Chemokines and chemokine receptors expression in the lesions of patients with American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) presents distinct active clinical forms with different grades of severity, known as localised (LCL), intermediate (ICL) and diffuse (DCL) cutaneous leishmaniasis. LCL and DCL are associated with a polarised T-helper (Th)1 and Th2 immune response, respectively, whereas ICL, or chronic cutaneous leishmaniasis, is associated with an exacerbated immune response and a mixed cytokine expression profile. Chemokines and chemokine receptors are involved in cellular migration and are critical in the inflammatory response. Therefore, we evaluated the expression of the chemokines CXCL10, CCL4, CCL8, CCL11 and CXCL8 and the chemokine receptors CCR3, CXCR3, CCR5 and CCR7 in the lesions of patients with different clinical forms of ACL using immunohistochemistry. LCL patients exhibited a high density of CXCL10+, CCL4+ and CCL8+ cells, indicating an important role for these chemokines in the local Th1 immune response and the migration of CXCR3+ cells. LCL patients showed a higher density of CCR7+ cells than ICL or DCL patients, suggesting major dendritic cell (DC) migration to lymph nodes. Furthermore, DCL was associated with low expression levels of Th1-associated chemokines and CCL11+ epidermal DCs, which contribute to the recruitment of CCR3+ cells. Our findings also suggest an important role for epidermal cells in the induction of skin immune responses through the production of chemokines, such as CXCL10, by keratinocytes.     

趋化因子及其受体基因家族的系统进化分析

通过分析现有的趋化因子和趋化因子受体的氨基酸序列,用距离法和最简约法构建了聚类图,探讨了趋化因子和趋化因子受体基因家族的系统演化特征。可见基因家族成员的分化早于脊椎动物的分化。不同物种的同一种基因的聚类关系能较好地反映物种经因子受体的进化速度不同,其中ＣＸＣＲ４的进化速率最低。趋化因子和趋化因子受体可能都起源于少数几个原始的基因,病毒编码与寄主相似的趋化因子或受体是进化过程中分子模拟的结果。     

趋化因子及其受体家族分子适应性进化分析

Branch-Site模型是检测基因序列中单个密码子位点是否具有选择作用的统计学方法。该模型能有效地检测基因在进化历程中是否受到选择作用, 并预测出那些在进化过程中对功能分化有重要贡献的、受正选择作用的密码子位点。趋化因子是一类控制免疫细胞定向迁移的细胞因子, 其功能行使由趋化因子受体介导。该文用Branch-Site模型分析趋化因子及其受体基因家族的分子适应性, 发现只有少数种类基因受到正选择作用, 如RANTES、CCR5等。并预测出一些可能受到正选择作用的位点, 蛋白3D分析显示, 它们均位于趋化因子和相应受体相互作用的结构区域。     

Differential upregulation of chemokine receptors on CD56+ NK cells and their transmigration to the site of infection in tuberculous pleurisy

Chemokines and their receptors orchestrate leukocyte recruitment and confer immunity during Mycobacterium tuberculosis infection. The immunoregulatory and cytotoxic activities of natural killer (NK) cells are essential at the site of infection during tuberculous pleurisy. The frequency, subtypes, and expression of phenotype markers and chemokine receptors on NK cells were assessed by flow cytometry in tuberculous (TB) and nontuberculous (NTB) pleural fluid (PF). Chemotaxis was also shown in response to chemokines. A significant decrease in CD56^dim with no change in CD56^bright NK cells was observed, while a significant increase in activation markers and Toll-like receptors (TLRs) was observed on TB-PF CD56^bright NK cells. Significantly increased expression of chemokine receptors CCR1, CCR2 and CCR7 on CD56^bright and CCR5 on CD56^dim NK cells was observed in the TB group. Transmigration of TB-PF NK cells was significantly high in response to IL-8, IP-10, MCP-1 and SLC. Transmigrated TB-NK cells showed a significant increase in CXCR2, CCR2 and CCR7 expression. The study suggests that CD56^bright NK cells may recognize M. tuberculosis directly using TLRs, HLA-DR and express CD69 as an early activation marker. In addition, CC chemokines induce activation signals in chemokine receptors mediating differential NK cell migration to the site. Thus, NK cells act as first direct sensors and effectors in mycobacterial infection.     

