PMN Adhesion Research Articles

Host defence against Staphylococcus aureus biofilms by polymorphonuclear neutrophils: Oxygen radical production but not phagocytosis depends on opsonisation with immunoglobulin G

Bacterial biofilms are increasingly recognised as a major cause of persistent infection and destructive inflammatory processes. In patients with biofilm infection, massive infiltration of leukocytes, particularly polymorphonuclear neutrophils is seen, and previous in vitro studies showed that PMN were able to phagocytose Staphylococcus aureus biofilms. We now addressed the question whether opsonisation of biofilms with immunoglobulin G and complement enhances the efficiency of phagocytosis, as it has been shown for “free-living” planktonic bacteria and other particulate materials. We found that incubation of biofilms with normal human serum resulted in IgG binding and in complement activation with deposits on the biofilm of C3bi. This “opsonisation”, however, did not affect the adherence of PMN to the biofilms nor did it enhance degranulation or phagocytosis. The clearance of biofilms, however, was increased, and the oxygen radical production by the PMN depended critically on the coating of biofilms with IgG.

Recruitment of Inflammatory cells to the Lung is Dependent upon Platelet Activating Factor production in Smokers

To recruit inflammatory cells to the airways, cells must move from the circulation across the endothelial and epithelial cell barriers. Platelet‐activating factor (PAF) is involved in transendothelial migration in several vascular beds. We hypothesize that an increase in PAF production and cell recruitment may exacerbate inflammatory disease in smokers. We incubated human lung microvascular endothelial cells (HMVEC‐L) with cigarette smoke extract (CSE). CSE (20 μg/ml) significantly inhibited PAF‐acetylhydrolase (PAF‐AH, hydrolyzes and inactivates PAF) activity after 12 hours of CSE incubation (2.1 ± 0.1 to 0.7 ± 0.1 nmol/mg protein/min, n=6, p<0.01). Inhibition of PAF‐AH resulted in increased PAF production (538 ± 60 to 1580 ± 241 dpm, n=6, p<0.01) and PMN adherence (14 ± 3 to 47 ± 3%, n=4, p<0.01). Pretreatment of PMN with the PAF receptor antagonist CV3988 (10μM for 10 min) resulted in complete inhibition of PMN adherence (0.4 ± 0.1, n=4, p<0.01). In the lung, PMN accumulation in the small airways represents one of the initial steps in inflammation and emphysema. In conclusion, these studies have shown that PAF likely plays a central role in the recruitment of inflammatory cells to the small airways of cigarette smokers via inactivation of PAF‐AH and may exacerbate or initiate inflammatory lung disease.

The novel non‐depolarizing, normokalemic cardioplegia formulation based on the combination of adenosine‐lidocaine (Adenocaine™) exerts superior anti‐neutrophil effects by synergistic actions of its components

ObjectivesWe tested the hypothesis that the combination of adenosine and lidocaine (AL) synergistically attenuates neutrophil (PMN) functions.Methods1. Superoxide anion (O2‐) generation, 2. PMN adherence, 3. Transmigration. Group differences (one‐way ANOVA) were accepted at P value<0.05.ResultsThe isolated porcine PMNs released superoxide dismutase (0.1mg/ml)‐sensitive O2‐ in a burst pattern upon activation. Both adenosine and lidocaine alone inhibited O2‐ production in a dose‐dependent pattern. AL further inhibited O2‐ generation in a synergistic manner. In addition, adenosine (0.1–10 μM) inhibited O2‐ generation by cytochalasin B (priming), whereas lidocaine (1–100 μM) inhibited O2‐ release stimulated by both cytochalasin B and fMLP (activation). Both adenosine and lidocaine alone inhibited PAF‐induced PMN adherence to endothelial cells. Adenosine (50μM) and lidocaine (100 μM) decreased adherence to 71.6±5.4% (n=8, P<0.05) and 73.2±8.1% (n=7, P<0.05) of stimulated control, respectively. AL further suppressed adherence to 47.2±4.3% (n=7, P<0.05). Adenosine (100μM) reduced PMN transendothelial migration to 80.1±6.7% of control (n=6, P<0.05), whereas lidocaine (100μM) showed no significant inhibition. AL did not further decrease transmigration.ConclusionsAL suppresses PMN O2‐ generation and adherence in a synergistic manner relative to adenosine or lidocaine alone.

