Early activation of the cardiac CX3CL1/CX3CR1 axis delays ß-adrenergic-induced heart failure.

We recently highlighted a novel potential protective paracrine role of cardiac myeloid CD11b/c cells improving resistance of adult hypertrophied cardiomyocytes to oxidative stress and potentially delaying evolution towards heart failure (HF) in response to early ß-adrenergic stimulation. Here we characterized macrophages (Mφ) in hearts early infused with isoproterenol as compared to control and failing hearts and evaluated the role of upregulated CX3CL1 in cardiac remodeling. Flow cytometry, immunohistology and Mφ-depletion experiments evidenced a transient increase in Mφ number in isoproterenol-infused hearts, proportional to early concentric hypertrophy (ECH) remodeling and limiting HF. Combining transcriptomic and secretomic approaches we characterized Mφ-enriched CD45+ cells from ECH hearts as CX3CL1- and TNFα-secreting cells. In-vivo experiments, using intramyocardial injection in ECH hearts of either Cx3cl1 or Cx3cr1 siRNA, or Cx3cr1-/- knockout mice, identified the CX3CL1/CX3CR1 axis as a protective pathway delaying transition to HF. In-vitro results showed that CX3CL1 not only enhanced ECH Mφ proliferation and expansion but also supported adult cardiomyocyte hypertrophy via a synergistic action with TNFα. Our data underscore the in-vivo transient protective role of the CX3CL1/CX3CR1 axis in ECH remodeling and suggest the participation of CX3CL1-secreting Mφ and their crosstalk with CX3CR1-expressing cardiomyocytes to delay HF.

Noninvasive Imaging of CCR2+ Cells in Ischemia-Reperfusion Injury After Lung Transplantation.

Ischemia-reperfusion injury-mediated primary graft dysfunction substantially hampers short- and long-term outcomes after lung transplantation. This condition continues to be diagnosed based on oxygen exchange parameters as well as radiological appearance, and therapeutic strategies are mostly supportive in nature. Identifying patients who may benefit from targeted therapy would therefore be highly desirable. Here, we show that C-C chemokine receptor type 2 (CCR2) expression in murine lung transplant recipients promotes monocyte infiltration into pulmonary grafts and mediates graft dysfunction. We have developed new positron emission tomography imaging agents using a CCR2 binding peptide, ECLi1, that can be used to monitor inflammatory responses after organ transplantation. Both 64 Cu-radiolabeled ECL1i peptide radiotracer (64 Cu-DOTA-ECL1i) and ECL1i-conjugated gold nanoclusters doped with 64 Cu (64 CuAuNCs-ECL1i) showed specific detection of CCR2, which is upregulated during ischemia-reperfusion injury after lung transplantation. Due to its fast pharmacokinetics, 64 Cu-DOTA-ECL1i functioned efficiently for rapid and serial imaging of CCR2. The multivalent 64 CuAuNCs-ECL1i with extended pharmacokinetics is favored for long-term CCR2 detection and potential targeted theranostics. This imaging may be applicable for diagnostic and therapeutic purposes for many immune-mediated diseases.

Role of chemokines polymorphisms in diseases.

The interest into chemokine polymorphisms came with the discovery of allelic variants of HIV co-receptors (CCR5 and CXCR4 mainly) that confers protection against virus entry into usual permissive cells. Since then, chemokines genetic background has been deeply studied in order to find associations between allelic variants and inflammation-related diseases as well as infectious diseases. In addition to HIV infection, chemokines genetic variations have been involved in other infectious diseases as HCV, Malaria and West Nile Virus, and also in a variety of non-infectious diseases such as cancer, auto-immune and cardiovascular diseases. This review aims to present genetic variations in chemokines encoding genes and discuss their role, sometimes controversial, in a variety of diseases.

[Polymorphisms of complement factor genes and age-related macular degeneration in a German population]. / Komplementfaktor-Polymorphismen und altersassoziierte Makuladegeneration in einer Gruppe deutscher Patienten.

