Enhancement of lymphocyte migration and cytokine production by ephrinB1 system in rheumatoid arthritis

Although the etiology of early events in rheumatoid arthritis (RA) remains undefined, an anomaly in T cell homeostasis and hyperproliferation of synovial-lining cells are involved in the disease process. Since it has been reported that the ephrin/Eph receptor system plays important signaling roles in inflammation processes, we attempted to examine ephrinB molecules in T cells and synovial cells derived from RA in this study. The expression level of ephrinB1 was significantly high in synovial fibroblasts and CD3-positive exudate lymphocytes in synovial tissues derived from patients with RA compared with those in osteoarthritis (OA). Protein and mRNA levels of ephrinB1 were also higher in peripheral blood lymphocytes (PBLs) prepared from patients with RA than those from normal controls. Similar results were obtained from an animal model of human RA, collagen antibody-induced arthritis mice. Moreover, a recombinant ephrinB1/Fc fusion protein stimulated normal PBLs to exhibit enhanced migration and production of TNF-alpha. EphrinB1/Fc also activated synovial cells established from patients with RA to produce IL-6. Tyrosine phosphorylation of EphB1 was induced in these cells by ephrinB1/Fc. The CpG islands in the 5' upstream regulatory region of the ephrinB1 gene were hypomethylated in RA patients compared with those of normal donors. These results suggest that ephrinB1 and EphB1 receptors play an important role in the inflammatory states of RA, especially by affecting the population and function of T cells. Inhibition of the ephrinB/EphB system might be a novel target for the treatment of RA.     

EphrinB2/EphB4 在血管生长中的作用及中医药研究展望*

近年来,有关ephrin及其Eph受体的作用研究已从神经系统方面逐步向血管生长扩展。已有研究表明ephrinB2/EphB4及其独特的双向信号转导几乎参与血管生长的每个方面,涉及血管发育过程中的动静脉分化、胚胎后血管新生包括内皮细胞增殖、迁移、粘附和分化等过程,且与VEGF、Notch等血管新生调控因子关系密切。另外,实验表明活血化瘀名方血府逐瘀汤显著的促血管新生作用与ephrinB2/EphB4相关,说明中医药促血管新生中ephrinB2/EphB4具有重要作用。本文部分总结了ephrinB2/EphB4在血管生长中的作用,并提出中医药在这方面的展望。     

Effect of a new monoclonal antibody, TA-2, that inhibits lymphocyte adherence to cytokine stimulated endothelium in the rat.

An important event in the migration of lymphocytes out of the blood is their adherence to endothelial cells (EC). In inflammatory sites cytokines activate EC and promote lymphocyte EC adherence and migration. Small peritoneal exudate lymphocytes (sPEL) preferentially migrate from the blood to cutaneous delayed-type hypersensitivity reactions and to sites injected with IFN-gamma, IFN-alpha/beta, and TNF-alpha, rather than to peripheral lymph nodes. The basis of this migration is sPEL adherence to cytokine-activated EC. To study this adhesion mAb to rat sPEL were screened for inhibition of sPEL adherence to IFN-gamma-stimulated EC. One mAb, TA-2, inhibited IFN-gamma-stimulated adherence to EC by 60%. This antibody had no effect on the baseline adherence of sPEL to unstimulated EC. Treatment of sPEL, but not EC, with TA-2-inhibited adhesion. TA-2 also inhibited adhesion to EC activated with mIL-1 alpha, TNF-alpha, and LPS, and the adhesion of spleen T cells to activated EC. The TA-2 Ag was expressed on virtually all lymph node, spleen, and sPEL lymphocytes but sPEL expressed two to three times higher levels than lymph node lymphocytes, and the highest levels were found on CD4+ and CD45R- memory T cells. TA-2 immunoprecipitated a group of four polypeptides with molecular mass of 150, 130, 83, and 66 kDa. Finally, TA-2 inhibited sPEL adhesion to TNF-alpha and IL-1 stimulated human umbilical vein EC to the same extent as an anti-human VCAM-1 mAb, and combinations of TA-2 and anti-VCAM-1 were not different from treatment with either antibody alone. Thus, TA-2 appears to recognize rat VLA-4 based on immunoprecipitation, immunofluorescence, and lymphocyte EC studies. VLA-4 mediates the adhesion of rat lymphocytes to rat microvascular EC stimulated with IFN-gamma, mIL-1 alpha, TNF-alpha, and LPS. VLA-4 is important in the increased adhesion of sPEL to EC and the enhanced sPEL migration to inflammation may in part be explained by increased expression of VLA-4 on these cells.     

