SDF-1α/CXCR4轴通过诱导胰腺癌上皮-间充质转化促进肿瘤迁移和侵袭

目的:探讨SDF-1α/CXCR4轴对胰腺癌细胞迁移和侵袭能力的影响及其作用机制。方法:应用RT-qPCR检测4种胰腺癌细胞株CXCR4 mRNA的表达。Transwell实验检测外源性SDF-1α及其受体CXCR4靶向抑制剂AMD3100对胰腺癌细胞迁移和侵袭能力的影响。MTS法检测外源性SDF-1α及AMD3100对胰腺癌细胞活力的影响。Western blot法检测外源性SDF-1α及AMD3100对胰腺癌细胞上皮-间充质转化(EMT)相关标志物表达的影响。结果:(1) 4种胰腺癌细胞株均不同程度地表达CXCR4 mRNA,其中PANC-1细胞株表达量最高。(2)外源性SDF-1α可增强PANC-1细胞的迁移和侵袭能力,该作用可被AMD3100所阻断。(3)外源性SDF-1α处理PANC-1细胞72 h可增强细胞活力,该作用可被AMD3100阻断。(4)外源性SDF-1α通过上调SNAIL和TWIST促使PANC-1细胞发生EMT,该作用可被AMD3100所阻断。结论:SDF-1/CXCR4轴通过促进胰腺癌细胞发生EMT而促进肿瘤迁移和侵袭。     

病毒巨噬细胞炎性蛋白Ⅱ N端肽调控SDF-1α/CXCR4诱导趋化机制

目的:评价病毒巨噬细胞炎性蛋白ⅡN端肽(NT21MP)是否通过干扰SDF-1α/CXCR4信号抑制人乳腺癌细胞株SKBR3细胞的趋化作用。方法:以RT-PCR和免疫组化检测SKBR3和MCF-7两种人乳腺癌细胞中CXCR4的表达;用细胞转移实验检测在NT21MP存在或缺乏的情况下SDF-1α诱导SKBR3细胞的趋化作用;以Fluo3/AM为细胞内游离钙离子的荧光指示剂,用激光扫描共聚焦显微镜测定NT21MP对SDF-1α诱导SKBR3细胞内游离钙浓度的影响;Western blot分析ERK1/2和FAK蛋白的磷酸化水平变化。结果:相对于MCF-7细胞,SKBR3细胞中CXCR4蛋白表达水平较高;经SDF-1α处理后,SKBR3的迁移能力提高,CXCR4抑制剂AMD3100可有效抑制SKBR3细胞的迁移,细胞经NT21MP预处理后,可剂量依赖性地抑制SKBR3细胞的迁移(P<0.05);NT21MP也可抑制由SDF-1α诱导的细胞内Ca2+峰值(P<0.05),而钙离子浓度升高是SKBR3细胞迁移的重要信号之一;另外,相对于阴性对照组,NT21MP也可下调SDF-1α诱导的SKBR3中信号蛋白ERK1/2和FAK的磷酸化水平(P<0.05)。结论:NT21MP可抑制SDF-1α诱导的SK-BR3细胞的迁移,可能与其上游钙离子释放和ERK1/2及FAK磷酸化阻断信号有关。     

CXCR4+骨髓间充质干细胞迁移与氧化型低密度脂蛋白致内皮细胞损伤

目的 研究内皮细胞分泌的基质细胞衍生因子1(SDF-1)是否影响CXCR4+干细胞迁移功能.方法 从骨髓中分离出CXCR4+的骨髓间充质干细胞(CXCR4+BMSC),用氧化型低密度脂蛋白(ox-LDL)刺激人脐静脉内皮细胞(HUVEC),用MTT法检测HUVEC细胞增殖,用RT-PCR检测SDF-1α mRNA的表达,用ELISA检测SDF-1α蛋白表达,用Transwell(R)检测CXCR4+BMSC迁移.结果 ox-LDL的刺激影响HUVEC增殖,并引起SDF-1α mRNA和蛋白表达升高;含SDF-1α培养基上清能促进CXCR4+BMSC迁移,这种迁移可被CXCR4抗体抑制.结论 CXCR4+BMSC可通过SDF-1α/CXCR4轴向引起内皮细胞发生迁移运动.     

