Parallel-plate Flow Chamber Assay Research Articles

Sphingosine kinase-1 mediates TNF activation of the integrin VLA-5 for leukocyte adhesion (94.26)

Abstract For leukocyte recruitment to sites of inflammation the classical paradigm states that selectins (eg P- and E-selectin) are important for initial tethering and rolling with subsequent interaction via integrins and cellular adhesion molecules (eg α4β1, VCAM-1 and ICAM-1) enabling their adhesion and emigration from the microvasculature. Previous studies have shown that the lipid enzyme sphingosine kinase-1 (SK-1) is essential for TNFα-induced E-selectin, VCAM-1 and ICAM-1 expression on endothelial cells (EC). However, its role in regulating the expression of other key adhesion molecules is still unclear. Herein we demonstrate that TNFα activates the integrin VLA-5 (α5β1) without altering total expression levels on human umbilical vein ECs. Moreover, inhibition studies with an SK-1 mutant suggest that TNFα-induced β1 activation is SK-1 dependent. Finally, a parallel plate flow chamber assay suggests that leukocyte adhesion to the TNFα-treated endothelium under shear stress can be attenuated by blocking VLA-5 or its ligand angiopoitein-2 on the ECs. Taken together, these results suggest that TNFα-induced activation of VLA-5 is SK-1 dependent and mediates, in part, leukocyte adhesion to the endothelium. Given that excessive leukocyte recruitment during inflammation results in chronic and fatal diseases, the regulation of integrins such as VLA-5 is key for developing successful therapeutics to alleviate disease. This work was supported by an NHMRC Project Grant, Florey Fellowship and Arthritis Australia.

Ethanol Enhances Neutrophil Membrane Tether Growth and Slows Rolling on P-Selectin but Reduces Capture from Flow and Firm Arrest on IL-1-Treated Endothelium

The effects of ethanol at physiological concentrations on neutrophil membrane tether pulling, adhesion lifetime, rolling, and firm arrest behavior were studied in parallel-plate flow chamber assays with adherent 1-microm-diameter P-selectin-coated beads, P-selectin-coated surfaces, or IL-1-stimulated human endothelium. Ethanol (0.3% by volume) had no effect on P-selectin glycoprotein ligand-1 (PSGL-1), L-selectin, or CD11b levels but caused PSGL-1 redistribution. Also, ethanol prevented fMLP-induced CD11b up-regulation. During neutrophil collisions with P-selectin-coated beads at venous wall shear rates of 25-100 s(-1), ethanol increased membrane tether length and membrane growth rate by 2- to 3-fold but reduced the adhesion efficiency (detectable bonding per total collisions) by 2- to 3-fold, compared with untreated neutrophils. Without ethanol treatment, adhesion efficiency and adhesion lifetime declined as wall shear rate was increased, whereas ethanol caused the adhesion lifetime over all events to increase from 0.1 s to 0.5 s as wall shear rate was increased, an example of pharmacologically induced hydrodynamic thresholding. Consistent with this increased membrane fluidity and reduced capture, ethanol reduced rolling velocity by 37% and rolling flux by 55% on P-selectin surfaces at 100 s(-1), compared with untreated neutrophils. On IL-1-stimulated endothelium, rolling velocity was unchanged by ethanol treatment, but the fraction of cells converting to firm arrest was reduced from 35% to 24% with ethanol. Overall, ethanol caused competing biophysical and biochemical effects that: 1) reduced capture due to PSGL-1 redistribution, 2) reduced rolling velocity due to increased membrane tether growth, and 3) reduced conversion to firm arrest.

Vascular Consequences of Cocaine Exposure.

