) and induce them to release large amounts of Th2 cytokines (IL-4, IL-5, IL-10, IL-13), thereby creating an allergy-like condition. The present review attempts to define the role of chemokine receptors like CCR5 and CXCR4, and especially fractalkine receptor CX3CR1 in the trafficking of lymphocytes in healthy individuals and HIV-1/AIDS patients. The role of chemokine receptors as co-receptors for HIV-1 virion gp120 glycoprotein has been defined, but the role of fractalkine and fractalkine receptor has been clarified only recently [9-19]. In healthy individuals fractalkine is expressed by blood vessel endothelial cells and the CX3CR1 receptors are expressed on leukocytes that migrate in the peripheral blood in the direction of increased fractalkine concentration. In HIV-1/AIDS patients the virus-infected CD4(+) Th2 cells migrate to organs that harbor the adaptive immune system cells in the thymus, genitals, gastrointestinal tract, and to the brain. A most significant finding which revealed the importance of the human CX3CR1 gene expression to the progression of the infection to the stage of AIDS was recently reported by Faure and collaborators [20, 21] who showed that the delayed or rapid progression to AIDS was affected in HIV-1-infected individuals who had inherited a fractalkine receptor gene with the polymorphisms V249I or T280M, respectively, located in the sixth and seventh transmembrane domains of CX3CR1 protein. The T280M mutation in the CX3CR1 gene caused a rapid progression to AIDS, while in patients with the V249I mutation progression to AIDS was much slower. These studies led to the idea that it might be possible to slow or prevent HIV-1/AIDS progression in HIV-1 patients by treating them with fractalkine antagonists that will bind to and inhibit the activity of the fractalkine receptor. It is hypothesized that treatment of HIV-1/AIDS patients with a combination of fractalkine antagonists, IL-4 antagonist IL-4delta2 and the adjuvant CpG ODN induced release of type I IFN from PDF, and may inhibit HIV-1 infection, especially in HAART-treated patients infected with drug-resistant HIV-1 mutants due to prevention of the availability of immune cells needed for the viral evasion of the immune response. The hypothesis implies that the advantage of the suggested mode of treatment of HIV-1-infected people is prevention of cellular processes that are used by the viral protein to cause immunodeficiency, and prevention of HIV-1 replication without induction of resistant mutants.

CD16+ monocytes produce IL-6, CCL2, and matrix metalloproteinase-9 upon interaction with CX3CL1-expressing endothelial cells.

The CD16+ subset of peripheral blood monocytes (Mo) is expanded dramatically during inflammatory conditions including sepsis, HIV-1 infection, and cancer. CD16+ express high levels of CX3CR1, which mediates arrest onto CX3CL1-expressing endothelial cells (EC) under flow conditions. In contrast, attachment of CD16- Mo onto cytokine-activated EC is independent of CX3CL1. Here, we investigate the ability of CD16+ and CD16- Mo to produce proinflammatory cytokines upon interaction with CX3CL1-expressing HUVEC. We demonstrate that CD16+ but not CD16- Mo produce high levels of IL-6, CCL2, and matrix metalloproteinase (MMP)-9 when cocultured with TNF/IFN-gamma-activated HUVEC or nonactivated HUVEC expressing CX3CL1. Furthermore, supernatants from Mo cocultured with cytokine-activated HUVEC induce neuronal death in vitro. These results suggest that membrane-bound CX3CL1 stimulates production of IL-6, CCL2, and MMP-9 by CD16+ Mo, likely via engagement of CX3CR1. Thus, expansion of CD16+ Mo and their accumulation onto CX3CL1-expressing EC may result in recruitment of Mo and T cell subsets at sites of inflammation in response to CCL2, IL-6-induced cell activation and/or differentiation, and MMP-9-mediated vascular and tissue injury.

The ascochlorin derivative, AS-6, inhibits TNF-alpha-induced adhesion molecule and chemokine expression in rat vascular smooth muscle cells.

