Kinetics of pulmonary in interleukin-I-induced MIN-FU
TSAN
AND JULIE
superoxide dismutase oxygen-tolerant rats E. WHITE
Research Service, Samuel S. Stratton Department of Veterans Affairs Medical Center, and Departments of Physiology and Medicine, Albany Medical College, Albany, New York 12208 Tsan, Min-Fu, and Julie E. White. Kinetics of pulmonary superoxide dismutase in interleukinl-induced oxygen-tolerant rats. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L342L347, 1992.-We have previously demonstrated that tracheal insufflation of interleukinla (IL- 1) enhances pulmonary Mn superoxide dismutase (Mn SOD) activity and protects rats against O2 toxicity (M. F. Tsan, C. Y. Lee, and J. E. White. J. Appl. Physiol. 71: 688-697, 1991). In this study, we investigated the kinetics of mRNA, specific (immunoreactive) proteins, and enzyme activities of pulmonary Mn SOD and Cu,Zn SOD in IL-l-induced O,-tolerant rats. At 1 day after IL-I (5 pg) insufflation and 0, exposure, levels of Mn SOD mRNA and specific protein, but not enzyme activity, were markedly elevated. At 2.3 and 7 days after O2 exposure, levels of Mn SOD mRNA, specific protein, and enzyme activity were all increased in IL-l-treated animals. In contrast, in control rats at 2.3 days after 0, exposure, level of Mn SOD mRNA was markedly elevated, whereas levels of specific protein and enzyme activity were decreased. Levels of pulmonary Cu,Zn SOD mRNA, specific protein, and enzyme activity were unchanged in control and IL-l-treated rats, except that in IL-l-induced long-term O,-tolerant rats (7 days after 0, exposure), they were all increased. Since at 7 days after IL-I insufflation, normoxia-exposed rats did not show increased levels of pulmonary Mn SOD or Cu,Zn SOD mRNA, the increased levels of pulmonary SOD seen in IL-l-induced long-term O,-tolerant rats are, at least in part, due to the effect of 0, exposure. superoxide dismutase mRNA; oxygen tolerance
(IL-l) and tumor necrosis factor (TNF) are multifunctional cytokines with widely overlapping activities involving immune response, inflammation, hematopoiesis, and tumoricidal effect (2, 6). Recent studies (20, 23, 26) have demonstrated that IL-l and TNF selectively induce Mn superoxide dismutase (Mn SOD) mRNA, leading to increased Mn SOD specific protein and enzyme activity without affecting the levels of Cu,Zn SOD, catalase, or glutathione (GSH) peroxidase. Since Mn SOD is strategically located in mitochondria, a major site of superoxide production under hyperoxic conditions (lo), enhancement of Mn SOD by IL-l or TNF may protect against O2 toxicity. We have previously demonstrated that although intraperitoneal administration of IL-l or TNF provides no protection against O2 toxicity, tracheal insufflation of a single dose (5 pg) of IL-l or TNF markedly prolongs the survival of rats exposed to 100% O2 (19, 21). These cytokine-treated rats are able to survive in an environment of 100% O2 for Xl days compared with control rats, which all die within 3 days of O2 exposure. These cytokine-induced, long-term, Oz-tolerant rats show no signs of respiratory distress and their lung histology reveals no evidence of pulmonary edema at 7 days of continuous O2 exposure. The protection against O2 toxicity by tracheal insufflation of IL-1 or TNF is associated with increased pulmonary SOD activities (19, 21,
INTERLEUKIN-1
22). To better understand the mechanism of increased pulmonary SOD activities in these cytokine-treated animals, in this study we investigated the kinetics of mRNA, specific proteins, and enzyme activities of pulmonary Mn SOD and Cu,Zn SOD in IL-l-induced 02tolerant rats. MATERIALS
AND METHODS
Materials. Recombinant human IL-la (IL-l) was obtained from Dr. Craig Reynolds of the Biological Response Modifiers Program, National Cancer Institute, Frederick, MD. The preparation had a specific activity of 3 x lo8 U/mg protein as measured by the DlO assay and contained 0.5 EU endotoxin/mg protein. Monospecific goat anti-rat Cu,Zn SOD antibody was a gift of Drs. Michael Hass and Donald Massaro, University of Miami, and anti-human Mn SOD antibody, which cross-reacts with rat Mn SOD, was obtained from Dr. Larry Oberley, University of Iowa. Mouse Mn and Cu,Zn SOD cDNA clones were kindly provided by Dr. Jacquelin B. Shaffer of the New York State Wadsworth Center for Laboratories and Research. Tracheal insufflation and exposure of rats to hyperoxia. These procedures were performed as described previously (19, 21). Briefly, male Sprague-Dawley rats (Harlan Sprague-Dawley, Altamont, NY) free of respiratory infections and weighing between 250 and 350 g, were anesthetized with methoxyflurane (Pitman-Moore, Washington Crossing, NJ) and intubated with a 16-gauge intravenous catheter (Angiocath, Becton-Dickinson, Sandy, UT). One milliliter of calcium-magnesium-free Hanks’ balanced salt solution (HBSS) containing 5 pg IL-l or 1 ml HBSS for control animals, followed by 2 ml of air was injected into the lungs through the intratracheal catheter. Auscultation of the chest was performed with a stethoscope to ensure that solution was insufflated into the lungs. After rats had recovered from the effect of anesthesia, they were placed in groups of five in a Lucite chamber (45 x 40 X 25 cm”) that was flushed with 100% O2 at 15 l/min for 15 min and then maintained at 3 l/min. The concentration of O2 in the chamber as monitored using an 0, analyzer (Hudson Oxygen Analyzer, Ventronics Products Division, Temecula, CA) was >99% at all times (21). The rats were given free access to water and diet. Control exposure (normoxia) was performed in room air. Measurement of pulmonary SOD enzyme activities and immunoreactive proteins. Preparation of lung extracts for enzyme assay and immunoblot analyses of SOD was performed as described previously (19, 22). At 1 day, 2.3 days (55 h), or 7 days after 0, exposure, rats were anesthetized with pentobarbital and ventilated using a Harvard rodent ventilator (Harvard Apparatus, South Natick, MA). After a midline thoracotomy, 150 U of heparin was given through intracardiac injection and animals were exsanguinated by transection of abdominal aorta. The main pulmonary artery was quickly cannulated and the left atrium was cut open. Lungs were flushed with 20 ml HBSS containing 4% Ficoll 70 followed by 5 ml potassium phosphate buffer (0.1 M, pH 7.4) containing 0.05 M KC1 using a peristaltic roller pump (Harvard Apparatus). Lungs were removed, trimmed, rinsed, blotted, and weighed. The right lungs were homogenized with a tissue homogenizer (Tissumizer, Tekmar,
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IL-l,
SOD
AND
OXYGEN
Cincinnati, OH) in 3 ml potassium phosphate buffer (0.005 M, pH 7.8) containing 0.1 mM EDTA. The homogenates were then twice sonicated on ice for I min using a sonic dismembrator (Fisher, Pittsburgh, PA) to disrupt mitochondria and intracellular granules, and the final volume was adjusted to IO ml. After centrifugation at 15,000 g for 10 min at 4°C the supernatant was dialyzed against homogenizing buffer for 16 h (12,000 mol wt cutoff, Spectrum Medical Industries, Los Angeles, CA) to remove low-molecularweight compounds that might nonspecifically reduce ferrichrome c and was then assayed for SOD activities. Left lungs were homogenized, sonicated, and extensively washed in perchloric acid (0.5 N) at 4” C; the precipitate was extracted in 10 ml 1.5 N perchloric acid by boiling for 20 min and was assayed for DNA contents. The SOD activity was assayed according to McCord and Fridovich (14) on the basis of the inhibitory effect of SOD on ferricytochrome c reduction by superoxide generated by the action of xanthine oxidase on xanthine. The total SOD activity was assayed in the presence of 10 PM KCN to inhibit cytochrome oxidase, whereas Mn SOD activity was assayed in the presence of 1 mM KCN to inhibit Cu,Zn SOD (15). The difference between the total SOD activity and Mn SOD activity was taken to be the Cu,Zn SOD activity. Protein contents were assayed using bicinchoninic acid according to Smith