BACKGROUND: An association of the Tyr402His variant of the complement factor H (CFH) gene with age-related macular degeneration (AMD) has been shown in several Caucasian populations, while studies for an association with other single nucleotide polymorphisms (SNP) of complement system genes have produced inconsistent results. We examined the distribution of several SNPs of complement system genes (CFH, C 2, C 3, factor B) in patients with exsudative AMD and healthy controls. PATIENTS/MATERIALS AND METHODS: 226 patients with exsudative AMD and 179 controls without AMD were included. Genomic DNA was extracted from saliva samples. RESULTS: A significant association with exsudative AMD was found only for SNP rs1061170 (Y402 H) in the CFH gene. For rs1047286 (P292L) and rs2230199 (R102G) in the C 3 gene, rs547154 (IVS10) and rs9332739 (E318D) in the C 2 gene and rs4151667 (L9 H) in CFB gene, no associations with exsudative AMD were found. CONCLUSIONS: We have replicated an association of the Y 402 H variant with exsudative AMD in our population. Although variants R 102G, IVS10, E 318D and L 9 H have been shown to be associated with AMD in earlier studies, we could not confirm these findings. The results show that AMD has variable association patterns with rare variants in different populations.

A polymorphism in the CCL2 chemokine gene is associated with asthma risk: a case-control and a family study in Tunisia.

Asthma is a complex genetic disorder characterized by chronic airway inflammation. We hypothesized that genetic polymorphisms in chemokines and their receptors alter leukocyte mobilization and may thus influence the risk and severity of childhood asthma. Distributions of the chemokine CCL2-2578G, CCL2-927C, CCR2-V64I, CX3CR1-V249I and CX3CR1-T280M receptor polymorphisms were examined in a case-control study of 121 children with asthma and 226 age-matched healthy controls and then replicated in a family study of 99 simplex families (297 individuals). The case-control study revealed that the CCL2-2578G allele was less frequent in children with than in those without asthma (P=0.0012). No association with asthma was found for the CCL2-927, CCR2 or CX3CR1 polymorphisms. The finding in the family study that the CCL2-2578G allele was transmitted less often by heterozygous parents to their children with asthma (P=0.0016) confirms the association of CCL2-2578G with asthma risk. Biochemical studies indicated that plasma CCL2 concentrations were higher in both patients (P=0.0214) and controls (P=0.001) carrying the G allele than in subjects with other polymorphisms. Both case-control and family-based studies suggest a protective effect of allele CCL2-2578G in Tunisian asthmatic children.

Lipid-bloated subretinal microglial cells are at the origin of drusen appearance in CX3CR1-deficient mice.

Drusen, the white yellowish deposits that can be seen in funduscopy, are a hallmark of age-related macular degeneration. Histologically, drusen are believed to be dome-shaped or more confluent lipid accumulations between the retinal pigment epithelium and the choriocapillaries. Recent advances in mouse funduscopy have revealed the presence of drusen-like structures in chemokine knockout animals in the absence of sizeable dome-shaped material below the retinal pigment epithelium. We show that aged CX3CR1-/- mice present with drusen-like appearance in funduscopy that is associated with a progressive age-related microglial cell accumulation in the subretinal space. We demonstrate that the anatomical equivalent of the drusen-like appearance in these mice are lipid-bloated subretinal microglial cells rather than subretinal pigment epithelium deposits [Combadière C, et al: J Clin Invest 2007;117:2920-2928].

Expression of chemokine receptors predicts the site of metastatic relapse in patients with axillary node positive primary breast cancer.

BACKGROUND: Recent studies have suggested that chemokine receptors are involved in development of organ-specific pattern of metastases. In the present study, we evaluated the association between the chemokine receptors expressed in primary tumor cells and the site of metastatic relapse in patients with breast cancer. METHODS: Primary tumors were obtained from 142 patients with axillary node-positive breast cancer and stained for CX3CR1, CXCR4, CCR6, and CCR7 expression. All statistical analyses were adjusted for systemic post-operative treatment. RESULTS: After a median follow-up of 13 years, none of the chemokine receptors was associated with overall survival or disease free survival. However, expression of chemokine receptors was found to be associated with increased risk of relapse in certain organs. By estimating the Mantel-Haenszel odds ratios (OR), CXCR4 was associated with increased risk of metastasis to the liver (OR = 3.71, P = 0.005), CX3CR1 was associated with metastasis to the brain (OR = 13.18, P = 0.01). Patients with CCR6 positivity were more likely to develop a first metastasis in the pleura (OR = 2.82, P = 0.06). In addition, CCR7 expression was associated with the occurrence of skin metastases (11% versus 0%, P = 0.017). INTERPRETATION: Expression of chemokine receptors in the primary tumor predicts the site of metastatic relapse in patients with axillary node positive breast cancer. This study, in concordance with the data obtained in animal models, suggests that the chemokine receptors family could be the biological support of the 'seed and soil' theory.

