PMN Adhesion Research Articles

Effects of poloxamer 188 on human PMN cells

Poloxamer 188 (P188), a nonionic block copolymer chemical surfactant known to have cytoprotective, rheologic, anti-inflammatory, and anti-thrombotic activity, has shown promise in the management of selected trauma patients. We studied human PMN oxidative burst and adhesion molecule expression when exposed to P188. After RBC lysis of whole blood samples, white blood cell components were primed with phosphotidylcholine, primed and activated with fMLP, primed and activated with PMA, or left unstimulated. Each group was treated with vehicle or P188 (0.005-15 mg/ml concentrations). Flow cytometry quantified: (1) PMN superoxide anion production and (2) PMN marker expression of CD11b and L-selectin. Among non-PMA activated PMNs, P188 increased superoxide anion production. PMA-activated PMNs decreased superoxide anion production, proportional to P188 dose. Among fMLP-activated PMNs, the highest P188 dose increased the expression of CD11b. Among PMA-activated PMNs, decreased CD11b expression was seen for the mid-range doses. PMNs altered their oxidative burst and marker expression after exposure to P188. When used at lower doses, P188 may increase the oxidative burst response and, when used at very high doses, increase CD11b expression. However, if PMNs are in a maximally activated state, a higher dose of P188 may decrease the oxidative burst response and decrease CD11b expression.

Leukotriene B4-induced hyperadhesiveness of endothelial cells for neutrophils: relation to CD54.

Leukotriene B4 (LTB4) induced an in vitro transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HUVEC), leading to a 2.2-fold increase in the binding of neutrophil granulocytes (PMN), which was less than that conferred by platelet activating factor (PAF) though more than thrombin did (3.4- or 2.0-fold increases, respectively). This study concerns the role of the adhesive molecules CD18 and CD54 for the LTB4- (as well as thrombin- and PAF-) induced endothelial hyperadhesiveness. The MoAbs 60.3 (to the CD18 molecule on PMN) and 84H10 (to one epitope of CD54 on the HUVEC) blocked the adherence of PMN to LTB4-treated HUVEC, whereas MoAb LB-2 (directed at another CD54 epitope) failed to do so. MoAb 84H10 blocked 43% of the thrombin-induced hyperadhesiveness, whereas the PAF response was unaffected. Thus, LTB4-induced HUVEC hyperadhesiveness may therefore be related to a specific domain on the CD54 (or on an antigenically related molecule) as well as being dependent on CD18, whereas the involvement of CD54 was much less or non-existent for the thrombin and PAF responses, respectively.

Neutrophil‐induced increase in vascular permeability involves activation of the contact system

Adhesion of polymorphonuclear leukocytes (PMN) to the endothelial cell (EC) lining leads to increased vascular permeability. Plasma prekallikrein (PK), factor XII (FXII) and H‐kininogen are principal components of the contact system assembling on the EC surface. Activated PK cleaves H‐kininogen, liberating bradykinin that triggers EC contraction and paracellular gap formation. Here, we investigated the role of the contact system in PMN‐evoked plasma extravasation. Thioglycollate‐induced pleurisy in the mouse was used to study PMN‐dependent plasma leakage. FITC‐dextran served as plasma marker. The fluorescence intensity and PMN content in the exudate was markedly increased 4 h after thioglycollate injection, whereas in neutropenic animals, the plasma leakage was abolished. Treatment with antagonists to bradykinin B1 and B2 receptors, or an inhibitor to PK/FXII largely reduced fluid extravasation by 79% and 74% respectively. Intravital microscopy of the hamster cheek pouch microcirculation revealed that treatment with the B1/B2 antagonists reduced plasma leakage without reducing PMN adhesion. These results demonstrate a critical role of contact system activation in PMN‐evoked increase in vascular permeability. Our data also indicate that PMN‐dependent permeability changes and PMN extravasation may be regulated independent of each other.

