The CC chemokine antagonist Met-RANTES inhibits eosinophil effector functions through the chemokine receptors CCR1 and CCR3

Eosinophils are predominant effector cells not only in allergic diseases but also in connective tissue diseases. The recruitment of eosinophils to the site of inflammation and release of reactive oxygen species leading to tissue damage and propagation of the inflammatory response are mediated by chemokines. Thus, agents that would be able to inhibit or antagonize chemokine-induced eosinophil activation are interesting as therapeutical agents. We describe the effect of a chemokine receptor antagonist, Met-RANTES, on human eosinophil effector functions in response to RANTES, monocyte chemoattractant protein (MCP)-3 and eotaxin. Met-RANTES was able to inhibit dose-dependently [Ca²⁺]_i transients in eosinophils following stimulation with RANTES, MCP-3 and eotaxin. Whereas maximal and half-maximal inhibitory effect of Met-RANTES following stimulation with RANTES and MCP-3 were observed at 2 μg/ml and 1 μg/ml, respectively, maximal and half-maximal inhibitory effects of Met-RANTES in response to eotaxin were detected at 10 μg/ml and 3 μg/ml. Moreover, eotaxin-induced [Ca²⁺]_i transients were only half reduced at a Met-RANTES concentration at which RANTES and MCP-3 were completely blocked. Besides its effect on [Ca²⁺]_i transients, Met-RANTES dose-dependently inhibited actin polymerization in eosinophils following chemokine stimulation. Whereas Met-RANTES totally inhibited RANTES- and MCP-3-induced actin polymerization at 5 μg/ml, the eotaxin-induced response was only reduced by 50%. However, Met-RANTES inhibited dose-dependently the release of reactive oxygen species in response to RANTES, MCP-3 and eotaxin. Again, eotaxin-induced release of reactive oxygen species, however, was only half reduced at a Met-RANTES concentration (10 μg/ml) at which RANTES and MCP-3 were completely blocked. The results of this study show that (1) Met-RANTES is an effective and powerful antagonist of effector functions of human eosinophils following stimulation with RANTES, MCP-3 and eotaxin; (2) Met-RANTES seems to be able to antagonize the response of eosinophils through chemokine receptor 1 (CCR1) preferentially to CCR3; (3) Met-RANTES antagonizes eosinophil but not neutrophil effector functions and might be therefore of interest for a new therapeutical approach to prevent the invasion and destructive power of eosinophils in diseases that are accompanied by eosinophil infiltration such as allergic asthma and connective tissue diseases.     

CCR7和VEGF-C蛋白与乳腺癌预后之间的关系

目的:探讨趋化因子受体7(CCR7)及血管内皮生长因子C(VEGF-C)蛋白在乳腺癌组织中的表达水平,并分析二者与乳腺癌预后的关系。方法:采用免疫组织化学技术,联合检测CCR7和VEGF-C蛋白分别在乳腺癌组织及正常乳腺组织中的表达差异情况,并分析二者与乳腺癌各相关临床病理特征之间的关系。采用Kaplan-Meier法来评估CCR7及VEGF-C蛋白的异常表达与乳腺癌患者生存期之间的关系。结果:CCR7蛋白在乳腺癌组织(68%)中的阳性表达率高于正常乳腺组织(30%),差异有统计学显著性(P0.01);而VEGF-C蛋白在乳腺癌组织(71%)中的阳性表达率也明显高于正常乳腺组织(24%),差异也有统计学显著性(P0.01)。且在乳腺癌组织中,CCR7与VEGF-C蛋白的表达呈正相关关系(r=0.613,P0.01)。CCR7和VEGF-C蛋白的高表达均与淋巴结转移和TNM分期有关(P0.05),而与年龄、肿瘤大小、雌激素受体和孕激素受体均无关。CCR7及VEGFC蛋白阳性表达者的生存期低于阴性表达者,两组比较差异有统计学显著性(P0.05)。结论:CCR7与VEGF-C的异常高表达可能与乳腺癌预后关系密切,二者可作为判断乳腺癌预后不良的重要指标之一。     