Evaluation of in-vitro anti-inflammatory activity of some 2-alkyl-4,6-dimethoxy-1,3,5-triazines

The ability of some 2-alkyl(aryl)-4,6-dimethoxy-1,3,5-triazine derivatives to interfere with production of reactive oxygen species (ROS) by human phagocytes was evaluated in an in-vitro cell model. Superoxide anion (O(2)(-*)) production by human polymorphonuclear cells (PMNs), challenged by the chemotactic agent N-formylmethionyl-leucyl-phenylalanine (FMLP), was inhibited in a dose-dependent manner by all the compounds tested, compounds 3, 4 and 5 being statistically the most active. Adhesion of PMNs to vascular endothelial cells (ECs) is a critical step in recruitment and infiltration of leucocytes into tissues during inflammation, and the effects of 1,3,5-triazine derivatives on PMN adhesion to ECs from the human umbilical vein (HUVEC) were also investigated. Triazines were incubated with PMNs and HUVEC; adhesion was quantitated by computerized micro-imaging fluorescence analysis. The 1,3,5-triazines tested inhibited the adhesion evoked by pro-inflammatory stimuli, such as platelet activating factor (PAF), FMLP, phorbol myristate acetate (PMA), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta(IL-1beta) in a dose-response manner over the concentration range 10(-9) to 10(-4)M, compounds 5 and 6 being the most active. Both of these compounds inhibited PMN adhesion to HUVEC, even when endothelial or PMN stimuli were used. Indeed, when both cell populations were activated contemporarily, the anti-adhesive effect was enhanced. The study suggests that 2-aryl-4,6-dimethoxy-1,3,5-triazines deserve further evaluation as anti-inflammatory agents.

Effects of sepsis on neutrophil chemotaxis

Neutrophil recruitment to sites of infection is a critical element of the innate immune response. In patients with sepsis, this response is dysregulated, with exuberant inflammation being followed by a state of profound immune suppression, including inhibition of neutrophil recruitment. This review examines mechanisms underlying suppression of neutrophil migration during sepsis. Mechanisms governing neutrophil chemotactic function in sepsis are complex. Bacterial products, cytokines, and chemokines can modulate neutrophil migratory responses during sepsis via induction of cytoskeletal changes, inhibition of polymorphonuclear leukocyte (PMN)-endothelial cell interactions, and alterations in G protein-coupled receptor expression or signaling. Impaired chemotactic responses can occur as a result of dysregulated PMN Toll-like receptor signaling. Other recently identified inhibitory mechanisms include exposure to elevated temperatures, activation of the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor-gamma, and suppression of PMN-endothelial interactions due to nitric oxide and its metabolites. Finally, circulating microparticles released in sepsis exert important immunomodulatory effects on PMN adherence and transmigration. Neutrophil recruitment is a coordinated process that is altered at multiple stages during sepsis, culminating in defective innate immunity and increased risk of infection in these patients. Defining mechanisms involved and strategies to interrupt these deleterious responses requires further investigation.

Mediators released from LPS-challenged lungs induce inflammatory responses in liver vascular endothelial cells and neutrophilic leukocytes