CD44 is highly expressed on milk neutrophils in bovine mastitis and plays a role in their adhesion to matrix and mammary epithelium

Mastitis, inflammation of the mammary gland, is a common and economically important disease in dairy animals. Mammary pathogenic organisms, such as Escherichia coli, invade the teat canal,milk ducts, and mammary alveolar space, replicate in mammary secretions, and elicit a local inflammatory response characterized by massive recruitment of blood polymorphonuclear neutrophil leukocytes (PMN) into the alveoli and milk ducts. CD44 is a trans-membrane glycoprotein previously shown to play a role in mediation and control of blood PMN recruitment in response to inflammatory signals. Here we show, for the first time, increased expression of CD44 on recruited milk PMN in bovine mastitis and the expression of a CD44 variant, CD44v10, on these PMN. Furthermore, we demonstrate that CD44 mediates specific adhesion of bovine blood PMN to hyaluronic acid and mammary epithelial cells. Our results suggest that in mastitis CD44 plays a role in recruiting blood PMN into the mammary glands, the exact nature of this role needs to be elucidated.

Subendothelial infiltration of neutrophil granulocytes and liberation of matrix-destabilizing enzymes in an experimental model of human neo-intima

It was the objective of this study to examine the role of human neutrophil granulocytes (PMN) in an in-vitro model of human neo-intima developed for the study of atherosclerosis. Human granulocytes were subjected to a co-culture model of human endothelial and smooth muscle cells. Subendothelial lipid accumulation was achieved by addition of native LDL to the culture medium. Tissue samples were analyzed by immunohistochemistry and scanning/transmission electron microscopy, and culture supernatants were examined for the presence of interleukin-8 (IL-8), MCP-1, GRO-alpha, elastase and matrixmetalloproteinase-8 (MMP-8). Following addition of 2 mg/ml LDL, adherence, transmigration and infiltration depth of PMN was increased significantly when compared to controls. LDL challenging was paralleled by a time- and dose-dependent secretion of IL-8 from intimal smooth muscle cells. PMN infiltration was mediated by the IL-8-signalling pathway and accompanied by release of elastase and MMP-8 into the supernatant and induction of endothelial cell apoptosis. In conclusion, LDL-induced secretion of IL-8 by intimal smooth muscle cells provides a potential mechanism of PMN-recruitment into culprit lesions. The concomitant release of potent matrix-degrading enzymes and the induction of EC apoptosis may have implications for plaque destabilization and cardiovascular events.

Preconditioning of Carbon Monoxide Releasing Molecule-derived CO Attenuates LPS-induced Activation of HUVEC

To investigate the effects and potential mechanisms of preconditioning of tricarbonyldichlororuthenium (III) dimer (CORM-2)-liberated CO on LPS-induced activation of endothelial cells (HUVEC). HUVEC were pretreated with CORM-2 at the concentration of 50 or 100 microM for 2 hrs, washed and stimulated with LPS (10 microg/ml) for additional 4 hrs. Activation (oxidative stress) of HUVEC was assessed by measuring intracellular oxidation of DHR 123 or nitration of DAF-FM, specific H(2)O(2) and NO fluorochromes, respectively. The expression of HO-1, iNOS (Western blot) and ICAM-1 (cell ELISA) proteins and activation of inflammation-relevant transcription factor, NF-kappaB (EMSA) were assessed. In addition, PMN adhesion to HUVEC was also assessed. The obtained data indicate that pretreatment of HUVEC with CORM-2 results in: 1) decrease of LPS-induced production of ROS and NO; 2) up-regulation of HO-1 but decrease in iNOS at the protein levels; 3) inhibition of LPS-induced activation of NF-kappaB; and 4) downregulation of expression of ICAM-1, and this was accompanied by a decrease of PMN adhesion to LPS-stimulated HUVEC. Preconditioning of CO liberated by CORM-2 elicited its anti-inflammatory effects by interfering with the induction of intracellular oxidative stress. In addition, it also supports the notion that CO is a potent inhibitor of iNOS and NF-kappaB.

Molecular mechanism of inhibition of early pulmonary injury and inflammatory response by exogenous carbon monoxide: experiment with mice

