Unstimulated Endothelium Research Articles

An Ex Vivo Test of Complement Activation on Endothelium for Individualized Eculizumab Therapy in Hemolytic Uremic Syndrome

Although primary atypical hemolytic uremic syndrome (aHUS) is associated with abnormalities in complement genes and antibodies to complement factor H, the role ofcomplement in secondary aHUS remains debatable. We evaluated the usefulness of an exvivo test to: (1) detect complement activation within the endothelium in primary and secondary aHUS, (2) differentiate active disease from remission, (3) monitor the effectiveness of eculizumab therapy, and (4) identify relapses during eculizumab dosage tapering and after discontinuation of treatment. Case series. 121 patients with primary aHUS and 28 with secondary aHUS. Serum samples were collected during acute episodes, following remission, and during eculizumab treatment and were assessed using a serum-induced exvivo C5b-9 endothelial deposition test. Serum-induced C5b-9 deposition on cultured microvascular endothelium was quantified by calculating the endothelial area covered by C5b-9 staining; values were expressed as percentage of C5b-9 deposits induced by a serum pool from healthy controls. Testing with adenosine diphosphate-activated endothelium demonstrated elevated C5b-9 deposits for all untreated patients with aHUS independent of disease activity, while testing with unstimulated endothelium demonstrated deposits only in active disease. Similar findings were observed in secondary aHUS. Serum-induced C5b-9 deposits on activated and unstimulated endothelium normalized during eculizumab treatment. 96% (22/23) of patients receiving eculizumab at extended 3- or 4-week dosing intervals demonstrated normal C5b-9 deposits on activated endothelium, despite most patients having CH50Eq (serum complement activity) > 20 UEq/mL, indicating that adequate complement control was achieved even with incomplete blockade of circulating C5. During eculizumab dosage tapering or after treatment discontinuation, all patients experiencing relapses versus only 6% (1/17) of those in stable remission had elevated C5b-9 deposits on unstimulated endothelium. The C5b-9 endothelial deposition test can be performed in only specialized laboratories. Findings on eculizumab dosage tapering need to be confirmed with longitudinal monitoring of C5b-9 deposition. The C5b-9 endothelial deposition assay may represent an advance in our ability to monitor aHUS activity and individualize therapy.

OR7 HLA expression on T and B cells – Comparisons to inflamed endothelium

Aim Crossmatches outcome is often interpreted as absolute proof of donor-specific reactivity that directly correlates to the risk of antibody-mediated rejection (ABMR); an immune attack on donor microvascular endothelium. However, direct comparisons of endothelial cell HLA expression to that of T and B cells have not been performed. We assessed HLA expression using global gene expression to compare HLA Class I and II expression in T cells, B cells, and human umbilical vein endothelial cells (HUVEC) under conditions that simulate immune attack. Methods CD4 and CD8 T cells and B cells were isolated from healthy volunteers. HUVECs were cultured under recommended culture conditions and remained unstimulated, or IFN-g treated (24 h), or IFN-g treated followed by the addition of polyclonal HLA antibodies and NK cells/monocytes to simulate inflamed ABMR immune attack. Expression was calculated as the geometric mean across each set of samples ( ⩾ 3 for each condition). Results HLA-A, -B, and -C levels were similar between CD4 and CD8 T cells but were higher than levels on B cells [Fig. 1A]. Unstimulated endothelium showed low Class I expression which was upregulated by IFN-g treatment, and most highly increased under inflamed conditions. HLA Class I levels on endothelium under inflamed conditions mirrored those in B cells but were >25% less than those on T cells for HLA-A and -B. B cells expressed high levels of HLA-DRB1, -DQB1, and -DPB1 [Fig. 1B]. However, even under intense immune attack, endothelial cells only expressed a fraction of B cell levels; 47% for -DQB1, 26% for -DRB1, and 17% for -DPB1. Conclusions Our data suggest that crossmatch interpretation derived from T cell reactivity may closely resemble what is predicted to be maximal endothelial HLA Class I expression. In contrast, reactivity observed against HLA Class II on B cells likely exaggerates the maximum levels observed on endothelium under immune attack.

IL-36γ Is a Strong Inducer of IL-23 in Psoriatic Cells and Activates Angiogenesis.

