VLA-4 (alpha(4)beta(1)) engagement defines a novel activation pathway for beta(2) integrin-dependent leukocyte adhesion involving the urokinase receptor

During acute inflammatory processes, beta(2) and beta(1) integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (alpha(4)beta(1)) engagement on beta(2) integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)-Fc or by 2 anti-CD29 (beta(1) chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via beta(2) integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of beta(2) integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1-transfected Chinese hamster ovary cells, but not to ligands of beta(1) integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1-Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1-Fc was required for beta(2) integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-beta(1) integrin mAb were ineffective. Activation of beta(2) integrins by alpha(4)beta(1) integrin ligation (VCAM-1-Fc or anti-beta(1) mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1-Fc or anti-beta(1) integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of beta(2) integrin-dependent cell-to-cell adhesion that requires alpha(4)beta(1) integrin ligation for initiation and uPAR as activation transducer. (Blood. 2000;96:506-513)     

Alpha-4 integrins and VCAM-1, but not MAdCAM-1, are essential for recruitment of mast cell progenitors to the inflamed lung

Normal mouse lungs lack appreciable numbers of mast cells (MCs) or MC progenitors (MCp's), yet the appearance of mature MCs in the tracheobronchial epithelial surface is a characteristic of allergic, T-cell-dependent pulmonary inflammation. We hypothesized that pulmonary inflammation would recruit MCp's to inflamed lungs and that this recruitment would be regulated by distinct adhesion pathways. Ovalbumin-sensitized and challenged mice had a greater than 28-fold increase in the number of MCp's in the lungs. In mice lacking endothelial vascular cell adhesion molecule 1 (VCAM-1) and in wild-type mice administered blocking monoclonal antibody (mAb) to VCAM-1 but not to mucosal addressin CAM-1 (MadCAM-1), recruitment of MCp's to the inflamed lung was reduced by greater than 75%. Analysis of the integrin receptors for VCAM-1 showed that in beta7 integrin-deficient mice, recruitment was reduced 73% relative to wild-type controls, and in either BALB/c or C57BL/6 mice, mAb blocking of alpha4, beta1, or beta7 integrins inhibited the recruitment of MCp's to the inflamed lung. Thus, VCAM-1 interactions with both alpha4beta1 and alpha4beta7 integrins are essential for the recruitment and expansion of the MCp populations in the lung during antigen-induced pulmonary inflammation. Furthermore, the MCp is currently unique among inflammatory cells in its partial dependence on alpha4beta7 integrins for lung recruitment.     

Interferon-gamma and interleukin-4 differentially regulate ICAM-1 and VCAM-1 expression on human lung fibroblasts.

The expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and more specifically vascular adhesion molecule-1 (VCAM-1) on lung fibroblasts may be important for migration of inflammatory cells through the submucosa to the airway lumen in the asthmatic inflammatory response. This study aimed to assess which cytokines are regulating ICAM-1 and VCAM-1 expression on human lung fibroblasts. For this purpose, confluent fibroblast cultures (derived from lung tissue from a nonasthmatic donor) were stimulated for 4 h with interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, interferon (IFN)gamma, IL-4, IL-5 or transforming growth factor (TGF)beta. IL-1beta (optimal concentration (OC) 1 U x mL(-1)) and TNFalpha (OC 100 U x mL(-1)) both increased ICAM-1 and VCAM-1 expression. IFNgamma (OC 2 U x mL(-1)) increased only ICAM-1 expression and IL-4 (OC 5 ng x mL(-1)) increased only VCAM-1 expression, whereas IL-5 (20 ng x mL(-1)) and TGFbeta (10 ng x mL(-1)) did not influence ICAM-1 or VCAM-1 expression. ICAM-1 expression reached a plateau at 8-12 h after cytokine stimulation and remained constant for at least 24 h. VCAM-1 showed a transient increased expression within 24 h after IL-1beta and TNFalpha stimulation. In contrast, VCAM-1 expression did not decrease after maximal expression at 4 h upon IL-4 stimulation. It is concluded that the Helper-1T-cell, type cytokine interferon gamma and the Helper-2 T-cell type cytokine interleukin-4 differentially regulate intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression on human lung fibroblasts. The proinflammatory cytokines interleukin-1beta and tumour necrosis factor alpha increase both intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, without differential regulation of the expression of these adhesion molecules.     

