Selective induction of peripheral and mucosal endothelial cell addressins with peripheral lymph nodes and Peyer's patch cell-conditioned media.

Vascular endothelial cell addressins play an important role in lymphocyte homing in secondary lymphoid organs and in chronic inflammatory areas. A SV40 large T antigen-immortalized cell line from peripheral lymph nodes, HECa1O [Bizouarne et al., 1993a], was used to characterize the location of addressins with regard to environmental factors and cytokines. For this purpose, two monoclonal antibodies, MECA 79 and MECA 367, specific for peripheral lymph node vascular addressin and for mucosal addressin (Peyer's patches), respectively, were bound to unstimulated HECa1O cells. Both mucosal and peripheral addressins were detected inside the cells and in cellular extracts of the resting cells. On the cell surface, both addressins could be evidenced on the same cells at a moderate level of expression. They partly mediate the EL4/EL4IL2 lymphoma cells' adhesion to HECa1O cells. Supernatants of cultured peripheral lymph node or Peyers' patch cells induced expression of MECA 79 or MECA 367 antigens, respectively, on the surface of HECa1O cells. Interleukins, IL-7, IL-3, and IL-8, induced the cell-surface appearance of MECA 79 but not of MECA 367 antigen. Therefore, the same cell type synthesizes both antigens, but the expression of these antigens on the cell surface is independently regulated, thus uncovering a characteristic tissue type-specific as well as environment-sensitive properties of microvascular endothelial cells.

Monoclonal antibodies to human lymphocyte homing receptors define a novel class of adhesion molecules on diverse cell types.

A 90-kD lymphocyte surface glycoprotein, defined by monoclonal antibodies of the Hermes series, is involved in lymphocyte recognition of high endothelial venules (HEV). Lymphocyte gp90Hermes binds in a saturable, reversible fashion to the mucosal vascular addressin (MAd), a tissue-specific endothelial cell adhesion molecule for lymphocytes. We and others have recently shown that the Hermes antigen is identical to or includes CD44 (In[Lu]-related p80), human Pgp-1, and extracellular matrix receptor III-molecules reportedly expressed on diverse cell types. Here, we examine the relationship between lymphoid and nonlymphoid Hermes antigens using serologic, biochemical, and, most importantly, functional assays. Consistent with studies using mAbs to CD44 or Pgp-1, mAbs against five different epitopes on lymphocyte gp90Hermes reacted with a wide variety of nonhematolymphoid cells in diverse normal human tissues, including many types of epithelium, mesenchymal elements such as fibroblasts and smooth muscle, and a subset of glia in the central nervous system. To ask whether these non-lymphoid molecules might also be functionally homologous to lymphocyte homing receptors, we assessed their ability to interact with purified MAd using fluorescence energy transfer techniques. The Hermes antigen isolated from both glial cells and fibroblasts--which express a predominant 90-kD form similar in relative molecular mass, isoelectric point, and protease sensitivity to lymphocyte gp90Hermes--was able to bind purified MAd. In contrast, a 140-160-kD form of the Hermes antigen isolated from squamous epithelial cells lacked this capability. Like lymphocyte binding to mucosal HEV, the interaction between glial gp90Hermes and MAd is inhibited by mAb Hermes-3, but not Hermes-1, suggesting that similar molecular domains are involved in the two binding events. The observation that the Hermes/CD44 molecules derived from several nonlymphoid cell types display binding domains homologous to those of lymphocyte homing receptors suggests that these glycoproteins represent a novel type of cell adhesion/recognition molecule (H-CAM) potentially mediating cell-cell or cell-matrix interactions in multiple tissues.

The mucosal vascular addressin is a tissue-specific endothelial cell adhesion molecule for circulating lymphocytes.

Tissue position-dependent or address-dependent expression of cell adhesion molecules has been proposed to play a part in cellular positioning in a variety of systems, for example during neural development, the metastasis of neoplasms, and the tissue-specific homing of lymphocytes. The extravasation of blood-borne lymphocytes is regulated by interactions with the endothelium of specialised venules, such as the high endothelial venules (HEV) in organized lymph nodes and mucosal lymphoid tissues. At least three separate lymphocyte-HEV recognition systems have been described, one mediating tissue-selective lymphocyte interactions with HEV in peripheral lymph nodes, another in mucosal lymphoid organs, and a third in inflamed synovium. We have previously identified a tissue-specific 'vascular addressin' in the mouse which is selectively expressed by venules mediating lymphocyte-homing into mucosal tissues. To determine whether this addressin is a specific adhesion molecule for lymphocytes, we have isolated it by monoclonal antibody-affinity chromatography and inserted it into supported phospholipid planar membranes. Lymphocytes bind to membranes containing the addressin, but not to phospholipid bilayers or to control glycophorin-reconstituted membranes. Only those lymphocytes and lymphoma cell lines capable of binding to mucosal HEV adhere well to the isolated addressin; peripheral lymph node HEV-specific or HEV-non-binding cell lines do not bind. Binding is blocked by anti-addressin antibody MECA-367. We conclude that the mucosal vascular addressin is a tissue-specific endothelial cell-adhesion molecule for lymphocytes, and suggest that it could regulate lymphocyte traffic into mucosal tissues by mediating attachment of blood-borne cells to endothelium.

