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Ibuprofen attenuates hypochlorous acid production from neutrophils in porcine acute lung injury
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OF SURGICAL
49,262-270
RESEARCH
(1990)
Ibuprofen Attenuates Hypochlorous Acid Production from Neutrophils in Porcine Acute Lung Injury1*2*3 P. DECLAN CAREY, F.R.C.S.I., KARL BYRNE, F.R.C.S.I., JOHN K. JENKINS, M.D., TIMOTHY D. SIELAFF, M.D., CIARAN J. WALSH, F.R.C.S.I., ALPHA A. FOWLER III, M.D., AND HARVEY J. SUGERMAN, M.D. Departments
of Surgery, Medicine,
and Pathology, Medical College of Virginia, Submitted
for publication
Academic
Press,
Inc.
INTRODUCTION Infusion of live Pseudomonas bacteria into the pig generates sepsis with an associated acute lung injury (ALI) i These experiments were supported in part by U.S. Army Contract DAMD 17-86-C-6168 and PHS HL35534-03. The views, opinions and/ or findings contained herein are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation. ‘These experiments were performed in accordance with the NIH Guidelines for the use of experimental animals. 3 Presented at the Annual Meeting of the Association for Academic Surgery, Louisville, KY, November 15-18, 1989.
0022~4804/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
262 Inc. reserved.
November
Commonwealth
University,
Richmond,
Virginia
23298
20. 1989
which closely simulates the lung injury seen in the adult respiratory distress syndrome (ARDS) in humans [l-3]. Multiple pathophysiological mechanisms of sepsis-induced acute lung injury have been proposed. Recent attention has focused upon the destructive capability of neutrophils (PMN) and their role in producing alveolarcapillary membrane injury following sequestration in the pulmonary microcirculation [4, 51. In a number of common human conditions (e.g., rheumatoid arthritis, ulcerative colitis, myocardial reperfusion, and adult respiratory distress syndrome) a vigorous inflammatory response is elicited from circulating phagocytes with subsequent host tissue damage [6-91. Current evidence supports the hypothesis that circulating PMNs become “primed” for reactive oxygen metabolite production following the onset of sepsis and, when sequestered in the microcirculation, may be responsible for widespread endothelial cell injury [ 10-121. Neutrophils stimulated by septic mediators undergo a “respiratory burst” which results in increased production of short-lived oxidant species such as superoxide anion (0;) [13, 141. Superoxide anion is rapidly metabolized to hydrogen peroxide by the action of superoxide dismutase (SOD) [ 13,151. In the presence of chloride ions (Cl-) the azurophilic granular enzyme myeloperoxidase (MPO) converts hydrogen peroxide to hypochlorous acid [9, 131. Hypochlorous acid is believed to be the most potent and destructive of the neutrophil-derived oxidants [9, 131. Cyclooxygenase metabolites of arachidonic acid are thought to play a major role in the physiological derangements in sepsis-induced ALI. Ibuprofen, a potent cyclooxygenase blocker, has been shown to be effective in attenuating alveolar-capillary membrane damage [2, 3, 16-181. The mechanism of this effect may be via alteration of certain neutrophil functions, such as down regulation of 0; generation [ 19-211. The present study was designed to determine the relationship between in viva production of HOC1 from circulating PMNs and various parameters of lung injury and to see whether the physiological improvement seen with ibuprofen therapy was associated with alterations in HOC1 production.
Hypochlorous acid (HOCl), a neutrophil-generated oxidant, has been implicated in tissue destruction in sepsisinduced acute lung injury (ALI). Ibuprofen, a cyclooxygenase inhibitor, successfully attenuates many of the physiological derangements in ALI. The aim of this study was to examine the role of PMN hypochlorous acid in sepsis-induced AL1 and evaluate the effect of ibuprofen therapy. Neutrophils from three groups of young (1525 kg), anesthetized swine were studied: controls (C, n = 7) received 0.9% NaCl, septic animals (Ps, n = 8) received live Pseudomonas aeruginosa (5 X lo8 CFU/ml at 0.3 ml/20 kg/min) for 60 min, and ibuprofen-treated animals (Ps + I, n = 6) received Ps plus ibuprofen 12.5 mg/kg administered at 0 and 120 min. Neutrophils were isolated from peripheral blood at 0,60, and 300 min and the rate and total production of HOC1 were assessed on the basis of the ability of the amino acid taurine to trap HOCl. Results: Septic (Ps) PMNs produce 32% more HOCl, P < 0.01, at 300 min than at baseline which was associated with a marked increase in both extravascular lung water (6.44 f 0.8 ml/kg, t = 0 vs 16.03 + 2.6 ml/ kg, t = 300, P < 0.01) and bronchoalveolar protein content (115 + 13 rg/ml, t = 0 vs 633 + 104 pg/ml, t = 300, P < 0.01). Ibuprofen significantly attenuated (P < 0.05) HOC1 production when compared to Ps, in conjunction with significantly (P < 0.05) reduced levels of extravascular lung water and bronchoalveolar lavage protein. 0 1990
Virginia
CAREY
MATERIALS Animal
ET AL.: HYPOCHLOROUS
ACID
AND METHODS
Conditioning
Yorkshire swine (15-30 kg) were obtained from a commercial vendor in the Richmond area and housed in the Virginia Commonwealth University vivarium for 3-5 days prior to use. Animals received benzathine and procaine penicillin G (300,000 Units each) intramuscularly 48 hr prior to use. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University and adhered to National Institutes of Health guidelines for the use of experimental animals. Animal
Preparation
On the day of the study, animals were preanesthetized (25 mg/kg ketamine hydrochloride, 0.4 mg atropine, im) and placed supine. General anesthesia was induced with sodium pentobarbital (10 mg/kg, iv) and maintained throughout the study with intermittent pentobarbital infusion. Endotracheal intubation using a cuffed endotracheal tube (National Catheter) was performed. Skeletal muscle paralysis was maintained by a continuous intravenous infusion of pancuronium bromide (0.2 mg/kg/ min). Animals were mechanically ventilated (Harvard large animal ventilator, Harvard Apparatus, South Natick, MA, 0.5 FiOz, 5 cm Hz0 PEEP; tidal volume, 15 ml/ kg) with the initial respiratory rate adjusted to produce a PaCOz of 40 Torr at the beginning of each experiment. Indwelling catheters were placed in the left common carotid artery for systemic arterial pressure (SAP) monitoring and arterial blood gas determination as well as the left external jugular vein for infusion of saline, Pseudomonas organisms, and ibuprofen (see below). An indwelling balloon-tipped pulmonary arterial catheter was inserted via the right internal jugular vein and positioned in the pulmonary artery via pressure monitoring for measurement of pulmonary arterial pressure (PAP), pulmonary arterial occlusion pressure (PAOP), central venous pressure (CVP), and thermodilution cardiac output (Edwards COMl). Measurements of Alveolar-Capillary Permeability: Thermal-Cardiogreen Extravascular Lung Water (E VL W) and Bronchoalveolar Lavage Protein Content (BAL-P) EVLW measurements were recorded every 60 min until the end of the 5-hr study period using the thermal cardiogreen double indicator dilution technique as previously described [22]. Bronchoalveolar lavage (BAL) was performed using a fiberoptic bronchoscope at baseline in the middle and lower lobes of the right lung. The distal end of a fiberoptic bronchoscope (Machita VT-5100C, 4mm) was wedged into a third- or fourth-order bronchus and a
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75-ml BAL was performed in each lobe using 25-ml aliquots of sterile 0.9% NaCl. Fluid was retrieved by gentle aspiration of the infusing syringe. The BAL procedure was repeated at 300 min in corresponding segments of the left lung. The procedures were performed by one of two technicians J.K.J. or A.A.F. and lavage return was consistently high, 75-80%. Cell free BAL fluid protein content was determined by a modification of the Lowry technique described by Markwell and associates for fluids containing lipids [23]. Results are expressed as micrograms protein per milliliter of recovered lavage fluid. Study Design Animals were randomly divided into three study groups. Control animals (C, n = 7) received an infusion of sterile 0.9% NaCl for 60 min. The Pseudomonas group (Ps, n = 8) was infused continuously for 1 hr with live Pseudomonas aeruginosa (PA0 strain, 5 X 10’ organisms/ml at 0.3 ml/20 kg/min). The treatment group (Ps -t I, n = 6) received an identical infusion of live organisms as well as ibuprofen (12.5 mg/kg infused at 0 and 120 min). Systemic arterial pressure (SAP), pulmonary artery pressure (PAP), pulmonary arterial occlusion pressure (PAOP), cardiac index (Cl), central venous pressure (CVP), and arterial blood samples for pH, P,Os and P,C!02 (pH/blood gas analyzer, Instrumentation Laboratories Model 1304) were measured at baseline and every 30 min during the experimental period. Systemic arterial blood samples were taken every 15 min for the first hour and every 30 min thereafter for measurement of peripheral total leukocyte count. Arterial blood samples were obtained at 0,60, and 300 min for neutrophil isolation. Previous studies from this laboratory using a similar model have shown that the CVP and PAOP do not significantly decline from baseline over the 300-min study period in septic animals; thus fluid was administered to each group only to replace those losses due to investigative phlebotomy [22]. Neutrophil Isolation Arterial blood was drawn into syringes containing sterile EDTA (15%, 0.1 ml/10 mls blood). Neutrophils were isolated by dextran sedimentation, Ficoll-Hypaque density gradient centrifugation, and hypotonic lysis [ 24, 251. Neutrophils were suspended in phosphate-buffered saline (PBS) at a concentration of 4 X lo6 cells/ml. Final preparations contained greater than 98% neutrophils by modified Wright’s Giemsa staining (Diff Quick, American Scientific) which were 99% viable by trypan blue dye exclusion. Peripheral white cell count was performed by hemocytometer on whole blood samples collected as outlined above. Measurement of HOC1 Production by Stimulated Neutrophils Neutrophil HOC1 production was determined by a modification of the chlorination of taurine method [26]
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1990
1
Time (min) 30
0
Mean pulmonary C Ps Ps + I
18.0 + 15.7 k 14.3 +
1.6 0.8 0.8
17.3 f 47.6 f 19.3 k
2.0 l.l* 1.8**
15.6 f 38.1 f 27.0 2
1.4 l.O* 3.3**** Mean arterial
C Ps Ps + I
215 247 22
+ 15 + 8 rf:12
224 183 250
+_16 f14 f 14**
244 157 241
180
120
60
519 *12* 2 27**
300
240
artery pressure (mm Hg) 16.6 f 29.8 + 24.8 +
1.5 0.9* 1.4*,**
16.9 iz 2.7 30.7 f 1.0* 23.5 + 1.9*,**
18.3 f 31.5 f 25.8 +
1.7 1.1* 2.4*
18.2 + 2.6 26.7 zb 3.1* 28.8 + 2.2*
oxygen tension (Torr) 259 140 253
+_16 f12, f 14**
243 118 197
220 +ll* + 38**
252 112 171
k19 f 13* f 33****
255 97 135
f12 *15* + 38*
l?r 6*** + 5% + 9**
131 82 104
f 7 k 6* f 12*
131 88 128
* * +
Mean systemic artery pressure (mm Hg) C Ps Ps + I
111 100 100
zk 8 +- 4 + 5
121 130 115
* 9 f 4*** + 10
* P < 0.05 vs C, **P < 0.05 vs Ps, ***P
138 103 125
+ 7+** * 3* f lo**
133 88 128
f f +
7*** 4+ 8**
136 76 115
5 7% 8**
< 0.05 vs baseline.
