Adhesion Of Human Neutrophils Research Articles

Adhesion of human neutrophils to fibronectin is inhibited by comaruman, pectin of marsh cinquefoil Comarum palustre L., and by its fragments

Earlier, we detected antiinflammatory action of comaruman, pectin of the marsh cinquefoil Comarum palustre L. This effect can be explained by new data concerning inhibition of adhesion of human neutrophils to fibronectin by comaruman and its fragments. The galacturonan backbone fragment of molecular mass >10 kD appears to be the active region of the comaruman macromolecule. Comaruman CP (50-200 microg/ml) was found to decrease adhesion of neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA, 1.625 microM) and by dithiothreitol (DTT, 0.5 mM). The fragments of comaruman CP-H (100 kD), CP-H1 (10-50 kD), and CP-H2 (100 kD) obtained by acidic hydrolysis and representing regions of linear polygalacturonan are shown to inhibit neutrophil adhesion more than the crude pectin. A fragment CP-E (<10 kD) obtained using pectinolysis and representing a branched region of the comaruman macromolecule failed to influence cell adhesion. The parent comaruman CP as well as fragments of its polygalacturonan backbone diminish PMA-initiated generation of oxygen radicals in neutrophils.

Adhesion of human neutrophils to fibronectin is inhibited by comaruman, pectin of marsh cinquefoil comarum palustre L., and by its fragments

Adhesion of human neutrophils to fibronectin is inhibited by comaruman, pectin of marsh cinquefoil comarum palustre L., and by its fragments

Adhesion of human neutrophils to fibronectin is inhibited by comaruman, pectin of marsh cinquefoil comarum palustre L., and by its fragments

Adhesion of human neutrophils to fibronectin is inhibited by comaruman, pectin of marsh cinquefoil comarum palustre L., and by its fragments

The inhibition of neutrophil-endothelial cell adhesion by hyaluronan independent of CD44

To study the effect of hyaluronan on cell adhesion and recruitment both in vitro and in vivo, since hyaluronan both inhibits restenosis and is anti-inflammatory. When administered to animals undergoing angioplasty the recruitment of cells into the restenotic plaque is inhibited, as well as into inflammatory lesions. The recent discovery that ICAM-1 binds hyaluronan and exhibits the B(X(7))B HA binding motif, led us also to investigate whether cell adhesion could be modulated by hyaluronan. Human neutrophils were adhered to human umbilical vein (HUVEC) or Ea.hy.926 HUVEC cells stimulated with phorbol myristate acetate (PMA) or tumour necrosis factor (TNFalpha). Neutrophil binding in vivo utilized FMLP-stimulated hamster cheek pouch post-capillary venules. Hyaluronan inhibited human neutrophil adhesion to both PMA and TNFalpha-stimulated HUVEC. Ea.hy.926 human immortal HUVECs expressed ICAM-1 in response to TNFalpha and PMA. E-selectin was also upregulated by 6 h with TNFalpha but not significantly with PMA. TNFalpha induced CD44 expression within 4 h, but PMA not significantly up to 6 h. However, specific binding of [125I]hyaluronan to Ea.hy.926 cells was increased by PMA-stimulation at 4 h. Neutrophil adhesion to PMA-stimulated Ea.hy.926 HUVECs was inhibited in a concentration dependent fashion by both anti-ICAM-1 and hyaluronan (1 ng/ml-10 microg/ml) at 4 h. At 1 mg/ml adhesion was stimulated by hyaluronan. Hyaluronan had no effect on neutrophil adhesion to resting Ea.hy.926 cells. Hyaluronan (25 mg/kg, i.v.) inhibited cell adhesion to FMLP-stimulated post capillary venules of the hamster cheek pouch, whilst leaving cell rolling unaffected. These results show that hyaluronan, at concentrations below those where intra-molecular associations occur, binds selectively to stimulated endothelial cells and inhibits neutrophil adhesion in vitro and in vivo via a mechanism which may involve molecules other than CD44, such as ICAM-1.

Cross-Species Chemoattraction and Xenograft Failure: Do Neutrophils Play a Role?

