Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Supplemental oxygen is commonly given to patients with cardiopulmonary disorders to enhance tissue oxygenation. Unfortunately, the prolonged administration of fractional inspired concentrations of oxygen greater than 50 to 60% leads to a variety of forms of tissue damage, including acute lung injury. The pathophysiology of this pulmonary toxicity has been characterized in animal models (1). These studies demonstrated that toxic concentrations of O₂ generate oxygen-derived free radicals that damage lung epithelial and endothelial cells, leading to a protein-rich fluid that floods the alveolar space. Recent researchers have also demonstrated that this injury is associated with a cell-death response with features of both cell necrosis and apoptosis (4, 5).

Interleukin (IL)-6 is a pleiotropic cytokine that is produced at sites of tissue inflammation. It is classified as an IL-6-type cytokine with IL-11, leukemia inhibitory factor, cardiotrophin-1, oncostatin M, and ciliary neurotrophic factor on the basis of the overlapping effector profiles of these cytokines and their shared use of gp130 as the -subunit in their multimeric receptor complexes (6). IL-6 induces fever, activates B and T lymphocytes, and stimulates hepatocytes to produce acute phase proteins. Recent studies have shown that IL-6 also has potent anti-inflammatory and protective properties. These include the ability to inhibit the production of tumor necrosis factor (TNF), IL-1, and macrophage inflammatory protein-2; decrease neutrophil sequestration; increase levels of IL-1 receptor antagonist and TNF soluble receptor; stimulate the production of metalloproteinase inhibitors; reduce intracellular superoxide production; reduce tissue matrix degradation; and inhibit cellular apoptosis (7- 14). Surprisingly, the ability of IL-6 to regulate hyperoxic lung injury has not been adequately investigated.

We hypothesized that the anti-inflammatory and cytoproptective effects of IL-6 could ameliorate hyperoxic lung injury. To test this hypothesis we compared the injury induced by 100% O₂ in transgenic mice in which IL-6 was selectively overexpressed in the lung (CC10-IL-6 mice) with appropriate transgene () littermate controls. These experiments demonstrated that CC10-IL-6 transgene (+) mice have an impressive ability to tolerate 100% O₂. This tolerance manifests as enhanced survival, decreased pulmonary edema and alveolar-capillary protein leakage, and decreased lung lipid peroxidation when compared with transgene () controls. Further, transgene () mice manifest an impressive cell-death response associated with DNA fragmentation which was inhibited in the IL-6 (+) animals. This protection was associated with the enhanced accumulation of the regulatory proteins Bcl-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of CC10-IL-6 Transgenic Mice

CC10-IL-6 transgenic mice were produced on a CBA × C57BL/6 background. In these mice the Clara cell 10-kD protein promoter (CC10) was used to overexpress IL-6 in a lung-specific fashion (in Clara cells and, to a lesser degree, alveolar epithelial cells). This results in 0.5 to 1.5 ng/ml levels of IL-6 in the animals' bronchoalveolar lavage (BAL) fluid (BALF). The methods used by our laboratory to generate these mice, the organ specificity of transgene targeting, and the pathologic alterations induced by IL-6 have been described elsewhere (15). Transgene () littermates were used as controls in all experiments.

Oxygen Exposure

Mice were exposed to 100% oxygen in a 50 × 50 × 30-cm airtight chamber and the fractional inspired O₂ concentration in the chamber was monitored with an inline oxygen analyzer (Vascular Technology, Inc., Chelmsford, MA) as described elsewhere (16).

Histologic Analysis

Transgenic and wild-type littermate control mice were killed, the right heart was perfused with saline, the trachea was cannulated, and the lungs were inflated to 25 cm of pressure with formalin. They were then fixed in 3% formalin and paraffin-embedded. Trichrome and hematoxylin and eosin stains were done on 5-µ sections in the Department of Pathology at Yale University School of Medicine.

Electron Microscopy

Lungs were isolated and the trachea was cannulated as described earlier. The lungs were then perfused in situ to 25 cm water pressure with 3.5% glutaraldehyde and postfixed for 24 h in 3.5% glutaraldehyde, and representative areas were embedded in epon. Ultrathin sections of embedded tissue were prepared, stained with 2% uranyl acetate and OsO₄, and examined using a Phillips EM300 electron microscope.