Essential role for Rap1 GTPase and its guanine exchange factor CalDAG-GEFI in LFA-1 but not VLA-4 integrin mediated human T-cell adhesion

Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3(+) human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1alpha (SDF-1alpha)-stimulated LFA-1-ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly, silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1alpha- and phorbol 12-myristate 13-acetate (PMA)-induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospholipase C (PLC) blocked Rap1 activation and inhibited cell adhesion and polarization on ICAM-1 and VCAM-1. Protein kinase C (PKC) inhibition led to enhanced levels of active Rap1 concomitantly with increased T-cell binding to ICAM-1, whereas adhesion to VCAM-1 was reduced. Thus, PLC/CalDAG-GEFI regulation of Rap1 is selectively required for chemokine- and PMA-induced LFA-1 activation in human T cells, whereas alternate PLC- and PKC-dependent mechanisms are involved in the regulation of VLA-4.     

Alpha4 and beta2 integrins have nonredundant roles for asthma development, but for optimal allergen sensitization only alpha4 is critical

OBJECTIVE: Recruitment of effector cell subsets to inflammatory lung, together with airway resident cells responsive to secreted products, play pivotal roles in developing and maintaining asthma. Differential use of adhesion molecules dictates the recruitment patterns of specific cell subsets, yet a clear understanding of the distinctive adhesive molecular pathways guiding them to lung is lacking. To provide further insight into the role of alpha4beta1/VCAM-1 pathway and to compare this to the role of beta2 integrin in the development of acute asthma phenotype, we used genetically deficient mice, in contrast to previous studies with anti-functional antibodies yielding ambiguous results. METHODS: Allergen-dependent airway inflammation and hyperresponsiveness was induced in conditional alpha4(Delta/Delta), VCAM-1(-/-), and beta2(-/-) mice. Cytology, immunocytochemistry, cytokine and immunoglobulin measurements, and cell type accumulation in lung, BAL fluid, plasma, and hemopoietic tissues were carried out. RESULTS: Asthma phenotype was totally abrogated in alpha4- or beta2-deficient mice. Adoptive transfer of sensitized alpha4(Delta/Delta) CD4(+) cells into challenged normal mice failed to induce asthma, whereas alpha4(+/+) CD4(+) cells were able to induce asthma in challenged alpha4(Delta/Delta) mice. Parallel studies with beta2(-/-) or VCAM-1(-/-) mice uncovered novel mechanistic insights in primary sensitization and into redundant or unique functional roles of these adhesion pathways in allergic asthma. CONCLUSIONS: The lack of alpha4 integrin not only impedes the migration of all white cell subsets to lung and airways, but also prevents upregulation of vascular cell adhesion molecule-1 (VCAM-1) in inflamed lung vasculature and, unlike beta2, attenuates optimal sensitization and ovalbumin-specific IgE production in vivo. As VCAM-1 deficiency did not protect mice from asthma, interactions of alpha4beta1(+) or alpha4beta7(+) cells with other ligands are suggested.     

Expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the pig coronary artery injury model: comparison of plain old balloon angioplasty and stent implantation

OBJECTIVES: Restenosis after coronary intervention usually occurs due to coronary remodeling or neointimal formation, but inflammation is also important especially after stent implantation. Adhesion molecules are important in the recruitment of inflammatory cells into the neointima and in the phenotypical changes of vascular smooth muscle cells. To examine the role of adhesion molecules in the pathogenesis of restenosis, immunohistochemical expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was investigated in the pig coronary injury model. METHODS: Left anterior descending coronary arteries of pigs were injured using a balloon. Two weeks after the injury, balloon injury was performed again in the balloon group and a Palmaz-Schatz stent was implanted in the stent group. Pigs were sacrificed at 1, 2 and 4 weeks. Immunohistochemical analysis was performed using ICAM-1, VCAM-1, macrophage and alpha-smooth muscle actin antibodies. RESULTS: In non-injured vessels, weak immunoreactivities of ICAM-1 and VCAM-1 were observed in the endothelium and media. In injured sites, ICAM-1 and VCAM-1 were found in the inflammatory cells and smooth muscle cells in the neointima from 1 week, and strong immunoreactivities were seen around the strut in the stent group. Although the immunoreactivities peaked at 2 weeks in the balloon group, strong immunoreactivities were still seen at 4 weeks in the stent group. Regenerated endothelial cells were positive for both antibodies from 2 weeks. CONCLUSIONS: The expression of ICAM-1 and VCAM-1 lasted longer in the stent group than in the balloon group, suggesting the occurrence of late restenosis after stent implantation. Control of the inflammatory response including adhesion molecules is essential for further reduction of restenosis after stent implantation.     

Expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 and homing factor CD44 after engraftment of Graves' lymphocytes in xenotransplanted human thyroid tissue in athymic nude mice.

The expression of adhesion molecules on thyrocytes and endothelium cells plays an important role in the pathogenesis of Graves' disease (GD). The intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1), and the homing receptor CD44 are responsible for the specific migration of lymphocytes in autoimmune thyroid diseases (AITD) (homing). Eight weeks after transplantation of thyroid tissue from 26 patients with nonautoimmune thyroid disease (nontoxic nodular goiter [NTG]) into nude mice, peripheral (PBL) and intrathyroidal lymphocytes (ITL) from 14 patients with NTG and 12 patients with GD were grafted into the animals. Two days after lymphocyte engraftment, the thyroid transplants were examined histologically (HE) and immunohistologically stained with monoclonal antibodies directed against ICAM-1, VCAM-1, and CD44. After injection of GD lymphocytes, thyroid transplants expressed significantly more ICAM-1, VCAM-1, and CD44 than after injection of NTG lymphocytes. This expression was even more pronounced after grafting of GD intrathyroidal lymphocytes. Our data demonstrate that only GD lymphocytes induce the expression of adhesion molecules and homing factor CD44, both of which play an important role in the migration of lymphocytes and induction of the autoimmune process.     

The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice

Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen-4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen-1 (LFA-1). Treatment of human CD34(+) cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti-LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34(+) cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule-1) and VLA-4/VCAM-1 (vascular adhesion molecule-1). Furthermore, SDF-1-induced polarization and extravasation of CD34(+)/CXCR4(+) cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation.     

Enhancement of G-CSF-induced stem cell mobilization by antibodies against the beta 2 integrins LFA-1 and Mac-1

The beta 2 integrins leukocyte function antigen-1 (LFA-1, CD11a) and macrophage antigen-1 (Mac-1, CD11b) have been reported to play a role in the attachment of CD34(+) cells to stromal cells in the bone marrow. When administered prior to interleukin-8 (IL-8), anti-LFA-1 antibodies completely prevent the IL-8-induced mobilization of hematopoietic stem cells in mice. Here, we studied the role of anti-beta 2 integrin antibodies in granulocyte colony-stimulating factor (G-CSF)-induced mobilization of hematopoietic progenitor cells. Administration of antibodies against the alpha chain of LFA-1 or against the alpha chain of Mac-1 followed by daily injections of G-CSF for more than 1 day resulted in a significant enhancement of mobilization of hematopoietic progenitor cells when compared with mobilization induced by G-CSF alone. Also, the number of late (day 28) cobblestone area-forming cells in vitro was significantly higher after mobilization with anti-LFA-1 antibodies followed by 5 microg G-CSF for 5 days than with G-CSF alone (119 +/- 34 days vs 17 +/- 14 days), indicating mobilization of repopulating stem cells. Pretreatment with blocking antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of LFA-1 and Mac-1, did not result in an effect on G-CSF-induced mobilization, suggesting that the enhancing effect required an interaction of the beta 2 integrins and one of their other ligands. Enhancement of mobilization was not observed in LFA-1-deficient (CD11a) mice, indicating that activated cells expressing LFA-1 mediate the synergistic effect, rather than LFA-1-mediated adhesion.     

