Polyethylene Glycol-conjugated Superoxide Dismutase Attenuates Septic Lung Injury in Guinea Pigs1- 3
YUKIO SUZUKI, TOSHIMORI TANIGAKI, DOV HEIMER, WEIZHENG WANG, WILLIAM G. ROSS, HOWARD H. SUSSMAN, and THOMAS A. RAFFIN
Introduction
Sepsis inducedby gram-negative bacteria is one of the most common predisposing factors for the development of the adult respiratory distress syndrome (ARDS) (I, 2). Gram-negative bacteria, in part through endotoxin and tumor necrosis factor, activate neutrophils to increaseoxygen consumption and produce reactive oxygen species (ROS) (3). ROS including superoxide anion (0 2-), hydrogen peroxide (H 202), hydroxyl radical (OH.), and singlet oxygen (02) playa key role in the pathogenesis of neutrophilmediated acute lung injury characteristic of ARDS (4-6). Many studies have shown that ROS have been implicated in various kinds of tissue injury. Some of the agents used to attenuate ROS-mediated tissue injury include superoxide dismutase (SOD) (7-9), catalase (10), N-acetylcysteine (11), and dimethylthiourea (12). SOD (molecular weight 32,600 D) is a specific enzymethat catalyzes the dismutation of superoxide anions to hydrogen peroxide (13) and is an essential enzyme for survival in an oxygenate atmosphere. The use of SOD is limited by its short circulating half-life (5 to 6 min) due to renal clearance (14, 15), limited cell membrane permeability (16), and its predisposition to elicitingimmunogenic and antigenic responses in humans (IS). Derivatives of sao have been developed by conjugation with polyethylene glycol (PEG-SOD) (17). The methoxypolyethylene glycols (molecular weight 5,000 D) with the general structure CH3(OCH2CH2)1140H are linear, uncharged, and water soluble by virtue of hydrogen bonding of three water molecules per ethylene oxide unit. PEG by itself is a surface-active molecule and has been used in molecular biology to promote cell fusion. PEG-SOD as compared with SOD has different physical and chemical properties including (1) extended blood circulating half-life (14, 17, 18), 388
SUMMARY Reactive oxygen species (ROS), Including superoxlde anions, play an Important role In mediating ecute lung Injury. We examined whether polyethylene glycol-conJugated superoxlde dlsmutase (PEG-SOD) attenuatas lung InJury In Escher/chl. coli-treated guinea pigs. 1Wenty-four guinea pigs were divided Into four groups: (1) control group; (2) septic group, In which live E. coli (2 )( 10'/kg) were Injected Intravenously; (3) pretreatment group, In which PEG-SOD (2,000 IU/kg) was Injected Intravenously 15 min before E. coli; and (4) posttreatment group, In which PEG-SOD (2,000 IU/kg) was Injected Intravenously 30 min after E. coli. Lung InJury wes aaaessed by the concentration ratio of "'I_labeled albumin In lung tlaaue and bronchoalveolar lavage (BAl) fluid relative to plasma (LIP and BALlP), lung wet-to-dry walght ratio, and the number of neutrophlls In BAl fluid. Plasma half-lite of PEG-SOD In normal guinea pigs was 13.5 h. LIp, lung wet-to-dry weight ratio, and the number of neutrophlls In BAl fluid decreased In both pretreatment and posttreatment groups compared with the septic group. BALIP decreased In the pretreatment group but not In the posttreatment group compared with the septic group. After the animal model stUdies, wa Investigated the effect of PEG-SOD on the human neutrophil extracellular generation of ROS stimulated by phorbol myrlstate acetate (PMA) In luclgenln-dependent chemllumlneecence (Cl). PEG·SOD at concentrations ~ 0.1 U/mllnhlblted PMA-Induced Cl In a dose-dependent manner. We also examined the effect of PEG-SOD on the neutrophil Intracellular generation of ROS using flow cytometry to a_ss Intracellular hydroethldlne oxidation. PEG-SOD at concentrations of 10 and 100 U/ml reduced the fluorescence caused by hydroethldlne oxidation In neutrophlls stimulated by PMA. These resulta suggest that (1) pretreatment and posttreatment with PEG·SODetlanuatad septic lung InJuryIn guinea pigs; (2) PEG-SOD Inhibited the extracellular generation of ROS In PMA-stlmulated neutrophlls; and (3) PEG-SOD reduced the Intracellular generation of ROS In PMA-stlmulated neutrophlls. AM REV RESPIR DIS 1992; 145:388-393
(2) decreased immunogenicity (19, 20), (3) greater uptake by endothelial cells