Cocaine use is associated with sudden cardiac death, cardiac ischemia, and stroke in patients with no additional risk factors and is a frequent cause of these syndromes in patients with sickle cell disease. Pathologic findings include platelet-rich microthrombi and an increased plasma concentration of von Willebrand Factor (VWF). These findings suggest that, in addition to the well-known cocaine effects of vasoconstriction and increased tissue oxygen demand, activation of platelets and/or endothelial cells contributes to cocaine-induced ischemia. However, studies investigating the effect of cocaine on platelet functions have been inconclusive, finding both platelet activation and inhibition depending on the assay used. Further, the ability of cocaine to activate the vascular endothelium has not been examined, in particular, the endothelial secretion of the most adhesive forms of VWF, the ultralarge forms (ULVWF). ULVWF are long VWF multimers that remain tethered to the endothelial surface upon secretion, extend into the blood vessel lumen under laminar flow in long strings measuring up to 0.5 cm in length, and have multiple exposed binding sites for receptors on platelets, erythrocytes, and leukocytes. Elevated levels of ULVWF, due either to enhanced secretion or defective processing, have been implicated in diseases such as thrombotic thrombocytopenic purpura (TTP) and sickle cell anemia. We hypothesized that a major consequence of cocaine exposure is activation of the vascular endothelium to secrete ULVWF, which would provide a platform for blood cell adhesion and subsequent thrombosis or vaso-occlusion. We evaluated the ability of cocaine to stimulate ULVWF from cultured endothelial cells in a parallel-plate flow chamber assay and found that 1 μg/ml cocaine, a level comparable to peak blood levels detectable in cocaine abusers, efficiently induced secretion of ULVWF capable of binding platelets under flow conditions similar to that induced by histamine 6 μg/ml (3.92 ULVWF strings/field with cocaine vs. 4.62 strings/field with histamine). We also assessed the activation of platelets exposed in vitro to cocaine by flow cytometry, using two markers of platelet activation: P-selectin expression (which signals a-granule release) and conformational activation of the platelet integrin aIIbb3, detected with the antibody PAC-1. We found that when platelet-rich plasma was incubated with cocaine at concentrations from 0.1 μg/ml to 10 μg/ml, there was no increase in P- selectin exposure or PAC-1 binding. Furthermore, pretreatment of platelets with cocaine inhibited the ability of platelets to subsequently be activated by ADP in a dose-dependent manner. We did not observe any increase in mean fluorescence above background in ADP stimulated platelets pre-incubated with 1 μg/ml cocaine for P-selectin or PAC-1 binding. However, exposure of platelets in whole blood to 1 μg/ml cocaine resulted in a 3.2-fold increase in P- selectin exposure and a 5.4-fold increase in PAC-1 binding. These results indicate that cocaine directly activates the vascular endothelium to secrete ULVWF, and activates platelets indirectly, involving as yet unknown factors in whole blood, resulting in the formation of microthrombi. These effects of cocaine are likely to have pathogenic roles in cardiovascular syndromes associated with cocaine use, including the triggering of vaso-occlusive crises in sickle cell anemia and may explain the observed association of cocaine use with TTP.

Suppressor and effector T cells exhibit differential migration capacity

Induction of immunological tolerance via anergic or regulatory T cells is a potential therapeutic strategy for immunological diseases such as allergy and autoimmunity. Despite extensive investigation into the mechanism of anergic and regulatory T cell-mediated immune suppression, the exact mechanism is still unclear. This project investigated the migration characteristics of anergic and regulatory T cells, in comparison to conventional T cells, by using a parallel-plate flow chamber assay and ova albumin (OVA)-TCR transgenic DO11.10 mice. Anergic T cells were generated by treating DO11.10 splenocytes with anti-CD80, anti-CD86 and anti-ICOSL blocking Abs before stimulation by OVA. On flowing through ICAM-1 and E-selectin at shear stress of 1.5 dynes/cm2, anergic T cells exhibited a significant reduction of adhesion as compared to OVA-stimulated effector T cells (71% reduction, P<0.001). In the same flow chamber system, activated CD4+CD25+ regulatory T cells isolated from DO11.10 mice also showed a significantly decreased adhesion compared to CD4+CD25− T cells (76% reduction, P<0.001). Reduced adhesion of the suppressor cells was also observed when using an endothelial cell-based flow chamber system. These findings demonstrate differential migration phenotypes of suppressor and effector T cells, suggesting distinct migration property may contribute to the execution of individual immune function. Funded by the Taiwan National Science Council and National Taiwan University Hospital

Evidence for a differential functional regulation of the two β3-integrins αVβ3 and αIIbβ3