Polymorphonuclear granulocytes (PMN, neutrophils) are well known to contribute to the pathogenesis of many congenital disorders (e.g., CR3 deficiency, chronic granulomatous disease, myeloperoxidase deficiency) and acquired disease states (e.g., inflammatory bowel disease, glomerulonephritis, immune vasculitis, rheumatoid arthritis, neutrophil dermatoses, sepsis). They play a key role in mediating ischemia-reperfusion injury following solid organ allotransplantation,andmorerecentevidencesuggeststhat,following early migration into the allograft, neutrophils are capable of amplifying T-cell mediated allograft rejection (1). In xenotransplantation, the major hurdle of hyperacute rejection (HAR) has led research efforts to focus on the role of natural xenoantibodies and complement activation, while the neutrophilwasconsideredtoplayasecondaryroleandwastherefore given little attention. Meanwhile, it seems that experimental prevention of HAR becomes feasible thanks to the genetic modification of donor animals (DAF/CD55 transgenicpigs,-Galknockoutpigsandmice)and,consequently, the mechanism of acute vascular rejection (AVR) and the cellular aspects of xenograft rejection now receive full attention. AVR is characterized by a dense cellular infiltrate, which is dominated not only by natural killer (NK) cells and macrophages, but also by PMN. It is therefore plausible to assume that neutrophils take an active part in the rejection process and this hypothesis is gaining increasing interest. Vercellotti et al. (2) showed that the adhesion of human neutrophils to porcine endothelial cells (PECs) was promoted by humannaturalxenoantibodiesandcomplement,and,particularly, that endothelial deposition of iC3b was critically involvedintheadhesionprocess.Ehrnfeltetal.(3)showedthat humanPMNsadheretoPECs,providedtheseareactivatedby human natural xenoantibodies. These studies indicate that the interaction between neutrophils and xenoendothelium requires preactivation of endothelial cells by complement and/or natural xenoantibodies and, consequently, that the PMN plays a secondary role in HAR. In contrast, others have providedevidencethathumanPMNhavetheinherentcapac

P-selectin binding to P-selectin glycoprotein ligand-1 induces an intermediate state of αMβ2 activation and acts cooperatively with extracellular stimuli to support maximal adhesion of human neutrophils

AbstractP-selectin glycoprotein ligand 1 (PSGL-1, CD162) and integrin αMβ2 (Mac-1, CD11bCD18) are leukocyte adhesion molecules essential for innate immunity and inflammation. The interaction of PSGL-1 with P-selectin (CD62P) mediates tethering, rolling, and weak adhesion of leukocytes, during which they become sufficiently activated in situ by locally released or displayed cytokines and chemoattractants for integrin-mediated firm adhesion. However, communication between P-selectin and the integrin, whether P-selectin can trigger β2-integrin activation, remains controversial. We found that P-selectin immunoglobulin chimera and PSGL-1 monoclonal antibodies (mAbs) increased adhesion of human neutrophils to immobilized, but not soluble, fibrinogen. This intermediate state of neutrophil adhesion was defined by moderate clustering of integrin αMβ2, no increase in CBRM1/5 (a mAb specific for the activation epitope on the αM subunit) recognition, and no increase in surface expression of αMβ2, whereas phorbol myristate acetate (PMA) induced extensive changes in these 3 parameters. Furthermore, platelet-activating factor or interleukin 8 acted in concert with P-selectin for further enhancing the activation of αMβ2. We thus propose a model in which P-selectin induces an intermediate state of integrin activation and then cooperates with other extracellular stimuli to support maximal adhesion of human neutrophils.

Role of Endothelial Mitochondria in Oxidant Production and Modulation of Neutrophil Adherence

This study is designed to test whether the postanoxic endothelial mitochondria is an important source of reactive oxygen species (ROS) using a chemical model of mitochondrial disruption to mimic the loss of mitochondrial integrity after anoxia/reoxygenation (A/R). The current objectives were to (1) determine the adhesion of human neutrophils to human umbilical vein endothelial cells exposed to antimycin A, a specific inhibitor of the mitochondrial cytochrome b–c₁ complex, and (2) define the mechanisms responsible for the early and late phases of neutrophil hyperadhesivity. Antimycin A caused a 5-fold increase in ROS generation and induced neutrophil adhesion at 30 min (phase 1) and 4 h (phase 2) that were quantitatively similar to that induced by A/R. Blockade of electron transport in antimycin A and A/R exposed cells with rotenone, amytal or thenoyltrifluoroacetate, but not myxothiazol, prevented neutrophil adhesion, confirming a role for mitochondrial ROS. Catalase inhibited phase 1 adhesion, indicating H₂O₂ involvement. Anti-ICAM-1 or anti-P-selectin monoclonal antibodies (mAbs) attenuated phase 1 adhesion, while anti-E-selectin mAb attenuated phase 2 adhesion, consistent with roles for constitutive ICAM-1 and preformed P-selectin in early and E-selectin in late phase responses. Actinomycin D and cycloheximide or competing ds-oligonucleotides containing cognate DNA sequences of the nuclear factor ĸB or activator protein-1 attenuated phase 2 adhesion, implicating a role for de novo protein synthesis. Peak surface expression of the endothelial cell adhesion molecules correlated with peak adhesions at phases 1 and 2. These results show that disruption of mitochondrial respiratory chain elicits ROS production that mediates transcription-independent and -dependent surface expression of various adhesion molecules that leads to a two-phase neutrophil-HUVEC interaction similar to that induced by A/R.