resulting in increased intracellular SOD activity (21,22), and (4) no inhibitory effect on neutrophil bactericidal activity in vitro and bacterial clearance in vivo (23). PEG-SOD has been found to attenuate ROS-mediated tissue injury in a variety of non-pulmonary animal models including brain edema in cats (24), myocardial ischemia in dogs (25), caeruleininduced acute pancreatitis in rats (26), and muscle injury in mice (27). PEGSOD has also attenuated ROS-mediated lung injury in the following animal models: xanthine-xanthine oxidase-induced bronchoconstriction in cats (28), reexpansion pulmonary edema in rabbits (29), and norepinephrine-induced pulmonary edema in rats (30). However, PEG-SOD did not attenuate lung injury in endotoxin-treated pigs (31). Additionally, no investigators have studied the effect of PEG-SOD on sepsis-induced lung injury or neutrophil ROS generation
either by chemiluminescence or flow cytometry. The aim of this study was to investigate the effect of PEG-SOD on sepsis-induced lung injury using our guinea pig model (32, 33) and to gain further insight into the mechanisms of action of PEG-SOD. We administered PEG-SOD to Escherichia coli-treated guinea pigs to investigate whether pretreatment and posttreatment with PEG-SOD could attenu-
(Received in original form April 30, 1991 and in revised form September 5,: 199J) I From the Departments of Medicine, Clinical Laboratory, and Pathology, Stanford University Medical Center, Stanford, California. • Supported by the Paul S. Lam Fund, the Samuel T. ReevesFund, and by Grant No. ROIHL-4S53301 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Thomas A. Raffin, M.D., Chief, Division of Pulmonary and Critical Care Medicine, Room H3149,Stanford University Medical Center, Stanford, CA 94305-5236.
389
PEG-80D ATTENUATES SEPSIS-INDUCED WNG INJURY
ate septic lung injury. Additionally, we examined the effect of PEG-SOD on human neutrophils, using chemiluminescence to assess extracellular generation of ROS and flow eytometry to assess intracellular generation of ROS. Methods
Preparation of Animals Specific pathogen-free guinea pigs (body weight 600 to 700 g, viral antigen free/caesarean obtained barrier sustained; Charles River Breeding Laboratories, Wilmington, MA) were used. The animals were anesthetized with 100 mg/kg of ketamine (Ketalar; Parke-Davis, Morris Plains, NJ), 5 mg/kg of xylazine (Rompun; Mobay Corporation, Shawnee, KS), and 0.5 mg/kg of acepromazine (TechAmerica Group, Inc., Elwood, KS) injected intramuscularly. Through a middle neck incision, a plastic catheter from a 21-g intermittent infusion set (Abbott Hospital, Inc., North Chicago, IL) was inserted into the right jugular vein, and an 18.5-g catheter (Delmed Inc., New Brunswick, NJ) was inserted into the common carotid artery. The animals recovered for 18 to 24 h before the experiment. All animal protocols were approved by the Laboratory Animal Care Panel of Stanford University. Preparation of E. coli and PEG-SOD The bacterial strain E. coli J96 (serotype 04:K6:H +), a human bacteremic-pyelonephritic isolate that has been used in our laboratory, was used (32-34). This bacteria was cultured on trypticase soy agar for 18 hand harvested into normal saline. The bacterial concentration of the inoculum was titrated by adjusting its absorbance at 600 nm using a spectrophotometer (Ultrospec II; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). PEG-SOD (Sigma Chemical Co., St. Louis, MO) was diluted with phosphate-buffered saline before use. In order to exclude the nonspecific protein scavenging of oxygen radicals, the control and septic groups were treated with the equivalent amount of PEG-SOD that had been inactivated by boiling at 100° C for 60 min (14)and sterilized by filtration (MiIlipore Corp., Bedford, MA). Preparation of Radioisotope-labeled Tracers To assess pulmonary permeability, we injected 125I-a1bumin and determined the concentration of 125I-albuminin lung tissue and in BAL fluid relative to that in plasma. 1251_ albumin was prepared from guinea pig albumin (Sigma) and iodine-125(lCN Radiochemicals, Irvine, CA) as previously described (32-34). The 125I-albuminbinding efficiency measured by thin-layer chromatography (DCPlastikifolien Kieselgel 60F254; EM Science, Cherry Hill, NJ) was 93070 or more. Erythrocytes were labeled with SlCr as previously described (32-34).