Paxillin selectively associates with constitutive and chemoattractant-induced high-affinity alpha4beta1 integrins: implications for integrin signaling

Leukocyte alpha4beta1 integrins regulate hematopoietic and lymphoid development, as well as the emigration of circulating cells to sites of inflammation. Because vascular cell adhesion molecule-1 (VCAM-1) binding to high-affinity alpha4beta1 is stable, these integrins can be detected and selectively precipitated from cell lysates using VCAM-1/Fc. With this approach, high-affinity alpha4beta1 integrin expression was demonstrated on lymphocytes in the bone marrow, thymus, spleen, and the peritoneal cavity of normal mice, but not in peripheral lymph nodes. Immature lymphocytes preferentially expressed high-affinity alpha4beta1 in the bone marrow and thymus. Paxillin is a cytoplasmic adaptor molecule that can bind to the alpha4 tail and initiate signaling. Paxillin was associated selectively with high-affinity integrins that were isolated from human Jurkat T cells or from murine tissues, and blotting with a phospho-specific antibody demonstrated that Ser988 in the alpha4 cytoplasmic tail was dephosphorylated in high-affinity but not low-affinity integrins. A rapid and transient alpha4beta1 affinity up-regulation in formyl peptide receptor-transfected U937 cells stimulated with N-formyl-methyonyl-leucyl-phenylalanine (fMLP) correlated temporally with induced paxillin binding to alpha4 integrins. These data suggest that ligand binding to high-affinity alpha4beta1 integrins may initiate outside-in signaling cascades through paxillin that regulate leukocyte maturation and emigration.     

Membrane type 1-matrix metalloproteinase is involved in migration of human monocytes and is regulated through their interaction with fibronectin or endothelium

Membrane type 1-matrix metalloproteinase (MT1-MMP) is involved in endothelial and tumor-cell migration, but its putative role in leukocyte migration has not been characterized yet. Here, we demonstrate that anti-MT1-MMP monoclonal antibody (mAb) impaired monocyte chemotactic protein-1 (MCP-1)-stimulated monocyte migration on fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). In addition, monocyte transmigration through tumor necrosis factor-alpha (TNF-alpha)-activated endothelium is also inhibited by anti-MT1-MMP mAb. Therefore, regulation of MT1-MMP in human peripheral blood monocytes was investigated. First, MT1-MMP clustering was observed at motility-associated membrane protrusions of MCP-1-stimulated monocytes migrating on FN, VCAM-1, or ICAM-1 and at the leading edge, together with profilin, of monocytes transmigrating through activated endothelial cells. In addition, up-regulation of MT1-MMP expression was induced in human monocytes upon attachment to FN in a manner dependent on alpha4beta1 and alpha5beta1 integrins. Binding of monocytes to TNF-alpha-activated human endothelial cells as well as to VCAM-1 or ICAM-1 also resulted in an increase of MT1-MMP expression. These findings correlated with an enhancement of MT1-MMP fibrinolytic activity in monocytes bound to FN, VCAM-1, or ICAM-1. Our data show that MT1-MMP is required during human monocyte migration and endothelial transmigration and that MT1-MMP localization, expression, and activity are regulated in monocytes upon contact with FN or endothelial ligands, pointing to a key role of MT1-MMP in monocyte recruitment during inflammation.     

Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture

Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34(+) progenitor cells were synchronized and assayed for adhesion and migration onto fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at different stages of a first cell cycle executed ex vivo. During S phase transit, adhesion to Fn was transiently increased while binding to VCAM-1 was reversibly decreased, after which adhesion to both ligands returned to baseline levels with cell cycle completion. Transmigration across Fn and VCAM-1 decreased irreversibly during S phase progression. The function of alpha4 and alpha5 integrins was assessed with specific neutralizing antibodies. In uncultured CD34(+) cells and long-term culture-initiating cells (LTC-ICs), both adhesion and migration on Fn were inhibited by anti-alpha4 but not by anti-alpha5 antibodies. In mitotically activated CD34(+) cells and LTC-ICs, adhesion and migration on Fn were mainly dependent on alpha5 integrin and to a lesser extent on alpha4 integrin. Changes in integrin function were not dependent on parallel modulation of integrin expression. In conclusion, Fn and VCAM-1 binding of progenitor cells fluctuates reversibly during cell cycle transit ex vivo. In addition, our data show that mitogenic activation induces a shift from a dominant alpha4 to a preferential alpha5 integrin-dependent interaction with Fn.     

