Showing papers by "Larry V. McIntire published in 1993"

E-selectin supports neutrophil rolling in vitro under conditions of flow.

Abstract: E-selectin was evaluated for its ability to support neutrophil adhesion under conditions of flow. At a wall shear stress of 1.85 dyn/cm2, neutrophils were found to attach to E-selectin expressed on the apical surface of L cell monolayers. The initial intercellular contact was most often evidenced by neutrophils rolling on the monolayer at a mean rate of congruent to 10 microns/s. Anti-E-selectin monoclonal antibody, CL2/6, inhibited this interaction by > 90%. Rolling neutrophils often transiently stopped, but in contrast to the behavior on stimulated endothelial cells, they remained spherical in shape and did not migrate on or beneath the monolayer. A possible contribution of neutrophil L-selectin to this interaction was indicated by the findings that anti-L-selectin monoclonal antibody, DREG-56, inhibited E-selectin-dependent adhesion under flow by > 65%, and there was a highly significant correlation between surface levels of L-selectin and E-selectin-dependent adhesion under flow. E-selectin also appeared to support neutrophil adhesion to IL-1 beta-stimulated endothelial cells under conditions of flow, but it accounted for only congruent to 30% of the level of adherence, in contrast to L-selectin which accounted for > 65%. Thus, both L-selectin and E-selectin can support neutrophil adhesion at wall shear stresses that preclude intercellular adhesion molecule-1-dependent adhesion, and they participate in neutrophil adherence to stimulated endothelial cells under conditions of flow.

Unusually large von Willebrand factor multimers preferentially promote young sickle and nonsickle erythrocyte adhesion to endothelial cells.

Abstract: Sickle red blood cells (RBC) suspended with endothelial cell (EC)-derived unusually large (UL) von Willebrand factor (vWF) multimers, but not large plasma vWF forms, adhered to human venous EC under shear flow conditions When sickle RBC were separated by density gradient centrifugation, fractions rich in less dense RBC were the most adhesive to EC in the presence of ULvWF Incubation of sickle RBC with monoclonal antibodies against platelet surface receptors GPIb or GPIIb/IIIa, or with the integrin receptor agonist Arg-Gly-Asp-Ser (RGDS) decreased the ULvWF-mediated sickle RBC adhesion to EC 84%, > 99%, and 90%, respectively When incubated with EC before the flow studies, anti-GPIb antibody and RGDS inhibited the ULvWF-mediated sickle RBC adhesion to EC ULvWF also promoted the adhesion to EC of nonsickle RBC (HbAA) from patients with an increased proportion of young erythrocytes When the EC supernatant was depleted of most vWF forms, young nonsickle RBC adhesion decreased by 90% Preincubation of young nonsickle RBC with anti-GPIb antibody, anti-GPIIb/IIIa antibody, or RGDS inhibited the ULvWF-mediated young RBC adhesion to EC by 47%, 88%, and 92%, respectively These data indicate that (1) low-density erythrocyte fractions enriched in young sickle or young nonsickle RBC are capable of binding ULvWF multimers via GPIb-like and GPIIb/IIIa-like receptors; (2) the RBC vWF receptors are lost or modified as erythrocytes age in the circulation; and (3) ULvWF/RBC complexes also bind to EC via a GPIb-like receptor

Effect of shear stress on86Rb+ efflux from calf pulmonary artery endothelial cells

Abstract: The effect of flow-induced shear stress on membrane K+ permeability was investigated by measuring86Rb+ efflux in cultured calf pulmonary artery endothelial cells. Cells were subjected to step changes in shear stress from 1 dyn/cm2 to 2.4, 4.8, or 10 dyn/cm2 in a parallel-plate flow chamber. Increasing shear stress produced a graded, transient increase in86Rb+ efflux which peaked within 1 min and subsequently declined rapidly toward pre-stimulus levels. Upon returning shear stress to 1 dyn/cm2,86Rb+ efflux initially decreased, but returned slowly to basal values. In contrast, application of bradykinin at a constant shear stress of 1 dyn/cm2 produced a transient increase in86Rb+ efflux that was followed by a sustained elevated phase during which time efflux gradually returned to pre-stimulus levels. In order to exclude the possibility that the transient increase in86Rb+ efflux with shear stress simply reflects a flow-dependent change in the washout of radiotracer, the transient convection-diffusion equation was solved using finite element simulation. When the flux of86Rb+ from the cell monolayer was assumed to be constant with time, the mathematical model predicted an increase in efflux rate coefficients upon step increases in flow that were only 7–19% of that observed experimentally. The numerical predictions correlated well with the experimentally obtained peaks when the flux of86Rb+ from the cell monolayer was simultaneously increased with flow to a new steady value. These simulations however, could not predict the transient nature of the response to increased shear stress. The results from the computer modeling suggest that the transient increase in86Rb+ efflux does not reflect a washout phenomenon and supports the hypothesis that shear stress produces a graded, transient increase in the K+ permeability of vascular endothelial cells.

Neutrophil adhesion to endothelial cells.

