Superoxide dismutase moderates basal and induced bacterial adherence and interleukin-8 expression in airway epithelial cells
Yuko Arita,
Ansamma Joseph,
Hshi-Chi Koo,
Yuchi Li,
Thomas A. Palaia,
Jonathan M. Davis, and
Jeffrey A. Kazzaz
CardioPulmonary Research Institute and the Departments of Medicine (Divisions of Pulmonary and Critical Care Medicine and Nephrology), Thoracic and Cardiovascular Surgery and Pediatrics (Neonatology), Winthrop-University Hospital, SUNY Stony Brook School of Medicine, Mineola, New York 11501
Submitted 31 December 2003
; accepted in final form 21 July 2004
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ABSTRACT
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Bacterial infection of the tracheobronchial tree is a frequent, serious complication in patients receiving treatment with oxygen and mechanical ventilation, resulting in increased morbidity and mortality. Using human airway epithelial cell culture models, we examined the effect of hyperoxia on bacterial adherence and the expression of interleukin-8 (IL-8), an important mediator involved in the inflammatory process. A 24-h exposure to 95% O2 increased Pseudomonas aeruginosa (PA) adherence 57% in A549 cells (P < 0.01) and 115% in 16HBE cells (P < 0.01) but had little effect on Staphylococcus aureus (SA) adherence. Exposure to hyperoxia, followed by a 1-h incubation with SA, further enhanced PA adherence (P < 0.01), suggesting that hyperoxia and SA colonization may enhance the susceptibility of lung epithelial cells to gram-negative infections. IL-8 expression was also increased in cells exposed to both hyperoxia and PA. Stable or transient overexpression of manganese superoxide dismutase reduced both basal and stimulated levels of PA adherence and IL-8 levels in response to exposure to either hyperoxia or PA. These data indicate that hyperoxia increases susceptibility to infection and that the pathways are mediated by reactive oxygen species. Therapeutic intervention strategies designed to prevent accumulation of intracellular reactive oxygen species may reduce opportunistic pulmonary infections.
bacteria; inflammation; cytokines; antioxidants; adenovirus
LUNG INFECTION IN CRITICALLY ill patients continues to be a major clinical problem. The estimated prevalence of nosocomial pneumonia ranges from 10 to 65%, with fatality rates >25% in most studies (21). The incidence of ventilator-associated pneumonia (VAP) is 4360% higher among patients with adult respiratory distress syndrome (2, 8, 29). Furthermore, pneumonia can be demonstrated in as many as 67% of lungs from mechanically ventilated patients histologically examined at autopsy (28).
Colonization of the upper airway by a variety of bacterial species, including gram-positive and enteric gram-negative bacteria, occurs before invasive pulmonary infection has developed. The most common pathogens in VAP are Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA), with some data suggesting a progression of infection with SA leading to subsequent PA infections (33). VAP usually develops within 10 days after the onset of adult respiratory distress syndrome, with colonization of the lower airway preceding VAP in two-thirds of cases (8). Adherence of bacteria to airway epithelial cells is a key element in the initial stages of infection, leading to colonization of the respiratory tract and to subsequent invasive pulmonary infections.
Severely ill hospitalized patients are often exposed to supraphysiological concentrations of oxygen. Reactive oxygen species (ROS) can injure the lung by overwhelming endogenous antioxidant defenses, causing the release of inflammatory mediators, damage to epithelial cell surfaces, and impairment of bacterial clearance (20, 27). Although numerous studies have demonstrated that damage to the epithelium exposes receptors on the basolateral surface, leading to increased bacterial adherence (7, 22, 32), no studies have examined the molecular pathways induced by hyperoxia on bacterial adherence.
Several studies have suggested that acute and chronic lung injury from hyperoxia and mechanical ventilation may be ameliorated by the administration of the antioxidant superoxide dismutase (SOD) (6, 17, 36). Three isoforms of SOD have been identified: cytosolic copper-zinc SOD (CuZnSOD), extracellular CuZnSOD, and mitochondrial manganese SOD (MnSOD). Studies in transgenic mice and cell culture models have demonstrated that targeted overexpression of MnSOD to airway epithelial cells confers protection from hyperoxia-induced lung injury (16, 38). Although these studies have shown benefits in the reduction of hyperoxic injury, there have been no studies addressing whether this strategy will help moderate or prevent hyperoxia-related infection.
