Expression of chemokine receptors CXCR4, CXCR5 and CCR7 on B and T lymphocytes from patients with primary antibody deficiency

The interaction of chemokines and their receptors directs lymphocyte migration, and is involved in the distribution and organization of lymphocytes within lymphoid tissues. We reasoned that abnormal chemokine receptor expression might give rise to defects of lymphocyte migration into and within lymphoid tissues, and consequently be associated with defective antibody production in primary antibody deficiencies. In this study, we have investigated the expression of chemokine receptors CXCR4, CXCR5 and CCR7 on lymphocyte subpopulations (naive and memory B cells; CD4⁺ and CD8⁺ T cells) in a cohort of patients with primary antibody deficiency (n = 23), and compared these with a group of healthy controls (n = 19). We show that there were significant differences in both the proportions of lymphocytes expressing, and the levels of expression of, specific chemokine receptors on individual lymphocyte subpopulations between patients and controls. Furthermore, these changes appeared more pronounced in patients with more severe antibody deficiency. These data support the hypothesis that abnormal lymphocyte trafficking may be involved in the pathogenesis of primary antibody deficiencies.     

Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst

Chemokines are implicated in the implantation process. The aim of this study was to investigate mRNA expression and protein levels of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle, during HRT and in the human blastocyst. The regulation of chemokine receptors in the endometrial epithelium was also studied using an in-vitro model for the apposition phase of human implantation. We found up-regulation of endometrial CXCR1 mRNA (419-fold increase), CCR5 mRNA (612-fold increase) and CCR2B mRNA (657 fold-increase) during the luteal phase peaking in the pre-menstrual endometrium. CXCR4 mRNA levels presented a specific although modest (18-fold increase) up-regulation during the implantation window. These findings were corroborated at the protein level in natural and HRT cycles. Immunoreactive CCR5 and CCR2B receptors were detected in human blastocysts whereas CXCR4 and CXCR1 were not present. Chemokine receptors in cultured endometrial epithelial cells showed an up-regulation and polarization of CXCR1, CXCR4 and CCR5 receptors when a human blastocyst was present. The specific distribution and regulation of chemokine receptors in the endometrial epithelium and the human blastocyst suggest a possible implication of these receptors in the apposition and adhesion phases of human implantation.     

HIV-1协同受体CXCR4、CCR5及相关趋化因子SDF-1在胎盘的表达

目的：研究HIV-1协同受体CXCR4、CCR5及CXCR4的特异性配体SDF-1在人胎盘组织的表达，探索HIV-1子宫内垂直传播的分子机制。方法：半定量RT-PCR检测早、中、晚孕期胎盘及早孕滋养细胞CXCR4、CCR5 mRNA水平；免疫组化和免疫细胞化学检测早孕胎盘及原代培养滋养细胞CXCR4、CCR5蛋白表达；原位杂交及免疫组化分析SDF-1在早孕胎盘的表达；ELISA测定滋养细胞SDF-1的动态分泌水平。结果：各孕期胎盘表达CXCR4及CCR5 mRNA；CXCR4蛋白定位于滋养细胞，而CCR5蛋白定位于绒毛基质中。滋养细胞可转录并翻译SDF-1，且能分泌可溶性SDF-1。结论：滋养细胞同时表达CXCR4及SDF-1，SDF-1可能通过降调CXCR4而拮抗X4-HIV-1感染胎儿细胞；R5-HIV-1或许能通过滋养层裂隙感染CCR5^#基质细胞和/或Hotbauer细胞，从而发生子宫内垂直传播。     

Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4

The homing and tissue-specific recruitment of bone marrow-derived progenitor cells is a major issue in stem cell research and therapy. Chemokine biology plays a central role in the homing and trafficking of leukocytes. Here we show functional expression of the chemokine receptors CCR1, CCR4, CCR7, CCR10, and CXCR5 on primary isolates of CD34- mesenchymal progenitor cells as well as immortalized mesenchymal stem cell (MSC) lines. Although mRNA expression of CXCR4 was detected in both primary cells and immortalized clones, the receptor was not expressed on the cell surface. On the basis of this expression profile, the MSC could potentially home to secondary lymphatic organs (CCR7, CXCR5), skin (CCR4, CCR10), small intestine (CCR10), and salivary glands (CCR10). To study tissue-specific homing, murine CD34- MSC lines showing concordant chemokine receptor expression were either transiently labeled with CMFDA, or were stably transfected with green fluorescent protein (GFP) expression plasmids. The MSC were then injected into syngeneic healthy mice, and the distribution of the cells determined. The injected cells efficiently homed to spleen, thymus, and lymph nodes. In addition, cells were found in the mucosa of the small intestine, skin, and salivary gland. No significant recruitment to bone marrow, liver, or kidney was seen. Chemokine biology may play an important role in the homeostasis and potentially tissue recruitment of early adult progenitor cells.     

Study of the HIV-1 receptors CD4, CXCR4, CCR5 and CCR3 in the human and rat testis

Sexual transmission of HIV is one of the main routes of transmission of AIDS. Despite the fact that the virus has been found in the semen and germ cells of patients with HIV, little is known about how the virus infects the cells of the genital tract. We studied the cellular distribution of CD4, a receptor necessary for HIV infection, and the major HIV co-receptors CCR3, CCR5 and CXCR4 in the rat and human testis. We used RT-PCR, Northern blotting and immunohistochemistry to demonstrate that CCR3 is absent from the testes of both species, whereas CCR5 and CXCR4 are present on the resident testicular macrophages in the interstitial space but not in the germ cell line. All of the human testicular macrophages expressed the markers CD45 and MAC387 and most also expressed CD4. Thus, our data suggest that macrophages in the testis may be infected by HIV and that these macrophages may be a site of early viral localization and a potential HIV reservoir. This may in turn alter the activity of Leydig cells and subsequently affect spermatogenesis.     

趋化因子受体CXCR4及CCR5真核表达载体的构建及其在卵巢癌细胞中的表达

目的:构建人CXCR4及CCR5真核表达重组质粒,转染人卵巢癌细胞SKOV3,建立稳定转染细胞系并观察其表达效果。方法:从人外周血单个核细胞中提取RNA,采用反转录PCR技术扩增CXCR4及CCR5的基因编码序列,将序列克隆至真核表达载体pEGFP,经酶切和测序鉴定后,应用脂质体转染技术将质粒cancer.pEGFP-CXCR4和pEGFP-CCR5分别导入不表达CXCR4及CCR5蛋白的SKOV3细胞,经G418抗性筛选得到阳性细胞克隆并扩大培养成系。分别采用免疫荧光染色和流式细胞术方法(FCM)检测稳定转染细胞株CXCR4和CCR5的表达。结果:构建了真核表达载体pEG-FP-CXCR4和pEGFP-CCR5;得到了抗G418阳性细胞克隆;免疫荧光染色和FCM检测结果显示,转染质粒的SKOV3细胞表达CXCR4和CCR5。结论:成功建立稳定表达趋化因子受体CXCR4和CCR5的卵巢癌细胞株,为CXCR4和CCR5在卵巢癌中的研究工作提供依据及平台。     

Expression of chemokine receptors CXCR4, CCR2, CCR5 and CX3CR1 in neural progenitor cells isolated from the subventricular zone of the adult rat brain

We have studied the expression of chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1 at the mRNA and protein levels in adult neural progenitor cells (NPCs) in neurosphere cultures using RT-PCR and immunocytochemistry methods. NPCs were isolated from the subventricular zone of adult rat brain and propagated in vitro as neurospheres. The neurospheres showed immunoactivity of nestin, an intermediate filament marker for NPCs. NPCs in the neurosphere cultures differentiated into NeuN-, GFAP-, or GalC-positive cells in vitro. Using cultured cortical microglial cells as positive control, we demonstrated the mRNA expression of CXCR4, CCR2, CCR5, and CX3CR1 in neurospheres by RT-PCR. Double immunofluorescent staining further confirmed the co-localization of nestin with either CXCR4, CCR2, CCR5, or CX3CR1 on neurospheres. These results suggest that adult NPCs in the neurosphere cultures express chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1.     