The IL-1 family member cytokine IL-36γ is recognised as key mediator in the immunopathology of psoriasis, hallmarks of which involve the activation of both resident and infiltrating inflammatory myeloid cells and aberrant angiogenesis. This research demonstrates a role for IL-36γ in both myeloid activation and angiogenesis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23 and TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. IL-36γ was also demonstrated to induce endothelial tube formation and branching, in a VEGF-A-dependent manner. Furthermore, IL-36γ-stimulated macrophages potently activated endothelial cells and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, psoriasis monocytes showed increased adherence to both the stimulated and unstimulated endothelium when compared with monocytes from healthy individuals. Collectively, these findings show that IL-36γ has the potential to enhance endothelium directed leucocyte infiltration into the skin and strengthen the IL-23/IL-17 pathway adding to the growing evidence of pathogenetic roles for IL-36γ in psoriatic responses. Our findings also point to a cellular response, which could potentially explain cardiovascular comorbidities in psoriasis in the form of endothelial activation and increased monocyte adherence.

Ca2 +-dependent potassium channels and cannabinoid signaling in the endothelium of apolipoprotein E knockout mice before plaque formation

Endothelial Ca2+-dependent K+ channels (KCa) regulate endothelial function. We also know that stimulation of type 2 cannabinoid (CB2) receptors ameliorates atherosclerosis. However, whether atherosclerosis is accompanied by altered endothelial KCa- and CB2 receptor-dependent signaling is unknown. By utilizing an in situ patch-clamp approach, we directly evaluated the KCa channel function and the CB2 receptor-dependent electrical responses in the endothelium of aortic strips from young ApoE−/− and C57Bl/6 mice. In the ApoE−/− group, the resting membrane potential (−30.1±1.1mV) was less negative (p<0.05) compared to WT (−38.9±1.4mV) and voltage ramps generated an overall KCa current of reduced amplitude. The peak hyperpolarization to 2μM Ach was not different between the groups. However, the sustained component was significantly reduced in ApoE−/− strips. In contrast, the peak hyperpolarization to 0.2μM Ach was increased in the ApoE−/− group, and SKA-31, a direct IKCa/SKCa channel opener, produced a hyperpolarization and whole-cell current of greater amplitude. The BKCa opener NS1619 produced hyperpolarization that was enhanced in ApoE−/− group. N-arachidonoyl glycine, a BKCa opener, produced a hyperpolarization of enhanced amplitude in ApoE−/− arteries. Selective CB2 receptor agonist AM1241 (5μM) had no effect on endothelial membrane potential in WT group; however, in ApoE−/− group, it elicited hyperpolarization that was inhibited by a selective CB2 receptor antagonist AM630. Conclusively, our data point to functional down-regulation of basal IKCa activity in unstimulated endothelium of ApoE−/− mice. Direct and indirect IKCa stimulation resulted in increased recruitment of the channels. In addition, our data point to up-regulation of endothelial BKCa channels and CB2 receptors in ApoE−/− arteries.

Regulation of the Micromechanical Properties of Pulmonary Endothelium by S1P and Thrombin: Role of Cortactin

Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as sepsis and acute respiratory disease stress syndrome. Actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. Here, we used atomic force microscopy to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing (induced by sphingosine 1-phosphate (S1P)) and barrier-disrupting (induced by thrombin) conditions. Atomic force microscopy elasticity measurements show differential effects: for the barrier protecting molecule S1P, the elastic modulus was elevated significantly on the periphery; for the barrier-disrupting molecule thrombin, on the other hand, it was elevated significantly in the central region of the cell. The force and elasticity maps correlate with F-actin rearrangements as identified by immunofluorescence analysis. Significantly, reduced expression (via siRNA) of cortactin, an actin-binding protein essential to EC barrier regulation, resulted in a shift in the S1P-mediated elasticity pattern to more closely resemble control, unstimulated endothelium.

Combinations of low concentrations of cytokines and acute agonists synergize in increasing the permeability of endothelial monolayers.