Effects of six different cytokines on lymphocyte adherence to microvascular endothelium and in vivo lymphocyte migration in the rat

The first step in the migration of lymphocytes out of the blood is adherence of lymphocytes to endothelial cells (EC) in the postcapillary venule. It is thought that in inflammatory reactions cytokines activate the endothelium to promote lymphocyte adherence and migration into the inflammatory site. Injection of IFN-gamma, IFN-alpha/beta, and TNF-alpha into the skin of rats stimulated the migration of small peritoneal exudate lymphocytes (sPEL) into the injection site, and these cytokines mediated lymphocyte recruitment to delayed-type hypersensitivity, sites of virus injection, and in part to LPS. The effect of cytokines on lymphocyte adherence to rat microvascular EC was examined. IFN-gamma, IFN-alpha/beta, IL-1, TNF-alpha, and TNF-beta increased the binding of small peritoneal exudate lymphocyte (sPEL) to EC. IFN-gamma was more effective and stimulated adherence at much lower concentrations than the other cytokines. IL-2 did not increase lymphocyte adherence. LPS strongly stimulated lymphocyte binding. Treatment of EC, but not sPEL, enhanced adhesion, and 24 h of treatment with IFN-gamma and IL-1 induced near maximal adhesion. Lymph node lymphocytes, which migrate poorly to inflammatory sites, adhered poorly to unstimulated and stimulated EC, whereas sPEL demonstrated significant spontaneous adhesion which was markedly increased by IFN-gamma, IL-1, and LPS. Spleen lymphocytes showed an intermediate pattern of adherence. Combinations of IFN-gamma and TNF-alpha were additive in stimulating sPEL-EC adhesion. Depletion of sPEL and spleen T cells by adherence to IFN-gamma stimulated EC decreased the in vivo migration of the lymphocytes to skin sites injected with IFN-gamma, IFN-alpha/beta, TNF-alpha, poly I:C, LPS, and to delayed-type hypersensitivity reactions by 50%, and significantly increased the migration of these cells to normal lymph nodes, as compared to unfractionated lymphocytes. Thus the cytokines and lymphocytes involved in migration to cutaneous inflammation in the rat stimulate lymphocyte adhesion to rat EC in vitro, and IFN-gamma stimulated EC appear to promote the selective adhesion of inflammatory site-seeking lymphocytes.     

The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.     

Dll4-selective Notch signaling induces ephrinB2 gene expression in endothelial cells

The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation. Here, we show that Notch stimulation instructively induces arterial characteristics in endothelial cells (EC). Forced expression of Notch intracellular domain (NICD, activated form of Notch) induced mRNA expression for a subset of arterial-specific markers such as ephrinB2, connexin40, and HERP1 only in EC but not other cell lines. In co-culture experiments using EC and either Dll4- or Jagged1-expressing cells, we found that Dll4 stimulation but not Jagged1 markedly induced ephrinB2 expression. An inducible expression of HERP1 and HERP2 by NICD has no measurable effects on expression of ephrinB2 and venous marker EphB4 although either HERP1 or HERP2 overexpression exerts potent inhibitory effects on EphB4 expression without ephrinB2 induction. We also found no functional interaction between Notch and TGF-beta-ALK1 signalings in an induction of ephrinB2 expression. These results suggest that Dll4-stimulated Notch signaling induces a part of arterial characteristics only in EC via HERP-independent mechanism. Our data provide new insight into the molecular mechanism of ligand-selective Notch activation during differentiation of arterial EC.     

Effect of antigen challenge on lymph node lymphocyte adhesion to vascular endothelial cells and the role of VLA-4 in the rat.

Lymphocytes from antigen-stimulated lymph nodes avidly migrate from the blood to cutaneous sites of inflammation such as DTH reactions or contact sensitivity. One of the initial steps in this migration is the adhesion of the lymphocyte to endothelial cells (EC); therefore, the adhesion of lymphocytes from antigen-stimulated lymph nodes to microvascular EC in the rat was examined. Two to five days after subcutaneous immunization with antigen, lymphocytes that adhered to unstimulated and IFN-gamma-, TNF-alpha-, IL-1 alpha-, and LPS-treated EC were increased in the regional lymph nodes. The enhanced adhesion was attributable to low-density lymphoblast-enriched lymph node cells while small high-density lymphocytes displayed little or no increase in their adhesion. Lymphoblast adhesion required the stimulation of the EC with 10 times the concentrations of IFN-gamma and TNF-alpha required for peritoneal exudate lymphocyte adhesion. There was a synergistic increase in the adhesion of the low-density lymphocytes to EC stimulated with combinations of IFN-gamma and TNF-alpha. Antibody to VLA-4 inhibited about 40% of the stimulated adhesion to EC treated with IFN-gamma, TNF-alpha, or LPS. In vivo anti-VLA-4 inhibited lymphoblast migration to IFN-gamma, TNF-alpha, LPS, and DTH reactions by 60%. Thus antigen stimulates the generation of low-density lymphoblasts that have an enhanced adherence to cytokine- and LPS-treated EC through a partially VLA-4-dependent mechanism and the migration of these cells to cutaneous inflammatory reactions is dependent upon VLA-4.     

