Regulation Of Leukocyte Adhesion Research Articles

ADAMTS13: a new link between thrombosis and inflammation

von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprotease with thrombospondin type I repeats–13 (ADAMTS13) activity is decreased in both acute and chronic inflammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and inflammation. Using intravital microscopy, we show that ADAMTS13 deficiency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in inflamed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13−/− mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13−/− compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13−/− but not in WT mice. Furthermore, in inflammatory models, ADAMTS13 deficiency resulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our findings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during inflammation.

Altered detection of molecules associated with leukocyte traffic in HUVECs derived from newborns with a strong family history of myocardial infarction

Atherosclerosis is a chronic inflammatory disease. As such, recruitment of immune cells is a significant event. Tightly controlled signaling molecules regulate leukocyte adhesion and migration to the tissues. The aim of this study was to determine if human umbilical vein endothelial cells (HUVECs) derived from healthy newborns with a strong family history of myocardial infarction (FHMI) showed variations in the presence of molecules related with leukocyte traffic and migration, in comparison to control healthy newborns. For this purpose, we evaluated the labeling of sialic acid containing glycoproteins, tight junction claudins and the cytoskeleton, using lectin- and immunocytochemistry in HUVECs from individuals with and without a strong FHMI. Our results show important differences in the labeling of alpha-2,3 or alpha-2,6 sialic acid-containing glycoconjugates, a disarrangement of actin filaments secondary to the absence of cytoplasmic claudin-5 immunopositivity and an increase in the binding of FHMI HUVECs to CD3+ Jurkat cells. It is possible that these differences relate to a predisposition for early appearance of atherosclerotic lesions.

Epac1-Rap1 signaling regulates monocyte adhesion and chemotaxis

Extravasation of leukocytes is a crucial process in the immunological defense. In response to a local concentration of chemokines, circulating leukocytes adhere to and migrate across the vascular endothelium toward the inflamed tissue. The small guanosinetriphosphatase Rap1 plays an important role in the regulation of leukocyte adhesion, polarization, and chemotaxis. We investigated the role of a guanine nucleotide exchange protein for Rap1 directly activated by cAMP (Epac1) in adhesion and chemotaxis in a promonocytic cell line and in primary monocytes. We found that Epac1 is expressed in primary leukocytes, platelets, CD34-positive hematopoietic cells, and the leukemic cell lines U937 and HL60. Epac activation with an Epac-specific cAMP analog induced Rap1 activation, beta1-integrin-dependent cell adhesion, and cell polarization. In addition, activated Epac1 enhanced chemotaxis of U937 cells and primary monocytes. Similar to activation of Epac1, stimulation of cells with serotonin to induce cAMP production resulted in Rap1 activation, increased cell adhesion and polarization, and enhanced chemotaxis. The effects of serotonin on U937 cell adhesion were dependent on cAMP production but could not be blocked by a protein kinase A inhibitor, implicating Epac in the regulation of serotonin-induced adhesion. In summary, our work reveals the existence of previously unrecognized cAMP-dependent signaling in leukocytes regulating cell adhesion and chemotaxis through the activation of Epac1.

Hepatocyte growth factor suppresses acute renal inflammation by inhibition of endothelial E-selectin

Vascular endothelial activation, marked by de novo expression of E-selectin, is an early and essential event in the process of leukocyte extravasation and inflammation. Evidence suggests that hepatocyte growth factor (HGF) ameliorates inflammation in animal models of renal disease, implying that HGF might inhibit specific components of the inflammatory response. This study examined the effect of HGF on endothelial E-selectin expression in acute inflammation induced by tumor necrosis factor (TNF)-α. In vitro, HGF suppressed TNF-α-induced cell surface expression of E-selectin in human umbilical vein endothelial cells (HUVEC) and inhibited E-selectin mediated monocytic adhesion to endothelial monolayers. HGF activated phosphatidylinositol 3-kinase (PI3K)–Akt that in turn inhibited its downstream transducer glycogen synthase kinase (GSK)3. Blockade of the PI3K–Akt pathway with specific inhibitors abrogated HGF induced inhibitory phosphorylation of GSK3 and suppression of E-selectin. In addition, selective inhibition of GSK3 activity by lithium suppressed TNF-α-induced E-selectin expression and monocytic adhesion, reminiscent of the action of HGF. Moreover, ectopic expression of an uninhibitable mutant GSK3β, in which the regulatory serine-9 is replaced by alanine, abolished HGF's suppressive effect on endothelial E-selectin. In vivo, administration of exogenous HGF reduced endothelial expression of E-selectin induced by bolus injection of TNF-α. This was associated with less sequestration of circulating fluorescence-labeled macrophages in the kidney. These findings suggest that HGF ameliorates acute renal inflammation in part by downregulating E-selectin mediated macrophage adhesion to the inflamed endothelium.