The deposition of circulating immune reactants in blood vessels, an important event in the pathogenesis of certain types of vasculitis, requires an increase in permeability in the endothelial monolayer. An in vitro model to examine the integrity of endothelial cell monolayers and their response to inflammatory mediators has been developed. Human umbilical vein endothelial cells were grown to confluence on an FITC-labelled matrix and monolayer integrity was assessed by the exclusion of a 125I-anti-FITC antibody. Alteration in endothelial monolayer permeability was associated with an increase in uptake of 125I-anti-FITC antibody, expressed as a percentage of the maximal uptake of antibody on to FITC-matrix from which endothelial cells had been stripped. We determined the effects on endothelial monolayer permeability of acute agonists (thrombin and histamine), cytokines (tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-1 and IL-4) and combinations of acute agonists and cytokines. Addition of thrombin in concentrations ranging from 0.5 to 15 U/ml led to an increased uptake of 125I-anti-FITC antibody from 2% to 15% relative to unstimulated endothelium. For other agonists and cytokines the increases in permeability were: (i) histamine (50-400 pmol/ml) increased uptake 5-22%; (ii) TNF (12.5-100 ng/ml) increased uptake 2-12%; (iii) IFN-gamma (125-250 U/ml) increased uptake 1.5-3%. IL-1 beta (50-100 U/ml) and IL-4 (50-100 U/ml) had no effect. Synergistic interactions on endothelial monolayer permeability were seen with the following combinations: (i) IL-4 (100 U/ml) and TNF (12.5 ng/ml) uptake 11%; (ii) IL-4 (100 U/ml) and IFN-gamma (125 U/ml) uptake 6.5%; (iii) TNF (12.5 ng/ml) and IFN-gamma (125 ng/ml) uptake 7%; (iv) thrombin (0.5 U/ml) and histamine (50 pmol/ml) uptake 13.5%; and (v) TNF (12.5 ng/ml) and thrombin (0.5 U/ml) uptake 8.5%. These observations suggest that interactions between cytokines and acute inflammatory mediators such as thrombin and histamine may be important in determining whether immune complexes are deposited in vessel walls. This model system may now be useful for the further investigation in vitro of the mechanisms involved in the pathogenesis of immune complex-mediated vascular damage.

Increased Leukocyte-Endothelial Interactions in Syndecan-1–Deficient Mice Involve Heparan Sulfate–Dependent and –Independent Steps

Purpose: Increased leukocyte-endothelial interactions and angiogenesis are observed in the ocular vasculature of mice lacking the cell surface heparan sulfate proteoglycan syndecan-1. Here we investigate the interaction of defined leukocyte populations of syndecan-1 knockout (KO) and wild-type mice with endothelial cells in vitro. Heparin is used to substitute for the lack of syndecan-1 heparan sulfate. Methods: The adhesion of polymorphonuclear cells and monocytes purified from syndecan-1 KO and wild-type mice to unstimulated and TNF-α–treated human umbilical vein endothelial cells (HUVECs) was measured in a static adhesion assay. Results: Adhesion of syndecan-1 KO leukocytes to HUVECs is increased relative to wild-type leukocytes, being more pronounced in TNF-α –stimulated HUVECs. Heparin reverted this adhesion to wild-type levels in unstimulated endothelium. Conclusions: Syndecan-1 acts as a negative regulator of polymorphonuclear leukocytes (PMNs) and monocyte adhesion to endothelial cells. Its heparan sulfate chains play different roles in this process in unstimulated endothelia compared to TNF-α –stimulated endothelia.

Methylprednisolone acts on peripheral blood mononuclear cells and endothelium in inhibiting migration phenomena in patients with multiple sclerosis.

Intravenous methylprednisolone hemisuccinate is administered to patients with multiple sclerosis (MS) during exacerbations to improve the rate of recovery. Corticosteroids could be beneficial in MS exacerbations also by decreasing transmigration of peripheral blood mononuclear cells (PBMNCs) through the blood-brain barrier. To evaluate how in vivo intravenous methylprednisolone treatment in patients with MS could influence transmigration of PBMNCs in an in vitro model; to perform transmigration experiments through a methylprednisolone-treated endothelium with PBMNCs from untreated healthy control subjects to evaluate putative selective effects of corticosteroids on endothelium; concomitantly, to quantify the concentration of matrix metalloproteinases 2 and 9 in supernatants of PBMNCs and in serum samples from methylprednisolone-treated patients with MS; to evaluate monokine induced by interferon-gamma release in the supernatants of human umbilical vein endothelial cells treated with interferon-gamma alone or interferon-gamma and methylprednisolone; and to perform gene expression studies of matrix metalloproteinases 2 and 9 in human umbilical vein endothelial cells and PBMNCs from methylprednisolone-treated patients with MS. Eight patients with MS in exacerbation were studied before and 3 and 24 hours after intravenous methylprednisolone treatment, 1 g. The absolute number of transmigrated PBMNCs from methylprednisolone-treated patients with MS significantly (P<.01) decreased at 3 hours and increased again at 24 hours, reaching values higher than those before treatment onset. Methylprednisolone was also able to significantly (P<.03) reduce the number of PBMNCs from healthy controls migrating through interferon-gamma-stimulated or unstimulated endothelium. In vitro methylprednisolone treatment decreased monokine induced by interferon-gamma production in human umbilical vein endothelial cells. Methylprednisolone may be able to decrease transmigration of PBMNCs through the blood-brain barrier, exerting its inhibitory effects on PBMNCs and endothelium. A "rebound" of transmigration at 24 hours suggests that a single infusion is not optimal for achieving a persistent reduction in transmigration.