Migration of lymphocytes through endothelial cell monolayers: augmentation by interferon-gamma

During normal lymphocyte recirculation and in chronic inflammation, lymphocytes emigrate from blood into the perivascular tissue. The mechanism of lymphocyte migration through the endothelial cell (EC) layer of blood vessels is poorly understood. To identify factors that control lymphocyte emigration, a method has been developed to measure human peripheral blood lymphocyte migration through monolayers of human umbilical vein EC and into nitrocellulose (NC) filters located below the EC monolayer. Counts were made of lymphocytes that had migrated into the NC filter using a particle counter. T lymphocytes attached to and migrated through EC monolayers in a T-cell-number- and time-dependent fashion. Migration required viable EC since lymphocytes failed to migrate through formaldehyde-fixed EC monolayers or monolayers of dermal fibroblasts. Interferon-gamma (IFN-gamma) markedly augmented the migration in a dose- and time-dependent manner when preincubated with the EC. When T lymphocytes were pretreated with IFN-gamma, no increase in migration was observed. Finally, IFN-gamma augmented the migration of T cells prebound to the EC, indicating that the IFN-gamma-enhanced migration was not due to increased binding of T cells to the EC, but rather to an action on the EC to facilitate subsequent migration.     

The EphB4 receptor-tyrosine kinase promotes the migration of melanoma cells through Rho-mediated actin cytoskeleton reorganization

Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrin-B ligands in a variety of tumors, suggesting a functional relation between EphB/ephrin-B signaling and tumor progression. The ability of the EphB receptors to regulate cell migration and promote angiogenesis likely contributes to tumor progression and metastasis. Here we show that EphB receptors, and especially EphB4, regulate the migration of murine melanoma cells. Highly malignant melanoma cells express the highest levels of EphB4 receptor and migrate faster than less malignant melanoma cells. Furthermore, inhibition of EphB receptor forward signaling by overexpression of a form of EphB4 lacking the cytoplasmic portion or by treatment with competitively acting soluble EphB2-Fc results in slower melanoma cell migration. In contrast, overexpression of active EphB4 significantly enhances cell migration. The effects of EphB4 receptor on cell migration and cell morphology require its kinase activity because the inhibition of EphB4 kinase activity by overexpression of kinase dead EphB4 inhibits cell migration and affects the organization of actin cytoskeleton. Activation of EphB4 receptor with its ligand ephrin-B2-Fc enhances the migratory ability of melanoma cells and increases RhoA activity, whereas inhibiting EphB receptor forward signaling decreases RhoA activity. Moreover, expression of dominant negative RhoA blocks the effects of active EphB4 on cell migration and actin organization. These data suggest that EphB4 forward signaling contributes to the high migratory ability of invasive melanoma cells by influencing RhoA-mediated actin cytoskeleton reorganization.     

The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis

The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. Here we investigated the role of EphB receptors and their ligands during embryonic blood vessel development in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, we detected expression of EphB4 receptors in the posterior cardinal veins and their derivatives, the intersomitic veins. Vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could however not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Furthermore, we found that ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. We performed RNA injection experiments to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos resulted in intersomitic veins growing abnormally into the adjacent somitic tissue. Our findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis possibly by mediating repulsive guidance cues to migrating endothelial cells.     

EphB4 promotes site-specific metastatic tumor cell dissemination by interacting with endothelial cell-expressed ephrinB2

The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4-expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. AACR.     

