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PubMed1995

Adhesion of head and neck squamous cell carcinoma to endothelial cells. The missing links.

OBJECTIVES: To examine the direct adhesion of head and neck squamous cell carcinoma (HNSCC) cells to basal and cytokine-activated human endothelial cells and to determine the cell adhesion molecules (CAMs) that mediate binding under these two conditions. DESIGN: Using an established model of tumor metastasis, the adhesion of four HNSCC cell lines to human umbilical vein endothelial cell (HUVEC) monolayers was examined, with and without pretreatment of HUVEC with tumor necrosis factor alpha (TNF-alpha). Surface CAM expression of HNSCC and HUVEC was determined by flow cytometry, and the results were used to direct studies of adhesion blocking using monoclonal antibodies. The contribution of various CAM to HNSCC binding of basal and cytokine-activated human endothelial cells in vitro was established. MAIN OUTCOME MEASURES: Adhesion of HNSCC to HUVEC monolayers was determined by a sodium chromate Cr 51-labeling assay in the presence or absence of monoclonal antibodies directed against specific CAMs. RESULTS: Four HNSCC cell lines were shown by flow cytometry to constitutively express the following CAMs: intercellular CAM-1, CD44, lymphocyte function-associated antigen-3, integrin chains alpha 6 and beta 1, and sialyl Lewis(x). No cell lines expressed lymphocyte function-associated antigen-1 or the integrin subunit alpha 4. Adhesion of JHU-011-SCC to TNF-alpha-activated HUVEC was enhanced above the untreated level in a time-dependent manner, with maximal adhesion at 12 hours. This increase correlated with endothelial-selectin expression by HUVEC and HNSCC expression of its ligand sialyl Lewis(x). Monoclonal antibody to sialyl Lewis(x) blocked the increased adhesion to TNF-alpha-activated HUVEC in two of four HNSCC cell lines. Monoclonal antibody to the alpha 6 integrin reduced binding to TNF-alpha-activated and non-activated HUVEC and to subendothelial matrix, but not to fibronectin. CONCLUSIONS: Studies of four HNSCC cell lines disclosed a consistent and distinctive pattern of adhesion molecule expression. The alpha 6 integrin subunit may be involved in direct adhesion to nonactivate and cytokine-activated endothelial cells or to laminin present on the endothelial surface. Sialyl Lewis(x) was more specifically involved in the increased adhesion to cytokine-activated HUVEC. This suggests that the sialyl Lewis(x)-endothelial-selectin ligand interaction may be important in facilitating HNSCC adhesion during metastasis to sites of active or chronic inflammation in vivo.

Carcinoma, Squamous Cell↗
PubMed1997

A quantitative immunofluorescence study of glomerular cell adhesion proteins in proteinuric states.

Whenever there is heavy proteinuria, the glomerular epithelial cells, the podocytes, show dramatic morphological changes which clearly demonstrate changes in cell adhesion. However, there is little information on the types of cell adhesion molecules expressed in the normal human glomerulus. Assessments of changes in cell adhesion molecules in human proteinuria have been confined to semi-quantitative immunostaining for integrins, and the results have not been entirely consistent. This study sought first to define which cell adhesion molecules are present in the normal glomerulus, using indirect immunofluorescence and a panel of antibodies directed against transmembrane adhesion proteins and against several cytoplasmic proteins which are known to be involved in adhesion. A wide variety of integrins were detected, the dominant form being alpha 3 beta 1. The cytoplasmic focal adhesion proteins vinculin, talin, paxillin, p130CAS, and pp125FAK were detected, although vinculin appeared to be confined mainly to the mesangium. The only intercellular adhesion molecule detected in the vicinity of the slit diaphragm was ZO-1; the results imply that the slit diaphragm does not bear a close relationship to any other form of intercellular junction. Changes in these adhesion components were also studied in proteinuria, using 18 cases each of minimal change nephropathy, 'early' membranous nephropathy, and normal controls. Fluorescence intensity was measured by image capture using a low light video camera and subsequent digital image analysis, an approach which demonstrated acceptable reproducibility. The most striking changes were an increase in phosphotyrosine and p130CAS in the nephrotic patients. Contrary to previous reports, little change was found in the expression of the most abundant integrins, nor did overall glomerular staining for ZO-1 alter. These results imply a controlled alteration in glomerular cell adhesion in nephrotic states in man, probable representing increased turnover of cell adhesion structures rather than the decrease which has been reported in short-term animal models. This is the first report of increased glomerular phosphotyrosine in man, which is associated with less stable adhesions and may be related to the loss of foot processes. Using human biopsy material, it was not possible to determine which proteins were phosphorylated, but the probable relationships to changes in cytoskeletal structure and slit diaphragm permeability justify further study.