Vascular inflammation induced by the proinflammatory cytokine/NF-kappaB pathway is one of the key mechanisms in the development of atherosclerosis. Peroxisome proliferators-activated receptor-gamma (PPARgamma) plays an important role in the prevention of arterial inflammation and formation of atherogenesis. Herein we examine the effects of a newly identified synthetic PPARgamma ligand, ascochlorin-6 (AS-6), on TNF-alpha-stimulated NF-kappaB activity and inflammatory molecule expression in vascular smooth muscle cells (VSMCs). AS-6 successfully inhibited TNF-alpha-stimulated NF-kappaB activity and inflammatory molecule expression, including vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and fractalkine (CX3CL1). Transient transfection with an [NF-kappaB]x4 luciferase reporter construct showed that AS-6 inhibition of TNF-alpha-stimulated NF-kappaB activation was PPARgamma-dependent. The effects of AS-6 on TNF-alpha-stimulated VCAM-1 and CX3CL1 expression were abolished in cells transfected with an adenovirus expressing dominant-negative PPARgamma and in cells treated with a PPARgamma specific inhibitor, GW9662, confirming again that the anti-inflammatory effect of AS-6 was PPARgamma-dependent. The inhibitory effects of AS-6 on TNF-alpha-stimulated inflammatory gene expression and NF-kappaB activation were more potent than those of rosiglitazone and pioglitazone. This study shows that AS-6 reduces the inflammatory response to TNF-alpha in VSMCs. The data suggest the possibility that AS-6 can be used to prevent the development and progression of atherosclerosis.

[Effect of dexamethasone on expression of fractalkine in lipopolysaccharide-induced acute lung injury].

OBJECTIVE: To investigate the effect of dexamethasone on the expression of fractalkine (FKN) in lipopolysaccharide (LPS)-induced acute lung injury (ALI). METHODS: The rat model of ALI was established by injection of LPS at the dose of 4 mg/kg. 42 Wistar rats were randomly divided into the normal group (n=6), LPS group (n=18), and dexamethasone (DEX) group (n = 18), and then the rats in both LPS and DEX groups were divided into three subgroups (1 h, 2 h and 4 h after injection of LPS), respectively. The pathological condition and the wet/dry ratio (W/D) of the lung were observed, and serum TNF-alpha level, and FKN mRNA of the lung were detected with ELISA and RT-PCR. RESULTS: The W/D, serum TNF-alpha level, and FKN mRNA of the lung were significantly increased in LPS group, compared with those in normal group (all P < 0.05), but the W/D, serum TNF-alpha level, and FKN mRNA of the lung in the DEX group were much more decreased than those in the LPS group (all P < 0.05). In addition, the expression of FKN mRNA in the lung tissue positively correlated with the concentration of TNF-alpha (r = 0.674, P <0.05). CONCLUSION: The findings suggested that pre-treatment with dexamethasone could inhibit the TNF-alpha level and prevent the increase of the expression of FKN mRNA, which may be one of the mechanisms by which DEX serves as a protection against LPS-induced lung injury.

Lipoxin A4 inhibits connective tissue growth factor-induced production of chemokines in rat mesangial cells.

Connective tissue growth factor (CTGF) is involved in mitogenesis, matrix production, and chemotaxis in mesenchymal cells. The effects of CTGF on the production of chemokines remain unclear. The present studies investigate the regulatory role of CTGF in the production of fractalkine, monocyte chemoattractant protein-1 (MCP-1), and RANTES (regulated upon activation, normal T cell expressed and secreted) in cultured mesangial cells of rats, and the modulatory effects of lipoxin A(4) (LXA(4)) on actions of CTGF. CTGF enhanced the mRNA expression and protein release of fractalkine, MCP-1, and RANTES, the expression of phospho (P)-p42/44 mitogen-activated protein kinase (MAPK), P-phosphoinositide 3-kinase (PI3-K), P-Akt, and activity of nuclear factor-kappaB (NF-kappaB) in mesangial cells. P-p42/44 MAPK blockade inhibited the CTGF-induced expression of P-p42/44 MAPK but not NF-kappaB, and partially decreased the levels of the above chemokines in supernatants. P-PI3-K blockade downregulated the CTGF-stimulated expression of P-PI3-K, P-Akt, and NF-kappaB but not P-p42/44 MAPK, and partially decreased the release of the above chemokines. NF-kappaB blockade abrogated the CTGF-activated NF-kappaB and partially decreased the secretion of the above chemokines. LXA(4) dose-dependently inhibited the CTGF-stimulated mRNA expression and protein release of the above chemokines, and the expression of P-p42/44MAPK, P-PI3-K, P-Akt, and NF-kappaB. In conclusion, these results demonstrate that CTGF induces production of fractalkine, MCP-1, and RANTES via the p42/44 MAPK-, PI3-K/Akt-, and NF-kappaB-dependent signal pathway, and LXA(4) downregulates the above effects of CTGF on rat mesangial cells.

