Leukocyte-endothelial Adhesive Interactions Research Articles

Elevated postischemic tissue injury and leukocyte-endothelial adhesive interactions in mice with global deficiency in caveolin-2: role of PAI-1.

Ischemia/reperfusion (I/R)-induced rapid inflammation involving activation of leukocyte-endothelial adhesive interactions and leukocyte infiltration into tissues is a major contributor to postischemic tissue injury. However, the molecular mediators involved in this pathological process are not fully known. We have previously reported that caveolin-2 (Cav-2), a protein component of plasma membrane caveolae, regulated leukocyte infiltration in mouse lung carcinoma tumors. The goal of the current study was to examine if Cav-2 plays a role in I/R injury and associated acute leukocyte-mediated inflammation. Using a mouse small intestinal I/R model, we demonstrated that I/R downregulates Cav-2 protein levels in the small bowel. Further study using Cav-2-deficient mice revealed aggravated postischemic tissue injury determined by scoring of villi length in H&E-stained tissue sections, which correlated with increased numbers of MPO-positive tissue-infiltrating leukocytes determined by IHC staining. Intravital microscopic analysis of upstream events relative to leukocyte transmigration and tissue infiltration revealed that leukocyte-endothelial cell adhesive interactions in postcapillary venules, namely leukocyte rolling and adhesion were also enhanced in Cav-2-deficient mice. Mechanistically, Cav-2 deficiency increased plasminogen activator inhibitor-1 (PAI-1) protein levels in the intestinal tissue and a pharmacological inhibition of PAI-1 had overall greater inhibitory effect on both aggravated I/R tissue injury and enhanced leukocyte-endothelial interactions in postcapillary venules in Cav-2-deficient mice. In conclusion, our data suggest that Cav-2 protein alleviates tissue injury in response to I/R by dampening PAI-1 protein levels and thereby reducing leukocyte-endothelial adhesive interactions.NEW & NOTEWORTHY The role of caveolin-2 in regulating ischemia/reperfusion (I/R) tissue injury and the mechanisms underlying its effects are unknown. This study uses caveolin-2-deficient mouse and small intestinal I/R injury models to examine the role of caveolin-2 in the leukocyte-dependent reperfusion injury. We demonstrate for the first time that caveolin-2 plays a protective role from the I/R-induced leukocyte-dependent reperfusion injury by reducing PAI-1 protein levels in intestinal tissue and leukocyte-endothelial adhesive interactions in postcapillary venules.

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

Atherosclerosis and leukocyte–endothelial adhesive interactions are increased following acute myocardial infarction in apolipoprotein E deficient mice

Objective To determine the effect of myocardial infarction (MI) on progression of atherosclerosis in apolipoprotein E deficient ( ApoE −/−) mice. Methods and results MI was induced following left anterior descending coronary artery (LAD) ligation in wild-type (WT) ( n = 9) and ApoE −/− ( n = 25) mice. Compared to sham-operated animals, MI mice demonstrated increased intravascular leukocyte rolling and firm adhesion by intravital microscopy, reflecting enhanced systemic leukocyte–endothelial interactions. To determine if MI was associated with accelerated atherogenesis, LAD ligation was performed in ApoE −/− mice. Six weeks following surgery, atherosclerosis was quantitated throughout the arterial tree by microdissection and Oil-Red-O staining. There was 1.6-fold greater atherosclerotic burden present in ApoE −/− MI mice compared to sham-operated mice. Conclusions Acute MI accelerates atherogenesis in mice. These results may be related to the increased risk of recurrent ischemic coronary events following MI in humans.