The systemic inflammatory response plays an important role in the progression of acute lung injury (ALI) to multiple organ dysfunction syndrome (MODS). However, the role of lung-derived inflammatory mediators in induction of the inflammatory response in remote organs is poorly understood. To address the above, we investigated the effects of lung inflammation on induction of inflammatory response(s) in the liver in vitro. Inflammation in mouse lungs was induced by intranasal administration of lipopolysaccharide (LPS; 1 mg/ml) followed by mechanical ventilation using the isolated perfused mouse lung method to obtain and characterize lung perfusate from the pulmonary circulation. LPS administration to mouse lungs resulted in an increased release of inflammation-relevant cytokines and chemokines into the perfusate (Luminex assay) compared with the saline-controls. Subsequently, primary mouse liver vascular endothelial cells (LVEC) or mouse polymorphonuclear leukocytes (PMN) in vitro were stimulated with the perfusate obtained from saline- or LPS-challenged lungs and assessed for various inflammation-relevant end points. The obtained results indicate that stimulation of LVEC with perfusate obtained from LPS-challenged lungs results in 1) reactive oxygen species (ROS) production; 2) activation of NF-kappaB; and 3) expression of E-selectin, ICAM-1, and VCAM-1 and a subsequent increase in PMN rolling and adhesion to LVEC. In addition, perfusate from LPS-challenged lung induced activation of PMN with respect to increased ROS production and upregulation of cell surface levels of adhesion molecules MAC-1 and VLA-4. Heat-inactivation of the perfusate obtained from LPS-challenged lungs was very effective in suppressing increased proadhesive phenotype (i.e., E-selectin and ICAM-1 expression) in LVEC, whereas targeted inhibition (immunoneutralization) of TNF-alpha and/or IL-6 in LPS-lung perfusate had no effect. Taken together, these findings indicate that multiple proinflammatory mediators (proteinaceous in nature) released from inflamed lungs act synergistically to induce systemic activation of circulating PMN and promote inflammatory responses in liver vascular endothelial cells.

Polymorphonuclear leukocytes isolated from umbilical cord blood as a useful research tool to study adherence to cell monolayers

One of the initial steps in the inflammatory process involves the adherence and transmigration of circulating polymorphonuclear leukocytes (PMN) across the endothelial cell monolayer. One of the main constituents of the neutrophil phagosome that contributes to bacterial killing is myeloperoxidase (MPO) which can be measured spectrophotometrically, using hydrogen peroxide as a substrate, and hence can be used as an index to quantify neutrophil adherence. To evaluate whether PMN isolated from umbilical cord blood could be used for in vitro experiments to monitor neutrophil adherence, we compared the adherence to confluent endothelial and epithelial cell monolayers using PMN isolated from umbilical cord and adult peripheral blood. The extent of PMN adherence was assessed by measuring MPO activity. In initial experiments, we isolated PMN from umbilical cord and adult peripheral blood and measured MPO activity with respect to cell number and assay incubation times. Our data demonstrate that PMN obtained from either source had similar MPO activity and similar adherence to endothelial or epithelial cells. In conclusion, our data suggest that umbilical cord blood is a suitable source of leukocytes to examine PMN adherence in the setting of inflammation in a variety of disease processes.

Phosphoinositide 3-kinase required for lipopolysaccharide-induced transepithelial neutrophil trafficking in the lung

Phosphoinositide 3-kinase gamma(PI3Kgamma) is a critical mediator of directional cell movement. Here, we sought to characterise the role of PI3Kgamma in mediating the different steps of polymorphonuclear leukocyte (PMN) trafficking in the lung. In a murine model of lipopolysaccharide (LPS)-induced lung injury, PMN migration into the different lung compartments was determined in PI3Kgamma gene-deficient (PI3Kgamma(-/-)) and wild-type mice. Bone marrow chimeras were created to characterise the role of PI3Kgamma on haematopoietic versus nonhaematopoietic cells. A small-molecule PI3Kgamma inhibitor was tested in vitro and in vivo. PMN adhesion to the pulmonary endothelium and transendothelial migration into the lung interstitium was enhanced in PI3Kgamma(-/-) mice. However, transepithelial migration into the alveolar space was reduced in these mice. When irradiated PI3Kgamma(-/-) mice were reconstituted with bone marrow from wild-type mice, migratory activity into the alveolar space was restored partially. A small-molecule PI3Kgamma inhibitor reduced chemokine-induced PMN migration in vitro when PMNs or epithelial cells, but not when endothelial cells, were treated. The inhibitor also reduced LPS-induced PMN migration in vivo. We conclude that PI3Kgamma is required for transepithelial but not for transendothelial migration in LPS-induced lung injury. Inhibition of PI3Kgamma activity may be effective at curbing excessive PMN infiltration in lung injury.