Alpha-lipoic acid inhibits fractalkine expression and prevents neointimal hyperplasia after balloon injury in rat carotid artery.

Vascular inflammation induced by the proinflammatory cytokine/NF-kappaB pathway is one of the key mechanisms in the development of neointimal hyperplasia. Accumulating evidence suggests that a recently identified chemokine, fractalkine, is involved in arterial inflammation and atherogenesis. However, no study has examined the expression of neointimal fractalkine and the effects of pharmacological agents on this process. The purposes of this study were to measure neointimal fractalkine expression in the rat carotid artery following balloon injury and to determine if alpha-lipoic acid (ALA) inhibits fractalkine expression and neointimal hyperplasia. Balloon injury of the rat carotid artery induced fractalkine expression in the medial as well as neointimal regions. ALA inhibited this expression and consequently prevented neoinitmal hyperplasia in a balloon-injured rat carotid artery. Additionally, ALA inhibited TNF-alpha-stimulated fractalkine expression in cultured vascular smooth muscle cells (VSMCs), a process which is mediated through the NF-kappaB pathway. In addition to fractalkine, ALA successfully inhibited TNF-alpha-stimulated expression of vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 in cultured VSMCs. These data suggest that the cytokine-fractalkine system is involved in the pathogenesis of restenosis. The present study supports the possibility that ALA, which inhibits the NF-kappaB/fractalkine pathway, may be used to prevent neointimal hyperplasia after angioplasty or stenting.

Respiratory syncytial virus G protein and G protein CX3C motif adversely affect CX3CR1+ T cell responses.

Interactions between fractalkine (CX3CL1) and its receptor, CX3CR1, mediate leukocyte adhesion, activation, and trafficking. The respiratory syncytial virus (RSV) G protein has a CX3C chemokine motif that can bind CX3CR1 and modify CXCL1-mediated responses. In this study, we show that expression of the RSV G protein or the G protein CX3C motif during infection is associated with reduced CX3CR1+ T cell trafficking to the lung, reduced frequencies of RSV-specific, MHC class I-restricted IFN-gamma-expressing cells, and lower numbers of IL-4- and CX3CL1-expressing cells. In addition, we show that CX3CR1+ cells constitute a major component of the cytotoxic response to RSV infection. These results suggest that G protein and the G protein CX3C motif reduce the antiviral T cell response to RSV infection.

Fraktalkine produced by airway smooth muscle cells contributes to mast cell recruitment in asthma.

Human airway smooth muscle cells (HASMC) secrete fractalkine (FKN), a chemokine the concentration of which is increased in asthmatic patients. HASMC also induce mast cell chemotaxis, as a component of asthma inflammation. We therefore evaluated the role of smooth muscle-derived FKN in mast cell migration. We assessed the capacity of recombinant FKN to induce human mast cell chemotaxis. This effect implicates a calcium-independent pathway involving actin reorganization and protein kinase C-delta. We found that HASMC constitutively produce FKN, the synthesis of which is reinforced upon proinflammatory stimulation. Under basal experimental conditions, FKN production by HASMC is not sufficient to induce mast cell chemotaxis. However, pretreatment of mast cells with the neuropeptide vasoactive intestinal peptide (VIP) increases FKN potency to attract mast cells. Since we observed, in asthmatic patients, an increase in both FKN and VIP expression by airway smooth muscle and a positive correlation between VIP staining and mast cell infiltration of the smooth muscle layer, we conclude that HASMC-derived FKN may contribute to mast cell recruitment in asthma.

Involvement of fractalkine/CX3CL1 expression by dendritic cells in the enhancement of host immunity against Legionella pneumophila.