Cell Adhesion↗
PubMed1992

Detection of circulating intercellular adhesion molecule-1 in chronic liver diseases.

The leucocyte adhesion molecule intercellular adhesion molecule-1 is induced on bile ducts in patients with primary biliary cirrhosis and primary sclerosing cholangitis and may be involved in targeting immune damage to these structures. It has recently been reported that, when activated, in vitro lymphocytes release a soluble form of intercellular adhesion molecule-1 that can also be detected in human serum. Because it is functionally active, this circulating intercellular adhesion molecule-1 might play a role in regulating inflammation by blocking adhesion. We used an enzyme-linked immunosorbent assay to detect circulating intercellular adhesion molecule-1 in the serum of patients with primary biliary cirrhosis and primary sclerosing cholangitis. Levels of circulating intercellular adhesion molecule-1 were markedly elevated in primary biliary cirrhosis and primary sclerosing cholangitis when compared with other chronic liver diseases. Circulating intercellular adhesion molecule-1 is probably derived from activated lymphocytes rather than from bile ducts because biliary epithelial cells from patients with primary biliary cirrhosis did not release circulating intercellular adhesion molecule-1 when stimulated to express the membrane-bound molecule in vitro. These studies are the first to demonstrate circulating intercellular adhesion molecule-1 in chronic inflammatory diseases that are characterized by strong tissue expression of intercellular adhesion molecule-1 and as such suggest a potential immunoregulatory role for circulating adhesion molecules. The very high levels detected in primary biliary cirrhosis and primary sclerosing cholangitis probably reflect lymphocyte activation, which is further evidence of immune pathogeneses for these diseases.

Bilirubin↗
PubMed1992

The neural cell adhesion molecule (NCAM) heparin binding domain binds to cell surface heparan sulfate proteoglycans.

The neural cell adhesion molecule (NCAM) has been strongly implicated in several aspects of neural development. NCAM mediated adhesion has been proposed to involve a homophilic interaction between NCAMs on adjacent cells. The heparin binding domain (HBD) is an amino acid sequence within NCAM and has been shown to be involved in NCAM mediated adhesion but the relationship of this domain to NCAM segments mediating homophilic adhesion has not been defined. In the present study, a synthetic peptide corresponding to the HBD has been used as a substrate to determine its role in NCAM mediated adhesion. A neural cell line expressing NCAM (B35) and its derived clone which does not express NCAM (B35 clone 3) adhered similarly to plates coated with HBD peptide. A polyclonal antiserum to NCAM inhibited B35 cell-HBD peptide adhesion by only 10%, a value not statistically different from inhibition caused by preimmune serum. Both these experiments suggested no direct NCAM-HBD interactions. To test whether the HBD peptide bound to cell surface heparan sulfate proteoglycans (HSPG), HSPG synthesis was inhibited using beta-D-xyloside. After treatment, B35 cell adhesion to the HBD peptide, but not to control substrates, was significantly decreased. B35 cell adhesion to the HBD peptide could be inhibited by 10(-7) M heparin but not chondroitin sulfate. Preincubation of the substrate (HBD peptide) with heparin caused dramatic reduction of B35 cell-HBD peptide adhesion whereas preincubation of B35 cells with heparin caused only modest reductions in cell-HBD adhesion. Furthermore, inhibition of HSPG sulfation with sodium chlorate also decreased the adhesion of B35 cells to the HBD peptide. These results strongly suggest that, within the assay system, the NCAM HBD does not participate in homophilic interactions but binds to cell surface heparan sulfate proteoglycan. This interaction potentially represents an important mechanism of NCAM adhesion and further supports the view that NCAM has multiple structurally independent binding sites.