as recently described by Kukreja et al. [27] where the ability of the amino acid taurine to act as a scavenger of HOC1 is utilized. The resulting chlorination complex (taurine chloramine), oxidizes 5-thio-2-nitrobenzoic acid (TNB) to its disulfide, 5-5’dithiobis-(2-nitrobenzoic acid) (DTNB) with a resultant decrease in absorbance which can be measured spectrophotometrically. Generation of HOC1 is indicated by taurine chloramine formation. TNB was prepared by sodium borohydride (Sigma Chemical Co.) reduction of DTNB (Sigma). Reagents were made up on the day of each assay. Isolated PMNs (2.6 X 106) were resuspended in PBS in the presence of 15 mM taurine and 0.77 mM TNB (pH 7.4) and placed in a cuvette at a final volume of 2.6 ml. Neutrophils were then activated by the addition of 100 nmole phorbol myristate acetate (PMA, Sigma). TNB oxidation was followed spectrophotometrically (Shimadzu UV-160) at 412 nm for 20 min at 37°C under continuous stirring. Assays were performed in duplicate and a reference cuvette containing cells and reaction mixture which remained unstimulated was used with each assay to adjust for spontaneous oxidation of the indicator. The amount of HOC1 generated per minute during the 20-min assay period was calculated from the absorbance change at 412 nm using an absorption coefficient of 1.36 X lo4 M-l cm-’ [28]. Data are expressed as nmole HOC1/106 PMNs/min. Cumulative production of HOC1 was determined for each cell sample from the total optical density change over 20 min. Results are expressed as nmole HOCl/106 PMNs/20 min. Statistics All results are expressed as means & SEM. The presence of significant differences within and between groups
was determined by analysis of variance (ANOVA) and repeated measures ANOVA. Differences between means were analyzed using Tukey-Kramer Studentized range test and Student’s t test where appropriate. The level of statistical significance was set at P < 0.05 for ANOVA. RESULTS
Physiological
Measurements
Physiological measurements for the three groups over the course of the study period are displayed in Table 1. Control animals exhibited minimal variation from baseline throughout the study period in any of the measured parameters. Animals in the Ps group showed an immediate and significant (P < 0.05) increase in pulmonary arterial pressure (PAP) following infusion of live organisms. PAP subsequently diminished somewhat from this initial peak but remained significantly higher than PAP in controls throughout the 300 min of the study. Ibuprofen pretreatment prevented the early phase pulmonary arterial hypertension. Furthermore, PAP following ibuprofen treatment was significantly decreased when compared to septic animals until 4 hr, but rose significantly above saline-infused controls from 90 min onward. Systemic arterial pressure (SAP) becomes significantly elevated (P < 0.05) in control animals at 60 min. In contrast, SAP in Pseudomonas-infused animals was elevated at 30 min and then fell progressively. Systemic arterial pressure was significantly lower than control values from 60 min onward. Ibuprofen maintained SAP at or near control levels from 0 to 300 min. Systemic arterial oxygen tension (P,O,) did not vary in control animals but underwent a steady decline in the Ps group, becoming significantly lower than
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2 Time (min)
0
60
120
240
180
300
Mean central venous pressure (mm Hg)
C PS Ps + I
4.8 rf 0.8 4.1 + 0.6 3.5 * 0.4
4.3 -c 1.1 4.4 -c 0.7 3.8 f 0.3
4.6 k 0.8 5.1 * 0.7 3.0 + 0.6
Mean pulmonary
C Ps Ps + I
6.0 + 1.0 5.6 + 0.6 6.1 + 1.1
7.7 k 0.8 7.7 * 1.0 7.0 t 1.0
capillary 6.8 k 0.8 7.1 f 0.8 6.4 f 0.9
Extravascular
C Ps Ps + I
6.84 k 1.1 6.44 rf- 0.8 7.51 AI 0.7
7.62 k 0.6 9.50 * 0.9 8.60 k 0.7
3.2 f 0.9 4.3 + 0.7 2.2 + 0.5
3.3 f 1.1 4.1 f 0.6 2.0 * 0.5
2.6 f 0.9 2.8 f 0.6 2.2 k 0.9
7.4 + 1.1 5.7 f 0.7 6.7 + 1.3
6.3 ” 1.0 6.1 ir 1.2 5.5 f 0.9
5.81 + 0.9 14.06 k 1.4* 10.24 f 0.9
6.78 f 0.1 16.03 + 2.6* 8.90 rLr1.5”
wedge pressure (mm Hg) 6.6 + 0.9 7.8 ?I 1.1 6.2 + 0.5
lung water (ml/kg)
6.05 k 0.9 10.60 * 0.9* 8.03 k 0.6
6.78 k 1.0 11.97 * 1.5* 9.59 k 1.1
* P < 0.05 “S c. **p < 0.05 “S Ps.
controls from 60 to 300 min. Arterial oxygen tension in ibuprofen-treated animals was not significantly different from control measurements until 240 min but in the last hour deteriorated toward levels observed in septic animals. Both PAOP and CVP levels remained at baseline levels in all groups throughout the study (Table 2). Peripheral
WBC Counts
Table 3 records the total peripheral WBC count measured at 0,60, and 300 min time points. A significant and progressive peripheral leukopenia was observed in group Ps following bacterial infusion. Leukopenia was well established by 60 min into the study becoming more profound by 300 min. A similar degree of leukopenia was unaltered by ibuprofen treatment in septic animals (Ps + I).
TABLE
3
WBC (X103 cells/mm3)
Control (n = 7) Pseudomonas (n = 8) Pseudomonas + ibuprofen (n = 6) * P < 0.05 vs control.
0 min
60 min
300 min
23.6 + 5.4
21.7 f 5.7
27.0 f 4.8
21.8 + 2.0
7.0 t 3.2*
4.1 k 1.3’
19.6 + 4.5
4.9 * 2.2*
5.2 f l.l*
Alveolar-Capillary
Membrane Permeability
Extravascular lung water (EVLW) measurements for the three groups are displayed in Table 1. Observed EVLW remained at baseline levels throughout the study in control animals. In septic animals, EVLW rose progressively, becoming significantly greater than control from 120 to 300 min, and reached a maximum of 16.03 f 2.7 ml/kg at 300 min. Ibuprofen treatment maintained EVLW within control limits at all time points with values significantly lower than those observed in the Ps-infused animals at 300 min. Bronchoalveolar lavage protein content in Ps animals was significantly greater than both C and Ps + I at 300 min. Bronchoalveolar lavage protein content in both C and Ps + I remained at baseline values throughout the study (Fig. 1). PMN Hypochlorous
Acid Production
Hypochlorous acid production from porcine PMNs in the three study groups is shown in Figs. 2-4. No differences were observed in the rate of production of HOC1 between PMNs at 0 and 60 min in all groups. In control animals, generation of taurine chloramine (HOCl) reached peak production at 8-10 min following PMA stimulation (Fig. 2). No differences in PMA-stimulated HOC1 production by neutrophils obtained at 0 or 300 min were observed (1.18 + 0.08 vs 1.2 * 0.1 nmole/lO” PMN, P = 0.29, ANOVA) in control animals. Unstimulated cells failed to produce significant quantities of HOC1 (Fig. 2). In contrast, PMNs from septic animals (Fig. 3) exhibited greater rates (P < 0.05) of HOC1 production up to 14 min following PMA stimulation when compared to PMN ob-
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200
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tained at baseline in the same animals. A 25% increase in maximum HOC1 production (1.7 nmole/106 PMN) occurred 10 min following PMA stimulation in septic PMNs (P < 0.01). Effect of Cyclooxygenase Inhibition Acid Production
on Hypochlorous
In Fig. 4 continuous production of HOC1 for groups Ps and Ps + I at 300 min are compared along with their respective baselines. Analysis of variance demonstrated overall differences between Ps 300 and Ps 0, Ps + I 0 and Ps + I 300 (P < 0.05). Despite significantly increased rates of HOC1 production in the first 5-6 min following PMA stimulation, the presence of ibuprofen in septic animals (Ps + I) resulted in a significant decrease in oxidant generating capacity at all assay time points when compared to septic PMNs alone (P < 0.05). Figure 5 compares the cumulative production of HOC1 over 20 min and includes each time point from the three groups. At 20 min following PMA stimulation, PMNs from Ps-infused animals obtained at 300 min generated significantly greater quantities of HOCl, 9.84 rt 0.4, 0 min vs. 13.02 + 0.3, 300 min nmole/106 PMNs (P < 0.01, Students t test) than all other groups. This quantitative increase in HOC1 pro-
8
16
20
Time’Tmin)
FIG. 2. Rate of HOC1 generation from PMNs following PMA stimulation in control animals (C, n = 7) at 0 and 300 min. Cells that remained unstimulated failed to produce appreciable quantities of HOCl. There was no significant differences between PMNs isolated at the two time points.