Intercellular Adhesion Molecule-1 Is Required for Chemoattractant-Induced Leukocyte Adhesion in Resting, but Not Inflamed, Venules in Vivo

The leukocyte integrins LFA-1 and Mac-1 bind to endothelial intercellular adhesion molecule-1 (ICAM-1). Leukocyte adhesion induced by micropipette injection of formylmethionylleucylphenylalanine (fMLP) or macrophage inflammatory protein 2 (MIP-2) next to a venule in the exteriorized mouse cremaster muscle was almost completely blocked after intravenous injection of the ICAM-1 mAb YN-1. In contrast, after 2-h pretreatment with TNF-α, leukocyte adhesion induced in postcapillary venules by fMLP or MIP-2 was not blocked by the ICAM-1 mAb. Leukocyte adhesion was significantly reduced by mAb GAME-46 to CD18 even after TNF-α treatment. We conclude that ICAM-1 is necessary for neutrophil adhesion to unstimulated endothelium, but not for adhesion to cytokine-stimulated endothelium. Although ICAM-1 is expressed at high levels after TNF-α, ICAM-1 either is not functional or is redundant with other endothelial ligands for β2 integrins.

Role of ICAM-1 and ICAM-2 and alternate CD11/CD18 ligands in neutrophil transendothelial migration.

We evaluated the relative contribution of ICAM-1 and ICAM-2, known ligands on endothelium for LFA-1 and Mac-1, in spontaneous neutrophil (PMN) transendothelial migration (TEM) across IL-1-activated HUVEC monolayers or TEM induced by C5a or IL-8 across unstimulated HUVEC grown on polycarbonate filters. Adhesion blocking mAb to ICAM-1 [R6.5 F(ab)2] or ICAM-2 [CBR IC2/2 F(ab)2] tended to inhibit TEM under each condition but, in general, inhibition was significant only with both ICAM-1 and ICAM-2 blockade. mAb to LFA-1 partially inhibited migration to C5a or IL-8 across unstimulated HUVEC and inhibition was not altered by additional treatment of HUVEC with mAbs to ICAM-1 and -2. In contrast, with IL-1 HUVEC, mAb to ICAM-1 significantly inhibited this LFA-1-independent TEM. mAb to Mac-1 alone partially inhibited TEM and, when combined with mAb to LFA-1, migration was almost completely blocked with all TEM conditions tested. The contribution of alternate ligands for Mac-1 in mediating Mac-1-dependent but ICAM-1/-2-independent C5a-induced TEM was examined using anti-LFA-1-treated PMN and anti-ICAM-treated resting HUVEC. Addition of RGD peptides, fibronectin, fibrinogen, heparins, collagens alone or in combination, even to heparinase-treated HUVEC, did not inhibit this Mac-1-mediated PMN TEM. The results indicate that: (1) LFA-1 mediates PMN TEM primarily by interaction with ICAM-1 and ICAM-2; (2) ICAM-2 may function in concert with ICAM-1 in this role, especially on unstimulated endothelium, and (3) Mac-1 on PMN also plays a major role in TEM and can utilize yet to be identified ligands distinct from ICAM-1 or -2, especially on unstimulated endothelium.

Adherence to Endothelial Cells Induces Release of Soluble Tumor Necrosis Factor (TNF) Receptor Forms from Neutrophil Granulocytes

The TNF receptors, TNF-R55 and TNF-R75, may undergo proteolytic cleavage and form soluble receptor forms, TNF-R55-BP and TNF-R75-BP. Neutrophils are abundant with both forms of TNF-receptors, while endothelial cells (ECV 304) only express TNF-R55. Human neutrophils were allowed to interact with an unstimulated or a IL-1β stimulated endothelium followed by determination of TNF-R75-BP with ELISA. Neutrophils in suspension or in contact with an unstimulated endothelium released only low amounts of TNF-R75-BP. However, neutrophils released significant amounts of TNF-R75-BP after adherence to an endothelium stimulated with IL-1β. Neutrophils were not generally activated during adherence since concomitant release of lactoferrin from neutrophils only reached levels of 1-5% compared with incubation with phorbolesters. Blocking integrins with antibodies to CD11/CD18 resulted in inhibition of both neutrophil adherence to an endothelium and shedding of TNF-R75. In addition, TNF-R55-BP decreased the production of TNF from IL-1β stimulated endothelial cells, suggesting that soluble TNF receptor forms are able to inhibit TNF production.