CORM-3-derived CO modulates polymorphonuclear leukocyte migration across the vascular endothelium by reducing levels of cell surface-bound elastase

Recently, it has been shown that carbon monoxide (CO)-releasing molecule (CORM)-released CO can suppress inflammation. In this study, we assessed the effects and potential mechanisms of a ruthenium-based water-soluble CO carrier [tricarbonylchloroglycinate-ruthenium(II) (CORM-3)] in the modulation of polymorphonuclear leukocyte (PMN) inflammatory responses in an experimental model of sepsis. Sepsis in mice was induced by cecal ligation and puncture. CORM-3 (3 mg/kg iv) was administered 15 min after the induction of cecal ligation and puncture. PMN accumulation in the lung (myeloperoxidase assay), bronchoalveolar lavage (BAL) fluid, and lung vascular permeability (protein content in BAL fluid) were assessed 6 h later. In in vitro experiments, human PMNs were primed with LPS (10 ng/ml) and subsequently stimulated with formyl-methionyl-leucylphenylalanine (fMLP; 100 nM). PMN production of ROS (L-012/dihydrorhodamine-123 oxidation), degranulation (release of elastase), and PMN rolling, adhesion, and migration to/across human umbilical vein endothelial cells (HUVECs) were assessed in the presence or absence of CORM-3 (1-100 muM). The obtained results indicated that systemically administered CORM-3 attenuates PMN accumulation and vascular permeability in the septic lung. Surprisingly, in in vitro experiments, treatment of PMNs with CORM-3 further augmented LPS/fMLP-induced ROS production and the release of elastase. The latter effects, however, were accompanied by an inability of PMNs to mobilize elastase to the cell surface (plasma membrane), an event required for efficient PMN transendothelial migration. The CORM-3-induced decrease in cell surface levels of elastase was followed by decreased PMN rolling/adhesion to HUVECs and complete prevention of PMN migration across HUVECs. In contrast, treatment of HUVECs with CORM-3 had no effect on PMN transendothelial migration. Taken together, these findings indicate that, in sepsis, CORM3-released CO, while further amplifying ROS production and degranulation of PMNs, concurrently reduces the levels of cell surface-bound elastase, which contributes to suppressed PMN transendothelial migration.

DARC on RBC limits lung injury by balancing compartmental distribution of CXC chemokines.

The Duffy antigen receptor for chemokines (DARC) has a high affinity for CC and CXC chemokines. However, it lacks the ability to induce cell responses that are typical for classical chemokine receptors. The role of DARC in inflammatory conditions remains to be elucidated. We studied the role of DARC in a murine model of acute lung injury. We found that in Darc-gene-deficient (Darc(-/-)) mice, LPS-induced PMN migration into the alveolar space was elevated more than twofold. In contrast, PMN adhesion to endothelial cells and within the interstitial space was reduced in Darc(-/-) mice. Darc(-/-) mice also exhibited increased microvascular permeability. Elevated PMN migration in Darc(-/-) mice was associated with increased concentrations of two essential CXCR2 ligands, CXCL1 and CXCL2/3 in the alveolar space. In the blood, CXCL1 was mostly associated with RBC in WT mice and with plasma in Darc(-/-) mice. We found that DARC on RBC prevented excessive PMN migration into the alveolar space. In contrast, DARC on non-hematopoietic cells appeared to have only minor effects on leukocyte trafficking in this model. These findings show how DARC regulates lung inflammation by controlling the distribution and presentation of chemokines that bind CXCR2.

