Reactive oxygen species and elastase mediate lung permeability after acid aspiration GIDEON GOLDMAN, RICHARD WELBOURN, LESTER DAVID SHEPRO, AND HERBERT B. HECHTMAN
KOBZIK,
C. R. VALERI,
Departments of Surgery and Pathology, Brigham and Women’s Hospital, and The Harvard Medical School, Boston 02115; and The Biological Science Center, Boston University, Boston, Massachusetts 02118 GOLDMAN,GIDEON,RICHARDWELBOURN,LESTERKOBZIK, C.R. VALERI,DAVID SHEPRO,ANDHERBERT B. HECHTMAN. Reactive oxygen species and elastase mediate lung permeability after acid aspiration. J. Appl. Physiol. 73(Z): 571-575, 1992.-
Acid aspiration leadsto increasedneutrophil (PMN) oxidative metabolism, an event associatedwith lung leukosequestration and permeability increase.Neutropenia protected the vascular barrier function against acid injury. This study tests whether active oxygen speciesand elastase(which are presumably releasedby adherent PMNs) affect the microvascular barrier. Anesthetized rats underwent tracheostomy and insertion of a cannula into a lung segment. This was followed by localized instillation of 0.1 N HCl (n = 18) or saline (n = 18). Sequestration of PMNs in acid-aspiratedand nonaspirated segmentswas 77 and 46 PMNs/high-power field (HPF), respectively, which was higher than control values of 11 and 8 PMNs/lO HPF in saline-aspiratedand nonaspirated regions(P < 0.05).Acid aspiration was associatedwith increased protein concentration in bronchoalveolar lavage (BAL) fluid to 3,550and 2,900pg/ml in the aspirated and nonaspirated lungs, respectively, which were higher than control values of 420 and 400 pg/ml (P < 0.05). Acid aspiration also led to increased lung wet-to-dry weight ratios (W/D) of 6.6 and 5.4, which were higher than control values of 3.4 and 3.3 (P < 0.05). Intravenous treatment of rats (n = 18) 90 min after aspiration with scavengersof reactive oxygen species,superoxide dismutase(1,500 U/kg), and catalase (5,000 U/kg), both conjugated to polyethylene glycol, did not reduce PMN sequestrationbut attenuated acid aspirationinduced increasein protein accumulation in BAL fluid in the aspirated and nonaspirated segments(990 and 610 pg/ml) as well as the increased lung W/D (4.6 and 4.0; all P < 0.05). Treatment of other rats at 90 min (n = 18) with the elastase inhibitor methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Val-chloromethylketone (0.4-mg/kg bolus, 0.4 mg kg-l h-l) protected only the nonaspirated lung segmentsfrom injury. Finally, because scavengersof reactive oxygen speciesinhibited thromboxane synthesis,another group wastreated at 90 min with the thromboxane synthase inhibitor OKY-046 (2 mg kg-l.0 h-l). There was a modestand significant decreasein BAL fluid protein and W/D of the aspirated and nonaspirated segments comparedwith untreated acid-aspirated animals, but this benefit was substantially lessthan treatment with superoxide dismutase-catalase(P < 0.05). The data indicate generalized lung PMN sequestration after localized acid aspiration. Reactive oxygen speciesand elastase,which are presumably releasedby these cells, decreasethe vascular barrier function. l
l
l
oxygen free radicals; neutrophil adhesion
ACID ASPIRATION INJURY is characterized
ane (TX) synthesis associated with neutrophil
by thrombox(PMN) ac-
tivation, generation of H,O,, and progressive PMN sequestration in the lungs (5, 19). Closely following PMN sequestration, pulmonary permeability and edema are noted. The importance of PMN in determining vascular barrier function in this setting is shown by the following observations: 1) a strong correlation exists between the number of sequestered neutrophils and the degree of lung edema; 2) neutropenia protects the vascular barrier against acid aspiration-induced permeability (4); and 3) inhibition of PMN activation and H,O, synthesis with TX antagonists reduces PMN sequestration and limits protein leak into bronchoalveolar lavage (BAL) fluid (591% The mechanism by which PMNs modify permeability has not been investigated in this setting. In other experimental models of the adult respiratory distress syndrome, indirect evidence has suggested that after adhesion, PMNs release their granular contents containing vasotoxic agents, which lead to lung edema. These agents include reactive oxygen species and elastase. Thus treatment with inhibitors of reactive oxygen species before ischemia and reperfusion or with an elastase inhibitor before perfusing a rat lung with H,O, prevented the subsequent pulmonary protein leak (2,9). Unfortunately interpretation of these latter observations is clouded by the fact that oxygen radical inhibition not only directly antagonizes action of oxygen radicals but will also act indirectly by limiting TX synthesis (10). TX inhibition reduces lung leukosequestration in acid aspiration (3). Neutrophils will release H,O, and elastase mainly after