The functional regulation of integrins is a major determinant of cell adhesion, migration and tissue maintenance. The binding of cytoskeletal proteins to various sites of integrin cytoplasmic domains is a key mechanism of this functional regulation. Expression of recombinant integrin αIIbβ3 and αMβ2 lacking the GFFKR-region in CHO cells results in constitutively activated integrins. In contrast, CHO cells stably expressing either a GFFKR-deleted αV(del)β3 or a FF to AA-substituted αV(AA)β3 do not reveal a constitutively activated integrin. Adhesion to immobilized fibrinogen is strongly impaired in αV(del)β3 or αV(AA)β3-expressing cells, whereas it is not impaired in αIIbβ3 and αMβ2, both lacking the GFFKR-region. In a parallel plate flow chamber assay, αVβ3-expressing cells adhere firmly to fibrinogen and spread even at shear rates of 15 to 20 dyn/cm2, whereas αV(del)β3 or αV(AA)β3 cells are detached at 15 dyn/cm2. Actin stress fiber formation and focal adhesion plaques containing αVβ3 are observed in αVβ3 cells but not in αV(del)β3 or αV(AA)β3-expressing cells. As an additional manifestation of impaired outside–in signaling, phosphorylation of pp125FAK was reduced in these cells. In summary, we report that the GFFKR-region of the αV-cytoplasmic domain and in particular two phenylalanines are essential for integrin αVβ3 function, especially for outside–in signaling. Our results suggest that the two β3-integrins αIIbβ3 and αVβ3 are differentially regulated via their GFFKR-region.

Effect of Hydroxyurea Treatment on In Vitro Adhesion of Sickle Erythrocytes to Sickle Leukocyte Subpopulations.

Microvascular vaso-occlusion in sickle cell disease is thought to involve adhesive interactions among erythrocytes (RBCs), leukocytes and vascular endothelial cells. Recent studies have demonstrated the presence of a significant inflammatory response in sickle cell disease, including changes in the cell surface adhesion molecules that mediate cell-cell interactions in the microvasculature. In this study, we used a parallel-plate flow chamber assay to determine the subpopulations of leukocytes that are involved in sickle leukocyte-RBC interactions. We also studied the effect of treatment with hydroxyurea (HU) on these adhesive interactions. Populations of monocytes, neutrophils (PMNs) and T cells were isolated by negative selection from the peripheral blood of untreated patients with sickle cell disease (SS), sickle patients receiving HU (SS-HU), and healthy control subjects (AA). Adhesive interactions involving these leukocyte subpopulations, human umbilical vein endothelial cells (HUVECs) pretreated with tumor necrosis factor-α (TNF-α ), and autologous RBCs were measured under a shear stress of 1 dyne/cm2. Compared to the corresponding cell populations from AA individuals, PMNs, monocytes, and T cells from SS individuals were significantly more adherent to TNF-α-treated HUVECs (774±59 vs. 502±27 cells/mm2, p=0.001; 533±66 vs. 348±36 cells/mm2, p=0.024; and 470±75 vs. 227±26 cells/mm2, p=0.009, respectively). HU therapy significantly decreased the adhesion of SS PMNs to HUVECs (774±59 cells/mm2 for SS vs. 604±36 for SS-HU, p=0.025). Compared to adherent AA leukocytes, adherent SS leukocytes exhibited greater participation in adhesive interactions with autologous RBCs (41±3% for SS vs. 27±3% for AA, p=0.002), and HU treatment decreased the fraction of leukocytes that captured autologous RBCs to the control level (29±3% for SS-HU, p=0.006 vs. SS). Compared to adherent PMNs from SS individuals, adherent PMNs from SS-HU individuals showed significantly reduced participation in the capture of RBCs (53±6% for SS vs. 35±5% for SS-HU, p=0.021). Although adherent T cells from SS individuals participated significantly more in RBC capture than adherent T cells from AA individuals (28±5% for SS vs. 10±2% for AA, p=0.007), HU therapy did not have a significant effect on this parameter (21±5% for SS-HU, p=0.373). Compared to AA leukocytes, SS leukocytes captured more RBCs per participating adherent leukocyte (2.8±0.2 vs. 1.9±0.1 RBCs/cell, p=0.001). HU therapy reduced the number of RBCs captured per PMN but not the number captured per T cell. Compared to AA T cells, SS T cells captured adherent RBCs for a significantly longer period of time (51±9 vs. 26±6 seconds, p=0.035). Our data suggest that sickle neutrophils, monocytes and T cells may all be involved in adhesive interactions with sickle RBCs. PMN-RBC and monocyte-RBC interactions appear to be more numerous than T cell-RBC interactions, although T cell-RBC interactions may be stronger. HU therapy appears to target PMN-RBC and monocyte-RBC interactions preferentially. Future studies will focus on the role of particular adhesion molecules in mediating these interactions and on the potential for therapeutic interventions targeting cell-cell adhesion.