Hydrodynamic hyperpolarization of endothelial cells.

The orientation and morphology of the endothelium lining the cardiovascular system may result from hemodynamic forces acting on the endothelial cells. To investigate the flow effects at the membrane level, we have examined the variations of the fluorescence intensity of two membrane-sensitive dyes, merocyanine 540 and bis(1,3-diethylthiobarbiturate)trimethineoxonol, (i) as a function of flow shear stress and (ii) with the onset or cessation of the flow. We found a time-dependent decrease in fluorescence intensity with the onset of the flow with an exponential approach to steady state of the order of 1 min. The process is reversible; when the flow is stopped the fluorescence intensity returns to its original value. The polarization of the endothelial cell membranes or, more precisely, the amplitude of the fluorescence intensity responses is an increasing function of the shear stress (up to 120 dynes/cm2). Assuming the equilibrium potential for K+ is more hyperpolarized than the resting potential and using valinomycin, we have deduced from the sign of the ionophore effects that the flow hyperpolarizes the endothelial cell membrane.

Topological and modulated distribution of surface markers on endothelial cells.

The mobility and distribution of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) and a specific endothelial cell surface protein was assessed by fluorescein-conjugated monoclonal antibodies on bovine and murine endothelial cells grown on their extracellular matrix. The combination of data obtained from fluorescence recovery after photobleaching measurements and observations under epifluorescence and total internal reflection fluorescence reveals a restriction of these protein markers to the apical membrane of endothelial cell. This asymmetry is evident both when cells are grown at a sparse density or at confluence. When cells are brought into suspension, the fluorescein-conjugated antibody is found over the entire cell surface. The fluorescence disappears from the basal part of the cell when the cells are again spread on coverslips coated with a layer of extracellular matrix. Conversely, cells spread on glass coverslips without extracellular matrix do not show this restriction phenomenon. It is suggested that the extracellular matrix provides the signal to induce the restricted topology of membrane protein markers on endothelial cells.

Heterogeneity of membrane phospholipid mobility in endothelial cells depends on cell substrate.

Cellular growth control and differentiation have been shown to be dependent on both cell-cell and cell-substrate contacts. Interactions of cells with extracellular material are critical events during embryonic development and maintenance of tissue function. Plasma membrane receptors have been described for components of the extracellular matrix such as fibronectin, laminin and various collagen types. Transmembrane signalling has been shown to be influenced by the lateral mobilities of the plasma membrane constituents. The interaction of cells with their extracellular matrix could thus have a significant effect on the mobility properties of the plasma membrane components. Here we have studied the dynamic properties of fluorescent membrane phospholipids in bovine endothelial cells using fluorescence recovery after photo-bleaching measurements. At this molecular level we find that the phospholipid lateral diffusion coefficient is dependent on the substrate upon which cells are allowed to adhere (collagen, fibronectin or a natural basement membrane) and on the topography of the cell (basal versus apical plasma membrane).

Topological analysis of wall mass transport using a luminescent immobilized enzymatic system.

A new technique of visualization of diffusion-convection phenomena at a solid-liquid interface using the luminol chemiluminescent reaction catalyzed by immobilized peroxidase has been previously described (Dimicoli, J.L., M. Nakache, and P. Peronneau, 1982, Biorheology, 19:281-300). We propose now a theoretical model that predicts quantitatively the light fluxes, JL, corresponding to the transfer J of the hydrogen peroxide substrate at the liquid-solid interface in a cylindrical tube for continuous flow experiments. A simple phenomenological relation, J alpha J1/mL (1 less than m less than 3) was first established for each point of the wall. Then, numerical integration showed that, independent of the laminar or turbulent character of the flow, J1/mL was proportional to (S1 Kideal)/(1 + Kideal/ET), where S1 is the bulk substrate concentration, Kideal is the ideal transport coefficient, and ET (in cm.S-1) a phenomenological first-order enzymatic rate constant per unit of wall surface. This relation proved to be satisfactory for all experimental conditions since a single mean value of ET takes into account the experimental data collected for a given enzymated tube in a large range of Reynolds number values (Re) (500 less than Re less than 9,000) and of distances from the entrance of the tube (chi greater than 0.3 cm). This quantitative analysis using a pseudo-first-order approximation interprets the observed great dependence of JL on Re(JL alpha Ren', with n' usually greater than 1/3 for laminar flows) and on S1 (JL alpha S1m). It predicts also that the laminar-to-turbulent transition can be evidenced for interfacial enzymatic activity, ET greater than 2.10(-4) cm.S-1, as observed with most of the tubes prepared by covalent binding of peroxidase on the acrylamide gel wall. The experiment had to be carried out at a pH value of 8, which corresponds to the fastest rate of the chemiluminescent reaction. The predicted entrance effects were also observed experimentally for the first time in an immobilized enzyme system. This technique appears therefore to be a valuable tool for the quantitative analysis of diffusion-convection phenomena at a liquid-solid interface with a good spatial resolution with a great range of flow rate.

