Cell adhesion and communication最新文献

Ligands for L-selectin are expressed not only on vascular endothelial cells but also in the extravascular tissues. In this article, we summarize the current understanding of the "vascular" ligands for L-selectin. We also describe identification of "non-vascular" ligands for L-selectin and discuss their possible biological significance.

Blood-borne lymphocytes migrate continuously to peripheral lymph nodes (PLN) and other organized lymphoid tissues where they are most likely to encounter their cognate antigen. Lymphocyte homing to PLN is a highly regulated process that occurs exclusively in specialized high endothelial venules (HEV) in the nodal paracortex. Recently, it has become possible to explore this vital aspect of peripheral immune surveillance by intravital microscopy of the subiliac lymph node microcirculation in anesthetized mice. This paper reviews technical and experimental aspects of the new model and summarizes recent advances in our understanding of the molecular mechanisms of lymphocyte homing to PLN which were derived from its use. Both lymphocytes and granulocytes initiate rolling interactions via L-selectin binding to the peripheral node addressin (PNAd) in PLN HEV. Subsequently, a G protein-coupled chemoattractant stimulus activates LFA-1 on rolling lymphocytes, but not on granulocytes. Thus, granulocytes continue to roll through the PLN, whereas LFA-1 activation allows lymphocytes to arrest and emigrate into the extravascular compartment. We have also identified a second homing pathway that allows L-selectin low/(activated/memory) lymphocytes to home to PLN. P-selectin on circulating activated platelets can mediate simultaneous platelet adhesion to PNAd in HEV and to P-selectin glycoprotein ligand (PSGL)-1 on lymphocytes. Through this mechanism, platelets can form a cellular bridge which can effectively substitute for the loss of L-selectin on memory cell subsets.

The principles determining the migration of different cell types may results from their differences in origin, size and shape, function of adhesion receptors, and environmental factors, including the extracellular matrix. Polarized leukocytes (T lymphocytes and dendritic cells) migrating in three-dimensional collagen lattices are small developing a highly dynamic leading edge and a trailing uropod, whereas invasive melanoma cells are larger, highly polarized and less dynamic. In contrast to leukocyte, tumor cells may additionally develop migrating cell clusters maintaining intense cell-cell interaction and cluster polarity. Leukocytes show a speed-oriented, oscillating and directionally unpredictable path profile strongly guided by matrix fibers, while melanoma cells and migrating cell clusters exhibit slow yet highly directional migration. Whereas leukocytes form short-lived interactions with collagen fibers in complete absence of tissue remodeling, melanoma cells and neoplastic cell clusters reorganize the matrix via profound pulling at attachment sites, limited fiber disruption upon detachment, and the shedding of cell surface determinants. Using blocking anti-integrin antibodies, tumor cell migration and migration-associated matrix reorganization were shown to be dependent on beta 1 integrin-mediated adhesion, whereas migrating T cells cannot be inhibited by a panel of anti-beta 1-, beta 2-, beta 3-, and alpha-integrin antibodies, either alone or in combination. Consequently, migrating melanoma cells use focal adhesions of integrins coclustered with cytoskeletal components at contacts with collagen fibers. T cells, however, lack typical focal adhesions, redistribute beta 1 integrins to the uropod and the focal adhesion kinase to the leading edge. In conclusion, an adhesion-dependent and reorganizing migration type employed by melanoma cells may be distinct from largely integrin-independent and non-reorganizing migration strategies used by leukocytes.

L-selectin plays a major role in leukocyte traffic through lymph node high endothelial venules (HEV). We have investigated the role of GlyCAM-1, a major L-selectin ligand produced by HEV, in mediating leukocyte rolling under in vitro flow conditions. Purified GlyCAM-1 was found to support tethering and rolling in physiological shear flow of both human and murine L-selectin expressing leukocytes at an efficiency comparable to the HEV-derived L-selectin ligands termed peripheral node addressin (PNAd). Major dynamic differences between L-selectin rolling of peripheral blood T lymphocytes and neutrophils expressing similar L-selectin level were observed on GlyCAM-1. Lymphocytes established slower and more shear resistant rolling than neutrophils and could roll on GlyCAM-1 at shear stresses lower than the threshold values required for L-selectin-mediated neutrophil rolling. Notably, high stability of L-selectin rolling of lymphocytes requires intact cellular energy, although initial lymphocyte tethering to L-selectin ligands is energy-independent. By contrast, L-selectin mediated rolling of neutrophils is insensitive to energy depletion. The distinct dynamic behavior and energy-dependence of L-selectin rolling in different leukocytes suggest that L-selectin adhesiveness in shear flow is regulated in a cell-type specific manner. The greater stability of L-selectin rolling of lymphocytes on surface-adsorbed GlyCAM-1 may contribute to their selective recruitment at peripheral lymph nodes.