Granulocyte-macrophage colony-stimulating factor regulates the functional adhesive state of very late antigen-4 expressed by eosinophils.

As very late antigen-4 (VLA-4) can exist in different functional states, we have sought to determine whether a cytokine expressed by inflamed endothelium (i.e., granulocyte-macrophage CSF (GM-CSF)) could regulate the functional state of VLA-4 expressed by eosinophils. Using a micropipette single cell adhesion assay able to measure the strength of adhesion forces, eosinophils exhibited low levels of basal adhesion to unstimulated endothelium (separation force, 0.022 +/- 0.003 mdynes). In contrast, individual eosinophils bound to IL-1beta-stimulated endothelium (0.49 +/- 0.02 mdynes), TNF-stimulated endothelium (0.62 +/- 0.05 mdynes), or IL-4-stimulated endothelium (0.11 +/- 0.01 mdynes) with increased avidity as assessed by separation force. Eosinophil binding to IL-4-stimulated endothelium was significantly inhibited by neutralizing Abs to either vascular cell adhesion molecule (VCAM) or VLA-4. The strength of eosinophil adhesion to VCAM (0.31 +/- 0.02 mdynes) or to connecting segment-1 (CS-1) (0.18 mdynes) was greater than the strength of eosinophil adhesion to unstimulated endothelium (0.02 mdynes), but was less than the strength of eosinophil adhesion to IL-1beta-stimulated endothelium (0.49 +/- 0.02 mdynes). After incubating eosinophils for 30 min with GM-CSF, the mean adhesion strength of eosinophils to CS-1 and VCAM increased significantly by 84 and 54%, respectively, compared with that of controls. This increased binding of eosinophils to VCAM or CS-1 was not due to alterations in VLA-4 receptor number (assessed by FACS analysis) or alterations in VLA-4 receptor distribution (assessed by confocal microscopy). These studies suggest that endothelial-derived cytokines such as GM-CSF have the potential to alter the functional state of eosinophil-expressed VLA-4 from a low affinity state to a high affinity state.

Integrin Function in Chronic Lymphocytic Leukemia

Integrins are central to many aspects of the tissue localization of normal and malignant lymphocytes. We examined how integrin function, rather than simple expression, might determine disease behavior in chronic lymphocyte leukemia (CLL). Using fluorescence-activated cell sorting (FACS) and immunoprecipitation, we first established the precise integrin heterodimer expression of a representative group of CLL patients (beta1 consistently present, with variable alpha 3, alpha 4, and alpha 5; alpha 4 beta 7 often expressed; alpha L beta 2 high; alpha V beta 3 absent). Regarding function, we initially examined the ability of CLL cells to interact with endothelium, because such interaction is the initial event determining the entry of CLL lymphocytes into tissues. The abnormal lymphocytes were shown to bind at low levels to unstimulated endothelium via beta 2/intercellular adhesion molecule (ICAM). However, when the endothelium was stimulated, markedly enhanced interaction with endothelium was observed in approximately half the cases; in these patients, the neoplastic population expressed alpha 4 beta 1, which conferred the ability to adhere strongly to stimulated endothelium via the alpha 4 beta 1 ligand, vascular cellular adhesion molecule-1 (VCAM-1). In relation to the migration of CLL cells within tissues, the abnormal lymphocytes showed differential binding to various adhesive proteins; they did not attach to basement membrane components, but displayed variable adhesion to fibronectin (FN). Finally, we examined the role of cell activation in these processes, and showed that activated CLL lymphocyte populations showed an increased capacity to adhere to both endothelium and matrix. Moreover, ex vivo CLL cells showed no capacity to migrate through endothelium/stroma, but were able to do so after cytokine stimulation. These studies show how the constitutive integrin expression/function, the intrinsic activation state of the cell, and the capacity of cytokines to modify integrin-mediated function all combine to determine the different patterns of clinical disease observed in CLL.