et al. (17). Measurement of DNA contents was made using the diphenylamine reaction according to the method of Richards (16). For immunoblot analysis, lung extracts (100 pg protein/lane for Mn SOD and 12.5 pug protein/lane for Cu,Zn SOD) were electrophoresed in denaturing sodium dodecyl sulfate (SDS) polyacrylamide (12%) gels according to Laemmli (13) and transferred electrophoretically to nitrocellulose membranes in a BioRad Transblot cell (Bio-Rad, Richmond, CA). After sample transfer was completed, membranes were blocked for 2 h in phosphate-buffered saline (PBS) containing 10% nonfat dry milk and 0.1% Tween 20 (BLOTTO). They were then incubated for 21 h with primary antibodies, which had been diluted in BLOTTO (anti-Cu,Zn SOD, I:400 and anti-Mn SOD, 1:lOOO). Membrane were then washed for 20 min in PBS containing 0.1% Tween (PBST) and incubated for 2 h in alkaline phosphatase-conjugated secondary antibodies (Boehringer-Mannhein, diluted 1:5000 in BLOTTO) that were directed against the immunoglobin G (IgG) fraction of the species that produced the primary antibody. At the end of the incubation, membranes were washed for 30 min in PBST and were equilibrated in 0.5 M tris(hydroxymethyl)aminomethane (Tris) (pH 9.5) for 5 min. To detect regions of bound antigen/primary antibody/alkaline phosphatase-conjugated secondary antibody on the nitrocellulose, membranes were stained for alkaline phosphatase activity using a detection kit purchased from Kirkegaard and Perry Lab., Gaithersburg, MD. Areas of alkaline phosphatase stain on individual blots were quantified using a computing densitometer (Molecular Dynamics, Sunnyvale, CA). Quantification of pulmonary SOD mRNA. This was performed as described previously (22). At 1, 2.3, or 7 days after hyperoxic or normoxic exposure, rats were anesthetized with pentobarbital and exsanguinated by transection of abdominal aorta. Lungs were quickly removed, trimmed, rinsed, blotted, and weighed. One gram of lung tissue was extracted for RNA by the acid quanidinium thiocyanate-phenol-chloroform method of Chromczynski and Sacchi (3). The remaining lung tissue was homogenized, extracted, and assayed for DNA contents as in Measurement of pulmonary SOD enzyme activities and immunoreactive proteins. For Northern blots, formamide-formaldehyde denatured RNA samples (15 or 25 pg total cellular RNA/lane) were electrophoresed in 1.2% agarose gels containing 2.2 M formaldehyde and stained with ethidium bromide to visualize the quality and size of 18s and 28s ribosomal RNA species. They were then
L343
TOLERANCE
transferred to nylon membrane (Micron Separations, Westborough, MA) by capillary blotting in 20x standard sodium citrate (SSC) (3 M NaCl, 0.3 M trisodium citrate, pH 7.0). Blots were prehybridized for 4-16 h at SZ*C in 50% formamide, 2~ Denhardt’s solution (0.04% Ficoll 400, 0.04% bovine serum albumin, and 0.04% polyvinylpyrrolidone), 0.1% SDS, 5~ SSPE (0.75 N NaCl, 0.05 M NaH2P04 and 0.005 M EDTA, pH 7.4), and 100 pg/ml denatured salmon sperm DNA. Hybridization was carried out in the same solution for 18-24 h with the addition of cDNA probes that had been labeled with [““P]CTP by random hexanucleotide priming (BRL, Gaithersburg, MD) to a specific activity of ~10~ counts per minute (cpm)/pg DNA. Blots were washed twice with 2~ SSPE, 0.1% SDS at room temperature, and twice with 0.1X SSPE, 0.1% SDS at 55°C. Radioactive signals were visualized by exposure of hybridized blots to Kodak X-Omat AR film (Eastman-Kodak, Rochester, NY) at -70” C with intensifying screens. Autoradiographs were quantified using a computing densitometer. Statistical anaZysis. Data from two groups were compared by a two-tailed t test and those from more than two groups were compared by one-way analysis of variance with correction for multiple comparisons (7). A difference is considered to be significant at P < 0.05. RESULTS
Effect of IL-l
insufflation on pulmonary SOD activities.
We have previously demonstrated that tracheal insufflation of IL-l (5 pg) results in 84% of rats surviving continuous 100% O2 exposure for more than 11 days compared with control (HBSS)- insufflated rats, which all die within 3 days of Oz exposure (19). In this study we determined pulmonary Mn SOD and Cu,Zn SOD activities at various intervals after Oz exposure. Compared with control rats, at 1 day after Oz exposure, IL- 1 -treated rats showed no increase in Mn . SOD activity, whereas at 2.3 days when control animals started to die of Oz toxicity, Mn SOD activity was markedly increased (100%) in ILl-insufflated rats (Fig. IA). This increase was due, in part, to a reduction (30%) of Mn SOD activity in control rats (compared with control rats 1 day after O2 exposure, P < 0.01). The Mn SOD activity in IL-1-insufflated rats that survived O2 exposure for 7 days was further increased to about three times that of l-day controls. In contrast, the Cu,Zn SOD activities of IL-1-insufflated rats at 1 and 2.3 days after O2 exposure were not significantly different from those of control rats (Fig. 1B).
5 300 F250
600 t,
I
I day
2.3day
7day
-
II- -I (5 )
500 I
I day
2.3day
-/day
Fig. 1. Effect of interleukin-1 (IL-l) insufflation on pulmonary superoxide dismutase (SOD) enzyme activities of rats exposed to hyperoxia. A: Mn SOD; B: Cu,Zn SOD. Rats were insufflated with IL-l (5 pg) or Hanks’ balanced salt solution, exposed to 100% O2 for 1, 2.3, or 7 days, and bloodless lung extracts were obtained. Mn SOD and Cu,Zn SOD enzyme activities were then determined and results were expressed as means f: SE for numbers of animals in parentheses. * P < 0.001 vs. all other values.
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
L344
IL-I, SOD AND OXYGEN
However, the Cu,Zn SOD activity of IL-l-treated rats that survived 7 days of Orzexposure was 2.4 times that of l-day controls. Effect of IL-1 insufflation on pulmonary SOD protein contents. To determine whether the increased SOD activities were due to increased numbers of SOD molecules or due to the activation of existing enzymes, we measured the levels of immunoreactive proteins of Mn SOD and Cu,Zn SOD. Figure 2 shows immunoblots of pulmonary Mn SOD and Cu,Zn SOD from control and IL-l-insufflated rats exposed to hyperoxia for 1, 2.3, or 7 days. These blots show single bands of proteins with molecular masses of -20 and 17.5 kDa, consistent with the subunit sizes of Mn SOD and Cu,Zn SOD, respectively. Quantitative analysis of immunoblots was performed using a computing densitometer. For the purpose of comparison, the results were expressed as relative units per milligram of DNA with the mean value of control animals exposed to Oz for 1 day normalized to 1 relative unit/mg DNA. As shown in Fig. 3A, in control rats exposed to O2 for 2.3 days, there was a 25% reduction in Mn SOD protein (P < 0.05). The levels of Mn SOD protein in IL-1-insufflated rats, compared with control rats, increased l- and 2.5-fold at 1 and 2.3 days after O2 exposure, respectively. At 7 days after O2 exposure, the level of Mn SOD protein was increased to about four times that of l-day controls. In contrast, levels of Cu,Zn SOD protein in IL-1-insufflated rats at 1 and 2.3 days after O2 exposure were not significantly different from those of controls, whereas the level of Cu,Zn SOD protein in IL1-insufflated rats that survived O2 exposure for 7 days was about twice that of l-day controls (Fig. 3B). Effect of IL-l insufflation on pulmonary SOD mRNA levels. We have also measured the steady-state mRNA levels of pulmonary SOD. Figure 4 shows the Northern
TOLERANCE
Fig. 3, Effect of interloukinl (IL-l) insufflation on levelx of pulmonary superoxide dixmutase (SOD) immunoreactive proteins of rats exposed to hyperoxia, A: Mn SOD; B: Cu,Zn SOD. Computing densitometric analysis was performed on immunoblots of pulmonary SOD. Results were expressed as relative units/mg DNA with the mean value of control rats exposed to O2 for 1 day normalized to 1 relative unit/mg DNA. Bars, means * SE Numbers in parentheses, number of animals. * I’ 4 0.001 vs. all other values.