Migration of circulating neutrophils occurs in several steps: capture and rolling adhesion are followed by activation of beta 2-integrins and immobilisation, and then neutrophils move over and through the endothelium. However, it is not clear how the underlying mechanisms and completion of each step depend on the concentration of stimulatory cytokines such as tumour necrosis factor-alpha (TNF). We therefore perfused neutrophils over human umbilical vein endothelial cells (HUVEC) which had been cultured with varying concentration of TNF (1-1000 U/ml) for 4 h, and recorded adhesion and migration by videomicroscopy. The number of adherent neutrophils increased with increasing TNF up to 5 U/ml, but changed little at higher concentrations. Interestingly, rolling adhesion at first predominated, but an increasing proportion of adherent cells became immobilised and migrated through the HUVEC monolayer over the complete TNF range. Immobilisation was inhibited by treating neutrophils with antibody against CD18, so that the major change in adhesive behaviour at higher levels of TNF occurred because the surface of the HUVEC presented agent(s) able to activate neutrophil beta 2-integrins. It was also evident that the selectins initiating capture of flowing neutrophils varied with concentration of TNF. At 100 U/ml TNF, both E-selectin and P-selectin supported capture and rolling adhesion, and antibody blockade of both receptors was required to inhibit adhesion. At lower dose (10 U/ml TNF), stable adhesion was blocked by antibody against E-selectin, although short-lived attachments could still be seen which were inhibited by antibody against P-selectin. Expression of sclectins increased with increasing concentration of TNF, judging from surface ELISA and reduction in the velocity of rolling adherent cells. Thus the efficiency of capture, the selectins mediating capture and the proportion of captured cells immobilised and migrating all depend on the concentration of TNF to which endothelial cells are exposed. These results suggest a model in which highly localised and efficient migration of neutrophils is achieved if a concentration gradient of TNF exists around an inflammatory locus.

Upon an increasing cell density human neutrophils develop more cell-to-cell contacts in conjunction with an increase in the pHi. These changes are accompanied by decreased superoxide formation after adherence, and a decrease in the total amount of 5-lipoxygenase products after various stimuli. Among the various arachidonate metabolites, leukotriene formation remained almost constant but the yield in 5-HETE decreased. This drop in could account for the decrease in total 5-lipoxygenase products observed when the cell density increased. We conclude that cellular signalling can be affected by an increase of cell-cell interactions. Whether the increase in cellular pH is a cause or consequence of such contact inhibition has yet be answered.

Integrins are a major class of cell surface receptors involved in cell-cell and cell-matrix adhesion and communication. Ha2/11 is a function-blocking anti-rat beta 1 integrin hamster IgM that should be a useful reagent for understanding beta 1 integrin function. We demonstrate that Ha2/11 cross reacts with human, Xenopus, and Drosophila beta 1 integrins, and use phage display to map the epitope for Ha2/11 to residues within the sequence LRSGEPQTF which lies 18 amino acids proximal to the putative I domain in beta 1 integrins. Monoclonal antibody mapping experiments, mutational analyses, and direct binding assays have implicated integrin I domains in both cation and ligand binding. Our data therefore suggest that Ha2/11 blocks beta 1 integrin function by interfering with I domain-mediated ligand binding.

L-selectin on lymphocytes reacts with glycosylated ligands on high endothelial venule walls in lymphoid organs. Through this carbohydrate-dependent interaction, rolling and initial attachment of lymphocytes to endothelium is mediated. Here we have studied an earlier described L-selectin-induced homotypic aggregation, to further elucidate the events that occur after engagement of L-selectin. It was found that the interaction of L-selectin with fucoidan, but not with other carbohydrates, or with monoclonal antibodies directed against the carbohydrate recognition domain of L-selectin, resulted in homotypic aggregation among both B- or T lymphocytes. Importantly, this aggregation was shown to be both lymphocyte function-associated antigen-1 (LFA-1) and calcium-independent. Furthermore, for aggregation metabolic energy was required, and signalling via protein tyrosine kinase appeared to be involved. Neither de novo protein synthesis, protein kinase C mediated signalling, Gi-protein mediated signal transduction, nor calcium mobilization were required for aggregation. During aggregation, L-selectin was not shed from the lymphocyte's cell surface. Finally, it was found that the lymphocyte binding capacity to high endothelial venules on cryostat sections was not altered upon triggering these lymphocytes via L-selectin. Interestingly, L-selectin-triggered cells showed increased binding to paracortical areas in peripheral lymph nodes. Our data suggest that signals via L-selectin, might lead to altered expression of cell surface molecules, important in interactions other than the first stage of lymphocyte rolling.

The adhesion integrin alpha(v)beta3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of alpha(v)beta3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of alpha(v)beta3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of alpha(v)beta3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of alpha(v)beta3. At both 2 and 4 wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of alpha(v)beta3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing alpha(v)beta3. These findings may explain the reduction in restenosis observed clinically following beta3 integrin blockade.

We have identified a novel gene, EMS1, that is consistently amplified and overexpressed in human carcinomas with an amplification of the chromosome 11q13 region. Comparisons of the EMS1 sequences with those present in the GenBank databases revealed a high identity with chicken cortactin. Southern and western blot analyses confirm the high sequence conservation during evolution. An antiserum specific for human cortactin, showed in gene transfer experiments that both human p80 and p85 isoforms are encoded by the EMS1 cDNA. Further comparisons demonstrated an high sequence and structural homology with HS1 that is implicated in signal transduction in lymphoid cells only. Expression of EMS1/cortactin mRNA was restricted to tumor cell lines derived from non-lymphoid origin. Cortactin contains (i) a filamentous actin binding tandem repeat domain, (ii) a proline-rich SH3-binding and (iii) a SH3 domain that is common in proteins involved in signal transduction. Our data suggest that human EMS1/cortactin has a function in signal transmission between cell-matrix contact sites and the cytoskeleton and, as such, its overexpression due to 11q13 amplification might effect adhesive properties of human carcinomas.