Amino Acid Sequence↗
PubMed1995

Control of neuronal morphology in vitro: interplay between adhesive substrate forces and molecular instruction.

Among the factors which influence neuronal morphology, the degree of substrate adhesivity has been suggested to play an important role in the growth and guidance of neurites. The present study was undertaken to investigate apparently contradictory results relating substrate adhesivity to the extent of neurite outgrowth. By using substrates coated with different concentrations of polyornithine to vary adhesivity, we could show that intermediate levels of neuron-to-substrate adhesive strength favored neurite outgrowth more than substrates of high or low adhesivity. However, when neurons were plated on substrates derived from the extracellular matrix, the strength of neuron-to-substrate adhesion was important for the growth of dendrite-like minor neurites, but not for the extension of axon-like major neurites, which grew independently of adhesive forces. On substrates of the cell adhesion molecule L1, growth of both major and minor neurites was adhesion-independent. Finally, in the presence of tenascin added to the culture medium, neurite growth was inhibited irrespective of the adhesivity of the substrate and the presence of substrate-bound extracellular matrix molecules or L1. These observations suggest that intermediate forces of adhesivity favor neurite growth in general, but that purely adhesive forces can be dominated by specific molecular instructions which differentially affect growth of major and minor neurites in positive and negative ways.

Animals↗
PubMed2000

E-selectin and very late activation antigen-4 mediate adhesion of hematopoietic progenitor cells to bone marrow endothelium.

Adhesion of CD34+ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34+ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34+ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34+ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34+ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1beta prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34+ cells to IL-1beta-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34+ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against beta2-integrins. Stimulation with the beta1-integrin-activating mAb, 8A2, induced adhesion of CD34+ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34+ HPCs is necessary for adhesion of CD34+ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion.

Antigens, CD34↗
PubMed2000

A surrogate method for assessment of beta(2)-integrin-dependent adhesion of human eosinophils to ICAM-1.

We have developed and validated an inexpensive and equivalent method for measuring eosinophil adhesion by beta(2)-integrin to endothelial ICAM-1 using bovine serum albumin (BSA) as a surrogate for the immunoglobulin supergene. The number of adherent eosinophils on BSA or ICAM-1 coated microplates was quantified by residual eosinophil peroxidase activity. Non-stimulated eosinophils did not adhere to either BSA or ICAM-1. However, after IL-5 stimulation, either BSA or ICAM-1 caused comparable and concentration-dependent adhesion of eosinophils. Eosinophil adhesion was rapid and occurred within 15 to 30 min of incubation for either BSA or ICAM-1. Preincubation of cells with CD11b or CD18 antibody specifically decreased adhesion to either BSA or ICAM-1. IL-5, PAF and fMLP all induced adhesion of eosinophils to either BSA or ICAM-1 in a concentration-dependent manner, and the optimal IL-5, fMLP and PAF concentrations for adhesion to BSA were the same as for adhesion to ICAM-1. BSA-binding was specific for beta(2)-integrin; neither alpha-CD49d mAb directed against the alpha(4)-chain or alpha-CD29 directed against the common beta(1)-chain of VLA-4 blocked adhesion to BSA or ICAM-1 controls. The protein tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, wortmanin, and mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, all inhibited IL-5-induced eosinophil adhesion to either BSA or ICAM-1 comparably. These results indicate that BSA is a reliable and economical surrogate ligand for ICAM-1 adhesion to beta(2)-integrin-dependent adhesion to ICAM-1. Ligation characteristics of BSA are identical to those for soluble ICAM-1, and the assay is suitable for assessment of signal transduction pathways mediating adhesion.

Androstadienes↗
PubMed2002

Venous shear stress enhances platelet mediated staphylococcal adhesion to artificial and damaged biological surfaces.

