Extracellular superoxide dismutase in the airways of transgenic mice reduces inflammation and attenuates lung toxicity following hyperoxia (original) (raw)
Characterization of the hEC-SOD Tg mice. The strategy used for creating Tg mice overexpressing hEC-SOD in the mouse lung is shown in Figure 1. We initially identified five founder strains by PCR analysis of tail DNA, and all subsequent work was performed using founder strain 32, whereas line 15 was used to rule out the possibility of a gene-insertion effect. Additionally, the hEC-SOD Tg mice appeared healthy throughout this study and showed no signs of accelerated senescence when compared with their Wt littermates.
The hEC-SOD transgene protein was only detected in the lungs of PCR-positive Tg mice and not in liver, heart, kidney, brain, skeletal muscle, small intestine, and blood, indicating a highly tissue-specific pattern of transgene expression (data not shown). The EC-SOD activity in the BALF and lung tissue of Tg mice was significantly higher than that of their Wt littermates (Table 1). In BALF, there was an approximate 2.9-fold increase, whereas in the lung tissue, there was a threefold increase in EC-SOD enzyme activity (Table 1). The increased expression of the human transgene did not measurably affect the basal expression of endogenous mouse lung EC-SOD (Figure 2). Additionally, no effect was observed in the expression of other BALF and/or lung tissue antioxidants — namely, Mn-SOD, CuZn-SOD, GPx, catalase, and GSH (Table 1).
Western blot analysis of human and mouse EC-SOD from the lungs of wild-type (Wt) and hEC-SOD transgenic (Tg) mice. Lung protein homogenates from Wt and Tg mice were electrophoresed in 12.5% SDS–polyacrylamide gel. The separated proteins were transferred to Immobilon-P and probed with the anti–hEC-SOD antibodies (WT474) or with anti–mouse EC-SOD (SS618) polyclonal antibodies. (a) Western blot of total lung protein (10 μg) probed with WT474. Lanes 1, 2, and 3 reflect lung homogenate from three independent Tg mice, whereas lanes 4, 5, and 6 are from Wt littermate controls. (b) An identical Western blot as in a was probed with SS618. Of note, hEC-SOD typically shows two bands with the lower-molecular-weight species representing a truncated COOH-terminus. Mouse EC-SOD typically shows three bands, with the lower two also representing COOH-terminal truncations (14).
Antioxidant content of BALF and lung tissue from wild-type and hEC-SOD transgenic mice under normal conditions while breathing room air
Immunohistochemical staining of mouse lung tissues using anti–hEC-SOD antibodies showed expression of the transgene only in type II alveolar epithelial cells (Figure 3a) and in nonciliated bronchial epithelial lining cells (Figure 3b). No staining was observed in the sections that were stained with the preimmune serum or the preabsorbed WT474 antibodies (data not shown).
Immunohistochemical staining of mouse lung tissues using rabbit anti–hEC-SOD antibodies. (a) Tg mouse lung demonstrating staining of type II cells with anti–hEC-SOD antisera. (b) Tg mouse showing hEC-SOD staining of the nonciliated bronchoepithelial lining cells. (c) Wt mouse lung did not stain with the hEC-SOD antibody. (d) Normal Wt mouse bronchoepithelial lining cells did not stain with anti–hEC-SOD antibody. ×225.
Hyperoxia exposure and mortality. Exposure to >99% oxygen was lethal to mice, especially after 84 hours. Hyperoxia was lethal to 40% of the exposed Wt mice (12/30) and 11.6% of the Tg mice (5/43). Using the rate and proportion z-test, the mortality rate was significantly higher in the Wt mice (P = 0.011).