Interleukin-8 (IL-8) is a potent neutrophil chemokine that has been associated with increased morbidity, mortality, and chronic lung inflammation. It is transcriptionally regulated and induced in response to a variety of stimuli, including infection and oxidant stress (see Ref. 31 for review). However, the potential interaction of hyperoxia and PA in the induction of IL-8 expression has not been examined. In this report, cell culture models of airway and alveolar epithelium were used to study the effects of hyperoxia on bacterial adherence, IL-8 expression, and the ability of MnSOD to modulate these processes.
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MATERIALS AND METHODS
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Generation of stable cell lines, cell culture, and cell viability assays.
Human adenocarcinoma alveolar epithelial cells, A549 (ATCC, Manassas, VA), were maintained in F12-K medium supplemented with 10% fetal bovine serum, 1% glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (GIBCO BRL, Rockville, MD) and maintained at 37°C in 5% CO2-95% room air (RA). Human bronchial epithelial cell line 16HBE (kindly provided by D. Gruenert, University of Vermont) were maintained in Dulbecco's modified medium supplemented with 10% fetal bovine serum, 1% glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (GIBCO BRL) at 37°C in 5% CO2-95% RA (3, 20).
Stable cell lines were generated by transfection of A549 cells with the pWESOD2 construct, which contains the full-length human MnSOD cDNA driven by the cytomegalovirus (CMV) promoter (16). Cells with genomic incorporation were initially selected based on antibiotic resistance (G418-sulfate, GIBCO Biochemical) and screened as previously described (16). All isolates expressed comparable amounts of MnSOD, and two independent cell lines (SOD2#3 and SOD2#5) were randomly selected for use in these studies. MnSOD activity in SOD2#3 and SOD2#5 was increased 1.7- and 1.4-fold, respectively, compared with the vector or wild-type A549 cells. These cell lines were maintained in media described below, supplemented with G418-sulfate (800 µg/ml). All experiments were performed in the absence of G418.
Hyperoxic conditions were generated in sealed humidified chambers flushed with 5% CO2-95% O2 (20). Membrane permeability was determined by exclusion of trypan blue dye, and cells were counted with a hemacytometer. Mitochondrial activity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma, St. Louis, MO) assay, which measures mitochondrial dehydrogenase activity (37) as recommended by the manufacturer.
Recombinant adenoviral-mediated expression of MnSOD.
Recombinant adenovirus (rAd) was used for the transient induction of MnSOD expression (transduction). Viral transduction was performed with a replication-deficient rAd, Ad.CMVMnSOD, constructed and grown at the University of Iowa as previously described (40). A rAd containing LacZ (Ad.CBlacZ) was constructed and grown in the Vector Core at the Institute for Human Gene Therapy (Philadelphia, PA) and used as transduction controls (11). Expression was induced 1 day posttransduction and remained elevated for 4 days posttransduction in A549 cells (data not shown). For this reason, A549 cells were seeded at subconfluence and then transduced with Ad.CMVMnSOD or Ad.CBlacZ viron particles in complete media. Cells were washed, and fresh media were added after 17 h. Cells were then harvested and seeded at 5.5 x 104/cm2 for adherence or IL-8 assays 4 days posttransduction (see below). MnSOD enzyme activity was assayed at the time of experiment in duplicate cultures.
Bacterial strains.
Nonmucoid laboratory strain of PA (PAO-1) and SA (ATCC 25903) were grown in tryptic soy broth. Bacteria were metabolically labeled with [35S]methionine for 18 h and then washed extensively with PBS before use.
Adherence assay.
Adherence to epithelial cells was assessed with a modified radiolabel assay as described previously (15). Briefly, A549 cells were seeded onto 12-well dishes at a density of 5.5 x 104/cm2 and allowed to adhere overnight. 16HBE cells were seeded onto 12-well dishes at a density of 8.8 x 104/cm2. Cells were then exposed to 95% O2 or RA at 37°C for 24 h. The radiolabeled bacteria (108 per well) were then added onto the cell layer and incubated for 1 h. Cells were washed with PBS to remove nonadherent bacteria and lysed with 1 N NaOH, and lysates were counted in a liquid scintillation counter. For pretreatment experiments, cells were exposed to either RA or hyperoxia for 24 h and then exposed to nonradiolabeled PA or SA (108 per well) for 1 h. The cells were extensively washed with PBS, and an adherence assay was performed.
