Specific inhibition of T-cell adhesion to extracellular matrix and proinflammatory cytokine secretion by human recombinant galectin-1

The migration of immune cells through the extracellular matrix (ECM) towards inflammatory sites is co-ordinated by receptors recognizing ECM glycoproteins, chemokines and proinflammatory cytokines. In this context, galectins are secreted to the extracellular milieu, where they recognize poly-N-acetyllactosamine chains on major ECM glycoproteins, such as fibronectin and laminin. We investigated the possibility that galectin-1 could modulate the adhesion of human T cells to ECM and ECM components. T cells were purified from human blood, activated with interleukin-2 (IL-2), labelled, and incubated further with intact immobilized ECM and ECM glycoproteins in the presence of increasing concentrations of human recombinant galectin-1, or its more stable, related, C2-S molecule obtained by site-directed mutagenesis. The presence of galectin-1 was shown to inhibit T-cell adhesion to intact ECM, laminin and fibronectin, and to a lesser extent to collagen type IV, in a dose-dependent manner. This effect was specifically blocked by anti-galectin-1 antibody and was dependent on the lectin's carbohydrate-binding properties. The inhibition of T-cell adhesion by galectin-1 correlates with the ability of this molecule to block the re-organization of the activated cell's actin cytoskeleton. Furthermore, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production was markedly reduced when IL-2-activated T cells were incubated with galectin-1 or its mutant. This effect was prevented by beta-galactoside-related sugars. The present study reveals an alternative inhibitory mechanism for explaining the suppressive properties of the galectin-1 subfamily on inflammatory and autoimmune processes.     

Fibronectin peptides mediate HMEC adhesion to porcine-derived extracellular matrix

Extracellular matrices (ECM) derived from porcine tissues have been shown to support the successful repair and remodeling of injured tissues when evaluated in animal models. Cell-matrix interactions, including ligand-integrin associations that facilitate endothelial cell adhesion, are clearly important in the tissue remodeling process. The goal of the present study was to identify the peptide sequences within the ubiquitous protein fibronectin (FN) that may be important in the initial interactions between the host endothelial cells and the ECM scaffold. Human microvascular endothelial cells (HMEC) were seeded upon porcine ECM after having been subjected to pretreatment with peptide ligands derived from tissue FN and were allowed to attach for 20 min. Non-adherent cells were removed and the remaining, tritium-labeled cells attached to the ECM were counted. Results showed that cyclo-RGD and REDV, but not LDV or PHSRN, play a role in mediating the attachment of HMEC to porcine ECM.     

Induction of T cell adhesion to extracellular matrix or endothelial cell ligands by soluble or matrix-bound interleukin-7

The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human IL-7 was found to bind ECM or fibronectin (FN) with IC₅₀ values of 10–100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4⁺ and CD8⁺ T cells in dose-dependent and β1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific β1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state.     

Bacterial binding to extracellular matrix proteins -- in vitro adhesion.

A binding assay was developed and used to study the binding of oral streptococcus to immobilized human fibronectin, laminin, vitronectin, fibrinogen, heparin, and collagen IV. The protein binding was dependent on the broth used for bacterial growth. The binding after growth in brain heart infusion broth, trypticase soy broth, Todd-Hewitt broth, and Dulbecco's modified Eagle's medium was examined. Most of the strains were able to bind to immobilized fibronectin and laminin, and to a minor extent vitronectin. Binding was not observed on immobilized fibrinogen, collagen IV, or heparin. Measured surface hydrophobicity correlated well with the bacterial binding strength to the proteins. Streptococcal incubation with putative inhibitors indicates multiple binding mechanisms of a lectin-like and protein nature, possibly involving protein receptors.     