基质细胞衍生因子-1_α/CXCR4/CXCR7轴对骨髓间充质干细胞迁移的影响

目的探讨基质细胞衍生因子-1α(SDF-1α)受体CXCR4、CXCR7在骨髓间充质干细胞(BMSCs)中蛋白和mRNA的表达;及SDF-1α/CXCR4/CXCR7轴对BMSCs迁移作用的可能机制。方法体外培养大鼠BMSCs,流式细胞术鉴定细胞表面抗原CD29、CD44和CD34。应用CXCR4特异性拮抗剂AMD3100及CXCR7中和抗体分别阻断CXCR4及CXCR7,通过Western blotting和RT-PCR分别检测BMSCs蛋白和mRNA的表达变化;Transwell法检测细胞迁移能力。本次实验分为单纯BMSCs组(A)、AMD3100预处理BMSCs组(B)、CXCR7中和抗体预处理BMSCs组(C)及AMD3100+CXCR7中和抗体预处理BMSCs组(D)。结果经鉴定第3代大鼠BMSCs中CD29和CD44均呈阳性表达,而CD34表达阴性。BMSCs中CXCR4、CXCR7蛋白和mRNA均有表达。与A组相比,B组及D组CXCR4及CXCR7蛋白表达明显受到抑制(P0.05),C组只有CXCR7蛋白表达降低(P0.05);各组CXCR4 mRNA和CXCR7 mRNA的表达差异均无显著性。SDF-1α可以诱导BMSCs迁移,与0μg/L组相比,10μg/L组和100μg/L组穿膜细胞数均显著增多(P0.01),与10μg/L组相比,100μg/L组穿膜细胞数亦明显增多(P0.01);AMD3100和CXCR7中和抗体均能抑制BMSCs的迁移作用(P0.05),当两者同时作用时,抑制效应更为显著(P0.05)。结论 BMSCs共表达CXCR4、CXCR7蛋白及mRNA;BMSCs的迁移具有SDF-1α浓度依赖性;SDF-1α/CXCR4/CXCR7轴介导BMSCs的迁移作用,CXCR4受体和CXCR7受体对BMSCs的迁移可能具有协同促进作用。     

HIF-1α/SDF-1信号轴在低氧诱导祖细胞迁移和黏附中的作用

目的: 分析低氧对大鼠肺动脉内皮细胞低氧诱导因子(HIF-1α)及间质细胞衍生因子(SDF-1)表达的影响,探讨二者的相互关系(即是否存在HIF-1α/SDF-1信号轴)及其在低氧诱导祖细胞迁移和黏附中的作用。方法: 免疫磁珠分离纯化SD大鼠外周血CD34/CXCR4阳性祖细胞,免疫荧光、Western blotting及ELISA法检测不同低氧时间HIF-1α和SDF-1在大鼠肺动脉内皮细胞的表达,迁移和黏附实验检测常氧组、低氧组、HIF-1α抑制剂2-甲氧雌二醇(2ME2)组、SDF-1中和抗体组及同时加2ME2和SDF-1中和抗体组祖细胞的迁移指数和黏附率。结果: HIF-1α和SDF-1的表达与低氧时间有关,低氧12 h时二者表达到峰值(均P<0.01),应用2ME2可使SDF-1表达下调(P<0.05),应用2ME2或SDF-1中和抗体后均下调祖细胞的迁移指数和黏附率(P<0.05)。结论: 低氧可诱导肺动脉内皮细胞HIF-1α及受其调控的下游因子SDF-1的表达,提示HIF-1α与SDF-1存在信号调节关系。HIF-1α/SDF-1信号轴在介导祖细胞迁移和黏附至肺动脉内皮细胞的过程中起重要作用。     