Experimental Protocol Circulating half-life ofPEG-SOD in normal guineapigs. To determine the circulating halflife of PEG-SOD, five guinea pigs received PEG-SOD (2,000 IU/kg) intravenously beginning at -15 min overa 6O-minperiod using an infusion pump (Harvard Apparatus, South Natick, MA). Normal saline (25 ml/kg) was administered intravenously to all animals hourly. Whole blood (1 ml) was drawn via the carotid catheter and put into heparinized tubes (Becton Dickinson Co., Rutherford, NJ) at -15 and 45 min, and 2, 4, 6, and 8 h, and the plasma was stored at - 20° C. The SOD activity in plasma was measured using the method of McCord and Fridovich (13). The mean value of triplicate determination was used to obtain plasma SOD activity in each animal. Circulating half-life was calculated from single linear regression between 2 and 8 h, when the mixing of PEG-SOD in blood was thought to be complete. Effect ofPEG-SOD on septic lung injury. Twenty-four guinea pigs were randomly divided into four groups (n = 6 in each group): a control group, a septic group, a pretreatment group, and a posttreatment group. E. coli (2 x 109/kg) in 2 ml of normal saline was injected intravenously into the septic, pretreatment, and posttreatment groups at time zero. The control group and the septic group received inactivated PEG-SOD intravenously beginning at -15 min over a 60-min period. The pretreatment and the posttreatment groups received PEG-SOD (2,000 IU/ kg) 15 min before and 30 min after E. coli injection over a 60-min period, respectively. Mean arterial blood pressure and heart rate were measured hourly through the carotid catheter connected to a pressure transducer (HP 78304A pressure recorder; Hewlett-Packard, Palo Alto, CA). Totreat hypotension and dehydration, 25 ml/kg of normal saline was administered intravenously to all animals hourly. At time 5 h, 5 IlCi of 125I-albumin were injected via the venous catheter. At 7 h, 2 IlCi of SlCr-labeled erythrocytes in 2 ml of whole blood wereinjected via the venous catheter. At 8 h, the animals were killed with 2 ml of pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL) followed by 6 mEq of KCI intravenously. The chest was opened, and each pulmonary hilus was double-clamped and transected. Six pieces of lung tissue, including bilateral middle lobes and lateral and medial bilateral caudal lobes, were placed in each preweighed glass tube for determination of radioactivity and water content. White Blood Cell (WBe) and Differential Counts WBC counts were determined using a cell counter (Cell-Dyn 610Hematology Analyzer; Sequoia Turner Corp., Mountain View,CA). Differential counts were performed on 200 cells from blood smears stained with a modified Wright's stain (Diff-Quik Stain Set; American Scientific Products, McGaw Park, IL).
Lung Water Measurement Lung tissue samples were dried in a vacuum oven (Model 5831; Napco Scientific Company, Tualatin, OR) at 65° C and 20 mm Hg for 48 h. The dry tissue weight was determined, and lung wet-to-dry weight ratios were calculated to assess pulmonary edema. The mean value of six lung tissue samples was used to obtain the lung wet-to-dry weight ratio in each animal. Bronchoalveolar Lavage (BAL) A BAL wasperformed on the left cranial lobe. The lobe was lavaged with two 5-ml aliquots of saline plus 1 ml of air and then rinsed with 1ml of saline. The BAL fluid was centrifuged at 400 x g and 4° C for 10 min. The cell pellet was resuspended in I ml of saline, and the total cell count was determined using a modified hemacytometer method (Unopette Microcollection System; Becton Dickinson). Differential counts were performed on 200 cells from BAL fluid smears stained with a Diff-Quik stain. The number of neutrophils in BAL fluid was determined to assess neutrophil infiltration into alveolar spaces and lung tissue. Permeability Measurements Transvascular flux of 125I-albumin into lung tissue and alveolar spaces was assessed using the concentration ratio of '2sl-albumin in lung tissue and in BAL fluid to plasma (LIP and BALlP). The 1251 and s'Cr activity of lung tissue, BAL fluid, and blood weredetermined in a gamma counter (Gamma 300 Radiation Counter; Beckman Instruments, Inc., Irvine, CA) with appropriate corrections for crossover. Blood contamination was determined by counting the s'Cr-erythrocyte activity of lung tissue, BAL fluid, and blood, and this contamination was subtracted (33, 34). The mean value of six lung tissue samples wasused to obtain the L/P in each animal. Preparation of Human Neutrophils Human peripheral blood neutrophils for the chemiluminescence assay and flow cytometric assay were obtained from healthy adult volunteers. Neutrophils were separated on a discontinuous gradient consisting of Histopaque 1077 and 1119 (Sigma) (35). The contaminating erythrocytes were removed with 0.83% ammonium chloride. Neutrophils were resuspended to 5 x 106/ml in Hanks' balanced salt solution (HBSS) without phenol red (Sigma). Greater than 99% of the cells obtained were neutrophils. The viability of cells by trypan blue exclusion was routinely ~ 98070. Chemiluminescence Assay (CL) Samples for CL were obtained by adding 100 IIIof neutrophil suspension (5 x lOS) to 700 III of 25 IlM lucigenin (bis-N-methylacridinium nitrate; Sigma) in HBSS. Then, 100 III of PEG-SOD or heat-inactivated PEGSOD were added and incubated for 15 min at 37° C in a dark room. CL was measured beginning with the addition of PMA (100 ng/ml). CL was monitored every 5 min up
390
SUZUKI, TANIGAKI, HEIMER, WANG, ROSS, SUSSMAN, AND RAFFIN
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to 30min with a chemiluminometer (1251 Luminometer; LKB Wallac, Turku, Finland) in a dark room. All experimentswereperformed in quadruplicate, and results were expressed as percentage of response compared with the control (0/0 control).