8 Time
16
, 20
(rn~$
FIG. 3. Rate of HOC1 generation from PMNs in septic animals (Ps, n = 8) at 0 and 300 min following PMA stimulation. Significant differences are indicated in the figure, *P < 0.05.
duction was not detected in PMNs from Ps-infused animals at 60 min. The quantitative increase in long-lived oxidant generation at 300 min in septic animals was 32% higher when compared to PMNs obtained at baseline. Pretreatment of animals with ibuprofen attenuated HOC1 production from PMNs obtained at 300 min and this was indistinguishable from that observed in control animals (9.79 * 0.4 vs 10.12 f 0.1 nmole/106). DISCUSSION The proposed aims of the present study were to examine neutrophil long-lived oxidant production during the evolution of Pseudomonas sepsis in the pig and to determine how cyclooxygenase inhibition would alter the production of this oxidant species. Neutrophils are thought to play a major role in sepsisinduced endothelial cell damage and increased alveolarcapillary membrane permeability by release of oxidants and proteolytic enzymes [ 15,291. Sequestration of PMNs in the pulmonary microvasculature of humans and animals following the onset of sepsis is well established [5, 301. Chemotaxins derived from macrophages, complement split products, (C5a, C3a) and altered pulmonary blood
0 4
4
(min)
FIG. 1. Estimated bronchoalveolar lavage protein content from harvested lavage fluid at 0 and 300 min showed significant influx (*P < 0.01) of protein to the alveolus in Ps animals. Ibuprofen pretreatment kept BAL-P at control levels.
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4
8
Time
12
16
20
(min)
FIG. 4. Rate of HOC1 production from isolated PMNs in group Ps and group Ps + I is compared. Included in this figure are the respective baseline (0 min) curves for the two groups. For statistical analysis each group was compared to its own baseline and 300 min time points were similarly compared (ANOVA). Individual time point variations are indicated in the figure (*P < 0.05 vs C, #P < 0.05 vs Ps + I).
CAREY
ET AL.: HYPOCHLOROUS
ACID
15 -,
F i I 0ml”
*
"010 i i I 0 $5 c (n=7) *pee 0, “5
Ps (n=8)
Ps+1
(n=6)
All
FIG. 5. The cumulative production of HOC1 from porcine PMNs following PMA stimulation. PMNs were harvested at 0, 60, and 300 min following the onset of saline or live Pseudomonas infusion. Cumulative production of HOC1 was calculated from the total change in optical density over the ZO-min assay period. PMNs harvested at 300 min in Ps animals exhibited significantly increased HOC1 generation compared to all other time points and cell groups (‘P < 0.01, Student’s t test).
flow all mediate neutrophil sequestration [31]. Thus, bacteria and endotoxin, both stimulators of C5a production, may be directly responsible for bringing PMNs into apposition with microvascular endothelium 1311. The mechanism by which PMNs effect pulmonary microvascular endothelial injury is unclear; however, the presence of “activated” PMNs in close approximation with pulmonary capillary endothelial cells could lead to host cell damage. Microvascular injury and host pulmonary dysfunction in sepsis may be associated with “priming” of the respiratory burst of circulating PMNs [ll, 121. Neutrophils are capable of generating large quantities of reactive oxygen metabolites which are important in defense against invading microorganisms but excessive production of toxic oxidants could lead to nonspecific destruction of host cells [32-341. Neutrophil excitation by a number of biochemical stimuli results in activation of a membrane bound NADPH oxidase system [13, 351. Electrons are shuttled from cytosolic NADPH to oxygen thus forming 0, (Fig. 6, Eq. (1)) [9]. Subsequently, a cascade of reactions occur leading to the production of a spectrum of potent oxidants [9]. The dismutation of 02 to hydrogen peroxide (H,O,) may occur spontaneously or may be rapidly catalyzed by superoxide dismutase (SOD) (Fig. 6, Eq. 2) [36]. Hydrogen peroxide in the presence of metal ions (e.g., Fe’+) may produce another potent oxidizing species, the hydroxyl radical (Fig. 6, Eq. 3) [15, 371. Hydrogen peroxide in the presence of myeloperoxidase (MPO) and halide ions may also be converted to the extremely toxic oxidant, hypochlorous acid (Fig. 6, Eq. 4) [38]. Alternatively, Hz02 may be scavenged by catalase or gluthathione peroxidase and rendered nontoxic [13, 39, 401. The majority of generated 0, is dismuted to HZ02 which serves as a branch point for production of further oxidants [9]. Weiss contends that the preferential pathway for the metabolism of Hz02 produced by PMNs occurs along the MPO-hypohalous acid pathway, leading to production of hypo-
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chlorous acid in the normal Cl- rich physiologic environment [9]. The use of the phorbol ester, phorbol myristate acetate (PMA) to activate the PMN oxidant generating mechanism is a standard technique. Intracellular activation of PMNs at the protein kinase C level consistently causes a maximal stimulation of the NADPH oxidase enzyme system. Neutrophil activation in Go, as in the present study, occurs by a number of routes, including phagocytosis of opsonized bacteria and by small bacterial peptides via ligand-receptor coupling [41]. However, previous studies have shown that septic stimulation of PMNs in uiuo results in unresponsiveness to the same stimulation following isolation [42]. It therefore becomes important, in studies such as the present one, to cause a maximal secondary stimulation of harvested cells which clearly demonstrates the nature of the “priming” response. Thus PMA serves as a probe to determine PMN “priming.” Hypochlorous acid, the active component in household bleaches, is a potent microbiocidal molecule [9]. It is widely believed that HOC1 participates in reducing inflammation by the inactivation of bacteria and fungi [38, 431. Low concentrations of HOC1 have been shown to exert a rapid and selective inhibition of bacterial growth and cell division [44]. Thus, HOC1 appears to be a critical component of the body’s defense against invading microorganisms. Antimicrobial function is generally confined to the phagocytic vacuole but extracellular release of HOC1 and other oxidants can occur during the formation and closure of vacuoles, during PMN phagocytosis [38]. The oxidant activities of HOC1 are nonselective and it has been shown to react avidly with all biomolecules [44]. Therefore, even though HOCl’s action is mainly protective, its potency and nonselectivity of action implicate it in host tissue damage in the inflammatory response [44]. Hypochlorous acid can directly attack cell membranes resulting in cell lysis and death [38, 441. Hypochlorous acid can potentiate the action of other oxidants by inactivating antioxidant mechanisms, in particular glutathione peroxidase and catalase [43]. Importantly, HOC1 (1)
(2)
o,+
e- ----------> o2
2 02-+ 2 H+ ---------->
HsOs+ 0,
(catalyzed by SOD)
(3)
0,. + ROOH -----------> RO + OH- + 0,
(4)
HrOs + Cl -----------a OCI + OH (catalysed bv MPO)
(catalysed by Fe++ or Cu’)
FIG. 6. The sequence of short-lived oxidant generation following neutrophil stimulation. SOD, superoxide dismutase; MPO, myeloperoxidase. (1) Superoxide anion generation. (2) Hydrogen peroxide generation from O;, catalyzed by SOD. (3) Hydroxyl radical generation. (4) Hypochlorous acid generation from hydrogen peroxide, catalyzed by MPO.