We investigated the role of blood components in the adhesion of staphylococci to biological and artificial surfaces under well-defined flow conditions by using the Cone and Plate(let) Analyzer. An enzyme-linked immunosorbent assay-like binding assay with biotinylated bacteria determined the extent of bacterial adhesion to subendothelial extracellular matrix (ECM), polystyrene (PS) and adult bovine aortic endothelial (ABAE) cell monolayer. The presence of adsorbed plasma proteins on PS and ECM did not increase and in some cases reduced staphylococcal adhesion under flow conditions (200s(-1)). However, their presence on ABAE cells increased bacterial adhesion but to a level still lower than the adhesion to PS and ECM. In contrast, adhered platelets significantly increased staphylococcal adhesion to both PS and ECM, but did not affect the adhesion to ABAE cells. Furthermore, bacterial adhesion to the platelets coated ECM and PS under flow conditions (200s(-1)) was increased by 1.4 to 2.6-fold compare to static conditions. The platelet-enhanced bacterial adhesion was markedly inhibited by blockade of the platelet GPIb receptor. In conclusion, staphylococcal extensive adhesion to ECM and PS surfaces is increased by venous flow and mediated by surface adhered activated platelets via a GPIb dependent mechanism. On the other hand, ABAE cells demonstrated limited bacterial adhesion that is mediated by adsorbed plasma proteins. Our results suggest that under physiological venous flow conditions the intact vessel wall is less prone for bacterial adhesion than damaged vessel wall.

Animals↗
PubMed1999

[Adhesion molecules and cancer].

INTRODUCTION: This review was aimed at summarizing recent advances in the understanding of cell adhesion in order to discuss the possible relevance of new knowledge to the exploration of cancer patients and elaboration of therapeutic strategies. CURRENT KNOWLEDGE AND KEY POINTS: During the last 10 years, many adhesion molecules were identified, thus allowing to determine their tissue distribution and functional regulation. The concept of adhesiveness was refined. It is now well known that adhesive rate (i.e., the minimal contact time required for bond formation) and binding strength (i.e., the minimal force required to detach bound cells) are distinct parameters. They may be regulated independently, and influence the cell behavior in different ways. It is now possible to achieve accurate control of tumor cell adhesiveness, either by inhibiting an adhesive mechanism (through monoclonal antibodies, competitive ligands, or inhibition of receptor expression with antisense strategy or gene knock-out) or by promoting a binding mechanism (with receptor transfection or pro-inflammatory stimulation). FUTURE PROSPECTS AND PROJECTS: Recent progress opens new possibilities for diagnosis and treatment. First, the interpretation of experimental data may be improved. Cell adhesive behavior is not entirely accounted for by the density of membrane adhesion receptors. Indeed, adhesion is influenced by receptor connection to the cytoskeleton and structure of the cell coat. An adhesion receptor may be anti-metastatic through an increase in tumor cohesion and cell differentiation, or pro-metastatic, through facilitation of cell migration towards a target tissue. New therapeutic strategies may include anti-adhesive procedure aimed at preventing metastasis formation. The potential importance of a better control of inflammatory processes is also emphasized in view of the influence of these processes on the expression of adhesion molecules.

Cell Adhesion↗
PubMed2006

Adhesion of antibody-functionalized polymersomes.

Polymersomes are vesicles made from synthetic block copolymers. The adhesiveness of micron-sized polymersomes, functionalized with antibodies that bind to vascular cell adhesion molecules, which could be useful for vascular targeting, was measured. Intercellular adhesion molecule-1 (ICAM-1) is an endothelial cell adhesion molecule whose expression increases during inflammatory disease, and is therefore a natural target for vascular delivery. We functionalized polymersomes with an anti-ICAM-1 antibody, using modular biotin-avidin chemistry. Micropipet aspiration was used to confirm specific adhesion and measure the adhesion strength between an anti-ICAM-1-coated polymersome and an ICAM-1-coated polystyrene microsphere at various surface densities of adhesion molecules. The adhesion is kinetically trapped, and adhesion strength is quantified by the critical tension for detachment. The adhesion strength increases in proportion to the surface density of anti-ICAM-1 molecules, in contrast to results seen previously when measuring adhesion between biotinylated vesicles and avidin-coated beads (Lin et al. Langmuir 2004, 20, 5493). The difference in dependence on the density of functional groups is likely due to the molecular presentation at the vesicle surface; in the current study, the presentation of biotinylated anti-ICAM-1 on a layer of avidin leads to the effective presentation of the anti-ICAM-1 and, thus, a monotonic increase in adhesiveness with antibody density.