Chemokine receptor CXCR4 and early-stage non-small cell lung cancer: pattern of expression and correlation with outcome.

BACKGROUND: The expression of CXCR4 has been implicated in metastatic dissemination in different models of breast cancer and melanoma. In the present study, we evaluated CXCR4 expression in non-small-cell lung cancer (NSCLC) and the relationship between CXCR4 expression and the prognosis of stage I disease. PATIENTS AND METHODS: Using immunohistochemical analysis, we retrospectively analyzed CXCR4 expression in specimens from 61 patients with completely resected pathologically confirmed stage I NSCLC for whom clinical follow-up data were available. RESULTS: In the present study, we have shown that: CXCR4 is expressed by tumor cells in stage I NSCLC; CXCR4 is located in the nucleus and/or in the cytoplasm of tumor cells; strong nuclear staining was observed in 17 cases (29.8%); patients whose tumors had CXCR4-positive nuclear staining had a significantly longer duration of survival than patients whose tumors had no nuclear expression (P = 0.039, log-rank test). Interestingly, the 5-year metastasis rates were 23.5% and 34.1% in patients with CXCR4-positive and CXCR4-negative nuclear expression, respectively (P = 0.2). CONCLUSION: Strong CXCR4-positive nuclear staining was associated with a significantly better outcome in early-stage NSCLC. The mechanisms underlying this clinically and biologically important finding need to be further explored.

[Chemokines and immunomodulation: applications for HIV infections]. / Chimiokines et immunomodulation: applications à l'infection VIH.

The genetic control of HIV infection by the host involves a certain number of genes, among which those which code for chemokines/chemokines receptors, cytokines, MHC. Genes such as CCR5, CCR2, SDF1, and more recently CX3CR1 received great attention from several laboratories including ours, since they play a role as HIV coreceptor and, as such, on the infectivity of the host. In addition, it was shown that the polymorphism of these genes influences the evolution of infection, whether they have a protective or deleterious effect. Results obtained by our laboratory on the genetic polymorphism and its implication in HIV infection will be reported herein. Furthermore, to better understand their role, we looked for the capacities that the chemokines may have to play an immunomodulatory function, independently of their chemoattractive effect. In two examples, we showed that chemokines influence notably the cellular immune functions, such as CD8 cytotoxicity (Rantes/CCR3) and gamma interferon production (fractalkine/CX3CR1). Globally, the results indicate that chemokines/chemokines receptors polymorphism represent important epidemiological factors, but also contributes to evaluate the prognosis of HIV infection, through a better understanding of the disease physiopathology.

Polymorphism in the fractalkine receptor CX3CR1 as a genetic risk factor for coronary artery disease.

Coronary atherosclerosis is a major cause of death in industrialized countries. Monocytes, which play a key role in atherosclerosis, migrate into the vessel wall, presumably guided by specific chemoattractant and adhesion molecules. A compelling candidate for this role is the chemokine receptor CX3CR1, which is expressed on monocytes and acts as either a chemotactic receptor or an adhesion molecule, depending on whether its ligand, fractalkine, is presented free or membrane bound. A common variant of CX3CR1 was recently identified, encoded by the alleles I249 and M280, which form a common I(249)M(280) haplotype. When CX3CR1 genotypes were analyzed in 151 patients with acute coronary syndromes and in 249 healthy controls, CX3CR1 I249 heterozygosity was associated with a markedly reduced risk of acute coronary events, independent of established acquired coronary risk factors (eg, smoking, diabetes). The adjusted odds ratio for this allele was 0.43 (95% confidence interval, 0.26-0.72; P =.001). Consistent with this, functional analysis of peripheral blood mononuclear cells showed that CX3CR1 I249 heterozygosity was associated with a significant decrease in the number of fractalkine binding sites per cell. The results show that CX3CR1 I249 is an independent genetic risk factor for coronary artery disease and that CX3CR1 may be involved in the pathogenesis of atherosclerotic disease. (Blood. 2001;97:1925-1928)

Relative diabetogenic properties of islet-specific Tc1 and Tc2 cells in immunocompetent hosts.