Antecedent Ethanol Attenuates Cerebral Ischemia/Reperfusion-induced Leukocyte-Endothelial Adhesive Interactions and Delayed Neuronal Death: Role of Large Conductance, Ca2+-activated K+Channels

EtOH-PC reduces postischemic neuronal injury in response to cerebral (I/R). We examined the mechanism underlying this protective effect by determining (i) whether it was associated with a decrease in I/R-induced leukocyte-endothelial adhesive interactions in postcapillary venules, and (ii) whether the protective effects were mediated by activation of large conductance, calcium-activated potassium (BK(Ca)) channels. Mice were administered ethanol by gavage or treated with the BK(Ca) channel opener, NS1619, 24 hours prior to I/R with or without prior treatment with the BK(Ca) channel blocker, PX. Both CCA were occluded for 20 minutes followed by two and three hours of reperfusion, and rolling (LR) and adherent (LA) leukocytes were quantified in pial venules using intravital microscopy. The extent of DND, apoptosis and glial activation in hippocampus were assessed four days after I/R. Compared with sham, I/R elicited increases in LR and LA in pial venules and DND and apoptosis as well as glial activation in the hippocampus. These effects were attenuated by EtOH-PC or antecedent NS1619 administration, and this protection was reversed by prior treatment with PX. Our results support a role for BK(Ca) channel activation in the neuroprotective effects of EtOH-PC in cerebral I/R.

Antecedent Ethanol Attenuates Cerebral Ischemia/Reperfusion‐induced Leukocyte‐Endothelial Adhesive Interactions: Role of Large Conductance, Ca2+‐activated K+ Channels (BKCa)

Our recent work indicates that ethanol ingestion (EtOH‐PC) 24 hrs prior to induction of cerebral ischemia/reperfusion (I/R) reduces postischemic neuronal injury. In this study, we investigated whether EtOH‐PC would reduce I/R‐induced leukocyte‐endothelial adhesive interactions in pial postcapillary venules, and if so, whether this protective effect was triggered by BKCa channel activation. To address this aim, C57BL6 mice were administered ethanol by gavage [(body weight (in g) ×0.6) + 0.3] or treated with the BKCa channel opener, NS‐1619 (0.1mg/kg, ip) 24 hrs prior to I/R in the absence or presence of the BKCa channel blocker, paxilline (PX; 2.5 mg/kg body wt, i.p., administered 10 min before ethanol or NS1619 administration). 24 hour later, both common carotid arteries (CCA) were occluded for 20 min followed by 2 and 3 hours of reperfusion, then the numbers of rolling (LR) and adherent (LA) leukocytes were quantified in pial venules using intravital microscopy. I/R induced large increases in LR and LA in pial venules compared with sham. The increased LR and LA were prevented by EtOH‐PC or antecedent NS1619 administration. These protective effects were attenuated by PX treatment coincident with ethanol or NS1619 administration. Our results indicate that EtOH‐PC may activate BKCa to prevent cerebral ischemia/reperfusion‐induced leukocyte‐endothelial adhesive interactions. Supported by R01AA014945 from NIH

Alveolar macrophages participate in the systemic inflammation of alveolar hypoxia

Alveolar hypoxia (AHx, FIO2 0.10) produces widespread inflammation in the systemic rat microcirculation. Dissociation between tissue PO2 and inflammation, plus the observation that plasma from hypoxic rats elicits inflammation in normoxic tissues suggest that AHx inflammation is triggered by an agent released from a distant site and carried by the circulation. These experiments tested the hypothesis that alveolar macrophages (AM) are the source of the agent that triggers AHx inflammation. AM of male rats were depleted by tracheal instillation of clodronate-containing liposomes. Controls received NaCl-containing liposomes. The cremaster muscle was studied with intravital microscopy 4 days after treatment. Clodronate reduced bronchoalveolar lavage AM count by 90%. AHx in controls significantly increased leukocyte-endothelial adhesive interactions, produced degranulation of perivascular mast cells, and increased vascular permeability. None of these effects were seen in AM-depleted AHx rats. While plasma obtained from control AHx rats (FIO2 0.10 for 5 min) induced inflammation in normoxic cremasters, plasma from AM-depleted AHx rats did not have these inflammatory effects. The data suggest that AM are the source of the agent that initiates AHx-induced systemic inflammation. Supported by HL39443 and HD043044.