SDF-1/CXCR4轴活化诱导内皮祖细胞增殖、迁移及管型形成

目的观察趋化因子SDF-1促内皮祖细胞增殖、迁移和管型形成的作用。方法用免疫细胞化学检测内皮祖细胞SDF-1和CXCR4表达;用MTT法、Millicell趋化法及Matrigel体外三维成型法分别检测不同浓度的趋化因子SDF-1促内皮祖细胞增殖、迁移和管型形成。并应用CXCR4受体抑制剂AMD3100观察上述指标的变化。结果免疫细胞化学显示内皮祖细胞表达SDF-1和CXCR4蛋白。SDF-1可促进内皮祖细胞的增殖、迁移和体外小管样结构的形成。AMD3100可抑制SDF-1的诱导作用。结论SDF-1/CXCR4轴在内皮祖细胞参与血管新生中可能发挥重要作用。     

趋化因子受体CXCR4在人肺癌高转移细胞株的表达和意义

目的：以人肺癌高、低转移细胞株95D、95C为研究对象，研究趋化因子受体CXCR4的表达及其在肿瘤细胞体外转移潜能中的作用和意义。方法：采用RT-PCR检测95D、95C细胞CXCR4 mRNA的表达情况；以PMA活化肿瘤细胞，研究CXCR4 mRNA表达水平与细胞活性状态的关系；应用钙离子内流实验验证其表达是否具有功能；通过趋化实验观察CXCR4特异性配件SDF-α和裸鼠组织匀浆液对95D细胞的趋化迁移作用；通过MTT法测定95D细胞对SDF-1α作用的增殖反应。结果：95D细胞功能性地高表达趋化因子受体CXCR4，且其表达水平与细胞活性状态有关；CXCR4特异性配件SDF-1α和裸鼠肺、淋巴结组织匀浆均可在体外趋化95D细胞的迁移，SDF-1α还可促进95D细胞的增殖。结论：95D细胞功能性高表达趋化因子受体CXCR4可能与人肺癌细胞株95D的体外高转移潜能有关。     

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。     

黄芪甲苷对EPCs中SDF-1α/CXCR4的调控作用

目的:探讨黄芪甲苷(AS-IV)对内皮祖细胞(EPCs)中CXC趋化因子受体4(CXCR4)和基质细胞衍生因子1α(SDF-1α)的调控作用及其作用机制。方法:体外培养大鼠骨髓源性EPCs,观察应用AS-IV及CXCR4的特异性阻断剂AMD3100后EPCs的增殖、黏附、迁移、凋亡和管状结构形成能力的变化,并分析AS-IV对EPCs中SDF-1α及CXCR4的mRNA和蛋白,以及p-CXCR4蛋白水平变化的影响。结果:AS-IV可以显著提升EPCs的增殖、黏附、迁移和管状结构形成能力,减轻EPCs的凋亡,上调EPCs中SDF-1α和CXCR4的mRNA和蛋白及p-CXCR4蛋白的水平(P0.05);AMD3100可以阻断AS-IV对CXCR4的mRNA和蛋白及p-CXCR4蛋白水平的上调作用,但不影响AS-IV对SDF-1α的mRNA和蛋白水平的上调作用。结论:AS-IV可能通过调控EPCs中SDF-1α/CXCR4的表达而增强EPCs的生物学作用。     

