Reperfusion-induced leukocyte infiltration: role of elastase

Abstract

Polymorphonuclear leukocytes are known to accumulate in tissues subjected to ischemia and reperfusion. Studies on endothelial cell monolayers suggest that limited release of elastase plays an important role, via basement membrane degradation, in the leukocyte diapedesis and extravasation elicited by proinflammatory mediators. Thus the objective of this study was to define the role of elastase in the leukocyte infiltration associated with reperfusion of the ischemic bowel. In one series of experiments the cat small intestine was subjected to 3 h of ischemia (blood flow reduced to 20% of base line) and 1 h of reperfusion. Neutrophil accumulation was quantified by measurement of tissue myeloperoxidase activity in mucosal biopsies obtained during base-line, ischemic, and reperfusion periods. Pretreatment with either of the elastase inhibitors Eglin C and L658,758 significantly attenuated the neutrophil infiltration induced by reperfusion but not by ischemia per se. In another series of experiments, leukocyte adherence and extravasation were monitored in cat mesenteric venules subjected to 1 h of ischemia and reperfusion. Pretreatment with L658,758 significantly attenuated the increased rates of leukocyte adherence and extravasation induced by reperfusion, with a proportionately greater reduction in leukocyte extravasation rate. These results indicate that elastase release is an important factor in reperfusion-induced neutrophil infiltration.

Lack of flagella disadvantages Salmonella enterica serovar Enteritidis during the early stages of infection in the rat

“…MPO levels in the rat small intestine were unchanged 24 h after infection with S. Enteritidis S1400 but were significantly elevated at 72 h. Intestine tissue water content and faecal elastase was also increased at 72 h. This suggests that active recruitment of neutrophils to the intestine (Darwin & Miller, 1999;Stein et al, 1998;Faro et al, 2000;Zimmerman & Granger, 1990;McCormick et al, 1995;Naughton et al, 1996a) may have occurred between 24 and 72 h p.i. These changes would be consistent with the infiltration of polymorphonuclear leukocytes and other inflammatory cells into gut tissue and the release of inflammatory cells into the lumen, previously observed in vivo (Naughton et al, 1995(Naughton et al, , 1996a.…”

“…They were left on ice for 60 min and then centrifuged (3000 g, 30 min, 4 8C). MPO activity in the extracts was determined by monitoring H 2 O 2 -dependent oxidation of 3,39,5,59-tetramethylbenzidine (TMB; Dynex Technologies) in 50 mM potassium phosphate buffer (pH 6·0) (Zimmerman & Granger, 1990). A 450 was measured after termination of the reaction with 0·18 M H 2 SO 4 .…”

“…Sample extract or leukocyte-elastase (Sigma-Aldrich) was serially diluted in 0·2 M Tris/ HCl (pH 8·0) containing 1 M NaCl and leupeptin (1 mg l À1 ) on a microtitre plate. Substrate (N-succinyl-Ala-Ala-Val p-nitroanilide, 0·2 g l À1 ; Sigma-Aldrich) was added and the A 405 was monitored immediately and at intervals up to 4 h during incubation at 37 8C (Zimmerman & Granger, 1990). Values were expressed as leukocyteelastase (ìg) equivalents.…”

Blood cells and endothelial barrier function

“…85 Neutrophils are also able to enhance transendothelial protein exchange by releasing proteases, like elastase and matrix metalloproteinases (MMP), which appear to alter barrier function by disrupting junctional complexes and inducing endothelial cell retraction. [86][87][88] Elastase has also been shown to promote the adhesion and transendothelial migration of leukocytes in the microcirculation, 89 suggesting that the permeability enhancing effect of the protease may also be related to an enhancement of neutrophilendothelial cell adhesion. This possibility is supported by reports describing diminished endothelial barrier function, resulting from junctional disassembly and cytoskeletal reorganization, following the ligation of neutrophil adhesion molecules with their counterreceptors on endothelial cells, such as the binding of b-2 integrins with either ICAM-1 or VCAM-1.…”

Intestinal Ischemia and Gangrene

“…Mesenteric ischemia and reperfusion leads to an increase in microvascular permeability and disruption of the intestinal mucosal barrier, primarily through the actions of activated polymorphonuclear neutrophils producing reactive oxygen species and other inflammatory mediators. The tissue damage induced by the oxygen free radicals is decreased in the presence of antioxidants, xanthine oxidase inhibitors, and free-radical scavenging substances; an observation, which conclusively establishes the role of reactive oxygen species in producing cellular injury during ischemia-reperfusion [17]. In addition, phospholipase A2 is activated during reperfusion, increasing the formation of cytotoxic lysophospholipids within the ischemic tissue and up-regulating the production of prostaglandins and leukotrienes [18].…”