SOD or inactivated PEG-SOD for 15min at 37° C, and further incubated with 100 ul of 160 ItM hydroethidine (Polysciences, Inc., Warrington, PA)in N,N-dimethylformamide (Sigma)for 15min at 37°C. The sampleswere incubated with phorbol myristate acetate (PMA) (100 ng/ml) for 15min at 37° C. The fluorescence and light-scattering properties of the cells weredetermined by using a FACScan flow cytometry system equipped with an argon laser (488 nm emission, 15mWoutput; Becton Dickinson Co., San Jose, CA). Parameters collectedincluded forward-angle light scatter (FSC), 90° light scatter (SSC), and ethidium bromide (EB)red fluorescence. EB red fluorescence was collected between564 and 606 nm using a bandpass filter. A total of 3,000 cells in each sample was examined. The list mode was evaluated by the Consort 30 program system (Becton Dickinson). All experiments wereperformed in quadruplicate, and the results wereexpressed as percentage of intensity of fluorescence compared with the control (% control).
Statistical Analysis All data werepresented as mean ± standard errors of the mean (SEM). One-way analysis of variance (ANOVA) and Fisher's PLSD test Flow Cytometric Assay were used to test for statistically significant Neutrophil suspensions (5 x 105 ) were in- differences between groups (StatViewII; cubated alone or with 100 ul of either PEG- . BrainPower, Inc, Calabasas, CA). A value
Fig. 2. Changes in mean arterial blood pressure in the control group (open circles). septic group (closed circles). pretreatment (triangles). and posttreatment (squares)groups. Values are mean ± SEM (n = e in each group). At time o h. E. coli (2 x 100/kg) was injected intravenously into the septic. pretreatment, and posttreatment groups. PEGSOD was administered 15 min before (pretreatment group) and 30 min after (posttreatment group) E. coli over a eomin period, respectively.
2
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8 6 4 2 o+---,----,---"-,--------,~__,-_.-_,_--,
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of p < 0.05 was used to determine significant differences between means. Results
Circulating Half-life of PEG-SOD in Normal Guinea Pigs The changes of plasma SOD activity are shown in figure l. SOD activity in plasma was 40.9 ± 3.4 IU/ml at 45 min and 27.6 ± 2.8 IVlml at 8 h. The circulating half-life of PEG-SOD from single linear regression analysis was 13.5 h (y = 38.6 - l.43x).
Effect of PEG-SOD on Septic Lung Injury in Guinea Pigs Changes in mean arterial blood pressure during the experiment in each group are shown in figure 2. The mean arterial blood pressure in the septic group fell at 1 and 2 h after E. coli injection, and then returned to the baseline. The trends of mean arterial blood pressure in both pretreatment and posttreatment groups did not change from that in the septic group. Changes in peripheral blood WBC counts are shown in figure 3. The WBC counts decreased 1 h after E. coli injection in septic, pretreatment, and posttreatment groups, and the leukopenia persisted throughout the experiment in these three groups. There were no significant changes among the septic, pretreatment, and posttreatment groups. The LIP is shown in figure 4. LIP increased in the septic (0.29 ± 0.04) and posttreatment groups (0.14 ± 0.05) compared with the control group (0.02 ± 0.01). LIP decreased in both pretreatment (0.03 ± 0.02) and posttreatment groups compared with the septic group. The lung wetto-dry weight ratio is shown in figure 5. The lung wet-to-dry weight ratio increased in the septic group (6.6 ± 0.3) compared with the control group (4.8 ± 0.1) and decreased in both pretreatment (5.3 ± 0.2) and posttreatment groups (5.4 ± 0.5) compared with the septic group. BALIP is shown in figure 6. BALIP increased in the septic group (0.021 ± 0.002) compared with the control group (0.010 ± 0.002). BALIP in the pretreatment group (0.012 ± 0.po2) but not the posttreatment group (0.015 ± 0.004) decreased compared with the septic group. The number of neutrophils in BAL fluid is shown in figure 7. The number of neutrophils in BAL fluid increased in the septic group (1,715 ± 579/mm 3 ) compared with the control group (100 ± 26/mm3 ) and decreased in both pretreatment (231 ± 53/mm 3 ) and posttreatment groups (572 ± 141/mm3 ) compared with the septic group.