Relaxin Stimulates Leukocyte Adhesion and Migration through a Relaxin Receptor LGR7-dependent Mechanism

Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration.

Effect of Olmesartan Medoxomil on Atherosclerosis

Angiotensin II is a potent vasoconstrictor and may also contribute to the progression of atherosclerosis. In atheromatous lesions located in human coronary and carotid arteries, angiotensin-converting enzyme and angiotensin II levels are significantly increased. Angiotensin II is known to have proinflammatory actions in vascular tissues, inducing inflammatory cytokines and oxidative stress. In particular, angiotensin II activates the potent cytoplasmic transcription factor nuclear factor-kappaB, which regulates leukocyte adhesion molecules, tumor necrosis factor (TNF)-alpha, monocyte chemotactic protein (MCP)-1, and interleukin (IL)-6, and contributes to the recruitment of circulating mononuclear leukocytes to the arterial intima. Olmesartan medoxomil has one of the highest degrees of antihypertensive efficacy among the angiotensin II type 1 receptor antagonists. In the EUTOPIA (EUropean Trial on Olmesartan and Pravastatin in Inflammation and Atherosclerosis) trial, olmesartan medoxomil significantly reduced serum levels of high-sensitivity C-reactive protein, high-sensitivity TNFalpha, IL-6, and MCP-1, all of which are involved in promoting atherosclerosis. In a monkey atherosclerotic model, a 3-D intravascular ultrasound analysis of aortas showed that serum levels of MCP-1 and the degree of intimal hyperplasia were significantly lower after treatment with olmesartan medoxomil than before treatment. In VIOS (Vascular Improvement with Olmesartan Medoxomil Study), treatment with olmesartan medoxomil for 1 year, significantly reduced the wall-to-lumen ratio in arteries, whereas atenolol did not. Thus, olmesartan medoxomil, which rapidly reduces inflammatory markers, may have a beneficial antiatherosclerotic effect.

Regulation of In Vitro and In Vivo Immune Functions by the Cytosolic Adaptor Protein SKAP-HOM

SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

A Direct Role for C1 Inhibitor in Regulation of Leukocyte Adhesion

Plasma C1 inhibitor (C1INH) is a natural inhibitor of complement and contact system proteases. Heterozygosity for C1INH deficiency results in hereditary angioedema, which is mediated by bradykinin. Treatment with plasma C1INH is effective not only in patients with hereditary angioedema, but also in a variety of other disease models, in which such therapy is accompanied by diminished neutrophil infiltration. The underlying mechanism has been explained primarily as a result of the inhibition of the complement and contact systems. We have shown that C1INH expresses the sialyl-Lewis(x) tetrasaccharide on its N-linked glycan, via which it binds to E- and P-selectins and interferes with leukocyte-endothelial adhesion in vitro. Here we show that both native C1INH and reactive center cleaved C1INH significantly inhibit selectin-mediated leukocyte adhesion in several in vitro and in vivo models, whereas N-deglycosylated C1INH loses such activities. The data support the hypothesis that C1INH plays a direct role in leukocyte-endothelial cell adhesion, that the activity is mediated by carbohydrate, and that it is independent of protease inhibitory activity. Direct involvement of C1INH in modulation of selectin-mediated cell adhesion may be an important mechanism in the physiologic suppression of inflammation, and may partially explain its utility in therapy of inflammatory diseases.

