SDF-1/CXCR4轴通过调控间充质干细胞定向分化修复缺氧缺血性脑损伤

目的:探讨基质细胞衍生因子-1(stromalcell-derivedfactor-1,SDF-1)/CXC趋化因子受体4(CXCchemokinereceptor4,CXCR4)轴调控间充质干细胞定向分化、修复缺氧缺血性脑损伤的作用。方法:大鼠间充质干细胞(ratmesenchymalstemcells,rMSCs)经缺氧培养不同时点(0h、6h、12h、24h、48h、72h)或SDF-1α(10μg/L)孵育后,采用RT-PCR、Westernblotting和流式细胞术检测其表面CXCR4表达的变化;建立大鼠缺氧缺血性脑损伤模型,运用RT-PCR和Westernblotting检测造模后不同时点(1d、3d、5d、7d、14d、21d)大鼠脑部海马组织中SDF-1αmRNA转录和蛋白表达的改变情况;用AMD3100(CXCR4拮抗剂)拮抗rMSCs表面CXCR4后,免疫细胞化学和Westernblotting检测rMSCs诱导向神经细胞分化中神经元特异性烯醇化酶(neuron-specificenolase,NSE)和胶质纤维酸性蛋白(glialfibrillaryacidicprotein,GFAP)等神经细胞特异性标志物的阳性率及表达变化情况。结果:低氧培养6h及12h的rMSCsCXCR4mRNA及蛋白表达水平均较常氧培养组明显增加(P<0.01),10μg/LSDF-1α孵育后rMSCs的CXCR4表达水平明显增加(P<0.01);缺氧缺血性脑损伤模型的大鼠脑内SDF-1α蛋白表达显著增加(P<0.01);5mg/LAMD3100处理后的rMSCs在向神经细胞诱导分化中NSE和GFAP蛋白的表达明显减少。结论:微小剂量的SDF-1α可诱导低氧培养的rMSCs表面CXCR4的表达,而在缺氧缺血性脑损伤模型的大鼠脑内SDF-1α表达增加,从而使SDF-1/CXCR4轴的生物学效应得以增强;CXCR4拮抗的rMSCs在分化中神经细胞特异性标志物NSE和GFAP的表达降低,表明SDF-1/CXCR4轴在rMSCs定向神经分化修复缺血缺氧脑损伤中具有重要的调控作用。     

趋化因子基质细胞衍生因子1(SDF-1)及其受体CXCR4

趋化因子及其受体在免疫和炎症反应、造血以及HIV感染等方面发挥重要作用,其中基质细胞衍生因子-1SDF-1及其受体CXCR4由于在造血干细胞迁移、归巢以及HIV感染中的作用而受到关注,并对其作用机制进行了探讨,现就SDF-1及其受体CXCR4的有关内容作一综述。     

sdf-1及其受体在成体干细胞迁移中的作用

基质细胞衍生因子是一种干细胞趋化因子,它可以通过与其受体CXCR4结合,调控干细胞定向迁移,发挥特定的生物学效应.SDF-1/CXCR4轴在调控骨髓造血干细胞归巢、调节干/前体细胞定向迁移至靶器官促进受损组织器官再生的病理生理过程中发挥着重要的作用.本文就SDF-1/CXCR4轴在成体干细胞迁移中的作用作简要综述.     