SDF-1/CXCR4信号轴通过NF-κB通路诱导人退变髓核细胞凋亡

目的探讨趋化因子SDF-1/CXCR4信号轴对人退变椎间盘髓核细胞凋亡的调控作用及其分子机制。方法将术中摘取的椎间盘标本根据Pfirrmann退变程度分为正常组和退变组。用免疫组织化学法、定量PCR和Western blot等检测SDF-1和CXCR4的表达。用退变椎间盘髓核原代细胞培养,取第3~5代的细胞给予10 ng/mL SDF-1刺激、CXCR4-siRNA转染及NF-κB特异性抑制剂PDTC(20μmol/L)等不同处理,Western blot和q-PCR验证转染效率和信号通路上靶蛋白(基因)的表达;Annexin V/PI检测细胞凋亡率;细胞免疫荧光检测NF-κB的重要基团P65的核转移情况。结果 SDF-1和CXCR4在退变椎间盘组织中表达显著升高(P0.05);SDF-1可以诱导退变髓核细胞的凋亡,但在CXCR4的表达受到沉默后,SDF-1的促凋亡作用被抑制(P0.05);加入SDF-1的诱导后,磷酸化P65的表达水平明显增高(P0.05),P65向核内移位;用PDTC抑制NF-κB活性后,SDF-1促凋亡作用明显减弱(P0.05)。结论 SDF-1/CXCR4信号轴促进髓核细胞凋亡的机制可能与Akt/NF-κB信号通路相关。     

Characterization of a Xenopus laevis CXC chemokine receptor 4: implications for hematopoietic cell development in the vertebrate embryo

Previous reports have shown that the Gi-protein-coupled CXC chemokine receptor 4 is activated by stromal cell-derived factor 1 (SDF-1). The receptor is present in many cell types and regulates a variety of cellular functions, including chemotaxis, adhesion, hematopoiesis, and organogenesis. To examine the role of CXCR4 as a regulator of organogenesis in the vertebrate embryo, we have isolated a cDNA encoding the Xenopus laevis homologue of CXCR4 (xCXCR4). The encoded polypeptide was functionally reconstituted with recombinant Gi2 in baculovirus-infected insect cells. Although xCXCR4 shares only 42% of its extracellular residues with mammalian CXCR4, it is indistinguishable from human CXCR4 in terms of its activation by human SDF-1alpha and SDF-1beta. The fact that only 19 of these residues are specifically present in the extracellular portions of CXCR4 suggests that these residues may be involved in recognizing SDF-1 and/or mediating CXCR4 activation by SDF-1. Xenopus CXCR4 mRNA expression was up-regulated during early neurula stages and remained high during early organogenesis. Whole mount in situ hybridization analysis showed abundant expression of xCXCR4 mRNA in the nervous system, including forebrain, hindbrain, and sensory organs, and in neural crest cells. xCXCR4 mRNA was also detected in the dorsal lateral plate, the first site of definitive hematopoiesis in the amphibian embryo corresponding to aorta-gonad-mesonephros or para-aortic splanchnopleura in mammals. This observation suggests that SDF-1 and CXCR4 are involved in regulating the migratory behavior of hematopoietic stem cells colonizing the larval or fetal liver. The hematopoietic defects observed in mice lacking SDF-1 or CXCR4 may, at least in part, be explained by a disturbance of this migration.     

Stromal-Cell Derived Factor Is Expressed by Dendritic Cells and Endothelium in Human Skin

Stromal-cell derived factor or SDF-1 is a CXC chemokine constitutively expressed by stromal bone marrow cell cultures that binds to the G-protein-coupled receptor CXCR4. SDF-1/CXCR4 represents a unique, nonpromiscuous ligand/receptor pair that plays an essential role in prenatal myelo- and lymphopoiesis as well as in cardiovascular and neural development. SDF-1 prevents entry of CXCR4-dependent (X4) HIV viruses in T lymphocytes, by binding and internalizing CXCR4. The expression pattern of SDF-1 protein in normal tissues is not known. Here we describe an analysis of SDF-1 mRNA and protein in normal and inflamed skin by in situ hybridization and immunohistochemistry, using a novel anti-SDF-1 monoclonal antibody. We also describe the expression pattern of CXCR4 receptor by immunohistochemistry. Our results show that SDF-1 protein and mRNA are normally expressed by endothelial cells, pericytes, and either resident or explanted CD1a+ dendritic cells. Epithelial cells of sweat glands but not keratinocytes also express SDF-1. In various inflammatory skin diseases, a large number of mononuclear cells and fibroblasts in close contact with CXCR4-positive lymphocytic infiltrates also express SDF-1. CXCR4 was also detected in many different normal cell types, including endothelial and epithelial cells, which points to a role for SDF-1/CXCR4 cell signaling in vascular and epithelial homeostasis. The demonstration of SDF-1 expression in dendritic and endothelial cells provides new insights into the mechanisms of normal and pathological lymphocyte circulation and makes it possible to envisage a role for locally secreted SDF-1 in the selective incapacity of mucosal dendritic cells to support and propagate infection by X4 HIV isolates.     