BAL

After anesthesia, a median sternotomy was performed and the trachea was transected. BAL was performed by instilling 0.6 ml of phosphate-buffered saline (PBS) and gently aspirating back. This was repeated three times. The samples were pooled and centrifuged, and the cell-free BALF was stored at 70°C until used. The total protein concentration in the BALF was measured using the Bio-Rad D.C. Protein Assay (Bio-Rad Labs, Hercules, CA).

Lung Tissue Homogenates

After anesthesia, a median sternotomy was performed, the aorta and inferior vena cava were transected, and the right ventricle was perfused with cold PBS to clear intravenous blood from the lungs. The lungs were then removed and homogenized, in either Tris-HCl (pH 7.4) or a potassium phosphate (pH 7.4) buffer in the presence of protease inhibitors, and centrifuged.

Lung Lipid Peroxidation

Oxidative stress can lead to lipid peroxidation and the subsequent generation of aldehydes that are formed when lipid hydroperoxides break down in biologic systems. The aldehydes most intensively studied so far are the 4-hydroxyalkenals (4-hydroxynonenal, 4-hydroxyhexenal) and malonaldehydes (such as malondialdehyde [MDA]). The assay that was used for tissue lipid peroxidation depends on the production of MDA, a three-carbon degradation product of lipid peroxidation. Detection of lung MDA was done colorimetrically by evaluating levels of thiobarbituric acid-reactive substances in whole-lung homogenates (17). Lungs were isolated and perfused with ice-cold 0.9% NaCl, and homogenates were prepared in ice-cold 20 mM Tris-HCl (pH 7.4). The homogenates were then centrifuged at 3,000 × g at 4°C for 10 min, and 200 µl of the supernatant were removed for the assay. Lipid peroxidation was assessed with a standard methodology that quantitates the interaction of a chromogenic reagent with malonaldehyde and 4-hydroxyalkenals (17) using the LPO-586 kit (Calbiochem, San Diego, CA). The LPO-586 Assay used in this analysis uses the chromogenic reagent N-methyl-2-phenylindole at 45°C. One molecule of either MDA or 4-hydroxyalkenal reacts with two molecules of N-methyl-2-phenylindole to yield a stable chromophore with maximal absorbance at 586 nm. The LPO-586 reaction, when catalyzed with methanesulfonic acid (as in our studies) can be used to assay MDA plus 4-hydroxyalkenals. When concentrated HCl is used as the catalyst, MDA alone, without interference with 4-hydroxyalkenals, is measured. Data are expressed as µM of products of lipid peroxidation in 200 µl of a 20% wt/vol solution of lung homogenate.

Superoxide Dismutase Analysis

The activities of copper/zinc superoxide dismutase (CuZnSOD) and manganese SOD (MnSOD) were assayed spectrophotometrically and by gel electrophoresis.

Spectrophotometric assay. Lung homogenates were prepared in a potassium phosphate (pH 7.4) buffer as described earlier, and cells were disrupted by sonication (Branson Sonifier; Branson Sonic Power, Danbury, CT) using three 15-s bursts at 4°C. A competitive inhibition assay was performed using hypoxanthine-xanthine oxidase-generated O₂· to reduce nitroblue tetrazolium (NBT) monitored spectrophotometrically at 560 nm. Inhibition of NBT reduction to 50% of maximal was defined as 1 unit of SOD activity (18). Inhibition of CuZnSOD activity by 5 mM potassium cyanide allowed differentiation of CuZnSOD and MnSOD.

Gel electrophoresis. The SODs were also assayed using the gel electrophoresis method of Beauchamp and Fridovich (19). This assay is based on the ability of SOD in a polyacrylamide gel to inhibit the reduction of NBT by superoxide anion generated using photochemically reduced riboflavin. Lung homogenates were prepared in 3 ml potassium phosphate buffer and 1.0 mM ethylenediaminetetraacetic acid, sonicated for 3 min, and centrifuged at 15,000 × g. Equal amounts of protein were run in a 10% polyacrylamide gel until the bromophenol blue marker dye came off the end of the apparatus. The gel was then placed in a solution of 2.45 × 10³ M NBT for 20 min; immersed in a N,N,N',N'-tetra-methyletholynediamine, riboflavin, and potassium phosphate solution for 15 min; and illuminated with a 15-W fluorescent light for 15 min. During illumination, the gels became uniformly blue except for areas containing SOD. Dilutions of purified Escherichia coli MnSOD and bovine erythrocyte CuZnSOD were used to generate standard curves, and the lung-extract MnSOD and CuZnSOD activities were measured from the linear regions of these curves. Assessments were done in the presence and absence of potassium cyanide to allow MnSOD and CuZnSOD to be differentiated.