blots of pulmonary Mn SOD and Cu,Zn- SOD mRNA from control and IL-1-insufflated rats exposed to O2 for 1, 2.3, or 7 days. These blots revealed that rat pulmonary Mn SOD mRNA was primarily of the l.O-kb species, whereas rat pulmonary Cu,Zn SOD mRNA was primarily of 0.7-kb species. The relative amounts of SOD encoding mRNA were quantified using a computing densitometer, and for the purpose of comparison, the results were expressed as relative units per milligram of DNA with the mean value of control rats exposed to O2 for 1 day normalized to 1 relative unit/mg DNA. As shown in Fig. 5A, the level of Mn SOD mRNA of control rats at 2.3 days after Oz exposure was markedly increased, e.g., 4.5fold higher than that of l-day controls. Increased levels of Mn SOD mRNA were noted in all IL-1-insufflated rats exposed to 0s for 1,2.3, or 7 days, e.g., nine-, six-, and twofold higher
A kDo 97.468
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1718
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2223
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Id
B
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12 13 14 15 161718
Fig. 2. Immunoblots of pulmonary superoxide dismutase (SOD). A: Mn SOD; B: Cu,Zn SOD. Bloodless lung extracts were obtained as in Fig. 1. Aliquots (100 pg protein/lane for Mn SOD and 12.5 pg protein/lane for Cu,Zn SOD) were subjected to immunoblot analysis. Each lane represents result from 1 animal. IL-l, interleukin-1.
l920212223242526272Ei
IrlIIIW Control-Id
IL-l-Id
Control-2.3d
IL-I-2.3d
IL- I-7d
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
IL-l,
11
u
C- Id
28s
SOD AND OXYGEN
{ v81 (9 “‘9 ,I I Qv13
IL-l-Id
C-2.3d
IL-l 2.3d
IL-l-
TOLERANCE
1,345
‘4 Fig. 4. Northern blots nf pulmonary nuperoxide dismutase (SOD) mRNA. A: Mn SOD; U: Cu,Zn SOD. Rats were insufflated with interleukin-1 (IL-l, 5 ag) or Hanks’ balanced salt solution and were exposed to I()()‘% O2 for 1, 23, or 7 days. Lung RNAs were then extracted and aliquots (25 pg RNA/lane) were subjected to Northern blot analysis. Each lane represents result of 1 animal.
7d
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1
I8S--'
I 2 3 4 5 6 7 8 9 1011 I2 1314 I’WLVJ~ C- Id IL-l-Id C-2.3d IL-l IL- I- 7d 2.3d A
B
-c
P 005
14
Y$
lday
0 Control
I
2.3days
7days
lday
2.3days
7doys
Fig. 5. Effect of interleukin-1 (IL-l) insufflation on levels of pulmonary superoxide dismutase (SOD) mRNA of rats exposed to hyperoxia. A: Mn SOD; B: Cu,Zn SOD. Computing densitometric analysis was performed on Northern blots of pulmonary SOD mRNA. Results were expressed as relative units/mg DNA with mean value of controls exposed to O2 for 1 day normalized to 1 relative unit/mg DNA. Bars, means + SE. Numbers in parentheses, number of animals. F’ values (vs. l-day control): *< 0.02, **< 0.05.
than that of l-day controls, respectively. In contrast, levels of Cu,Zn SOD mRNA were unaltered in control and IL-1-insufflated rats at 1 and 2.3 days after O2 exposure (Fig. 5B). The level of Cu,Zn SOD mRNA in IL-l-insufflated rats at 7 days after O2 exposure was slightly, but significantly, increased, e.g., 100% over l-day controls. Since 0s exposure alone caused an increase in pulmonary Mn SOD mRNA, the enhanced levels of pulmonary Mn SOD mRNA observed in IL-1-insufflated and 02exposed rats (Figs. 4A and 5A) could be due to the com-
bined effects of O2 and IL-l. To determine the effect of IL-l alone, we have studied the levels of pulmonary Mn SOD mRNA in IL-1-insufflated rats exposed to normoxia. For the purpose of comparison, we have included samples from control and IL-1-insufflated rats exposed to O2 for 1 day. The results were quantified using a computing densitometer and were expressed as relative units per milligram of DNA with the mean value of control rats exposed to O2 for 1 day normalized to 1 relative unit/mg DNA. As shown in Fig. 6, IL-l markedly enhanced the level of pulmonary Mn SOD mRNA at 1 day, but not at 2.3 or 7 days, after insufflation. Compared with normoxia-exposed controls, exposure to O2 for 1 day did not augment pulmonary Mn SOD mRNA in control rats. Furthermore, the levels of pulmonary Mn SOD mRNA in IL-1-insufflated rats exposed to hyperoxia or normoxia for 1 day were not significantly different, suggesting that the Mn SOD mRNA enhancing effect observed in IL-linsufflated rats exposed to O2 for 1 day was primarily due to the effect of IL-l. Similar studies were also performed for Cu,Zn SOD mRNA, and the results revealed that IL-l had no effect in the levels of pulmonary Cu,Zn SOD mRNA in rats exposed to normoxia for 1, 2.3, or 7 days (data not shown). DISCUSSION
The results presented in this study demonstrate that IL-l selectively enhances pulmonary Mn SOD, but not Cu,Zn SOD, mRNA. In normoxia-exposed rats, the level
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IL-I, SOD AND OXYGEN
TOLERANCE
of Mn SOD protein and enzyme activity were slightly, but significantly, reduced in control rats exposed to O2 for 2.3 days compared with those of l-day 0s control rats (22, 28Sand this study). Ho et al. (11) have also reported that the ISSlevel of rat pulmonary Mn SOD mRNA increases 5- to lo-fold after 3-5 days of exposure to 85% Oz without a concomitant increase in Mn SOD protein and enzyme 1 I t 3, ,4 5, 6, ,7 8, 9, I’0 1’ ‘2, activity. The mechanism for the failure of hyperoxiaexposed rats to coordinately increase levels of Mn SOD AIR-C AIR-IL-I AIR-C AIR-IL-I protein is not clear. However, it may be due to the proId Id 2.3d 2.3d duction of defective (nontranslatable) Mn SOD mRNA, 28San inhibition of Mn SOD protein synthesis, and/or an enhancement of Mn SOD degradation. ISSThe effect of hyperoxia on pulmonary antioxidant enzymes has been extensively studied. Exposure of adult rats to lethal doses (>95%) of O:! does not enhance pul13 14 I5 16 17 18 19 20 21 22 2324 25 26 27 monary antioxidant enzyme activities, and in general UU they die within 3 days of O2 exposure (8, 19, 21). On the AIR-C AIR-IL-I o-c’- 2 2other hand, exposure of adult rats to sublethal doses (85 7d 7d Id or 90%) of O2 for 5-7 days results in significant increases in pulmonary Mn SOD, Cu,Zn SOD, and GSH peroxidase activities (5, 12). These antioxidant enzyme-augmented P95%) of Oz. In T-l contrast, neonatal rats are known to be resistant to lethal 0 Control (3) doses of Oz. They respond to lethal doses of O2 by inw IL -I creasing pulmonary SOD, catalase, and GSH peroxidase activities within 24 h of exposure (1,8,18). The increased pulmonary SOD activity within 24 h of Oz exposure in neonatal animals is accounted for solely by an increase in 16) Mn SOD activity (18). Based on the above reported ef131(3) c3j3’ ,i fects of O2 on pulmonary antioxidant enzymes and our , my 7days ldoy 2 3 days observations made in the current and previous (22) studies, we suggest that the rapid and selective induction of pulmonary Mn SOD mRNA in IL- 1-insufflated rats leadFig. 