Promotion of leukocyte adhesion by a novel interaction between vitronectin and the beta2 integrin Mac-1 (alphaMbeta2, CD11b/CD18).

The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) binds a number of ligands and counter-receptors and thereby is a major determinant in regulation of leukocyte adhesion and extravasation. Vitronectin (VN) is an adhesion-promoting factor that is abundantly present as matrix molecule in vascular diseases such as atherosclerosis. Until now, only an indirect interaction between Mac-1 and VN via the urokinase receptor (urokinase plasminogen activator receptor) was known. We now propose that Mac-1 and VN can directly interact with each other. In an in vitro system with purified components, Mac-1 specifically bound the multimeric matrix form of VN but not the monomeric plasma form. Using various competitors, the interaction domains in Mac-1 and VN were localized. Mac-1-expressing but not untransfected Chinese hamster ovary cells adhered strongly on VN. Introduction of a GFFKR deletion in the alphaM subunit of Mac-1, which increases the constitutive activation of the integrin, led to increased adhesion on VN. Peripheral human blood neutrophils adhered and migrated on multimeric VN in a Mac-1-dependent manner. These results show that there is a specific integrin-affinity-regulated interaction between Mac-1 and the matrix form but not the plasma form of VN that may significantly participate in leukocyte adhesion and extravasation.

Blood coagulation in cystic fibrosis: modulating inflammation?

Cystic fibrosis (CF) is a devastating chronic inflammatory disease caused by a mutation in the gene encoding cystic fibrosis transmembrane regulator (CFTR), which is a chloride channel in epithelia cell membranes [1]. The phenotype is dominated by pathological changes occurring in organs that express CFTR, particularly the airways, where infection contributes to severe chronic pulmonary disease. The still unavoidable result is a markedly reduced life expectancy (median age of survival 32 years in 2000). Chronic infection is a hallmark of this disease, so that the question whether infection elicits inflammation or vice versa has never been answered unequivocally. Colonization with specific pathogens, particularly Pseudomonas aeruginosa ,o ccurs in about 100% of cases. Contributing factors are reduced interleukin-10 production in the lungs and reduced sialysation of glycoconjugates on epithelial cell membranes. CFTR specifically acts as receptor for P. aeruginosa, facilitating its clearance by internalization under normal conditions. The F508-deleted CFTR cannot bind this bacterium and impaired clearance is the result. Finally, epithelial cells produce defensins which are only partially active in a high salt milieu which is typical of CF, diminishing the antimicrobial barrier [1]. All these factors probably contribute to the chronic infection in CF. The resulting inflammatory state triggers a host response reaction, which also involves the blood coagulation system. In past decades much attention has been given to the possible role that blood coagulation might play in the inflammatory response, as an integral part of the innate immune system [2]. In experimental models of sepsis it has been demonstrated that proinflammatory mediators including tumor necrosis factor (TNF)-a, interleukin (IL)-1 and interleukin (IL)-6 induce tissue factor expression and subsequent thrombin generation. TNF-a impairs fibrinolytic activity by inducing plasminogen activator inhibitor-1 (PAI-1), while the same mediators downregulate the level of expression of natural

Vascular elastic laminae: anti-inflammatory properties and potential applications to arterial reconstruction.

Biomaterials, including non-biodegradable and biodegradable polymers, and collagen and fibrin matrices, have been used in experimental and clinical arterial reconstruction. While these biomaterials exhibit various characteristics suitable for arterial reconstruction, the patency of biomaterial-based arterial substitutes remains problematic because of inflammation and thrombogenesis. Endothelial cell seeding of biomaterials has been proposed and used for reducing the thrombogenicity of biomaterials. However, difficulties in cell retention hamper the application of such an approach. Although autogenous vein grafts offer satisfactory results, not all patients possess veins available for arterial replacements. Thus, a critical issue in arterial reconstruction is developing arterial substitutes that are inflammation/thrombosis-resistant while possessing the characteristics of natural arteries. Here we show that allogenic vascular elastic laminae exhibit anti-inflammatory properties and may be considered a potential material for arterial reconstruction. In this article, we briefly review the composition, structure, and function of vascular elastic laminae, summarize recent discoveries on the role of elastic laminae in regulating leukocyte adhesion and vascular smooth muscle cell proliferation and migration, and discuss potential applications of allogenic elastic laminae to arterial reconstruction.

Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration.

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phospho inositide-dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein-protein interactions.

Molecular Mechanisms Involved in CD43-mediated Apoptosis of TF-1 Cells: ROLES OF TRANSCRIPTION, Daxx EXPRESSION, AND ADHESION MOLECULES

CD43 (leukosialin, sialophorin), an abundant leukocyte surface sialoglycoprotein, regulates leukocyte adhesion and transmits activating signals in T cells and dendritic cells. Immobilized anti-CD43 monoclonal antibody (mAb) MEM-59 has been previously shown to induce apoptosis of hematopoietic progenitors. In this study we show that it also triggers apoptosis of the myeloid progenitor-derived cell line TF-1. The kinetics of the MEM-59-induced apoptosis were unusually slow, with the first apoptotic cells appearing 36-48 h after their contact with the immobilized antibody; in 5 days, 90% of the cells were dead. CD43-mediated apoptosis was enhanced by coimmobilized anti-CD45 mAb and partly suppressed by coimmobilized anti-CD50 (ICAM-3) or anti-CD99 mAb. The MEM-59-triggered apoptosis of TF-1 cells was also inhibited by the overexpression of an apoptotic regulator, Daxx. CD43-mediated apoptosis was preceded by the repression of the DNA binding activity of the transcription factor AP-1. DNA array screening revealed that the expression of several genes encoding apoptosis-regulating proteins, including 14-3-3 proteins and the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor beta-subunit, was repressed in TF-1 cells bound to immobilized MEM-59. The down-regulation of 14-3-3 proteins and GM-CSF receptor beta was accompanied by translocation of the proapoptotic protein Bad to the mitochondria. These results suggest that engagement of CD43 may, presumably through the repressing transcription, initiate a Bad-dependent apoptotic pathway.

Novel chemokine functions in lymphocyte migration through vascular endothelium under shear flow

The recruitment of circulating leukocytes at vascular sites in target tissue has been linked to activation of Gi-protein signaling in leukocytes by endothelial chemokines. The mechanisms by which apical and subendothelial chemokines regulate leukocyte adhesion to and migration across endothelial barriers have been elusive. We recently found that endothelial chemokines not only stimulate integrin-mediated arrest on vascular endothelial ligands but also trigger earlier very late antigen (VLA)-4 integrin-mediated capture (tethering) of lymphocytes to vascular cell adhesion molecule 1 (VCAM-1)-bearing surfaces by extremely rapid modulation of integrin clustering at adhesive contact zones. This rapid modulation of integrin avidity requires chemokine immobilization in juxtaposition with the VLA-4 ligand VCAM-1. We also observed that endothelial-bound chemokines promote massive lymphocyte transendothelial migration (TEM). It is interesting that chemokine-promoted lymphocyte TEM requires continuous exposure of lymphocytes but not of the endothelial barrier to fluid shear. It is noteworthy that lymphocyte stimulation by soluble chemokines did not promote lymphocyte TEM. Our results suggest new roles for apical endothelial chemokines both in triggering lymphocyte capture to the endothelial surface and in driving post-arrest events that promote lymphocyte transmigration across endothelial barriers under shear flow.

Regulation of leukocyte adhesion to heart by the tripeptides feG and feG(NH<sub>2</sub>)

Regulation of leukocyte adhesion to heart by the tripeptides feG and feG(NH<sub>2</sub>)

Leukocyte adhesion and microvessel permeability.