TUNEL Analysis

Terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) analysis was performed as described by our laboratories (20). In brief, tissue sections (4 to 5 µm) were mounted onto slides pretreated with 3-aminopropylethoxysilane. Slides were baked and washed in fresh xylene, rehydrated through a series of graded alcohols, and washed in distilled water. For TUNEL assay, the tissue sections were treated with proteinase K and terminal transferase and then labeled with rhodamine-conjugated antidigoxigenin. All reagents were obtained from Boehringer Mannheim (Indianapolis, IN). Tissue sections were counterstained with 4', 6-diamidine-2-phenylindole-dihydrochloride (DAPI). To quantify the extent of TUNEL- positive cells in the mouse lung, samples were illuminated with ultraviolet light to visualize either TUNEL-positive nuclei (590 nm) or total DAPI-stained nuclei (420 nm). Images were captured with a CCD video camera. Uniform camera control settings were used for image capture, and image threshholding was identical for all images. The captured images were analyzed using the Metamorph system (Universal Imaging, West Chester, PA) on a personal computer. At least 25 fields were analyzed from at least three individuals at each time point. The TUNEL-positive index was calculated as the percent of TUNEL-positive nuclei divided by the DAPI-staining nuclei.

Western Blots

Western blot analysis was performed using the protein fraction of lung homogenates. Equal quantities of test and control samples (20 to 50 µg) and recombinant protein standards were fractionated by electrophoresis in a 5% stacking and 12% resolving sodium dodecyl sulfate polyacrylamide gel for 1 h at 110 mV. The proteins were then transferred onto nitrocellulose membranes, which were blocked overnight with 10% nonfat milk, washed with 0.05% Tween in PBS, and incubated with appropriate primary antibodies. Monoclonal antibodies (mAbs) against Bcl-2 were obtained from Trevigen, Inc. (Gaithersburg, MD) and Zymed Labs (San Francisco, CA) and used at a dilution of 1:5,000. The positive control protein for mouse Bcl-2 was a mouse M1 cell lysate obtained from PharMingen (San Diego, CA). mAb against mouse TIMP-1 was obtained from Oncogene Research Products (Cambridge, MA) and used at a dilution of 1 µg/ml. The secondary antibody was goat antimouse immunoglobulin G (Pierce Labs, Rockford, IL) (1:10,000; 1 h). The protein bands were imaged using an enhanced chemiluminescence kit (Pierce Labs) and exposed for 30 to 120 s on Kodak Biomax MR film (Eastman Kodak, Rochester, NY). Equal protein loading was confirmed by Coomassie blue staining of all gels.

Statistical Analysis

Data are expressed as means ± standard error of the mean (SEM). Normally distributed data were assessed for significance by Student's t test or analysis of variance, as appropriate. Data that were not normally distributed were assessed for significance by the Wilcoxon rank sum test. Differences in survival between transgene (+) and () mice were assessed using ² analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of IL-6 on Survival

To determine if IL-6 has the ability to ameliorate oxygen toxicity, we compared the survival in 100% O₂ of transgene (+) CC10-IL-6 mice with their wild-type littermate controls. In keeping with observations from our laboratory and others (1, 16, 21), all wild-type mice died within 72 to 96 h of 100% O₂ exposure. In contrast, the transgene (+) mice were remarkably resistant to hyperoxia (Figure 1). All transgene (+) mice lived for more than 8 d, and more than 90% survived for longer than 12 d (Figure 1). During this exposure interval, the transgene (+) mice demonstrated normal behavior and normal levels of activity.

View larger version (12K): [in this window] [in a new window]   Figure 1.   Survival of CC10-IL-6 mice exposed to 100% O2. Comparisons are made of transgene (+) (triangles) and () (squares) littermate animals. Each value illustrates the percent survival of 20 mice.