Adhesiveness↗
PubMed1997

Adhesion of leukocytes to dermal endothelial cells is induced after single-dose, but reduced after repeated doses of UVA.

Approximately 20-50% of ultraviolet A (UVA) irradiation delivered to the skin surface may reach the human dermal microvascular endothelial cells (HDMEC) that play a pivotal role in cellular inflammatory tissue; however, the pathophysiologic role of HDMEC in UVA-induced skin changes is largely unknown. Based on previous in vivo and in vitro studies revealing UVA-induced expression of endothelial adhesion molecules, we studied isolated HDMEC under various conditions in order to further delineate the impact of UVA on these cells. The expression of cell adhesion molecules was determined by flow cytometry and the resulting changes of stable adhesion of leukocytes to endothelial cells were quantitated for granulocytes, lymphocytes, and monocytes using a newly developed multicellular adhesion assay. Additionally, antibody blocking experiments were performed to delineate the role of individual cell adhesion molecules in UVA-induced leukocyte adherence. High-dose polychromatic UVA (25 J per cm2, maximal emission at 375 nm) induced intercellular adhesion molecule-1 and E-selectin with different kinetics but correlating the adhesion of leukocyte subsets. This effect subsided, however, in the course of 3-6 daily applied UVA doses. Moreover, pro-inflammatory cytokine challenge by tumor necrosis factor-alpha and interleukin-1-alpha resulted in significantly weaker induction of intercellular adhesion molecule-1 and E-selectin in repeatedly UVA-exposed HDMEC. Differential quantitation of peripheral blood derived granulocytes, lymphocytes, and monocytes revealed reduced adhesion particularly of lymphocytes followed by monocytes and granulocytes compared with leukocyte adhesion to nonirradiated but cytokine-stimulated HDMEC. It is concluded that UVA substantially influences endothelial cell adhesion molecules expression and thus directly interferes with leukocyte adhesion to endothelial cells. Divergent UVA-induced effects in this respect can be attributed to the mode of UV exposure as well as to the condition of endothelial cells prior to UVA exposure.

Cell Adhesion↗
PubMed2004

Colorectal surgery: the risk and burden of adhesion-related complications.

OBJECTIVES: Adhesions are associated with serious medical complications. This study examines the real-time burden of adhesion-related readmissions following colorectal surgery and assesses the impact of previous surgery on adhesion-related outcomes. PATIENTS AND METHODS: The study used data from the Scottish National Health Service Medical Record Linkage Database to identify three cohorts of patients who had undergone open colorectal surgery during the financial years 1996-97, 1997-98 and 1998-99. Each cohort was followed up for at least 2 years and the number and category of adhesion-related readmissions was recorded. The influence of any previous operations on adhesion-related readmissions was also determined by performing a subanalysis within the 1996-97 cohort of patients who had no record of abdominal surgery within either the previous 5 or 15 years. The relative risk of adhesion-related readmissions was also assessed. RESULTS: In the 1996-97 cohort, 9.0% of patients were readmitted within a year after surgery; 2.1% had complications directly related to adhesions and 6.9% had complications that were possibly related. After 4 years, 19.0% of patients were readmitted for reasons directly or possibly related to adhesions. Many patients were readmitted on more than one occasion and the relative risk of adhesion-related complications was 29.7 per 100 initial procedures over 4 years. In the subgroups that had no record of abdominal surgery within the previous 5 or 15 years, the relative risks of adhesion-related complications were 24.8% and 23.5%, respectively. There was no change in the rate of adhesion-related readmissions following colorectal surgery between 1996 and 1999. CONCLUSION: Colorectal surgery is associated with a considerable rate of adhesion-related readmissions. Preventative measures should be considered to reduce this risk.