Anti-adhesive glycoproteins in echinoderm mucus secretions

Marine invertebrates produce a large variety of mucus secretions which are rich in glycoproteins. As part of our studies of natural antifouling mechanisms, mucus secretions from the starfish Marthasterias glacialis and Porania pulvillus and the brittlestar Ophiocomina nigra have been used to characterise the structure and function of some of the glycoproteins present in these secretions. Mucus was collected from all three species and fractionated by size exclusion chromatography. A high molecular weight glycoprotein fraction was collected from each species. Monosaccharide analysis and FTIR demonstrated a composition consistent with a mucin-type glycoprotein. The mucin from M. glacialis and O. nigra inhibited in vitro bacterial adhesion in a dose-dependent manner. In contrast, the mucin from P. pulvillus promoted bacterial adhesion in a dose-dependent manner. All of the mucins inhibited the adhesion of human neutrophils to cultured human vascular endothelial cells (HUVECs) and had no anticoagulant activity. The mucins described here have adhesion-regulating functions that may have a role in the antifouling or feeding mechanisms of the organisms that produce them. These mucins may also be of therapeutic value through their ability to regulate human neutrophil adhesion or bacterial adhesion.

Tick saliva reduces adherence and area of human neutrophils.

During natural infection with the agent of Lyme disease, Borrelia burgdorferi, spirochetes are delivered with vector saliva, which contains anti-inflammatory and antihemostatic activities. We show here that the saliva of ixodid ticks reduces polymorphonuclear leukocyte (PMN) adhesion via downregulation of beta2-integrins and decreases the efficiency of PMN in the uptake and killing of spirochetes. Inhibition of integrin adhesion and signaling reduces anti-inflammatory functions of PMN. These effects may favor the initial survival of spirochetes in vivo.

The molecular basis for adhesion-mediated suppression of reactive oxygen species generation by human neutrophils.

Human neutrophil adherence to ECMs induces an initial inhibition of stimulated reactive oxygen species (ROS) formation, followed by an enhanced phase of oxidant production. The initial integrin-mediated suppression of ROS constitutes a mechanism to prevent inappropriate tissue damage as leukocytes migrate to inflammatory sites. The Rac2 guanosine 5'-triphosphatase (GTPase) is a critical regulatory component of the phagocyte NADPH oxidase. We show that activation of Rac2 is inhibited in adherent neutrophils, correlating with inhibition of ROS formation. Conversely, NADPH oxidase components p47 and p67 assemble normally, suggesting a specific action of adhesion on the Rac2 molecular switch. Reconstitution with activated Rac2 restored rapid NADPH oxidase activation kinetics to adherent neutrophils, establishing that inhibition was due to defective Rac2 activity. We provide evidence that integrins inhibit Rac2 activation via a membrane-associated guanine nucleotide exchange factor, likely to be Vav1. Activation of Vav1, but not its upstream activator, Syk, is suppressed by cell adhesion. Vav1 activity is inhibited due to dephosphorylation of the regulatory Tyr174 via enhanced tyrosine phosphatase activity in adherent cells. These studies identify an integrin-mediated pathway in which Vav1 is as a strong candidate for the critical regulatory point in suppression of Rac2 activation and ROS generation during inflammatory responses.

Inhibitory effect of salmeterol on the respiratory burst of adherent human neutrophils.