To investigate the direct effect of leukocyte adherence to microvessel walls on microvessel permeability, we developed a method to measure changes in hydraulic conductivity (L(p)) before and after leukocyte adhesion in individually perfused venular microvessels in frog mesentery. In 19 microvessels that were initially free of leukocyte sticking or rolling along the vessel wall, control L(p) was measured first with Ringer-albumin perfusate. Blood flow was then restored in each vessel with a reduced flow rate in the range of 30-116 microm/s to facilitate leukocyte adhesion. Each vessel was recannulated in 45 min. The mean number of leukocytes adhering to the vessel wall was 237 +/- 22 leukocytes/mm(2). At the same time, L(p) increased to 4.7 +/- 0.5 times the control value. Superfusion of isoproterenol (10 microM) after leukocyte adhesion brought the increased L(p) back to 1.1 +/- 0.2 times the control in 5-10 min (n = 9). Superfusing isoproterenol before leukocyte adhesion prevented the increase in L(p) (n = 6). However, the number of leukocytes adhering to the vessel wall was not significantly affected. These results demonstrated that leukocyte adhesion caused an increase in microvessel permeability that could be prevented or restored by increasing cAMP levels in endothelial cells using isoproterenol. Thus cAMP-dependent mechanisms that regulate inflammatory agent-induced increases in permeability also modulate leukocyte adhesion-induced increases in permeability but act independently of mechanisms that regulate leukocyte adhesion to the microvessel wall. Application of ketotifen, a mast cell stabilizer, and desferrioxamine mesylate, an iron-chelating reagent, attenuated the increase in L(p) induced by leukocyte adhesion, suggesting the involvement of oxidants and the activation of mast cells in leukocyte adhesion-induced permeability increase. Furthermore, with the use of an in vivo silver stain technique, the locations of the adherent leukocytes on the microvessel wall were identified quantitatively in intact microvessels.

A role for the actin-bundling protein L-plastin in the regulation of leukocyte integrin function.

Regulation of leukocyte integrin avidity is a crucial aspect of inflammation and immunity. The actin cytoskeleton has an important role in the regulation of integrin function, but the cytoskeletal proteins involved are largely unknown. Because inflammatory stimuli that activate integrin-mediated adhesion in human polymorphonuclear neutrophils (PMN) and monocytes cause phosphorylation of the actin-bundling protein L-plastin, we tested whether L-plastin phosphorylation was involved in integrin activation. L-plastin-derived peptides that included the phosphorylation site (Ser-5) rapidly induced leukocyte integrin-mediated adhesion when introduced into the cytosol of freshly isolated primary human PMN and monocytes. Substitution of Ala for Ser-5 abolished the ability of the peptide to induce adhesion. Peptide-induced adhesion was sensitive to pharmacologic inhibition of phosphoinositol 3-kinase and protein kinase C, but adhesion induced by a peptide containing a phosphoserine at position 5 was insensitive to inhibition. These data establish a novel role for L-plastin in the regulation of leukocyte adhesion and suggest that many signaling events implicated in integrin regulation act via induction of L-plastin phosphorylation.

Nitric Oxide Inhibition Decreases Neutrophil Adhesion at the Inflammatory Site, While Increasing Adhesion in Remote Organs in Peritonitis

Nitric oxide (NO) is a regulator of leukocyte adhesion in the microcirculation. This study was designed to examine the effects of a NO synthase inhibitor on neutrophil adhesion in the peritoneum, lung, liver, and kidney in a rat peritonitis model using a fluorescence microscopic method. Sprague–Dawley rats were given normal saline (control) orNω-nitro-l-arginine methyl ester (L-NAME) at dosages of 10 mg/kg (N10) or 100 mg/kg (N100) (n= 66) intraperitoneally. One hour after pretreatment fluorescein-labeled neutrophils were infused without bacterial challenge (0 hr). Other rats received an injection of 107Escherichia coliinto the peritoneal cavity 1 hr after pretreatment. Labeled neutrophils were infused 1 and 5 hr after bacterial challenge. Just 2 min after neutrophil injection, blood samples were obtained and the animals were killed. Five peritoneal samples (omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. Combined plasma nitrite/nitrate levels were determined. In another set of rats (n= 36), an arterial catheter was inserted after L-NAME treatment and bacterial challenge. At 0, 1, 5, and 12 hr after challenge, blood pressure, heart rate, and arterial blood gas data were measured. One hour afterE. colichallenge, the number of neutrophils in the peritoneum was significantly lower in both L-NAME-treated groups than in the control group. In contrast, the number of labeled neutrophils in the lungs was significantly higher in the N100 group than in the control group. Neutrophil accumulation in the lungs and peritoneum at 0 and 5 hr and in the liver and kidney at 0, 1, and 5 hr did not differ among groups, nor did combined plasma nitrite/nitrate levels. L-NAME treatment had no influence on either hemodynamic or blood gas data. In conclusion, administration of L-NAME increases neutrophil adhesion in the lung, while decreasing that in the peritoneum. NO plays an important role in neutrophil adhesion at the inflammatory site, as well as in remote organs, during peritonitis. NO inhibition may be detrimental, due to neutrophil sequestration, in this peritonitis model.