Direct visualization of diffusion convection phenomena at liquid solid interfaces by the use of a chemiluminescent enzymatic immobilized system.

A new method is developed for direct visualization of the local mass transfer at solid liquid interfaces. Peroxidase is immobilized by entrapment in a polyacrylamide gel coating the interior surface of a glass tube. The reaction of oxidation of luminol by H2O2 catalyzed by this enzyme involves light emission. Furthermore at low H2O2 concentration (less than or equal to 5. x 10(-5) M), this reaction is controlled by the diffusion of H2O2 from the bulk flow to the wall, as evidenced by the Re1/3 dependence of the light flux V measured in the laminar case. It is possible in these conditions to directly relate V as measured at each point of the wall, to the local properties of the flow : (i) a decrease of V is always observed when moving downstream from the input of the tube, but it is much more pronounced for laminar flows than for turbulent ones, as theoretically expected; (ii) the sensitivity of the method has been tested for evaluating the diffusion convection phenomena at the wall downstream from a stenosis. Furthermore the local hydrodynamic properties have been characterized by measuring through pulsed Doppler velocimetry the velocity of the moving liquid phase as a function of the position in the flow. The data obtained show the presence of a maximum of V in the vicinity of the reattachment point of the liquid streamlines at the wall. This constitutes the first experimental confirmation of calculations on diffusion convection phenomena downstream from stenoses. These first experiments show one the ability of the method to detect the local properties of the parietal mass transfer phenomena, as a function of the geometry of the wall and the hydrodynamic characteristics of the flow.

Visualization of the parietal mass transfer by using immobilized peroxidase.

A new method is developed for direct visualization of the local mass transfer at solid liquid interfaces involving the chemiluminescent oxidation of luminol by H2O2 catalyzed by immobilized peroxidase. At low concentration of H2O2 (C less than 5 x 10(-5) M) this reaction is controlled by diffusion and it is possible to characterize the diffusion convection of H2O2 at each point of the tube as a function of the local hydrodynamic properties. These properties are characterized by pulsed Doppler ultrasound velocimetry. 1) The rate of light emission depends on Re1/3 for the laminar case and decreases when going downstream in accordance with the known theories of diffusion convection along tubes. 2)Downstream to a stenosis, a maximum of light appears which depends on the input Reynolds number in a manner similar to the reattachment point of the flow. This constitutes the first experimental confirmation of calculations on diffusion convection downstream to stenoses. These first experiments show the capacity of the method to detect the local properties of the parietal mass transfer phenomenon as a function of the geometry of the wall and the hydrodynamic properties of the flow.

[Biomaterials]. / Les biomatériaux.

Biomaterials--materials used for the elaboration of systems designed for human implantation or organ substitutes--can be classified as metals and alloys, ceramics and polymers. Their uses are largely diversified, for soft and hard tissues replacement. Interactions rise between biological environment and implants, the mechanisms of them not always known: inflammatory response, corrosion and degradation of materials leading to leaching of some constituents possibly toxic and alteration of their mechanical properties. Blood interfacing materials introduce some particular problems of hemocompatibility. The matching of implant to biological medium, in other words, its biocompatibility has to be a priori evaluated, but until now no in vitro or in vivo evaluation method is fully reliable.

ESR study of free and immobilized elastase.

Porcine pancreatic elastase (EC 3.4.21.11) has been immobilized on polyacrylamide beads using glutaraldehyde ad bridging reagent without important loss of catalytic activity. A nitroxide spin label, 1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate, reacting covalently with the serine-195 residue of the active centre of free elastase was used as a conformational and dynamical electron spin resonance probe. This signal is quenched by (Cu2+) which bind specifically at the active site at a distance of 7 A from the nitroxide group. This distance is not significantly affected by the fixation on the solid support. The electron spin resonance lineshape analysis indicates some mobility of the spin label with respect to the native protein. This restricted motion, which is pH dependent, is not noticeably modified by the immobilization of the enzyme. This immobilization has therefore induced no large conformational change of the protein in the vicinity of the active centre. Thermal denaturation of elastase in homogeneous solution is irreversible. Immobilization on the polyacrylamide beads results in 70% reversibility, but the temperature of denaturation is not modified.