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 beta 1 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 beta 1 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.     

Serum levels of endothelial and neural cell adhesion molecules in prostate cancer

The purpose of this study was to describe the longitudinal development of running economy [defined as the oxygen uptake (VO2) at a submaximal running speed] in males and females from teenage to young adult age using data from the Amsterdam Growth and Health Study. Submaximal VO2 (in ml.kg-1.min-1) was measured in 84 males and 98 females while they ran on a treadmill at a constant speed of 8 km.h-1 for 6 min at three different treadmill slopes (0%, 2.5% and 5%). This test was carried out six times, on the same subjects at the ages of 13, 14, 15, 16, 21, and 27 years. The longitudinal development of running economy in males and females was analysed using a two-way analysis of variance for repeated measurements. At all three slopes, a significant decrease in VO2 with increasing age was found for both males and females, implying a significant increase in running economy for both sexes. Males showed significantly higher VO2 values than females at all ages measured and for all three slopes, suggesting that females have a significantly higher running economy than males. In order to make a better comparison of the VO2 of individuals of different sizes, allometric models were used; power function ratios were constructed in which body mass was expressed to an exponential power. Following this analysis the difference in submaximal VO2 and running economy between males and females appeared even larger.     

Direct cell/cell contact with stimulated T lymphocytes induces the expression of cell adhesion molecules and cytokines by human brain microvascular endothelial cells

Upon inflammation, stimulated, but not resting T lymphocytes cross the blood-brain barrier and migrate into the central nervous system. This study shows that direct contact between stimulated T lymphocytes and human brain microvascular endothelial cells (HB-MVEC) induces phenotypic and functional changes on the latter cells. Plasma membranes isolated from stimulated T lymphocytes (S-PM) up-regulated the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on isolated HB-MVEC. In addition, HB-MVEC activated by S-PM secreted interleukin (IL)-6 and IL-8. The levels of ICAM-1, E-selectin, IL-6, and IL-8 expressed in S-PM-activated HB-MVEC were similar to those observed with 1000 U/ml tumor necrosis factor (TNF). In contrast, VCAM-1 expression was 15% of that induced by TNF. Inhibitors of TNF diminished (< or = 45%), but did not abolish the expression of cell adhesion molecules and IL-6 induced by S-PM, IL-8 production being insignificantly affected (< or = 10%). This suggests that membrane-associated TNF was partially involved in HB-MVEC activation. The present study demonstrates that stimulated T lymphocytes are able to activate HB-MVEC upon direct cell contact. This novel mechanism of inducing the expression of cell adhesion molecules may prompt the initial adhesion of stimulated T lymphocytes to brain endothelium.     

Expression of adhesion molecules by lp(a): a potential novel mechanism for its atherogenicity

Lp(a) is a major inherited risk factor for premature atherosclerosis. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to interfere with plasminogen activation and its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that Lp(a) stimulates production of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin in cultured human coronary artery endothelial cells (HCAEC). This effect resulted from a rise in intracellular free calcium induced by Lp(a) and could be inhibited by the intracellular calcium chelator, BAPTA/AM. The involvement of the LDL and VLDL receptors in Lp(a) activation of HCAEC were ruled out since Lp(a) induction of adhesion molecules was not prevented by an antibody (IgGC7) to the LDL receptor or by receptor-activating protein, an antagonist of ligand binding to the VLDL receptor. Addition of alpha2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease levels of VCAM-1 and E-selectin stimulated by Lp(a), suggesting that neither the low density lipoprotein receptor-related protein nor cell-surface proteoglycans are involved in Lp(a)-induced adhesion molecule production. Neither does the binding site on HCAEC responsible for adhesion molecule production by Lp(a) appear to involve plasminogen receptors, as levels of VCAM-1 and E-selectin were not significantly decreased by the addition of glu-plasminogen, the lysine analog epsilon-aminocaproic acid, or by trans-4-(aminomethyl)-cyclohexanecarboxymethylic acid (tranexamic acid), which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen. In contrast, recombinant apolipoprotein (a) [r-apo(a)] competed with Lp(a) and attenuated the expression of VCAM-1 and E-selectin. In summary, we have identified a calcium-dependent interaction of Lp(a) with HCAEC capable of inducing potent surface expression of VCAM-1 and E-selectin that does not appear to involve any of the known potential Lp(a) binding sites. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell-activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.     