Double-Stranded RNA Induces Sickle Erythrocyte Adherence to Endothelium: A Potential Role for Viral Infection in Vaso-Occlusive Pain Episodes in Sickle Cell Anemia

Vaso-occlusive pain episodes in sickle cell anemia are hypothesized to be precipitated by adherence of sickle erythrocytes to vascular endothelium in the microcirculation. Febrile episodes, thought to be viral in etiology, are frequently associated with vaso-occlusion; however, a direct link between viral infection and vascular occlusion has not yet been established. Many pathogenic viruses contain double-stranded RNA or replicate through double-stranded RNA intermediates. Double-stranded RNA has been shown to induce vascular cell adhesion molecule-1 (VCAM-1) protein expression on endothelial cells. Recently, a new adhesion pathway has been described between VCAM-1 expressed on cytokine stimulated endothelium and the alpha 4 beta 1 integrin complex expressed on sickle reticulocytes. Based on these observations, the hypothesis was developed that viral infection, through double-stranded RNA intermediates, increases endothelial VCAM-1 expression leading to sickle erythrocyte adhesion to endothelium via an alpha 4 beta 1-VCAM-1--dependent mechanism. In support of this hypothesis, endothelial cells exposed to the synthetic double-stranded RNA poly(I:C) or the RNA virus parainfluenza 1 (Sendai virus) express increased levels of VCAM-1 and support increased sickle erythrocyte adherence under continuous flow at 1.0 dyne/cm2 shear stress as compared with unstimulated endothelium. Blocking antibodies directed against either VCAM-1 on the endothelium or alpha 4 beta 1 on sickle erythrocytes inhibit nearly all of the increased sickle cell adherence caused by poly(I:C) or Sendai virus. These results support the hypothesis that viruses, through double-stranded RNA elements, can induce sickle erythrocyte adherence to endothelium through alpha 4 beta 1-VCAM-1--mediated adhesion and provide a potential link between viral infection and microvascular occlusion precipitating sickle cell pain episodes.

The adhesion molecules used by monocytes for migration across endothelium include CD11a/CD18, CD11b/CD18, and VLA-4 on monocytes and ICAM-1, VCAM-1, and other ligands on endothelium.

CD11/CD18 and VLA-4 integrins mediate interactions of monocytes with HUVEC cultured on human amniotic tissue. In the present study, the roles of individual CD11/CD18 integrins and endothelial adhesion molecules were examined using blocking mAbs and peptides. After 20 min of incubation, monocyte adhesion to and migration across unstimulated endothelium was dependent primarily on CD11a/CD18. When incubation was extended to 2 h to allow for completion of migration, either CD11a/CD18 or CD11b/CD18 could be used. Similarly, either CD11a/CD18 or CD11b/CD18 could be used by monocytes to bind to and traverse IL-1 beta-stimulated endothelium. Although both CD11a/CD18 and CD11b/CD18 are known to bind to ICAM-1, results of Ab-mixing experiments suggest that alternative ligands on HUVEC for CD11/CD18 integrins also may be used during transendothelial migration of monocytes. Our previous studies indicate that VLA-4 on monocytes interacts primarily with VCAM-1 on unstimulated endothelium. In contrast, migration of monocytes across IL-1 beta-stimulated endothelium was less dependent on VCAM-1. mAbs directed against binding sites for VLA-4 in domain 1 and domain 4 of VCAM-1 did not, by themselves, inhibit interactions of monocytes with stimulated HUVEC. VLA-4-dependent migration across IL-1 beta-stimulated endothelium was markedly inhibited only when mAbs to VCAM-1 were added in combination with peptides of fibronectin. Therefore, VLA-4 can interact with either VCAM-1 or alternative ligands on IL-1 beta-stimulated HUVEC-amnion cultures to mediate transendothelial migration of monocytes.

Two independent sets of monoclonal antibodies define neoepitopes linked to soluble ligand binding and leukocyte adhesion functions of activated alpha M beta 2.