Constitutive association of cell surface CCR5 and CXCR4 in the presence of CD4

Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV-1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF-1alpha. CCR5 was resistant to ligand-dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co-immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV-1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine-chemokine receptor interactions and HIV-1 coreceptor functions.     

pH-independent endocytic cycling of the chemokine receptor CCR5

Following agonist activation, the chemokine receptor CCR5 is internalised through clathrin-coated pits and delivered to recycling endosomes. Subsequently, ligand- free and resensitised receptors are recycled to the cell surface. Currently little is known of the mechanisms regulating resensitisation and recycling of this G-protein coupled receptor. Here we show that raising the pH of endocytic compartments, using bafilomycin A, monensin or NH(4)Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well-characterised G protein coupled receptor, the beta(2)-adrenergic receptor, following agonist-induced internalisation. CCR5-bound RANTES (CCL5) and MIP-1beta (CCL4) only exhibit pH-dependent dissociation at pH < 4.0, below the values normally found in endocytic organelles. Although receptor-agonist dissociation is not dependent on low pH, the subsequent degradation of released chemokine is inhibited in the presence of reagents that raise endosomal pH. Our data show that exposure to low pH is not required for RANTES or MIP-1beta dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.     

Functional defect of peripheral neutrophils in mice with induced deletion of CXCR2

Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis, and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and nonhematopoietic cells. Germline CXCR2 deficient (Cxcr2^‐/‐) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell‐types. Here, we describe Cxcr2^fl/fl mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2^fl/fl mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2^fl/fl mice were crossed to Mx‐Cre mice in which Cre recombinase is induced by Type I interferons, elicited by injection with polyinosinic‐polycytidylic acid (poly(I:C)). CXCR2‐deficient neutrophils were observed in poly(I:C) treated Cxcr2^fl/fl::Mx‐Cre⁺ (Cxcr2‐CKO) mice, but not in poly(I:C) treated Cxcr2^f//+::Mx‐Cre⁺ mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2‐CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2‐CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2^fl/fl mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations. genesis 51:587–595. © 2013 Wiley Periodicals, Inc.     

趋化因子及其受体在神经系统发育中的作用

趋化因子是具有趋化作用的一类细胞因子,参与白细胞迁移的调控,在炎症中诱导性表达,与炎症过程密切相关,最初的研究主要局限于免疫系统。近几年来研究发现,趋化因子不仅参与神经系统疾病的炎症过程,而且在神经细胞成熟、发育等生理情况下组成性表达,发挥重要的生理调节作用,这一有趣的现象日益成为关注的焦点。本文主要针对趋化因子及其受体在神经系统发育中的作用及相关机制的研究成果予以综述,将有助于深入理解趋化因子与神经系统发育的关系,为进一步的研究提供依据。     

趋化因子及其受体介导的细胞迁移与移植物抗宿主病

移植物抗宿主病(graft-versus-hostdisease,GVHD)是同种异基因骨髓移植中的重要并发征。供者T细胞在输注入受者体内后迁移进入淋巴组织,识别受者同种异基因抗原,被受者抗原递呈细胞(antigenpresentingcell,APC)激活,进而活化、增殖分化,介导急性GVHD的发生。现有的研究已表明,活化的异体效应性T细胞经淋巴组织迁移进入黏膜组织以及实质性靶器官,如消化道、肝脏、肺脏和皮肤,进而造成这些器官和组织的损伤。因此,分子间相互作用尤其是趋化因子及其受体介导的效应性细胞的迁移是GVHD发生发展过程中关键的一环,受到了广泛的关注。进一步以趋化因子及其受体为靶标,亦可能形成有效的免疫生物学治疗,具有广阔的应用前景。     

Chemokine receptor-usage of clinical HIV-1 isolates obtained from patients with HIV-1 infection in late clinical stages using PHA-blast