Effect of IL-6 on Lung Histology

To further understand the mechanism(s) of IL-6-induced protection in the setting of hyperoxia, we compared the gross and microscopic features of the lungs of transgene (+) and () mice after exposure to 100% O₂ for 72 h. On gross examination, the lungs of the transgene () mice appeared boggy and hemorrhagic with copious edema fluid upon transection of the trachea. In contrast, the transgene (+) lungs were unchanged from baseline. Microscopically, 72 h of 100% O₂ exposure induced a mild neutrophilic infiltrate, capillary congestion, and alveolar hemorrhage in transgene () mice, but not in the transgene (+) littermates (data not shown).

Ultrastructurally, 48 h of 100% O₂ exposure caused extensive cell damage, manifest as membrane swelling, ruffling, and bleb formation in the lungs of the transgene () animals. These changes were even more apparent after 72 h of hyperoxia (Figure 2). In contrast, the membranes of the transgene (+) animals had a normal ultrastructural appearance, even after 8 to 10 d of 100% O₂ exposure (Figure 2 and data not shown).

View larger version (128K): [in this window] [in a new window]   Figure 2.   Ultrastructural comparisons of transgene () and transgene (+) animals after exposure to 100% O2 for 72 h. (A) Bleb formation and membrane ruffling (solid arrows) in the lungs of transgene () animals. (B) In contrast, alveolar type II (solid arrow) and type I (white arrow) cells from transgene (+) animals have normal-appearing membranes. Both panels are shown at the same original magnification: ×9,200.

Effect of IL-6 on Alveolar-Capillary Protein Leakage

To further characterize the effects of IL-6, we compared the levels of protein in the BALF from transgene (+) mice and wild-type littermate controls. At baseline, similar levels of BALF protein were detected in transgene (+) and wild-type mice. In contrast, exposure to 100% O₂ caused a significant increase in alveolar-capillary leakage and BALF protein. This response was significantly greater in transgene () than in the transgene (+) mice. After exposure to 100% O₂ the BALF from transgene () and (+) mice contained 3.89 ± 0.57 and 1.17 ± 0.17 mg/ml of protein, respectively (P < 0.05) (Figure 3). These studies demonstrate that IL-6 decreases 100% O₂-induced alveolar-capillary protein leakage.

View larger version (13K): [in this window] [in a new window]   Figure 3.   BALF protein content of transgene () and (+) mice. BAL was performed on CC10-IL-6 transgene (+) (open bars) and transgene () littermate control (closed bars) mice at baseline and after 72 h of exposure to 100% O2. The levels of protein were assessed as described in MATERIALS AND METHODS. The values represent the means ± SEM of determinations performed on six animals at each time point (*P < 0.05 versus transgene [+] animals at the same time point).

Effect of IL-6 on Lung Lipid Peroxidation

Because toxic concentrations of oxygen mediate their effects, in part via lipid peroxidation, we compared the levels of lipid peroxidation in lung homogenates from transgene (+) mice and transgene () littermate controls both before and after exposure to hyperoxia. At baseline, the levels of lipid peroxidation by-products in the lungs of transgene () mice and transgene (+) mice were similar (Figure 4). After 72 h of exposure to 100% oxygen there was a significant increase in the level of lung lipid peroxidation in the transgene () mice (P < 0.001). In contrast, the transgene (+) mice did not show a statistically significant increase in the level of lipid peroxidation (P = 0.06). In addition, after 72 h of 100% O₂ exposure, the levels of lipid peroxidation by-products in lungs from transgene () animals were significantly greater than in lungs from transgene (+) animals (P < 0.008) (Figure 4). This demonstrates that the transgenic overexpression of IL-6 blunts 100% O₂-induced membrane lipid peroxidation.

View larger version (12K): [in this window] [in a new window]   Figure 4.   Lung lipid peroxidation in transgene () and transgene (+) mice. CC10-IL-6 transgene (+) mice (open bars) and transgene () littermate controls (filled bars) were evaluated before and after exposure to 100% O2 for 72 h. Total lung lipid peroxidation was assessed as described in MATERIALS AND METHODS. Values represent the means ± SEM of 10 animals at each time point (*P < 0.008 comparing transgene [] and [+] animals at this time point).