Legionnaires' disease is clinically manifested as severe pneumonia caused by Legionella pneumophila. However, the dendritic cell (DC)-centered immunological framework of the host defense against L. pneumophila has not been fully delineated. For this study, we focused on a potent chemoattractant for lymphocytes, fractalkine/CX3CL1, and observed that the fractalkine expression of DCs was somewhat up-regulated when they encountered L. pneumophila. We therefore hypothesized that fractalkine expressed by Legionella-capturing DCs is involved in the induction of T-cell-mediated immune responses against Legionella, which would be enhanced by a genetic modulation of DCs to overexpress fractalkine. In vivo immunization-challenge experiments demonstrated that DCs modified with a recombinant adenovirus vector to overexpress fractalkine (AdFKN) and pulsed with heat-killed Legionella protected immunized mice from a lethal Legionella infection and that the generation of in vivo protective immunity depended on the host lymphocyte subsets, including CD4(+) T cells, CD8(+) T cells, and B cells. Consistent with this, immunization with AdFKN/Legionella/DC induced significantly higher levels of serum anti-Legionella antibodies of several isotypes than those induced by control immunizations. Further analysis of spleen cells from the immunized mice indicated that the AdFKN/Legionella/DC immunization elicited Th1-dominated immune responses to L. pneumophila. These observations suggest that fractalkine may play an important role in the DC-mediated host defense against intracellular pathogens such as L. pneumophila.

Antagonist of fractalkine (CX3CL1) delays the initiation and ameliorates the progression of lupus nephritis in MRL/lpr mice.

OBJECTIVE: Lupus nephritis is characterized by immune complex deposition and inflammatory cell infiltration into the renal glomeruli. Local generation of chemokines and the presence of chemokine receptors on the infiltrating cells may be involved in this process. Fractalkine (Fkn)/CX3CL1 and its receptor, CX3CR1, form one such chemokine system. We therefore undertook this study to investigate whether Fkn antagonist inhibits the initiation and progression of lupus nephritis in MRL/lpr mice. METHODS: NH(2)-terminally truncated Fkn/CX3CL1 analogs were transfected into a nonmetastatic fibroblastoid cell line, MRL/N-1, and injected subcutaneously into MRL/lpr mice. RESULTS: Fkn analogs truncated by >/=4 amino acid residues from the N-terminus failed to induce chemotaxis and calcium influx by CX3CR1-expressing cells. Of these, the most potent antagonist (Fkn-AT) lacked the 4 N-terminal amino acid residues. Fkn expression in the glomerulus was significantly increased in 12-week-old MRL/lpr mice. Expression was localized predominantly in the glomerular endothelial cells, but was occasionally observed in the mesangial cells and, to a lesser extent, in the interstitial microvasculature. Inoculation of MRL/lpr mice with Fkn-AT before the onset or during the early stages of lupus nephritis significantly reduced glomerular hypercellularity, glomerulosclerosis, crescent formation, and vasculitis compared with control mice. This seemed to be due to a marked reduction in macrophage accumulation. In contrast, Fkn antagonist did not affect pneumonitis, sialadenitis, lymphadenopathy, or splenomegaly. CONCLUSION: We prepared a novel potent Fkn antagonist and demonstrated its ability to delay the initiation and ameliorate the progression of lupus nephritis. This agent may therefore provide a new therapeutic approach to lupus nephritis.

T cell costimulation by fractalkine-expressing synoviocytes in rheumatoid arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) accumulate prematurely aged T cells that have acquired a new profile of regulatory receptors. Many of the de novo-expressed receptors are typically found on natural killer cells, including CX(3)CR1, the receptor for the chemokine fractalkine (FKN). This study explored whether interactions between CX(3)CR1 and FKN are relevant for T cell functions in rheumatoid synovitis. METHODS: FKN expression was examined by real-time polymerase chain reaction and immunohistochemistry. CX(3)CR1 expression on peripheral blood T cells was analyzed by flow cytometry. T cell activation was quantified by determining proliferative responses, interferon-gamma (IFNgamma) secretion, and granule release. Fibroblast-like synoviocyte (FLS)/T cell adhesion was measured by the retention of 5-carboxyfluorescein diacetate succinimidyl ester-labeled T cells on FLS monolayers. RESULTS: FKN was expressed on cultured synovial fibroblasts and hyperplastic synoviocytes in the rheumatoid tissue. Among CD4+ T cells, only senescent CD28- T cells were positive for CX(3)CR1 (P < 0.001). Such CD4+,CD28-,CX(3)CR1+ T cells strongly adhered to FLS, with soluble FKN blocking the interaction. FKN expressed on FLS costimulated T cell-activating signals and amplified proliferation, IFNgamma production, and expulsion of cytoplasmic granules. CONCLUSION: Senescent CD4+ T cells that accumulate in rheumatoid arthritis aberrantly express CX(3)CR1. FKN, which is membrane-anchored on synoviocytes, enhances CD4+ T cell adhesion, provides survival signals, and costimulates the production of proinflammatory cytokines as well as the release of granules. By virtue of their altered receptor profile, senescent CD4+ T cells receive strong stimulatory signals from nonprofessional antigen-presenting cells in the synovial microenvironment.