In the present sudy, chemokine receptor-usage of primary HIV-1 isolates was examined using U87-CD4 cells expressing chemokine receptors CCR3, CCR5 and CXCR4. HIV-1 was isolated from the peripheral blood mononuclear cells (PBMC) and/or plasma of eight HIV-1-infected individuals in late CDC-II and CDC-IV clinical stages using PHA-blast prepared from the PBMC of healthy blood donors. The primary HIV-1 isolates from patients in late CDC-II stage rarely infected monocyte-derived macrophages in the present study, whereas most isolates from patients in the CDC-IV stage infected the macrophages. In the experiments using U87-CD4 cells expressing chemokine receptors, the isolates from patients in the late CDC-II stage infected U87-CD4 cells expressing CXCR4, but not U87-CD4 cells expressing CCR5. In contrast, most isolates from patients in the CDC-IV stage infected both U87-CD4 cells expressing CXCR4 or CCR5. The isolates which infected both U87-CD4 cells were supposed to contain dual tropic HIV-1 or a mixture of CXCR4-tropic and CCR5-tropic HIV-1s. Analysis of the deduced amino acid sequence of the V3 region in proviral env gene showed that the V3 region in U87-CD4 cells infected with CXCR4-tropic HIV-1 isolates was largely different from that in the cells infected with CCR5-tropic isolates, but were highly similar to that in cells infected with dual tropic isolates. These results suggest that PHA-blasts may preferentially support the replication of the CXCR4-tropic and dual tropic HIV-1s, and that CXCR4-tropic and dual tropic HIV-1s are also present in peripheral blood from patients in the late stage of the asymptomatic phase.     

The good and the bad of chemokines/chemokine receptors in melanoma

Chemokine ligand/receptor interactions affect melanoma cell growth, stimulate or inhibit angiogenesis, recruit leukocytes, promote metastasis, and alter the gene expression profile of the melanoma associated fibroblasts. Chemokine/chemokine receptor interactions can protect against tumor development/growth or can stimulate melanoma tumor progression, tumor growth and metastasis. Metastatic melanoma cells express chemokine receptors that play a major role in the specifying the organ site for metastasis, based upon receptor detection of the chemokine gradient elaborated by a specific organ/tissue. A therapeutic approach that utilizes the protective benefit of chemokines involves delivery of angiostatic chemokines or chemokines that stimulate the infiltration of cytotoxic T cells and natural killer T cells into the tumor microenvironment. An alternative approach that tackles the tumorigenic property of chemokines uses chemokine antibodies or chemokine receptor antagonists to target the growth and metastatic properties of these interactions. Based upon our current understanding of the role of chemokine‐mediated inflammation in cancer, it is important that we learn to appropriately regulate the chemokine contribution to the tumorigenic ‘cytokine/chemokine storm’, and to metastasis.     

The role of CXCR7 on the adhesion, proliferation and angiogenesis of endothelial progenitor cells

Previous studies confirmed that stromal cell-derived factor 1 (SDF-1) was a principal regulator of retention, migration and mobilization of haematopoietic stem cells and endothelial progenitor cells (EPCs) during steady-state homeostasis and injury. CXC chemokine receptor 4 (CXCR4) has been considered as the unique receptor of SDF-1 and as the only mediator of SDF-1-induced biological effects for many years. However, recent studies found that SDF-1 could bind to not only CXCR4 but also CXC chemokine receptor 7 (CXCR7). The evidence that SDF-1 binds to the CXCR7 raises a concern how to distinguish the potential contribution of the SDF-1/CXCR7 pathway from SDF-1/CXCR4 pathway in all the processes that were previously attributed to SDF-1/CXCR4. In this study, the role of CXCR7 in EPCs was investigated in vitro. RT-PCR, Western blot and flow cytometry assay demonstrate that both CXCR4 and CXCR7 were expressed highly in EPCs. The adhesion of EPCs induced by SDF-1 was inhibited by blocking either CXCR4 or CXCR7 with their antibodies or antagonists. SDF-1 regulated the migration of EPCs via CXCR4 but not CXCR7. However, the transendothelial migration of EPCs was inhibited by either blocking of CXCR4 or CXCR7. Both CXCR7 and CXCR4 are essential for the tube formation of EPCs induced by SDF-1. These results suggested that both CXCR7 and CXCR4 are important for EPCs in response to SDF-1, indicating that CXCR7 may be another potential target molecule for angiogenesis-dependent diseases.     

Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases

Stromal chemokine gradients within the breast tissue microenvironment play a critical role in breast cancer cell invasion, a prerequisite to metastasis. To elucidate which chemokines and mechanisms are involved in mammary cell migration we determined whether mesenchymal D1 stem cells secreted specific chemokines that differentially promoted the invasion of mammary tumor cells in vitro. Results indicate that mesenchymal D1 cells produced concentrations of CCL5 and CCL9 4- to 5-fold higher than the concentrations secreted by 4T1 tumor cells (P < 0.01). Moreover, 4T1 tumor cell invasion toward D1 mesenchymal stem cell conditioned media (D1CM), CCL5 alone, CCL9 alone or a combination CCL5 and CCL9 was observed. The invasion of 4T1 cells toward D1 mesenchymal stem CM was dose-dependently suppressed by pre-incubation with the CCR1/CCR5 antagonist met-CCL5 (P < 0.01). Furthermore, the invasion of 4T1 cells toward these chemokines was prevented by incubation with the broad-spectrum MMP inhibitor GM6001. Additionally, the addition of specific MMP9/MMP13 and MMP14 inhibitors prevented the MMP activities of supernatants collected from 4T1 cells incubated with D1CM, CCL5 or CCL9. Taken together these data highlight the role of CCL5 and CCL9 produced by mesenchymal stem cells in mammary tumor cell invasion.     

Chemokine receptor CCR5 functionally couples to inhibitory G proteins and undergoes desensitization

Chemokine receptor CCR5 is not only essential for chemotaxis of leukocytes but also has been shown to be a key coreceptor for HIV-1 infection. In the present study, hemagglutinin epitope-tagged human CCR5 receptor was stably expressed in Chinese hamster ovary cells or transiently expressed in NG108–15 cells to investigate CCR5-mediated signaling events. The surface expression of CCR5 was confirmed by flow cytometry analysis. The CCR5 agonist RANTES stimulated [³⁵S]GTPγS binding to the cell membranes and induced inhibition on adenylyl cyclase activity in cells expressing CCR5. The effects of RANTES were CCR5 dependent and could be blocked by pertussis toxin. Furthermore, overexpression of Giα2 strongly increased both RANTES-dependent G-protein activation and inhibition on adenylyl cyclase in cells cotransfected with CCR5. These data demonstrated directly that activation of CCR5 stimulated membrane-associated inhibitory G proteins and indicated that CCR5 could functionally couple to G-protein subtype Giα2. The abilities of CCR5 to activate G protein and to inhibit cellular cAMP accumulation were significantly diminished after a brief prechallenge with RANTES, showing rapid desensitization of the receptor-mediated responsiveness. Prolonged exposure of the cells to RANTES caused significant reduction of surface CCR5 as measured by flow cytometry, indicative of agonist-dependent receptor internalization. Our data thus demonstrated that CCR5 functionally couples to membrane-associated inhibitory G proteins and undergoes agonist-dependent desensitization and internalization. J. Cell. Biochem. 71:36–45, 1998. © 1998 Wiley-Liss, Inc.     

Angiotensin II–C–C chemokine receptor2/5 axis‐dependent monocyte/macrophage recruitment contributes to progression of experimental autoimmune myocarditis

Angiotensin II (ANG II) plays critical roles in modulation of circulatory homeostasis and activation of innate and adaptive immunity and has also been implicated in several mouse models of autoimmune disease. However, how ANG II regulates macrophages and is involved in development of experimental autoimmune myocarditis (EAM) remains unclear. Therefore, the present study aimed to address the above question and explore possible mechanisms. EAM was induced in BALB/c mice. ANG II was quantitated by ELISA and hematoxylin and eosin staining was employed to analyze pathological changes and macrophage infiltration. The chemotactic ability of ANG II was assessed by using a Transwell system. It was found that ANG II is up‐regulated in serum and heart tissues of mice with EAM and that ANG II significantly drives monocyte/macrophage infiltration through the C‐C chemokine receptor 2/5 (CCR2/5) axis. CCR2/5 antagonists and ANG II receptor inhibitor could all abrogate monocyte/macrophage infiltration and ameliorate development of EAM. Our results have firstly identified a novel function of ANG II: that it is a critical chemokine for monocyte/macrophage recruitment. Furthermore, our results indicate that ANG II is a potential candidate for treatment of inflammatory diseases.