Granulocyte chemotactic protein 2 acts via both IL- 8 receptors,CXCR1 and CXCR2

Interleukin-8 (IL-γ) acts on human neutrophils via two receptors, CXCR1 and CXCR2. It shares CXCR2 with all neutrophil-activating chemokines, which like IL-8 have a conserved Glu-Leu-Arg (ELR) N-terminal motif, but is generally considered to be the only relevant agonist for CXCR1. IL-8 has a basic residue at the sixth position after the second cysteine, which was suggested to contribute to CXCR1 specificity. Among the other ELR chemokines, only granulocyte chemotactic protein 2 (GCP-2) has such a basic determinant. Using Jurkat cells that stably express either CXCR1 or CXCR2, we studied receptor activation by IL-8, GCP-2 epithelial neutrophil-activating protein 2 (ENA-78) (which shares 77 % identical amino acids with GCP-2) and growth-regulated oncogene α (GROα). At 10 nM and higher concentrations, GCP-2 and IL-8 induced significant activation of CXCR1-expressing cells, but no activity was found with GROα and ENA-78. As expected, however, all four chemokines had similar activities on CXCR2-expressing cells. A variant of GCP-2 in which the basic residue, Arg20, was replaced by a glycine was synthesized. This derivative was ineffective on CXCR1, but was as active as wild-type GCP-2 in CXCR2-expressing cells. GCP-2 displaced radiolabeled IL-8 from both receptors with low affinity, and in this respect resembled ENA-78 and GROα. Our data show that GCP-2 acts via both IL-8 receptors and thus appears to be functionally more similar to IL-8 than to the other ELR chemokines. Activation of CXCR1 appears to depend significantly on the presence of a basic binding determinant close to the second cysteine.     

红细胞生成素对慢性肾衰竭大鼠外周血内皮祖细胞SDF-1及其受体表达的影响*

 目的：观察红细胞生成素（EPO）对慢性肾衰竭（CRF）大鼠外周血内皮祖细胞（EPCs）归巢因子表达的影响。方法：采用分阶段5/6肾切除制备大鼠CRF模型。实验动物随机分为3组：假手术组、CRF组和EPO治疗组。从第3周开始,治疗组大鼠每次皮下注射重组人EPO 50 U/kg,每周3次,共6周。8周时取其外周血分离与培养EPCs,并检测EPCs的功能。采用实时荧光定量PCR和Western blotting检测外周血EPCs基质细胞衍生因子1 （SDF-1）及其受体（CXCR4）、EPO及其受体（EPOR）mRNA和蛋白的表达。结果：与CRF组比较,EPO治疗可上调外周血EPCs中SDF-1和CXCR4 mRNA及蛋白的表达（均P<0.05）;此外,EPO还可上调CRF大鼠外周血EPCs的 EPO及EPOR mRNA和蛋白的表达（均P<0.05）。结论：EPO对慢性肾衰竭大鼠外周血EPCs的动员可能与其增加外周血EPCs的 SDF-1及其受体表达有关。     

The chemokine SDF-1α triggers a chemotactic response and induces cell polarization in human B lymphocytes

We studied the expression and possible functional role of chemokine receptors CXCR3, CXCR4 and CCR5 in normal human B lymphocytes. B cells from both peripheral blood and tonsils expressed high levels of CXCR4 but not the other chemokine receptors tested. CXCR4 ligand, stromal cell-derived factor (SDF)-1α, elicited a potent chemotactic response and induced a polarized motile phenotype in B cells, resulting in redistribution of the adhesion molecule ICAM-3 to a posterior appendage of the cell, termed uropod, and of CXCR4 receptor to the leading edge of migrating B cells. Time-lapse videomicroscopy studies revealed that SDF-1α-treated cells recruited additional bystander B cells through the uropod. SDF-1α induced levels of cellular recruitment comparable to those elicited by polarization-inducing anti-ICAM-3 monoclonal antibody, in an LFA-1/ICAM-1, −3-dependent fashion. Moreover, this chemokine increased intracellular Ca²⁺ levels in B lymphocytes, and induced a rapid CXCR4 receptor down-regulation on the cell surface membrane. These results provide new insight into the important biological role of SDF-1α in physiological processes in which B cells participate, and suggest a key role for chemokines in normal B cell trafficking and recirculation.     