The effect of Toxoplasma gondii infection on expression of chemokines by rat retinal vascular endothelial cells.

Cells infected by Toxoplasma gondii undergo up-regulation of proinflammatory cytokines, organelle redistribution, and protection from apoptosis. During infection in man, the parasite encysts within the retina, a process that results in retinochoroiditis which can lead to permanent loss of sight. The reasons for the parasite to infect retinal tissue and the mechanisms by which it encysts are not clearly understood. We studied the effect of infection with T. gondii of retinal vascular endothelial cells using the Clontech Atlastrade mark array system in order to elucidate changes in gene expression. We compared hybridization of RNA to the array from infected and uninfected cells at two time points; 2 and 24 h. Exposure to T. gondii after 2 h resulted in change of expression of approximately 6% of genes on the array, including those involved in cell structure, protein and vesicle trafficking, cell-cycle regulation, transcriptional and translational machinery, and apoptosis. Among the genes involved in the inflammatory response, chemokine genes such as GRO1 (Growth Regulated Oncogene 1), MCP-1 (Monocyte Chemotactic Protein-1), FKN (Fractalkine) and RANTES (Regulated upon Activation, Normal T Cell Expressed and Secreted) were found to be up-regulated and protein production was confirmed by ELISA. However after 24 h of infection, GRO1, MCP-1 and FKN were down-regulated, confirmed by RT-PCR. Thus, invasion of retinal vascular endothelium (RVE) cells by T. gondii leads to the production of chemokines important in directing the traffic of inflammatory cells to the infected area.

Fractalkine reduces N-methyl-d-aspartate-induced calcium flux and apoptosis in human neurons through extracellular signal-regulated kinase activation.

Our purpose was to investigate in human neurons the neuroprotective pathways induced by Fractalkine (FKN) against glutamate receptor-induced excitotoxicity. CX(3)CR1 and FKN are expressed constitutively in the tested human embryonic primary neurons and SK-N-SH, a human neuroblastoma cell line. Microfluorometry assay demonstrated that CX(3)CR1 was functional in 44% of primary neurons and in 70% of SK-N-SH. Fractalkine induced ERK1/2 phosphorylation within 1 min and Akt phosphorylation after 10 min, and both phosphorylation decreased after 20 min. No p38 and SAPK/JNK activation was observed after FKN treatment. Application of FKN triggered a 53% reduction of the NMDA-induced neuronal calcium influx, which was insensitive to pertussis toxin and LY294002 an inhibitor of Akt pathway, but abolished by PD98059, an ERK1/2 pathway inhibitor. Moreover, FKN significantly reduced neuronal NMDA-induced apoptosis, which was pertussis toxin insensitive and abolished in presence of PD98059 and LY294002. In conclusion, FKN protected human neurons from NMDA-mediated excitotoxicity in at least two ways with different kinetics: (i) an early ERK1/2 activation which reduced NMDA-mediated calcium flux; and (ii), a late Akt activation associated with the previously induced ERK1/2 activation.

Coexpression of fractalkine and its receptor in normal human endometrium and in endometrium from users of progestin-only contraception supports a role for fractalkine in leukocyte recruitment and endometrial remodeling.

Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyte subpopulations enter the uterus are currently unknown. Endometrial leukocytes have no genomic progesterone receptors; thus, we hypothesized that leukocyte migration is induced indirectly by progesterone-regulated chemokines. Fractalkine (CX3CL1), a chemotactic membrane-bound adhesion factor, and its receptor (CX3CR1) were assessed by immunohistochemistry in endometrial samples across the menstrual cycle, in early pregnancy, and in women using progestin-only contraceptives. Fractalkine was localized predominantly to glandular epithelial and decidualized stromal cells, with the highest staining intensity in the secretory phase and early pregnancy. It was also detected in subpopulations of endometrial leukocytes (macrophages and uterine NK cells), with maximal numbers during the proliferative phase and early pregnancy. CX3CR1 was similarly colocalized to the glandular epithelium and decidualized stromal cells, with the highest expression in the secretory phase. CX3CR1-positive leukocytes (macrophages and neutrophils) were in greatest abundance during the menstrual phase. In the endometrium of women using progestin-only contraceptives, immunoreactive fractalkine was markedly reduced in the glandular epithelium, but was increased in decidualized stroma and infiltrating leukocytes. These findings support a number of roles for fractalkine in the endometrium, in the secretory phase, in early pregnancy, and when influenced by progestin-only contraceptives.

Induction of Fractalkine and CX3CR1 mediated by host CD8+ T cells in allograft tolerance induced by donor specific blood transfusion.

BACKGROUND: Donor-specific tolerance to heart allografts in the rat can be achieved by donor-specific blood transfusions (DST) before transplantation. This tolerance induction requires the presence of host CD8 T cells and is characterized by the infiltration of numerous leukocytes. METHODS: To identify new mediators involved in tolerance induction, gene searching was performed and resulted in the identification of the Fractalkine receptor, CX3CR1, as being highly expressed in tolerated allografts. RESULTS: We showed that the high CX3CR1 mRNA accumulation found in tolerated allografts was related to the active recruitment of monocytes/macrophages. CX3CR1 transcript accumulation was preceded by an early expression of its ligand, Fractalkine, by graft endothelial cells. Interestingly, depletion of recipient CD8 cells led to a dramatic decrease in both CX3CR1 and Fractalkine mRNA levels. Moreover, in vitro, CD8 T cells from DST-primed animals were found to strongly induce Fractalkine expression in an allogeneic endothelial cell line. CONCLUSION: This is the first report describing Fractalkine, a chemokine usually described in inflammatory processes, as being expressed in a model of allograft tolerance.

Human cytomegalovirus-specific CD4+-T-cell cytokine response induces fractalkine in endothelial cells.

Cytomegalovirus (CMV) infection has been linked to inflammation-related disease processes in the human host, including vascular diseases and chronic transplant rejection. The mechanisms through which CMV affects the pathogenesis of these diseases are for the most part unknown. To study the contributing role of the host immune response to CMV in these chronic inflammatory processes, we examined endothelial cell interactions with peripheral blood mononuclear cells (PBMC). Endothelial cultures were monitored for levels of fractalkine induction as a marker for initiating the host inflammatory response. Our results demonstrate that in the presence of CMV antigen PBMC from normal healthy CMV-seropositive donors produce soluble factors that induce fractalkine in endothelial cells. This was not observed in parallel assays with PBMC from seronegative donors. Examination of subset populations within the PBMC further revealed that CMV antigen-stimulated CD4(+) T cells were the source of the factors, gamma interferon and tumor necrosis factor alpha, driving fractalkine induction. Direct contact between CD4(+) cells and the endothelial monolayers is required for this fractalkine induction, where the endothelial cells appear to provide antigen presentation functions. These findings indicate that CMV may represent one member of a class of persistent pathogens where the antigen-specific T-cell response can result in the induction of fractalkine, leading to chronic inflammation and endothelial cell injury.