IL-8 assay.
The cell culture supernatant from the control and experimental groups were collected, and particulate was removed by centrifugation (350 g for 10 min). IL-8 expression was assayed colorimetrically with the IL-8 ELISA kit (Beckman Coulter, Brea, CA) at an optical density of 450 nm using 550 nm as a reference filter with a Beckman ELISA reader. Samples were diluted to ensure that the readings were within the linear range of the assay.
Electron microscopy.
Cells were seeded onto 35-mm dishes, incubated in either RA or 95% O2, and fixed in 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h at 4°C. The cells were then postfixed in buffered 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in LX112 (Ladd, Burlington, VT). Thin sections were stained with uranyl acetated and lead citrate and examined on a Zeiss EM 10 transmission electron microscope.
Statistical analysis.
All data are reported as means ± SD. Statistical analysis was performed using Student's t-test or ANOVA with Fisher's paired least-significant difference post hoc analysis using StatView version 5.01 (SAS Institute, Cary, NC). P values were considered significant at <0.05.
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RESULTS
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Effects of hyperoxia on mitochondrial structure and bacterial adherence.
To determine whether oxidant-induced stress pathways also affect bacterial adherence, we exposed A549 and 16HBE cells to 95% O2 for 24 h and assayed for the adherence of PA and SA. Previous studies have established that this is a nonlethal exposure (20). Exposure of A549 cells to hyperoxia resulted in a 57.1 ± 2.7% increase of PA adhesion (Table 1) (P < 0.01, ANOVA). This effect was even greater with exposure of 16HBE cells (115.4 ± 20.4%, P < 0.001, ANOVA). In contrast, hyperoxic exposure resulted in only a modest increase in SA adhesion to either A549 cells (16 ± 0.3%, P < 0.01) or 16HBE cells (6.5 ± 1.8%, P = not significant). These results demonstrate that hyperoxia increases the adherence of PA to lung epithelial cells and that the increase was significantly greater in airway compared with alveolar cells. Furthermore, hyperoxia induced more of an increase in PA adherence than in SA adherence in both epithelial cell types.
Several studies have addressed the effect of prolonged exposure to hyperoxia on epithelial cells (1, 14, 20, 24). These studies have demonstrated that hyperoxia inactivates mitochondrial enzymes (e.g., succinate dehydrogenase, aconitase, etc.), inducing growth arrest and cell death. The majority of these studies have focused on subconfluent cultures that allowed assessment of both growth arrest and cell death. However, the experiments described here were performed with confluent cultures, which precludes our ability to assess the effect on growth arrest; therefore, the effect of a short (24 h) exposure on mitochondrial enzymes and cell death was assessed. The MTT assay, which assesses mitochondrial dehydrogenase activity, was used as an early marker of oxidant stress in mitochondria. As shown in Table 1, MTT activity decreased to 67.4% (P < 0.01) of baseline in A549 and 56.2% (P < 0.001) in 16HBE cells compared with RA controls after 24 h of hyperoxia exposure. The effect on viability was examined next. A 24-h exposure to 95% O2 minimally reduced cell viability in both cell types (Table 2). These data indicate that hyperoxia induces oxidant stress but not widespread damage during a 24-h exposure. Several reports suggest that damage to the epithelial layer results in unmasking of receptors on the basolateral surface. To determine the effect of these exposures on the ultrastructure of the cell, hyperoxia- and RA-exposed A549 and 16HBE cells were examined by electron microscopy. In a previous study, we (20) had demonstrated that exposure of subconfluent A549 cells to 95% O2 for 6 days resulted in cellular and nuclear swelling. As shown in Fig. 1, a 24-h exposure induced some mitochondrial swelling (see arrows) relative to RA controls; however, there were no disruptions in the tight junctions noted (see Fig. 1, insets). Although not conclusive, these data suggest that the majority of the damage is limited to the mitochondria.