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has been shown to inhibit the activity of circulating alprotease inhibitor. Diminished a,-antiprotease function could lead to significant regional or widespread host tissue damage due to unrestricted activity of PMN-derived proteases (e.g., elastase). Hypochlorous acid also activates latent PMN degranulation enzymes, collagenase and gelatinase. A combined assault of these PMN serine proteases could thus lead to significant degradation of components of basement membrane and interstitium [9, 38, 451. This study suggests that development of sepsis may be associated with enhanced intravascular chlorinated-oxidant production. Neutrophils isolated at 300 min in septic animals exhibited enhanced rates of HOC1 generation (up to 25%) immediately following PMA stimulation (Fig. 3). Cumulative production of HOC1 from 300-min PMNs was increased by 32% above baseline (P < 0.01; Fig. 5). Increasing disruption of the alveolar-capillary membrane occurred in conjunction with these changes. Evidence of membrane damage in septic animals first appeared at 120 min. Extravascular lung water was significantly and progressively elevated above control from 120 to 300 min. Bronchoalveolar lavage protein at 300 min was four times baseline levels in septic animals, P < 0.01. In uiuo administration of the cyclooxygenase inhibitor, ibuprofen, dramatically attenuated the upregulation of HOC1 production from PMNs harvested at 300 min when compared to untreated septic animals (Figs. 4 and 5). Ibuprofen treatment also attenuated increased alveolar-capillary membrane permeability. Bronchoalveolar lavage protein was maintained at control levels in ibuprofen animals and EVLW was maintained at control levels until 300 min when it was significantly lower than that observed in septic animals. Thus, PMNs exhibiting enhanced oxidant generation are present in the peripheral circulation at a time when increasing disruption of the alveolar-capillary membrane is occurring. These data suggest that neutrophi1 HOC1 may play a role in the production of alveolarcapillary membrane damage in this model of sepsis-induced ALI. The significance of increased PMN production of HOC1 during the development of sepsis relates in part to the ability of the oxidant to react with endogenous amines producing hydrophilic and lipophilic chloramines [40]. Monochloramines exhibit a half-life in PMN culture supernatants of approximately 18 hr [40]. Although monochloramines are weaker oxidants than HOCl, lipophilic chloramines freely diffuse across lipoprotein bilayer of cell membranes and react with cytosolic components, potentiating a direct cell membrane attack by HOC1 [ 381. Thus, short-lived nonspecific reactivity of HOC1 is converted into a highly specific long-acting cytotoxic effect [38]. The evidence reported here of sustained circulatory oxidant stress due to the increased presence of HOC1 could provide key information needed to understand the im-
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portance of the neutrophil in effecting widespread microvascular injury following the onset of sepsis. The potency of extracellular HOC1 can be appreciated by understanding the efficiency with which this oxidant induces lysis of bacteria. Test and Weiss showed that lo6 human PMN are capable of generating 2 X 10M7mole of HOC1 during a 2-hr incubation which was adequate to destroy 150 X lo6 Escherichia coli organisms in milliseconds at physiological pH [38]. In further experiments, the authors suggested that HOCl-derived oxidants were much more reactive in producing tissue damage than generally thought. This was evidenced by the report that lo6 PMNs under optimum conditions could produce enough HOC1 to induce lysis of 2.4 X lo5 human tumor cells (approximately 180 nmole) [38,46]. In the current study total production from 300min Ps-infused PMNs over 20 min was 13.02 nmole/106 cells. Assuming a constant rate of HOC1 production for up to 2 hr [38] this quantity amounts to a total of 78.12 nmole/106 PMNs during a 2-hr period. Therefore, approximately 320 nmole HOC1 could be produced per unit volume of plasma in the septic pig as compared with 180 nmole from baseline human cells (pigs possess approximately 4X the number of granulocytes per ml than humans). The means by which cyclooxygenase inhibition attenuates PMN long-lived oxidant production is not clear. Ibuprofen is known to attenuate the “priming” response of porcine PMN for 0, generation 1191. Minta and Williams have suggested that nonsteroidal anti-inflammatory drugs block ligand-receptor interactions on PMN surfaces and prevent upregulation of 0; generation, thus reducing the substrate for HOC1 production [ 201. Wasil et al. demonstrated that a wide range of commonly used anti-inflammatory agents, including ibuprofen, scavenge HOCl, but they concluded that these reactions were insufficiently rapid under physiological conditions to protect al-antiproteases [6]. Hypochlorous acid assay in the present study was performed on isolated PMNs; therefore, scavenging effects of the drug were eliminated and a true assessment of the activity of the enzyme generating system could be made. Alternatively, cyclooxygenase metabolites may act to reduce MPO secretion from neutrophil azurophilic granules. Although this hypothesis has not been proven, cyclooxygenase inhibition may alter second messenger systems intracellularly, leading to decreased release of MPO from PMN following exposure to PMA, a known stimulator of protein kinase C. In conclusion, this study provides evidence which suggests that development of sepsis is associated with enhanced intravascular generation of chlorinated oxidants by PMNs. This phenomenom is closely linked with the development of pulmonary microvascular injury. The study also provides new evidence which suggests that improved physiological outcome following cyclooxygenase blockade may be related to containment of the production of PMN halogenated oxidants. However, results of stud-
CAREY
ET AL.: HYPOCHLOROUS
ACID
ies, such as this, which demonstrate changes in function of circulating PMNs in sepsis must be interpreted with caution since such data may not totally reflect the activity of PMNs sequestered in the pulmonary microcirculation.
IN PORCINE
4.
5.
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18.
Gnidec, A. G., Sibbald, W. J., Cheung, H., and Metz, C. A. Ibuprofen reduces the progression of permeability edema in an animal model of hyperdynamic sepsis. J. Appl. Physiol. 65(3): 1024, 1988.
19.
Carey, P. D., Jenkins, J. K., Byrne, K., Schneider, A., Sielaff, T. D., Fowler, A. A., and Sugerman, H. J. Ibuprofen attenuates the enhanced respiratory burst and alveolar-capillary membrane permeability in porcine ARDS. Surg. Forum 40: 83, 1989.
Brigham, K. L., Woolverton, W. C., Blake, L. H., and Staub, N. C. Increased sheep lung vascular permeability caused by Pseudomonas bacteremia. J. Clin. Invest. 54: 792, 1974. Kopolovic, R., Thraikill, K. M., Martin, D. T., Ambrose, T., Vento, M., Carey, L. C., and Cloutier, C. T. Effects of ibuprofen on a porcine model of acute respiratory failure. J. Surg. Res. 36: 300, 1984.
20.
Minta, J. O., and Williams, M. D. Some nonsteroidal antiinflammatory drugs inhibit the generation of superoxide anions by activated polymorphs by blocking ligand-receptor interactions. J. Rheumatol. 12:751,1985.
21.
Rampart, M., and Williams, T. J. Suppression of inflammatory oedema by ibuprofen involving a mechanism independent of cyclooxygenase blockade. Biochem. Pharmacol. 35(4): 581, 1986.
Lee, C. C., Sugerman, H. J., Tatum, J. L., Wright, T. P., Hirsh, P. D., and Hirsch, J. I. Effects of ibuprofen on a pig Pseudomonas ARDS model. J. Surg. Res. 40: 438, 1986. Heflin, A. C., and Brigham, K. Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia. J. Clin. Invest. 68: 1253, 1981. Till, G. O., Johnson, K. L., Kunkel, R., and Ward, P. A. Intravascular activation of complement and acute lung injury. Dependancy on neutrophils and toxic oxygen metabolites. J. Clin. Invest. 69: 1126, 1982. Wasil, M., Halliwell, B., Moorhouse, C. P., Hutchison, D. C., and Baum, H. Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by some anti-inflammatory drugs. Biochem. Pharmac. 36: 3847, 1987.
22.
Byrne, K., Cooper, K. R., Carey, P. D., Berlin, A., Sielaff, T. D., Blocher, C. R., Jenkins, J., Fisher, B. J., Tatum, J. L., Fowler, A. A., and Sugerman, H. J. Pulmonary compliance: Early assessment of evolving lung injury following onset of sepsis. J. Appl. Physiol., in press.
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Markwell, M. A., Haas, S. M., Bieber, L. L., and Tolbert, N. E. A modification of the Lowry procedure to simplify protein determination in lipoprotein samples. Anal. Biol. 87: 206, 1978.
24.
Boyum, A. Isolation of mononuclear cells and granulocytes from human blood. Scan. J. Clin. Lab. Invest. 97(Suppl.): 77, 1968.
25.
Barbour, A. G., Allred, C. D., Solberg, C. O., and Hill, H. R. Chemiluminescence by polymorphonuclear leukocytes from patients with active bacterial infections. J. Infect. Dis 141(l): 14, 1980.
26.
Weiss, S. J., Klein, R., Slivka, A., and Wei, M. Chlorination taurine by human neutrophils. J. Clin. Znuest. 70: 598, 1982.
27.
Kukreja, R. C., Weaver, A. B., and Hess, M. L. Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants. Biochim. Biophys. Acta 990: 198, 1989.
28.
Ellman, G. L. Tissue sulfhydryl 82: 70,1959.
29.
Schraufstltter, I. U., Revak, S. D., and Cochrane, C. G. Proteases and oxidants in experimental pulmonary inflammatory injury. J. Clin. Invest. 73: 1175, 1984.
30.
Reide, U. N., Joachim, H., Hassenstein, J., Costabel, U., Sandritter, W., Augustin, P., and Mittermayer, C. H. Pulmonary air-blood flow barrier of human shock lungs: A clinical, ultrastructural and morphometric study. Pathol. Res. Pratt. 162: 41, 1978.
31.
Thommasen, H. V. The role of the polymorphonuclear leukocyte in the pathogenesis of the adult respiratory distress syndrome. Clin. Invest. Med. 8(2): 185, 1985.
32.
Del Maestro, R. F., Thaw, H. H., Bjfk, J., Planker, M., and Arfors, K. E. Free radicals as mediators of tissue injury. Acta Physiol. Stand. (Suppl.) 492: 43, 1980.
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Newburger, P. E., Chovaniec, M. E., and Cohen, H. J. Activity and activation of the granulocyte superoxide-generating system. Blood 55(l): 85, 1980.
34.