BAL cell counts. There was no difference in total cell counts between Wt and Tg mice after 48 hours of hyperoxia (Figure 4a). However, the total cell count increased significantly by 72 hours in the Wt mice when compared with the Tg mice (4.5 × 105 ± 0.42 vs. 2.5 × 105 ± 0.29; P < 0.05) and remained elevated even at 84 hours (5.6 × 105 ± 0.54 vs. 3.6 × 105 ± 0.38; P < 0.05). This increase in the total BAL cell count was mainly due to the disproportionate recruitment and influx of the polymorphonuclear cells (PMNs) (Figure 4b). The Wt mice showed an approximate 4.8-fold increase in PMNs at 72 hours and a 4.7-fold increase at 84 hours over that of Tg littermates (P < 0.05; Figure 4b). ND significantly (P < 0.05) suppressed neutrophil migration into the alveolar spaces in hyperoxic mice, and overexpression of hEC-SOD further suppressed PMN BALF counts (Figure 4c).
Total cell and PMN counts in the BALF from wild-type (Wt) and transgenic (Tg) mice exposed to hyperoxia. (a) Total cell counts at 48, 72, and 84 h of exposure to >99% oxygen. (b) PMN cell counts at 48, 72, and 84 h of exposure to >99% oxygen. Both total cell and PMN counts significantly increased with time of exposure. (c) Tg and Wt animals were neutrophil-depleted (ND) or not (placebo), and were subsequently exposed to >99% oxygen or room air (RA) for 72 h followed by BALF analysis of total PMN cell counts. The Wt oxygen-exposed groups showed significant increases in PMN cell counts when compared with the Tg oxygen-exposed groups. In addition, in Wt animals, neutrophil depletion alone significantly reduced the BALF PMN cell counts vs. the oxygen–placebo (O-placebo) group. Values are expressed as mean ± SEM (n = 6). *P < 0.05 between groups; **P < 0.01 between groups. BALF, bronchoalveolar lavage fluid; PMN, polymorphonuclear neutrophil.
Markers of lung injury and edema. The amount of acute lung damage elicited by hyperoxia was measured by an increase in BALF total protein and BALF LDH. Hyperoxia exposure resulted in significant increases with time of exposure for both parameters tested (Figure 5, a and b). However, the EC-SOD Tg mouse strain showed a marked attenuation of these lung injury parameters, at both 72 and 84 hours, when compared with their Wt littermates (P < 0.05). ND in Wt mice significantly lessened hyperoxic damage to the lung by 72 hours of exposure as assessed by the total protein and LDH in the BALF (Figure 5, c and d). ND in the Tg mice showed a trend to decrease further acute lung damage, but this difference was not found to be significant (Figure 5, c and d).
Markers of lung injury. (a) Total protein in the BALF from mice exposed to hyperoxia at 48, 72, and 84 h. The total protein concentration increased significantly in the wild-type (Wt) group at 72 h. (b) LDH activity in the BALF from mice exposed to hyperoxia at 48, 72, and 84 h. LDH increased with time and was significantly higher in Wt mice. (c) The total protein concentration in the BALF of neutrophil-depleted (ND) animals was significantly reduced compared with non-ND oxygen-exposed mice. For a given oxygen exposure group, overexpressing hEC-SOD further reduced the BALF total protein. (d) LDH activity was decreased significantly in the ND mice. Values are expressed as mean ± SEM. (n = 6). *P < 0.05 Wt vs. Tg group; **P < 0.01 Wt vs. Tg group. LDH, lactate dehydrogenase.
We used the lung wet/dry ratio in an attempt to probe pulmonary capillary permeability differences between the two groups of mice. The length of hyperoxia exposure correlated with an increase in pulmonary edema fluid as measured by the wet/dry ratio (Figure 6a). The edema was significantly higher in the Wt mice at 84 hours when compared with EC-SOD Tg littermates (P < 0.05). Furthermore, ND decreased the lung permeability significantly (P < 0.05) in the Wt mice. However, no significant difference was noted between the ND and non-ND Tg mice (Figure 6b).