Improving endothelial cell adhesion to vascular graft surfaces: clinical need and strategies

Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of anticoagulation to maintain patency. Preseeding synthetic graft materials such as expanded polytetrafluoroethylene (ePTFE) and polyethylene terephthalate (PET) with endothelial cells (EC) has been examined in various in vitro and in vivo models. Although various studies provide encouraging results, clinical trials for EC seeding on synthetic grafts have not been equally successful. This paper provides a brief review of the various reports on EC seeding in animal and clinical studies. We discuss the inefficiencies associated with the EC seeding process and examine plasma protein treatment of the graft surfaces as a viable option for improving EC attachment, retention and spreading. As an alternative to existing therapies we present data on a heterogeneous ligand treatment of fibronectin (Fn) and avidin-biotin for enhanced human umbilical vein endothelial cell (HUVEC) adhesion to ePTFE graft surfaces. Control consisted of HUVECs seeded on Fn treated ePTFE graft surfaces. Functionality of HUVECs was assessed by measuring prostacyclin production of cells on both homogeneous and heterogeneous ligand treated surfaces. Laminar flow studies with a variable width flow chamber and scanning electron microscopy were used to measure initial cell retention and observe initial cell spreading on ePTFE surfaces, respectively. HUVEC retention on heterogeneous ligand treated graft surface was significantly (p < 0.001) higher compared to homogeneous ligand treated surfaces for shear stress in the range of 10-30 dyn cm(-2). HUVEC showed more cellular spreading on the heterogeneous ligand treated surface after seeding for 1-2 h. In vivo experimentation was performed in immune deficient (nude) rats by replacing a section of both the femoral arteries with 8 mnm long, 1 mm internal diameter denucleated ePTFE grafts treated with homogeneous and heterogeneous ligands respectively. Both grafts were seeded with similar cell density for 15 min prior to implantation. EC attachment and retention was measured by staining EC with hematoxylin and counting the cells before and after flow using light microscopy. The results indicate that a heterogeneous ligand treatment of graft surfaces using avidin-biotin and Fn-integrin attachment mechanisms increase cell seeding efficiency, initial cell retention and cellular spreading.     

Expression of cell adhesion molecules in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth retardation

Platelets and neutrophils are involved in maternal placental vascular damage in pre-eclampsia. Recruitment of these cells is probably mediated by cell adhesion molecules expressed at the uteroplacental bed. It remains controversial as to whether platelets and neutrophils mediate damage to trophoblast or villous vasculature. The purpose of this study was to determine the expression of cell adhesion molecules in placentae from normal pregnancies and pregnancies complicated by pre-eclampsia and intrauterine growth retardation (IUGR). Immunostaining for platelet endothelial cell adhesion molecule (PECAM) and intercellular adhesion molecule-1 (ICAM-1) was localized mainly to the endothelium of stem villi, intermediate villi, terminal villi and decidual vessels. Scattered staining for ICAM-1 was also evident in the stroma and fetal membranes. The endothelium of stem villi, intermediate villi and terminal villi were all negative for vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin. PECAM, ICAM-1 and ICAM-2 mRNA were all detectable in normal placentae using northern blotting analysis whereas mRNA for E-Selectin and VCAM-1 were both undetectable. There were no differences in cell adhesion molecule immunostaining or mRNA expression in placentae from pregnancies complicated by pre-eclampsia and IUGR inconclusion, expression of cell adhesion molecules in placentae from pre-eclampsia and IUGR are consistent with a normal physiological role in vascular function.     

Cytoskeleton and cell adhesion molecules: critical targets of toxic agents

Normal development of the nervous system depends upon complex physical interactions between cells and their local environment. These interactions are mediated by several families of cell adhesion molecules (CAMs). Differential expression and function of CAMs are operative in neural tube formation, neuron migration, in post-migratory differentiation, and maintenance of mature neural structure. CAMs also facilitate contact-dependent cell processes, such as formation of cell junctions. Temporal regulation of these molecules during development may provide "windows of vulnerability" to toxicants. In addition to their extracellular binding activities, some CAMs have membrane-spanning domains by which they communicate directly with the cytoskeleton, permitting extracellular signals to be rapidly translated into cell responses via modifications in cytoskeletal organization. These cytologic changes are particularly critical during migration, neurite formation and synaptogenesis. Toxic perturbation of adhesion molecules can have catastrophic effects on morphogenetic processes both directly and via events which depend upon cytoskeletal rearrangement. Toxicants can also act directly upon the cytoskeleton, resulting secondarily in changes of the membrane distribution and function of CAMs. Toxicant-induced changes in CAMs and cytoskeleton may occur contemporaneously. Interference of cell adhesion-cytoskeleton interactions may be a pivotal molecular event dictating developmental consequences of neurotoxicant exposure.     

Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat

AIMS: To investigate the longevity and reproducibility of choroidal neovascularisation (CNV) induced by krypton laser photocoagulation in the rat. The presence of cell adhesion molecules (CAMs) and vascular endothelial growth factor (VEGF) during the development of CNV was also studied. METHODS: 67 pigmented rats underwent retinal photocoagulation by krypton laser. The eyes were examined by either single or serial fluorescein angiography at 3 days, 1, 2-3, 4-5, 7-8, and 12 weeks post photocoagulation. The expression of CAMs (ICAM-1, E-selectin, and CD44) and VEGF post photocoagulation was studied by immunohistochemistry. RESULTS: CNV related fluorescein leakage appeared in 46.4% of 766 laser spots delivered to the 58 eyes that were tested at 2-3 weeks post treatment. The ratio of hyperfluorescent laser sites did not change significantly at 8 weeks post laser. The number of leaky spots was independent of the total number of lesions delivered to each eye (at 2-3 weeks post laser 10-15 spots/eye: 44% and 25-30 spots/eye: 49%; t = 0.7673; p = 0.3903). Nine eyes were followed by serial angiography between 2 and 12 weeks. The laser spots with fluorescein leakage at 2 weeks (51.5%) remained leaky at 12 weeks (51.5%). Histopathologically, macrophage accumulation peaked at 5 days and CNV was firstly observed at 1 week post photocoagulation. ICAM-1, E-selectin, CD44, and VEGF were maximally induced at 3-5 days post laser photocoagulation, and were localised to RPE, choroidal vascular endothelial, and inflammatory cells. VEGF was also detected in intravascular leucocytes at the sites of laser lesions. CONCLUSIONS: These studies demonstrated that krypton laser photocoagulation can be successfully used to produce lesions similar to those of human CNV. The response induced remained present for an extended period of time (12 weeks), thus offering a potential model to screen candidate CNV inhibitory agents. In addition, it is proposed that the expression of ICAM-1, E-selectin, CD44, and VEGF before new vessel formation might be linked to the initiation of CNV.     

Differential regulation of tissue-specific lymph node high endothelial venule cell adhesion molecules by tumour necrosis factor and transforming growth factor-beta 1

Lymphocytes migrate from blood into lymph nodes (LN) of rats specifically at segments of venules lined by high endothelium (HEV). We have previously shown that pretreatment of LN HEV cells with pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), augments their adhesiveness for thoracic duct lymphocytes (TDL). Here we report that a mouse monoclonal antibody, 3C10, recognized tissue-specific endothelial determinants on rat LN HEV cells and blocked their adhesiveness for TDL and EL-4J cells transfected with rat L-selectin. In contrast, 3C10 antibody did not inhibit lymphocyte attachment to Peyer's patch (PP) frozen sections or cultured PP HEV cells. The antibody immunoprecipitated from LN HEV cells two proteins with apparent molecular weights of 90,000 and 50,000. The expression of 3C10 antigen on LN HEV cells was increased by incubation with TNF-alpha or IFN-gamma. Furthermore, pretreatment of cytokine-stimulated LN HEV cells with 3C10 antibody blocked TDL binding in a dose-dependent manner. In contrast, 3C10 antigen expression on LN HEV cells was significantly decreased following incubation of cells with transforming growth factor-beta 1 (TGF-beta 1). In addition, TGF-beta 1 also abrogated the adhesiveness of LN HEV cells stimulated with TNF-alpha, IFN-gamma or both cytokines. Together, these data suggest that endothelial determinants recognized by the 3C10 antibody are tissue-specific ligands for lymphocyte adhesion and cytokines such as TNF-alpha and TGF-beta differentially regulate their expression and function.     

Growth cone interactions with purified cell and substrate adhesion molecules visualized by interference reflection microscopy

The migration of growth cones on substrates consisting of naturally occurring cell adhesion molecules has been extensively studied in cell culture. However, relatively little is known about how growth cones contact the substrate or how the patterns of contact change as growth cones move forward. We have examined the interactions of chick retinal ganglion cell growth cones with laminin, merosin, N-cadherin, L1 and poly-L-lysine by time-lapse interference reflection microscopy (IRM) using a laser scanning confocal microscope. In images obtained by IRM, areas of a cell that are closely apposed to the substrate appear dark whereas areas that are farther away appear light. Growth cones on Jaminin and merosin were almost uniformly light, indicating that very little of the membrane was in close contact with the substrate. Growth cones on N-cadherin had a mottled appearance with some relatively large dark gray areas. The proximal portions of filopodia often were dark, in contrast to those on laminin and merosin which were light. In addition, growth cones on N-cadherin had numerous dark gray punctate regions of close association with the substrate. Growth cones on L1 had darker regions than growth cones on other substrates and these comprised a larger fraction of their area. There also were differences in the temporal dynamics of growth cone interactions with different substrates and these differences correlated with differences in rates of growth. None of the contacts observed in growth cones were as dark or stable as focal contacts of fibroblasts.     