Characterization of two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes

Two cDNAs, encoding the stress-inducible 70-kDa heat shock protein (Hsp70) and the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), were isolated from grass carp. The Hsp70 and Hsc70 cDNAs were 2250 bp and 2449 bp in length and contained 1932 bp and 1953 bp open reading frames, respectively. Tissue distribution results showed that Hsp70/Hsc70 was highly expressed in gill, kidney, head kidney and peripheral blood lymphocytes (PBLs). Using grass carp PBLs as a cell model, effects of lipopolysaccharide (LPS) on the mRNA and protein levels of Hsp70/Hsc70 were examined. In this case, LPS increased the mRNA expression of Hsp70 in a time- and dose-dependent manner, but had no effect on Hsc70 mRNA expression. In agreement with this, LPS elevated the intracellular Hsp70 markedly, but not the Hsc70 protein levels in parallel experiments. Furthermore, Hsp70 protein was also detected in culture medium. Moreover, inhibition of LPS on Hsp70 release in a time-dependent manner was observed, indicating that there may be a dynamic balance between Hsp70 stores and Hsp70 release in grass carp PBLs following exposure to LPS. Taken together, these results not only shed new insights into the different regulations of LPS on Hsp70/Hsc70 gene expression, protein synthesis and release, but also provide a basis for further study on the functional role of Hsp70 in fish immune response.     

Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes.

Human T-cell leukemia virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). Lymphocytes from patients with ATL or TSP/HAM display abnormal proliferation properties in culture. Here we report that purified, soluble Tax1 protein can be taken up by, and stimulate proliferation of, uninfected human peripheral blood lymphocytes (PBLs) that have been stimulated with phytohemagglutinin (PHA). Tax1 was 40 to 70% as active as interleukin-2 (IL-2) in stimulating proliferation of PBLs. Heat inactivation, chloroform extraction, and immunoprecipitation with antisera specific for Tax1 each abolished the ability of the protein to stimulate lymphocyte proliferation. Tax1 failed to stimulate PBL proliferation in the absence of PHA. After an initial round of cell division, Tax1-treated PBLs exhibited prolonged sensitivity to IL-2-induced proliferation. These results indicate that Tax1 can stimulate lymphocyte proliferation in culture and imply that extracellular Tax1 may be involved in the spontaneous proliferation of TSP/HAM lymphocytes and the IL-2-dependent proliferation of ATL lymphocytes.     

Differential induction of gelatinase B (MMP-9) and gelatinase A (MMP-2) in T lymphocytes upon alpha(4)beta(1)-mediated adhesion to VCAM-1 and the CS-1 peptide of fibronectin

Integrin alpha(4)beta(1) on the surface of T lymphocytes interacts with vascular cell adhesion molecule-1 (VCAM-1) and fibronectin during migration of lymphocytes from the blood to sites of inflammation. Migrating lymphocytes actively modify their environment through a number of mechanisms including proteolysis of the extracellular matrix by matrix metalloproteinases (MMP) synthesized by the cells. In this study, expression of MMP upon alpha(4)beta(1)-mediated adhesion of leukocytes to two major ligands, the IIICS-1 domain of fibronectin and VCAM-1, has been examined. Adhesion of T lymphoblastoid Jurkat cells to the CS-1 peptide induced expression of mRNA for two MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9). As evaluated by relative RT-PCR and Northern blot analyses, the level of mRNA was upregulated about 4- to 5-fold for both MMPs compared to control cells maintained in suspension. With time, both enzymes were detected in conditioned media and inside the cells, and their identities were verified by Western blotting and gelatin zymography. Adhesion of Jurkat cells to the second major alpha(4)beta(1) ligand, VCAM-1, upregulated mRNA for MMP-2 (3.5-fold) and failed to induce expression of mRNA for MMP-9. Accordingly, only MMP-2 protein was detected in conditioned media of cells adherent to VCAM-1. Occupancy of alpha(4)beta(1) on the surface of suspended cells with soluble CS-1 peptide or VCAM-1 did not upregulate synthesis and release of MMPs. A similar pattern of induction of MMPs after adhesion to CS-1 and VCAM-1 was observed in T lymphocytes isolated from human blood. These results demonstrate that adhesion of T lymphocytes through alpha(4)beta(1) to different ligands, which bind to similar or overlapping sites in the integrin, induces intracellular events leading to distinct patterns of MMPs biosynthesis.     

梅山猪胚胎附植期EphB2的组织表达及RNA-seq分析

猪产仔数是一个重要的经济和繁殖性状, 而胚胎附植是影响猪产仔数的重要因素。为了研究促红细胞生成素产生肝细胞受体B2 (EphB2)对猪胚胎附植过程中子宫内膜迁移和粘附活动的影响, 文章以太湖流域梅山猪为研究对象, 利用实时荧光定量PCR (qRT-PCR)和蛋白免疫印迹(Western blot)方法, 检测EphB2基因在梅山猪胚胎附植前、中、后期子宫内膜附植点/非附植点和卵巢组织的mRNA和蛋白表达谱, 并用转录组测序(RNA-seq)方法分析了不同附植时期子宫内膜附植点和卵巢组织的差异表达基因。qRT-PCR和Western blot结果表明, EphB2在胚胎附植前、中、后期子宫内膜附植点和非附植点的mRNA和蛋白均呈现先升高后降低的表达趋势, 且附植中期的表达量显著高于前期和后期(P<0.01);EphB2在附植前、中、后期卵巢中的mRNA和蛋白表达趋势则相反, 为先降低后升高, 且不同时期间表达差异显著(P<0.05)。RNA-seq结果表明, EphB2在子宫内膜附植点的mRNA表达, 附植中期极显著高于附植前期(P<0.01);EphB2在卵巢的mRNA表达, 附植中期显著高于附植后期(P<0.05)。综上所述, EphB2很可能在猪胚胎附植过程中发挥着重要的调控作用, 为潜在的猪产仔数性状候选基因。     