CD8(+) T cells are important effectors, as well as regulators, of organ-specific autoimmunity. Compared with Tc1-type CD8(+) cells, Tc2 cells have impaired anti-viral and anti-tumor effector functions, although no data are yet available on their pathogenic role in autoimmunity. Our aim was to explore the role of autoreactive Tc1 and Tc2 cells in autoimmune diabetes. We set up an adoptive transfer model in which the recipients were transgenic mice expressing influenza virus hemagglutinin (HA) specifically in their pancreatic ss islet cells (rat insulin promoter-HA mice) and islet-specific Tc1 and Tc2 cells were generated in vitro from HA-specific CD8(+) cells of TCR transgenic mice (CL4-TCR mice). One million Tc1 cells, differentiated in vitro in the presence of IL-12, transferred diabetes in 100% of nonirradiated adult rat insulin promoter-HA recipients; the 50% diabetogenic dose was 5 x 10(5). Highly polarized Tc2 cells generated in the presence of IL-4, IL-10, and anti-IFN-gamma mAb had a relatively low, but definite, diabetogenic potential. Thus, 5 x 10(6) Tc2 cells caused diabetes in 6 of 18 recipients, while the same dose of naive CD8(+) cells did not cause diabetes. Looking for the cause of the different diabetogenic potential of Tc1 and Tc2 cells, we found that Tc2 cells are at least as cytotoxic as Tc1 cells but their accumulation in the pancreas is slower, a possible consequence of differential chemokine receptor expression. The diabetogenicity of autoreactive Tc2 cells, most likely caused by their cytotoxic activity, precludes their therapeutic use as regulators of autoimmunity.

Rapid progression to AIDS in HIV+ individuals with a structural variant of the chemokine receptor CX3CR1.

Human immunodeficiency virus (HIV) enters cells in vitro via CD4 and a coreceptor. Which of 15 known coreceptors are important in vivo is poorly defined but may be inferred from disease-modifying mutations, as for CCR5. Here two single nucleotide polymorphisms are described in Caucasians in CX3CR1, an HIV coreceptor and leukocyte chemotactic/adhesion receptor for the chemokine fractalkine. HIV-infected patients homozygous for CX3CR1-I249 M280, a variant haplotype affecting two amino acids (isoleucine-249 and methionine-280), progressed to AIDS more rapidly than those with other haplotypes. Functional CX3CR1 analysis showed that fractalkine binding is reduced among patients homozygous for this particular haplotype. Thus, CX3CR1-I249 M280 is a recessive genetic risk factor in HIV/AIDS.

Identification of CX3CR1. A chemotactic receptor for the human CX3C chemokine fractalkine and a fusion coreceptor for HIV-1.

Fractalkine is a multimodular human leukocyte chemoattractant protein and a member of the chemokine superfamily. Unlike other human chemokines, the chemokine domain of fractalkine has three amino acids between two conserved cysteines, referred to as the CX3C motif. Both plasma membrane-associated and shed forms of fractalkine have been identified. Here, we show that the recombinant 76-amino acid chemokine domain of fractalkine is a potent and highly specific chemotactic agonist at a human orphan receptor previously named V28 or alternatively CMKBRL1 (chemokine beta receptor-like 1), which was shown previously to be expressed in neutrophils, monocytes, T lymphocytes, and several solid organs, including brain. CMKBRL1/V28 also functioned with CD4 as a coreceptor for the envelope protein from a primary isolate of HIV-1 in a cell-cell fusion assay, and fusion was potently and specifically inhibited by fractalkine. Thus CMKBRL1/V28 is a specific receptor for fractalkine, and we propose to rename it CX3CR1 (CX3C chemokine receptor 1), according to an accepted nomenclature system.

CC chemokine receptors 1 and 3 are differentially regulated by IL-5 during maturation of eosinophilic HL-60 cells.

CC chemokine receptors 1 and 3 (CCR1 and CCR3) are expressed by eosinophils; however, factors regulating their expression and function have not previously been defined. Here we analyze chemokine receptor expression and function during eosinophil differentiation, using the eosinophilic cell line HL-60 clone 15 as a model system. RNA for CCR1, -3, -4, and -5 was not detectable in the parental cells, and the cells did not specifically bind CC chemokines. Cells treated with butyric acid acquired eosinophil characteristics; expressed mRNA for CCR1 and CCR3, but not for CCR4 or CCR5; acquired specific binding sites for macrophage-inflammatory protein-1alpha and eotaxin (the selective ligands for CCR1 and CCR3, respectively); and exhibited specific calcium flux and chemotaxis responses to macrophage-inflammatory protein-1alpha, eotaxin, and other known CCR1 and CCR3 agonists. CCR3 was expressed later and at lower levels than CCR1 and could be further induced by IL-5, whereas IL-5 had little or no effect on CCR1 expression. Consistent with the HIV-1 coreceptor activity of CCR3, HL-60 clone 15 cells induced with butyric acid and IL-5 fused with HeLa cells expressing CCR3-tropic HIV-1 envelope glycoproteins, and fusion was blocked specifically by eotaxin or an anti-CCR3 mAb. These data suggest that CCR1 and CCR3 are markers of late eosinophil differentiation that are differentially regulated by IL-5 in this model.

