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.

Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses.

Fractalkine (FKN, CX3CL1) is a membrane-bound CX3C chemokine induced by primary proinflammatory signals in vascular endothelial cells (ECs). Here we examined the role of FKN in polarized Th1 or Th2 responses. Proinflammatory signals, including LPS, IL-1, TNF, and CD40 ligand, induced FKN, as did IFN-gamma, which had synergistic activity with TNF. IL-4 and IL-13 did not stimulate the expression of FKN and markedly reduced induction by TNF and IFN-gamma. TNF alone or combined with IFN-gamma also induced release of soluble FKN, which was inhibited by IL-4 and IL-13. In light of this differential regulation of FKN by the master cytokines that control polarized responses, we analyzed the interaction of FKN with natural killer (NK) cells and polarized T-cell populations. NK cells expressed high levels of the FKN receptor CX3CR1 and responded to FKN. CX3CR1 was preferentially expressed in Th1 compared with Th2 cells. Th1 but not Th2 cells responded to FKN. By immunohistochemistry, FKN was expressed on ECs in psoriasis, a Th1-dominated skin disorder, but not in Th2-driven atopic dermatitis. Similarly, ECs in Mycobacterium tuberculosis granulomatous lymphadenitis, but not those in reactive lymph node hyperplasia or in Castelman's disease, showed immunoreactive FKN. These results indicate that regulated expression of FKN in ECs participates in an amplification circuit of polarized type I responses.

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.

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.

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.

Fractalkine mediates natural killer-dependent antitumor responses in vivo.

CX3CR1 has been described previously as a marker of human cytotoxic effector cells. We evaluated the possibility of using its ligand, CX3CL1, to redirect immune response against tumors. When murine lymphoma cell lines (EL4 and its derivative EG7) stably transfected with human-CX3CL1 were injected s.c. into C57BL/6 mice, the tumor growth was severely impaired when compared with the growth of control cell lines. This antitumor effect of CX3CL1 was also found in T- and B-cell-deficient Rag1-/- mice but vanished in natural killer (NK) cell-deficient beige mice and in CX3CR1-/- mice, suggesting the involvement of CX3CR1-expressing NK cells. In addition, increased NK cell infiltration was observed in CX3CL1-producing tumors compared with controls. The effect of CX3CR1 on tumor growth required host cytotoxic effector cell functions because both IFNgamma-/- and perforin-/- mice were resistant to CX3CL1 antitumor effect. Finally, intratumoral injection of DNA plasmid coding for a chimeric immunoglobulin presenting the CX3CL1 chemokine domain provided strong antitumor activity. Together, these data demonstrate that the CX3CL1 can reduce incidence and size of lymphoma in vivo through increased recruitment of activated NK cytotoxic cells. These findings offer the first evidence of the potential of chimeric immunoglobulin-chemokines in anticancer therapy.

[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.

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.

Linked chromosome 16q13 chemokines, macrophage-derived chemokine, fractalkine, and thymus- and activation-regulated chemokine, are expressed in human atherosclerotic lesions.

Chemokines are important mediators of macrophage and T-cell recruitment in a number of inflammatory pathologies, and chemokines expressed in atherosclerotic lesions may play an important role in mononuclear cell recruitment and macrophage differentiation. We have analyzed the expression of the linked chromosome 16q13 genes that encode macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17), and the CX(3)C chemokine fractalkine (CX(3)CL1) in primary macrophages and human atherosclerotic lesions by reverse transcription-polymerase chain reaction and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine, and TARC is upregulated by treatment with the Th2-type cytokines interleukin-4 and interleukin-13. High levels of MDC, TARC, and fractalkine mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine, and TARC are expressed by a subset of macrophages within regions of plaques that contain plaque microvessels. We conclude that MDC, fractalkine, and TARC, which are chromosome 16q13 chemokines, could play a role in mononuclear cell recruitment into atherosclerotic lesions and influence the subsequent inflammatory response. Macrophage-expressed chemokines upregulated by interleukin-4 may be useful surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions.

Inflammatory agents regulate in vivo expression of fractalkine in endothelial cells of the rat heart.