391
PEQ.SOD ATTENUATES SEPSIS-INDUCED UlNQ INJURY
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Fig, 4. Effectof pretreatmentand posttreatmentwithPEG-SOD on 12"I-albumin lung-to-plasma ratios (LJP). Valuesare mean ± SEM (n = 6 in each group). Asterisk indicates p < 0.05 compared with the controlgroup;daggerindicates p < 0.05compared withthesepticgroup.
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YUKIO SUZUKI, TOSHIMORI TANIGAKI, DOV HEIMER, WEIZHENG WANG, WILLIAM G. ROSS, HOWARD H. SUSSMAN, and THOMAS A. RAFFIN
Introduction
Sepsis inducedby gram-negative bacteria is one of the most common predisposing factors for the development of the adult respiratory distress syndrome (ARDS) (I, 2). Gram-negative bacteria, in part through endotoxin and tumor necrosis factor, activate neutrophils to increaseoxygen consumption and produce reactive oxygen species (ROS) (3). ROS including superoxide anion (0 2-), hydrogen peroxide (H 202), hydroxyl radical (OH.), and singlet oxygen (02) playa key role in the pathogenesis of neutrophilmediated acute lung injury characteristic of ARDS (4-6). Many studies have shown that ROS have been implicated in various kinds of tissue injury. Some of the agents used to attenuate ROS-mediated tissue injury include superoxide dismutase (SOD) (7-9), catalase (10), N-acetylcysteine (11), and dimethylthiourea (12). SOD (molecular weight 32,600 D) is a specific enzymethat catalyzes the dismutation of superoxide anions to hydrogen peroxide (13) and is an essential enzyme for survival in an oxygenate atmosphere. The use of SOD is limited by its short circulating half-life (5 to 6 min) due to renal clearance (14, 15), limited cell membrane permeability (16), and its predisposition to elicitingimmunogenic and antigenic responses in humans (IS). Derivatives of sao have been developed by conjugation with polyethylene glycol (PEG-SOD) (17). The methoxypolyethylene glycols (molecular weight 5,000 D) with the general structure CH3(OCH2CH2)1140H are linear, uncharged, and water soluble by virtue of hydrogen bonding of three water molecules per ethylene oxide unit. PEG by itself is a surface-active molecule and has been used in molecular biology to promote cell fusion. PEG-SOD as compared with SOD has different physical and chemical properties including (1) extended blood circulating half-life (14, 17, 18), 388
SUMMARY Reactive oxygen species (ROS), Including superoxlde anions, play an Important role In mediating ecute lung Injury. We examined whether polyethylene glycol-conJugated superoxlde dlsmutase (PEG-SOD) attenuatas lung InJury In Escher/chl. coli-treated guinea pigs. 1Wenty-four guinea pigs were divided Into four groups: (1) control group; (2) septic group, In which live E. coli (2 )( 10'/kg) were Injected Intravenously; (3) pretreatment group, In which PEG-SOD (2,000 IU/kg) was Injected Intravenously 15 min before E. coli; and (4) posttreatment group, In which PEG-SOD (2,000 IU/kg) was Injected Intravenously 30 min after E. coli. Lung InJury wes aaaessed by the concentration ratio of "'I_labeled albumin In lung tlaaue and bronchoalveolar lavage (BAl) fluid relative to plasma (LIP and BALlP), lung wet-to-dry walght ratio, and the number of neutrophlls In BAl fluid. Plasma half-lite of PEG-SOD In normal guinea pigs was 13.5 h. LIp, lung wet-to-dry weight ratio, and the number of neutrophlls In BAl fluid decreased In both pretreatment and posttreatment groups compared with the septic group. BALIP decreased In the pretreatment group but not In the posttreatment group compared with the septic group. After the animal model stUdies, wa Investigated the effect of PEG-SOD on the human neutrophil extracellular generation of ROS stimulated by phorbol myrlstate acetate (PMA) In luclgenln-dependent chemllumlneecence (Cl). PEG·SOD at concentrations ~ 0.1 U/mllnhlblted PMA-Induced Cl In a dose-dependent manner. We also examined the effect of PEG-SOD on the neutrophil Intracellular generation of ROS using flow cytometry to a_ss Intracellular hydroethldlne oxidation. PEG-SOD at concentrations of 10 and 100 U/ml reduced the fluorescence caused by hydroethldlne oxidation In neutrophlls stimulated by PMA. These resulta suggest that (1) pretreatment and posttreatment with PEG·SODetlanuatad septic lung InJuryIn guinea pigs; (2) PEG-SOD Inhibited the extracellular generation of ROS In PMA-stlmulated neutrophlls; and (3) PEG-SOD reduced the Intracellular generation of ROS In PMA-stlmulated neutrophlls. AM REV RESPIR DIS 1992; 145:388-393