6. Effect of interleukin-1 (IL-l) insufflation on levels of pulmonary Mn-superoxide dismutase (Mn SOD) mRNA of rats exposed to noring to an increase in Mn SOD protein and enzyme activmoxia. A: Northern blot; B: computing densitometric analysis. Rats ity within 2.3 days of 0s exposure, protects rats from the were insufflated with IL-l (5 rg) or Hanks’ balanced salt solution and were exposed to normoxia for 1, 2.3, or 7 days, or 100% Oe for 1 day. acute toxicity of lethal hyperoxia. This enables the aniLung RNAs were then extracted and aliquots (15 rg RNA/lane) were mals to survive long enough to respond to hyperoxia by subjected to Northern blot analysis (A). Each lane represents result increasing pulmonary antioxidant enzymes including Mn from 1 animal. Computing densitometric analysis was performed on SOD, Cu,Zn SOD, catalase, and GSH peroxidase as demNorthern blots and results were expressed as relative units/mg DNA with mean value of control rats exposed to Oe for 1 day normalized to 1 onstrated in these rats at 7 days after O2 exposure (22, and this study). The markedly increased pulmonary anrelative unit/mg DNA (B). Bars, means + SE. Numbers in parentheses, tioxidant enzymes are then responsible for the chronic number of animals. adaptation of these animals to an environment of 100% of pulmonary Mn SOD mRNA was markedly elevated 1 02. The level of pulmonary Mn SOD mRNA at 7 days after day after IL-l insufflation. However, it returned to the O2 exposure in IL-1-insufflated rats was much lower than control level by 2.3 days. In contrast, the level of pulmothose of rats exposed to O2 for 1 or 2.3 days. However, the nary Mn SOD mRNA remained elevated, though to a lesser extent, through day 7 in IL-1-insufflated and 02- levels of Mn SOD protein and enzyme activity were much exposed animals. This suggests that the prolonged in- higher at 7 days of O2 exposure. The reason for this crease of pulmonary Mn SOD mRNA levels observed at discrepancy is not clear, but it may be due to the differ2.3 and 7 days was, at least in part, due to the effect of ence in pulmonary Mn SOD protein synthesis and/or hyperoxia. This conclusion is further supported by the degradation. In addition, we have also observed a discrepancy between the levels of pulmonary Mn SOD protein observation that the level of pulmonary Mn SOD mRNA rats at 1 day after in control rats exposed to Oz for 2.3 days was enhanced to and enzyme activity in IL-1-insufflated the same extent as that of IL-1-insufflated rats exposed to O2 exposure, e. g., an increased level of Mn SOD immunoreactive protein, but not the enzyme activity compared O2 for the same period. As we have previously reported (22), the increased level with l-day O2 controls. It is possible that the increased Mn SOD protein may represent some of pulmonary Mn SOD mRNA in control rats exposed to immunoreactive However, it is unlikely that the O2 for 2.3 days was not associated with an increased level kind of proenzyme( of Mn SOD protein or enzyme activity. In fact, the levels discrepancy is due to technical reasons, since we have
A
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IL-l,
SOD
AND
OXYGEN
measured the Mn SOD immunoreactive protein and enzyme activity several times with similar results. Furthermore, the results were obtained from five animals in each group. In the current study, the levels of pulmonary Mn SOD and Cu,Zn SOD proteins and enzyme activities were determined from perfused lung h.omogenates without correction for the contributi .on by infiltrating neutrophils. However, we have previously demonstrated that under these experimental conditions neutrophils contribute to < 2% of the total pulmonary Mn SOD and Cu,Zn SOD activities (19). Recently, White et al. (24) have demonstrated that a combination of TNF and IL-l given in split doses by intraperitoneal and intravenous injections markedly prolongs the survival of rats exposed to >95% Oz. No enhancement of pulmonary antioxidant enzyme activities were observed at 52 h when control rats started to die of O2 toxicity, however, pulmonary Mn SOD activ ity was not measured. A trans ient increase of pulmonary antioxidant enzyme activities was observed at 72 h, but not at 7 days after O2 exposure (25). Thus it is not clear how these cytokine-treated rats adapt to 100% O2 environment. Lung is a complex organ consisting of many different cell types with varying sensitivity to O2 toxicity (4). Since our measurements (mRNA, immunoreactive protein, and enzyme activity) of Mn SOD and Cu,Zn SOD were performed using lung extracts, the results represented overall changes of the lung. It is not clear whether the observed alterations in pulmonary Mn SOD and Cu,Zn SOD are uniform phenomena throughout the various cell types present in the lung or selective responses of particular cell types. Further studies using techniques such as immunohistochemistry or in situ hybridization are necessary to define these changes at the cellular level. The authors thank Alexander Durant and Willie care of the animals and Rhoda Hamid for secretarial This work was supported by the Department of Medical Research Service. Address for reprint requests: M.-F. Tsan, Research VA Medical Center, Albany, NY 12208. Received
9 March
1992; accepted
in final
form
13 May
Boykin for their assistance. Veterans Affairs Service,
Stratton
1992.
REFERENCES 1. Autor, A. P., L. Frank, and R. J. Roberts. Developmental characteristics of pulmonary superoxide dismutase: relationship to idiopathic respiratory distress syndrome. Pediatr. Res. 10: 154158, 1976. 2. Beutler, B., and A. Cerami. Cachectin: more than a tumor necrosis factor. N. Engl. J. Med. 316: 375-385, 1987. 3. Chromczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid quanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 4. Crapo, J. D., B. E. Barry, H. A. Fescue, and J. Shelburne. Structural and biochemical changes in rat lungs occurring during exposure to lethal and adaptive doses of oxygen. Am. Rev. Respir. Dis. 122: 123-143, 1980. 5. Crapo, J. D., and D. F. Tierney. Superoxide dismutase and pulmonary oxygen toxicity. Am. J. PhysioZ. 226: 1401-1407, 1974. 6. Dinarello, C. A. Interleukin-1. Rev. Infect. Dis. 6: 51-95, 1984.