     

Chemokine receptor 5 blockade modulates macrophage trafficking in renal ischaemic-reperfusion injury

Chemokine receptor 5 (CCR5) is a pivotal regulator of macrophage trafficking in the kidneys in response to an inflammatory cascade. We investigated the role of CCR5 in experimental ischaemic-reperfusion injury (IRI) pathogenesis. To establish IRI, we clamped the bilateral renal artery pedicle for 30 min and then reperfused the kidney. We performed adoptive transfer of lipopolysaccharide (LPS)-treated RAW 264.7 macrophages following macrophage depletion in mice. B6.CCR5^−/− mice showed less severe IRI based on tubular epithelial cell apoptosis than did wild-type mice. CXCR3 expression in CD11b⁺ cells and inducible nitric oxide synthase levels were more attenuated in B6.CCR5^−/− mice. B6.CCR5^−/− mice showed increased arginase-1 and CD206 expression. Macrophage-depleted wild-type mice showed more injury than B6.CCR5^−/− mice after M1 macrophage transfer. Adoptive transfer of LPS-treated RAW 264.7 macrophages reversed the protection against IRI in wild-type, but not B6.CCR5^−/− mice. Upon knocking out CCR5 in macrophages, migration of bone marrow-derived macrophages from wild-type mice towards primary tubular epithelial cells with recombinant CCR5 increased. Phospho-CCR5 expression in renal tissues of patients with acute tubular necrosis was increased, showing a positive correlation with tubular inflammation. In conclusion, CCR5 deficiency favours M2 macrophage activation, and blocking CCR5 might aid in treating acute kidney injury.     

重组人β趋化因子RANTES基因融合表达产物活性分析

为研制基于病毒受体的抗艾滋病毒感染基因药物 ,将重组人 β趋化因子RANTES基因导入大肠杆菌中表达谷胱甘肽S 转移酶 (GST) RANTES融合蛋白 ,并回收携带末端延伸肽 (TEP)的RANTES修饰蛋白 .荧光免疫化学分析表明 ,2种非天然RANTES蛋白均显示对人外周血淋巴细胞的结合活性 .暗示在细胞表面可能已发生RANTES与CCR5之间的相互作用 .2种修饰RANTES蛋白都能使人外周血细胞的过氧化物酶活性显著升高 ,提示这 2个人造蛋白可能仍存在对淋巴细胞的趋化性诱导 .     

Effects of blocking chemokine receptor CCR1 with BX471 in two models of fibrosis prevention and rescue in mice

BackgroundThe induction, progression and resolution of liver fibrosis are influenced by multiple chemokines. The inhibition of CCR1 signalling by a specific non-peptide inhibitor (BX471) reduces kidney fibrosis after unilateral ureteral obstruction via suppression of leukocyte recruitment in mice. However, it remains unclear whether selective CCR1 inhibition also affects hepatic fibrogenesis. Therefore we aimed to study the effect of this intervention on liver fibrosis in prevention (CCl₄ administration) and rescue (ABCB4-deficient mice) mouse models.MethodsIn the prevention model, hepatic fibrosis was induced by repeated injections of CCl₄. Additionally, the verum group was treated with subcutaneous injections of BX471, while controls received vehicle only. ABCB4 deficient mice (on the BALB/c-background) with sclerosing cholangitis and biliary fibrosis received BX471 or vehicle, respectively (rescue model). Liver histopathology was assessed after Sirius red staining of collagen, and hepatic collagen contents were measured. In addition, we performed gene expression analyses of fibrosis-related genes.ResultsBX471 injections were tolerated moderately well by all mice, and all mice developed hepatic fibrosis. Significant differences were neither observed in serum aminotransferase activities after 6 weeks of treatment between the two groups in the prevention nor in the rescue model. Interestingly, hepatic collagen contents were significantly higher in mice treated with BX471 in the prevention model as compared to controls but histological stages of liver sections did not differ. Of note, we observed only moderate effects on liver fibrosis in the ABCB4 knock-out model.ConclusionsOur data indicate that BX471 treatment did neither affect serum and tissue markers of liver injury and fibrosis in the CCl₄ model and only moderately in the Abcb4^-/- model of biliary fibrosis. The animal models indicate that treatment with BX471 alone is unlikely to exert major beneficial effects in chronic liver disease.     

趋化因子SDF-1及受体CXCR4研究进展

趋化因子(chemokine)是一类一级结构相似,以对白细胞等多种细胞具有趋化定向运动作用为特征的小分子蛋白。功能研究表明,趋化因子在胚胎发育、血管生成、炎症、肿瘤、艾滋病等机体多种生理和病理过程中发挥重要作用,部分趋化因子的衍生物或抑制物具有潜在的临床应用前景。不久的将来,趋化因子及其受体可能成为疾病治疗的分子靶点。