Neutrophil Adhesion to Periostin is Mediated by Integrins

Neutrophil (PMN) localization at the lumenal epithelium surface is a key histological feature of the large intestine of inflammatory bowel disease ((IBD), Crohn's Disease and Ulcerative Colitis) patients. Extracellular matrix proteins (ECM) can affect PMN recruitment and other functions. Periostin, an ECM protein secreted by fibroblasts during cellular injury and tissue repair, was investigated as an adhesive molecule for PMN. Immunofluorescence reveals that periostin similarly to the subepithelial fibroblasts is confined to the base of the crypt epithelium. Flow Cytometry and adhesion results show that intestinal fibroblasts and T84 epithelial cells adhere to periostin and express periostin on the cell surface. Anti‐αvβ3 and anti‐αvβ5 (known integrin receptors for periostin) antibodies inhibit periostin adhesion to fibroblasts and T84 cells. In particular, anti‐αvβ3 and anti‐αvβ5 antibodies inhibit 50% of adhesion. However, anti‐CD11b antibodies inhibit 95% of this adhesion. PMN surface expression of periostin is reduced when PMN are exposed to microbial products and anti‐periostin antibodies inhibit PMN adhesion to fibrinogen (a known ligand for CD11b, a precursor to fibrin, and important in the inflammatory response) by 85%. Thus, PMN adhesion to periostin can be modulated by αvβ3, αvβ5 and CD11b integrins and may play a role in PMN‐epithelial interactions in IBD.

Inhibition of Neutrophil Function by Two Tick Salivary Proteins

The saliva of hematophagous arthropods contains potent anti-inflammatory and antihemostatic activities that promote acquisition of the blood meal and enhance infection with pathogens. We have shown that polymorphonuclear leukocytes (PMN) treated with the saliva of the tick Ixodes scapularis have reduced expression of beta(2) integrins, impaired PMN adherence, and reduced killing of Borrelia burgdorferi, the causative agent of Lyme disease. Here we describe two Ixodes proteins that are induced upon tick feeding and expressed predominantly in the salivary glands. Using saliva harvested from ticks with reduced levels of ISL 929 and ISL 1373 through targeted RNA interference knockdown, as well as purified recombinant proteins, we show the effects of these proteins on downregulation of PMN integrins and inhibition of the production of O(2)(-) by PMN in vitro. Mice immunized with ISL 929/1373 had increased numbers of PMN at the site of tick attachment and a lower spirochete burden in the skin and joints 21 days after infection compared to control-immunized animals. Our results suggest that ISL 929 and ISL 1373 contribute to the inhibition of PMN functions shown previously with tick saliva and support important roles for these inhibitory proteins in the modulation of PMN function in vivo.

Effects of Propyl Gallate on adhesion of polymorphonuclear leukocytes to human endothelial cells induced by tumor necrosis factor alpha