The allergy-associated chemokine receptors CCR3 and CCR5 can be inactivated by the modified chemokine NNY-CCL11

BACKGROUND: CC chemokine ligand 11 (CCL11) is the outstanding member of all described CC chemokine receptor 3 (CCR3) ligands and is shown to be selective for this receptor. However, it also activates CCR5 but only in the micromolar range. The in vivo activity of CCL11 is expected to be temporally restricted, as it is degraded by specific proteases such as the dipeptidyl-peptidase IV (DP4), also termed CD26. Based on the approach to inactivate chemokine receptors in allergic disease models as has been demonstrated for DP4-resistant n-nonanoyl (NNY)-CCL14 and for amino-oxypentane (AOP)-CCL5, it is tempting to study similar compounds derived from CCL11. METHODS: Synthesis of NNY-CCL11 was performed and it was characterized for biological functions in human and mouse eosinophils as well as in cell lines stably transfected either with human CCR3 or CCR5. Resistance to DP4 treatment was also investigated. RESULTS: The functional activities of NNY-CCL11 mediated via CCR3 show an almost identical pattern to CCL11 with respect to intracellular calcium mobilization and CCR3 internalization. N-terminal cleavage of CCL11 by preincubation with DP4 results in a reduced capacity to internalize CCR3, while preincubation of NNY-CCL11 shows no influence. In contrast to CCL11, NNY-CCL11 also activates CCR5+ cell lines and human monocytes in the nanomolar range, being about 100 times more potent than CCL11. CONCLUSIONS: n-Nonanoyl-CCL11 represents a compound with dual activity restricted to CCR3 and CCR5. Because of its receptor-inactivating capacity and stability against DP4 degradation, NNY-CCL11 is a suitable tool for the decoding of the pathophysiological mechanisms of allergic diseases.     

趋化因子受体CCR5、CCR7、CXCR3和CXCR6在丙肝患者肝内及外周血CD4+ T淋巴细胞上的表达及其意义

目的比较趋化因子受体CCR5、CCR7、CXCR3和CXCR6在丙肝患者肝内和外周血CD4^＋T淋巴细胞表面表达水平及其意义,同时进一步了解其与肝脏组织学炎症反应的关系.方法采用荧光标记抗趋化因子受体的单克隆抗体对肝内及外周血中CD4^＋T淋巴细胞表面的趋化因子受体进行染色后,采用9色11参数流式细胞仪LSRⅡ进行检测分析.结果（1）肝内CCR5^＋、CXCR3^＋或/和CXCR6^＋的CD4^＋T淋巴细胞频数高于外周血（P＜0.001）,而CCR7^＋CD4^＋T淋巴细胞频数低于外周血（P＜0.001）;（2）肝内CCR5^＋或CXCR6^＋的活性（CD38^＋）CD4^＋T淋巴细胞频数高于外周血（P＜0.05）;（3）肝内表达2种或2种以上趋化因子受体CCR5、CXCR3和CXCR6的CD4^＋T淋巴细胞频数明显高于外周血（P＜0.001）,而不表达或仅表达一种上述趋化因子受体CD4^＋T淋巴细胞频数明显低于外周血（P＜0.001）;（3）CCR5和CXCR6在肝内CD4^＋T淋巴细胞表面的表达有中等度相关;（4）肝内组织学炎症明显组表达趋化因子受体CCR5、CXCR3或CXCR6的CD4^＋T淋巴细胞频数高于炎症轻微组.结论趋化因子受体CCR5、CXCR3和CXCR6可能介导CD4^＋T淋巴细胞向肝内迁徙定植,并参与肝脏炎症的病理免疫学反应过程.     