To investigate the effect of determine whether the CO-releasing molecules-liberated CO could attenuate leukocytes sequestration and the inflammatory response in the lung of thermally injured mice. Thirty-six C57BL/6 mice were randomly divided into 3 groups: burn group, burned with hot water on the back skin with an area as large as 15% of the total body surface area with the hair shed so as o cause full-thickness thermal injury, CORM-2 group, undergoing the same thermal injury and then receiving intravenous injection of CORM-2 immediately, and sham operation group, undergoing sham thermal injury. Twenty-four hours later the mice were killed. The myeloperoxidase enzyme (MPO) level in lung tissue was detected. Evans blue test and lung wet/dry weight ratio were used to examine the lung edema degree. Bronchoalveolar lavage (BAL) fluid was collected to undergo ELISA to detect the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Activation of nuclear factor (NF)-kappaB was detected with electrophoretic mobility shift assay. The expression level of intercellular adhesion molecule-1, ICAM-1) in the lung was assessed by Western blotting. Whole blood samples were collected from the left ventricles. Serum was isolated and used to stimulated lung endothelial cells for 4 h. Polymorphonuclear neutrophilic leukocytes (PMNs) were isolated from mice bone marrow, labeled with Na(51)CrO(4), cultured, and added with the murine lung endothelial cells (MLECs) stimulated by serums from the mice of the 3 groups so as to measure leukocyte adhesion. The MPO activity of the CORM-2 group was (42 +/- 7) U/g tissue, significantly lower than that of the burn group [(87 +/- 11) U/g tissue, P < 0.05]. The Evans blue extraction level of the CORM-1 group was (53.1 +/- 4.6), not significantly different from that of the burn group [(55.1 +/- 3.8), P > 0.05], however, still significantly higher than that of the sham group [(8.8 +/- 1.3), P < 0.05]. The wet/dry weight ratio of the CORM-2 group was 4.80 +/- 0.11, significantly higher than that of the sham group (3.20 +/- 0.07, P < 0.05), but not significantly different from that of the burn group (4.70 +/- 0.18, P > 0.05). The TNF-alpha and IL-1beta levels of the CORM-2 group were (92 +/- 4) pg/ml and (27.2 +/- 2.9) pg/ml respectively, both significantly higher than those of the sham group [(24 +/- 4) pg/ml and (6.6 +/- 1.0) pg/ml respectively, both P < 0.05], but significantly lower that those of the burn group [(160 +/- 9) pg/ml and (27.2 +/- 2.9) pg/ml respectively, both P < 0.05]. The A value for the lung ICAM-1 protein level of the CORM-1 group was (2.4 +/- 0.4), significantly higher than that of the sham group [(1.4 +/- 0.6)], however, significantly lower than that of the burn group [(3.5 +/- 1.1), P < 0.05]. The lung NF-kappaB activity of the CORM-1 group was significantly lower than that of the burn group. The PMN adhesion to the MLECs stimulated by the CORM-2-treated thermally injured mice serum was (25.4 +/- 5.6)%, significantly lower than that of the burn group [(46.5 +/- 8.5)%, P < 0.05]. Also, CORM-2 markedly decreased the production of inflammatory mediators in BAL fluid without suppressing the permeability of pulmonary microcirculation. CORM-released CO attenuates the inflammatory response in the lung of thermally injured mice by decreasing leukocyte sequestration and interfering with NF-kappaB activation, protein expression of ICAM-1, thus suppressing endothelial cells pro-adhesive phenotype.

Carbon monoxide liberated from carbon monoxide-releasing molecule CORM-2 attenuates inflammation in the liver of septic mice

Recent studies suggest that exogenously administered CO is beneficial for the resolution of acute inflammation. In this study, we assessed the role of CO liberated from a systemically administered tricarbonyldichlororuthenium-(II)-dimer (CORM-2) on modulation of liver inflammation during sepsis. Polymicrobial sepsis in mice was induced by cecal ligation and perforation (CLP). CORM-2 (8 mg/kg iv) was administered immediately after CLP induction, and neutrophil [polymorphonuclear leukocyte (PMN)] tissue accumulation, activation of transcription factor, NF-kappaB, and changes in adhesion molecule ICAM-1 expression (inflammation-relevant markers) were assessed in murine liver 24 h later. In addition, the effects and potential mechanisms of CORM-2-released CO in modulation of vascular endothelial cell proinflammatory responses were assessed in vitro. To this end, human umbilical vein endothelial cells (HUVEC) were stimulated with LPS (1 microg/ml) in the presence or absence of CORM-2 (10-100 microM) and production of intracellular reactive oxygen species (ROS), (DHR123 oxidation) and NO (DAF-FM nitrosation) and subsequent activation of NF-kappaB were assessed 4 h later. In parallel, expression of ICAM-1 and inducible NO synthase (iNOS) proteins along with PMN adhesion to LPS-challenged HUVEC were also assessed. Induction of CLP resulted in increased PMN accumulation, ICAM-1 expression, and activation of NF-kappaB in the liver of septic mice. These effects were significantly attenuated by systemic administration of CORM-2. In in vitro experiments, CORM-2-released CO attenuated LPS-induced production of ROS and NO, activation of NF-kappaB, increase in ICAM-1 and iNOS protein expression and PMN adhesion to LPS-stimulated HUVEC. Taken together, these findings indicate that CO released from systemically administered CORM-2 provides anti-inflammatory effects by interfering with NF-kappaB activation and subsequent downregulation of proadhesive vascular endothelial cell phenotype in the liver of septic mice.

