Initial Adhesive Interactions Research Articles

Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells

Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.

ERYTHROPOIETIN ENHANCES OXYGENATION IN CRITICALLY PERFUSED TISSUE THROUGH MODULATION OF NITRIC OXIDE SYNTHASE

The aim of this study was to investigate the effect of human recombinant erythropoietin (EPO) on the microcirculation and oxygenation of critically ischemic tissue and to elucidate the role of endothelial NO synthase in EPO-mediated tissue protection. Island flaps were dissected from the back skin of anesthetized male Syrian golden hamsters including a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. Before ischemia, animals received an injection of epoetin beta at a dose of 5,000 U/kg body weight with (n = 7) or without (n = 7) blocking NO synthase by 30 mg/kg body weight L-NAME (Nomega-nitro-L-arginine methyl ester hydrochloride). Saline-treated animals served as control (n = 7). Ischemic tissue damage was characterized by severe hypoperfusion and inflammation, hypoxia, and accumulation of apoptotic cell nuclei after 5 h of collateralization. Erythropoietin pretreatment increased arteriolar and venular blood flow by 33% and 37%, respectively (P < 0.05), and attenuated leukocytic inflammation by approximately 75% (P < 0.05). Furthermore, partial tissue oxygen tension in the ischemic tissue increased from 8.2 to 15.8 mmHg (P < 0.05), which was paralleled by a 21% increased density of patent capillaries (P < 0.05) and a 50% reduced apoptotic cell count (P < 0.05). The improved microcirculation and oxygenation were associated with a 2.2-fold (P < 0.05) increase of endothelial NO synthase protein expression. Of interest, L-NAME completely abolished all the beneficial effects of EPO pretreatment. Our study demonstrates that, in critically ischemic and hypoxic collateralized tissue, EPO pretreatment improves tissue perfusion and oxygenation in vivo. This effect may be attributed to NO-dependent vasodilative effects and anti-inflammatory actions on the altered vascular endothelium.

Dynamic study of the transition from hyaluronan- to integrin-mediated adhesion in chondrocytes

Membrane-bound hyaluronan mediates the initial adhesive interactions between many cell types and external surfaces. In RCJ-P chondrocytes, such early contacts are mediated through a thick hyaluronidase-sensitive coat. The early adhesion is followed by integrin-mediated interactions and the formation of stable focal adhesions. During this process, the distance between the cell membrane and the surface is reduced from micrometers to few tens of nanometers. The transition from hyaluronan- to integrin-mediated adhesion was studied on glass surfaces by total internal reflection fluorescence microscopy. Hyaluronan-mediated adhesion precedes focal adhesions formation by 2-10 min. After these initial interactions, the pericellular hyaluronan remains sequestered into discrete pockets between the cell and the surface, which are a few hundreds nanometers thick and a few micrometers wide, and are flanked by focal adhesions. The hyaluronan coat facilitates the nucleation of small paxillin-rich contacts, which later mature into focal adhesions. These dynamic studies demonstrate that pericellular hyaluronan mediates initial cell-surface adhesion, and regulates the formation of focal adhesions.

Integrity of actin fibers and microtubules influences metastatic tumor cell adhesion

Tumor cell adhesion within host organ microvasculature, its stabilization and invasion into host organ parenchyma appear to be important steps during formation of distant metastasis. These interactions of circulating tumor cells with the host organs occur in the presence of fluid shear forces and soluble and cellular environmental conditions of the blood that can modulate their cellular responses and possibly their metastatic efficiency. Cytoskeletal components, such as actin filaments and microtubules, can regulate biophysical characteristics and cellular signaling of the circulating cells. Therefore, we investigated the role of these cytoskeletal structures for early steps during metastasis formation in vivo and in vitro. Using an intravital observation technique, tumor cell adhesion of colon carcinoma cells within the hepatic microcirculation of rats and their invasion into liver parenchyma was observed. Disruption of actin filaments increased cell adhesion, whereas tubulin disruption inhibited adhesive interactions in vivo. The impairment of the cytoskeleton modulated adhesion-mediated cell signaling via focal adhesion kinase (FAK) and paxillin under flow conditions in vitro. In the presence of fluid flow, focal adhesions were enlarged and hyperphosphorylated, whereas stress fibers were reduced compared to static cell adhesion. Disruption of microtubules, however, partially inhibited these effects. Combining the in vivo and in vitro results, our study suggested that changes in cell rigidity and avidity of cell adhesion molecules after disruption of cytoskeletal components appear to be more important for initial adhesive interactions in vivo than their interference with adhesion-mediated cellular signal transduction.