Soluble VCAM-1 binding to alpha4 integrins is cell-type specific and activation dependent and is disrupted during apoptosis in T cells

Soluble vascular cell adhesion molecule-1 (sVCAM-1) is generated during inflammation and can alter lymphocyte functions. The authors report that the binding of sVCAM-1 to alpha4 integrin-bearing cells is a dynamically regulated, active cellular process. Binding of recombinant sVCAM-1 to alpha4 integrins on peripheral blood mononuclear cells was cell-type specific. Circulating CD16+ NK cells constitutively bound sVCAM-1 with high affinity, whereas a subpopulation of T-lymphocytes, primarily CD45RO+ (memory), bound sVCAM-1 only after phorbol ester stimulation. sVCAM-1 binding to homogenous stable cell lines was also cell-type specific, and required active cellular processes because it was blocked by the inhibition of ATP synthesis and by Fas-induced apoptosis. Indeed, the loss of high-affinity VCAM-1 binding was an early event in apoptosis. Furthermore, an H-Ras/Raf-initiated signaling pathway also suppressed sVCAM-1 binding to alpha4beta1 integrins. Collectively, these results showed that the capacity of alpha4 integrins to bind VCAM-1 is actively regulated and that this regulation may control alpha4 integrin-dependent cellular functions. (Blood. 2000;95:602-609)     

Infection of endothelium with E1(-)E4(+), but not E1(-)E4(-), adenovirus gene transfer vectors enhances leukocyte adhesion and migration by modulation of ICAM-1, VCAM-1, CD34, and chemokine expression

Intravascular introduction of replication-deficient adenoviral vectors (Advectors) provides an ideal model of delivery of transgenes for the treatment of various vascular abnormalities. On the basis of the knowledge that Advectors can induce inflammatory responses after intravascular administration, we speculated that cellular activation by Advector infection could directly modulate the endothelial cell (EC) adhesion molecule/chemokine expression repertoire. Infection of human umbilical vein ECs or bone marrow microvascular ECs with an E1(-)E4(+) Advector resulted in the upregulation of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD34, but not E-selectin, P-selectin, CD36, CD13, CD44, HLA-DR or PECAM. Upregulation of ICAM-1, VCAM-1, and CD34 was apparent 12 hours after infection and persisted for weeks after infection. Selective induction of adhesion molecules was mediated by the presence of the E4 gene in the Advector, because infection of ECs with an E1(-)E4(-) Advector had no effect on adhesion molecule expression. ECs infected with E1(-)E4(+) Advector, but not those infected with E1(-)E4(-) Advector, supported the adhesion of leukocytes. Monoclonal antibodies to ICAM-1 and VCAM-1 inhibited adhesion of leukocytes to E1(-)E4(+)-infected ECS: Infection of the ECs with E1(-)E4(+) Advector, but not E1(-)E4(-) Advector, resulted in downregulation of expression of chemocytokines, including interleukin-8, MCP-1, RANTES, and GM-CSF. Nonetheless, a large number of leukocytes migrated through ECs infected with E1(-)E4(+), but not those infected with E1(-)E4(l-), in response to exogenous chemokines. These results demonstrate that infection of ECs with E1(-)E4(+) Advectors, but not E1(-)E4(-) Advectors, may directly augment inflammatory responses by upregulating expression of adhesion molecules and enhancing migration through Advector-infected ECs and suggest that E1(-)E4(-) Advectors may be a better choice for gene-transfer strategies directed to the ECS:     

Lymphocyte function-associated antigen-1 binding residues in intercellular adhesion molecule-2 (ICAM-2) and the integrin binding surface in the ICAM subfamily

The crystal structure of intercellular adhesion molecule-2 (ICAM-2) revealed significant differences in the presentation of the critical acidic residue important for integrin binding between I and non-I-domain integrin ligands. Based on this crystal structure, we mutagenized ICAM-2 to localize the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1). The integrin binding site runs diagonally across the GFC beta-sheet and includes residues on the CD edge of the beta-sandwich. The site is oblong and runs along a flat ridge on the upper half of domain 1, which is proposed to dock to a groove in the I domain of LFA-1, with the critical Glu-37 residue ligating the Mg2+ in the I domain. Previous mutagenesis of ICAM-1 and ICAM-3, interpreted in light of the recently determined ICAM-1 and ICAM-2 structures, suggests similar binding sites. By contrast, major differences are seen with vascular cell adhesion molecule-1 (VCAM-1), which binds alpha4 integrins that lack an I domain. The binding site on VCAM-1 includes the lower portion of domain 1 and the upper part of domain 2, whereas the LFA-1 binding site on ICAM is confined to the upper part of domain 1.     