Shepherd, V. L. The role of the respiratory burst of phagocytes in host defense. Semin. Respir. Infect. l(2): 99, 1986.
35.
Fantone, J. C., and Ward, P. A. Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Amer. J. Pathol. 107: 397, 1982.
Henson, P. M., and Johnston, R. B. Tissue injury in inflammation. Oxidants, proteinases and cationic proteins. J. Clin. Invest. 79: 669, 1987. Brigham, K. L., and Meyrick, B. Interactions of granulocytes with the lungs. Circ. Res. 54(6): 623, 1984. Weiss, S. J. Tissue destruction by neutrophils. N. Engl. J. Med. 320: 365,1989. Goris, R. J. A., te Boekhorst, T. P. A., Nuytinck, J. K. S., and Gimbrere, J. S. F. Multiple organ failure. Arch. Surg. 20: 1109, 1985.
11.
Zimmerman, G. A., Renzetti, A. D., and Hill, H. R. Functional and metabolic activity of granulocytes from patients with adult respiratory distress syndrome. Amer. Rev. Respir. Dis. 127: 290,1983.
12.
Holman, R. G., and Maier, R. V. Superoxide production by neutrophils in a model of adult respiratory distress syndrome. Arch. Surg. 123: 1491,1988. Weiss, S. J., and LoBuglio, A. F. Biology of disease. Phagocytegenerated oxygen metabolites and cellular injury. Lab. Znuest. 47(l): 5, 1982. Babior, B. M., Curnutte, J. T., and Okamura, N. The respiratory burst oxidase of the human neutrophil. In B. Halliwell (Ed.), Oxygen Radicals and Tissue Injury. Bethesda, MD: Upjohn Co., 1988. Pp. 43-48. Fantone, J. C., Feltner, D. E., Brieland, J. K., and Ward, P. A. Phagocytic cell-derived inflammatory mediators and lung disease. Chest. 91(3): 428, 1987. Byrne, K., Carey, P. D., Berlin, A., Jenkins, J. K., Cooper, K. R., Fowler, A. A., and Sugerman, H. J. Ibuprofen prevents deterioration
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Balk, R. A., Jacobs, R. F., Tryka, A. F., Townsend, J. W., Walls, R. C., and Bone, R. C. Effects of ibuprofen on neutrophil function and acute lung injury in canine endotoxin shock. Crit. Care. Med.
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REFERENCES
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The authors acknowledge the invaluable technical contributions of Charles R. Blocher and Bernard J. Fisher and the kind gift of ibuprofen from the Upjohn Co., Kalamazoo, Michigan.
2.
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in static transpulmonary compliance and transalveolar protein flux in a septic porcine ARDS model. J. Trauma. 29(7): 1026, 1989. [Abstract]
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Fridovich, I. Biological effects of the superoxide radical. Arch. B&hem. Biophys. 247: 1,1986. Bertrand, Y. Oxygen-free radicals and lipid peroxidation in adult respiratory distress syndrome. Intensive Cure Med. 11: 56, 1985. Test, S. T., and Weiss, S. J. The generation of utilization of chlorinated oxidants by human neutrophils. Adu. Free Radical Biol. Med. 2: 91,1986. Suttorp, N., Toepper, W., and Roka, L. Antioxidant defense mechanisms of endothelial cells: Gluthathione redox cycle versus catalase. Amer. J. Physiol. 251: C671, 1986. Weiss, S. J., Lampert, M. B., and Test, S. T. Long-lived oxidants generated by human neutrophils: Characterization and bioactivity. Science 222: 625, 1983. Omann, G. M., Allen, R. A., Bokoch, G. M., Painter, R. G., Traynor, A. E., and Sklar, L. A. Signal transduction and cytoskeletal activation in the neutrophil. Physiol. Reu. 67: 285, 1987.
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Tennenberg, S. D., and Solomkin, J. S. Neutrophil activation in sepsis: The relationship between fMet-Leu-Phe receptor mobilization and oxidative activity. Arch. Surg. 123: 171, 1987. Aruoma, 0. I., and Halliwell, B. Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and glutathione peroxidase. Biochem. J. 248: 973, 1987.
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McKenna, S. M., and Davies, K. J. A. The inhibition of bacterial growth by hypochlorous acid. Biochem. J. 254: 685, 1988.
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van Zyl, J. M., Basson, K., and van der Walt, B. J. The inhibitory effect of acetaminophen on the myeloperoxidase-induced antimicrobial system of the polymorphonuclear leukocyte. Biochem. Pharmacol. 38: 161, 1989.
46.
Lampert, M. B., and Weiss, S. J. The chlorinating human monocyte. Blood 62: 645,1983.
potential
of the
OF SURGICAL
49,262-270
RESEARCH
(1990)
Ibuprofen Attenuates Hypochlorous Acid Production from Neutrophils in Porcine Acute Lung Injury1*2*3 P. DECLAN CAREY, F.R.C.S.I., KARL BYRNE, F.R.C.S.I., JOHN K. JENKINS, M.D., TIMOTHY D. SIELAFF, M.D., CIARAN J. WALSH, F.R.C.S.I., ALPHA A. FOWLER III, M.D., AND HARVEY J. SUGERMAN, M.D. Departments
of Surgery, Medicine,
and Pathology, Medical College of Virginia, Submitted
for publication
Academic
Press,
Inc.
INTRODUCTION Infusion of live Pseudomonas bacteria into the pig generates sepsis with an associated acute lung injury (ALI) i These experiments were supported in part by U.S. Army Contract DAMD 17-86-C-6168 and PHS HL35534-03. The views, opinions and/ or findings contained herein are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation. ‘These experiments were performed in accordance with the NIH Guidelines for the use of experimental animals. 3 Presented at the Annual Meeting of the Association for Academic Surgery, Louisville, KY, November 15-18, 1989.
0022~4804/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
262 Inc. reserved.
November
Commonwealth
University,
Richmond,
Virginia
23298
20. 1989
which closely simulates the lung injury seen in the adult respiratory distress syndrome (ARDS) in humans [l-3]. Multiple pathophysiological mechanisms of sepsis-induced acute lung injury have been proposed. Recent attention has focused upon the destructive capability of neutrophils (PMN) and their role in producing alveolarcapillary membrane injury following sequestration in the pulmonary microcirculation [4, 51. In a number of common human conditions (e.g., rheumatoid arthritis, ulcerative colitis, myocardial reperfusion, and adult respiratory distress syndrome) a vigorous inflammatory response is elicited from circulating phagocytes with subsequent host tissue damage [6-91. Current evidence supports the hypothesis that circulating PMNs become “primed” for reactive oxygen metabolite production following the onset of sepsis and, when sequestered in the microcirculation, may be responsible for widespread endothelial cell injury [ 10-121. Neutrophils stimulated by septic mediators undergo a “respiratory burst” which results in increased production of short-lived oxidant species such as superoxide anion (0;) [13, 141. Superoxide anion is rapidly metabolized to hydrogen peroxide by the action of superoxide dismutase (SOD) [ 13,151. In the presence of chloride ions (Cl-) the azurophilic granular enzyme myeloperoxidase (MPO) converts hydrogen peroxide to hypochlorous acid [9, 131. Hypochlorous acid is believed to be the most potent and destructive of the neutrophil-derived oxidants [9, 131. Cyclooxygenase metabolites of arachidonic acid are thought to play a major role in the physiological derangements in sepsis-induced ALI. Ibuprofen, a potent cyclooxygenase blocker, has been shown to be effective in attenuating alveolar-capillary membrane damage [2, 3, 16-181. The mechanism of this effect may be via alteration of certain neutrophil functions, such as down regulation of 0; generation [ 19-211. The present study was designed to determine the relationship between in viva production of HOC1 from circulating PMNs and various parameters of lung injury and to see whether the physiological improvement seen with ibuprofen therapy was associated with alterations in HOC1 production.