T lymphocyte expression of thrombospondin-1 and adhesion to extracellular matrix components

The mechanisms controlling the formation of pseudopodia and other active cell edges in T lymphocytes are not understood. We show here that T lymphocytes express thrombospondin-1 (TSP-1). TSP-1 in T lymphocytes has a high turnover as shown by the fact that brefeldin and monensin rapidly increase while cycloheximide tend to decrease the cellular TSP-1 content. T cell TSP-1 is preferentially stored intracellularly and shows variable cell surface expression. T lymphocyte adhesion to fibronectin and collagen type IV induces TSP-1 expression on the cell surface via a brefeldin sensitive mechanism. A monoclonal antibody to TSP-1 inhibits the flattening and pseudopodia formation of the adherent T cells. Furthermore, the same antibody to TSP-1 also exerts an inhibitory effect on T cell migration in the absence of exogenous TSP-1. These results indicate that endogenous TSP-1 is part of an adhesion-dependent mechanism controlling cytoplasmic spreading and migration in T lymphocytes.     

Chondroitin sulfate B and heparin mediate adhesion of Penicillium marneffei conidia to host extracellular matrices

Penicilliosis is a disseminated infection in immunocompromised individuals caused by the dimorphic fungus, Penicillium marneffei. Very little is known about its route of infection, however, it is thought that initial infection occurs through inhalation of conidia. We investigated the role played by various extracellular matrix glycosaminoglycans (GAGs) in the initial adherence of P. marneffei conidia using a direct adhesion assay. GAGs were further used to block the binding of fungal spores to human lung epithelial cells and highly sulfated GAGs were tested for their inhibitory effects owing to their degree of sulfation. Our results demonstrated high levels of conidial adhesion to chondroitin sulfate B, heparin and highly sulfated chitosan (CP-3). No direct adherence was observed to immobilized chondroitin sulfate (CS) A, CSC, CSD and hyaluronic acid, as well as chitosans with low sulfate content. The results suggested that P. marneffei conidia bind to iduronic acid (IdoA) of the polysaccharide chains. Involvement of negatively charged sulfate groups in adhesion was also indicated. Furthermore, significant inhibition of conidial adherence to A549 cells was observed in the presence of CSB, heparan sulfate (HS), heparin and CP-3. It was further demonstrated that GAGs can affect the adhesion of conidia to fibronectin and laminin, glycoproteins that have previously been implicated as adhesive receptors for fungal conidia. CSB and HS could partially inhibit the adhesion of fungal conidia to laminin and fibronectin implying that conidia can weakly interact with the IdoA GAG-binding domain(s) of these molecules. The data indicated that, in addition to fibronectin and laminin, IdoA-containing GAGs may play an important role in fungal adherence to the surface of human lung epithelium.     

Engineered matrix coatings to modulate the adhesion of CD133+ human hematopoietic progenitor cells

Interactions of hematopoietic progenitor cells (HPC) with their local microenvironments in the bone marrow are thought to control homing, differentiation, and self-renewal of the cells. To dissect the role of extracellular matrix (ECM) components of the niche microenvironment, a set of well-defined ECM coatings including fibronectin, heparin, heparan sulphate, hyaluronic acid, tropocollagen I, and co-fibrils of collagen I with heparin or hyaluronic acid was prepared and analysed with respect to the attachment of human CD133+ HPC in vitro. The extension of the adhesion areas of individual cells as well as the fraction of adherent cells were assessed by reflection interference contrast microscopy (RICM). Intense cell-matrix interactions were found on surfaces coated with fibronectin, heparin, heparan sulphate, and on the collagen I based co-fibrils. Insignificant adhesion was found for tropocollagen I and hyaluronic acid. The strongest adhesion of HPC was observed on fibronectin with contact areas of about 7 microm(2). Interaction of HPC with coatings consisting of heparin, heparan sulphate, and co-fibrils result in small circular shaped contact zones of 3 microm(2) pointing to another, less efficient, adhesion mechanism. Analysing the specificity of cell-matrix interaction by antibody blocking experiments suggests an integrin(alpha(5)beta(1))-specific adhesion on fibronectin, while adhesion on heparin was shown to be mediated by selectins (CD62L). Taken together, our data provide a basis for the design of advanced culture carriers supporting site-specific proliferation or differentiation of HPC.     

Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins

This study examines the spreading and adhesion of human umbilical vein endothelial cells (HUVEC) on artificial extracellular matrix (aECM) proteins containing sequences derived from elastin and fibronectin. Three aECM variants were studied: aECM 1 contains lysine residues periodically spaced within the protein sequence and three repeats of the CS5 domain of fibronectin, aECM 2 contains periodically spaced lysines and three repeats of a scrambled CS5 sequence, and aECM 3 contains lysines at the protein termini and five CS5 repeats. Comparative cell binding and peptide inhibition assays confirm that the tetrapeptide sequence REDV is responsible for HUVEC adhesion to aECM proteins that contain the CS5 domain. Furthermore, more than 60% of adherent HUVEC were retained on aECM 1 after exposure to physiologically relevant shear stresses (相似文献   

Effects of nitric oxide on the adhesion of human melanocytes to extracellular matrix components

The aim of the present study was to explore whether nitric oxide (NO) interferes with the attachment of human melanocytes to the extracellular matrix (ECM) components. Consequently, the effects have been investigated of the NO-releasing compounds 3-morpholino-sydnonimine (SIN-1) and S-nitroso-glutathione (GSNO) on the in vitro adhesion of human melanocytic cells to fibronectin. The NO donors induced a concentration-dependent reduction in the adhesion of both ⁵¹CrO₄²⁻-labelled melanocytes and melanoma cells to fibronectin. Pigmented M14 melanoma cells were more susceptible to the effect of SIN-1 (half-maximal inhibiting effect at about 0·5 mm) than normal human melanocytes and also than the non-pigmented melanoma cells Mel57 (half-maximal inhibiting effects between 0·9 and 2 mm). This effect of SIN-1 also appeared to be related to the melanin content of normal melanocytes, whereas GSNO was significantly less active. Both flow cytometric analysis and immunocytochemical staining showed expression of neuronal NO synthase in all cell lines. The results of this study suggest that aberrant in vivo production of NO during infection and inflammation may contribute to loss of melanocytes in, for example, vitiligo, by reducing de novo attachment of melanocytes to the ECM. These findings could also be important for understanding the process of metastasis. © 1997 John Wiley & Sons, Ltd.     

The influence of extracellular matrix proteins on T-cell proliferation and apoptosis in women with endometriosis or uterine leiomyoma

PROBLEM: Interactions between the extracellular matrix (ECM) and peripheral blood T cells in women with endometriosis and leiomyoma are hardly unknown. We have investigated the influence of two major ECM components, collagen IV (C-IV) and fibronectin (Fn), on T-cell proliferation and apoptosis in women with endometriosis and uterine leiomyoma. beta1 integrin expression, responsible for interactions with ECM proteins, was also studied. METHOD OF STUDY: Peripheral blood lymphocytes were obtained from 53 women (17 with uterine leiomyomas, 18 with endometriosis, and 18 from healthy donors). T cells were exposed to ECM proteins co-immobilized with monoclonal antibody anti-CD3 for 72 hr. Apoptosis and S phase of the cell cycle of the T cells were studied by DNA analysis using flow cytometry. The proliferation of T cells was evaluated by MTT assay. The percentage of CD3+ cells expressing CD29 (beta1 integrin chain) was evaluated by double-color flow cytometry. Results were analyzed statistically using the Mann-Whitney test. RESULTS AND CONCLUSIONS: (1) A general increase in the percentage of T cells in S phase could be seen in women with endometriosis and uterine leiomyoma in all culture conditions what may suggest general activation of T cells. (2) A significant increase in the percentage of cells in S phase was shown only in the case of T cells exposed to anti-CD3 + C-IV in both women with uterine leiomyoma and endometriosis. (3) However, no apoptotic cells were observed. (4) T cells from patients with uterine leiomyoma exhibited significantly increased level of proliferation after culture with anti-CD3 + C-IV. (5) More T cells expressed beta1 integrin in women with endometriosis or uterine leiomyoma than in healthy donors. Our data may suggest that increased beta1 integrin expression may enhance T-cell-ECM interactions, which may be responsible for the increased proliferation of T cells but not for apoptosis. Therefore, it is possible that interactions of T cells with ECM proteins, especially with C-IV, may contribute to the pathogenesis of endometriosis and uterine leiomyoma.     