Lung edema. (a) Lung wet/dry ratios at 48, 72, and 84 h of exposure to hyperoxia. A significant increase in the wet weight of wild-type (Wt) lungs was observed at 84 h of hyperoxic exposure. (b) After 72 h of hyperoxia exposure, neutrophil depletion and overexpression of hEC-SOD decreased lung wet/dry ratio significantly in the Wt vs. transgenic (Tg) group (*P < 0.05). Values are expressed as mean ± SEM (n = 6).
Specificity of EC-SOD expression in Tg mice strains. Studies were undertaken to determine whether the tolerance of Tg animals to hyperoxia was the direct result of overexpressing hEC-SOD in the mouse lung or simply due to the transgene’s random insertion into the mouse genome. A second mouse Tg founder line, line 15, was partly characterized and shown to have similar levels of hEC-SOD in the mouse lung as was found in line 32. Six animals each from line 15, line 32, and Wt were exposed to >99% O2 for 72 hours, and the development of acute lung injury was determined as measured by the total protein and LDH in the BALF. We found Tg line 15 and line 32 to have similar levels of protection from hyperoxia, as judged by BALF protein, and both were significantly less than Wt (288 ± 182, 220 ± 101, 633 ± 218 μg/ml, respectively; P < 0.05). Parallel findings were also noted for LDH levels in BALF (153 ± 35, 132 ± 42, 314 ± 63 U/l, respectively; P < 0.05). We interpret these findings to mean that the tolerance to hyperoxia in the hEC-SOD Tg mice is the result of overexpression of the transgene and is not due to local gene effects of inserting DNA into the mouse genome.
Lung antioxidants. At each of the time points indicated, the total SOD activity was significantly higher in BALF and lung homogenate in Tg vs. Wt mice (Figure 7, a and b) and was mainly due to increases in the hEC-SOD isoenzyme (data not shown). Although the total SOD activity showed a trend to increasing with time of hyperoxia exposure in the Wt animals, this was not significant (Figure 7, a and b).
Total SOD in the lung and BALF from mice exposed to hyperoxia. (a) SOD activity (U/ml BALF) in the BALF of the transgenic (Tg), but not wild-type (Wt), mice increased significantly with time. There was a trend to increasing with time in Wt, but this was not significant. (b) The total SOD activity (U/mg lung protein) in the lung homogenate of both Wt and Tg mice showed a trend to increasing with time of oxygen exposure, but this was not significant. At the three time points tested, Tg mice showed significantly higher enzyme activities compared with Wt controls. Values are expressed as mean ± SEM (n = 6). *P < 0.05 Tg vs. Wt.
The total glutathione content in the lung tissue (Wt: 9.7 ± 1.5, 14.7 ± 1.3, 17.5 ± 1.4 μmol/mg protein; Tg: 10.4 ± 2.0, 14.5 ± 1.0, and 18.0 ± 1.0 μmol/mg protein at 48, 72, and 84 hours, respectively) and the BALF (Wt: 4.8 ± 1.1, 6.3 ± 1.0, 8.6 ± 0.8 nmol/ml; Tg: 4.6 ± 1.0, 4.9 ± 0.6, and 7.0 ± 0.9 nmol/ml at 48, 72, and 84 hours, respectively) increased significantly with time of hyperoxia exposure but did not differ between the two groups of mice.
The level of catalase activity was significantly increased (P < 0.05) in the hyperoxia-exposed Tg mice, most prominently during the 72-hour adaptation period, and began to decrease by 84 hours in the two groups of mice (Figure 8a). The GPx activity in BALF increased significantly (P < 0.05) with time of exposure in both groups of animals but showed significantly higher levels (P < 0.05) in Tg mice at 84 hours compared with Wt littermates (Figure 8b). There was no discernible pattern of GPx activity in the total lung preparations in the two groups of mice (Figure 8c).