(2) decreased immunogenicity (19, 20), (3) greater uptake by endothelial cells
resulting in increased intracellular SOD activity (21,22), and (4) no inhibitory effect on neutrophil bactericidal activity in vitro and bacterial clearance in vivo (23). PEG-SOD has been found to attenuate ROS-mediated tissue injury in a variety of non-pulmonary animal models including brain edema in cats (24), myocardial ischemia in dogs (25), caeruleininduced acute pancreatitis in rats (26), and muscle injury in mice (27). PEGSOD has also attenuated ROS-mediated lung injury in the following animal models: xanthine-xanthine oxidase-induced bronchoconstriction in cats (28), reexpansion pulmonary edema in rabbits (29), and norepinephrine-induced pulmonary edema in rats (30). However, PEG-SOD did not attenuate lung injury in endotoxin-treated pigs (31). Additionally, no investigators have studied the effect of PEG-SOD on sepsis-induced lung injury or neutrophil ROS generation
either by chemiluminescence or flow cytometry. The aim of this study was to investigate the effect of PEG-SOD on sepsis-induced lung injury using our guinea pig model (32, 33) and to gain further insight into the mechanisms of action of PEG-SOD. We administered PEG-SOD to Escherichia coli-treated guinea pigs to investigate whether pretreatment and posttreatment with PEG-SOD could attenu-
(Received in original form April 30, 1991 and in revised form September 5,: 199J) I From the Departments of Medicine, Clinical Laboratory, and Pathology, Stanford University Medical Center, Stanford, California. • Supported by the Paul S. Lam Fund, the Samuel T. ReevesFund, and by Grant No. ROIHL-4S53301 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Thomas A. Raffin, M.D., Chief, Division of Pulmonary and Critical Care Medicine, Room H3149,Stanford University Medical Center, Stanford, CA 94305-5236.
389
PEG-80D ATTENUATES SEPSIS-INDUCED WNG INJURY
ate septic lung injury. Additionally, we examined the effect of PEG-SOD on human neutrophils, using chemiluminescence to assess extracellular generation of ROS and flow eytometry to assess intracellular generation of ROS. Methods
Preparation of Animals Specific pathogen-free guinea pigs (body weight 600 to 700 g, viral antigen free/caesarean obtained barrier sustained; Charles River Breeding Laboratories, Wilmington, MA) were used. The animals were anesthetized with 100 mg/kg of ketamine (Ketalar; Parke-Davis, Morris Plains, NJ), 5 mg/kg of xylazine (Rompun; Mobay Corporation, Shawnee, KS), and 0.5 mg/kg of acepromazine (TechAmerica Group, Inc., Elwood, KS) injected intramuscularly. Through a middle neck incision, a plastic catheter from a 21-g intermittent infusion set (Abbott Hospital, Inc., North Chicago, IL) was inserted into the right jugular vein, and an 18.5-g catheter (Delmed Inc., New Brunswick, NJ) was inserted into the common carotid artery. The animals recovered for 18 to 24 h before the experiment. All animal protocols were approved by the Laboratory Animal Care Panel of Stanford University. Preparation of E. coli and PEG-SOD The bacterial strain E. coli J96 (serotype 04:K6:H +), a human bacteremic-pyelonephritic isolate that has been used in our laboratory, was used (32-34). This bacteria was cultured on trypticase soy agar for 18 hand harvested into normal saline. The bacterial concentration of the inoculum was titrated by adjusting its absorbance at 600 nm using a spectrophotometer (Ultrospec II; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). PEG-SOD (Sigma Chemical Co., St. Louis, MO) was diluted with phosphate-buffered saline before use. In order to exclude the nonspecific protein scavenging of oxygen radicals, the control and septic groups were treated with the equivalent amount of PEG-SOD that had been inactivated by boiling at 100° C for 60 min (14)and sterilized by filtration (MiIlipore Corp., Bedford, MA). Preparation of Radioisotope-labeled Tracers To assess pulmonary permeability, we injected 125I-a1bumin and determined the concentration of 125I-albuminin lung tissue and in BAL fluid relative to that in plasma. 1251_ albumin was prepared from guinea pig albumin (Sigma) and iodine-125(lCN Radiochemicals, Irvine, CA) as previously described (32-34). The 125I-albuminbinding efficiency measured by thin-layer chromatography (DCPlastikifolien Kieselgel 60F254; EM Science, Cherry Hill, NJ) was 93070 or more. Erythrocytes were labeled with SlCr as previously described (32-34).