High prevalence and usual persistence of serum monoclonal immunoglobulins evidenced by sensitive methods in renal transplant recipients

BACKGROUND: The occurrence of serum monoclonal immunoglobulins in kidney transplant recipients is well known but their significance and predictive value for the occurrence of lymphoma are a matter of debate. We therefore conducted a study of monoclonal immunoglobulins by a sensitive method during the long-term follow up of grafted patients. METHODS: Monoclonal immunoglobulins were characterized by high-resolution electrophoresis, conventional immunoelectrophoretic analysis, and a sensitive Western blotting procedure in the serum from 84 renal transplant recipients prior to grafting and subsequently, with a 1-8-year follow-up and excluding the patients who developed posttransplant lymphoma. RESULTS: Low abundance monoclonal immunoglobulins were detectable prior to transplantation in 56 cases (66.6%) and after graft in 72 cases (85.5%) (and in 1 case (1.2%) and 18 cases (21.4%) of cases respectively, by immunoelectrophoresis). These abnormalities were often multiple in individual sera. Monoclonal components detected by immunoblotting were transient in 23.8% of patients only (whereas those evidenced by immunoelectrophoresis usually became undetectable by this method) and their pattern was remarkably stable in the majority of cases. The frequency of post-transplant monoclonal immunoglobulins was higher in patients of more than 50 years of age than in younger patients. The appearance of monoclonal components after grafting and their transient character correlated with CMV infections. No correlation was found with various other parameters. The isotypic distribution of monoclonal immunoglobulins with an IgM, IgG3, and IgG1 predominance and an abnormally low kappa/lambda ratio was the same as that observed in various immunodeficiency states. The monoclonal immunoglobulin pattern in three further patients who developed post-transplant lymphoma was unremarkable. CONCLUSION: Monoclonal immunoglobulins hence are not discriminant for lymphoma and their characterization does not appear to be necessary in the evaluation of followed up grafted patients, at least for a prediction of post-transplant lymphoma.     

Polymorphonuclear leukocytes induce PDGF release from IL-1beta-treated endothelial cells: role of adhesion molecules and serine proteases

Polymorphonuclear leukocytes (PMNs) and endothelial cells interact at sites of vascular injury during inflammatory response and during the development of atherosclerotic lesions. Such close proximity leads to the modulation of several of the biological functions of the 2 cell types. Because we have shown previously that PMNs enhance release of growth factors from resting endothelial cells, we decided to evaluate whether coincubation of PMNs with interleukin-1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) could further modulate mitogen release from HUVEC. We found that PMN-HUVEC coincubation resulted in a 10-fold increase in mitogen release, compared with HUVEC alone (14+/-6 versus 1.3+/-0.1). When PMNs were incubated with IL-1beta-treated HUVEC, a further increase in mitogen release (up to 35-fold) was observed. The mitogenic activity was immunologically related to platelet-derived growth factor (PDGF) because the activity was abolished by an anti-PDGF antibody. PDGF-AB antigen, detected in low concentrations in conditioned medium from HUVEC alone, was increased 4-fold when IL-1beta or PMNs were incubated with HUVEC and dramatically upregulated (up to 40-fold) when PMNs were cocultured with IL-1beta-treated HUVEC. The presence of the protease inhibitor eglin C abolished mitogenic activity generation, suggesting a role for PMN-derived elastase and cathepsin G. Indeed, purified elastase and cathepsin G mimicked PMN-induced mitogen release from HUVEC. Because PMNs firmly adhered to IL-1beta-treated HUVEC, we investigated the role of cell-cell adhesion in mitogen release. Adhesion and PDGF release were inhibited by approximately 60% in the presence of anti-CD11a/CD18 and anti-intercellular adhesion molecule-1 monoclonal antibodies. This study suggests a new role for PMNs and their interaction with endothelium in pathological conditions in which intimal hyperplasia is a common feature.