Inhibition of Hydrogen Sulfide Generation Contributes to Gastric Injury Caused by Anti-Inflammatory Nonsteroidal Drugs

Hydrogen sulfide (H(2)S), an endogenous gaseous mediator that causes vasodilation, is generated in mammalian tissues by cystathionine beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). Here, we have investigated the role of H(2)S in a rodent model of nonsteroidal anti-inflammatory drug (NSAID) gastropathy. Rats were given acetyl salycilic acid (ASA) or an NSAID alone or in combination with NaHS, an H(2)S donor, and killed 3 hours later. Gastric blood flow was measured by laser-Doppler flowmetry, whereas intravital microscopy was used to quantify adhesion of leukocytes to mesenteric postcapillary endothelium. At a dose of 100 micromol/kg, NaHS attenuated by 60%-70% the gastric mucosal injury, and tumor necrosis factor (TNF)-alpha, intercellular adhesion molecule (ICAM)-1, and lymphocyte function-associated antigen (LFA)-1 mRNA up-regulation induced by NSAIDs (P < .05) NaHS administration prevented the associated reduction of gastric mucosal blood flow (P < .05) and reduced ASA-induced leukocyte adherence in mesenteric venules. NaHS did not affect suppression of prostaglandin E(2) (PGE(2)) synthesis by NSAIDs. Glibenclamide, a K(ATP) channel inhibitor, and DL-propargylglycine, a CSE inhibitor, exacerbated, whereas pinacidil, a K(ATP) opener, attenuated gastric injury caused by ASA. Exposure to NSAIDs reduced H(2)S formation and CSE expression (mRNA and protein) and activity by 60%-70%. By promoter deletion and mutation analysis, an Sp1 consensus site was identified in the CSE promoter. Exposure to NSAIDs inhibits Sp1 binding to its promoter and abrogates CSE expression in HEK-293 cells transfected with a vector containing the core CSE promoter. Exposure to NSAIDs inhibits Sp1 and ERK phosphorylation. These data establish a physiologic role for H(2)S in regulating the gastric microcirculation and identify CSE as a novel target for ASA/NSAIDs.

Therapeutic Intervention in Inflammatory Diseases: A Time and Place for Anti‐Adhesion Therapy

The recruitment of leukocytes from the blood into tissue is central to the development and maintenance of the majority of inflammatory diseases. This multistep process requires a series of leukocyte-endothelial adhesive interactions, involving several families of adhesion molecules. Molecules that block these interactions have been targeted as potential therapeutic treatments for acute and chronic inflammatory diseases. However, many of the anti-adhesion therapy clinical trials have yielded disappointing outcomes. This review discusses some of the animal models that raise questions about the suitability of anti-adhesion therapy to treat certain inflammatory diseases. The authors suggest that it is crucial to understand the underlying mechanisms and time lines of leukocyte recruitment in each affected tissue and inflammatory disease to develop more effective anti-adhesion therapy.

Effect of anti-CD11b (alphaM-MAC-1) and anti-CD54 (ICAM-1) monoclonal antibodies on indomethacin induced chronic ileitis in rats.