Experimental Protocol Circulating half-life ofPEG-SOD in normal guineapigs. To determine the circulating halflife of PEG-SOD, five guinea pigs received PEG-SOD (2,000 IU/kg) intravenously beginning at -15 min overa 6O-minperiod using an infusion pump (Harvard Apparatus, South Natick, MA). Normal saline (25 ml/kg) was administered intravenously to all animals hourly. Whole blood (1 ml) was drawn via the carotid catheter and put into heparinized tubes (Becton Dickinson Co., Rutherford, NJ) at -15 and 45 min, and 2, 4, 6, and 8 h, and the plasma was stored at - 20° C. The SOD activity in plasma was measured using the method of McCord and Fridovich (13). The mean value of triplicate determination was used to obtain plasma SOD activity in each animal. Circulating half-life was calculated from single linear regression between 2 and 8 h, when the mixing of PEG-SOD in blood was thought to be complete. Effect ofPEG-SOD on septic lung injury. Twenty-four guinea pigs were randomly divided into four groups (n = 6 in each group): a control group, a septic group, a pretreatment group, and a posttreatment group. E. coli (2 x 109/kg) in 2 ml of normal saline was injected intravenously into the septic, pretreatment, and posttreatment groups at time zero. The control group and the septic group received inactivated PEG-SOD intravenously beginning at -15 min over a 60-min period. The pretreatment and the posttreatment groups received PEG-SOD (2,000 IU/ kg) 15 min before and 30 min after E. coli injection over a 60-min period, respectively. Mean arterial blood pressure and heart rate were measured hourly through the carotid catheter connected to a pressure transducer (HP 78304A pressure recorder; Hewlett-Packard, Palo Alto, CA). Totreat hypotension and dehydration, 25 ml/kg of normal saline was administered intravenously to all animals hourly. At time 5 h, 5 IlCi of 125I-albumin were injected via the venous catheter. At 7 h, 2 IlCi of SlCr-labeled erythrocytes in 2 ml of whole blood wereinjected via the venous catheter. At 8 h, the animals were killed with 2 ml of pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL) followed by 6 mEq of KCI intravenously. The chest was opened, and each pulmonary hilus was double-clamped and transected. Six pieces of lung tissue, including bilateral middle lobes and lateral and medial bilateral caudal lobes, were placed in each preweighed glass tube for determination of radioactivity and water content. White Blood Cell (WBe) and Differential Counts WBC counts were determined using a cell counter (Cell-Dyn 610Hematology Analyzer; Sequoia Turner Corp., Mountain View,CA). Differential counts were performed on 200 cells from blood smears stained with a modified Wright's stain (Diff-Quik Stain Set; American Scientific Products, McGaw Park, IL).
Lung Water Measurement Lung tissue samples were dried in a vacuum oven (Model 5831; Napco Scientific Company, Tualatin, OR) at 65° C and 20 mm Hg for 48 h. The dry tissue weight was determined, and lung wet-to-dry weight ratios were calculated to assess pulmonary edema. The mean value of six lung tissue samples was used to obtain the lung wet-to-dry weight ratio in each animal. Bronchoalveolar Lavage (BAL) A BAL wasperformed on the left cranial lobe. The lobe was lavaged with two 5-ml aliquots of saline plus 1 ml of air and then rinsed with 1ml of saline. The BAL fluid was centrifuged at 400 x g and 4° C for 10 min. The cell pellet was resuspended in I ml of saline, and the total cell count was determined using a modified hemacytometer method (Unopette Microcollection System; Becton Dickinson). Differential counts were performed on 200 cells from BAL fluid smears stained with a Diff-Quik stain. The number of neutrophils in BAL fluid was determined to assess neutrophil infiltration into alveolar spaces and lung tissue. Permeability Measurements Transvascular flux of 125I-albumin into lung tissue and alveolar spaces was assessed using the concentration ratio of '2sl-albumin in lung tissue and in BAL fluid to plasma (LIP and BALlP). The 1251 and s'Cr activity of lung tissue, BAL fluid, and blood weredetermined in a gamma counter (Gamma 300 Radiation Counter; Beckman Instruments, Inc., Irvine, CA) with appropriate corrections for crossover. Blood contamination was determined by counting the s'Cr-erythrocyte activity of lung tissue, BAL fluid, and blood, and this contamination was subtracted (33, 34). The mean value of six lung tissue samples wasused to obtain the L/P in each animal. Preparation of Human Neutrophils Human peripheral blood neutrophils for the chemiluminescence assay and flow cytometric assay were obtained from healthy adult volunteers. Neutrophils were separated on a discontinuous gradient consisting of Histopaque 1077 and 1119 (Sigma) (35). The contaminating erythrocytes were removed with 0.83% ammonium chloride. Neutrophils were resuspended to 5 x 106/ml in Hanks' balanced salt solution (HBSS) without phenol red (Sigma). Greater than 99% of the cells obtained were neutrophils. The viability of cells by trypan blue exclusion was routinely ~ 98070. Chemiluminescence Assay (CL) Samples for CL were obtained by adding 100 IIIof neutrophil suspension (5 x lOS) to 700 III of 25 IlM lucigenin (bis-N-methylacridinium nitrate; Sigma) in HBSS. Then, 100 III of PEG-SOD or heat-inactivated PEGSOD were added and incubated for 15 min at 37° C in a dark room. CL was measured beginning with the addition of PMA (100 ng/ml). CL was monitored every 5 min up
390
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to 30min with a chemiluminometer (1251 Luminometer; LKB Wallac, Turku, Finland) in a dark room. All experimentswereperformed in quadruplicate, and results were expressed as percentage of response compared with the control (0/0 control).