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7. Einot, I., and K. R. Gabriel. A study of the power of several methods of multiple comparisons. J. Am. Stat. Assoc. 70: 574-583, 1975. 8. Frank, L., J. R. Bucher, and R. J. Roberts. Oxygen toxicity in neonatal and adult animals of various species. J. Appl. Physiol. 45: 699-704, 1978. 9. Frank, L., J. Yam, and R. J. Roberts. The role of endotoxin in protection of adult rats from oxygen-induced lung toxicity. J. Clin. Invest. 61: 269-275, 1978. 10. Freeman, B. A., and J. D. Crapo. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J. Biol. Chem. 256: 10986-10992, 1981. 11. Ho, Y. S., M. S. Dey, and J. D. Crapo. Modulation of lung antioxidant enzyme expression by hyperoxia (Abstract). Am. Rev. Respir. Dis. 141: A821, 1990. 12. Kimball, R. E., K. Reddy, T. H. Peirce, L. W. Schwartz, M. G. Mustafa, and C. E. Cross. Oxygen toxicity: augmentation of antioxidant defense mechanisms in rat lung. Am. J. Physiol. 230: 1425-1431, 1976. 13. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of the bacteriophage T,. Nature Lond. 227: 680-682, 1970. 14. McCord, J. M., and I. Fridovich. Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244: 6049-6055, 1969. 15. Ody, C., Y. Bach-Dieterle, I. Wand, and A. F. Junod. Effect of hyperoxia on superoxide dismutase content of pig pulmonary artery and aortic endothelial cells in culture. Exp. Lung Res. 1: 271-279, 1980. 16. Richards, G. M. Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal. Biochem. 57: 369-376, 1974. 17. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85, 1985. 18. Stevens, J. B., and A. P. Autor. Induction of superoxide dismutase by oxygen in neonatal rat lung. J. Biol. Chem. 252: 35093514, 1977. 19. Tsan, M. F., C. Y. Lee, and J. E. White. Interleukin 1 protects rats against oxygen toxicity. J. Appl. Physiol. 71: 688-697, 1991. 20. Tsan, M. F., J. E. White, P. J. Del Vecchio, and J. B. Shaffer. IL-6 enhances TNF- and IL-l-induced increase of Mnsuperoxide dismutase mRNA and oxygen toxicity. Am. J. Physiol. 263 (Lung Cell. MOL. Physiol. 7): L22-L26, 1992. 21. Tsan, M. F., J. E. White, T. A. Santana, and C. Y. Lee. Tracheal insufflation of tumor necrosis factor protects rats against oxygen toxicity. J. Appl. Physiol. 68: 1211-1219, 1990. 22. Tsan, M. F., J. E. White, C. Treanor, and J. B. Shaffer. Molecular basis for tumor necrosis factor-induced increase in pulmonary superoxide dismutase activities. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L506-L512, 1990. 23. Visner, G. A., W. C. Dougall, J. M. Wilson, I. A. Burr, and H. S. Nick. Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin 1, and tumor necrosis factor. J. Biol. Chem. 265: 2856-2864, 1990. 24. White, C. W., P. Ghezzi, C. A. Dinarello, S. A. Caldwell, I. F. McMurty, and J. E. Repine. Recombinant tumor necrosis factor/cachectin and interleukin 1 pretreatment decreases lung oxidized glutathione accumulation, lung injury, and mortality in rats exposed to hyperoxia. J. Clin. Invest. 79: 1868-1873, 1987. 25. White, C. W., P. Ghezzi, S. McMahan, C. A. Dinarello, and J. E. Repine. Cytokines increase rat lung antioxidant enzyme during exposure to hyperoxia. J. AppZ. Physiol. 66: 1003-1007, 1989. 26. Wong, G. H. W., and D. V. Goeddel. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science Wash. DC 242: 941-944, 1988.
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
TSAN
AND JULIE
superoxide dismutase oxygen-tolerant rats E. WHITE
Research Service, Samuel S. Stratton Department of Veterans Affairs Medical Center, and Departments of Physiology and Medicine, Albany Medical College, Albany, New York 12208 Tsan, Min-Fu, and Julie E. White. Kinetics of pulmonary superoxide dismutase in interleukinl-induced oxygen-tolerant rats. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L342L347, 1992.-We have previously demonstrated that tracheal insufflation of interleukinla (IL- 1) enhances pulmonary Mn superoxide dismutase (Mn SOD) activity and protects rats against O2 toxicity (M. F. Tsan, C. Y. Lee, and J. E. White. J. Appl. Physiol. 71: 688-697, 1991). In this study, we investigated the kinetics of mRNA, specific (immunoreactive) proteins, and enzyme activities of pulmonary Mn SOD and Cu,Zn SOD in IL-l-induced O,-tolerant rats. At 1 day after IL-I (5 pg) insufflation and 0, exposure, levels of Mn SOD mRNA and specific protein, but not enzyme activity, were markedly elevated. At 2.3 and 7 days after O2 exposure, levels of Mn SOD mRNA, specific protein, and enzyme activity were all increased in IL-l-treated animals. In contrast, in control rats at 2.3 days after 0, exposure, level of Mn SOD mRNA was markedly elevated, whereas levels of specific protein and enzyme activity were decreased. Levels of pulmonary Cu,Zn SOD mRNA, specific protein, and enzyme activity were unchanged in control and IL-l-treated rats, except that in IL-l-induced long-term O,-tolerant rats (7 days after 0, exposure), they were all increased. Since at 7 days after IL-I insufflation, normoxia-exposed rats did not show increased levels of pulmonary Mn SOD or Cu,Zn SOD mRNA, the increased levels of pulmonary SOD seen in IL-l-induced long-term O,-tolerant rats are, at least in part, due to the effect of 0, exposure. superoxide dismutase mRNA; oxygen tolerance
(IL-l) and tumor necrosis factor (TNF) are multifunctional cytokines with widely overlapping activities involving immune response, inflammation, hematopoiesis, and tumoricidal effect (2, 6). Recent studies (20, 23, 26) have demonstrated that IL-l and TNF selectively induce Mn superoxide dismutase (Mn SOD) mRNA, leading to increased Mn SOD specific protein and enzyme activity without affecting the levels of Cu,Zn SOD, catalase, or glutathione (GSH) peroxidase. Since Mn SOD is strategically located in mitochondria, a major site of superoxide production under hyperoxic conditions (lo), enhancement of Mn SOD by IL-l or TNF may protect against O2 toxicity. We have previously demonstrated that although intraperitoneal administration of IL-l or TNF provides no protection against O2 toxicity, tracheal insufflation of a single dose (5 pg) of IL-l or TNF markedly prolongs the survival of rats exposed to 100% O2 (19, 21). These cytokine-treated rats are able to survive in an environment of 100% O2 for Xl days compared with control rats, which all die within 3 days of O2 exposure. These cytokine-induced, long-term, Oz-tolerant rats show no signs of respiratory distress and their lung histology reveals no evidence of pulmonary edema at 7 days of continuous O2 exposure. The protection against O2 toxicity by tracheal insufflation of IL-1 or TNF is associated with increased pulmonary SOD activities (19, 21,
INTERLEUKIN-1
22). To better understand the mechanism of increased pulmonary SOD activities in these cytokine-treated animals, in this study we investigated the kinetics of mRNA, specific proteins, and enzyme activities of pulmonary Mn SOD and Cu,Zn SOD in IL-l-induced 02tolerant rats. MATERIALS
AND METHODS