Bovine recombinant IFNγ induces endothelial cell gene transcription of immunoregulatory molecules and upregulates PMN and PBMC adhesion on bovine endothelial cells

Interferon gamma (IFNgamma) is an important modulator of immune responses acting on multiple cell types, such as lymphocytes, macrophages or endothelial cells. We investigated the effects of recombinant bovine IFNgamma on bovine umbilical vein endothelial cells (BUVEC) for the level of polymorphonuclear neutrophil cell (PMN)- and peripheral blood mononuclear cell (PBMC)-adhesion as well as the gene transcription of endothelial cell-derived adhesion molecules (E-selectin, P-selectin, VCAM-1, ICAM-1), chemokines (CXCL1, CXCL8, CXCL10, CCL2, CCL5), GM-CSF, iNOS and COX-2 in comparison to TNFalpha-stimulation. IFNgamma strongly induced PMN and PBMC adhesion on BUVEC involving CD4(+), CD8(+) and gammadelta-TCR(+) (WC1(+)) lymphocytes. Furthermore, IFNgamma-stimulation led to a strong upregulation in the transcription of VCAM-1, ICAM-1, CXCL10 and CCL2 genes and to a low to moderate increase in the E- and P-selectin, CXCL1, CXCL8, CCL5, COX-2 and iNOS gene transcripts, but failed to enhance GM-CSF gene transcription. These results indicate that IFNgamma can be considered an important activator of endothelial cells in the bovine system, most probably by influencing the outcome of inflammatory responses through selective upregulation of immunoregulatory molecules.

Antiendothelial cell antibodies mediate enhanced leukocyte adhesion to cytokine-activated endothelial cells through a novel mechanism requiring cooperation between FcγRIIa and CXCR1/2

AbstractAntiendothelial cell antibodies (AECAs) are commonly detectable in diseases associated with vascular injury, including systemic lupus erythematosus (SLE), systemic sclerosis, Takayasu arteritis, Wegener granulomatosis, Behçet syndrome, and transplant arteriosclerosis. Here, we explore the hypothesis that these antibodies might augment polymorphonuclear leukocyte (PMN) adhesion to endothelium in inflammation. Initially, we established that a mouse IgG mAb bound to endothelial cells (ECs) significantly increased PMN adhesion to cytokine-stimulated endothelium in an FcγRIIa-dependent manner. Neutralizing antibodies, and adenoviral transduction of resting ECs, demonstrated that the combination of E-selectin, CXCR1/2, and β2 integrins is both necessary and sufficient for this process. We observed an identical mechanism using AECA IgG isolated directly from patients with SLE. Assembled immune complexes also enhanced PMN adhesion to endothelium, but, in contrast to adhesion because of AECAs, this process did not require CXCR1/2, was not inhibited by pertussis toxin, and was FcγRIIIb rather than FcγRIIa dependent. These data are the first to demonstrate separate nonredundant FcγRIIa and FcγRIIIb-mediated mechanisms by which EC-bound monomeric IgG and assembled immune complexes amplify leukocyte adhesion under dynamic conditions. Furthermore, the observation that FcγRIIa and CXCR1/2 cooperate to enhance PMN recruitment in the presence of AECAs suggests a mechanism whereby AECAs may augment tissue injury during inflammatory responses.

Src family kinases mediate neutrophil adhesion to adherent platelets

AbstractPolymorphonuclear leukocyte (PMN)–platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of “outside-in” signaling by Src-family tyrosine kinases (SFKs) in the regulation of αMβ2-integrin–dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in β2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from αMβ2−/−, hck−/−fgr−/−, and hck−/−fgr−/−lyn−/− mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of αMβ2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for αMβ2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin–PSGL-1–mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease.