T细胞相关趋化因子及受体的研究进展

T细胞在发育、成熟、活化及发挥生物学效应的各个阶段表达不同的趋化因子受体。T细胞相关趋化因子及其受体的表达在不同的细胞类群上具有时相和分布的差异,并通过趋化因子与其受体特异性结合的模式,参与T细胞的发育过程,调控细胞的定向迁移,从而影响局部甚至整个机体的免疫状态。此外,它还在炎症、感染、肿瘤、自身免疫疾病等众多病理生理的过程中发挥重要作用。在这一领域的深入研究将为相关疾病的预防和治疗提供新的思路和途径。     

The carboxyl terminus of the chemokine receptor CCR3 contains distinct domains which regulate chemotactic signaling and receptor down-regulation in a ligand-dependent manner

The chemokine receptor CCR3 regulates the chemotaxis of leukocytes implicated in allergic disease, such as eosinophils. Incubation of eosinophils with CCL11, CCL13 or CCL5 resulted in a rapid decrease of cell-surface CCR3 which was replicated using CCR3 transfectants. Progressive truncation of the CCR3 C terminus by 15 amino acids produced three constructs, Delta340, Delta325 and Delta310. Delta340 and Delta325 were able to bind CCL11 with affinities similar to wild-type CCR3. Delta340 transfectants exhibited enhanced migration and reduced receptor down-regulation in response to CCL11 and CCL13. Delta325 transfectants displayed chemotactic responses to CCL11 and CCL13 similar to wild-type CCR3, and had impaired down-regulation when stimulated with CCL13 but not CCL11. In contrast, neither the Delta325 nor Delta340 truncation affected chemotaxis or receptor down-regulation induced by CCL5. Delta310 transfectants bound CCL11 poorly and were biologically inactive. Inhibitors of p38 mitogen-activated protein kinase and PI3-kinase antagonized eosinophil shape change responses and chemotaxis of transfectants to CCL11 and CCL13. In contrast, shape change but not chemotaxis was sensitive to inhibition of the extracellular signal-regulated kinase kinase pathway suggesting differential regulation of the two responses. Thus, the CCR3 C terminus contains distinct domains responsible for the regulation of receptor desensitization and for coupling to chemotactic responses.     

A specific CCR3 chemokine receptor antagonist inhibits both early and late phase allergic inflammation in the conjunctiva

Allergic inflammation manifests as one of a number of diseases, including asthma, dermatitis, food allergy, vernal keratoconjunctivitis, and systemic anaphylaxis. Together these diseases affect nearly 25% of the Western world and are a leading health-care problem. The diseases are often biphasic, with an early phase driven primarily by mast cell degranulation and a late phase characterized by leukocyte recruitment. While chemokines are well known to be critical for leukocyte recruitment, their importance in early-phase reactions is poorly defined. We show here that administration of a single oral dose of a high affinity and highly selective CCR3 antagonist ablates both the early and late phase reactions in a mouse model of allergic conjunctivitis. A direct analysis of mast cells in the conjunctiva demonstrates that antagonism of the CCR3 receptor stabilizes the mast cell in vivo, thereby leading to the impaired early phase reaction. The late phase reaction is also strongly inhibited as characterized by both reduced eosinophilia and neutrophilia. These results constitute the first direct evidence that antagonism of CCR3 has clear potential for the treatment of allergic diseases.     

Lipopolysaccharide binding protein is an essential component of the innate immune response to Escherichia coli peritonitis in mice

Lipopolysaccharide (LPS) binding protein (LBP) is an acute-phase protein that enhances the responsiveness of immune cells to LPS by virtue of its capacity to transfer LPS to CD14. To determine the role of LBP in the innate immune response to peritonitis, LBP gene-deficient (LBP(-/-)) and normal wild-type mice were intraperitoneally infected with Escherichia coli, the most common causative pathogen of this disease. LBP was detected at low concentrations in peritoneal fluid of healthy wild-type mice, and the local LBP levels increased rapidly upon induction of peritonitis. LBP(-/-) mice were highly susceptible to E. coli peritonitis, as indicated by accelerated mortality, earlier bacterial dissemination to the blood, impaired bacterial clearance in the peritoneal cavity, and more severe remote organ damage. LBP(-/-) mice displayed diminished early tumor necrosis factor alpha, interleukin-6, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein 2 production and attenuated recruitment of polymorphonuclear leukocytes to the site of infection, indicating that acute inflammation was promoted by LBP. Locally produced LBP is an essential component of an effective innate immune response to E. coli peritonitis.     