Materials. Recombinant human IL-la (IL-l) was obtained from Dr. Craig Reynolds of the Biological Response Modifiers Program, National Cancer Institute, Frederick, MD. The preparation had a specific activity of 3 x lo8 U/mg protein as measured by the DlO assay and contained 0.5 EU endotoxin/mg protein. Monospecific goat anti-rat Cu,Zn SOD antibody was a gift of Drs. Michael Hass and Donald Massaro, University of Miami, and anti-human Mn SOD antibody, which cross-reacts with rat Mn SOD, was obtained from Dr. Larry Oberley, University of Iowa. Mouse Mn and Cu,Zn SOD cDNA clones were kindly provided by Dr. Jacquelin B. Shaffer of the New York State Wadsworth Center for Laboratories and Research. Tracheal insufflation and exposure of rats to hyperoxia. These procedures were performed as described previously (19, 21). Briefly, male Sprague-Dawley rats (Harlan Sprague-Dawley, Altamont, NY) free of respiratory infections and weighing between 250 and 350 g, were anesthetized with methoxyflurane (Pitman-Moore, Washington Crossing, NJ) and intubated with a 16-gauge intravenous catheter (Angiocath, Becton-Dickinson, Sandy, UT). One milliliter of calcium-magnesium-free Hanks’ balanced salt solution (HBSS) containing 5 pg IL-l or 1 ml HBSS for control animals, followed by 2 ml of air was injected into the lungs through the intratracheal catheter. Auscultation of the chest was performed with a stethoscope to ensure that solution was insufflated into the lungs. After rats had recovered from the effect of anesthesia, they were placed in groups of five in a Lucite chamber (45 x 40 X 25 cm”) that was flushed with 100% O2 at 15 l/min for 15 min and then maintained at 3 l/min. The concentration of O2 in the chamber as monitored using an 0, analyzer (Hudson Oxygen Analyzer, Ventronics Products Division, Temecula, CA) was >99% at all times (21). The rats were given free access to water and diet. Control exposure (normoxia) was performed in room air. Measurement of pulmonary SOD enzyme activities and immunoreactive proteins. Preparation of lung extracts for enzyme assay and immunoblot analyses of SOD was performed as described previously (19, 22). At 1 day, 2.3 days (55 h), or 7 days after 0, exposure, rats were anesthetized with pentobarbital and ventilated using a Harvard rodent ventilator (Harvard Apparatus, South Natick, MA). After a midline thoracotomy, 150 U of heparin was given through intracardiac injection and animals were exsanguinated by transection of abdominal aorta. The main pulmonary artery was quickly cannulated and the left atrium was cut open. Lungs were flushed with 20 ml HBSS containing 4% Ficoll 70 followed by 5 ml potassium phosphate buffer (0.1 M, pH 7.4) containing 0.05 M KC1 using a peristaltic roller pump (Harvard Apparatus). Lungs were removed, trimmed, rinsed, blotted, and weighed. The right lungs were homogenized with a tissue homogenizer (Tissumizer, Tekmar,
L342 Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
IL-l,
SOD
AND
OXYGEN
Cincinnati, OH) in 3 ml potassium phosphate buffer (0.005 M, pH 7.8) containing 0.1 mM EDTA. The homogenates were then twice sonicated on ice for I min using a sonic dismembrator (Fisher, Pittsburgh, PA) to disrupt mitochondria and intracellular granules, and the final volume was adjusted to IO ml. After centrifugation at 15,000 g for 10 min at 4°C the supernatant was dialyzed against homogenizing buffer for 16 h (12,000 mol wt cutoff, Spectrum Medical Industries, Los Angeles, CA) to remove low-molecularweight compounds that might nonspecifically reduce ferrichrome c and was then assayed for SOD activities. Left lungs were homogenized, sonicated, and extensively washed in perchloric acid (0.5 N) at 4” C; the precipitate was extracted in 10 ml 1.5 N perchloric acid by boiling for 20 min and was assayed for DNA contents. The SOD activity was assayed according to McCord and Fridovich (14) on the basis of the inhibitory effect of SOD on ferricytochrome c reduction by superoxide generated by the action of xanthine oxidase on xanthine. The total SOD activity was assayed in the presence of 10 PM KCN to inhibit cytochrome oxidase, whereas Mn SOD activity was assayed in the presence of 1 mM KCN to inhibit Cu,Zn SOD (15). The difference between the total SOD activity and Mn SOD activity was taken to be the Cu,Zn SOD activity. Protein contents were assayed using bicinchoninic acid according to Smith et al. (17). Measurement of DNA contents was made using the diphenylamine reaction according to the method of Richards (16). For immunoblot analysis, lung extracts (100 pg protein/lane for Mn SOD and 12.5 pug protein/lane for Cu,Zn SOD) were electrophoresed in denaturing sodium dodecyl sulfate (SDS) polyacrylamide (12%) gels according to Laemmli (13) and transferred electrophoretically to nitrocellulose membranes in a BioRad Transblot cell (Bio-Rad, Richmond, CA). After sample transfer was completed, membranes were blocked for 2 h in phosphate-buffered saline (PBS) containing 10% nonfat dry milk and 0.1% Tween 20 (BLOTTO). They were then incubated for 21 h with primary antibodies, which had been diluted in BLOTTO (anti-Cu,Zn SOD, I:400 and anti-Mn SOD, 1:lOOO). Membrane were then washed for 20 min in PBS containing 0.1% Tween (PBST) and incubated for 2 h in alkaline phosphatase-conjugated secondary antibodies (Boehringer-Mannhein, diluted 1:5000 in BLOTTO) that were directed against the immunoglobin G (IgG) fraction of the species that produced the primary antibody. At the end of the incubation, membranes were washed for 30 min in PBST and were equilibrated in 0.5 M tris(hydroxymethyl)aminomethane (Tris) (pH 9.5) for 5 min. To detect regions of bound antigen/primary antibody/alkaline phosphatase-conjugated secondary antibody on the nitrocellulose, membranes were stained for alkaline phosphatase activity using a detection kit purchased from Kirkegaard and Perry Lab., Gaithersburg, MD. Areas of alkaline phosphatase stain on individual blots were quantified using a computing densitometer (Molecular Dynamics, Sunnyvale, CA). Quantification of pulmonary SOD mRNA. This was performed as described previously (22). At 1, 2.3, or 7 days after hyperoxic or normoxic exposure, rats were anesthetized with pentobarbital and exsanguinated by transection of abdominal aorta. Lungs were quickly removed, trimmed, rinsed, blotted, and weighed. One gram of lung tissue was extracted for RNA by the acid quanidinium thiocyanate-phenol-chloroform method of Chromczynski and Sacchi (3). The remaining lung tissue was homogenized, extracted, and assayed for DNA contents as in Measurement of pulmonary SOD enzyme activities and immunoreactive proteins. For Northern blots, formamide-formaldehyde denatured RNA samples (15 or 25 pg total cellular RNA/lane) were electrophoresed in 1.2% agarose gels containing 2.2 M formaldehyde and stained with ethidium bromide to visualize the quality and size of 18s and 28s ribosomal RNA species. They were then
L343
TOLERANCE
transferred to nylon membrane (Micron Separations, Westborough, MA) by capillary blotting in 20x standard sodium citrate (SSC) (3 M NaCl, 0.3 M trisodium citrate, pH 7.0). Blots were prehybridized for 4-16 h at SZ*C in 50% formamide, 2~ Denhardt’s solution (0.04% Ficoll 400, 0.04% bovine serum albumin, and 0.04% polyvinylpyrrolidone), 0.1% SDS, 5~ SSPE (0.75 N NaCl, 0.05 M NaH2P04 and 0.005 M EDTA, pH 7.4), and 100 pg/ml denatured salmon sperm DNA. Hybridization was carried out in the same solution for 18-24 h with the addition of cDNA probes that had been labeled with [““P]CTP by random hexanucleotide priming (BRL, Gaithersburg, MD) to a specific activity of ~10~ counts per minute (cpm)/pg DNA. Blots were washed twice with 2~ SSPE, 0.1% SDS at room temperature, and twice with 0.1X SSPE, 0.1% SDS at 55°C. Radioactive signals were visualized by exposure of hybridized blots to Kodak X-Omat AR film (Eastman-Kodak, Rochester, NY) at -70” C with intensifying screens. Autoradiographs were quantified using a computing densitometer. Statistical anaZysis. Data from two groups were compared by a two-tailed t test and those from more than two groups were compared by one-way analysis of variance with correction for multiple comparisons (7). A difference is considered to be significant at P < 0.05. RESULTS
Effect of IL-l
insufflation on pulmonary SOD activities.
We have previously demonstrated that tracheal insufflation of IL-l (5 pg) results in 84% of rats surviving continuous 100% O2 exposure for more than 11 days compared with control (HBSS)- insufflated rats, which all die within 3 days of Oz exposure (19). In this study we determined pulmonary Mn SOD and Cu,Zn SOD activities at various intervals after Oz exposure. Compared with control rats, at 1 day after Oz exposure, IL- 1 -treated rats showed no increase in Mn . SOD activity, whereas at 2.3 days when control animals started to die of Oz toxicity, Mn SOD activity was markedly increased (100%) in ILl-insufflated rats (Fig. IA). This increase was due, in part, to a reduction (30%) of Mn SOD activity in control rats (compared with control rats 1 day after O2 exposure, P < 0.01). The Mn SOD activity in IL-1-insufflated rats that survived O2 exposure for 7 days was further increased to about three times that of l-day controls. In contrast, the Cu,Zn SOD activities of IL-1-insufflated rats at 1 and 2.3 days after O2 exposure were not significantly different from those of control rats (Fig. 1B).