SOD or inactivated PEG-SOD for 15min at 37° C, and further incubated with 100 ul of 160 ItM hydroethidine (Polysciences, Inc., Warrington, PA)in N,N-dimethylformamide (Sigma)for 15min at 37°C. The sampleswere incubated with phorbol myristate acetate (PMA) (100 ng/ml) for 15min at 37° C. The fluorescence and light-scattering properties of the cells weredetermined by using a FACScan flow cytometry system equipped with an argon laser (488 nm emission, 15mWoutput; Becton Dickinson Co., San Jose, CA). Parameters collectedincluded forward-angle light scatter (FSC), 90° light scatter (SSC), and ethidium bromide (EB)red fluorescence. EB red fluorescence was collected between564 and 606 nm using a bandpass filter. A total of 3,000 cells in each sample was examined. The list mode was evaluated by the Consort 30 program system (Becton Dickinson). All experiments wereperformed in quadruplicate, and the results wereexpressed as percentage of intensity of fluorescence compared with the control (% control).
Statistical Analysis All data werepresented as mean ± standard errors of the mean (SEM). One-way analysis of variance (ANOVA) and Fisher's PLSD test Flow Cytometric Assay were used to test for statistically significant Neutrophil suspensions (5 x 105 ) were in- differences between groups (StatViewII; cubated alone or with 100 ul of either PEG- . BrainPower, Inc, Calabasas, CA). A value
Fig. 2. Changes in mean arterial blood pressure in the control group (open circles). septic group (closed circles). pretreatment (triangles). and posttreatment (squares)groups. Values are mean ± SEM (n = e in each group). At time o h. E. coli (2 x 100/kg) was injected intravenously into the septic. pretreatment, and posttreatment groups. PEGSOD was administered 15 min before (pretreatment group) and 30 min after (posttreatment group) E. coli over a eomin period, respectively.
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Circulating Half-life of PEG-SOD in Normal Guinea Pigs The changes of plasma SOD activity are shown in figure l. SOD activity in plasma was 40.9 ± 3.4 IU/ml at 45 min and 27.6 ± 2.8 IVlml at 8 h. The circulating half-life of PEG-SOD from single linear regression analysis was 13.5 h (y = 38.6 - l.43x).
Effect of PEG-SOD on Septic Lung Injury in Guinea Pigs Changes in mean arterial blood pressure during the experiment in each group are shown in figure 2. The mean arterial blood pressure in the septic group fell at 1 and 2 h after E. coli injection, and then returned to the baseline. The trends of mean arterial blood pressure in both pretreatment and posttreatment groups did not change from that in the septic group. Changes in peripheral blood WBC counts are shown in figure 3. The WBC counts decreased 1 h after E. coli injection in septic, pretreatment, and posttreatment groups, and the leukopenia persisted throughout the experiment in these three groups. There were no significant changes among the septic, pretreatment, and posttreatment groups. The LIP is shown in figure 4. LIP increased in the septic (0.29 ± 0.04) and posttreatment groups (0.14 ± 0.05) compared with the control group (0.02 ± 0.01). LIP decreased in both pretreatment (0.03 ± 0.02) and posttreatment groups compared with the septic group. The lung wetto-dry weight ratio is shown in figure 5. The lung wet-to-dry weight ratio increased in the septic group (6.6 ± 0.3) compared with the control group (4.8 ± 0.1) and decreased in both pretreatment (5.3 ± 0.2) and posttreatment groups (5.4 ± 0.5) compared with the septic group. BALIP is shown in figure 6. BALIP increased in the septic group (0.021 ± 0.002) compared with the control group (0.010 ± 0.002). BALIP in the pretreatment group (0.012 ± 0.po2) but not the posttreatment group (0.015 ± 0.004) decreased compared with the septic group. The number of neutrophils in BAL fluid is shown in figure 7. The number of neutrophils in BAL fluid increased in the septic group (1,715 ± 579/mm 3 ) compared with the control group (100 ± 26/mm3 ) and decreased in both pretreatment (231 ± 53/mm 3 ) and posttreatment groups (572 ± 141/mm3 ) compared with the septic group.
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