Adult↗
PubMed1995

An in vivo evaluation of adhesives used in extraoral maxillofacial prostheses.

PURPOSE: This investigation introduces percent adherent area and 10-minute and 8-hour peel-strength values as an in vivo evaluation of three popular maxillofacial prosthetic adhesives when used with three extraoral prosthetic polymers. MATERIALS AND METHODS: Specimens of Silastic MDX 4-4210, Silastic Medical Adhesive A (Dow Corning Corporation, Midland, MI), and Silicone A-2186 (Factor II, Inc, Lakeside, AZ) were adhered to 25 subjects' arms using Secure Medical Adhesive (Factor II, Inc), Pros-Aide Adhesive (ADM Tronics Inc, Northvale, NJ), and Hollister Colostomy Adhesive (Hollister Inc, Libertyville, IL). After 10 minutes, the peel strength required to remove the specimens was measured. Additional specimens were then adhered to the subjects and worn for 8 hours. The nonadherent areas of the specimens were then marked, the peel strength was measured again, and then the percent adherent area was calculated. RESULTS: Hollister Colostomy Adhesive showed greater 10-minute peel-strength values when used with Silastic Medical Adhesive A. The peel-strength values of the Hollister Colostomy Adhesive when used with Silastic MDX 4-4210 increased with time. The greatest percent adherent area was obtained with Silastic MDX 4-4210 using Pros-Aide Adhesive, and the lowest was obtained with Pros-Aide Adhesive using Silicone A-2186 and Silastic Medical Adhesive A. CONCLUSIONS: There was no clearly superior adhesive/prosthetic material combination based on the tests used in this study; however, individual skin variations between patients affect the adhesive properties.

Adhesiveness↗
PubMed2004

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin.

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.

Cell Adhesion↗
PubMed1998

A CD18/ICAM-1-dependent pathway mediates eosinophil adhesion to human bronchial epithelial cells.

Eosinophil adhesion to airway epithelium is believed to facilitate eosinophil accumulation and retention in asthmatic airways. Monoclonal antibodies (mAb) against intercellular adhesion molecule-1 (ICAM-1) and its CD18 leukocyte integrin ligands have been shown to inhibit airway eosinophilia in animal models of asthma, although the role of this pathway in eosinophil-epithelial adhesion is not fully understood. To investigate the role in vitro of CD18 and ICAM-1, we measured adhesion of fluorescently labeled human eosinophils to normal human bronchial epithelial cell (NHBEC) monolayers pretreated for 24 h with culture medium (low constitutive ICAM-1) or tumor necrosis factor-alpha (TNF-alpha; 1 ng/ml) and interferon-gamma (IFN-gamma) (10 ng/ml; increased ICAM-1). Stimulation of eosinophils with C5a (10(-7) M) increased adhesion measured at 30 min to unactivated NHBEC from 11.4 +/- 0.7 to 15.5 +/- 0.4% (n = 4), and this increase was CD18/ICAM-1-independent, whereas phorbolmyristate acetate (PMA) (10(-8) M)-induced adhesion (20.7 +/- 1.7%) was abolished by anti-CD18 and reduced by anti-ICAM-1. In contrast, C5a- and PMA-induced adhesion to TNF-alpha/IFN-gamma-activated NHBEC (increased from 11.1 +/- 1.3% to 21.9 +/- 1.0% and 27.6 +/- 1.9%, respectively) was CD18- and ICAM-1-dependent. Eotaxin, but not regulated on activation normal T cells expressed and secreted, macrophage inflammatory protein-1, formyl methionyl leucyl phenylalanine, leukotriene B4 or platelet-activating factor, also induced CD18/ICAM-1-dependent adhesion to activated NHBEC. In the absence of added chemoattractants, eosinophil adhesion to NHBEC increased with time and, at 120 min, was significantly greater (P < 0.01) to activated NHBEC (37.3 +/- 2.4%, n = 5) than to unactivated monolayers (24.3 +/- 1.9%); mAb against CD18 or ICAM-1 abolished increased, but not basal, adhesion. These results suggest that CD18/ICAM-1 mediated eosinophil adhesion to activated NHBEC but that adhesion to resting NHBEC was largely independent of this pathway.