To investigate the effects of Propyl Gallate (PrG) on cellular adhesion between human To investigate the effects of Propyl Gallate (PrG) on cellular adhesion between human umbilical vein endothelial cells (HUVEC) and polymorphonuclear leukocytes (PMN) as well as the expression umbilical vein endothelial cells (HUVEC) and polymorphonuclear leukocytes (PMN) as well as the expression of intercellular adhesion molecule-1 (ICAM-1, CD54) and E-selectin (CD62E) on the VEC surface. of intercellular adhesion molecule-1 (ICAM-1, CD54) and E-selectin (CD62E) on the VEC surface. A human VEC inflammation model was induced by tumor necrosis factor alpha (TNF-alpha). VECs were pre- A human VEC inflammation model was induced by tumor necrosis factor alpha (TNF-alpha). VECs were preincubated with varying concentrations of PrG (0.001-5 mmol/L) or 1 per thousand DMSO (v:v) or 10 mmol/L acetylsalicylic incubated with varying concentrations of PrG (0.001-5 mmol/L) or 1 per thousand DMSO (v:v) or 10 mmol/L acetylsalicylic acid (ASA) for 1 h, and then were stimulated with 10 ng/mL TNF-alpha for 6 h. Rose bengal vital staining method acid (ASA) for 1 h, and then were stimulated with 10 ng/mL TNF-alpha for 6 h. Rose bengal vital staining method was used to measure the adherence rate of PMN to VEC, while flow cytometry was used to determine the was used to measure the adherence rate of PMN to VEC, while flow cytometry was used to determine the expression of CD54 and CD62E on the VEC surface. expression of CD54 and CD62E on the VEC surface. After 6 h of incubation with TNF-alpha, the adherence After 6 h of incubation with TNF-alpha, the adherence of PMN to HUVECs as well as the percentage of fluorescence-positive cells and mean fluorescence intensity of PMN to HUVECs as well as the percentage of fluorescence-positive cells and mean fluorescence intensity (MFI) of surface CD54 and CD62E in HUVECs increased significantly ( (MFI) of surface CD54 and CD62E in HUVECs increased significantly (P<0.01). Pre-treatment of HUVECs with <0.01). Pre-treatment of HUVECs with PrG (0.1-5 mmol/L) significantly suppressed the adherence of PMN to VECs induced by TNF-alpha (PrG (0.1-5 mmol/L) significantly suppressed the adherence of PMN to VECs induced by TNF-alpha (P<0.05). PrG <0.05). PrG (1-5 mmol/L) inhibited the VEC surface expression of CD62E and CD54 in a dose-dependent way ( (1-5 mmol/L) inhibited the VEC surface expression of CD62E and CD54 in a dose-dependent way (P<0.05). PrG <0.05). PrG at lower concentrations (0.001-0.1 mmol/L) showed no effect on CD54 expression, while it showed a slightly at lower concentrations (0.001-0.1 mmol/L) showed no effect on CD54 expression, while it showed a slightly increasing trend in CD62E expression (increasing trend in CD62E expression (P>0.05). ASA at 10 mmol/L had no obvious effect on the positive rate of >0.05). ASA at 10 mmol/L had no obvious effect on the positive rate of CD62E and CD54. CD62E and CD54. High concentrations of PrG (0.1-5 mmol/L) exert its inhibitory effect on cellular High concentrations of PrG (0.1-5 mmol/L) exert its inhibitory effect on cellular adherence of PMN to HUVECs, and its mechanism may be related to inhibiting surface expression of CD54 and adherence of PMN to HUVECs, and its mechanism may be related to inhibiting surface expression of CD54 and CD62E in HUVECs. Its action concentration was lower than that of ASA. CD62E in HUVECs. Its action concentration was lower than that of ASA.

Different amplifying mechanisms of interleukin‐17 and interferon‐γ in Fcγ receptor–mediated cartilage destruction in murine immune complex–mediated arthritis

Previously, we reported that interferon-gamma (IFNgamma) aggravates cartilage destruction in immune complex (IC)-mediated arthritis via up-regulation of activating Fcgamma receptors (FcgammaR). Recently, we found that interleukin-17 (IL-17) also aggravates cartilage destruction in arthritis models in which ICs are involved, but the underlying mechanism remains unknown. This study was undertaken to determine the role of IL-17 in FcgammaR-mediated cartilage destruction in IC-mediated arthritis and to compare its effect with that of IFNgamma. IC-mediated arthritis was passively induced in gamma-chain(-/-) mice, which lack functional activating FcgammaR, and in wild-type controls. AdIL-17 or a control vector was injected into the knee joints 1 day prior to induction of IC-mediated arthritis. Knee joints were isolated for histologic analysis, and synovium samples were obtained for reverse transcriptase-polymerase chain reaction (RT-PCR). Macrophage (RAW 264.7) cell lines and polymorphonuclear cell (PMN; 32Dcl3) lines were stimulated with IFNgamma or IL-17 for analysis of FcgammaR expression using RT-PCR and fluorescence-activated cell sorting. IL-17 overexpression prior to induction of IC-mediated arthritis significantly aggravated cartilage destruction and inflammation, characterized by a massive influx of PMNs, which adhered to the cartilage surface. Although IL-17 overexpression increased FcgammaR messenger RNA levels in the synovium, in vitro stimulation of macrophages and PMNs revealed that, in contrast to IFNgamma, IL-17 did not directly regulate FcgammaR expression. Despite similar inflammation in AdIL-17-enhanced IC-mediated arthritis in gamma-chain(-/-) mice and wild-type controls, severe cartilage destruction and PMN adherence were completely absent in gamma-chain(-/-) mice. Our findings indicate that IL-17-mediated aggravation of cartilage destruction in IC-mediated arthritis is FcgammaR dependent. However, in contrast to IFNgamma, which directly up-regulates FcgammaR expression on macrophages and PMNs, IL-17 enhances cartilage destruction by increasing the local amount of FcgammaR-bearing neutrophils.