Role of the cytoskeleton in adhesion stabilization of human colorectal carcinoma cells to extracellular matrix components under dynamic conditions of laminar flow.

Adhesion stabilization of malignant cells in the microcirculation is necessary for successful metastasis formation. The adhesion of colon carcinoma cells to microcirculation extracellular matrix (ECM) components is mediated, in part, by integrins that can be intracellularly linked to cytoskeletal proteins. Thus the functional status of at least certain integrins can be regulated by complex interactions with cytosolic, cytoskeletal and membrane-bound proteins. Wall shear stress caused by fluid flow also influences cellular functions, such as cell morphology, cytoskeletal arrangements and cell signaling. Using a parallel plate laminar flow chamber dynamic adhesion of human HT-29 colon carcinoma cells to collagen was investigated and compared with cell adhesion under static conditions. Cells were pretreated with cytochalasin D, nocodazole, colchicine or acrylamide to disrupt actin filaments, microtubules or intermediate filaments. Disruption of actin filaments completely inhibited all types of adhesive interactions. In contrast, impairment of tubulin polymerization or disruption of intermediate filaments resulted in different effects on static and dynamic adhesion. Treatment with acrylamide did not interfere with dynamic cell adhesion, whereas under static conditions it partially reduced adhesion rates. Under dynamic conditions increased initial adhesive interactions between HT-29 cells and collagen were found after disruption of microtubules, and the adherent cells demonstrated extensive crawling on collagen surfaces. In contrast, under static adhesion disrupting microtubules did not affect cell adhesion rates. Cytochalasin D and acrylamide were found to inhibit Tyr-phosphorylation of FAK and paxillin, whereas microtubule disrupting agents at low but not high concentrations increased phosphorylation of these focal adhesion proteins. Our results revealed that cytoskeletal components appear to be involved in adhesion stabilization of HT-29 cells to ECM components, and hydrodynamic shear forces modulate this involvement. Tyr-phosphorylation of focal adhesion proteins, such as paxillin and FAK, appears to be a part of this cytoskeleton-mediated process.

Beta1-integrin-mediated dynamic adhesion of colon carcinoma cells to extracellular matrix under laminar flow.

To resist substantial wall shear stress exerted by blood flow metastasizing colon carcinoma cells have to form adhesive contacts with endothelial cells and subendothelial extracellular matrix (ECM). At secondary sites tumor cells have to stabilize these initial adhesive interactions to prevent detachment and recirculation. Previously we found that adhesion of colon carcinoma cells to ECM components under static conditions is mediated, in part, by various beta1-integrins. Since other malignant cells possess adhesive properties that are different under static and dynamic conditions, we analyzed human colon carcinoma cell adhesion under flow by decreasing the flow (wall shear stress, WSS) of cell suspensions and allowing cells to interact with collagen-coated surfaces in a laminar flow chamber. HT-29 colon carcinoma cells were used to study wall shear adhesion threshold (WSAT), dynamic adhesion rate (DAR) and adhesion stabilization rate (ASR). DAR was determined after a low flow period using a WSS set at 50% of WSAT. ASR was calculated 60 sec after reestablishment of high WSS. Glass slides were coated with collagen I (C I) or bovine serum albumin (BSA, negative control). In some experiments cells were pretreated with function-blocking anti-beta1 or nonspecific IgG. Rolling of cells occurred on C I- and BSA-coated surfaces at high WSS. By decreasing WSS cell sticking without definite adhesion was found, and cells stuck to BSA at WSS lower than that found for C I. Further decreasing WSS below WSAT enabled stable cell adhesion to C I, but only a few cells adhered to BSA. ASR was found to be 73% of primarily adherent cells (to C I). Pretreatment with anti-beta1 did not affect cell rolling but did inhibit cell sticking and adhesion completely, whereas nonspecific IgG was without effect. Activation of PKC using phorbol ester resulted in an increase of adhesive interactions under dynamic and static conditions, whereas its inhibition reduced adhesion. Adhesive interactions of HT-29 colon carcinoma cells with ECM-coated surfaces under laminar flow conditions occurred in various steps: (1) rolling, (2) sticking or initial adhesion, and (3) stabilization of adhesion. Under shear flow rolling of tumor cells on ECM-coated surfaces appeared to be mediated mainly by physical/mechanical and nonspecific surface-cell membrane interactions, whereas stabilized adhesion to ECM was specifically mediated by beta1-integrin binding to ECM components. PKC seems to be involved in the regulation of adhesion stabilization under static and flow conditions.