Antibodies, Monoclonal↗
PubMed2004

Role of Rap1 in promoting sickle red blood cell adhesion to laminin via BCAM/LU.

Vaso-occlusion is a hallmark of sickle cell disease. Agonist-induced activation of sickle red blood cells (SS RBCs) promotes their adhesion to vascular proteins, potentially contributing to vasoocclusion. Previously, we described a cyclic adenosine monophosphate (cAMP)-dependent increase in SS RBC adhesion to laminin. Here, we investigated whether Rap1, a small guanosine triphosphatase (GTPase) known to promote integrin-mediated adhesion in other cells, was involved in this signaling pathway. We found that agonists known to induce cAMP signaling promoted the GTP-bound, active state of Rap1 in SS RBCs. The cAMP-dependent exchange factor Epac (exchange protein directly activated by cAMP) is a likely upstream activator of Rap1, since Epac is present in these cells and the Epac-specific cAMP analog 8CPT-2-Me (8-(4-cholorophenylthio)-2'-O-methyl-cAMP) activated Rap1 and promoted SS RBC adhesion to laminin. This 8CPT-2-Me-stimulated adhesion was integrin independent, since it was insensitive to RGD peptide or antibodies against the only known integrin on SS RBCs, alpha4beta1. However, this adhesion was completely inhibited by either a soluble version of basal cell adhesion molecule/Lutheran (BCAM/LU) or a BCAM/LU adhesion-blocking anti-body. Surprisingly, 8CPT-2-Me-activated Rap1 did not promote SS RBC adhesion to a known alpha4beta1 ligand, vascular cell adhesion molecule 1 (VCAM-1). These results demonstrate that Epac-induced Rap1 activation in SS RBCs promotes BCAM/LU-mediated adhesion to laminin. Thus, Epac-mediated Rap1 activation may represent an important signaling pathway for promoting SS RBC adhesion.

Anemia, Sickle Cell↗
PubMed2003

Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption.

Tyrosine phosphorylation of focal adhesion components is involved in the regulation of focal adhesion formation and turnover, yet the underlying molecular mechanisms are still poorly defined. In the present study, we have used quantitative fluorescence microscopy to investigate the dynamic relationships between the incorporation of new components into growing focal adhesions and tyrosine phosphorylation of these sites. For this purpose, a new approach for monitoring phosphotyrosine levels in live cells was developed, based on a 'phosphotyrosine reporter' consisting of yellow fluorescent protein fused to two consecutive phosphotyrosine-binding Src-homology 2 (SH2)-domains derived from pp60(c-Src). This YFP-dSH2 localized to cell-matrix adhesions and its intensity was linearly correlated with that of an anti-phosphotyrosine antibody labeling. The differential increase in vinculin and phosphotyrosine levels was examined in live cells by two-color time-lapse movies of CFP-vinculin and YFP-dSH2. In this study, focal adhesion growth was triggered by microtubule disruption, which was previously shown to stimulate focal adhesion development by inducing cellular contraction. We show here that, 2 minutes after addition of the microtubule-disrupting drug nocodazole, the local densities of the focal adhesion-associated proteins vinculin, paxillin and focal adhesion kinase (FAK) are significantly elevated and the focal adhesion area is increased, whereas elevation in tyrosine phosphorylation inside the growing adhesions occurs only a few minutes later. Phosphotyrosine and FAK density reach their maximum levels after 10 minutes of treatment, whereas vinculin and paxillin levels as well as focal adhesion size continue to grow, reaching a plateau at about 30 minutes. Our findings suggest that protein recruitment and growth of focal adhesions are an immediate and direct result of increased contractility induced by microtubule disruption, whereas tyrosine phosphorylation is activated later.

Amino Acid Sequence↗
PubMed1998

EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway.

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

Animals↗