Blockade of Class IA Phosphoinositide 3-Kinase in Neutrophils Prevents NADPH Oxidase Activation- and Adhesion-dependent Inflammation

We examined the role of class IA phosphoinositide 3-kinase (PI3K) in the regulation of activation of NADPH oxidase in PMNs and the mechanism of PMN-dependent lung inflammation and microvessel injury induced by the pro-inflammatory cytokine TNF-alpha. TNF-alpha stimulation of PMNs resulted in superoxide production that was dependent on CD11b/CD18-mediated PMN adhesion. Additionally, TNF-alpha induced the association of CD11b/CD18 with the NADPH oxidase subunit Nox2 (gp91(phox)) and phosphorylation of p47(phox), indicating the CD11b/CD18 dependence of NADPH oxidase activation. Transduction of wild-type PMNs with Deltap85 protein, a dominant-negative form of the class IA PI3K regulatory subunit, p85alpha, fused to HIV-TAT (TAT-Deltap85) prevented (i) CD11b/CD18-dependent PMN adhesion, (ii) interaction of CD11b/CD18 with Nox2 and phosphorylation of p47(phox), and (iii) PMN oxidant production. Furthermore, studies in mice showed that i.v. infusion of TAT-Deltap85 significantly reduced the recruitment of PMNs in lungs and increase in lung microvascular permeability induced by TNF-alpha. We conclude that class IA PI3K serves as a nodal point regulating CD11b/CD18-integrin-dependent PMN adhesion and activation of NADPH oxidase, and leads to oxidant production at sites of PMN adhesion, and the resultant lung microvascular injury in mice.

Role of CO-Releasing Molecules Liberated CO in Attenuating Leukocytes Sequestration and Inflammatory Responses in the Lung of Thermally Injured Mice

Acute lung injury and pulmonary inflammatory responses are important complications most frequently encountered in severely burned patients. Polymorphonuclear leukocyte (PMN) sequestration and the subsequent generation of oxidants and inflammatory mediators play the key roles in the pathogenesis of acute lung injury. In this study, we used CO-releasing molecules (CORM-2) to determine whether the CO-releasing molecules-liberated CO could attenuate leukocytes sequestration and the inflammatory response in the lung of thermally injured mice. Fifty-four mice were assigned to three groups in three respective experiments. In each experiment, mice in sham group (n=6) underwent sham thermal injury, whereas mice in the burn group (n=6) received 15% total body surface area (TBSA) full-thickness thermal injury and mice in CORM-2 group (n=6) underwent the same thermal injury with immediate administration of CORM-2 (8 mg/kg, i.v.). PMN accumulation (MPO assay) in mice lungs and tumor necrosis factor-alpha and interleukin-1beta in BAL fluid, pulmonary edema formation, and wet/dry weight ratios of lung were determined. Activation of NF-kappaB and expression level of ICAM-1 in the lung was assessed. In in vitro experiment, PMN adhesion to experimental mice serum-stimulated mouse lung endothelial cells (MLEC) was assessed. Treatment of thermally injured mice with CORM-2 attenuated PMN accumulation and prevented activation of NF-kappaB in the lung. This was accompanied by a decrease of the expression of ICAM-1. In parallel, PMN adhesion to MLEC stimulated by CORM-2-treated thermally injured mice serum was markedly decreased. Also, CORM-2 markedly decreased the production of inflammatory mediators in BAL fluid without suppressing the permeability of pulmonary microcirculation. CORM-released CO attenuates the inflammatory response in the lung of thermally injured mice by decreasing leukocyte sequestration and interfering with NF-kappaB activation, protein expression of ICAM-1, and therefore, suppressing endothelial cells' pro-adhesive phenotype.

