Role of neutrophil elastase in LTB₄‐induced neutrophil transmigration in vivo assessed with a specific inhibitor and neutrophil elastase deficient mice

Abstract

Background and purpose: 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 (NEExperimental approach: 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. Key results: 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 À/À 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 À/À mice. Conclusions and implications: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 À/À mice, highlighting the added value of investigating pharmacological blockers in parallel with genetic deletion.

NOD2 Signaling Contributes to Host Defense in the Lungs against Escherichia coli Infection

“…N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline (Sigma Chemical) was used as the substrate for neutrophil elastase (NE), as this compound is not hydrolyzed by cathepsin G. Hydrolysis of the substrate by NE was determined spectrophotometrically at 405 nm. In brief, 1 mM substrate was incubated with 0.2 ml of sample in 0.1 M Tris (hydroxyl-methyl) aminomethane-HCl buffer (pH 8.0) containing 0.5 M NaCl at 37°C for 24 h. Optical density was determined and activity of NE was determined, where one enzyme unit is defined as the quantity of enzyme that liberated 1 mol of p-nitroaniline in 24 h (65).…”

Cleavage of the CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis

“…A direct cleavage assay using purified, functionally active CD11b/CD18 further confirmed the specific cleavage of CD11b by neutrophil serprocidins (Figures 5-6). The finding that NE, NCG and NPR-3 can all contribute to CD11b shedding and PMN detachment may also provide clues that explain the controversial reports [18][19][20][22][23][24][25][26] on the roles of serprocidins in modulating PMN recruitment. Because each of the 3 serprocidins can effectively cleave CD11b, the combination of NE, NPR-3, and NCG forms a highly efficient mechanism for the rapid shedding of neutrophil CD11b during cell detachment and subsequent migration.…”

“…Using NE-deficient mice and various inflammatory mouse models, systematic studies from Nourshargh's laboratory demonstrated the essential role of NE in mediating PMN transmigration. 19,20,22 In contrast, Hirche et al 23 reported no significant defects of leukocyte transmigration in mice with individual NE knockouts. Similarly, the inhibition of NE by a specific inhibitor did not affect leukocyte transendothelial migration under flow.…”

“…[18][19][20][21] Although it is generally believed that leukocyte serprocidins promote PMN transmigration by cleaving off the adhesion molecules that hold PMN on the cell migration path, the molecular basis of this role for serprocidins is still unclear, and the reports about the functions of serprocidins in leukocyte recruitment remain controversial. Using NE-deficient mice and various inflammatory mouse models, systematic studies from Nourshargh's laboratory demonstrated the essential role of NE in mediating PMN transmigration.…”

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

“…First recognized as degradative enzymes able to kill pathogens and cleave extracellular matrix components, NSPs have been attributed a potential role in chemotaxis and migration through cleavage of adhesion molecules at intercellular junctions (Cepinskas et al, 1999;Hermant et al, 2003). This function, however, remains debated because some studies demonstrated decreased neutrophil chemotaxis when using NSP inhibitors (Stockley et al, 1990;Lomas et al, 1995;Young et al, 2007), whereas others have revealed no alteration of neutrophil migration in response to neutrophil-specific chemotactic stimuli in animal models deficient in NSPs (MacIvor et al, 1999;Adkison et al, 2002;Allport et al, 2002).…”