Human neutrophils, plated in fibronectin-coated wells and stimulated with N-formyl-methionylleucyl-phenylalanine (fMLP), were found to undergo a massive and prolonged respiratory burst, as measured by monitoring superoxide production. The beta 2-agonist salmeterol inhibited the respiratory burst in a dose-dependent manner. In contrast, salbutamol was ineffective. Moreover, the neutrophil respiratory burst was partially suppressed by prostaglandin E2 (PGE2) and the phosphodiesterase type IV (PDE-IV) inhibitor RO 20-1724. When salmeterol was used in combination with PGE2 or RO 20-1724, additive inhibitory effects were observed. The inhibitory activity of salmeterol was not reversed in the presence of the beta-blocker propranolol, and did not correlate with its ability of increasing cyclic AMP (cAMP) levels. Finally, the compounds used did not affect neutrophil adherence to fibronectin-coated wells. The results suggest that salmeterol is capable of down-regulating the neutrophil oxidative response to fMLP, also of co-operating with PGE2 and PDE-IV inhibitor RO 20-1724 in a manner not related to its beta 2-receptor binding activity. In other words, salmeterol displays neutrophil-directed effects, susceptible to be amplified by natural mediators such as PGE2 or PDE-IV inhibitors, consistent with possible anti-inflammatory properties of the drug.

Porcine endothelium activated by anti-alpha-GAL antibody binding mediates increased human neutrophil adhesion under flow.

Neutrophils participate in acute vascular rejection (AVR) of organ xenografts. Induced antibodies (Abs), including anti-Galalpha1,3Gal (alpha-Gal) Abs, have been suggested to cause AVR. We investigated the adhesion of naive human neutrophils to porcine aortic endothelial cells (PAECs) stimulated with anti-alpha-Gal Abs under conditions of flow. In addition, the ability of human neutrophils to adhere to human and porcine endothelium under static and flow conditions was evaluated. In a flow-adhesion assay, a significant increase in adhesion of human neutrophils to PAECs, but not to human aortic endothelial cells (HAECs), was detected 6 hours after anti-alpha-Gal Ab-binding. After Ab stimulation, PAECs expressed CD62E and increased levels of CD106, indicating an activated endothelial cell (EC) phenotype. In a migration assay, supernatants from Ab-stimulated PAECs induced migration of human neutrophils, which was partially blocked by anti-porcine (p) interleukin (IL)-8 Abs and an antagonist to platelet-activating factor (PAF). In static and flow-adhesion assays, no difference in adhesion of human neutrophils to unstimulated or tumor necrosis factor (TNF)-alpha-stimulated HAECs and PAECs could be detected. Our data suggest that anti-alpha-Gal Abs play an important role in the initiation of AVR by mediating adhesion and recruitment of neutrophils within an organ xenograft. In contrast with previous investigations, our data argues against a differential recognition of PAECs and HAECs by human neutrophils. Thus, to prevent AVR and accomplish long-term xenograft survival, it will be important to remove anti-alpha-Gal Abs before and after pig-to-human transplantation.

The pivotal role of scavenger receptor CD36 and phagocyte-derived oxidants in oxidized low density lipoprotein-induced adhesion to endothelial cells

Adhesion of phagocytes to endothelial cells constitutes a crucial step in atherogenesis. Oxidized low density lipoproteins (LDL) are supposed to facilitate the adhesion process. We investigated the molecular mechanisms by which mildly and extensively hypochlorite-oxidized LDL force adhesion of murine macrophages and human neutrophils to human umbilical venous endothelial cells. After 1 h of co-incubation of macrophages, endothelial cells, and lipoproteins adhesion significantly increased to 160±13% (S.E.M., n=5) in the presence of mildly oxidized lipoprotein, and 210±11% (S.E.M., n=5) in the presence of extensively oxidized lipoprotein. Similar results were obtained with neutrophils. CD36 antibody (FA6-152) significantly reduced adhesion to 102±7% (S.E.M., n=5) using mildly oxidized low density lipoprotein and to 179±16% (S.E.M., n=5) using extensively oxidized low density lipoprotein. Native high density lipoprotein and to a lesser extent methionine-oxidized high density lipoprotein significantly counteracted the effects of low density lipoprotein. Prior incubation of endothelial cells with modified lipoproteins followed by their removal and subsequent incubation with macrophages or neutrophils resulted in only minor changes of adhesion. This suggests that the direct contact of low density lipoprotein with phagocytes followed by activation of a respiratory burst with release of reactive oxygen species facilitates the adhesion process. Accordingly, the addition of antioxidants (superoxide dismutase and catalase) to the co-incubation medium was followed by a significant decrease in phagocyte adhesion. It is concluded that oxidized low density lipoprotein-induced respiratory burst activation of phagocytes with subsequent release of oxidants constitutes a crucial step in promoting the adhesion of phagocytes to endothelial cells.