Adhesion and migration of polymorphonuclear leukocytes across human brain microvessel endothelial cells are differentially regulated by endothelial cell adhesion molecules and modulate monolayer permeability

The mechanisms by which polymorphonuclear leukocytes (PMN) cross the human blood–brain barrier have not been fully elucidated. Using a well characterized in vitro model of the human BBB, we examined the role of endothelial cell adhesion molecules on the adhesion and transendothelial migration of PMN across primary cultures of human brain microvessel endothelial cells (HBMEC). A small number of PMN (0.06%) adhered to unstimulated HBMEC, and the basal adhesion was not affected by anti-adhesion molecule antibodies. Treatment of HBMEC with tumor necrosis factor (TNF)-α resulted in increased PMN adhesion that was significantly inhibited by blocking antibodies to E-selectin and ICAM-1, but not VCAM-1 or PECAM-1. A very small number of adherent PMN migrated across unstimulated HBMEC monolayers. Migration increased 2 to 20 fold following stimulation of HBMEC with TNF-α. Monoclonal antibody blocking studies showed that PMN used ICAM-1, but not VCAM-1, E-selectin or PECAM-1 to move across activated monolayers. Anti-adhesion molecule antibodies did not diminish the basal PMN migration. Ultrastructurally, PMN often aggregated on top and between adjacent endothelial cells and adhered by first extending pseudopodia along the apical endothelial surface. They then flattened and inserted themselves between endothelial cells in order to migrate across the monolayers. At the end of the migration period, the cultures resumed their continuity with no evidence of disruption. Transendothelial migration of PMN decreased the transendothelial electrical resistance and increased the permeability to horseradish peroxidase, which penetrated alongside the migrating leukocytes. A blocking antibody to ICAM-1 that greatly decreased migration, had no effect on the permeability changes. These studies provide insights into the mechanisms that regulate the entry of PMN into the brain and the increased permeability of the BBB in CNS inflammation.

The Neutrophil-Activating Protein of Helicobacter pylori Crosses Endothelia to Promote Neutrophil Adhesion In Vivo

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of beta(2) integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection.

Effects of Nitrogen Dioxide on the Expression of Intercellular Adhesion Molecule-1, Neutrophil Adhesion, and Cytotoxicity: Studies in Human Bronchial Epithelial Cells

Nitrogen Dioxide (NO2) is a product of high-temperature combustion and an environmental oxidant of concern. We have recently reported that early changes in NO2-exposed human bronchial epithelial cells are causally linked to increased generation of proinflammatory mediators, such as nitric oxide/nitrite and cytokines like interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-8. The objective of the present in vitro study was to further delineate the cellular mechanisms of NO2-mediated toxicity, and to define the nature of cell death that ensues upon exposure of normal human bronchial epithelial (NHBE) cells to a brief high dose of NO2. Our results demonstrate that the NHBE cells undergo apoptotic cell death during the early post-NO2 period, but this is independent of any significant increase in caspase-3 activity. However, necrotic cell death was more prevalent at later time intervals. Interestingly, an increased expression of HO-1, a redox-sensitive stress protein, was observed in NO2-exposed NHBE cells at 24 h. Since neutrophils (PMNs) play an active role in acute lung inflammation and resultant oxidative injury, we also investigated changes in human PMN–NHBE cell interactions. As compared to normal cells, increased adhesion of PMNs to NO2-exposed cells was observed, which resulted in an increased NHBE cell death. The latter was also increased in the presence of IL-8 and TNF-α + interferon (IFN)-γ, which correlated with upregulation of intercellular adhesion molecule-1 (ICAM-1). Our results confirmed an involvement of nitric oxide (NO) in NO2-induced cytotoxicity. By using NO synthase inhibitors such as L-NAME and 3-aminoguanidine (AG), a significant decrease in cell death, PMN adhesion, and ICAM-1 expression was observed. These findings indicate a role for the L-arginine/NO synthase pathway in the observed NO2-mediated toxicity in NHBE cells. Therapeutic strategies aimed at controlling excess generation of NO and/or inflammatory cytokines may be useful in alleviating NO2-mediated adverse effects on the bronchial epithelium.