Antioxidant enzymes levels after hyperoxia exposure. (a) Catalase activity was measured in the total lung homogenate from mice exposed to hyperoxia. The enzyme activity increased significantly with time in transgenic (Tg) and wild-type (Wt) mice. (b) GPx activity was measured in the BALF of mice after exposure to hyperoxia. The enzyme increased significantly by 84 h of exposure. (c) GPx activity in the total lung homogenate from mice after exposure to hyperoxia. Values are expressed as mean ± SEM (n = 6). *P < 0.05 Tg vs. Wt. GPx, glutathione peroxidase.
Oxidation of glutathione and lipids. The percentage of the GSSG was significantly increased in the lung tissues of Wt and Tg mice by 84 hours of exposure (Wt: 19.6 ± 2.4, 53.0 ± 4.5, 71.0 ± 6.1; Tg: 21.0 ± 2.1, 66 ± 8.4, 86.0 ± 9.5 at 48, 72, and 84 hours, respectively). In the BALF, the percentage of oxidized GSSG also increased significantly with time of exposure (Wt: 66.2 ± 4.7, 75.2 ± 7.7, 82.1 ± 7.0; Tg: 45.3 ± 7.0, 69.6 ± 13.5, 85.5 ± 5.8 at 48, 72, and 84 hours, respectively). At 72 hours of hyperoxia, ND independently and significantly reduced the GSSG levels in BALF (Figure 9a).
Markers of oxidative stress in the BALF after 72 h of hyperoxia exposure. (a) The level of oxidized GSH was significantly elevated in all groups after 72 h of >99% oxygen exposure, particularly in the oxygen–placebo (O-placebo) wild-type (Wt) mice in which the level of oxidized glutathione (GSSG) exceeds the GSSG levels in the other groups of mice. (b) The level of lipid peroxides measured as malondialdehyde was significantly attenuated in the neutropenic animals and hEC-SOD transgenic (Tg) mice. Values are expressed as mean ± SEM (n = 6). *P < 0.05 Tg vs. Wt, and neutrophil-depleted (ND) vs. controls; **P < 0.01 Tg vs. Wt, and ND vs. controls. GSH, reduced glutathione.
Exposure to hyperoxia resulted in enhanced lipid peroxidation, as measured by levels of MDA in both the BALF and lung tissue and increased with the time of exposure. In the Wt BALF, MDA levels measured 43.5 ± 4.1, 305.6 ± 41.5, and 417.2 ± 49.8 pmol/ml at 48, 72, and 84 hours of exposure, respectively, whereas BALF, MDA levels in Tg mice measured 19 ± 1.3, 123.7 ± 14, and 396.6 ± 33.7 pmol/ml. The difference between Wt and Tg animals was significant (P < 0.05) at 72 hours of exposure. When total lung MDA levels were measured, they showed an increase with time of exposure in the two groups but no significant differences between them (Wt: 9.4 ± 1.4, 16.7 ± 2.5, 21.2 ± 2.4 pmol/mg protein at 48, 72, and 84 hours; Tg: 8.8 ± 10, 20.5 ± 1.9, 24.7 ± 1.6 pmol/mg protein at 48, 72, and 84 hours, respectively). After 72 hours of hyperoxia exposure, there was a significant attenuation of BALF lipid peroxidation in ND Wt mice when compared with non-ND animals (Figure 9b). A further attenuation of BALF MDA levels was seen in ND Tg mice (Figure 9b).
Effect of hyperoxia on cytokine release in the BALF. The levels of TNF-α and MIP-2 in the BALF from ND and non-ND Wt and Tg mice were measured after exposure to either 72 hours of >99% O2 or to room air. With room air exposure, the levels of TNF-α and MIP-2 in the BALF of Wt and Tg mice did not differ from each other and were near the limits of detection of our assays (Figure 10, a and b). The two cytokines levels were significantly increased in the hyperoxia-exposed Wt animals, whereas these responses were blunted in the ND mice and even further so in the Tg animals.