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Fig. 1. Electron micrographs of adjacent A549 and 16HBE cells in room air (RA) and hyperoxia. Confluent cultures of A549 and 16HBE cells were exposed to either RA or 95% O2 for 24 h as indicated. Cells were fixed, sectioned, and examined by transmission electron microscopy. Mitochondrial swelling (see arrows) is evident. Boxes outline areas that are magnified in the insets, demonstrating that the integrity of tight junctions (arrowheads) is maintained in hyperoxia-exposed cells. Scale bar = 1 µm.
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Next, we determined whether hyperoxia-induced pathways interact synergistically with bacterial colonization to further predispose epithelial cells to gram-negative infection. A549 and 16HBE cells were exposed to hyperoxia for 24 h and treated with either unlabeled PA or SA for 1 h, and then the adhesion assay was performed with labeled PA for 1 h as described above. Exposure of A549 cells to hyperoxia followed by preincubation with PA led to an additional 22.9% increase in PA adherence (P < 0.01) compared with cells exposed to hyperoxia alone (Fig. 2A). When A549 cells were preincubated with SA, PA adherence increased by 30.0% (P < 0.01) compared with cells exposed to hyperoxia alone (Fig. 2A). Similarly, PA adhesion increased by 13.7% in 16HBE cells (P < 0.001) with PA pretreatment, whereas preincubation with SA increased subsequent PA adherence by 30.6% (P < 0.0001) compared with cells treated with hyperoxia alone (Fig. 2B). In contrast, preincubation with PA did not enhance SA adherence in either cell line in RA or in hyperoxia (data not shown). In RA control cells, bacterial pretreatment did not increase subsequent PA adhesion (data not shown). Together, these results suggest that SA colonization promotes subsequent PA colonization only when epithelial cells are exposed to hyperoxia, presumably due to an upregulation of PA receptors by ROS.

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Fig. 2. Hyperoxia induces bacterial adhesion in lung epithelial cells. A549 (A) and 16HBE (B) cells were exposed to 95% O2 for 24 h. A subset of cells were then exposed to either no bacteria, Pseudomonas aeruginosa (PA), or Staphylococcus aureus (SA) for 1 h in RA at 37°C. After these pretreatments, adherence of PA was measured by radiolabeled PA as described (39). Adherent PA (dpm) were normalized to the epithelial cell number, and numbers were plotted by average of 6 experiment points. *P < 0.01, **P < 0.001, and #P < 0.0001, relative to RA control.
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Overexpression of MnSOD improves mitochondrial function.
A549 cells with chromosomal integration of an exogenous copy of the MnSOD cDNA driven by the CMV promoter were generated. The resulting stable cell lines (SOD2#3 and SOD2#5) were then tested for their ability to protect cells exposed to 95% O2. Because hyperoxia inactivates mitochondrial enzymes and MnSOD is localized in the mitochondria, the effect of MnSOD overexpression on mitochondrial function was assessed with the MTT assay. Mitochondrial dehydrogenase activity decreased to 77.2% compared with RA control (P < 0.001) in empty vector controls when exposed to 95% O2 (Fig. 3). The MnSOD-overexpressing SOD2 cell lines had improved dehydrogenase activity compared with empty vector controls (85.8%, P < 0.01 in SOD2#3 and 90.6%, P < 0.02 in SOD2#5 cell lines, Fig. 3). To determine whether similar results would be achieved by transient expression, expression was induced with Ad.CMVMnSOD with activity measured at comparable levels to stable cell lines. Protection against hyperoxic injury was comparable in cells undergoing transient and stable expression (data not shown). Taken together, these data indicate that overexpression of MnSOD ameliorated the effects of hyperoxia, reflected by the improvement in mitochondrial dehydrogenase activity.

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Fig. 3. Manganese SOD (MnSOD) prevents hyperoxia-induced inhibition of mitochondrial activity in A549 cells. pWE4#5, SOD2#3, and SOD2#5 cell lines were exposed to either RA or 95% O2 for 24 h. Dehydrogenase activity was determined by the MTT assay per manufacturer's instructions. Values represent percentage of dehydrogenase activity in hyperoxia-treated cells relative to the RA control. Values were plotted by average of 8 experiment points from 2 independent experiments. *P < 0.05 and #P < 0.001, relative to empty vector control (ANOVA).