NADPH oxidase activation in fibronectin adherent human neutrophils: A potential role for β1 integrin ligation

Background. Hydrogen peroxide production by human neutrophils is tightly coupled to integrin ligation. This provides a means for spatial localization of oxidant production. However, integrin specificity and consequent signaling mechanisms for this process remain undefined. In the present study we demonstrate that otherwise unstimulated neutrophils adherent to fibronectin, a β1 ligand, but not fibrinogen, a β2 ligand, produce hydrogen peroxide in a time-dependent fashion. We hypothesized that signaling proceeded through focal, adhesionlike structures. Methods. Triton-X insoluble (actin cytoskeleton) fractions from suspension phase cells and cells adherent to the β1 integrin ligand fibronectin were assessed for the presence of the integrin-associated kinase Pyk2, the scaffolding protein paxillin, and the downstream Src family kinase Lyn. Lysates were subjected to immunoprecipitation with anti-phosphotyrosine and probed for Pyk2, paxillin, and Lyn. Associations between focal adhesion kinase (FAK) and paxillin were determined by immunoprecipitation with anti-FAK and probing with anti-paxillin antibody. Activation of NADPH oxidase was determined by demonstration of redistribution of p47phox in Triton-X insoluble fractions. Results. NADPH oxidase activation, as judged by H2O2 production, occurred with fibronectin-, but not in suspension or fibrinogen-adherent cells. Cells adherent to fibronectin for 20 minutes demonstrated marked increases in Pyk2, paxillin, and Lyn activation in comparison to fibronectin-adherent and suspension phase cells. Conclusion. This study demonstrates that fibronectin adherence is a significant initiating factor for NADPH oxidase assembly in human neutrophils. This appears to be mediated by β1 integrins. We demonstrate formation of focal adhesions containing Pyk2/FAK, paxillin, and Lyn, and translocation of p47phox. (Surgery 2003;133:378-83.)

Expression of IL-2 receptor beta and gamma chains by human gingival fibroblasts and up-regulation of adhesion to neutrophils in response to IL-2.

To investigate the role of human gingival fibroblasts (HGF), the major constituents of gingival tissue in periodontal inflammatory disease, the expression of interleukin-2 receptor (IL-2R) alpha, beta, and gamma chains was examined. Immunohistochemistry showed a pronounced accumulation of CD8(+) T cells in the inflamed lamina propria of gingival tissue from patients with adult periodontitis. HGF express IL-2Rbeta and IL-2Rgamma at mRNA and protein levels, but the expression of IL-2Ralpha could not be detected, as assessed by reverse transcriptase-polymerase chain reaction and flow cytometry. IL-2Rbeta, and -gamma expressed on HGF were functionally active, as addition of neutralizing anti-IL-2Rbeta and -gamma antibodies caused inhibition of the IL-2-induced production of monocyte chemoattractant protein-1 (MCP-1), and addition of IL-2 induced phosphorylation of Janus tyrosine kinase 3, which is critical in signaling through IL-2Rgamma in HGF. The IL-2-induced MCP-1 production was significantly inhibited by pretreatment with neutralizing antibody to IL-15. Addition of IL-2 also induced a marked up-regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of HGF, which in turn, significantly augmented the adhesion of human neutrophils, which were inhibited by an anti-ICAM-1 antibody. These results suggest that HGF express functional IL-2Rbetagamma, respond to IL-2 from infiltrated T cells, and actively participate in the inflammatory process in the periodontal region and that IL-15 produced by HGF sustains IL-2-mediated signaling in HGF.

Down-regulation of Rac Activity during β2 Integrin-mediated Adhesion of Human Neutrophils

In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.

A role for Syk-kinase in the control of the binding cycle of the beta2 integrins (CD11/CD18) in human polymorphonuclear neutrophils.