Phenotypic Knockout of CXCR4 on Molt-4 with SDF-1α/54 Attached with KDEL

Objective :To investigate the mechanism of phenotypic knockout of CXCR4 on T-cell leukemia cell line Molt-4 via SDF-1α/54/KDEL intrakine technology, which the mutant SDF-1α/54, human stromal cell-derived Faceor-1 (SDF-1α) was deleted its Cterminal α-helix and attached with a endoplasimc reticulum retention signal 4-peptide-KDEL encoding gene, so that retain the newly synthesized receptor CXCR4 within the Molt-4 cells endoplasmic reticulum. Methods: The recombinant vector pEGFP-C3/SDF-1α/54/KDEL were transfected into Cos-7 cells by liposome, SDF-1α/54/KDEL fusion protein was confirmed with western blot. The recombinant plasmids were transfected transiently into Molt-4 by electroporation. Results:Western blot confirmed SDF-1α/54/KDEL expression in Cos-7. A dramatic downregulation of CXCR4 expression on Molt-4 was demonstrated by flow cytometric (FCM) analysis. Conclusion:SDF-1α/54/KDEL and SDF-1αKDEL have no significant deviation for phenotypic knockout of CXCR4. These suggest that the phenotypic knockout effects of SDF-1α/54 against CXCR4 are not influenced by deleting of SDF-1α helix in the C-terminal.     

Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis

The alpha-chemokine stromal-derived factor (SDF)-1 and the G-protein-coupled seven-span transmembrane receptor CXCR4 axis regulates the trafficking of various cell types. In this review, we present the concept that the SDF-1-CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells. Supporting this is growing evidence that SDF-1 plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells (HSCs) and their homing/retention in bone marrow. Moreover, functional CXCR4 is also expressed on nonhematopoietic tissue-committed stem/progenitor cells (TCSCs); hence, the SDF-1-CXCR4 axis emerges as a pivotal regulator of trafficking of various types of stem cells in the body. Furthermore, because most if not all malignancies originate in the stem/progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and, as a result, the SDF-1-CXCR4 axis is also involved in directing their trafficking/metastasis to organs that highly express SDF-1 (e.g., lymph nodes, lungs, liver, and bones). Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, and we discuss here the common molecular mechanisms involved in these processes. Finally, the responsiveness of CXCR4+ normal and malignant stem cells to an SDF-1 gradient may be regulated positively/primed by several small molecules related to inflammation which enhance incorporation of CXCR4 into membrane lipid rafts, or may be inhibited/blocked by small CXCR4 antagonist peptides. Consequently, strategies aimed at modulating the SDF-1-CXCR4 axis could have important clinical applications both in regenerative medicine to deliver normal stem cells to the tissues/organs and in clinical hematology/oncology to inhibit metastasis of cancer stem cells.     