Human fractalkine mediates leukocyte adhesion but not capture under physiological shear conditions; a mechanism for selective monocyte recruitment.

Fractalkine is a unique chemokine possessing a long mucin-like stalk and a transmembrane region that has been proposed to act as an adhesion molecule. We investigated the ability of fractalkine to recruit leukocytes from whole blood, using an immobilized fractalkine fusion protein in the parallel-plate flow-chamber assay. Significant adhesion of leukocytes to fractalkine peaked at 2 dynes/cm(2) but was minimal at 10 dynes/cm(2). In contrast, VCAM-1 could recruit cells from whole blood at 10 dynes/cm(2). Co-immobilization of fractalkine and VCAM-1 at 10 dynes/cm(2) resulted in a twofold increase in adherent cells compared with VCAM-1 alone, suggesting that fractalkine can mediate adhesion at high shear if combined with a molecule that can mediate leukocyte tethering. Pretreatment of blood with pertussis toxin eliminated this increase in adhesion, implicating intracellular signaling in fractalkine-mediated mechanisms of adhesion to co-immobilized fractalkine/VCAM-1. Analysis of the cell types recruited to fractalkine alone at low shear, or to fractalkine and VCAM-1 at 10 dynes/cm(2), revealed that monocytes were recruited to fractalkine with the highest specificity. In conclusion, fractalkine is unlikely to act alone at shear forces found in most vascular beds where it most likely co-operates with tethering molecules, e.g. VCAM-1, in the recruitment of monocytes.

Differential L-Selectin Binding Activities of Human Hematopoietic Cell L-Selectin Ligands, HCELL and PSGL-1

Expression of L-selectin on human hematopoietic cells (HC) is associated with a higher proliferative activity and a more rapid engraftment after hematopoietic stem cell transplantation. Two L-selectin ligands are expressed on human HCs, P-selectin glycoprotein ligand-1 (PSGL-1) and a specialized glycoform of CD44 (hematopoietic cell E- and L-selectin ligand, HCELL). Although the structural biochemistry of HCELL and PSGL-1 is well characterized, the relative capacity of these molecules to mediate L-selectin-dependent adhesion has not been explored. In this study, we examined under shear stress conditions L-selectin-dependent leukocyte adhesive interactions mediated by HCELL and PSGL-1, both as naturally expressed on human HC membranes and as purified molecules. By utilizing both Stamper-Woodruff and parallel-plate flow chamber assays, we found that HCELL displayed a 5-fold greater capacity to support L-selectin-dependent leukocyte adherence across a broad range of shear stresses compared with that of PSGL-1. Moreover, L-selectin-mediated leukocyte binding to immunopurified HCELL was consistently >5-fold higher than leukocyte binding to equivalent amounts of PSGL-1. Taken together, these data indicate that HCELL is a more avid L-selectin ligand than PSGL-1 and may be the preferential mediator of L-selectin-dependent adhesive interactions among human HCs in the bone marrow.