A fine control of beta(2) integrin (CD11/CD18)-mediated firm adhesion of human neutrophils to the endothelial cell monolayer is required to allow ordered emigration. To elucidate the molecular mechanisms that control this process, intracellular protein tyrosine signaling subsequent to beta(2) integrin-mediated ligand binding was studied by immunoprecipitation and Western blotting techniques. The 72-kDa Syk-kinase, which was tyrosine-phosphorylated upon adhesion, was found to coprecipitate with CD18, the beta-subunit of the beta(2) integrins. Moreover, inhibition of Syk-kinase by piceatannol enhanced adhesion and spreading but diminished N-formyl-Met-Leu-Phe-induced chemotactic migration. The enhancement of adhesiveness was associated with integrin clustering, which results in increased integrin avidity. In contrast, piceatannol had no effect on the surface expression or on the affinity of beta(2) integrins. Altogether, this suggests that Syk-kinase controls alternation of beta(2) integrin-mediated ligand binding with integrin detachment.

Expression of Functional ICAM-1 on Cultured Human Keratocytes Induced by Tumor Necrosis Factor- α

Purpose: Leukocytes such as neutrophils contribute to the pathogenesis of corneal ulcer. The effect of the proinflammatory cytokine tumor necrosis factor (TNF)-α on the expression of intercellular adhesion molecule (ICAM)-1 by cultured human keratocytes was investigated because the interaction of leukocytes with ICAM-1 expressed on the surface of structural cells mediates leukocyte infiltration into tissue at sites of inflammation. Methods: Cultured human keratocytes were incubated with various concentrations of TNF-α. The surface expression of ICAM-1 was evaluated by whole-cell enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry. The abundance of ICAM-1 mRNA in cell lysate was determined by quantitative reverse transcription and polymerase chain reaction analysis. Adhesion of neutrophils to corneal fibroblasts was assayed by measuring the fluorescence of Calcein-AM-labeled neutrophils. Results: Incubation of keratocytes with TNF-α induced increased expression of ICAM-1 on the surface of keratocytes in a dose- and time-dependent manner. The abundance of ICAM-1 mRNA in keratocytes was increased by the incubation of cells with TNF-α. Exposure of keratocytes to TNF-α increased the adherence of human neutrophils to these cells. Conclusions: Stimulation of keratocytes with TNF-α resulted in an increase in the abundance of ICAM-1 mRNA, the cell surface expression of ICAM-1 protein, and enhanced adhesion of neutrophils to these cells. The expression of ICAM-1 on human keratocytes may thus contribute to leukocyte infiltration into the corneal stroma of individuals with inflammatory ocular diseases.

Fgr but not Syk tyrosine kinase is a target for beta 2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils.

An early and critical event in beta(2) integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent beta(2) integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) beta(2) integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3',5'-tetrahydroxy- trans -stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected beta(2) integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in beta(2) integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.

Syndecan-4-dependent signaling in the inhibition of endotoxin-induced endothelial adherence of neutrophils by antithrombin.

Circulating endotoxin is elevated in sepsis and plays a role in endothelial dysfunction whereas antithrombin is decreased by virtue of its consumption during complex formation with clotting factors and by proteolytic degradation by granulocyte elastase. Dysfunction of endothelium results in enhanced leukocyte rolling and diapedesis into tissues leading to edema formation and injury. Antithrombin exerts beneficial effects on endothelial function in sepsis. A direct anti-inflammatory action of anti-thrombin in inflammatory cells is exerted via heparan sulfate proteoglycans. In this study, we investigated whether antithrombin affects endotoxin-induced adhesion of neutrophils to human endothelial cells in vitro and whether glycosaminoglycans are involved in its signaling. Adhesion of human neutrophils to monolayers of umbilical vein endothelial cells was tested under static conditions. Endothelial cells were pretreated with endotoxin, interleukin-1, heparinase-I, chondroitinase-ABC or anti-syndecan-4-antibody. Endotoxin and interleukin-1 increased neutrophil adherence to human umbilical vein endothelial cells which was inhibited by antithrombin. Concomitant incubation with pentasaccharide abolished this effect of antithrombin. Treatment of endothelial cells with heparinase or chondroitinase led to higher adhesion and prevented effects of antithrombin. With antibodies to syndecan-4, enhanced adhesion of neutrophils was observed. As studied by Western blotting, endotoxin-induced signaling was diminished by antithrombin and the effect was reversible by chondroitinase or heparinase. From our results, we can conclude that endotoxin-induced adhesion of leukocytes to endothelium can be reversed by ligation of syndecan-4 with antithrombin's heparin-binding site and interferences with stress response signaling events in endothelium.