Natural truncation of RANTES abolishes signaling through the CC chemokine receptors CCR1 and CCR3, impairs its chemotactic potency and generates a CC chemokine inhibitor

Selective leukocyte trafficking towards sites of inflammation is mediated by chemokines. RANTES is a CC chemokine that attracts lymphocytes, monocytes, dendritic cells, eosinophils, basophils and NK cells. A natural form of human RANTES lacking two N-terminal residues was isolated from stimulated sarcoma cells, fibroblasts, and leukocytes. RANTES(3 – 68) showed a more than tenfold reduction in chemotactic potency for monocytes and eosinophils. To elucidate the mechanism involved, receptor recognition studies were performed. In cells transfected with the CC chemokine receptor (CCR) 5, the major co-receptor for macrophage-tropic HIV-1 strains, RANTES(3 – 68) mobilized calcium and desensitized RANTES(1 – 68)-induced calcium fluxes equally well as RANTES(1 – 68). However, RANTES(3 – 68) was ineffective on CCR1 and CCR3 transfectants. The reduced potency of natural RANTES(3 – 68) by selective loss of receptor-activating characteristics was confirmed with recombinant RANTES(3 – 68). In chemotaxis assays using monocytic cells, RANTES(3 – 68) inhibited RANTES(1 – 68), macrophage inflammatory protein-1α (MIP-1α), MIP-1β or monocyte chemotactic protein-3 (MCP-3), but not MCP-1- or MCP-2-induced chemotaxis. Thus, a minor post-translational modification has a remarkable impact on the biological activities of RANTES and a pathophysiologically induced change in the relative amounts of intact and truncated RANTES might affect the outcome of inflammation or HIV infection.     

靶向趋化因子受体CCR7的反义肽核酸对树突状细胞凋亡的影响

目的研究靶向趋化因子受体CCR7的反义肽核酸（PNA）对树突状细胞（DC）CCR7蛋白表达及凋亡的影响，并探讨其凋亡机制。方法体外培养大鼠骨髓来源的Dc，脂多糖（LPS）诱导成熟。设计靶向CCR7mRNA翻译起始区的反义PNA，以随机PNA和空白组为对照处理体外培养7d的未成熟Dc，脂多糖诱导48h后收集成熟Dc，免疫细胞化学染色法检测CCR7蛋白表达，流式细胞术检测细胞凋亡率，Westernblot检测磷酸化Akt和Akt蛋白表达。应用渥曼青霉素（wortmannin）抑制磷脂酰肌醇-3激酶（phosphatidylinositol3-kinase，P13K）／Akt信号通路进一步观察磷酸化Akt蛋白表达和细胞凋亡率的变化。结果反义PNA组DCCCR7蛋白表达明显低于随机PNA组和空白组，而细胞凋亡率却明显增高，差异有统计学意义（P〈0．05），Westernblot检测显示反义PNA组Akt蛋白磷酸化水平明显低于随机PNA组和空白组，差异有统计学意义（P〈0．05）。DC经P13K抑制剂预处理后，CCR7介导的Akt蛋白磷酸化及抗凋亡作用被阻断。结论CCR7反义PNA能够有效抑制体外培养的大鼠DC趋化因子受体CCR7的表达并导致其发生凋亡，其机制可能是阻断了CCR7介导P13K／Akt信号转导通路的活化。     

The chemokine CCL5 induces selective migration of bovine classical monocytes and drives their differentiation into LPS-hyporesponsive macrophages in vitro