Genotypes of CCR2 and CCR5 chemokine receptors in human myasthenia gravis

The aim of this study was to examine the association of human autoimmune myasthenia gravis (MG) with two DNA polymorphisms of the chemokine receptors CCR5-Delta 32 and CCR2-64I. CCR2 and CCR5 interact primarily with the human CC family ligands CCL2 (formerly called monocyte chemoattractant protein; MCP-1), CCL3 and CCL4 (macrophage inflammatory protein-1 alpha and -1 beta; MIP-1 alpha/beta), and their main function is to recruit leukocytes from circulation into the tissues, thus playing an important role in human inflammatory disorders. A PCR-based genotyping method was used to determine the genetic variation at the CCR5 gene and an automated real-time Pyrosequencing technology was employed for the analysis of G right curved arrow A point mutation at the CCR2 gene. Results obtained from 158 patients and 272 healthy controls demonstrate no evidence of association between genetic variants of CCR2 and CCR5 with MG and its clinical manifestations. CCR2-64I and CCR5-Delta 32 genotypes are thus unlikely to be involved in protection or predisposition to MG.     

Expression of chemokine receptors CCR5 and CXCR4 on CD4+ T cells and plasma chemokine levels during treatment of active tuberculosis in HIV-1-coinfected patients

The pathogenesis of persistently elevated plasma HIV viremia in patients coinfected with tuberculosis (TB) during anti-TB treatment in Africans remains unknown. We examined the expression of chemokine receptors CCR5 and CXCR4 on CD4+ T cells and plasma chemokine levels of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation normal T expressed and secreted (RANTES), and stromal cell-derived factor (SDF)-1alpha among TB patients with HIV coinfection during the first 2 months of anti-TB treatment. During treatment of TB, the plasma HIV-1 load and CD4+ T-cell count remained unchanged. Levels of CCR5 and CXCR4 expression on CD4+ T cells as well as plasma levels of chemokines remained persistently elevated during anti-TB treatment. Persistently elevated plasma HIV viremia also paralleled persistently elevated expressions of activated CCR5+ or CXCR4+ CD4+ T cells. These results suggest that increased expression of CCR5 and CXCR4 on an activated CD4+ T-cell population coupled with persistently elevated chemokines may provide a suitable condition for continuous replication of HIV associated with TB coinfection. This, in turn, may contribute, at least in part, to the observed persistently elevated plasma HIV viremia in coinfected patients despite anti-TB treatment.     

Potential role of the chemokine receptors CXCR3, CCR4, and the integrin alphaEbeta7 in the pathogenesis of psoriasis vulgaris

Various adhesion molecules have been implicated in T lymphocyte binding to dermal vascular endothelium in psoriasis vulgaris, but the chemotactic signals that promote subsequent homing into the adjacent dermis and overlying epidermis are poorly defined. We studied chemokine receptor (CCR1-CCR5, CXCR1-CXCR3), chemokine (interferon-gamma inducible protein 10 [IP-10]), monokine induced by interferon-gamma (MIG), thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and adhesion molecule (cutaneous lymphocyte antigen [CLA], E-selectin, lymphocyte function-associated antigen-1 [LFA-1], intercellular adhesion molecule-1 [ICAM-1], very late antigen 4 [VLA-4], vascular cell adhesion molecule-1 [VCAM-1], alphaEbeta7, and E-cadherin) expression in psoriasis by immunohistology, flow cytometry, and molecular techniques. CXCR3 and CCR4 were expressed by dermal CD3+ lymphocytes, and their chemokine ligands, IP-10, MIG, TARC, and MDC, were up-regulated in psoriatic lesions. Keratinocytes stimulated with tumor necrosis factor-alpha and interferon-gamma up-regulated expression of IP-10, MIG, and MDC mRNA, whereas dermal endothelial cells, similarly stimulated, up-regulated expression of IP-10, MDC, and TARC mRNA, suggesting that these cell types were sources of the chemokines detected in biopsies. There was enhanced expression of E-selectin, CLA, LFA-1, ICAM-1, VLA-4, VCAM-1, and alphaEbeta7 in psoriatic lesions versus nonlesional skin. Finally, intra-epidermal CLA+ and alphaEbeta7+ T lymphocytes selectively expressed the chemokine receptor CXCR3. Collectively, these data suggest that CXCR3 and CCR4 may be involved in T lymphocyte trafficking to the psoriatic dermis and that CXCR3 is selectively involved in subsequent T cell homing to the overlying epidermis.     