Hypochlorous acid (HOCl), a neutrophil-generated oxidant, has been implicated in tissue destruction in sepsisinduced acute lung injury (ALI). Ibuprofen, a cyclooxygenase inhibitor, successfully attenuates many of the physiological derangements in ALI. The aim of this study was to examine the role of PMN hypochlorous acid in sepsis-induced AL1 and evaluate the effect of ibuprofen therapy. Neutrophils from three groups of young (1525 kg), anesthetized swine were studied: controls (C, n = 7) received 0.9% NaCl, septic animals (Ps, n = 8) received live Pseudomonas aeruginosa (5 X lo8 CFU/ml at 0.3 ml/20 kg/min) for 60 min, and ibuprofen-treated animals (Ps + I, n = 6) received Ps plus ibuprofen 12.5 mg/kg administered at 0 and 120 min. Neutrophils were isolated from peripheral blood at 0,60, and 300 min and the rate and total production of HOC1 were assessed on the basis of the ability of the amino acid taurine to trap HOCl. Results: Septic (Ps) PMNs produce 32% more HOCl, P < 0.01, at 300 min than at baseline which was associated with a marked increase in both extravascular lung water (6.44 f 0.8 ml/kg, t = 0 vs 16.03 + 2.6 ml/ kg, t = 300, P < 0.01) and bronchoalveolar protein content (115 + 13 rg/ml, t = 0 vs 633 + 104 pg/ml, t = 300, P < 0.01). Ibuprofen significantly attenuated (P < 0.05) HOC1 production when compared to Ps, in conjunction with significantly (P < 0.05) reduced levels of extravascular lung water and bronchoalveolar lavage protein. 0 1990
Virginia
CAREY
MATERIALS Animal
ET AL.: HYPOCHLOROUS
ACID
AND METHODS
Conditioning
Yorkshire swine (15-30 kg) were obtained from a commercial vendor in the Richmond area and housed in the Virginia Commonwealth University vivarium for 3-5 days prior to use. Animals received benzathine and procaine penicillin G (300,000 Units each) intramuscularly 48 hr prior to use. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University and adhered to National Institutes of Health guidelines for the use of experimental animals. Animal
Preparation
On the day of the study, animals were preanesthetized (25 mg/kg ketamine hydrochloride, 0.4 mg atropine, im) and placed supine. General anesthesia was induced with sodium pentobarbital (10 mg/kg, iv) and maintained throughout the study with intermittent pentobarbital infusion. Endotracheal intubation using a cuffed endotracheal tube (National Catheter) was performed. Skeletal muscle paralysis was maintained by a continuous intravenous infusion of pancuronium bromide (0.2 mg/kg/ min). Animals were mechanically ventilated (Harvard large animal ventilator, Harvard Apparatus, South Natick, MA, 0.5 FiOz, 5 cm Hz0 PEEP; tidal volume, 15 ml/ kg) with the initial respiratory rate adjusted to produce a PaCOz of 40 Torr at the beginning of each experiment. Indwelling catheters were placed in the left common carotid artery for systemic arterial pressure (SAP) monitoring and arterial blood gas determination as well as the left external jugular vein for infusion of saline, Pseudomonas organisms, and ibuprofen (see below). An indwelling balloon-tipped pulmonary arterial catheter was inserted via the right internal jugular vein and positioned in the pulmonary artery via pressure monitoring for measurement of pulmonary arterial pressure (PAP), pulmonary arterial occlusion pressure (PAOP), central venous pressure (CVP), and thermodilution cardiac output (Edwards COMl). Measurements of Alveolar-Capillary Permeability: Thermal-Cardiogreen Extravascular Lung Water (E VL W) and Bronchoalveolar Lavage Protein Content (BAL-P) EVLW measurements were recorded every 60 min until the end of the 5-hr study period using the thermal cardiogreen double indicator dilution technique as previously described [22]. Bronchoalveolar lavage (BAL) was performed using a fiberoptic bronchoscope at baseline in the middle and lower lobes of the right lung. The distal end of a fiberoptic bronchoscope (Machita VT-5100C, 4mm) was wedged into a third- or fourth-order bronchus and a
IN PORCINE
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LUNG
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263
75-ml BAL was performed in each lobe using 25-ml aliquots of sterile 0.9% NaCl. Fluid was retrieved by gentle aspiration of the infusing syringe. The BAL procedure was repeated at 300 min in corresponding segments of the left lung. The procedures were performed by one of two technicians J.K.J. or A.A.F. and lavage return was consistently high, 75-80%. Cell free BAL fluid protein content was determined by a modification of the Lowry technique described by Markwell and associates for fluids containing lipids [23]. Results are expressed as micrograms protein per milliliter of recovered lavage fluid. Study Design Animals were randomly divided into three study groups. Control animals (C, n = 7) received an infusion of sterile 0.9% NaCl for 60 min. The Pseudomonas group (Ps, n = 8) was infused continuously for 1 hr with live Pseudomonas aeruginosa (PA0 strain, 5 X 10’ organisms/ml at 0.3 ml/20 kg/min). The treatment group (Ps -t I, n = 6) received an identical infusion of live organisms as well as ibuprofen (12.5 mg/kg infused at 0 and 120 min). Systemic arterial pressure (SAP), pulmonary artery pressure (PAP), pulmonary arterial occlusion pressure (PAOP), cardiac index (Cl), central venous pressure (CVP), and arterial blood samples for pH, P,Os and P,C!02 (pH/blood gas analyzer, Instrumentation Laboratories Model 1304) were measured at baseline and every 30 min during the experimental period. Systemic arterial blood samples were taken every 15 min for the first hour and every 30 min thereafter for measurement of peripheral total leukocyte count. Arterial blood samples were obtained at 0,60, and 300 min for neutrophil isolation. Previous studies from this laboratory using a similar model have shown that the CVP and PAOP do not significantly decline from baseline over the 300-min study period in septic animals; thus fluid was administered to each group only to replace those losses due to investigative phlebotomy [22]. Neutrophil Isolation Arterial blood was drawn into syringes containing sterile EDTA (15%, 0.1 ml/10 mls blood). Neutrophils were isolated by dextran sedimentation, Ficoll-Hypaque density gradient centrifugation, and hypotonic lysis [ 24, 251. Neutrophils were suspended in phosphate-buffered saline (PBS) at a concentration of 4 X lo6 cells/ml. Final preparations contained greater than 98% neutrophils by modified Wright’s Giemsa staining (Diff Quick, American Scientific) which were 99% viable by trypan blue dye exclusion. Peripheral white cell count was performed by hemocytometer on whole blood samples collected as outlined above. Measurement of HOC1 Production by Stimulated Neutrophils Neutrophil HOC1 production was determined by a modification of the chlorination of taurine method [26]
264
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VOL. 49, NO. 3, SEPTEMBER
TABLE
1990
1
Time (min) 30
0
Mean pulmonary C Ps Ps + I
18.0 + 15.7 k 14.3 +
1.6 0.8 0.8
17.3 f 47.6 f 19.3 k
2.0 l.l* 1.8**
15.6 f 38.1 f 27.0 2
1.4 l.O* 3.3**** Mean arterial
C Ps Ps + I
215 247 22
+ 15 + 8 rf:12
224 183 250
+_16 f14 f 14**
244 157 241
180
120
60
519 *12* 2 27**
300
240
artery pressure (mm Hg) 16.6 f 29.8 + 24.8 +
1.5 0.9* 1.4*,**
16.9 iz 2.7 30.7 f 1.0* 23.5 + 1.9*,**
18.3 f 31.5 f 25.8 +
1.7 1.1* 2.4*
18.2 + 2.6 26.7 zb 3.1* 28.8 + 2.2*
oxygen tension (Torr) 259 140 253
+_16 f12, f 14**
243 118 197
220 +ll* + 38**
252 112 171
k19 f 13* f 33****
255 97 135
f12 *15* + 38*
l?r 6*** + 5% + 9**
131 82 104
f 7 k 6* f 12*
131 88 128
* * +
Mean systemic artery pressure (mm Hg) C Ps Ps + I
111 100 100
zk 8 +- 4 + 5
121 130 115
* 9 f 4*** + 10
* P < 0.05 vs C, **P < 0.05 vs Ps, ***P
138 103 125
+ 7+** * 3* f lo**
133 88 128
f f +
7*** 4+ 8**
136 76 115
5 7% 8**
< 0.05 vs baseline.
as recently described by Kukreja et al. [27] where the ability of the amino acid taurine to act as a scavenger of HOC1 is utilized. The resulting chlorination complex (taurine chloramine), oxidizes 5-thio-2-nitrobenzoic acid (TNB) to its disulfide, 5-5’dithiobis-(2-nitrobenzoic acid) (DTNB) with a resultant decrease in absorbance which can be measured spectrophotometrically. Generation of HOC1 is indicated by taurine chloramine formation. TNB was prepared by sodium borohydride (Sigma Chemical Co.) reduction of DTNB (Sigma). Reagents were made up on the day of each assay. Isolated PMNs (2.6 X 106) were resuspended in PBS in the presence of 15 mM taurine and 0.77 mM TNB (pH 7.4) and placed in a cuvette at a final volume of 2.6 ml. Neutrophils were then activated by the addition of 100 nmole phorbol myristate acetate (PMA, Sigma). TNB oxidation was followed spectrophotometrically (Shimadzu UV-160) at 412 nm for 20 min at 37°C under continuous stirring. Assays were performed in duplicate and a reference cuvette containing cells and reaction mixture which remained unstimulated was used with each assay to adjust for spontaneous oxidation of the indicator. The amount of HOC1 generated per minute during the 20-min assay period was calculated from the absorbance change at 412 nm using an absorption coefficient of 1.36 X lo4 M-l cm-’ [28]. Data are expressed as nmole HOC1/106 PMNs/min. Cumulative production of HOC1 was determined for each cell sample from the total optical density change over 20 min. Results are expressed as nmole HOCl/106 PMNs/20 min. Statistics All results are expressed as means & SEM. The presence of significant differences within and between groups
was determined by analysis of variance (ANOVA) and repeated measures ANOVA. Differences between means were analyzed using Tukey-Kramer Studentized range test and Student’s t test where appropriate. The level of statistical significance was set at P < 0.05 for ANOVA. RESULTS
Physiological
Measurements
Physiological measurements for the three groups over the course of the study period are displayed in Table 1. Control animals exhibited minimal variation from baseline throughout the study period in any of the measured parameters. Animals in the Ps group showed an immediate and significant (P < 0.05) increase in pulmonary arterial pressure (PAP) following infusion of live organisms. PAP subsequently diminished somewhat from this initial peak but remained significantly higher than PAP in controls throughout the 300 min of the study. Ibuprofen pretreatment prevented the early phase pulmonary arterial hypertension. Furthermore, PAP following ibuprofen treatment was significantly decreased when compared to septic animals until 4 hr, but rose significantly above saline-infused controls from 90 min onward. Systemic arterial pressure (SAP) becomes significantly elevated (P < 0.05) in control animals at 60 min. In contrast, SAP in Pseudomonas-infused animals was elevated at 30 min and then fell progressively. Systemic arterial pressure was significantly lower than control values from 60 min onward. Ibuprofen maintained SAP at or near control levels from 0 to 300 min. Systemic arterial oxygen tension (P,O,) did not vary in control animals but underwent a steady decline in the Ps group, becoming significantly lower than
CAREY
ET AL.: HYPOCHLOROUS
ACID
TABLE
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265
INJURY
2 Time (min)
0
60
120
240
180
300
Mean central venous pressure (mm Hg)
C PS Ps + I
4.8 rf 0.8 4.1 + 0.6 3.5 * 0.4
4.3 -c 1.1 4.4 -c 0.7 3.8 f 0.3
4.6 k 0.8 5.1 * 0.7 3.0 + 0.6
Mean pulmonary
C Ps Ps + I
6.0 + 1.0 5.6 + 0.6 6.1 + 1.1
7.7 k 0.8 7.7 * 1.0 7.0 t 1.0
capillary 6.8 k 0.8 7.1 f 0.8 6.4 f 0.9
Extravascular
C Ps Ps + I
6.84 k 1.1 6.44 rf- 0.8 7.51 AI 0.7
7.62 k 0.6 9.50 * 0.9 8.60 k 0.7
3.2 f 0.9 4.3 + 0.7 2.2 + 0.5
3.3 f 1.1 4.1 f 0.6 2.0 * 0.5
2.6 f 0.9 2.8 f 0.6 2.2 k 0.9
7.4 + 1.1 5.7 f 0.7 6.7 + 1.3
6.3 ” 1.0 6.1 ir 1.2 5.5 f 0.9
5.81 + 0.9 14.06 k 1.4* 10.24 f 0.9
6.78 f 0.1 16.03 + 2.6* 8.90 rLr1.5”
wedge pressure (mm Hg) 6.6 + 0.9 7.8 ?I 1.1 6.2 + 0.5
lung water (ml/kg)
6.05 k 0.9 10.60 * 0.9* 8.03 k 0.6
6.78 k 1.0 11.97 * 1.5* 9.59 k 1.1
* P < 0.05 “S c. **p < 0.05 “S Ps.