The integrin alpha M beta 2 mediates a variety of events, adhesive, phagocytic, and inflammatory. Evidence has suggested that the functional events may be mediated by the "activated" conformational forms of alpha M beta 2 produced by appropriate stimulation of myeloid and monocytic lineage. The activation of alpha M beta 2 may be associated with new epitopes on alpha M beta 2, sites that may be related to the acquired receptor functions. Monoclonal antibodies were produced that preferentially bind neoepitopes expressed by activated alpha M beta 2. These anti-neo antibodies each inhibited three activation-associated specific receptor alpha M beta 2 functions, though to different extents. One set of anti-neo antibodies inhibited in a concordant manner the binding of factor X and of fibrinogen by > 90%, abolished the alpha M beta 2-initiated cellular coagulant response, and inhibited monocyte adhesion to unstimulated endothelial monolayers. A second set of anti-neo antibodies only diminished factor X and fibrinogen binding by approximately 40% to 50% but markedly suppressed Xa generation and only partially inhibited monocyte adherence to unstimulated endothelium. Concordance was observed between binding of factor X or fibrinogen and competence for leukocyte adhesion to unstimulated endothelium. Antibody competition assays segregated the anti-neo antibodies into the same two distinct sets, consistent with recognition of separate neoepitopes that are linked to alpha M beta 2 function. These data support the conclusion that the activated conformer of alpha M beta 2 that binds fibrinogen and factor X also mediates monocyte-endothelial interactions as well as the alternative cellular coagulation pathway.

Monoclonal antibody to an activation neoepitope of alpha M beta 2 inhibits multiple alpha M beta 2 functions.

alpha M beta 2, a beta 2 integrin expressed on cells of myelomonocytic differentiation, functions as a receptor for factor X (X) and fibrinogen (Fg) and participates in leukocyte adhesion to vascular endothelium. Acquisition of high affinity ligand binding has been suggested to result from a conformational change of alpha M beta 2 in response to agonist-induced leukocyte stimulation. We now describe mAb 7A10, which preferentially binds to alpha M beta 2 on activated cells and reports the functional activation of this integrin. Agonist-stimulated monocytes and monocytic cells, but not resting cells, maximally expressed the 7A10 neoepitope, whereas expression of other selected alpha M beta 2 epitopes remained unchanged. The neoepitope was elicited equally by exposure of cells to either ADP or FMLP and did not require divalent cations for expression. Saturation of the 7A10 neoepitope by this Ab on stimulated THP-1 cells inhibited both X and Fg binding and abolished the alpha M beta 2-driven cellular coagulant response. Stimulated monocytic cells, which bound X and/or Fg, exhibited a sustained adhesion to unstimulated endothelial cell monolayers and Ab 7A10 inhibited this sustained adhesion. We conclude that activated conformers of alpha M beta 2 mediate X and Fg binding, that assembly of either X or Fg or both on alpha M beta 2 mediates leukocyte adhesion to unstimulated endothelium through sparse ICAM-1, and that mAb 7A10 can report and functionally inhibit this pathway of leukocyte adhesion.

Monocytes use either CD11/CD18 or VLA-4 to migrate across human endothelium in vitro.

Monocytes traverse the endothelial lining of blood vessels and migrate into both normal and inflamed tissues. An in vitro model of a vascular wall, consisting of HUVEC cultured on acellular human amniotic tissue, was employed to examine the roles of several adhesion molecules in diapedesis of monocytes. Approximately half of the monocytes added to this system traversed the endothelium in a time-dependent fashion, completing their migration within 2 h. Pretreatment of HUVEC with IL-1 beta for 4 h increased the rate of adhesion of monocytes, but did not alter the number that ultimately migrated. A mAb to CD18, ts1/18, greatly inhibited adhesion and migration of monocytes when the monocytes were incubated with unstimulated HUVEC monolayers for 20 min. Much less inhibition was observed when the incubation period was increased to 2 h or when HUVEC were pretreated with IL-1 beta. A mAb to VLA-4, HP1/2, had little or no inhibitory effect in all cases. The combination of ts1/18 and HP1/2 greatly inhibited (up to 98%) adhesion and migration of monocytes across both unstimulated and IL-1 beta-stimulated monolayers. Additional inhibition experiments indicated that VLA-4 interacted with unstimulated endothelium by binding to VCAM-1 and, to a much lesser extent, fibronectin. These results suggest that monocytes are capable of interacting with endothelium during diapedesis via either CD11/CD18- or VLA-4-dependent pathways.

Granulocyte-macrophage-colony-stimulating factor differentially regulates neutrophil migration across IL-1-activated and nonactivated human endothelium.