Effect of IL-6 on Antioxidant Enzymes

SOD enzymes are believed to play a significant role in the protection of the lung from hyperoxia (22). To address the possibility that IL-6-induced protection is due to IL-6-induced alterations in SOD activity, we measured the activities of MnSOD and CuZnSOD in equal amounts of lung tissue from transgene () and (+) mice before and after exposure to 100% oxygen. Both gel electrophoresis and spectrophotometric approaches failed to reveal significant differences in MnSOD or CuZnSOD activities before and after 72 h of 100% oxygen exposure (data not shown). Thus, IL-6 confers protection in the setting of hyperoxic lung injury via a mechanism that is largely independent of the major protective enzymes MnSOD and CuZnSOD.

Effect of IL-6 on Nuclear DNA Fragmentation

Hyperoxic lung injury is characterized by a cell-death response with features of apoptosis and necrosis (4, 5). Because both can be associated with DNA fragmentation (27) we used TUNEL assays to assess the levels of DNA fragmentation in transgene () and (+) mice before and after exposure to 100% O₂. In these studies, the data are expressed as a fragmentation index, which is the percentage of total nuclei that were TUNEL-positive. At baseline, only rare TUNEL-positive cells were noted and similar numbers of TUNEL-positive cells were noted in transgene () and (+) mice (data not shown). Exposure to 100% O₂ for 72 h caused a significant increase in the labeling of stromal cells in transgene () and (+) mice (Figure 5). Interestingly, the fragmentation index of transgene () mice was significantly greater than that of transgene (+) animals (27.3 versus 15.1, respectively) (Figure 5) (P < 0.05). This demonstrates that the protective effects of IL-6 are associated with decreased cell death and DNA fragmentation.

View larger version (38K): [in this window] [in a new window]   View larger version (7K): [in this window] [in a new window]   Figure 5.   Effect of IL-6 on cellular DNA fragmentation. CC10- IL-6 transgene () and transgene (+) mice were exposed to 100% O2 for 72 h. DNA fragmentation was then evaluated with the TUNEL assay as described in MATERIALS AND METHODS. A and B are representative photomicrographs illustrating the levels of nuclear staining in transgene () and (+) lungs, respectively, at low power (×40). Insets show DAPI staining of all nuclei. (C) Percent of TUNEL-positive nuclei in lungs from transgene () (filled bars) and (+) (open bars) mice. *P < 0.05.

Effect of IL-6 on Bcl-2 and BAX

The Bcl-2 family of proteins are potent regulators of many pathways of apoptosis (reviewed in References 28 and 29) and necrosis (30, 31). Bcl-2 also has antioxidant properties in some, but not all, experimental systems (32). This led us to speculate that Bcl-2 proteins might play a role in the IL-6-induced protection that was noted. To further investigate this possibility, we used immunoblot analysis to characterize the accumulation of selected Bcl-2 proteins in transgene (+) and () mice before and after exposure to 100% O₂. Comparisons of Bcl-2 and BAX were undertaken because of their known abilities to inhibit and stimulate cell-death responses, respectively (28, 29). Our data demonstrate impressive differences in the levels of Bcl-2 in transgene () and (+) mice (Figure 6). At baseline, the levels of Bcl-2 were significantly greater in (+) than in () mice. Exposure to 100% O₂ for 72 h did not significantly alter this relationship. In contrast, the levels of BAX were similar in transgene () and (+) mice before and after O₂ exposure (Figure 6). These data demonstrate that the CC-10-IL-6 transgenic mice have higher levels of Bcl-2, but not BAX, in lung tissue compared with transgene () littermate controls.

View larger version (21K): [in this window] [in a new window]   Figure 6.   Bcl-2 and BAX expression in CC10-IL-6 transgenic mice. Lungs were obtained from transgene () and (+) mice before and after exposure to 100% oxygen, and the protein fractions of the whole-lung homogenates were assessed for Bcl-2 and BAX protein using Western blot analysis as described in MATERIALS AND METHODS. Representative transgene () and (+) animals (n = 9) are illustrated.