The proline-rich region of HIV-1 Nef affects CXCR4-mediated chemotaxis in Jurkat T cells

T-cell chemotaxis constitutes an essential function of the immune response, since active secretion of chemokines controls homing and recruitment of leukocytes into tissues. Modification of chemotactic responses by HIV-1 may provide a mechanism to increase viral spread, and may be an important factor in HIV-1 disease progression and pathogenesis. One potent T-cell chemoattractant is SDF-1 alpha, the natural ligand for the HIV-1 co-receptor CXCR4. In addition, the HIV-1 gp120 molecule shares the chemotactic properties of several chemokines, including SDF-1 alpha. HIV-1 Nef is a pathogenic determinant and a virulence factor that has pleiotropic effects on immune cell processes and receptor signaling. In this study, the effects of Nef on T-cell migration to SDF-1 alpha, and on CXCR4 receptor signaling were examined. We report that disruption of the proline-rich region of Nef inhibits T-cell migration to SDF-1 alpha. This dominant negative effect indicates that Nef occupies a position in the CXCR4-mediated signaling pathway that is upstream of an SH3-dependent pathway. The results suggest that Nef may play an important role in homing of T cells during viral invasion in HIV-1 disease.     

Caspase-dependent apoptosis of cells expressing the chemokine receptor CXCR4 is induced by cell membrane-associated human immunodeficiency virus type 1 envelope glycoprotein (gp120)

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins interact with CD4 and chemokine receptors on T cells to deliver signals that trigger either activation, anergy, or apoptosis. However, the molecular mechanisms driving these responses remain poorly understood. In this study we demonstrate that apoptosis is induced upon HIV-1 envelope binding to the chemokine receptor CXCR4. Cells expressing a mutant form of CXCR4 with a C-terminal deletion were also sensitive to HIV-1 envelope-mediated apoptosis, indicating that the cytoplasmic tail of CXCR4 is not required to induce the apoptotic pathway. The specificity of this process was analyzed using several inhibitors of gp120-CD4-CXCR4 interaction. Monoclonal antibodies directed against the gp120-binding site on CD4 (ST4) and against CXCR4 (MAB173) prevented the apoptotic signal in a dose-dependent manner. The cell death program was also inhibited by SDF-1alpha, the natural ligand of CXCR4, and by suramin, a G protein inhibitor that binds with a high affinity to the V3 loop of HIV-1 gp120 envelope protein. These results highlight the role played by gp120-binding on CXCR4 to trigger programmed cell death. Next, we investigated the intracellular signal involved in gp120-induced apoptosis. This cell death program was insensitive to pertussis toxin and did not involve activation of the stress- and apoptosis-related MAP kinases p38(MAPK) and SAPK/JNK but was inhibited by a broad spectrum caspase inhibitor (z-VAD.fmk) and a relatively selective inhibitor of caspase 3 (z-DEVD.fmk). Altogether, our results demonstrate that HIV induces a caspase-dependent apoptotic signaling pathway through CXCR4.     

SDF-1alpha regulates HIV-1-gp120-induced changes in CD79b surface expression and Ig production in activated human B cells

Binding of HIV-1 glycoprotein (gp120) to activated B cells of HIV-infected and HIV-uninfected subjects induces increased cell proliferation, cAMP generation, immunoglobulin (Ig) production and downregulation of the invariant chain, CD79b, of the B-cell receptor. We present evidence that the stromal cell-derived factor-1alpha (SDF-1alpha), itself a B-cell stimulant, reversed gp120-driven downregulation of CD79b in CD40- and IL-4-activated purified HIV-1 seronegative human peripheral blood B cells. SDF-1alpha augmented gp120-induced Ig production, downregulated CXCR4 receptor expression, and alone, exerted no effect on CD79b surface expression, reversed the gp120-induced downregulation of CD79b. These SDF-1alpha-modulated B-cell responses were specifically abrogated by an anti-SDF-1alpha antibody. These data suggest that SDF-1alpha plays an important regulatory role in the altered B-cell responses seen in HIV-1 infection. Further, these findings may enhance the understanding of the pathophysiology of HIV-1 infection and suggest a strategy utilizing SDF-1alpha or related molecules as an anti-HIV therapy.     