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。     

基质细胞衍生因子免疫阳性细胞在正常成年大鼠脑中的分布

目的观察SDF-1免疫阳性细胞在正常SD大鼠脑中的分布,为开展SDF-1研究提供基础资料。方法对10只正常雄性SD大鼠脑进行免疫组织化学染色,显示SDF-1免疫阳性细胞的形态和分布。结果SDF-1免疫阳性细胞见于大脑皮质、海马、扣带、穹窿、缰内侧核、菱形核、弓状核、丘脑后正中核、中央内侧核、丘脑外侧后核、丘脑外侧背核等处,细胞形态以中等大小圆形为主。结论SDF-1免疫阳性细胞在成年SD大鼠脑内分布广泛,它们可能参与中枢神经系统中的多种生理功能。     

慢性乙肝患者外周血单个核细胞中CXCR1、CXCR2及IL-8 mRNA表达与干扰素治疗关系

目的:了解乙型肝炎病毒(HBV)慢性感染患者外周血单个核细胞(PBMC)中CXCR1、CXCR2及IL-8 mRNA表达水平及与α干扰素(IFN-α)治疗的关系。方法:采用实时定量PCR法动态观察30例慢性乙型肝炎患者接受IFN-α治疗前、治疗3个月、6个月后其外周血单个核细胞CXCR1、CXCR2及IL-8 mRNA表达水平。结果:治疗前慢性乙肝患者CXCR1、CXCR2及IL-8 mRNA表达水平分别为(0.44740.0386)、(0.4720 0.0458)、(1.1897 0.1028),均高于正常对照组(n=36),其中CXCR1及IL-8 mRNA水平升高显著,差异有统计学意义(P<0.01)。治疗过程中CXCR1、CXCR2及IL-8表达水平均显著下降。IFN-α治疗6个月后CXCR1、CXCR2及IL-8 mRNA表达水平分别为(0.41290.0395)、(0.4461 0.0477)、(0.8660 0.1307),与治疗前相比,差异有显著性(P<0.01或P<0.05)。治疗前的CX-CR1、CXCR2及IL-8的表达水平在HBV高复制组(HBV-DNA>106,n=22)明显高于HBV低复制组(HBV-DNA<106,n=8),差异有统计学意义(P<0.05)。结论:慢性乙肝患者外周血单个核细胞中CXCR1和IL-8表达水平显著升高,在干扰素治疗后,其表达水平下调,证明其可能与慢性乙肝炎症的发生机制相关。     

Chemokine receptor CX3CR1 promotes dendritic cell development under steady-state conditions

Expression of CX3CR1 is an attribute of myeloid precursors committed to the monocyte/macrophage (Mφ)/DC lineages and is maintained during all stages of DC differentiation. Nevertheless, the exact role of this molecule during developmental progression of myeloid precursors towards the DC lineage remains elusive. To overcome potential compensatory mechanisms and issues of redundancy, we employed competitive adoptive transfer experiments to assess a possible function of CX3CR1 in DC and monocyte/Mφ differentiation in vivo. We show here that expression of CX3CR1 promotes the generation of DCs and monocytes/Mφ under steady-state conditions and during compensatory expansion after selective depletion of DCs, but not under inflammatory conditions evoked by sub-lethal irradiation. Direct administration of CX3CR1-deficient and CX3CR1-sufficient precursors into the spleen or the thymus resulted in a similar competitive advantage of WT over CX3CR1-deficient precursors as i.v. transfer, suggesting that CX3CR1-mediated survival rather than recruitment to lymphoid organs is critical for DC/Mφ differentiation. In conclusion, our data support the hypothesis that CX3CR1 promotes proper development of myeloid precursors into DCs and monocytes/Mφs under steady-state conditions, possibly by providing survival signals or mediating accessibility to organ-specific niches, rather than acting as a mediator of homing to the spleen or the thymus.     

A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5

We have developed a long-term culture system using the murine bone marrow stromal cells MS-5 to support the growth of progenitor B cells with CD34^–, CD10⁺, CD19⁺, and cytoplasmic μ chain (Cμ)-negative surface phenotype from human CD34⁺ cells purified from umbilical cord blood (CB). When 10³ CB CD34⁺ cells/well were seeded on MS-5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony-stimulating factor (rhG-CSF), but not rhIL-7, strikingly enhanced the growth of progenitor B cells from CB CD34⁺ population cultured on MS-5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a Cμ⁺ phenotype, and a small number of cells had immunoglobulin μ chain on their cell surface in the presence of both rhSCF and rhG-CSF. When CD34⁺ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS-5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34⁺ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG-CSF, and that a species-cross-reactive membrane-bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS-5 stromal cells. Further studies are required to investigate the mechanism how rhG-CSF acts on progenitor B cells to allow their proliferation and differentiation.     