Cell adhesion and migration. I. Neutrophil adhesive interactions with intestinal epithelium.

In many inflammatory conditions of the gastrointestinal tract, disease activity and patient symptoms correlate with the histological finding of neutrophil (PMN) migration across the epithelium. PMN interactions with intestinal epithelium can influence epithelial functions ranging from barrier maintenance to electrolyte secretion. Additionally, PMN recruitment to the epithelium can be modulated by epithelial interactions with luminal enteric pathogens. Adhesive interactions between PMN and intestinal epithelial cells have been shown to be distinct from interactions of PMN with endothelia. In particular, PMN transepithelial migration is modulated by a distinct array of cytokines including interferon-gamma and interleukin-4 and requires the PMN beta2-integrin CD11b/CD18 but is independent of CD11a/CD18, selectins, and intercellular adhesion molecule 1. Additionally, an integral membrane protein termed CD47 has recently been shown to play an important role in PMN transepithelial migration at point(s) subsequent to initial adhesive interactions. This article provides a brief overview of PMN interactions with epithelia and their functional consequences in relation to inflammatory disease.

Adhesion of L1210 cells to sulfonated styrene copolymer surfaces in the absence of serum

The initial adhesive interaction of living L1210 cells with sulfonated surfaces of (styrene/methyl methacrylate) copolymers (S/MMA), in the absence of serum in the medium, was examined. The surfaces of S/MMA copolymers were modified by sulfonation with sulfur trioxide at the gas/copolymer interface. The variation in styrene content in the copolymer (from 5 to 50 mol% of styrene) causes a change of the content of sulfonic groups bonded to the aromatic rings of styrene at the copolymer surface. It was shown by means of X-ray photoelectron spectroscopy that the sulfur atomic concentration S increases linearly with the styrene mer content in the copolymer. It was also shown that the contact angle θ of water on the surfaces is a decreasing linear function of S. Cells were allowed to interact with the surfaces, under stationary conditions for time t int up to the moment at which the shearing force F, caused by the medium flow, was started. The early adhesion of cells to the surfaces was examined ( t int was within the range of seconds), and the results were expressed by means of a quantity A as a function of one variable, for various empirical variables. The quantity A is defined as a ratio of the number of cells adhering to the surface during t int and remaining adherent despite the application of F, to the number of sedimented cells. The following results were obtained. (1) The function A( t int) increases and reaches a plateau at 100% adhesion. For the surfaces of wettability characterized by θ = 58°, 52° and 12°, the times t int of reaching the plateau are in the ratio of 12:9:1, respectively. (2) The function A( F) decreases for the surfaces of low wettability. (3) The function A( S) linearly increases and the function A( θ) linearly decreases, owing to the increase in the SO 3H group content at the surfaces. Results (1) and (3) are obtained at F = 3.9 × 10 −9 N, and results (2) and (3) at t int = 10 s. The Brownian motion of cells was examined. For its duration d, from the moment of cell sedimentation onto the surface to the moment of the absence of determinable cell oscillations, it was shown that the function d( S) linearly increases while the function d( θ) linearly decreases. It was shown that the initial contact of living L1210 cells with the substratum, in the absence of serum proteins, is affected by sulfonic groups. The number of cells strongly adhering, within a few seconds, to the S/MMA copolymer surfaces increases linearly with the content of the sulfonic groups at these surfaces.