Leukocyte emigration from blood to sites of inflammation involves sequential interaction of specific adhesion molecules expressed by both leukocytes and endothelial cells. The central steps in leukocyte-endothelial adhesive interactions are leukocyte rolling, sticking, and transmigration. This study investigated the effect of monoclonal antibodies against CD54 (ICAM-1) and CD11b (alphaM-chain of MAC-1) on intestinal inflammation. Anti-CD54 and anti-CD11b were tested in rats with indomethacin-induced chronic ileitis. Macroscopic changes were assessed by a modified version of the Wallace et al. score. Leukocyte rolling and sticking were investigated by intravital microscopy. Results show that indomethacin administration led to a chronic inflammatory response characterized by significant increase (P<0.05) in rolling (from 5.41+/-2.87 to 32.41+/-15.03 100 microm(-1) s(-1)) and sticking (from 0.16+/-0.18 to 9.11+/-5.3 100 microm(-1) s(-1)) leukocytes. After antibody treatment only the anti-CD11b group showed significant (P<0.05) reduction in rolling (from 32.41+/-15.03 to 6.6+/-2.7 100 microm(-1) s-1) and sticking (from 9.11+/-5.3 to 0.07+/-0.09 100 microm(-1) s-1) leukocytes. This was also the case for macroscopic changes. Indomethacin led to a rise in the Wallace score from 0 to 4.29+/-0.76 points (P<0.05) and anti-CD11b to a reduction from 4.29+/-0.76 to 1.29+/-1.11 points (P<0.05). Anti-CD54 and combined anti-CD11b/CD54 administration was not followed by significant changes. Therefore we suggest that leukocyte-based CD11b but not endothelial-based CD54 contributes most to leukocyte adhesion in the setting of indomethacin-induced ileitis in rats.

Systemic hypoxia promotes leukocyte-endothelial adherence via reactive oxidant generation.

We recently demonstrated that systemic hypoxia during reduced inspired PO(2) produces a rapid increase in leukocyte adherence to rat mesenteric venules. Evidence suggests that the mechanism of this response involves decreased nitric oxide (NO) levels. One possible pathway for NO depletion could involve increased reactive oxygen species (ROS) generation resulting in inactivation of NO. The overall goal of the present study was to examine the role of ROS in promoting leukocyte-endothelial adherence during systemic hypoxia. Experiments were designed to 1) evaluate changes in ROS generation in the mesenteric microcirculation during systemic hypoxia, 2) determine how the ROS signal changes when PO(2) levels return to normal after a period of systemic hypoxia, 3) assess the effect of antioxidants on ROS generation during hypoxia, and 4) utilize antioxidants to examine the functional relationship between ROS generation and leukocyte adherence during hypoxia. The major findings from this study are that systemic hypoxia increases ROS generation within the mesenteric microcirculation and that antioxidants prevent the increase in leukocyte-endothelial adhesive interactions observed in hypoxia.

Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells.

The objective of this study was to assess the effects of two structurally distinct yet selective proteasome inhibitors (PS-341 and lactacystin) on leukocyte adhesion, endothelial cell adhesion molecule (ECAM) expression, and nuclear factor-kappaB (NF-kappaB) activation in tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells (HUVEC) and the transformed, HUVEC-derived, ECV cell line. We found that TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclear neutrophil (PMN) adhesion to HUVEC but not to ECV; TNF also significantly enhanced surface expression of vascular cell adhesion molecule 1 and E-selectin (in HUVEC only), as well as intercellular adhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVEC with lactacystin completely blocked TNF-stimulated PMN adhesion, partially blocked U-937 adhesion, and completely blocked TNF-stimulated ECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1 expression in ECV. Pretreatment of HUVEC with PS-341 partially blocked TNF-stimulated leukocyte adhesion and ECAM expression. These effects of lactacystin and PS-341 were associated with inhibitory effects on TNF-stimulated NF-kappaB activation in both HUVEC and ECV. Our results demonstrate the importance of the 26S proteasome in TNF-induced activation of NF-kappaB, ECAM expression, and leukocyte-endothelial adhesive interactions in vitro.