The directed migration of neutrophils across vascular endothelium to localize in inflammatory tissues is controlled by soluble mediators, including cytokines and growth factors. Granulocyte-macrophage CSF (GM-CSF) enhances and primes neutrophil functions, but its specific role in the movement and localization of neutrophils to infective sites has not been clarified. We demonstrate, using an in vitro model of the vascular endothelial barrier, that GM-CSF enhances neutrophil migration across unstimulated endothelium, increasing the percentage of migrating cells from 7.7 +/- 0.9% (mean +/- SE) in controls to 12.5 +/- 1.5% in the presence of GM-CSF (100 ng/ml) (n = 14, p < 0.0005). This effect is dose dependent, with maximal effects achieved at concentrations of 10 ng/ml or greater, and is independent of concentration gradients of GM-CSF. Transendothelial migration of neutrophils can also be increased by cytokine treatment of the endothelial cells. Preincubation of endothelial monolayers with IL-1 (10 U/ml) for 4 h increases the percentage of migrating cells to 16.8 +/- 1.4% (238 +/- 25% of base line, n = 7, p < 0.005). In the presence of GM-CSF, however, neutrophil migration across IL-1 treated endothelium (12.5 +/- 1.6%, n = 7) is no different from that across resting endothelium (11.6 +/- 1.6% in the same seven experiments). Hence GM-CSF acts to inhibit neutrophil migration across IL-1-activated endothelium, and almost completely abolishes IL-1-induced migration (n = 7, p < 0.0005). This differential effect of GM-CSF on neutrophil migration, depending upon the conditions of endothelial activation, does not relate to an effect on adhesion, because GM-CSF has no effect on the increased adhesion of neutrophils to IL-1-treated endothelium. The effect of GM-CSF on neutrophil migration across activated endothelium may be relevant to the clinical administration of human rGM-CSF.

Electrical properties of resting and acetylcholine‐stimulated endothelium in intact rat aorta.

1. The passive electrical properties and the effects of acetylcholine on the membrane potential of the endothelium of intact rat aorta were investigated using the whole cell mode of the patch clamp technique. 2. Unstimulated endothelium had a membrane potential of -58 +/- 8 mV (S.E.M., n = 193; range -47 to -76 mV). The input resistance was 43 +/- 13 M omega (S.E.M., n = 8; range 26-64 M omega). KCl and BaCl2, but not tetraethylammonium (2 mM), 4-aminopyridine (5 mM) or 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 microM) depolarized the endothelium. 3. Acetylcholine (0.2-4 microM) evoked in most preparations a biphasic response with a transient hyperpolarization to a value close to the K+ reversal potential, followed by depolarization beyond the resting potential. In 46% of recordings, the depolarization was followed by oscillations in membrane potential. The duration of the hyperpolarization and magnitude of the depolarization was similar in all recordings from a given aorta, but varied greatly between different preparations. 4. Hyperpolarization of the endothelium below the K+ reversal potential reversed the direction of the first phase of the acetylcholine-evoked response, which was unaffected by tetraethylammonium, 4-aminopyridine, or DIDS. 5. The removal of extracellular Ca2+ evoked a depolarization of the endothelium from -61 +/- 3 to -34 +/- 3 mV (S.E.M., n = 9) over 2-15 min. Restoration of external Ca2+ evoked a transient hyperpolarization. 6. ACh applied in nominally Ca(2+)-free medium shortly after Ca2+ removal evoked only a transient hyperpolarization. After the establishment of a stable membrane potential in Ca(2+)-free medium, acetylcholine was without effect. 7. NiCl2 (2 mM) evoked a small depolarization of the endothelium (6 +/- 2 mV; S.E.M., n = 7). The subsequent removal of Ni2+ evoked a transient hyperpolarization. 8. In the presence of Ni2+, acetylcholine evoked a short-lived hyperpolarization. Both the application of Ni2+ and the removal of extracellular Ca2+ immediately blocked oscillations in membrane potential evoked by acetylcholine. 9. The blockers of voltage-operated Ca2+ channels, nifedipine (1-10 microM) and verapamil (20 microM) were without effect on the biphasic acetylcholine-evoked responses. 10. In preparations in which acetylcholine evoked large (20-45 mV) oscillations in membrane potential, depolarization of the endothelium alone, by current injection or application of KCl, did not evoke oscillations. 11. The activator of protein kinase C, phorbol 12, 13-dibutyrate (200 nM) depolarized and greatly increased the input resistance of the endothelium, presumably due to an effect on gap junctions.(ABSTRACT TRUNCATED AT 400 WORDS)