Exclusive Neutrophil Recruitment with Oncostatin M in a Human System

Oncostatin M (OSM), a member of the IL-6 family has been postulated to be a potent recruiter of leukocytes, however information regarding the molecular mechanism(s) underlying this event is extremely limited. Therefore, the aim of this study was to investigate the role of OSM-mediated leukocyte recruitment in a human system in vitro under flow conditions. A parallel-plate flow chamber assay was used to examine leukocyte recruitment from whole blood by human umbilical vein endothelium treated for 24 hours with OSM. OSM in a dose-response manner revealed very significant leukocyte rolling and adhesion reaching optimal levels at a very low concentration of OSM (10 ng/ml). The OSM-induced leukocyte rolling and adhesion was comparable to levels seen with tumor necrosis factor. OSM was extremely selective for neutrophil recruitment (96%) with <3% lymphocyte recruitment. By contrast, tumor necrosis factor-alpha revealed no such selectivity, recruiting 70% neutrophils and at least 25% lymphocytes and detectable levels of eosinophils at 24 hours. The molecular mechanism underlying the leukocyte recruitment seemed to be entirely dependent on P-selectin as leukocyte recruitment could be completely blocked by the addition of a P-selectin-blocking antibody. An elevation in both P-selectin message and protein was observed with 24 hours of OSM stimulation of endothelium. By contrast, E-selectin and VCAM-1 were not detectable after OSM stimulation. Similar results were seen with passaged dermal microvascular endothelium that does not have a prestored pool of P-selectin. Based on these results, we conclude that OSM may be a very selective potent recruiter of neutrophils in more prolonged inflammatory conditions, an event exclusively dependent on P-selectin.

Bartonella henselae induces NF-kappaB-dependent upregulation of adhesion molecules in cultured human endothelial cells: possible role of outer membrane proteins as pathogenic factors.

The endothelium is a specific target for Bartonella henselae, and endothelial cell infection represents an important step in the pathogenesis of cat scratch disease and bacillary angiomatosis. Mechanisms of Bartonella-endothelial cell interaction as well as signaling pathways involved in target cell activation were analyzed. B. henselae strain Berlin-1, isolated from bacillary angiomatosis lesions of a human immunodeficiency virus-infected patient, potently stimulated human umbilical cord vein endothelial cells (HUVEC), as determined by NF-kappaB activation and enhanced adhesion molecule expression. These effects were accompanied by increased PMN rolling on and adhesion to infected endothelial cell monolayers, as measured in a parallel-plate flow chamber assay. Monoclonal antibodies against E-selectin significantly reduced PMN rolling and adhesion. In our hands, B. henselae Berlin-1 was substantially more active than the typing strain B. henselae ATCC 49882. E-selectin and ICAM-1 upregulation occurred for up to 9 days, as verified by Northern blotting and cell surface enzyme-linked immunosorbent assay. Induction of adhesion molecules was mediated via NF-kappaB activation and could be blocked by a specific NF-kappaB inhibitor. Additional studies indicated that B. henselae-induced effects did not require living bacteria or Bartonella lipopolysaccharides. Exposure of HUVEC to purified B. henselae outer membrane proteins (OMPs), however, reproduced all aspects of endothelial cell activation. In conclusion, B. henselae, the causative agent of cat scratch disease and bacillary angiomatosis, infects and activates endothelial cells. B. henselae OMPs are sufficient to induce NF-kappaB activation and adhesion molecule expression followed by enhanced rolling and adhesion of leukocytes. These observations identify important new properties of B. henselae, demonstrating its capacity to initiate a cascade of events culminating in a proinflammatory phenotype of infected endothelial cells.

Chemoattractants induce a rapid and transient upregulation of monocyte alpha4 integrin affinity for vascular cell adhesion molecule 1 which mediates arrest: an early step in the process of emigration.

Chemoattractants and chemokines induce arrest of rolling monocytes during emigration from blood into tissues. In this study, we demonstrated that alpha4 integrin affinity for vascular cell adhesion molecule (VCAM)-1 was upregulated rapidly and transiently by chemoattractants and stromal cell-derived factor (SDF)-1alpha and mediated monocyte arrest. alpha4 integrin affinity changes were detected and blocked using soluble VCAM-1/Fc (sVCAM-1/Fc). In a flow cytometry assay, markedly increased sVCAM-1/Fc binding to human blood monocytes or U937 cells transfected with formyl peptide (FP) receptor was detected 30 s after FP or SDF-1alpha treatment and declined after 2 min. In a parallel plate flow chamber assay, FP, C5a, platelet-activating factor, or SDF-1alpha coimmobilized with VCAM-1 induced leukocyte arrest, which was blocked by inclusion of sVCAM-1/Fc but not soluble nonimmune immunoglobulin G in the assay buffer.

A numerical analysis of forces exerted by laminar flow on spreading cells in a parallel plate flow chamber assay

Exposure of spreading anchorage-dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two-dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm(2), whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area.