5 300 F250
600 t,
I
I day
2.3day
7day
-
II- -I (5 )
500 I
I day
2.3day
-/day
Fig. 1. Effect of interleukin-1 (IL-l) insufflation on pulmonary superoxide dismutase (SOD) enzyme activities of rats exposed to hyperoxia. A: Mn SOD; B: Cu,Zn SOD. Rats were insufflated with IL-l (5 pg) or Hanks’ balanced salt solution, exposed to 100% O2 for 1, 2.3, or 7 days, and bloodless lung extracts were obtained. Mn SOD and Cu,Zn SOD enzyme activities were then determined and results were expressed as means f: SE for numbers of animals in parentheses. * P < 0.001 vs. all other values.
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
L344
IL-I, SOD AND OXYGEN
However, the Cu,Zn SOD activity of IL-l-treated rats that survived 7 days of Orzexposure was 2.4 times that of l-day controls. Effect of IL-1 insufflation on pulmonary SOD protein contents. To determine whether the increased SOD activities were due to increased numbers of SOD molecules or due to the activation of existing enzymes, we measured the levels of immunoreactive proteins of Mn SOD and Cu,Zn SOD. Figure 2 shows immunoblots of pulmonary Mn SOD and Cu,Zn SOD from control and IL-l-insufflated rats exposed to hyperoxia for 1, 2.3, or 7 days. These blots show single bands of proteins with molecular masses of -20 and 17.5 kDa, consistent with the subunit sizes of Mn SOD and Cu,Zn SOD, respectively. Quantitative analysis of immunoblots was performed using a computing densitometer. For the purpose of comparison, the results were expressed as relative units per milligram of DNA with the mean value of control animals exposed to Oz for 1 day normalized to 1 relative unit/mg DNA. As shown in Fig. 3A, in control rats exposed to O2 for 2.3 days, there was a 25% reduction in Mn SOD protein (P < 0.05). The levels of Mn SOD protein in IL-1-insufflated rats, compared with control rats, increased l- and 2.5-fold at 1 and 2.3 days after O2 exposure, respectively. At 7 days after O2 exposure, the level of Mn SOD protein was increased to about four times that of l-day controls. In contrast, levels of Cu,Zn SOD protein in IL-1-insufflated rats at 1 and 2.3 days after O2 exposure were not significantly different from those of controls, whereas the level of Cu,Zn SOD protein in IL1-insufflated rats that survived O2 exposure for 7 days was about twice that of l-day controls (Fig. 3B). Effect of IL-l insufflation on pulmonary SOD mRNA levels. We have also measured the steady-state mRNA levels of pulmonary SOD. Figure 4 shows the Northern
TOLERANCE
Fig. 3, Effect of interloukinl (IL-l) insufflation on levelx of pulmonary superoxide dixmutase (SOD) immunoreactive proteins of rats exposed to hyperoxia, A: Mn SOD; B: Cu,Zn SOD. Computing densitometric analysis was performed on immunoblots of pulmonary SOD. Results were expressed as relative units/mg DNA with the mean value of control rats exposed to O2 for 1 day normalized to 1 relative unit/mg DNA. Bars, means * SE Numbers in parentheses, number of animals. * I’ 4 0.001 vs. all other values.
blots of pulmonary Mn SOD and Cu,Zn- SOD mRNA from control and IL-1-insufflated rats exposed to O2 for 1, 2.3, or 7 days. These blots revealed that rat pulmonary Mn SOD mRNA was primarily of the l.O-kb species, whereas rat pulmonary Cu,Zn SOD mRNA was primarily of 0.7-kb species. The relative amounts of SOD encoding mRNA were quantified using a computing densitometer, and for the purpose of comparison, the results were expressed as relative units per milligram of DNA with the mean value of control rats exposed to O2 for 1 day normalized to 1 relative unit/mg DNA. As shown in Fig. 5A, the level of Mn SOD mRNA of control rats at 2.3 days after Oz exposure was markedly increased, e.g., 4.5fold higher than that of l-day controls. Increased levels of Mn SOD mRNA were noted in all IL-1-insufflated rats exposed to 0s for 1,2.3, or 7 days, e.g., nine-, six-, and twofold higher
A kDo 97.468
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-
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Fig. 2. Immunoblots of pulmonary superoxide dismutase (SOD). A: Mn SOD; B: Cu,Zn SOD. Bloodless lung extracts were obtained as in Fig. 1. Aliquots (100 pg protein/lane for Mn SOD and 12.5 pg protein/lane for Cu,Zn SOD) were subjected to immunoblot analysis. Each lane represents result from 1 animal. IL-l, interleukin-1.
l920212223242526272Ei
IrlIIIW Control-Id
IL-l-Id
Control-2.3d
IL-I-2.3d
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Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
IL-l,
11
u
C- Id
28s
SOD AND OXYGEN
{ v81 (9 “‘9 ,I I Qv13
IL-l-Id
C-2.3d
IL-l 2.3d
IL-l-
TOLERANCE
1,345
‘4 Fig. 4. Northern blots nf pulmonary nuperoxide dismutase (SOD) mRNA. A: Mn SOD; U: Cu,Zn SOD. Rats were insufflated with interleukin-1 (IL-l, 5 ag) or Hanks’ balanced salt solution and were exposed to I()()‘% O2 for 1, 23, or 7 days. Lung RNAs were then extracted and aliquots (25 pg RNA/lane) were subjected to Northern blot analysis. Each lane represents result of 1 animal.
7d
_/
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B
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lday
0 Control
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2.3days
7days
lday
2.3days
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Fig. 5. Effect of interleukin-1 (IL-l) insufflation on levels of pulmonary superoxide dismutase (SOD) mRNA of rats exposed to hyperoxia. A: Mn SOD; B: Cu,Zn SOD. Computing densitometric analysis was performed on Northern blots of pulmonary SOD mRNA. Results were expressed as relative units/mg DNA with mean value of controls exposed to O2 for 1 day normalized to 1 relative unit/mg DNA. Bars, means + SE. Numbers in parentheses, number of animals. F’ values (vs. l-day control): *< 0.02, **< 0.05.