Expression cloning of a functional glycoprotein ligand for P-selectin

The initial adhesive interactions between circulating leukocytes and endothelia are mediated, in part, by P-selectin. We now report the expression cloning of a functional ligand for P-selectin from an HL-60 cDNA library. The predicted amino acid sequence reveals a novel mucin-like transmembrane protein. Significant binding of transfected COS cells to P-selectin requires coexpression of both the protein ligand and a fucosyl-transferase. This binding is calcium dependent and can be inhibited by a neutralizing monoclonal antibody to P-selectin. Cotransfected COS cells express the ligand as a homodimer of 220 kd. A soluble ligand construct, when coexpressed with fucosyltransferase in COS cells, also mediates P-selectin binding and is immunocrossreactive with the major HL-60 glycoprotein that specifically binds P-selectin.

Down-regulation of Homing Receptors

Receptors known as DREG adhesion molecules on human neutrophils and monocytes provide for homing of these phagocytic leukocytes to sites of inflammation. They mediate the initial adhesive interaction of the leukocytes to vascular endothelial cells and are then shed from the cell surface in response to chemotactic factors and inflammatory mediators. Systemic accumulation of these agents following major injury or sepsis may therefore promote shedding of DREG receptors from circulating leukocytes and impair their recruitment to sites of inflammation. To test this hypothesis, we have analyzed the expression of DREG receptors on neutrophils and monocytes from 25 patients admitted to the Surgical Intensive Care Unit. Receptor expression was measured by flow cytometry of cells stained with murine monoclonal DREG-56 anti-DREG antibody. For 14 nonseptic patients, mean monocyte positivity for DREG was reduced from 64% to 40%. For 11 septic patients, mean neutrophil and monocyte positivity for DREG was reduced from 94% to 82% and 64% to 34%, respectively. These results suggest that monocytes are more affected than neutrophils in vivo by conditions expected to stimulate shedding of DREG and that sepsis promotes shedding of these adherence receptors. Accumulation of DREG-negative monocytes in association with sepsis may be sufficient to impair their recruitment to inflammatory sites and limit their contribution to host defense against infection and tissue repair.

Role of endothelium in chronic inflammation

The character of the immunologically stimulated chronic inflammatory infiltrate is to a considerable extent determined by nonspecific factors governing mononuclear cell traffic. The volume, composition and distribution of this traffic is strongly dependent on an initial adhesive interaction between circulating mononuclear cells and the EC of the PCV of the involved tissues. The emigration of lymphocytes from the PCV is preceded by binding of the lymphocytes to the endothelial lining cell. This binding is enhanced by lymphokines (IFN-gamma, TNF-beta) and monokines (IL-1, TNF-alpha), secreted by perivascular inflammatory cells and acting on the EC. This enhancement may permit an initial, immunologically generated small focus of mononuclear cells to amplify itself to a larger infiltrate. Movement of the EC-bound lymphocyte through the endothelium may be facilitated by IFN-gamma, as suggested by evidence that T cells, bound to the surface of EC monolayers overlying nitrocellulose filters, migrate through these monolayers in increased numbers in the presence of IFN-gamma. Movement of the lymphocyte through the wall of the PCV and subsequently through the perivascular space toward the inflammatory focus may be enhanced by the chemotactic action of IL-1, generated by the macrophages of the perivascular infiltrate. The lining layer of the synovial membrane may play an important role in the development of synovitis. Hyperplasia of the synovial lining layer is associated with the secretion of chemotactic agents which can attract monocytes from the PCV toward the lining. The continuous movement of monocytes from PCV to lining may establish a steady-state population of macrophage-like cells in the interstitium of the synovium. These may provide the accessory cell population required for the immune responses which give rise to the various chronic inflammatory synovitides. The sublining layer of the rheumatoid synovium does, in fact, contain large numbers of DR+macrophage+ cells which may represent this accessory cell population. Since the cytokines mediate the inflammatory effects of the cellular immune response, it follows that when injected intraarticularly they should produce synovial inflammation. Such inflammation has, in fact, been produced in experimental animals by the intraarticular injection of supernatants of antigen-stimulated PBMC or of rIL-1. A cytokine-mediated chronic inflammatory reaction may be expected to assemble a polyclonal population of lymphocytes around the PCV. However, the fraction of the accumulated T cells which is antigen reactive is not entirely a random one as indicated by the following.(ABSTRACT TRUNCATED AT 400 WORDS)