The comparison of IL-6, elastase and α-PI expressions in human chronic periodontitis with type 2 diabetes mellitus

Diabetes mellitus is an important risk factor of periodontal diseases. It is one of the main contributing factors for periodontal disease and also limiting factor for periodontal treatments such as implant therapy. Although diabetes itself does not cause periodontitis, periodontal disease progresses more rapidly and leads to more tooth losses in patients with poorly controlled blood glucose. Severe periodontitis has been associated with an increased risk of poor glycemic control and, in turn untreated advanced periodontal disease can deteriorate the metabolic control of diabetes. Various pathogenetic factors have been suggested to explain the increased prevalence and severity of periodontitis in diabetes.1) Reduced polymorphonuclear leukocyte(PMN) function has been found in patients with diabetes. This impairment of function was noted in assays of PMN chemotaxis, adherence and phagocytosis. Studies of PMN defects suggest that this dysfunction could lead to impaired host resistance to infection. Gingival fibroblasts from diabetic patients synthesize less collagen compared to non-diabetic subjects. In addition to decreased collagen production, crevicular fluid collagenolytic activity also was increased in diabetic patients. This increased crevicular fluid collagenase activity appears to be primarily of neutrophil origin. Non-enzymatic glycosylation process results in increased cross-linking between collagen molecules. This cross-linking of collagen significantly contributes to reduced solubility and de-

β2‐integrin affinity and valence in binding ICAM‐1 regulates contact mediated emigration of PMN in shear flow.

Polymorphonuclear leukocytes (PMN) circulate to inflamed vascular endothelium by homing to a gradient of chemokine activators secreted from injured tissue. During activation, the β2-integrin Leukocyte Functional Antigen-1 (LFA-1) shifts conformation into a high affinity state primed for binding its primary ligand on the endothelium, Intercellular Adhesion Molecule-1 (ICAM-1). After neutrophil capture by the endothelium, LFA-1 is redistributed on the cellular membrane into high density clusters, a harbinger to cellular polarization where LFA-1 is found at the leading and trailing regions of membrane contact. We have previously reported that dimeric clusters of LFA-1 bind 10-fold longer to ICAM-1 dimers than monomers. Further, the affinity state of LFA-1 regulates the lifetime and stability of PMN adhesion. We show here that formation of dimeric LFA-1/ICAM-1 clusters can trigger outside-in signaling through LFA-1. Capture of dimeric ICAM-1 beads by PMN induced active CD18, F-actin, and Rap-1 clustering at the bead/cell interface much more efficiently than monomer. Outside-in signaling via LFA-1 was supported by an intermediate or high affinity state, but not a low affinity conformation. Activated Src-family kinases were also involved in transmembrane signaling and colocalized with LFA-1/ICAM-1 clusters. We conclude that assembly of an inflammatory synapse involves dimerization of high affinity LFA-1 with ICAM-1, thereby providing a substrate for direction signaling of cytoskeletal mediated cell polarization and transmigration.

Endothelial Cell Cortactin Coordinates Intercellular Adhesion Molecule-1 Clustering and Actin Cytoskeleton Remodeling during Polymorphonuclear Leukocyte Adhesion and Transmigration

Endothelial cell ICAM-1 interacts with leukocyte beta(2) integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.

Lewis x Antigen Mediates Adhesion of Human Breast Carcinoma Cells to Activated Endothelium. Possible Involvement of the Endothelial Scavenger Receptor C-type Lectin

Lewis x (Le(x), CD15), also known as SSEA-1 (stage specific embryonic antigen-1), is a trisaccharide with the structure Galbeta(1-4)Fucalpha(1-3)GlcNAc, which is expressed on glycoconjugates in human polymorphonuclear granulocytes and various tumors such as colon and breast carcinoma. We have investigated the role of Le(x) in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti-Le(x) mAbs (FC-2.15 and MCS-1) on this adhesion. We also analyzed the cytolysis of Le(x+)-cells induced by anti-Le(x) mAbs and complement when cells were adhered to the endothelium, and the effect of these antibodies on HUVEC. The results indicate that MCF-7 cells can bind to HUVEC, and that MCS-1 but not FC-2.15 mAb inhibit this interaction. Both mAbs can efficiently lyse MCF-7 cells bound to HUVEC in the presence of complement without damaging endothelial cells. We also found a Le(x)-dependent PMN interaction with HUVEC. Although both anti-Le(x) mAbs lysed PMN in suspension and adhered to HUVEC, PMN aggregation was only induced by mAb FC-2.15. Blotting studies revealed that the endothelial scavenger receptor C-type lectin (SRCL), which binds Le(x)-trisaccharide, interacts with specific glycoproteins of M (r ) approximately 28 kD and 10 kD from MCF-7 cells. The interaction between Le(x+)-cancer cells and vascular endothelium is a potential target for cancer treatment.