their adhesion to endothelium (13, 24). In initial experiments, we noted that administration of oxygen radical scavengers before acid aspiration prevented TX generation, PMN sequestration, and limited lung edema. Thus the beneficial effects of antagonists to reactive oxygen species could be by TX inhibition, thereby preventing PMN adhesion and release of toxic neutrophil products as well as by directly neutralizing the effects of these toxic agents after their release. To evaluate how neutrophils injure the vascular barrier, we waited 90 min after acid aspiration until the process of adhesion in the lungs had occurred. At this point, after peak plasma TX levels, we gave antagonists to reactive oxygen species and an elastase inhibitor. Such a strategy presumably allowed PMN to become sequestered in the lungs, at which point the importance of toxic oxygen products in mediating lung injury could be studied. To control for any possible effect of TX inhibition at
0161-7567/92 $2.00 Copyright 0 1992 the AmericanPhysiological
Society
571
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 2, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
572
OXYGEN
SPECIES
AND
ELASTASE
90 min, another experimental group was given the TX synthase inhibitor, OKY-046. The results indicate that after localized acid aspiration there is generalized lung PMN sequestration. Despite continued adherence of neutrophils, inhibition of oxygen free radicals or elastase (presumably released by adherent PMNs) prevented a decrease in vascular barrier function. METHODS
Animal
Preparation
Ninety adult male Sprague-Dawley rats (Charles River Lab, Wilmington, MA) weighing -500 g were anesthetized with pentobarbital sodium (60 mg/kg ip). A jugular venous catheter was aseptically inserted for fluid or drug infusion (1 ml/h) and hourly anesthetic dosing (10 mg/kg iv). A tracheostomy was performed with a 15-gauge tube. Through this tube, a fine-bore polyester cannula (0.61 mm OD and 0.28 mm ID) was introduced into the anterior inferior segment of the left lung, which represented approximately one-third the weight of the left lung. All animals were maintained supine for the duration of the experiment. Preparation
of Solutions and Drugs
Hydrochloric acid. 0.1 ml of 33% HCl (McGaw Park, IL) was mixed with 9 ml of 0.9% NaCl. The final concentration was 0.1 N. When 0.1 ml of the solution was introduced into the anterior-inferior segment of the left lung, the result was generalized pulmonary edema with a mortality rate of
KOBZIK,
C. R. VALERI,
Departments of Surgery and Pathology, Brigham and Women’s Hospital, and The Harvard Medical School, Boston 02115; and The Biological Science Center, Boston University, Boston, Massachusetts 02118 GOLDMAN,GIDEON,RICHARDWELBOURN,LESTERKOBZIK, C.R. VALERI,DAVID SHEPRO,ANDHERBERT B. HECHTMAN. Reactive oxygen species and elastase mediate lung permeability after acid aspiration. J. Appl. Physiol. 73(Z): 571-575, 1992.-
Acid aspiration leadsto increasedneutrophil (PMN) oxidative metabolism, an event associatedwith lung leukosequestration and permeability increase.Neutropenia protected the vascular barrier function against acid injury. This study tests whether active oxygen speciesand elastase(which are presumably releasedby adherent PMNs) affect the microvascular barrier. Anesthetized rats underwent tracheostomy and insertion of a cannula into a lung segment. This was followed by localized instillation of 0.1 N HCl (n = 18) or saline (n = 18). Sequestration of PMNs in acid-aspiratedand nonaspirated segmentswas 77 and 46 PMNs/high-power field (HPF), respectively, which was higher than control values of 11 and 8 PMNs/lO HPF in saline-aspiratedand nonaspirated regions(P < 0.05).Acid aspiration was associatedwith increased protein concentration in bronchoalveolar lavage (BAL) fluid to 3,550and 2,900pg/ml in the aspirated and nonaspirated lungs, respectively, which were higher than control values of 420 and 400 pg/ml (P < 0.05). Acid aspiration also led to increased lung wet-to-dry weight ratios (W/D) of 6.6 and 5.4, which were higher than control values of 3.4 and 3.3 (P < 0.05). Intravenous treatment of rats (n = 18) 90 min after aspiration with scavengersof reactive oxygen species,superoxide dismutase(1,500 U/kg), and catalase (5,000 U/kg), both conjugated to polyethylene glycol, did not reduce PMN sequestrationbut attenuated acid aspirationinduced increasein protein accumulation in BAL fluid in the aspirated and nonaspirated segments(990 and 610 pg/ml) as well as the increased lung W/D (4.6 and 4.0; all P < 0.05). Treatment of other rats at 90 min (n = 18) with the elastase inhibitor methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Val-chloromethylketone (0.4-mg/kg bolus, 0.4 mg kg-l h-l) protected only the nonaspirated lung segmentsfrom injury. Finally, because scavengersof reactive oxygen speciesinhibited thromboxane synthesis,another group wastreated at 90 min with the thromboxane synthase inhibitor OKY-046 (2 mg kg-l.0 h-l). There was a modestand significant decreasein BAL fluid protein and W/D of the aspirated and nonaspirated segments comparedwith untreated acid-aspirated animals, but this benefit was substantially lessthan treatment with superoxide dismutase-catalase(P < 0.05). The data indicate generalized lung PMN sequestration after localized acid aspiration. Reactive oxygen speciesand elastase,which are presumably releasedby these cells, decreasethe vascular barrier function. l