Immunohistochemical analysis of CCR2, CCR3, CCR5, and CXCR4 in the human brain: potential mechanisms for HIV dementia

The CXC chemokine receptor CXCR4 was the first molecule identified as a coreceptor working in conjunction with CD4 to mediate cellular entry for the human immunodeficiency virus (HIV-1). Since that original discovery, 11 other seven-mtransmembrane domain molecules, many of which are chemokine receptors, have been shown to facilitate HIV entry into cells. These include CCR5, CCR3, CCR2, CCR1, CCR8, CX3CR1, STRL33 (BONZO), GPR15 (BOB), GPR1, US28, and APJ. In studies done by this and other labs, CCR3, CCR5, and CXCR4 have been identified in CNS microglia and several laboratories, including ours, have shown that CXCR4 is expressed in neurons. Neuronal expression of CCR2, CCR3, and CCR5 has been less consistent. We performed a semiquantitative immunohistochemical analysis of the expression of CCR2, CCR3, CCR5, and CXCR4 in 23 regions of the brain and in two sections of the spinal cord. Hippocampal neurons were positive for CCR2, CCR3, and CXCR4, but not for CCR5. In other regions of the brain, neurons, and glial cells reacted with anti-CCR2, anti-CCR3, and anti-CXCR4 antibodies, whereas only glial cells (primarily microglia) were positive for CCR5. The areas of highest expression, however, seem to be subcortical regions and the limbic system. The limbic system plays a key role in memory, and the presence of CXCR4-which can bind the viral envelope protein gp120-min a subset of neurons from this system may play a role in the development of HIV-related dementia.     

The unique target specificity of a nonpeptide chemokine receptor antagonist: selective blockade of two Th1 chemokine receptors CCR5 and CXCR3

CC chemokine receptor (CCR) 5 and CXC chemokine receptor (CXCR)3 are expressed on T helper cell type 1 cells and have been implicated in their migration to sites of inflammation. Our preceding study demonstrated that a nonpeptide synthetic CCR5 antagonist, TAK-779 (N, N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6, 7-dihydro-5H-benzocyclohepten-8-yl]carbon-yl]amino]benzyl]-tetrahydro-2H-pyran4-aminium chloride, inhibits the development of experimentally induced arthritis by modulating the migration of CCR5(+)/CXCR3(+) T cells to joints. The present study investigated the functional properties of TAK-779, including the effect of this antagonist on CXCR3 function. For this purpose, transfectants expressing mouse CCR5 (mCCR5) or mCXCR3 and expressing mCCR4 or mCXCR4 as controls were established by introducing each relevant gene into 2B4 T cells and were subjected to the following assays. First, the ligand binding to chemokine receptors was assayed by incubating transfectants with [(125)I]-labeled relevant ligand or with the unlabeled relevant ligand followed by staining with anti-ligand antibody. Second, chemokine-induced lymphocyte function-associated antigen-1 (LFA-1) activation was assayed by measuring the adhesion of cells to microculture plates coated with purified intercellular adhesion molecule-1. Third, chemokine-stimulated chemotaxis was assayed by observing the cell migration through transwells. In these assays, TAK-779 blocked the ligand binding as well as LFA-1 up-regulating and chemotactic function of mCXCR3 and mCCR5 but did not elicit a biologically significant inhibition of those functions of mCCR4 and mCXCR4. These observations indicate the unique target specificity of TAK-779 and explain why this antagonist efficiently blocks the migration of T cells expressing CCR5 and CXCR3 to sites of inflammation.     