Human and mouse studies indicate distinct roles of selected chemokines for monocyte subset attraction. We therefore analyzed the still unknown sensitivity and response of bovine monocyte subsets toward two monocyte-attracting chemokines (CCL2, CCL5). Only CCL5 induced a significant Ca²⁺influx and migration response in bovine monocytes, with classical and intermediate monocytes being significantly stimulated and attracted compared to nonclassical monocytes. The presence of CCL5 during in vitro macrophage differentiation did not alter their capacity to phagocytize or to generate reactive oxygen species upon stimulation with E. coli. However, macrophages differentiated in the presence of CCL5 displayed an altered phenotype with significantly less expressed CD14 and MHC class II molecules, whereas CD16 was upregulated. Moreover, CCL5-differentiated macrophages displayed a reduced upregulation of CXCL8, ARG1, IL6 and IL10 mRNA. Taken together, CCL5 but not CCL2 mainly attract bovine classical monocytes and promote their differentiation into LPS-hypo-responsive macrophages.     

Increased inflammation in mice deficient for the chemokine decoy receptor D6

Chemokines are chemotactic cytokines with a key role in the control of cell trafficking and positioning under homeostatic and inflammatory conditions. D6 is a promiscuous 7-transmembrane-domain receptor expressed on lymphatic vessels which recognizes most inflammatory, but not homeostatic, CC chemokines. In vitro experiments demonstrated that D6 is unable to signal after ligand engagement, and it is structurally adapted to sustain rapid and efficient ligand internalization and degradation. These unique functional properties lead to the hypothesis that D6 may be involved in the control of inflammation by acting as a decoy and scavenger receptor for inflammatory chemokines. Consistent with this hypothesis, here we report that D6(-/-) mice showed an anticipated and exacerbated inflammatory response in a model of skin inflammation. Moreover, the absence of D6 resulted in increase cellularity and inflammatory-chemokine levels in draining lymph nodes. Thus, D6 is a decoy receptor structurally adapted and strategically located to tune tissue inflammation and control transfer of inflammatory chemokines to draining lymph nodes.     

CCR6 is a functional chemokine receptor that serves to identify select B-cell non-Hodgkin's lymphomas

Several recent studies have revealed important contributions of chemokines and their receptors to the development and progression of both hematopoietic and nonhematopoietic neoplasms. The chemokine receptor CCR6 is unusual in that it mediates leukocyte chemotaxis in response to a single chemokine, CCL20 (macrophage inhibitory factor-3alpha), as well as in response to a family of antimicrobial peptides termed "beta-defensins." CCR6 is critical for mucosal immunity, and expression of the receptor is tightly regulated on hematopoietic cells. Here we characterize the expression of CCR6 on B cells and B-cell non-Hodgkin's lymphomas. We demonstrate that CCR6 expression is limited to cells comprising the mantle and marginal zones of the secondary lymphoid tissues and serves to identify the majority of mantle cell, marginal zone, and mucosa-associated lymphoid tissue lymphomas. Furthermore, we show that CCR6 serves as a functional chemokine receptor when expressed by neoplastic cells. Finally, we establish that the cognate ligand for CCR6 is present on mucosal epithelium infiltrated by neoplastic cells in select extranodal lymphomas. Thus, CCR6 is a useful new marker identifying a subset of B-cell non-Hodgkin's lymphomas and likely contributes to the localization of select extranodal lymphomas at mucosal sites.     

The chemokine SLC is expressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts activated T cells via CCR7