Effect of IL-6 on TIMP-1

TIMP-1 is an important inhibitor of matrix metalloproteinases (MMPs) in many tissue-inflammatory responses (reviewed in Reference 35). Recently, TIMP-1 has also been shown to have antioxidant properties and to inhibit cellular apoptosis (36). To investigate the possible role of TIMP-1 in the IL-6-induced protection that was noted, we used immunoblot analysis to characterize the accumulation of TIMP-1 protein in the lungs from transgene () and (+) mice before and after 72 h of 100% O₂ exposure. These studies demonstrated the heightened accumulation of TIMP-1 protein in the lungs from transgene (+) as versus the transgene () mice (Figure 7). Exposure to 100% O₂ did not significantly alter this relationship (Figure 7).

View larger version (7K): [in this window] [in a new window]   Figure 7.   TIMP-1 expression in CC10-IL-6 transgenic mice. Homogenates were prepared from lungs of transgene (+) and () mice before and after exposure to 100% oxygen, and the levels of TIMP-1 accumulation were evaluated by Western analysis. They are compared with recombinant TIMP-1. Representative transgene (+) and () animals (n = 9) are illustrated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To determine whether exogenous IL-6 has protective effects in the setting of hyperoxic lung injury we compared the survival and indices of pulmonary injury in transgenic mice that overexpress IL-6 in a lung-specific manner with their transgene () littermate controls. These studies demonstrated that IL-6 diminishes hyperoxic lung injury. This protection was manifest as significantly enhanced survival and decreased tissue injury, alveolar-capillary protein leakage, and lung lipid peroxidation. These studies also showed that exposure to 100% O₂ causes a prominent TUNEL-positive cell-death response and that IL-6 inhibits this cytopathic reaction. Lastly, our investigations demonstrated that this IL-6-induced effect is not associated with alterations in SOD activity that can account for this protection, but is associated with elevated levels of the cell-death inhibitors Bcl-2 and TIMP-1.

IL-6 is a well-recognized mediator of both acute and chronic tissue inflammation that is induced during acute lung injury (37, 38). It also has anti-inflammatory properties, is protective in models of tissue injury, and can ameliorate tissue damage in a variety of lung injury systems (7). Our studies are the first to show that the expression of IL-6 can minimize the injury caused by 100% O₂. Since IL-1 and TNF induce IL-6 elaboration by a variety of lung stromal cells (39), these observations suggest that IL-6 may be an important mediator of the protective effects of IL-1 and TNF in hyperoxic lung injury (40). These observations also complement those of Tsan and colleagues (42), who demonstrated that IL-6 can interact with IL-1 and TNF to mediate protection from hyperoxic lung injury. There are, however, a number of important differences between our findings and those of Tsan and associates that deserve comment. First, in contrast to Tsan and coworkers, our studies showed that IL-6 expression by itself is sufficient to induce this protective state. In addition, the protection that we noted did not appear to be mediated by MnSOD, whereas that described by Tsan and colleagues was associated with the induction of and potentially mediated by this important antioxidant enzyme. The explanation for these different results is not fully clear. They may, however, reflect the different experimental protocols that were used. Our studies used overexpression transgenic technology which, by definition, exposes pulmonary tissues to IL-6 for a prolonged time period before the initiation of hyperoxic conditions. Tsan and associates (42) administered IL-6 via tracheal insufflation, and administered the cytokine and initiated the hyperoxic exposure simultaneously. Therefore, their animals were exposed to IL-6 for shorter intervals and probably had lower concentrations of IL-6 at sites of lung injury. When viewed in combination, however, both studies support the contention that IL-6 has important protective effects in this injury state.

Pulmonary oxygen toxicity is mediated by reactive oxygen intermediates (ROIs), which damage membrane proteins and lipids, polysaccharides, and DNA. Thus, to protect lung tissue from hyperoxic injury, one needs to limit the generation of ROIs, augment antioxidant defenses, or induce cytoprotective responses that diminish tissue susceptibility to ROI-induced damage. Our studies showed that IL-6 decreases 100% O₂-induced tissue injury and begins to investigate the mechanism(s) of this protective response. The mechanistic studies do not address the possibility that IL-6 might limit ROI generation. They do, however, demonstrate that IL-6-induced protection is not associated with alterations in the levels of MnSOD or CuZnSOD that could explain this protective response. This is in keeping with earlier studies from our laboratory showing that the protection induced by IL-11 in the setting of 100% O₂ exposure is mediated by a SOD-independent mechanism (16) and studies that demonstrate that MnSOD is an important, but not the only, protective mechanism controlling oxidant-mediated lung injury (25, 26, 43, 44). Importantly, these studies show that IL-6 is a prominent inhibitor of 100% O₂-induced cell death and DNA fragmentation. This observation is in accord with documented ability of IL-6-type cytokines to inhibit apoptotic cell death in a number of experimental systems (14, 45). When viewed in combination, these studies suggest that the protective effects of IL-6 are mediated, at least partially, by the ability of IL-6 to induce cytoprotective responses in end-organ tissues that diminish their susceptibility to ROI-induced injury.