Tenascin-C modulates adhesion of cardiomyocytes to extracellular matrix during tissue remodeling after myocardial infarction.

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays important roles in tissue remodeling. TNC is not normally expressed in adults but reappears under pathologic conditions. The present study was designed to clarify the contribution of TNC to ventricular remodeling after myocardial infarction. We examined the expression of TNC after experimental myocardial infarction in the rat by immunohistochemistry and in situ hybridization. Within 24 hours of permanent coronary ligation, interstitial fibroblasts in the border zone started to express TNC mRNA. The expression of TNC was down-regulated on Day 7 and was no longer apparent by Day 14 after infarction. During the healing process, TNC protein and TNC-producing cells were found at the edges of the residual myocardium. Some of the TNC-producing cells were immunoreactive for alpha-smooth muscle actin. In culture, TNC increased the number of cardiomyocytes attached to laminin but inhibited the formation of focal contacts at costameres. The results indicate that during the acute phase after myocardial infarction, interstitial cells in the border zone synthesize TNC, which may loosen the strong adhesion of surviving cardiomyocytes to connective tissue and thereby facilitate tissue reorganization.     

IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components

Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, beta(1) integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-gamma (INF-gamma) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-gamma production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites.     

Signaling by epidermal growth factor differentially affects integrin-mediated adhesion of tumor cells to extracellular matrix proteins

 The adhesion of different epidermal growth factor (EGF) receptor (EGFR) expressing cell lines to various extracellular matrix (ECM) proteins is influenced by EGF. To investigate a putative receptor cross-talk between EGFR and integrins we chose two cell lines for a more detailed analysis: the highly metastatic rat mammary carcinoma clone MTLn3 that showed increased adhesion to a panel of ECM proteins in the presence of 10 ng/ml EGF and the nonmetastatic human vulva carcinoma cell line A431 which showed a decreased adhesion under the same conditions. These EGF-mediated stimulatory or inhibitory effects on adhesion were observed within a few minutes. On human A431 cells the inhibitory effect was blocked by an EGFR-specific antibody that interferes with ligand binding. In cell adhesion assays performed in the presence of divalent cations MTLn3 and A431 cells exhibited the typical behavior described for integrin-dependent matrix adhesion: Mn²⁺ enhanced binding to collagen IV and fibronectin whereas Ca²⁺ inhibited adhesion to collagen IV but not to fibronectin. Adhesion-inhibition assays with anti-human integrin antibodies revealed that A431 cells adhere to collagen via α₁β₁ and α₂β₁, and that adhesion to fibronectin is mediated predominantly through α₅β₁. The interaction of MTLn3 cells with fibronectin was in part RGD dependent, indicating the involvement of either α₃β₁ or α₅β₁. Addition of EGF in these assays showed that affecting the integrin extracellular domains by addition of either bivalent cations, RGD peptides, or function-blocking integrin antibodies did not prevent the effects mediated by EGF. We conclude that signals downstream of EGFR can modulate integrin-mediated adhesion to ECM proteins in both an inhibitory and a stimulatory manner. Received: 8 May 1996 / Accepted: 1 July 1996     

Expression of adhesion molecules and extracellular matrix proteins in glioblastomas: relation to angiogenesis and spread