Secondary lymphoid-tissue chemokine, SLC, also known as exodus-2 and 6Ckine, is a novel CC chemokine with selectivity for T lymphocytes and preferential expression in lymphoid tissues. We have studied its production, receptor usage and biological activities. High levels of SLC mRNA were detected in lymph nodes, the gastrointestinal tract and several gland tissues, but no expression was found by Northern blot analysis in freshly isolated or stimulated blood monocytes and lymphocytes, or neutrophils and eosinophils. In situ hybridization revealed constitutive expression of SLC in the T cell areas and the marginal zone of follicles in lymph nodes and the mucosa-associated lymphoid tissue, but not in B cell areas or sinuses. Comparison with immunocytochemical staining showed similarity between the in situ expression of SLC and the distribution of interdigitating dendritic cells but not with sinus-lining dendritic cells, macrophages or T lymphocytes. SLC induced chemotaxis of T lymphocytes and its activity increased considerably when the cells were conditioned with IL-2 or phytohemagglutinin (PHA). Under optimal conditions SLC had unusually high efficacy and induced the migration of up to 50 % of input T lymphocytes. SLC also induced Ca²⁺ mobilization in these cells. Similar responses were obtained with EBI1 ligand chemokine (ELC), and sequential stimulation with both chemokines led to cross-desensitization, suggesting that SLC acts via the ELC receptor, CCR7. This was confirmed using murine pre-B cells stably transfected with CCR7 which bound SLC with high affinity and showed chemotaxis and Ca²⁺ mobilization in response to both SLC and ELC. In T lymphocytes PHA and IL-2, which enhanced chemotactic responsiveness, also markedly enhanced CCR7 expression. In contrast to all known chemokine receptors, up-regulation of CCR7 by IL-2 was transient. A maximum was reached in 2 – 3 days and expression returned to initial levels within 8 – 10 days. The present study shows that SLC is constitutively produced within the T cell areas of secondary lymphoid organs and attracts T lymphocytes via CCR7.     

The chemokine receptor D6 limits the inflammatory response in vivo

How the inflammatory response is initiated has been well defined but relatively little is known about how such responses are resolved. Here we show that the D6 chemokine receptor is involved in the post-inflammatory clearance of beta-chemokines from cutaneous sites. After induction of inflammation by phorbol esters, wild-type mice showed a transient inflammatory response. However, in D6-deficient mice, an excess concentration of residual chemokines caused a notable inflammatory pathology with similarities to human psoriasis. These results suggest that D6 is involved in the resolution of the cutaneous inflammatory response.     

MARCO, an innate activation marker of macrophages, is a class A scavenger receptor for Neisseria meningitidis

The scavenger receptor-A I/II (SR-A) and macrophage receptor with collagenous domain (MARCO) share a common domain organisation and ligand repertoire, including selected polyanions and gram-positive and -negative organisms, but differ in fine specificity of ligand binding, tissue distribution and regulation. Neisseria meningitidis (NM) is a selective ligand for SR-A, but there is evidence for an additional SR-A-independent, polyanion-sensitive component for NM recognition. We therefore studied the relative contribution of MARCO and SR-A to binding of NM by resident and elicited peritoneal macrophages obtained from MARCO-/-, SR-A-/- and SR-A-MARCO-/- mice. Results confirmed that both mouse and human MARCO are able to bind NM independently of NM LPS. MARCO and SR-A contributed independently to NM binding, correlating with their expression levels in different cell populations, but neither of these two molecules was required for release of TNF-alpha and nitric oxide. We propose that the TLR-dependent induction of MARCO by innate immune stimulation enhances recognition and uptake of pathogenic organisms such as NM, thus contributing to host defence against infection.     

The chemokine receptor CCR5 in the central nervous system

The expression and the role of the chemokine receptor CCR5 have been mainly studied in the context of HIV infection. However, this protein is also expressed in the brain, where it can be crucial in determining the outcome in response to different insults. CCR5 expression can be deleterious or protective in controlling the progression of certain infections in the CNS, but it is also emerging that it could play a role in non-infectious diseases. In particular, it appears that, in addition to modulating immune responses, CCR5 can influence neuronal survival. Here, we summarize the present knowledge about the expression of CCR5 in the brain and highlight recent findings suggesting its possible involvement in neuroprotective mechanisms.     

天然免疫记忆研究进展

经典免疫学理论认为只有获得性免疫能建立免疫记忆.现研究发现天然免疫细胞包括NK细胞、单核细胞、巨噬细胞等经过驯化后能在再次刺激后产生增强的免疫反应,这种现象被称为天然免疫记忆或训练免疫.天然免疫记忆主要由代谢及表观遗传重编程而存储记忆.天然免疫记忆为感染性疾病、癌症及炎症等疾病的发病机理及治疗提供了新的研究视角.