Rapid up-regulation of alpha4 integrin-mediated leukocyte adhesion by transforming growth factor-beta1

The alpha4 integrins (alpha4beta1 and alpha4beta7) are cell surface heterodimers expressed mostly on leukocytes that mediate cell-cell and cell-extracellular matrix adhesion. A characteristic feature of alpha4 integrins is that their adhesive activity can be subjected to rapid modulation during the process of cell migration. Herein, we show that transforming growth factor-beta1 (TGF-beta1) rapidly (0.5-5 min) and transiently up-regulated alpha4 integrin-dependent adhesion of different human leukocyte cell lines and human peripheral blood lymphocytes (PBLs) to their ligands vascular cell adhesion molecule-1 (VCAM-1) and connecting segment-1/fibronectin. In addition, TGF-beta1 enhanced the alpha4 integrin-mediated adhesion of PBLs to tumor necrosis factor-alpha-treated human umbilical vein endothelial cells, indicating the stimulation of alpha4beta1/VCAM-1 interaction. Although TGF-beta1 rapidly activated the small GTPase RhoA and the p38 mitogen-activated protein kinase, enhanced adhesion did not require activation of both signaling molecules. Instead, polymerization of actin cytoskeleton triggered by TGF-beta1 was necessary for alpha4 integrin-dependent up-regulated adhesion, and elevation of intracellular cAMP opposed this up-regulation. Moreover, TGF-beta1 further increased cell adhesion mediated by alpha4 integrins in response to the chemokine stromal cell-derived factor-1alpha. These data suggest that TGF-beta1 can potentially contribute to cell migration by dynamically regulating cell adhesion mediated by alpha4 integrins.     

Live Brugia malayi Microfilariae Inhibit Transendothelial Migration of Neutrophils and Monocytes

Lymphatic filariasis is a major tropical disease caused by the parasite Brugia malayi. Microfilariae (Mf) circulate in the peripheral blood for 2–3 hours in synchronisation with maximal feeding of the mosquito vector. When absent from the peripheral blood, Mf sequester in the capillaries of the lungs. Mf are therefore in close contact with vascular endothelial cells (EC) and may induce EC immune function and/or wound repair mechanisms such as angiogenesis. In this study, Mf were co-cultured with human umbilical vein EC (HUVEC) or human lung microvascular EC (HLMVEC) and the transendothelial migration of leukocyte subsets was analysed. In addition, the protein and/or mRNA expression of chemokine, cytokine and angiogenic mediators in endothelial cells in the presence of live microfilariae were measured by a combination of cDNA arrays, protein arrays, ELISA and fluorescence antibody tests.Surprisingly, our findings indicate that Mf presence partially blocked transendothelial migration of monocytes and neutrophils, but not lymphocytes. However, Mf exposure did not result in altered vascular EC expression of key mediators of the tethering stage of extravasation, such as ICAM-1, VCAM-1 and various chemokines. To further analyse the immunological function of vascular EC in the presence of Mf, we measured the mRNA and/or protein expression of a number of pro-inflammatory mediators. We found that expression levels of the mediators tested were predominantly unaltered upon B. malayi Mf exposure. In addition, a comparison of angiogenic mediators induced by intact Mf and Wolbachia-depleted Mf revealed that even intact Mf induce the expression of remarkably few angiogenic mediators in vascular EC. Our study suggests that live microfilariae are remarkably inert in their induction and/or activation of vascular cells in their immediate local environment. Overall, this work presents important insights into the immunological function of the vascular endothelium during an infection with B. malayi.     

EphB receptors interact with NMDA receptors and regulate excitatory synapse formation

EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. We find that EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB. The kinase activity of EphB may be important for subsequent steps in synapse formation, as perturbation of EphB tyrosine kinase activity affects the number of synaptic specializations that form in cultured neurons. These findings indicate that EphrinB activation of EphB promotes an association of EphB with NMDA receptors that may be critical for synapse development or function.