Inhibition of fractalkine ameliorates murine collagen-induced arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive infiltration of inflammatory cells in the synovium of multiple joints. We and others have shown that fractalkine (FKN/CX3CL1), a chemokine expressed on fibroblast-like synoviocytes and endothelial cells in RA synovium, may contribute to the accumulation of T cells, macrophages, and dendritic cells, which express CX3CR1, the receptor for FKN. This interaction might be involved in adhesion of the inflammatory cells to endothelial cells, migration into the synovium, and cytokine production. In this study, we examined the effect of FKN inhibition on murine collagen-induced arthritis. Anti-FKN mAb significantly lowered clinical arthritis score compared with control Ab, and reduced infiltration of inflammatory cells and bone erosion in the synovium. However, anti-FKN mAb did not affect the production of either serum anti-collagen type II (CII) IgG or IFN-gamma by CII-stimulated splenic T cells. Furthermore, treatment with anti-FKN mAb inhibited migration of adoptively transferred splenic macrophages into the inflamed synovium. Our results suggest that anti-FKN mAb ameliorates arthritis by inhibiting infiltration of inflammatory cells into the synovium. Thus, FKN can be a new target molecule for the treatment of RA.

Mycobacterium tuberculosis-induced CXCR4 and chemokine expression leads to preferential X4 HIV-1 replication in human macrophages.

Opportunistic infections such as pulmonary tuberculosis (TB) increase local HIV-1 replication and mutation. As AIDS progresses, alteration of the HIV-1 gp120 V3 sequence is associated with a shift in viral coreceptor use from CCR5 (CD195) to CXCR4 (CD184). To better understand the effect of HIV/TB coinfection, we screened transcripts from bronchoalveolar lavage cells with high density cDNA arrays and found that CXCR4 mRNA is increased in patients with TB. Surprisingly, CXCR4 was predominately expressed on alveolar macrophages (AM). Mycobacterium tuberculosis infection of macrophages in vitro increased CXCR4 surface expression, whereas amelioration of disease reduced CXCR4 expression in vivo. Bronchoalveolar lavage fluid from TB patients had elevated levels of CCL4 (macrophage inflammatory protein-1beta), CCL5 (RANTES), and CX3CL1 (fractalkine), but not CXCL12 (stromal-derived factor-1alpha). We found that M. tuberculosis infection of macrophages in vitro increased viral entry and RT of CXCR4-using [corrected] HIV-1, but not of CCR5-using [corrected] HIV-1. Lastly, HIV-1 derived from the lung contains CD14, suggesting that they were produced in AM. Our results demonstrate that TB produces a permissive environment for replication of CXCR4-using virus by increasing CXCR4 expression in AM and for suppression of CCR5-using HIV-1 by increasing CC chemokine expression. These changes explain in part why TB accelerates the course of AIDS. CXCR4 inhibitors are a rational therapeutic approach in HIV/TB coinfection.

Fractalkine and its receptor, CX3CR1, upregulation in streptozotocin-induced diabetic kidneys.

BACKGROUND: Fractalkine is induced on activated endothelial cells and promotes strong adhesion of T cells and monocytes via its receptor CX3CR1. In kidney, fractalkine expression might be induced by high shear stress and play an important role in prolonged glomerular diseases. We examined whether fractalkine and CX3CR1 upregulation are found in streptozotocin-induced diabetic kidneys. METHODS: Diabetic rats were randomized to receive an angiotensin-converting enzyme inhibitor (temocapril), aminoguanidine or no treatment. Reverse transcription-competitive polymerase chain reaction, Western blot analysis and immunohistochemistry were used. RESULTS: At 4 weeks, fractalkine and CX3CR1 mRNA expression in diabetic kidneys increased compared with that in controls. Fractalkine staining in diabetic kidneys was clearly detected, along with glomerular capillary lumen and peritubular capillaries. A few CX3CR1 positive cell infiltration in diabetic glomeruli were found. Treatment with temocapril or aminoguanidine did not affect these changes. At 8 weeks, fractalkine and CX3CR1 mRNA expression in untreated diabetic kidneys markedly increased compared with that in controls. Membrane-anchored fractalkine protein expression in untreated diabetic rats also increased. The increased expression was suppressed by the treatment with temocapril and aminoguanidine. Increased CX3CR1-positive cell infiltration in diabetic glomeruli was also inhibited by both treatments. Some CX3CR1-positive cells were ED3 positive. CONCLUSIONS: Fractalkine and CX3CR1 upregulation were demonstrated in an early stage of diabetic kidney. These upregulation, as well as urinary albumin excretion, were suppressed by treatments with temocapril and aminoguanidine for 8 weeks. These findings suggest that fractalkine expression and CX3CR1-positive cell infiltration in diabetic kidneys might play an important role for progression of diabetic nephropathy.