B lymphocytes that migrate to tuberculous pleural fluid via the SDF-1/CXCR4 axis actively respond to antigens specific for Mycobacterium tuberculosis

B-cell biology has been largely uncharacterized in the field of tuberculosis (TB). In this study, we investigated the immunophenotypical and functional characteristics of B cells obtained from the pleural fluid (PF) and peripheral blood of patients with tuberculous pleuritis (TP). Our results indicated that the total numbers of B cells, CD27(+) memory B cells and plasmablasts were clearly lower in the PF than in peripheral blood. Furthermore, we found significantly higher expression of CXCR4 on B cells in the PF, and a chemotaxis assay showed that B cells in the PF were more responsive to stromal cell-derived factor-1 (SDF-1) than B cells from peripheral blood. In addition, SDF-1 levels in PF were remarkably high compared with SDF-1 levels in plasma, suggesting that the SDF-1/CXCR4 axis might facilitate the migration of circulating B cells into tuberculous pleural space. Importantly, we observed that significantly more antibodies were produced by B cells in the PF following stimulation with BCG, early secretory antigenic target (ESAT-6)/culture filtrate protein-10 (CFP-10) or ESAT-6 protein. Collectively, these data demonstrate that Mycobacterium tuberculosis-specific B cells exist at local sites of infection in TP patients and this localization might influence the immune response to M. tuberculosis.     

趋化因子受体CXCR4及其配体SDF-1与强直性脊柱炎发病的相关性研究

目的：探讨趋化因子及其受体在强直性脊柱炎（AS）时的变化及其在关节炎发病机制中的作用。方法： 用含588个基因的cDNA微阵列方法比较13例AS患者和7例健康志愿者外周血单个核细胞（PBMC）基因的表达水平；用流式细胞术对PBMC表面C-X-C趋化因子受体4(CXCR4)蛋白表达水平进行检测, 并以免疫组化方法和ELISA方法检测其配体SDF-1（基质来源因子）在滑膜成纤维细胞和滑膜组织的表达情况。结果： AS的588 个基因谱和健康志愿者有明显区别，其中趋化因子受体(CXCR4)在AS外周血的单核细胞和CD8+的T淋巴细胞表达显著高于健康志愿者组(P＜0.05）。 SDF-1在AS病人的PBMC、滑液单个核细胞（SFMC）、关节液成纤维细胞和关节滑膜衬里层细胞的表达也增高。 结论： 趋化因子受体CXCR4及其产物在AS患者的PBMC表达明显增多, SDF-1在AS关节炎患者滑膜成纤维细胞和滑膜组织表达增多，提示CXCR4及其配体SDF-1这一信息通道在AS炎症病变的发展与持续中可能起重要作用。     

SDF-1/CXCR4增强骨髓间质干细胞的造血支持作用

目的探讨基质细胞衍生因子-1(SDF-1)/CXCR4在骨髓间质干细胞(MSCs)支持CD34 造血干/祖细胞(HSPCs)扩增中的作用。方法在长期培养基(LTC)中,以大鼠骨髓MSCs作为饲养层体外扩增骨髓CD34 细胞,每周分别加入SDF-1、SDF-1抗体或CXCR4抗体至5周。计算CD34 细胞数和集落形成细胞(CFC)数,以评价造血支持功能。为评估SDF-1/CXCR4对CD34 细胞增殖周期的影响,进行了杀伤试验以计算增殖指数。流式细胞术检测MSCs和CD34 细胞中SDF-1与CXCR4的表达;ELISA检测MSCs和CD34 细胞培养基中SDF-1的含量。结果CD34 细胞数、CFC数和增殖指数在加入SDF-1后明显增加(P<0.01),加入SDF-1抗体或CXCR4抗体后明显减少(分别为P<0.05,P<0.01)。CD34 细胞表面表达CXCR4,MSCs则不表达;MSCs细胞内表达SDF-1,而CD34 细胞不表达。在MSCs培养基中检测到SDF-1,在CD34 细胞培养基中未发现。结论SDF-1/CXCR4在骨髓MSCs支持HSPCs扩增中起重要作用。     