Expression of the chemokine receptor CCR3 on human mast cells

BACKGROUND: The aim of this study was to investigate whether human mast cells express functional active CCR3 receptors, which are activated by CC chemokines. These ligands include the CCR3-selective chemokines eotaxin and eotaxin-2 and the more promiscuous CC chemokines, MCP-4, MCP-3, MCP-2 and RANTES. METHODS: Immunohistochemical analysis was performed on skin, gut and lung specimens. Double immunostaining was performed with anti-CCR3 and antitryptase, and anti-CCR3 and antichymase antibody (Ab) by using the avidin-biotin-peroxidase system with two different substrates. Mast cells were isolated and purified from human lung parenchyma (HLMC) by countercurrent elutriation followed by discontinuous Percoll density gradient. Flow-cytometric analysis of HLMC surface CCR3 expression was performed with the monoclonal Ab anti-CCR3 (7B11). Functional activation of HLMC was verified by the ability of cells to release histamine and/or migrate in response to eotaxin. RESULTS: High percentages (>70%) of tryptase-positive cells showing CCR3 expression were found in the skin and in the intestinal submucosa, whereas much lower percentages (< or = 20%) were found in the intestinal mucosa and in the lung interstitium. Eotaxin (1-100 nM) neither induced histamine release from HLMC nor enhanced anti-IgE-induced histamine release. In contrast, eotaxin (10-100 nM) and RANTES (10-100 nM) induced HLMC chemotaxis in vitro. Preincubation of HLMC with antibody anti-CCR3 (5 microg/ml) before loading into the chemotaxis chamber abrogated chemotaxis elicited by eotaxin. Double immunostaining with anti-CCR3 and anti-chymase antibody showed that the vast majority of CCR3-expressing mast cells in the various human tissues examined were tryptase-chymase double-positive. CONCLUSIONS: These results indicate that CCR3 is expressed on human mast cells and that these cells are attracted by CCR3-binding chemokines.     

人趋化因子受体CCR5胞外片段的融合表达与纯化

目的 对人趋化因子受体CCR5的N端胞外部分与第二个胞外环(extracellular loop-2,ECL-2)进行融合表达与纯化.方法 分别扩增人趋化因子受体CCR5的胞外N.端部分与ECL-2部分相应的编码序列,通过重叠延伸拼接(splicing byoverlapping extension,SOE)PCR实现两片段DNA的拼接后(命名为CCR5-N-E2)克隆人质粒pBlueScript M13中,同时构建原核表达载体pET-21b(+)-CCR5-N-E2,转入表达菌株BL21(DE3),IVIG诱导表达重组蛋白CCR5-N-E2,表达产物通过蛋白免疫印迹进行鉴定,并采用金属螯合层析法对重组表达产物进行纯化.结果 经测序,构建的重组原核表达载体与预期完全一致,相对分子质量为8 500的日的蛋白在BL21(DE3)菌株中得到高效表达,表达量约占总蛋白的50%,表达产物以包涵体形式存在.蛋白免疫印迹实验表明该重组蛋白与抗CCR5 N.端序列的单克隆抗体发生特异性结合.通过Ni2+亲和层析,目的 蛋白的纯度可达95%以上.结论 实现CCR5-N-E2编码序列的拼接、融合蛋白的表达以及纯化,为广泛筛选以CCR5为靶点的临床治疗药物奠定了基础.     

Antagonism of the chemokine receptors CXCR3 and CXCR4 reduces the pathology of experimental autoimmune encephalomyelitis

Abstract Chemokines regulate lymphocyte trafficking under physiologic and pathologic conditions. In this study, we have investigated the role of CXCR3 and CXCR4 in the activation of T lymphocytes and their migration to the central nervous system (CNS) using novel mutant chemokines to antagonize CXCR3 and CXCR4 specifically. A series of truncation mutants of CXCL11, which has the highest affinity for CXCR3, were synthesized, and an antagonist, CXCL11((4-79)), was obtained. CXCL11((4-79)) strongly inhibited the migration of activated mouse T cells in response to all three high-affinity CXCR3 ligands, CXCL9, 10 and 11. CXCL12((P2G2)), while exhibiting minimal agonistic activity, potently inhibited the migration of activated mouse T cells in response to CXCL12. Interfering with the action of CXCR3 and CXCR4 with these synthetic receptor antagonists inhibited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis and reduced the accumulation of CD4(+) T cells in the CNS. Further investigation demonstrated that CXCL12((P2G2)) inhibited the sensitization phase, whereas CXCL11((4-79)) inhibited the effector phase of the immune response. Our data suggest that simultaneous targeting of CXCR4 and CXCR3 may be of benefit in the treatment of the CNS autoimmune disease.