We studied the immunohistochemical expression of inducible adhesion molecules, integrins and extracellular matrix proteins in 10 cases of glioblastoma multiforme in order to investigate their angiogenesis, local invasiveness, poor metastasizing properties and their lack of tumour infiltrating leukocytes. In glioblastomas endothelial proliferations represent the majority of vascular structures; they were positive for endothelial markers (vWF, CD31, VE-cadherin) and negative for macrophage markers (CD68, PAM-1). Immunohistologically, they were subtyped into: 1 solid-glomeruloid ICAM-1, α₂β₁, α₃β₁, α₅β₁ negative; 2 channelled-branching ICAM-1 negative and α₂β₁, α₃β₁, α₅β₁ positive; 3 channelled-telangiectatic ICAM-1, α₂β₁, α₃β₁, α₅β₁ positive. In channelled proliferations, the expression and distribution of tenascin and merosin in the basal membrane was similar to that of normal brain vessels. The expression of all these molecules might indicate different steps of maturation of endothelial proliferations. The majority of endothelial proliferations may be immunohistologically considered as incomplete vascular structures; this might account for the low metastasizing tendency and low recruitment of leukocytes by these tumours. Neoplastic astrocytes were GFAP-1, ICAM-1, VCAM-1, α₂β₁, α₃β₁ and α₅β₁ immunoreactive and α₆β₄ negative; this allows them to interact with extracellular matrix proteins and might, in part, explain the tendency of glioblastomas to infiltrate locally.     

Expression and role of integrins in adhesion of human colonic carcinoma cells to extracellular matrix components

We have examined integrin expression and function in the human colon carcinoma cell line HT29, and in clonal sublines derived from the HT29 line. These cells express several different integrin subunits including beta 1, alpha 2,3, 6 and alpha_v, but do not express the classic alpha S/beta 1 fibronectin receptor. Clonal variation in the pattern of integrin expression was quite limited. The profile of integrin expression correlates well with the adhesive behavior of HT29 cells. Thus the cells adhere well to vitronectin, laminin and type IV collagen, but not at all to fibronectin. Adhesion to collagen was completely blocked by an antibeta 1 monoclonal antibody, indicating that beta 1 integrins mediate this process. Adhesion to laminin was strongly blocked by anti-beta 1 monoclonal or anti-beta 6 monoclonal, suggesting that the alpha 6/beta 1 complex functions in attachment to laminin; this was somewhat surprising since immunoprecipitation experiments indicate that most of the alpha 6 subunit seems to be associated with the beta 4 subunit. Despite their strong adherence to laminin, collagen and vitronectin, HT29 cells are not very motile and, in response to gradients of these proteins, do not migrate nearly as well as CHO cells tested under similar conditions. Since HT29 cells can undergo an enterocyte-like differentiation in glucose-free medium, we compared integrin expression in HT29 and its subclones during the process of differentiation. There was no correlation between the state of differentiation, as assessed by expression of brush-border hydrolases, and the level of expression of any of the integrin subunits measured. Thus the pattern of integrin expression in these colonic tumor cells seems to be a characteristic of the cell line, and is not readily modified by changes in cell growth or differentiation.     

siRNA干扰CD73表达抑制人乳腺癌细胞MB-MDA-231与细胞外基质的黏附

 目的： 探讨CD73对乳腺癌细胞MB-MDA-231与细胞外基质黏附的影响及其机制。方法： ① 构建CD73 siRNA表达载体，脂质体介导转染MB-MDA-231细胞。② 流式细胞仪检测瞬时转染效率，G418筛选稳定转染克隆。③ 半定量RT-PCR和Western blotting检测转染细胞 CD73 mRNA及蛋白表达。④ 细胞黏附实验检测MB-MDA-231细胞CD73基因表达抑制后与细胞外基质黏附力的改变。结果： ① CD73 siRNA表达载体转染MB-MDA-231可有效抑制CD73 mRNA表达(抑制效率=91.0%), P<0.01。② CD73 siRNA表达载体转染 MB-MDA-231可有效抑制CD73蛋白表达(抑制效率=79.3%), P<0.01。③ 干扰MB-MDA-231细胞CD73表达可显著抑制细胞与细胞外基质的黏附（P<0.01）。结论： ① CD73siRNA表达载体可有效干扰MB-MDA-231细胞CD73基因表达。② MB-MDA-231细胞CD73基因表达抑制可显著降低细胞与细胞外基质的黏附。