黏附分子及CD40配体与急性心肌梗死的关系

目的观察青年急性心肌梗死患者淋巴细胞的血管间黏附分子、淋巴细胞黏附分子(VCAM-1及ICAM-1)及CD40配体(CD40L)的表达,探讨其急性心肌梗死炎症免疫学发病机制。方法将年龄≤45岁,与年龄>45岁急性心肌梗死患者上述指标比较,体检正常者作为对照。用流式细胞仪检测淋巴细胞VCAM-1、ICAM-1及CD40L阳性表达率。结果≥45岁组VCAM-1、ICAM-1及CD40L明显高于正常对照组(P<0.05),但与老年心肌梗死组对比差异无显著意义(P>0.05)。≤45岁心肌梗死组中VCAM-1、ICAM-1与CD40L显著相关。结论VCAM-1、ICAM-1及CD40L的高表达是急性心肌梗死发生和发展的重要炎症免疫学机制之一。     

Role of vascular cell adhesion molecule-1 and fibronectin connecting segment-1 in monocyte rolling and adhesion on early atherosclerotic lesions

Atherosclerotic lesion development seems to be inflammatory in nature and involves the recruitment of monocytes to the vessel wall. In this study, we investigated the role of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) connecting segment-1 containing the amino acid sequence ILDV as functional ligands for alpha(4)beta(1) integrin (VLA-4) in monocyte rolling and adherence to early atherosclerotic lesions. Carotid arteries of apolipoprotein E-deficient mice were isolated and perfused with monocytes or U937 cells. Cell adhesion was reduced 95+/-4% by monoclonal antibodies HP1/2 and HP2/1, which block VLA-4 binding to both VCAM-1 and FN connecting segment-1. mAb HP1/3 preferentially blocked interaction of VLA-4 with FN but not VCAM-1 and decreased adhesion by 30+/-8%. In contrast, blocking VCAM-1 by perfusing the isolated carotid artery with mAb MK-2.7 reduced adhesion by 75+/-12%. Mononuclear cell adhesion to the early atherosclerotic endothelium was inhibited by 68+/-10% in the presence of EILDVPST but not in the presence of control peptide EIDVLPST. When VLA-4 or VCAM-1 was blocked, more mononuclear cells rolled on early lesions at significantly higher (approximately doubled) rolling velocities. These data demonstrate that (1) blockade of VCAM-1 can abrogate the majority (75+/-12%) of VLA-4-dependent monocyte adhesion on early atherosclerotic endothelia and (2) ILDV peptide interferes with VLA-4 binding to both VCAM-1 and FN and may be useful in limiting monocyte adhesion to atherosclerotic lesions.     

Thrombogenic effects of antiphospholipid antibodies are mediated by intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and P-selectin