Neutrophils launch monocyte extravasation by release of granule proteins

During their journey from the blood stream to sites of inflammation polymorphonuclear leukocytes (PMN) release a wide panoply of granule proteins. Shortly after the PMN efflux, the extravasation of monocytes sets in and recent research provides evidence that the release of PMN granule proteins and monocyte extravasation are causally interrelated. Granule proteins seeded on the endothelium by adherent PMN allow direct activation and subsequent adhesion of monocytes. In addition, PMN granule components enhance the endothelial expression of cell adhesion molecules, efficiently supporting the arrest of monocytes at inflamed vessels. Moreover, granule proteins contribute to the fine tuning of the local chemokine network. Proteolytic modification of chemokines as well as enhancement of local chemokine synthesis lead to increased monocyte extravasation. Finally, PMN granule proteins exert direct chemotactic effects, a mechanism which is of special importance in the early recruitment of inflammatory monocytes. Hence, granule proteins modify the monocyte extravasation cascade in a multifaceted manner ensuring the efficiency of these mechanisms.

Chronic increases in sphingosine kinase-1 activity induce a pro-inflammatory, pro-angiogenic phenotype in endothelial cells

Sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which has potent pro-inflammatory and pro-angiogenic effects. We investigated the effects of raised SK1 levels on endothelial cell function and the possibility that this signaling pathway is activated in rheumatoid arthritis. Human umbilical vein endothelial cells with 3- to 5-fold SK1 (ECSK) overexpression were generated by adenoviral and retroviralmediated gene delivery. The activation state of these cells and their ability to undergo angiogenesis was determined. S1P was measured in synovial fluid from patients with RA and OA. ECSK showed an enhanced migratory capacity and a stimulated rate of capillary tube formation. The cells showed constitutive activation as evidenced by the induction of basal VCAM-1 expression, and further showed a more augmented VCAM-1 and E selectin response to TNF compared with empty vector control cells (ECEV). These changes had functional consequences in terms of enhanced neutrophil binding in the basal and TNFstimulated states in ECSK. By contrast, over-expression of a dominant-negative SK inhibited the TNF-induced VCAM-1 and E selectin and inhibited PMN adhesion, confirming that the observed effects were specifically mediated by SK. The synovial fluid levels of S1P were significantly higher in patients with RA than in those with OA. Small chronic increases in SK1 activity in the endothelial cells enhance the ability of the cells to support inflammation and undergo angiogenesis, and sensitize the cells to inflammatory cytokines. The SK1 signaling pathway is activated in RA, suggesting that manipulation of SK1 activity in diseases of aberrant inflammation and angiogenesis may be beneficial.

Transmigration of Neutrophils across Inflamed Endothelium Is Signaled through LFA-1 and Src Family Kinase

Leukocyte capture on inflamed endothelium is facilitated by a shift in LFA-1 from low to high affinity that supports binding to ICAM-1. LFA-1 bonds help anchor polymorphonuclear leukocytes (PMN) to inflamed endothelium in shear flow, and their redistribution to the leading edge guides pseudopod formation, migration, and extravasation. These events can be disrupted at the plasma membrane by stabilizing LFA-1 in a low- or intermediate-affinity state with allosteric small molecules. We hypothesized that a minimum dimeric bond formation between high-affinity LFA-1 and ICAM-1 under shear stress is necessary to catalyze transmembrane signaling of directed cell migration. Microspheres and substrates were derivatized with monomeric or dimeric ICAM-1 to simulate the surface of inflamed endothelium under defined ligand valence. Binding to dimeric ICAM-1, and not monomeric ICAM-1, was sufficient to elicit assembly of F-actin and phosphorylation of Src family kinases that colocalized with LFA-1 on adherent PMN. Genetic deletion or small molecule inhibition of Src family kinases disrupted their association with LFA-1 that correlated with diminished polarization of arrested PMN and abrogation of transmigration on inflamed endothelium. We conclude that dimeric bond clusters of LFA-1/ICAM-1 provide a key outside-in signal for orienting cytoskeletal dynamics that direct PMN extravasation at sites of inflammation.

Annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles

AbstractPolymorphonuclear leukocyte (PMN)–derived microparticles display inhibitory properties on target cells as assessed in vitro; since PMNs contain abundant amounts of the endogenous anti-inflammatory protein annexin 1 (AnxA1), we tested here whether biologically active AnxA1 could be present in PMN-derived microparticles. PMN adhesion to human umbilical vein endothelial cell (HUVEC) monolayers led to the generation of microparticles that contained AnxA1, as detected by Western blotting, flow cytometry, and mass spectrometry analyses. Addition of these microparticles to recipient PMNs prior to flow over HUVEC monolayers significantly inhibited cell adhesion, an effect abrogated by a neutralizing anti-AnxA1 antibody, or an antibody raised against the AnxA1 receptor, that is termed lipoxin A4 receptor or ALX. Intravenous delivery of human PMN–derived microparticles markedly inhibited PMN recruitment to an air pouch inflamed with IL-1β. This anti-inflammatory effect was also dependent on endogenous AnxA1, since injection of microparticles produced from wild-type PMNs (bone marrow derived), but not from AnxA1-null PMNs, inhibited IL-1β–induced leukocyte trafficking. In conclusion, PMN-derived microparticles contain functionally active AnxA1 that confers them anti-inflammatory properties; generation of these microparticles in the microcirculation could promote inflammatory resolution by time-dependent dampening of cell recruitment.

Anti-inflammatory actions of aprotinin provide dose-dependent cardioprotection from reperfusion injury.

Myocardial injury following ischaemia and reperfusion has been attributed to activation and transmigration of polymorphonuclear leukocytes (PMNs) with release of mediators including oxygen-derived radicals and proteases causing damage. We studied the serine protease inhibitor aprotinin in an in vivo rabbit model of 1 h of myocardial ischaemia followed by 3 h of reperfusion (MI+R). Aprotinin (10,000 Ukg(-1)) or its vehicle were injected 5 min prior to the start of reperfusion. Myocardial injury was significantly reduced with aprotinin treatment as indicated by a reduced necrotic area (11+/-2.7% necrosis as percentage of area at risk after aprotinin; 24+/-3.1% after vehicle; P<0.05) and plasma creatine kinase activity (12.2+/-1.5 and 17.3+/-2.3 IU g(-1) protein in aprotinin and vehicle groups, respectively, P<0.05). PMN infiltration (assessed by myeloperoxidase activity) was significantly decreased in aprotinin-treated animals compared to vehicle (P<0.01). Histological analysis also revealed a substantial increase in PMN infiltration following MI+R and this was significantly reduced by aprotinin therapy (44+/-15 vs 102+/-2 PMN mm2 in aprotinin vs vehicle-treated animals, P<0.05). In parallel in vitro experiments, aprotinin inhibited neutrophil-endothelium interaction by reducing PMN adhesion on isolated, activated aortic endothelium. Finally, immunohistochemical analysis illustrated aprotinin significantly reduced myocardial apoptosis following MI+R. Inhibition of serine proteases by aprotinin inhibits an inflammatory cascade initiated by MI+R. The cardioprotective effect appears to be at least partly due to reduced PMN adhesion and infiltration with subsequently reduced myocardial necrosis and apoptosis.

Expression of sialyltransferase activity on intact human neutrophils

Endogenous polymorphonuclear leukocyte (PMN)-associated sialidase activity enhances PMN adhesion to and migration across the endothelium through the removal of sialylated cell-surface residues. We tested the hypothesis that PMNs also express sialyltransferase (ST) activity that restores sialyl residues to the PMN surface. We developed a highly sensitive fluorometric assay to demonstrate that intact human PMNs can mediate and accept sialyl residue transfer. This ST activity is inhibited by a ST inhibitor, CMP, which also inhibits the transendothelial migration of PMNs in response to IL-8 in vitro and in vivo. We conclude that intact PMNs express sialidase and ST activities that permit rapid modulation of their surface sialylation and their ability to adhere to and migrate across the endothelium.