l
l
oxygen free radicals; neutrophil adhesion
ACID ASPIRATION INJURY is characterized
ane (TX) synthesis associated with neutrophil
by thrombox(PMN) ac-
tivation, generation of H,O,, and progressive PMN sequestration in the lungs (5, 19). Closely following PMN sequestration, pulmonary permeability and edema are noted. The importance of PMN in determining vascular barrier function in this setting is shown by the following observations: 1) a strong correlation exists between the number of sequestered neutrophils and the degree of lung edema; 2) neutropenia protects the vascular barrier against acid aspiration-induced permeability (4); and 3) inhibition of PMN activation and H,O, synthesis with TX antagonists reduces PMN sequestration and limits protein leak into bronchoalveolar lavage (BAL) fluid (591% The mechanism by which PMNs modify permeability has not been investigated in this setting. In other experimental models of the adult respiratory distress syndrome, indirect evidence has suggested that after adhesion, PMNs release their granular contents containing vasotoxic agents, which lead to lung edema. These agents include reactive oxygen species and elastase. Thus treatment with inhibitors of reactive oxygen species before ischemia and reperfusion or with an elastase inhibitor before perfusing a rat lung with H,O, prevented the subsequent pulmonary protein leak (2,9). Unfortunately interpretation of these latter observations is clouded by the fact that oxygen radical inhibition not only directly antagonizes action of oxygen radicals but will also act indirectly by limiting TX synthesis (10). TX inhibition reduces lung leukosequestration in acid aspiration (3). Neutrophils will release H,O, and elastase mainly after their adhesion to endothelium (13, 24). In initial experiments, we noted that administration of oxygen radical scavengers before acid aspiration prevented TX generation, PMN sequestration, and limited lung edema. Thus the beneficial effects of antagonists to reactive oxygen species could be by TX inhibition, thereby preventing PMN adhesion and release of toxic neutrophil products as well as by directly neutralizing the effects of these toxic agents after their release. To evaluate how neutrophils injure the vascular barrier, we waited 90 min after acid aspiration until the process of adhesion in the lungs had occurred. At this point, after peak plasma TX levels, we gave antagonists to reactive oxygen species and an elastase inhibitor. Such a strategy presumably allowed PMN to become sequestered in the lungs, at which point the importance of toxic oxygen products in mediating lung injury could be studied. To control for any possible effect of TX inhibition at
0161-7567/92 $2.00 Copyright 0 1992 the AmericanPhysiological
Society
571
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 2, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
572
OXYGEN
SPECIES
AND
ELASTASE
90 min, another experimental group was given the TX synthase inhibitor, OKY-046. The results indicate that after localized acid aspiration there is generalized lung PMN sequestration. Despite continued adherence of neutrophils, inhibition of oxygen free radicals or elastase (presumably released by adherent PMNs) prevented a decrease in vascular barrier function. METHODS
Animal
Preparation
Ninety adult male Sprague-Dawley rats (Charles River Lab, Wilmington, MA) weighing -500 g were anesthetized with pentobarbital sodium (60 mg/kg ip). A jugular venous catheter was aseptically inserted for fluid or drug infusion (1 ml/h) and hourly anesthetic dosing (10 mg/kg iv). A tracheostomy was performed with a 15-gauge tube. Through this tube, a fine-bore polyester cannula (0.61 mm OD and 0.28 mm ID) was introduced into the anterior inferior segment of the left lung, which represented approximately one-third the weight of the left lung. All animals were maintained supine for the duration of the experiment. Preparation
of Solutions and Drugs
Hydrochloric acid. 0.1 ml of 33% HCl (McGaw Park, IL) was mixed with 9 ml of 0.9% NaCl. The final concentration was 0.1 N. When 0.1 ml of the solution was introduced into the anterior-inferior segment of the left lung, the result was generalized pulmonary edema with a mortality rate of