Inflammatory cytokine levels in BALF after 72 h of hyperoxia exposure. (a) The level of TNF-α measured in the BALF after 72 h of >99% oxygen exposure increased significantly in all hyperoxic (O) groups. In the oxygen-exposed groups, both neutrophil depletion and overexpressing hEC-SOD resulted in significantly lower TNF-α. The levels of TNF-α in the room air–exposed mice were below the limits of detection. (b) The levels of MIP-2 were measured in BALF after 72 h of >99% oxygen exposure and demonstrated a pattern similar to TNF-α. The levels of MIP-2 in the BALF of room air–exposed mice were very low and typically below the limits of detection. Values are expressed as mean ± SEM (n = 6). *P < 0.05 transgenic (Tg) vs. wild-type (Wt), and neutrophil-depleted (ND) vs. controls; **P < 0.01 Tg vs. Wt, and ND vs. controls. MIP-2, macrophage inflammatory protein-2; TNF-α, tumor necrosis factor-α.
ICAM-1 expression. To characterize further the mechanisms that underlie low neutrophil influx in the Tg mice lungs in response to hyperoxia, ICAM-1 mRNA levels were determined by RT-PCR analysis. The amount of GAPDH mRNA transcripts was used as a control for the RT-PCR reactions. The level of ICAM-1 mRNA was significantly different between the two groups after hyperoxia exposure (Figure 11). Hyperoxia induced a twofold increase in ICAM-1 mRNA in the lungs of Wt mice within 84 hours of exposure compared with Tg animals. The level of ICAM-1 mRNA was not significantly different in the two groups of animals at 48 hours of exposure, whereas 72 hours of hyperoxia elicits an increase in ICAM-1 mRNA in the lungs of both groups of animals.
Intercellular adhesion molecule-1 (ICAM-1) mRNA levels in the lung. After exposure to 48, 72, and 84 h of >99% hyperoxia, total RNA was extracted from both wild-type (Wt) and transgenic (Tg) mouse lungs and subjected to reverse transcriptase–PCR with primers specific for ICAM-1 and glyceraldehyde phosphate dehydrogenase (GAPDH) as outlined in the text. Aliquots of each PCR reaction were electrophoresed on agarose gels, stained with ethidium bromide, and signal intensity determined. Four animals were used at each time point. The top graph shows the raw agarose gel, and the bottom graph shows the level of ICAM-1 mRNA after normalization to GAPDH mRNA levels. *P < 0.05.
Histopathology. At each time point (48, 72, and 84 hours), the lungs of both Wt and Tg mice were fixed, sectioned, stained, and examined for evidence of lung injury. Using a previously described scoring scheme (27), pathological changes were recorded based on the degree of pulmonary edema, hyaline membrane formation, alveolar septal thickening, and inflammatory cell infiltration. Evidence of vascular injury was detected in the Wt mice as early as after 48 hours of hyperoxic exposure (data not shown). After 72 hours of hyperoxia exposure, the majority of lungs examined from Wt mice showed evidence for decreased pulmonary compliance as indicated by increased edema, thickening of the alveolar septal walls, hyaline membrane formation, vascular and interstitial congestion, hemorrhage, and diffuse inflammatory cell infiltration (Figure 12a), whereas mild histopathologic changes were seen in the Tg mice (Figure 12b). Increased microscopic lung damage was also observed in Tg mice after 84 hours of hyperoxia exposure; however, it was less severe than the injury seen in the Wt animals (data not shown).
Light microscopy of paraffin-embedded and hematoxylin and eosin–stained section of representative lungs from mice exposed to hyperoxia. (a) Representative lung sections from Wt mice exposed to 72 h of >99% oxygen showing hyaline membrane formation (open arrows), alveolar septal thickening, and cellular infiltration composed mainly of macrophages and PMNs (filled arrows). (b) Representative lung sections from Tg mice exposed to 72 h of >99% oxygen showing modest alveolar septal thickness and fewer numbers of alveolar macrophages (filled arrows) and PMNs. Original magnification: ×132.