Regulation of leukocyte adhesion and signaling in inflammation and disease.

Cell adhesion molecules provide the foundation for cell communication, trafficking, and immune surveillance central to host defense. These adhesion molecules which include selectins, integrins and members of the Ig superfamily, provide a recognition system between leukocytes, endothelial cells and matrix molecules. Leukocyte-endothelial interactions initiate recruitment at sites of injury, infection and inflammation. Cell-cell and cell-matrix interactions also influence leukocyte phenotype and function. Dysregulation of these adhesion and signal transduction pathways can contribute to continued recruitment and persistent leukocyte activation with unresolved inflammation. Based on the pivotal role adhesive interactions play, the adhesion molecules provide potential targets for intervention. Selected synthetic fibronectin peptides, which inhibit leukocyte integrin binding and signal transduction in vitro, block recruitment and activation to limit inflammation in vivo.

Platelet-Activating Factor Is Involved in the Regulation of Pathological Leukocyte Adhesion after Liver Transplantation

An increased leukocyte adhesion and loss of integrity of endothelial cells is known to be a critical step in the reperfusion period after cold ischemia of transplanted livers. Platelet-activating factor (PAF) is discussed as one of the inflammatory mediators involved in mediating reperfusion injury, e.g., by regulation of leukocyte adhesion. In this study we investigated the effect of a synthetic PAF receptor antagonist, WEB 2086 (Boehringer Ingelheim, FRG), on microcirculation and adhesion of leukocytes to sinusoidal endothelium after liver transplantation in the rat. Livers from SPRD rats (n= 6 per group) were stored for 5 hr in ice-cold UW solution and orthotopically transplanted using the cuff technique. In a randomized and blinded fashion WEB 2086 (1 mg/kg body wt) or placebo was given twice intravenously, at the beginning of the recipient operation and 1 min prior to reperfusion of the liver graft. Afterin vivostaining of leukocytes with acridine orange, microcirculation and leukocyte–endothelium interaction of transplanted livers and livers from sham-operated animals were investigated 90 min and 5 hr after surgery by means of intravital microscopy. After 90 min of reperfusion, temporary leukocyte adhesion in periportal regions was increased after liver transplantation (22.0 ± 2.5%; mean ± SEM) in contrast to the sham group (14.2 ± 1.8%). Administration of WEB 2086 reduced temporary leukocyte adhesion significantly (11.5 ± 2.5%;P< 0.05), whereas no significant differences were observed with respect to permanent leukocyte adhesion. Five hours after recipient operation, a significant increase of permanent leukocyte adhesion was observed after transplantation (21.0 ± 2.6%;P< 0.05) compared to sham-operated animals (12.1 ± 1.7%). Application of WEB 2086 diminished permanent leukocyte adhesion significantly (12.2 ± 1.3%;P< 0.05), whereas no differences were noticed between the different groups with respect to temporary leukocyte adhesion at this time. The results indicate that reduction of temporary leukocyte adhesion in the early reperfusion period by administration of PAF receptor antagonist WEB 2086 was followed by a reduction of permanent leukocyte adhesion in the late reperfusion period, e.g., at 5 hr. Thus, it is concluded that PAF is one of the mediators which is involved in the regulation of leukocyte adhesion after liver transplantation.