The chemokine receptor CXCR4 is expressed within the mast cell lineage and its ligand stromal cell-derived factor-1alpha acts as a mast cell chemotaxin

In this study we provide evidence that the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) acts as a mast cell chemoattractant through interactions with its receptor CXCR4 expressed on mast cell progenitors in the blood as well as on in vitro-developed and leukemic mast cells. We found expression of CXCR4 on cord blood-derived mast cells (CBMC) and on the human mast cell line HMC-1, analyzed by RNAse protection assay and flow cytometry. SDF-1alpha induced intracellular calcium mobilization in HMC-1 cells and was chemotactic for both HMC-1 cells and CBMC. The activity of SDF-1alpha was completely blocked by treating the cells with pertussis toxin, indicating the involvement of Gi-proteins in the signaling. By applying a transwell assay we could show that SDF-1alpha induces migration of a cell population in peripheral blood that is enriched for cells with the capacity to differentiate into mast cells. These findings thus suggest a mechanism by which human mast cell progenitors may be recruited from circulation into the tissue.     

Divergent chemokine receptor expression and the consequence for human IgG4 B cell responses

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of B cells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 B cells, circulating IgG4 B cells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive B cells with an IgG4-inducing combination of T_FH/T_H2 cytokines. Consistent with these observations, we found a low abundance of IgG4 B cells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20⁺ B cell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 B cells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.     

Desensitization of chemokine receptor CCR5 in dendritic cells at the early stage of differentiation by activation of formyl peptide receptors

Chemokine receptors are subjected to heterologous desensitization by activation of formyl peptide receptors. We investigated the cross-talk between formyl peptide receptors and the chemokine receptor CCR5 in human monocyte-differentiated immature dendritic cells (iDC). Monocytes cultured with GM-CSF and IL-4 for 4 days exhibit markers characteristic of iDC and maintain the expression of both formyl peptide receptors FPR and FPRL1, as well as CCR5. Pretreatment of iDC with W peptide (WKYMVm), a potent agonist for FPR and FPRL1 but with preference for FPRL1, resulted in down-regulation of CCR5 from the cell surface and reduced cell response to the CCR5 ligands through a PKC-dependent pathway. Furthermore, W peptide induced a PKC-dependent phosphorylation of CCR5 and inhibited infection of iDC by R5 HIV-1. Our results indicate that the expression and functions of CCR5 in iDC can be attenuated by W peptide, which activates formyl peptide receptors, and suggest an approach to the design of novel anti-HIV-1 agents.     

Increased expression of SDF-1/CXCR4 is associated with lymph node metastasis of invasive micropapillary carcinoma of the breast

Aims:  Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are implicated in tumour chemotaxis and metastasis. The aim was to examine their roles in the metastasis of invasive micropapillary carcinoma (IMPC) of the breast, a tumour with a high propensity for nodal spread.
Methods and results:  We compared the expression of SDF-1 and CXCR4 in 103 cases of breast cancer containing IMPC components with a control group of 96 cases of invasive ductal carcinoma (IDC), not otherwise specified type by immunohistochemistry and chemical in situ hybridization (CISH). The results showed that the predominant cytoplasmic expression of both SDF-1 and CXCR4 was greater in tumour cells of the IMPC components than in those of the non-IMPC components and the control IDC cases, and was correlated significantly with the number of positive lymph nodes ( P  < 0.05). SDF-1 expression on cell membranes was less frequently identified in IMPC than IDC ( P  = 0.021). Immunohistochemical detection of SDF-1 in endothelial cells of lymphatic vessels was more common in IMPC ( P   =  0.007) and correlated significantly with lymph node status ( P  = 0.002), although SDF-1 mRNA was rarely detected by CISH.
Conclusions:  This study suggests that up-regulation of cytoplasmic expression of SDF-1/CXCR4 might be one of the molecular mechanisms facilitating lymph node metastasis of IMPC.