At sites of tissue injury, cells can die via necrosis or apoptosis. Traditionally, these processes have been considered operationally and mechanistically distinct cell-death responses (49, 50). However, recent studies have demonstrated that the distinction between apoptosis and necrosis may not be as crisp as once thought. This blurring is the result of studies showing that apoptosis-like DNA laddering can be seen in cells undergoing necrosis, that known inducers of apoptosis can cause cells to die via necrosis, that apoptosis and necrosis can be induced by the same agent in the same cell population, and that apoptosis and necrosis can be present simultaneously in tissues experiencing the same injury (4, 5, 27, 49, 51). Early studies of pulmonary oxygen toxicity demonstrated cell swelling and other features compatible with cellular necrosis (1, 52, 53). More recent studies have demonstrated DNA fragmentation compatible with apoptosis and suggested that the cell-death response in oxygen toxicity has features of both processes (4, 5). Although the exact nature of this cell-death response is still being investigated, it is clear from our studies that IL-6 has impressive cytoprotective properties in this injury system. To gain insight into the mechanism(s) of this protection, we determined whether altered IL-6 altered the accumulation of proteins that regulate these responses. Bcl-2 was a particularly attractive candidate because it has been shown to inhibit apoptosis and necrosis (28, 49, 54). Our studies demonstrate that IL-6 is a potent inducer of pulmonary Bcl-2 accumulation. This supports the hypothesis that IL-6- induced Bcl-2 may play an important role in the protection that was noted. Additional investigation will be required to test the validity of this hypothesis. If Bcl-2 is shown to play an important role in this protective response, additional investigations will also be required to determine whether the beneficial effects of Bcl-2 are the result of its ability to alter cell-death responses and/or is ability to act as an antioxidant, as has been demonstrated in some, but not all, experimental systems (32).

The matrix-degrading activities of MMPs are controlled by a variety of inhibitors, including the members of the TIMP family. Studies of TIMP-1, the original member of this family, have focused almost exclusively on its ability to inhibit MMP activity. More recent studies, however, have shown that TIMP-1 may have a number of other important biologic roles at sites of injury. Particularly relevant to the present investigations are the studies of Guedez and colleagues (36) demonstrating that TIMP-1 can inhibit apoptosis via a mechanism that is independent of its anti-MMP activity. Because it is reasonable to believe that an agent that preserves lung scaffolding while decreasing cell death could be protective in the setting of oxygen toxicity, studies were undertaken to determine whether IL-6 regulated TIMP-1 in lung structures. These studies show that IL-6 is a potent stimulator of pulmonary TIMP-1 accumulation. This observation is in accord with prior in vitro investigations demonstrating that IL-6-type cytokines are potent inducers of TIMP-1 elaboration (55). These findings support the hypothesis that IL-6-induced protection in the setting of 100% O₂ exposure is mediated, in part, by the induction of TIMP-1. When the Bcl-2 and TIMP-1 data are viewed in combination, it is reasonable to speculate that IL-6-induced protection is mediated via a number of different mechanisms and that the multimechanistic nature of the effects of IL-6 are responsible, at least in part, for the impressively potent protection that is conferred by IL-6 in this injury setting.

In summary, our studies show that IL-6 has impressive protective effects in the setting of 100% O₂-induced acute lung injury. Toxic concentrations of oxygen are given to large numbers of patients throughout the world on a daily basis. Oxidant-induced organ damage also plays a major role in the pathogenesis of a variety of other pulmonary and nonpulmonary disorders. Our studies suggest that IL-6-type cytokines can improve the therapeutic index of hyperoxic O₂ administration and favorably alter the course of oxidant-mediated disorders. Additional investigation is warranted to determine whether exogenously administered IL-6 is useful therapeutically or prophylactically in the setting of hyperoxia-induced lung injury and other oxidant-induced injury states.