Abstract: The emigration of neutrophils at sites of inflammation apparently requires intercellular adhesion. Initially, leukocyte adherence is observed in postcapillary venules where neutrophils roll along the luminal surface of the endothelial cells before stopping, changing shape, and migrating into the perivascular tissue. Recent evidence indicates that the adhesion molecules supporting the rolling phenomenon are distinct from those required for stopping and transmigration. The contribution of E-selectin (ELAM-1) to neutrophil adhesion and rolling was investigated using anti-E-selectin monoclonal antibody in an in vitro adhesion assay of isolated human neutrophils to murine L-cell monolayers stably transfected with human E-selectin cDNA. Isolated human neutrophils adhered to E-selectin expressing L-cell monolayers under physiological wall shear stress of 1.85 dynes/cm2 but not to untransfected L-cells or ICAM-1 expressing L-cells. 47.8 +/- 6.0% of these adherent cells were rolling at an average rolling velocity of 10.6 +/- 1.7 microns/second. This adhesion and rolling was almost completely blocked by anti-E-selectin monoclonal antibody. Monoclonal antibody to L-selectin also reduced adhesion to E-selectin expressing cells by 70%. Chemotactic stimulation of neutrophils reduced both the number of adherent and rolling cells and the average velocity of the rolling cells without influencing the percentage of attached cells that were rolling. Pretreatment with anti-CD18 monoclonal antibody did not reduce the adhesion of the activated neutrophils but reversed the reduction in velocity caused by activation of these cells. The inhibitory potential of the anti-E-selectin monoclonal antibody was much less pronounced in adhesion of isolated neutrophils to human umbilical vein endothelial cell monolayers under conditions of flow and was limited to one third of the total adhesion. The proportion of the adherent neutrophils which transmigrated to the subluminal space of the endothelial cell monolayers was independent of pretreatment with anti-E-selectin monoclonal antibody.

Computerized Analysis of Tumor Cells Flowing in a Parallel Plate Chamber to Determine Their Adhesion Stabilization Lag Time

Abstract: The importance of cell adhesion in a variety of physiological phenomena requires development of an understanding of the factors and molecular mechanisms underlying these behaviors. Cell adhesion is a multistep process involving primary receptor-ligand interactions followed by secondary events that may lead to the formation of focal contacts. Due to the lack of well-defined assays to study adhesion stabilization, little is known about this process, except that it may involve signaling events, receptor recruitment, and, as we have demonstrated, covalent peptide cross-linking by cell membrane-associated transglutaminase [Menter et al.: Cell Biophys. 18:123–143, 1992]. To study the stabilization process we have developed a dynamic assay employing a parallel plate flow chamber coupled with video microscopy and digital image processing. Our studies utilize wheat germ agglutinin-selected human metastatic melanoma cell variants that exhibit differences in their experimental metastatic potential and expression of transglutaminase. Using this assay, quantifying cell-substrate stabilization was found to be quick, reliable, reproducible, and useful in evaluating agents that block this process. © 1993 Wiley-Liss, Inc.

Tumor Cell - Substrate Stabilization Mediated by Integrins

Abstract: Metastasis formation is dependent on the arrest and stabilization of adhesive interactions to prevent detachment from secondary sites. Primary receptor-ligand interactions are not sufficient to maintain prolonged adhesive contacts without secondary events that lead to stabilization. Tumor cell arrest and stabilization were studied under physiologically relevant shear conditions. We used a parallel-plate flow chamber with surfaces coated with human plasma fibronectin or vitronectin. Our previous work suggested that stabilization of cells to immobilized proteins is in part attributed to transglutaminase covalently cross-linking cytoskeletal-integrin-fibronectin multiprotein complexes via lysine-glutamine linkages. To study the role of integrins in mediating arrest and initiating stabilization we used a human melanoma line (70w) and polyclonal antibodies that inhibit the function of the fibronectin (α 5 β 1 ) and vitronectin (α v β 3 /β 5 ) integrin receptors. To confirm the role of integrins in initiating stabilization we used CHO (Chinese hamster ovary) cells selected for low levels of α 5 β 1 integrin expression and integrin transfected CHO cells selected for α 5 β l overexpression. The level of fibronectin receptor surface expression was inversely related to the adhesion stabilization lag time. These studies confirmed that integrins are essential for mediating arrest and initiating stabilization. They also confirm that secondary events are necessary for complete stabilization to occur. Finally, it is important to note that the arrest and stabilization methods we have developed are capable of detecting biologic effects at far greater sensitivity than static adhesion assays. Some examples of pharmacologic agents or biomaterials effects that can be detected using stabilization assays include: 1) very low drug doses, 2) very low levels of peptide, carbohydrate, and antibody inhibitors, 3) slight modification of endogenous protein expression by antisense oligonucleotides or transfected genetic expression constructs.

Adhesive Interactions of Neutrophils with Endothelial Cells

Abstract: The adhesion of neutrophils to endothelial cells appears to be necessary for their localization at sites of inflammation, and numerous studies show that the β 2 (CD18) integrins are involved in this process. For example, patients with CD18 deficiency have markedly reduced or absent β 2 integrins on their leukocytes, and biopsies of infected lesions reveal few if any neutrophils in extravascular tissues [1]. Additionally, it is now clear that anti-CD 18 monoclonal antibodies (MAb) profoundly inhibit the extravasation of neutrophils at many different sites of inflammation in animal models [2]. Furthermore, a member of another family of adhesion molecules [3], LECAM-1 (also known as the MEL-14 antigen or peripheral lymph node homing receptor) was shown to participate in the extravasation of neutrophils in a murine peritonitis model [4]. LECAM-1 is a member of the LECAM family [3] and is constitutively expressed on the neutrophil surface.