controls from 60 to 300 min. Arterial oxygen tension in ibuprofen-treated animals was not significantly different from control measurements until 240 min but in the last hour deteriorated toward levels observed in septic animals. Both PAOP and CVP levels remained at baseline levels in all groups throughout the study (Table 2). Peripheral
WBC Counts
Table 3 records the total peripheral WBC count measured at 0,60, and 300 min time points. A significant and progressive peripheral leukopenia was observed in group Ps following bacterial infusion. Leukopenia was well established by 60 min into the study becoming more profound by 300 min. A similar degree of leukopenia was unaltered by ibuprofen treatment in septic animals (Ps + I).
TABLE
3
WBC (X103 cells/mm3)
Control (n = 7) Pseudomonas (n = 8) Pseudomonas + ibuprofen (n = 6) * P < 0.05 vs control.
0 min
60 min
300 min
23.6 + 5.4
21.7 f 5.7
27.0 f 4.8
21.8 + 2.0
7.0 t 3.2*
4.1 k 1.3’
19.6 + 4.5
4.9 * 2.2*
5.2 f l.l*
Alveolar-Capillary
Membrane Permeability
Extravascular lung water (EVLW) measurements for the three groups are displayed in Table 1. Observed EVLW remained at baseline levels throughout the study in control animals. In septic animals, EVLW rose progressively, becoming significantly greater than control from 120 to 300 min, and reached a maximum of 16.03 f 2.7 ml/kg at 300 min. Ibuprofen treatment maintained EVLW within control limits at all time points with values significantly lower than those observed in the Ps-infused animals at 300 min. Bronchoalveolar lavage protein content in Ps animals was significantly greater than both C and Ps + I at 300 min. Bronchoalveolar lavage protein content in both C and Ps + I remained at baseline values throughout the study (Fig. 1). PMN Hypochlorous
Acid Production
Hypochlorous acid production from porcine PMNs in the three study groups is shown in Figs. 2-4. No differences were observed in the rate of production of HOC1 between PMNs at 0 and 60 min in all groups. In control animals, generation of taurine chloramine (HOCl) reached peak production at 8-10 min following PMA stimulation (Fig. 2). No differences in PMA-stimulated HOC1 production by neutrophils obtained at 0 or 300 min were observed (1.18 + 0.08 vs 1.2 * 0.1 nmole/lO” PMN, P = 0.29, ANOVA) in control animals. Unstimulated cells failed to produce significant quantities of HOC1 (Fig. 2). In contrast, PMNs from septic animals (Fig. 3) exhibited greater rates (P < 0.05) of HOC1 production up to 14 min following PMA stimulation when compared to PMN ob-
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800 o-0Control m-0 Ps 1 A-&P5 + lb”
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tained at baseline in the same animals. A 25% increase in maximum HOC1 production (1.7 nmole/106 PMN) occurred 10 min following PMA stimulation in septic PMNs (P < 0.01). Effect of Cyclooxygenase Inhibition Acid Production
on Hypochlorous
In Fig. 4 continuous production of HOC1 for groups Ps and Ps + I at 300 min are compared along with their respective baselines. Analysis of variance demonstrated overall differences between Ps 300 and Ps 0, Ps + I 0 and Ps + I 300 (P < 0.05). Despite significantly increased rates of HOC1 production in the first 5-6 min following PMA stimulation, the presence of ibuprofen in septic animals (Ps + I) resulted in a significant decrease in oxidant generating capacity at all assay time points when compared to septic PMNs alone (P < 0.05). Figure 5 compares the cumulative production of HOC1 over 20 min and includes each time point from the three groups. At 20 min following PMA stimulation, PMNs from Ps-infused animals obtained at 300 min generated significantly greater quantities of HOCl, 9.84 rt 0.4, 0 min vs. 13.02 + 0.3, 300 min nmole/106 PMNs (P < 0.01, Students t test) than all other groups. This quantitative increase in HOC1 pro-
8
16
20
Time’Tmin)
FIG. 2. Rate of HOC1 generation from PMNs following PMA stimulation in control animals (C, n = 7) at 0 and 300 min. Cells that remained unstimulated failed to produce appreciable quantities of HOCl. There was no significant differences between PMNs isolated at the two time points.
8 Time
16
, 20
(rn~$
FIG. 3. Rate of HOC1 generation from PMNs in septic animals (Ps, n = 8) at 0 and 300 min following PMA stimulation. Significant differences are indicated in the figure, *P < 0.05.
duction was not detected in PMNs from Ps-infused animals at 60 min. The quantitative increase in long-lived oxidant generation at 300 min in septic animals was 32% higher when compared to PMNs obtained at baseline. Pretreatment of animals with ibuprofen attenuated HOC1 production from PMNs obtained at 300 min and this was indistinguishable from that observed in control animals (9.79 * 0.4 vs 10.12 f 0.1 nmole/106). DISCUSSION The proposed aims of the present study were to examine neutrophil long-lived oxidant production during the evolution of Pseudomonas sepsis in the pig and to determine how cyclooxygenase inhibition would alter the production of this oxidant species. Neutrophils are thought to play a major role in sepsisinduced endothelial cell damage and increased alveolarcapillary membrane permeability by release of oxidants and proteolytic enzymes [ 15,291. Sequestration of PMNs in the pulmonary microvasculature of humans and animals following the onset of sepsis is well established [5, 301. Chemotaxins derived from macrophages, complement split products, (C5a, C3a) and altered pulmonary blood
0 4
4
(min)
FIG. 1. Estimated bronchoalveolar lavage protein content from harvested lavage fluid at 0 and 300 min showed significant influx (*P < 0.01) of protein to the alveolus in Ps animals. Ibuprofen pretreatment kept BAL-P at control levels.
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4
8
Time
12
16
20
(min)
FIG. 4. Rate of HOC1 production from isolated PMNs in group Ps and group Ps + I is compared. Included in this figure are the respective baseline (0 min) curves for the two groups. For statistical analysis each group was compared to its own baseline and 300 min time points were similarly compared (ANOVA). Individual time point variations are indicated in the figure (*P < 0.05 vs C, #P < 0.05 vs Ps + I).