Recent studies have shown that antiphospholipid (aPL) enhances expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on endothelial cells (ECs) and that these effects are correlated with increased adhesion of leukocytes to endothelium in cremaster muscle in vivo and with thrombosis in a mouse model. Activation of ECs by aPL may create a hypercoagulable state that precedes and contributes to thrombosis in patients with aPL syndrome (APS). This study proposed to examine whether this in vivo activation of ECs and enhanced thrombosis by aPL are mediated by ICAM-1, P-selectin, or VCAM-1. The dynamics of thrombus formation and the number of adhering leukocytes were studied in ICAM-1-deficient (ICAM-1(-/-)) mice or ICAM-1-/P-selectin-deficient (ICAM-1(-/-)/P-selectin(-/-)) mice treated with affinity-purified aPL antibodies (ap IgG-APS) or with control IgG and compared with wild-type mice treated in a similar fashion. In another set of experiments, the adhesion of leukocytes to cremaster muscle and the dynamics of thrombus formation were studied in CD1 mice treated with aPL or control IgG before and 30 minutes after intravenous infusion with 100 microg monoclonal antibody anti-VCAM-1. The results indicate that the enhanced adhesion of leukocytes to endothelium in wild-type mice was significantly reduced in ICAM-1(-/-) and completely abrogated in ICAM-1(-/-)/P-selectin(-/-) mice treated with ap IgG-APS compared with wild-type mice treated with ap IgG-APS (6.9+/-2.3, 0.4+/-0.4 versus 35+/-12, respectively). More importantly, this correlated with a significant reduction in thrombus size compared with wild-type mice treated with ap IgG-APS (895+/-259 microm(2), 859+/-243 microm(2) versus 3816+/-672 microm(2), respectively). Infusion of the mice with anti-VCAM-1 antibodies significantly reversed the enhanced adhesion of leukocytes (14.9+/-3 to 11.3+/-2.1) and thrombus size 3830+/-1008 microm(2) versus 876+/-548 microm(2)) in mice treated with ap IgG-APS. The data indicate that ICAM-1, P-selectin, and VCAM-1 expression are important in thrombotic complications by aPL antibodies and may provide novel targets for therapy in patients with APS.     

Regulation of ICAM-1-mediated fibroblast-T cell reciprocal interaction: implications for modulation of gut inflammation.

BACKGROUND & AIMS: Immune-nonimmune cell interactions modulate mucosal immunity. We investigated the expression of adhesion molecules by intestinal fibroblasts, the effect of immune cell-derived factor on fibroblast binding of T cells, and the consequences of interfering with adhesion molecule expression on fibroblast-T cell interaction. METHODS: Expression of fibroblast intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 surface and messenger RNA (mRNA) was measured before and after exposure to immune cell-derived supernatants. Fibroblasts were treated with antibodies to ICAM-1 or VCAM-1, or ICAM-1 antisense oligonucleotide Isis 2302, before a T-cell adhesion assay. RESULTS: Fibroblast activation by immune cell-derived cytokines enhanced ICAM-1 and VCAM-1 surface expression and mRNA as well as adhesiveness for T cells. Blockade with neutralizing antibodies showed that binding was almost exclusively dependent on ICAM-1. Isis 2302 specifically reduced fibroblast ICAM-1 mRNA and dose-dependently inhibited ICAM-1 surface expression and T-cell binding. CONCLUSIONS: ICAM-1 is essential for intestinal fibroblast binding of T cells, a phenomenon that is efficiently and specifically disrupted by ICAM-1 antisense oligonucleotides. These observations emphasize the crucial regulatory role of fibroblasts in mucosal immunity and their potential as targets for therapeutic intervention in intestinal inflammation.     

Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium

Myocardial damage due to reperfusion of ischemic tissue is caused primarily by infiltrating neutrophils. Although leukocyte beta2 integrins (CD18) play a critical role, significant neutrophil emigration persists when CD18 is neutralized or absent. This study examined the role of leukocyte beta1 integrin (alpha4) and its endothelial ligand VCAM-1 in CD18-independent neutrophil migration across cardiac endothelium. In a mouse model of myocardial ischemia and reperfusion, we show that compared with wild-type mice, neutrophil infiltration efficiency was reduced by 50% in CD18-null mice; in both types of mice, myocardial VCAM-1 staining increased after reperfusion. In wild-type mice, antibodies against CD18, ICAM-1 (an endothelial ligand for CD18), or VCAM-1 given 30 minutes before ischemia did not block neutrophil emigration at 3 hours reperfusion. Although anti-VCAM-1 attenuated neutrophil emigration by 90% in CD18-null mice, it did not diminish myocardial injury. To determine if CD18-independent neutrophil emigration was a tissue-specific response, we used isolated peripheral blood neutrophils from wild-type or CD18-null mice and showed neutrophil migration across lipopolysaccharide-activated cultured cardiac endothelium is CD18-independent, whereas migration across endothelium obtained from inferior vena cava is CD18-dependent. Consistent with our in vivo findings, migration of CD18-deficient neutrophils on cardiac endothelial monolayers is blocked by antibodies against alpha4 integrin or VCAM-1. We conclude tissue-specific differences in endothelial cells account, at least partially, for CD18-independent neutrophil infiltration in the heart.