than that of l-day controls, respectively. In contrast, levels of Cu,Zn SOD mRNA were unaltered in control and IL-1-insufflated rats at 1 and 2.3 days after O2 exposure (Fig. 5B). The level of Cu,Zn SOD mRNA in IL-l-insufflated rats at 7 days after O2 exposure was slightly, but significantly, increased, e.g., 100% over l-day controls. Since 0s exposure alone caused an increase in pulmonary Mn SOD mRNA, the enhanced levels of pulmonary Mn SOD mRNA observed in IL-1-insufflated and 02exposed rats (Figs. 4A and 5A) could be due to the com-
bined effects of O2 and IL-l. To determine the effect of IL-l alone, we have studied the levels of pulmonary Mn SOD mRNA in IL-1-insufflated rats exposed to normoxia. For the purpose of comparison, we have included samples from control and IL-1-insufflated rats exposed to O2 for 1 day. The results were quantified using a computing densitometer and were expressed as relative units per milligram of DNA with the mean value of control rats exposed to O2 for 1 day normalized to 1 relative unit/mg DNA. As shown in Fig. 6, IL-l markedly enhanced the level of pulmonary Mn SOD mRNA at 1 day, but not at 2.3 or 7 days, after insufflation. Compared with normoxia-exposed controls, exposure to O2 for 1 day did not augment pulmonary Mn SOD mRNA in control rats. Furthermore, the levels of pulmonary Mn SOD mRNA in IL-1-insufflated rats exposed to hyperoxia or normoxia for 1 day were not significantly different, suggesting that the Mn SOD mRNA enhancing effect observed in IL-linsufflated rats exposed to O2 for 1 day was primarily due to the effect of IL-l. Similar studies were also performed for Cu,Zn SOD mRNA, and the results revealed that IL-l had no effect in the levels of pulmonary Cu,Zn SOD mRNA in rats exposed to normoxia for 1, 2.3, or 7 days (data not shown). DISCUSSION
The results presented in this study demonstrate that IL-l selectively enhances pulmonary Mn SOD, but not Cu,Zn SOD, mRNA. In normoxia-exposed rats, the level
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L346
IL-I, SOD AND OXYGEN
TOLERANCE
of Mn SOD protein and enzyme activity were slightly, but significantly, reduced in control rats exposed to O2 for 2.3 days compared with those of l-day 0s control rats (22, 28Sand this study). Ho et al. (11) have also reported that the ISSlevel of rat pulmonary Mn SOD mRNA increases 5- to lo-fold after 3-5 days of exposure to 85% Oz without a concomitant increase in Mn SOD protein and enzyme 1 I t 3, ,4 5, 6, ,7 8, 9, I’0 1’ ‘2, activity. The mechanism for the failure of hyperoxiaexposed rats to coordinately increase levels of Mn SOD AIR-C AIR-IL-I AIR-C AIR-IL-I protein is not clear. However, it may be due to the proId Id 2.3d 2.3d duction of defective (nontranslatable) Mn SOD mRNA, 28San inhibition of Mn SOD protein synthesis, and/or an enhancement of Mn SOD degradation. ISSThe effect of hyperoxia on pulmonary antioxidant enzymes has been extensively studied. Exposure of adult rats to lethal doses (>95%) of O:! does not enhance pul13 14 I5 16 17 18 19 20 21 22 2324 25 26 27 monary antioxidant enzyme activities, and in general UU they die within 3 days of O2 exposure (8, 19, 21). On the AIR-C AIR-IL-I o-c’- 2 2other hand, exposure of adult rats to sublethal doses (85 7d 7d Id or 90%) of O2 for 5-7 days results in significant increases in pulmonary Mn SOD, Cu,Zn SOD, and GSH peroxidase activities (5, 12). These antioxidant enzyme-augmented P95%) of Oz. In T-l contrast, neonatal rats are known to be resistant to lethal 0 Control (3) doses of Oz. They respond to lethal doses of O2 by inw IL -I creasing pulmonary SOD, catalase, and GSH peroxidase activities within 24 h of exposure (1,8,18). The increased pulmonary SOD activity within 24 h of Oz exposure in neonatal animals is accounted for solely by an increase in 16) Mn SOD activity (18). Based on the above reported ef131(3) c3j3’ ,i fects of O2 on pulmonary antioxidant enzymes and our , my 7days ldoy 2 3 days observations made in the current and previous (22) studies, we suggest that the rapid and selective induction of pulmonary Mn SOD mRNA in IL- 1-insufflated rats leadFig. 6. Effect of interleukin-1 (IL-l) insufflation on levels of pulmonary Mn-superoxide dismutase (Mn SOD) mRNA of rats exposed to noring to an increase in Mn SOD protein and enzyme activmoxia. A: Northern blot; B: computing densitometric analysis. Rats ity within 2.3 days of 0s exposure, protects rats from the were insufflated with IL-l (5 rg) or Hanks’ balanced salt solution and were exposed to normoxia for 1, 2.3, or 7 days, or 100% Oe for 1 day. acute toxicity of lethal hyperoxia. This enables the aniLung RNAs were then extracted and aliquots (15 rg RNA/lane) were mals to survive long enough to respond to hyperoxia by subjected to Northern blot analysis (A). Each lane represents result increasing pulmonary antioxidant enzymes including Mn from 1 animal. Computing densitometric analysis was performed on SOD, Cu,Zn SOD, catalase, and GSH peroxidase as demNorthern blots and results were expressed as relative units/mg DNA with mean value of control rats exposed to Oe for 1 day normalized to 1 onstrated in these rats at 7 days after O2 exposure (22, and this study). The markedly increased pulmonary anrelative unit/mg DNA (B). Bars, means + SE. Numbers in parentheses, tioxidant enzymes are then responsible for the chronic number of animals. adaptation of these animals to an environment of 100% of pulmonary Mn SOD mRNA was markedly elevated 1 02. The level of pulmonary Mn SOD mRNA at 7 days after day after IL-l insufflation. However, it returned to the O2 exposure in IL-1-insufflated rats was much lower than control level by 2.3 days. In contrast, the level of pulmothose of rats exposed to O2 for 1 or 2.3 days. However, the nary Mn SOD mRNA remained elevated, though to a lesser extent, through day 7 in IL-1-insufflated and 02- levels of Mn SOD protein and enzyme activity were much exposed animals. This suggests that the prolonged in- higher at 7 days of O2 exposure. The reason for this crease of pulmonary Mn SOD mRNA levels observed at discrepancy is not clear, but it may be due to the differ2.3 and 7 days was, at least in part, due to the effect of ence in pulmonary Mn SOD protein synthesis and/or hyperoxia. This conclusion is further supported by the degradation. In addition, we have also observed a discrepancy between the levels of pulmonary Mn SOD protein observation that the level of pulmonary Mn SOD mRNA rats at 1 day after in control rats exposed to Oz for 2.3 days was enhanced to and enzyme activity in IL-1-insufflated the same extent as that of IL-1-insufflated rats exposed to O2 exposure, e. g., an increased level of Mn SOD immunoreactive protein, but not the enzyme activity compared O2 for the same period. As we have previously reported (22), the increased level with l-day O2 controls. It is possible that the increased Mn SOD protein may represent some of pulmonary Mn SOD mRNA in control rats exposed to immunoreactive However, it is unlikely that the O2 for 2.3 days was not associated with an increased level kind of proenzyme( of Mn SOD protein or enzyme activity. In fact, the levels discrepancy is due to technical reasons, since we have
A
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IL-l,
SOD
AND
OXYGEN
measured the Mn SOD immunoreactive protein and enzyme activity several times with similar results. Furthermore, the results were obtained from five animals in each group. In the current study, the levels of pulmonary Mn SOD and Cu,Zn SOD proteins and enzyme activities were determined from perfused lung h.omogenates without correction for the contributi .on by infiltrating neutrophils. However, we have previously demonstrated that under these experimental conditions neutrophils contribute to < 2% of the total pulmonary Mn SOD and Cu,Zn SOD activities (19). Recently, White et al. (24) have demonstrated that a combination of TNF and IL-l given in split doses by intraperitoneal and intravenous injections markedly prolongs the survival of rats exposed to >95% Oz. No enhancement of pulmonary antioxidant enzyme activities were observed at 52 h when control rats started to die of O2 toxicity, however, pulmonary Mn SOD activ ity was not measured. A trans ient increase of pulmonary antioxidant enzyme activities was observed at 72 h, but not at 7 days after O2 exposure (25). Thus it is not clear how these cytokine-treated rats adapt to 100% O2 environment. Lung is a complex organ consisting of many different cell types with varying sensitivity to O2 toxicity (4). Since our measurements (mRNA, immunoreactive protein, and enzyme activity) of Mn SOD and Cu,Zn SOD were performed using lung extracts, the results represented overall changes of the lung. It is not clear whether the observed alterations in pulmonary Mn SOD and Cu,Zn SOD are uniform phenomena throughout the various cell types present in the lung or selective responses of particular cell types. Further studies using techniques such as immunohistochemistry or in situ hybridization are necessary to define these changes at the cellular level. The authors thank Alexander Durant and Willie care of the animals and Rhoda Hamid for secretarial This work was supported by the Department of Medical Research Service. Address for reprint requests: M.-F. Tsan, Research VA Medical Center, Albany, NY 12208. Received
9 March
1992; accepted
in final
form
13 May
Boykin for their assistance. Veterans Affairs Service,
Stratton
1992.
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