Mechanisms and Consequences of Phagocyte Adhesion to Endothelium

Leukocyte adhesion to endothelium is essential for the development of an appropriate immune-inflammatory response. The vital importance of leukocyte-endothelial adhesive interactions in host defense and homeostasis is illustrated by the clinical manifestations of patients with congenital defects of leukocyte adhesion functions. However, under some circumstances leukocyte adhesion to endothelium may instead lead to vascular and tissue damage. In recent years, there has been remarkable progress in the understanding of the molecular basis of leukocyte adhesion to endothelium, and this knowledge has led to a new approach to immunomodulation in human disease, ie ‘antiadhesion’ therapy. This review focuses on cell adhesion molecules mediating adhesion of circulating phagocytes to vascular endothelium, on congenital defects of phagocyte adhesion in man, and on the current status of antiadhesion therapy directed towards phagocyte and endothelial adhesion molecules. We will also consider markers of phagocyte activation, which may provide a means to identify those patients who would benefit most from antiadhesion therapy.

Ethanol‐Induced Leukocyte Adherence and Albumin Leakage in Rat Mesenteric Venules: Role of CD18/Intercellular Adhesion Molecule‐1

The adherence and emigration of leukocytes have been implicated as a rate-limiting step in the microvascular disturbance in a variety of pathogenic events. The objective of the present study was to investigate leukocyte-endothelial cell adhesion and endothelial barrier function in rat mesenteric microvessels exposed to ethanol, which is known to cause inflammation and injury in various organs. Mesentery of male Wistar rats was used for intravital microscopic observations. Leukocyte adherence and albumin leakage were monitored in single postcapillary venules using the intravital fluorescence microscope. Superfusion of 50 mM ethanol elicited the leukocyte adherence and albumin leakage within 60 min. Pretreatment with a monoclonal antibody directed against either CD18 or intercellular adhesion molecule-1 (ICAM-1) significantly prevented the ethanol-induced increase in leukocyte adherence and decrease in barrier function of endothelium. These results suggest that ethanol-induced leukocyte adherence is mediated by CD18 on leukocytes and ICAM-1 on endothelial cells. The present study further supports that CD18/ ICAM-1-dependent leukocyte-endothelial adhesive interactions lead to macromolecular leakage in the postcapillary venules exposed to ethanol.

Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton.

We have examined functions of the cytoplasmic domain of E-selectin, an inducible endothelial transmembrane protein, especially its ability to associate with the cytoskeleton during leukocyte adhesion. Confocal microscopy of interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells (HUVEC) visualized clustering of E-selectin molecules in the vicinity of leukocyte-endothelial cell attachment sites. A detergent based extraction and Western blotting procedure demonstrated an association of E-selectin with the insoluble (cytoskeletal) fraction of endothelial monolayers that correlated with adhesion of leukocytes via an E-selectin-dependent mechanism. A mutant form of E-selectin lacking the cytoplasmic domain (tailless E-selectin) was expressed in COS-7 cell and supported leukocyte attachment (in a nonstatic adhesion assay) in a fashion similar to the native E-selectin molecule, but failed to become associated with the cytoskeletal fraction. To identify the cytoskeletal components that associate with the cytoplasmic domain of E-selectin, paramagnetic beads coated with the adhesion-blocking anti-E-selectin monoclonal antibody H18/7 were incubated with IL-1 beta-activated HUVEC, and then subjected to detergent extraction and magnetic separation. Certain actin-associated proteins, including alpha-actinin, vinculin, filamin, paxillin, as well as focal adhesion kinase (FAK), were copurified by this procedure, however talin was not. When a mechanical stress was applied to H18/7-coated ferromagnetic beads bound to the surface of IL-1 beta-activated HUVEC, using a magnetical twisting cytometer, the observed resistance to the applied stress was inhibited by cytochalasin D, thus demonstrating transmembrane cytoskeletal mechanical linkage. COS-7 cells transfected with the tailless E-selectin failed to show resistance to the twisting stress. Taken together, these data indicate that leukocyte adhesion to cytokine-activated HUVEC induces transmembrane cytoskeletal linkage of E-selectin through its cytoplasmic domain, a process which may have important implications for cell-cell signaling as well as mechanical anchoring during leukocyte-endothelial adhesive interactions.