Stromal cell-derived factor-1 from biliary epithelial cells recruits CXCR4-positive cells: implications for inflammatory liver diseases

Although stromal cell-derived factor-1 (SDF-1) plays an important role in hematopoiesis in the fetal liver, the role after birth remains to be clarified. We investigated the role of SDF-1 and its receptor, CXCR4, in 75 patients; this included controls and patients with viral hepatitis, liver cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. Interestingly, SDF-1 appeared up-regulated in biliary epithelial cells (BEC) of inflammatory liver disease. Furthermore, in inflammatory liver diseases, SDF-1 was expressed by BEC of interlobular and septal bile ducts and by proliferated bile ductules. The message expression of SDF-1 in BEC was confirmed at a single-cell level by RT-PCR and laser capture microdissection. The plasma levels of SDF-1 were significantly higher in patients with liver diseases than in normal controls. Flow cytometric analysis of the surface expression of CXCR4 showed that most liver-infiltrating lymphocytes express CXCR4 and the intensity was up-regulated more significantly in liver-infiltrating lymphocytes than in peripheral blood lymphocytes. These results suggest that increased SDF-1 production by BEC may play an important role in the recruitment of CXCR4-positive inflammatory cells into the diseased livers. These data are significant because modulation of the SDF-1/CXCR4 interaction has therapeutic implications for inflammatory liver diseases.     

Functional expression of chemokine receptor CXCR4 on human epithelial cells

Chemokines and their receptors play an important role in the process of leucocyte recruitment at sites of inflammation. However, recent evidence suggests that these proteins can also regulate non-leucocyte cell functions such as angiogenesis, migration and proliferation. We have investigated the expression of the CXC chemokine receptor 4 (CXCR4) on primary cultures of type II alveolar epithelial cells, their transformed counterpart, the A549 cell line and also on other epithelial cell lines from various tissues. We found that all epithelial cell types tested express mRNA for CXCR4. Flow cytometric analysis and immunocytochemical staining shows that CXCR4 chemokine receptor is abundantly expressed on the surface of A549 epithelial cells. Furthermore, A549 cells responded to the CXCR4 ligand, stromal-derived factor-1alpha (SDF-1alpha) with a rapid and robust calcium mobilization and not to other CXC chemokines, suggesting that CXCR4 is functionally active and is able to couple to G-protein signalling mechanisms. A549 cells did not proliferate in response to either SDF-1alpha or interleukin-8 (IL-8) CXC chemokines. These findings may have important implications for epithelial physiology and pathology.     

Glycans and proteoglycans are involved in the interactions of human immunodeficiency virus type 1 envelope glycoprotein and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells

We demonstrate that human immunodeficiency virus HIV-1(LAI) envelope glycoprotein 120 (gp120(LAi)) specifically interacts with several membrane ligands on lymphoid CEM or monocytic U937 cells in addition to its previously identified receptor, CD4, and CXCR4, its coreceptor. In its native state, gp120(LAI) is able to elicit specific multimolecular complexes with these membrane ligands at the surface of the cells; most of the interactions are abolished by mannan or heparin but not by dextran. Similarly, stromal cell-derived factor (SDF)-1alpha interacts not only with CXCR4 expressed by CXCR4(+) CD4(+) U937, CEM, and HOS-CD4(+) CXCR4(+) cells but also with CD4 expressed by intact U937, CEM, and HOS-CD4(+) CXCR4(+/-) cells or electroblotted onto Immobilon. SDF-1alpha binding to CD4(+) CXCR4(+/-) cells, or soluble CD4 electroblotted onto Immobilon, is significantly inhibited by sCD4, whereas truncated sCD4 lacking D3 and D4 domains had no significant effect, which indicates that SDF-1 binds to CD4 but at regions different from the HIV-gp120-binding site. Heparin and mannan also inhibit SDF-1alpha binding to intact CD4(+) CXCR4(+/-) cells, and electroblotted soluble CD4. Heparitinase treatment of such cells reduced SDF-1alpha binding. These data demonstrate that glycans and glycosaminoglycans are directly or indirectly involved in the interactions of HIV-1 gp120(LAI) and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells and thus could play a role both in HIV-1 infection and in the physiology of SDF-1alpha.