leukotriene-b4 and sivelestat

The serine protease neutrophil elastase (NE) appears to regulate inflammatory responses at multiple levels but its role in leukocyte transmigration in vivo remains unclear. The present study aimed to address this issue by using both an NE inhibitor (ONO-5046) and NE deficient (NE(-/-)) mice.. A number of inflammatory mediators (LTB(4), KC and PAF) were investigated in vitro for their ability to stimulate the release and the surface expression of NE by neutrophils. In addition, the role of NE in leukocyte migration elicited by topical LTB(4) was investigated in vivo in mouse cremasteric venules as observed by intravital microscopy.. Amongst the mediators tested in vitro, LTB(4) was found to be a highly potent and efficacious inducer of NE cell surface expression on murine neutrophils. Furthermore, in wild-type mice (WT), LTB(4)-induced leukocyte transmigration was reduced by intravenous ONO-5046 (66% inhibition), an effect that appeared to occur at the level of the perivascular basement membrane. Interestingly, LTB(4)-induced responses were normal in NE(-/-) mice and, while ONO-5046 had no inhibitory effect in these animals, the broad-spectrum serine protease inhibitor aprotinin suppressed leukocyte transmigration in both WT and NE(-/-) mice.. The findings demonstrate the potent ability of LTB(4) to induce cell-surface expression of NE and provide evidence for the involvement of NE in LTB(4)-induced neutrophil transmigration in vivo. The results also suggest the existence of compensatory mechanisms in NE(-/-) mice, highlighting the added value of investigating pharmacological blockers in parallel with genetic deletion.

Topics: Animals; Basement Membrane; Bone Marrow Cells; Cell Movement; Chemotaxis, Leukocyte; Enzyme Inhibitors; Glycine; Leukocyte Elastase; Leukotriene B4; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Neutrophils; Platelet Activating Factor; Sulfonamides; Venules

Although neutrophil migration from the systemic circulation involves the beta2- (or CD18) integrin family, the existence of an alternative, CD18-independent route of neutrophil extravasation to tissues has been demonstrated in animal models. The molecular interactions involved in this alternative migratory route have not yet been characterized. The objective of this study was to assess the CD18-dependency of neutrophil migration across human endothelial cells from an organ known to support CD18-independent migration, the lung, with a view to establishing an in vitro model to facilitate study of CD18-independent migration. Neutrophil migration across human pulmonary artery endothelial cells (HPAECs) in response to three different chemoattractants, formylmethionyl leucylphenyl-alanine (FMLP), interleukin (IL)-8, and leukotriene (LT) B(4), was examined. Results demonstrated that a function-blocking antibody to CD18 decreased FMLP-stimulated migration by 71.7 +/- 4.4% (P < 0.001). In contrast, migration in response to LTB(4) was decreased by only 20.5 +/- 10.2% (P < 0.01), and no significant decrease was observed with migration to IL-8. Neutrophils that migrated to FMLP had 1.7-fold more surface CD11b/CD18 compared with nonmigrated neutrophils (P < 0.01), whereas this integrin complex was not significantly upregulated on neutrophils that had migrated to IL-8 or LTB(4). Further investigation of this migratory route indicated that it did not involve the beta1 integrins (CD29) or the endothelial selectins, E- or P-selectin, nor did it require the activity of either metalloproteinases or neutrophil elastase. These results indicate that neutrophil migration across HPAECs in vitro to IL-8 and LTB(4) is predominantly CD18-independent and provides a much-needed in vitro system for examination of the neutrophil-endothelial interactions involved in this alternative migratory route.

Topics: CD11 Antigens; CD18 Antigens; Cell Line; Cell Movement; Chemotactic Factors; Dose-Response Relationship, Drug; Endothelium, Vascular; Glycine; Humans; Hydroxamic Acids; Interleukin-8; L-Selectin; Leukotriene B4; Lung; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Permeability; Phenylalanine; Protease Inhibitors; Pyrazines; Sulfonamides; Thiophenes