CAREY
ET AL.: HYPOCHLOROUS
ACID
15 -,
F i I 0ml”
*
"010 i i I 0 $5 c (n=7) *pee 0, “5
Ps (n=8)
Ps+1
(n=6)
All
FIG. 5. The cumulative production of HOC1 from porcine PMNs following PMA stimulation. PMNs were harvested at 0, 60, and 300 min following the onset of saline or live Pseudomonas infusion. Cumulative production of HOC1 was calculated from the total change in optical density over the ZO-min assay period. PMNs harvested at 300 min in Ps animals exhibited significantly increased HOC1 generation compared to all other time points and cell groups (‘P < 0.01, Student’s t test).
flow all mediate neutrophil sequestration [31]. Thus, bacteria and endotoxin, both stimulators of C5a production, may be directly responsible for bringing PMNs into apposition with microvascular endothelium 1311. The mechanism by which PMNs effect pulmonary microvascular endothelial injury is unclear; however, the presence of “activated” PMNs in close approximation with pulmonary capillary endothelial cells could lead to host cell damage. Microvascular injury and host pulmonary dysfunction in sepsis may be associated with “priming” of the respiratory burst of circulating PMNs [ll, 121. Neutrophils are capable of generating large quantities of reactive oxygen metabolites which are important in defense against invading microorganisms but excessive production of toxic oxidants could lead to nonspecific destruction of host cells [32-341. Neutrophil excitation by a number of biochemical stimuli results in activation of a membrane bound NADPH oxidase system [13, 351. Electrons are shuttled from cytosolic NADPH to oxygen thus forming 0, (Fig. 6, Eq. (1)) [9]. Subsequently, a cascade of reactions occur leading to the production of a spectrum of potent oxidants [9]. The dismutation of 02 to hydrogen peroxide (H,O,) may occur spontaneously or may be rapidly catalyzed by superoxide dismutase (SOD) (Fig. 6, Eq. 2) [36]. Hydrogen peroxide in the presence of metal ions (e.g., Fe’+) may produce another potent oxidizing species, the hydroxyl radical (Fig. 6, Eq. 3) [15, 371. Hydrogen peroxide in the presence of myeloperoxidase (MPO) and halide ions may also be converted to the extremely toxic oxidant, hypochlorous acid (Fig. 6, Eq. 4) [38]. Alternatively, Hz02 may be scavenged by catalase or gluthathione peroxidase and rendered nontoxic [13, 39, 401. The majority of generated 0, is dismuted to HZ02 which serves as a branch point for production of further oxidants [9]. Weiss contends that the preferential pathway for the metabolism of Hz02 produced by PMNs occurs along the MPO-hypohalous acid pathway, leading to production of hypo-
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chlorous acid in the normal Cl- rich physiologic environment [9]. The use of the phorbol ester, phorbol myristate acetate (PMA) to activate the PMN oxidant generating mechanism is a standard technique. Intracellular activation of PMNs at the protein kinase C level consistently causes a maximal stimulation of the NADPH oxidase enzyme system. Neutrophil activation in Go, as in the present study, occurs by a number of routes, including phagocytosis of opsonized bacteria and by small bacterial peptides via ligand-receptor coupling [41]. However, previous studies have shown that septic stimulation of PMNs in uiuo results in unresponsiveness to the same stimulation following isolation [42]. It therefore becomes important, in studies such as the present one, to cause a maximal secondary stimulation of harvested cells which clearly demonstrates the nature of the “priming” response. Thus PMA serves as a probe to determine PMN “priming.” Hypochlorous acid, the active component in household bleaches, is a potent microbiocidal molecule [9]. It is widely believed that HOC1 participates in reducing inflammation by the inactivation of bacteria and fungi [38, 431. Low concentrations of HOC1 have been shown to exert a rapid and selective inhibition of bacterial growth and cell division [44]. Thus, HOC1 appears to be a critical component of the body’s defense against invading microorganisms. Antimicrobial function is generally confined to the phagocytic vacuole but extracellular release of HOC1 and other oxidants can occur during the formation and closure of vacuoles, during PMN phagocytosis [38]. The oxidant activities of HOC1 are nonselective and it has been shown to react avidly with all biomolecules [44]. Therefore, even though HOCl’s action is mainly protective, its potency and nonselectivity of action implicate it in host tissue damage in the inflammatory response [44]. Hypochlorous acid can directly attack cell membranes resulting in cell lysis and death [38, 441. Hypochlorous acid can potentiate the action of other oxidants by inactivating antioxidant mechanisms, in particular glutathione peroxidase and catalase [43]. Importantly, HOC1 (1)
(2)
o,+
e- ----------> o2
2 02-+ 2 H+ ---------->
HsOs+ 0,
(catalyzed by SOD)
(3)
0,. + ROOH -----------> RO + OH- + 0,
(4)
HrOs + Cl -----------a OCI + OH (catalysed bv MPO)
(catalysed by Fe++ or Cu’)
FIG. 6. The sequence of short-lived oxidant generation following neutrophil stimulation. SOD, superoxide dismutase; MPO, myeloperoxidase. (1) Superoxide anion generation. (2) Hydrogen peroxide generation from O;, catalyzed by SOD. (3) Hydroxyl radical generation. (4) Hypochlorous acid generation from hydrogen peroxide, catalyzed by MPO.
268
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has been shown to inhibit the activity of circulating alprotease inhibitor. Diminished a,-antiprotease function could lead to significant regional or widespread host tissue damage due to unrestricted activity of PMN-derived proteases (e.g., elastase). Hypochlorous acid also activates latent PMN degranulation enzymes, collagenase and gelatinase. A combined assault of these PMN serine proteases could thus lead to significant degradation of components of basement membrane and interstitium [9, 38, 451. This study suggests that development of sepsis may be associated with enhanced intravascular chlorinated-oxidant production. Neutrophils isolated at 300 min in septic animals exhibited enhanced rates of HOC1 generation (up to 25%) immediately following PMA stimulation (Fig. 3). Cumulative production of HOC1 from 300-min PMNs was increased by 32% above baseline (P < 0.01; Fig. 5). Increasing disruption of the alveolar-capillary membrane occurred in conjunction with these changes. Evidence of membrane damage in septic animals first appeared at 120 min. Extravascular lung water was significantly and progressively elevated above control from 120 to 300 min. Bronchoalveolar lavage protein at 300 min was four times baseline levels in septic animals, P < 0.01. In uiuo administration of the cyclooxygenase inhibitor, ibuprofen, dramatically attenuated the upregulation of HOC1 production from PMNs harvested at 300 min when compared to untreated septic animals (Figs. 4 and 5). Ibuprofen treatment also attenuated increased alveolar-capillary membrane permeability. Bronchoalveolar lavage protein was maintained at control levels in ibuprofen animals and EVLW was maintained at control levels until 300 min when it was significantly lower than that observed in septic animals. Thus, PMNs exhibiting enhanced oxidant generation are present in the peripheral circulation at a time when increasing disruption of the alveolar-capillary membrane is occurring. These data suggest that neutrophi1 HOC1 may play a role in the production of alveolarcapillary membrane damage in this model of sepsis-induced ALI. The significance of increased PMN production of HOC1 during the development of sepsis relates in part to the ability of the oxidant to react with endogenous amines producing hydrophilic and lipophilic chloramines [40]. Monochloramines exhibit a half-life in PMN culture supernatants of approximately 18 hr [40]. Although monochloramines are weaker oxidants than HOCl, lipophilic chloramines freely diffuse across lipoprotein bilayer of cell membranes and react with cytosolic components, potentiating a direct cell membrane attack by HOC1 [ 381. Thus, short-lived nonspecific reactivity of HOC1 is converted into a highly specific long-acting cytotoxic effect [38]. The evidence reported here of sustained circulatory oxidant stress due to the increased presence of HOC1 could provide key information needed to understand the im-
VOL. 49, NO. 3, SEPTEMBER
1990
portance of the neutrophil in effecting widespread microvascular injury following the onset of sepsis. The potency of extracellular HOC1 can be appreciated by understanding the efficiency with which this oxidant induces lysis of bacteria. Test and Weiss showed that lo6 human PMN are capable of generating 2 X 10M7mole of HOC1 during a 2-hr incubation which was adequate to destroy 150 X lo6 Escherichia coli organisms in milliseconds at physiological pH [38]. In further experiments, the authors suggested that HOCl-derived oxidants were much more reactive in producing tissue damage than generally thought. This was evidenced by the report that lo6 PMNs under optimum conditions could produce enough HOC1 to induce lysis of 2.4 X lo5 human tumor cells (approximately 180 nmole) [38,46]. In the current study total production from 300min Ps-infused PMNs over 20 min was 13.02 nmole/106 cells. Assuming a constant rate of HOC1 production for up to 2 hr [38] this quantity amounts to a total of 78.12 nmole/106 PMNs during a 2-hr period. Therefore, approximately 320 nmole HOC1 could be produced per unit volume of plasma in the septic pig as compared with 180 nmole from baseline human cells (pigs possess approximately 4X the number of granulocytes per ml than humans). The means by which cyclooxygenase inhibition attenuates PMN long-lived oxidant production is not clear. Ibuprofen is known to attenuate the “priming” response of porcine PMN for 0, generation 1191. Minta and Williams have suggested that nonsteroidal anti-inflammatory drugs block ligand-receptor interactions on PMN surfaces and prevent upregulation of 0; generation, thus reducing the substrate for HOC1 production [ 201. Wasil et al. demonstrated that a wide range of commonly used anti-inflammatory agents, including ibuprofen, scavenge HOCl, but they concluded that these reactions were insufficiently rapid under physiological conditions to protect al-antiproteases [6]. Hypochlorous acid assay in the present study was performed on isolated PMNs; therefore, scavenging effects of the drug were eliminated and a true assessment of the activity of the enzyme generating system could be made. Alternatively, cyclooxygenase metabolites may act to reduce MPO secretion from neutrophil azurophilic granules. Although this hypothesis has not been proven, cyclooxygenase inhibition may alter second messenger systems intracellularly, leading to decreased release of MPO from PMN following exposure to PMA, a known stimulator of protein kinase C. In conclusion, this study provides evidence which suggests that development of sepsis is associated with enhanced intravascular generation of chlorinated oxidants by PMNs. This phenomenom is closely linked with the development of pulmonary microvascular injury. The study also provides new evidence which suggests that improved physiological outcome following cyclooxygenase blockade may be related to containment of the production of PMN halogenated oxidants. However, results of stud-
CAREY
ET AL.: HYPOCHLOROUS
ACID
ies, such as this, which demonstrate changes in function of circulating PMNs in sepsis must be interpreted with caution since such data may not totally reflect the activity of PMNs sequestered in the pulmonary microcirculation.
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potential
of the