Gene organization and promoter function for CC chemokine receptor 5 (CCR5).

CC chemokine receptor 5 (CCR5) functions physiologically as a receptor for the leukocyte chemoattractants macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and RANTES, and functions pathologically as a key cell entry coreceptor for HIV-1. The factors that regulate CCR5 expression may be useful therapeutic targets for HIV-1 infection. To identify nuclear regulatory factors, we have located and functionally characterized the CCR5 gene promoter. The gene consists of two exons separated by a 1.9-kb intron. Exon 1 contains 43 bp of the 5'-untranslated region; exon 2 contains 11 bp of the 5'-untranslated region and the complete open reading frame. Primer extension analysis identified two adjacent transcriptional start points (tsp) that map to the first 2 bp found in the longest known CCR5 cDNA sequence. A TATA box is present 31 bp upstream from the first tsp. CCR5 mRNA was detected constitutively in both primary human myeloid and lymphoid cells by Northern blot hybridization. Consistent with this, transcription of a chloramphenicol acetyltransferase reporter gene was constitutively activated in both transiently transfected myeloid and lymphoid cell lines by the 80-bp gene fragment located immediately upstream of the tsp. Deletion analysis located a strong silencer element between nucleotides -244 and -80, and a strong enhancer element between -486 and -244. These results suggest that the gene region between -486 and -1 may regulate the expression of CCR5 in monocyte/macrophages and T lymphocytes.

Dendritic cells express multiple chemokine receptors used as coreceptors for HIV entry.

Cells of the dendritic lineage are thought to be among the first cells infected after mucosal exposure to HIV. In this study, we have identified the presence of multiple chemokine receptors on dendritic cells (DC) that may function as coreceptors for HIV entry. DC effectively used CCR5 for entry of macrophage (M)-tropic isolates. CCR3, the eotaxin receptor, initially identified on eosinophils, is expressed on DC and may be used as an entry coreceptor by certain dual-tropic strains. CXCR4 was not expressed on DC, although SDF-1 induced a calcium flux and DC could be infected by T cell line (T)-tropic HIV. Our findings provide evidence for the presence of a non-CXCR4 SDF-1 receptor on DC that is used mainly by T-tropic strains of HIV. DC from individuals homozygous for a 32-bp deletion of the CCR5 gene are also infectable with M-tropic strains of HIV-1, and this infection is inhibited by stromal cell-derived factor (SDF)1, suggesting that this receptor can also be used by M-tropic HIV for entry. Delineation of the spectrum of coreceptor usage on DC may offer new approaches to interfere with the initiation and propagation of HIV infection.

Gene cloning, RNA distribution, and functional expression of mCX3CR1, a mouse chemotactic receptor for the CX3C chemokine fractalkine.

Human fractalkine and its apparent murine counterpart neurotactin are the only members identified so far of the CX3C subfamily of chemokines. Recently, a human fractalkine receptor was identified and named CX3CR1. Here we have identified a mouse counterpart of this receptor. The receptor was identified by analysis of a mouse genomic clone named PC2 isolated by homology hybridization using CX3CR1 as probe. Clone PC2 has a 354-codon open reading frame that has 83% amino acid identity to CX3CR1. PC2 RNA was abundant in brain and lung and comparatively less abundant in lung, liver, kidney, testis, and peripheral blood leukocytes, a pattern similar to that found for CX3CR1. The recombinant fractalkine, but no other chemokines tested, induced chemotaxis and transient increases in [Ca2+]i in HEK 293 cells transfected with PC2, whereas untransfected cells did not respond. Furthermore, fractalkine bound specifically to the transfected cells (Kd=4 nM). Thus, fractalkine is a functional ligand for this receptor and we propose to name it mCX3CR1 for murine CX3C chemokine receptor 1.

Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309.

The human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre-B cell line exhibited calcium flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors. CCR8 may regulate monocyte chemotaxis and thymic cell line apoptosis.