Repeated intranasal administration of interleukin 8 (IL-8) induces bronchial hyperresponsiveness (BHR) accompanied by lower airway neutrophil accumulation (ANA) in guinea-pigs. Leukotriene B4 (LTB4) is a chemotactic factor for neutrophils. To elucidate whether LTB4 and neutrophil elastase are involved in the IL-8-induced BHR and ANA, the effects of a LTB4 antagonist (ONO-4057) and a neutrophil elastase inhibitor (ONO-5046) on the responses were examined. IL-8 (5 microg x kg[-1]) was administered intranasally to guinea-pigs twice weekly for 3 weeks. One day after the last administration, animals were anaesthetized and artificially ventilated through tracheal cannulae, and lateral pressure at the tracheal cannula (Pao) was measured as an overall index of airway responses to inhaled histamine. ONO-4057 (2 or 20 mg x kg[-1]) or ONO-5046 (30 or 300 mg x kg[-1]) was administered intraperitoneally 24 and 1 h before anaesthesia. ONO-4057, but not ONO-5046, significantly inhibited the IL8-induced BHR and ANA, assessed by bronchoalveolar lavage, in a dose-dependent manner. These findings suggest that interleukin 8 causes bronchial hyperresponsiveness and airway neutrophil accumulation in guinea-pigs in vivo. In part this appears to be due to release of leukotriene B4, whereas it may not be mediated by neutrophil elastase.

Topics: Animals; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Movement; Glycine; Guinea Pigs; Immunosuppressive Agents; Interleukin-8; Leukocyte Elastase; Leukotriene B4; Male; Neutrophils; Phenylpropionates; Sulfonamides

To determine the role of neutrophil elastase in asthmatic responses, we studied the effect of ONO-5046, a specific neutrophil elastase inhibitor, on antigen-induced asthmatic responses in allergic sheep. Pulmonary resistance (RL) was measured for 8 h after antigen challenge. Measurements of airway responsiveness to methacholine and bronchoalveolar lavage fluid (BALF) were obtained 8 h after challenge. Antigen challenge caused early and late increases in RL, airway hyperresponsiveness (AHR), and recruitment of neutrophils and eosinophils along with increases in TXB2 and LTB4 in BALF. ONO-5046 treatment significantly reduced both early and late bronchoconstriction, neutrophil recruitment, increases in LTB4 in BALF, and AHR. ONO-5046 post-treatment significantly reduced the increase in RL 8 h after antigen challenge. Another neutrophil elastase inhibitor, FR 134043, significantly reduced both early and late bronchoconstriction. ONO-5046 had little effect on calcium ionophore-induced LTB4 release from isolated neutrophils and whole blood obtained from drug-treated sheep. These findings suggest that neutrophil elastase is involved in antigen-induced bronchoconstriction and AHR mediated by neutrophil accumulation and 5-lipoxygenase products in allergic sheep.

Topics: Airway Resistance; Animals; Antigens; Asthma; Benzoquinones; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Calcimycin; Glycine; Heterocyclic Compounds; Leukocyte Elastase; Leukocytes; Leukotriene B4; Masoprocol; Methacholine Chloride; Pancreatic Elastase; Polycyclic Compounds; Sheep; Sulfonamides; Thromboxane B2

The effects of a competitive neutrophil elastase (NE) inhibitor, ONO-5046, and a recombinant human superoxide dismutase on leukotriene B4 (LTB4)-induced polymorphonuclear leukocyte (PMN)-mediated increase in microvascular permeability in isolated non-blood-perfused rabbit lungs were studied. Pulmonary microvascular permeability and lung edema were evaluated by use of the fluid filtration coefficient (Kf) and the wet-to-dry lung weight ratio (W/D), respectively. Pulmonary capillary pressure was estimated by the double occlusion technique. NE activity in the perfusate was determined using a spectrophotometric method. The PMNs (2-3 x 10(8) cells) were added into the perfusate in all groups of lungs. Injection of LTB4 (5 micrograms) increased Kf and W/D without affecting pulmonary arterial or capillary pressure. The LTB4-induced lung injury was closely associated with the increase in NE activity in the perfusate. Infusion of ONO-5046 (1 or 10 mg.kg-1 x h-1) inhibited the LTB4-induced increases in Kf, W/D, and perfusate NE activity in a dose-dependent fashion. Infusion of recombinant human superoxide dismutase (80,000 U.kg-1 x h-1) attenuated the LTB4-induced increases in Kf and W/D, although it did not influence the elevation of perfusate NE activity induced by LTB4. These results suggest that both NE and superoxide anion play important roles in the LTB4-induced PMN-mediated increase in pulmonary microvascular permeability.

Topics: Animals; Capillary Permeability; Female; Glycine; In Vitro Techniques; Leukocyte Elastase; Leukotriene B4; Lung Diseases; Male; Neutrophils; Organ Size; Pancreatic Elastase; Pulmonary Circulation; Pulmonary Edema; Rabbits; Sulfonamides; Superoxide Dismutase; Superoxides