Fractalkine is distinguished structurally from other chemokines in that it contains a mucin-like stalk that tethers a CX3C chemokine module to a transmembrane-spanning region; its expression in cultured endothelial cells has been shown to be up-regulated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1). The purpose of this study was to determine whether fractalkine is expressed, in a proinflammatory agent-regulated manner, by cardiac endothelial cells in vivo. Steady state levels of fractalkine mRNA were increased in rat cardiac tissues after in vivo treatment with lipopolysaccharide (LPS), IL-1, or TNF-alpha. In situ hybridization and immunohistochemical analysis revealed that endothelial cells of the coronary vasculature and endocardium were the principal source of proinflammatory agent-inducible fractalkine, although some fractalkine immunoreactivity was also found on the myocytes. These data are the first demonstration of in vivo cardiac endothelial cell fractalkine expression and regulation by proinflammatory agents such as LPS, IL-1, or TNF-alpha. Cardiac endothelial cell-expressed fractalkine may contribute to the influx of leukocytes into the heart during inflammation.

CX3CL1 and CX3CR1 expression in human brain tissue: noninflammatory control versus multiple sclerosis.

An important role for CX3CL1 in neuroinflammation and neurodegeneration has been suggested in recent publications. In this study, we compared the expression of CX3CL1 and its receptor CX3CR1 in human brain tissue derived from control patients without neurological complications and in multiple sclerosis (MS) patients. Results from this study demonstrate that CX3CL1 is constitutively expressed in human central nervous system (CNS) astrocytes in vivo and under basal conditions in human adult astrocyte cultures. CX3CR1 is expressed on astrocytes and microglial cells both in vivo and in vitro. Chemotaxis assay shows a functional response upon CX3CR1 signaling in microglial cells. Although CX3CL1 expression is upregulated in cultured astrocytes in response to proinflammatory cytokines, no evidence for expression differences of CX3CL1 between control patients and MS patients was found. Our data suggest that CX3CL1 has more general physiological functions, which occur also in the absence of proinflammatory conditions.

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.

TNF-alpha and IL-4 regulate expression of fractalkine (CX3CL1) as a membrane-anchored proadhesive protein and soluble chemotactic peptide on human fibroblasts.

The CX(3)C chemokine, fractalkine (FKN, CX(3)CL1), has multiple functions and exists as two distinct forms, a membrane-anchored protein and a soluble chemotactic peptide that cleaves from the cell surface FKN. In this study, we first demonstrated the expression of FKN in tumor necrosis factor (TNF)-alpha- and interleukin (IL)-4-stimulated human fibroblasts. The induction of FKN was observed for both forms. We also demonstrated monocyte chemotactic activity in the culture supernatant from the fibroblasts stimulated with these cytokines. These results suggest that TNF-alpha- and IL-4-stimulated fibroblasts may play an important role in accumulation of monocytes at inflammatory sites.

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.

Neuronal injury regulates fractalkine: relevance for HIV-1 associated dementia.

Fractalkine (FKN), a chemokine highly expressed in the central nervous system, participates in inflammatory responses operative in many brain disorders including HIV-1 associated dementia (HAD). In this report, HIV-1 progeny virions and pro-inflammatory products led to FKN production associated with neuronal injury and apoptosis. FKN was produced by neurons and astrocytes; but differentially produced by the two cell types. Laboratory tests paralleled those in infected people where cerebrospinal fluid FKN levels in HIV-1 infected cognitively impaired (n=16) patients were found to be increased when compared to infected patients without cognitive impairment (n=8, P=0.0345). These results demonstrate a possible role of FKN in HAD pathogenesis.

Inhibition by pentoxifylline of TNF-alpha-stimulated fractalkine production in vascular smooth muscle cells: evidence for mediation by NF-kappa B down-regulation.

(1) Fractalkine is a CX(3)C chemokine for mononuclear leukocytes that is expressed mainly by vascular cells, and regulated by pro-inflammatory cytokines. This study investigated signal transduction mechanisms by which tumor necrosis factor (TNF)-alpha stimulated fractalkine expression in cultured rat vascular smooth muscle cells (VSMCs), and the modulatory effect of a haemorrheologic agent, pentoxifylline, on its production. (2) TNF-alpha (1-50 ng ml(-1)) stimulated fractalkine mRNA and protein expression in concentration- and time-dependent manners. Pretreatment with calphostin C (0.4 micro M, a selective inhibitor of protein kinase C (PKC), and PD98059 (40 micro M), a specific inhibitor of p42/44 mitogen-activated protein kinase (MAPK) kinase, attenuated TNF-alpha-stimulated fractalkine mRNA and protein expression. In contrast, H-89 (2 micro M), a selective inhibitor of cAMP-dependent protein kinase, wortmannin (0.5 micro M), a selective inhibitor of phosphatidylinositol 3-kinase, and SB203580 (40 micro M), a specific inhibitor of p38 MAPK, had no discernible effect. (3) The ubiquitin/proteosome inhibitors, MG132 (10 micro M) and pyrrolidine dithiocarbamate (200 micro M), suppressed activation of NF-kappaB as well as stimulation of fractalkine mRNA and protein expression by TNF-alpha. (4) TNF-alpha-activated phosphorylation of PKC was blocked by calphostin C, whereas TNF-alpha-augmented phospho-p42/44 MAPK and phospho-c-Jun levels were reduced by PD98059. Neither calphostin C nor PD98059 affected TNF-alpha-induced degradation of I-kappaBalpha or p65 nuclear translocation. (5) Pretreatment with pentoxifylline (0.1-1 mg ml(-1)) decreased TNF-alpha-stimulated fractalkine mRNA and protein expression, which was preceded by a reduction in TNF-alpha-activated phosphorylation of PKC, p42/44 MAPK and c-Jun as well as degradation of I-kappaBalpha and p65/NF-kappaB nuclear translocation. (6) These data indicate that activation of PKC, p42/44 MAPK kinase, and NF-kappaB are involved in TNF-alpha-stimulated fractalkine production in VSMCs. Down-regulation of the PKC, p42/44 MAPK, and p65/NF-kappaB signals by PTX may be therapeutically relevant and provide an explanation for the anti-fractalkine effect of this drug.

Production and neuroprotective functions of fractalkine in the central nervous system.

The CX3C-chemokine, fractalkine is reportedly to be expressed in the central nervous system, and up-regulated in certain pathological conditions, such as HIV encephalopathy and multiple sclerosis. In the present study, we examined the production of fractalkine and the expression of its receptor, CX3CR1 in murine glial and neuronal cell in vitro, and investigated its neuroprotective functions. Both fractalkine and CX3CR1 were expressed constitutively in neurons, microglia, and astrocytes. Neither the production of fractalkine nor its receptor expression was up-regulated by lipopolysaccharide (LPS), as measured by mRNA expression and protein synthesis. Fractalkine dose-dependently suppressed the production of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha with activated microglia. It also significantly suppressed neuronal cell death induced by microglia activated with LPS and interferon-gamma, in a dose-dependent manner. These results suggest the possible functions of fractalkine as an intrinsic inhibitor against neurotoxicity by activated microglia.

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.

Heparin inhibits IFN-gamma-induced fractalkine/CX3CL1 expression in human endothelial cells.

Heparin is primarily used as an anticoagulant but has many biological functions as well. It binds with high affinity to a range of cytokines including interferon-gamma (IFN-gamma) and members of chemokine superfamily. IFN-gamma is a proinflammatory cytokine that plays a pivotal role in immune and inflammatory responses; and in endothelial cells, it regulates the expression of fractalkine/CX3CL1 that is a potent agonist for the chemotaxis and adhesion of monocytes and lymphocytes. We have investigated the effect of heparin on the fractalkine expression in human umbilical vein endothelial cells (HUVEC) in culture. HUVEC were treated with approximately 100 mg/mL heparin and the expression of the IFN-gamma-induced fractalkine mRNA and protein were measured by reverse transcription-PCR and western blotting. The IFN-gamma-induced expressions of fractalkine mRNA and protein were inhibited by heparin in a concentration-dependent manner. Heparin also inhibited adhesion of mononuclear cells (MNC) to HUVEC monolayers stimulated with IFN-gamma, but it did not inhibit the MNC adhesion to the monolayers stimulated with interleukin-1beta. Electrophoretic analysis demonstrated direct binding of heparin to IFN-gamma and heparin was found to partially block the binding of IFN-gamma to IFN-gamma receptor (IFN-gamma R). Heparin may play a regulatory role in inflammatory and immune responses by modulating the interaction between leukocytes and the vascular endothelium.