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MnSOD reduces O2-induced increases in bacterial adhesion.
As illustrated in Fig. 4A, stable cell lines overexpressing MnSOD had a significant reduction of bacterial adherence in RA (2148%, P < 0.02) compared with vector controls. Although PA adherence did increase when the SOD cells were exposed to hyperoxia, it was reduced by an average of 55% (P < 0.0001) compared with vector controls exposed to hyperoxia. To confirm that these effects were not a result of clonal differences, A549 cells were transduced with Ad.CMVMnSODto increase expression transiently. MnSOD activity at a multiplicity of infection of 75 viral particles per cell resulted in a twofold higher level of expression compared with nontransduced cells. In transduced cells, basal (RA) levels of bacterial adhesion were reduced by an average of 58% in SOD transduced cells compared with controls (P < 0.001, ANOVA) (Fig. 4B). When exposed to 95% O2, bacterial adhesion was reduced by an average of 50.8% in SOD-transduced cells relative to Ad.CBLacZ controls (P < 0.001, ANOVA). These results with two different methods demonstrate that a moderate increase of MnSOD activity can effectively reduce PA adherence in lung epithelial cells both in RA and after prolonged exposure to hyperoxia. The reduction of adherence detected in RA-exposed cells overexpressing MnSOD suggests that reducing the basal level of ROS production helps moderate the subsequent response in hyperoxia.

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Fig. 4. MnSOD reduces the hyperoxia-induced PA adhesion. A: empty vector (pWE4#5) and MnSOD cell lines (SOD2#3 and SOD2#5). B: Ad.CBLacZ-transduced (LacZ) and Ad.CMVMnSOD-transduced (MnSOD) A549 cells. Cells were exposed to either RA or 95% O2 for 24 h. Solid bars represent RA, and the open bars represent hyperoxia. PA adherence was normalized to the radioactivity (dpm) of bacteria per cell number. *P < 0.01, **P < 0.001, ***P < 0.0001, and #P < 0.02, relative to empty vector or LacZ controls.
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Effect of hyperoxia and MnSOD on IL-8 levels.
Because IL-8 is an important proinflammatory chemokine associated with lung inflammation and infection, we hypothesized that hyperoxia would induce IL-8 expression. To assess IL-8 expression, 16HBE and A549 cells were exposed to hyperoxia and IL-8 levels were assayed by ELISA. In 16HBE cells, basal levels of IL-8 were 72.4 ± 0.1 pg/ml, with hyperoxia inducing expression by 127.4% (P < 0.0001, Fig. 5A). The basal level of expression in A549 cells was 22.1 ± 1.1 pg/ml, and levels increased by 61.3% after hyperoxic exposure (P < 0.001) (Fig. 5B). In 16HBE cells, both basal and induced levels of IL-8 per cell were significantly higher than in A549 cells (P < 0.0001).

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Fig. 5. Hyperoxia induces interleukin-8 (IL-8) production and PA binding to epithelial cells further increases IL-8 production. 16HBE cells (A) and A549 cells (B) were cultured in either RA or 95% O2 for 24 h. A subset of cultures were then exposed to PAO-1 for 1 h, and IL-8 levels were determined by ELISA. Values were normalized against cell number and represented as pg/ml of culture medium of 104 cells. Values represent mean of 4 samples from 2 independent experiments (n = 8) ± SD. *P < 0.01, **P < 0.001, and #P < 0.0001, relative to RA control [Fisher's paired least-significant difference (PLSD)].
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Because PA adherence increases after exposure to hyperoxia and PA adherence induces an IL-8 response (9), we predicted that the combination of PA and hyperoxia would result in an additive response. To test this hypothesis, 16HBE and A549 cells were exposed to either RA or 95% O2 for 24 h followed by a 1-h incubation with PA. As shown in Fig. 5A, the addition of PA to 16HBE cells (RA) induced a 50% increase in IL-8 expression (from 72.4 to 108.8 pg/ml, P < 0.01). Exposure to hyperoxia followed by PA exposure increased IL-8 production by 180% (269.2 pg/ml) relative to RA controls and 124.5% relative to cells exposed to hyperoxia alone (216.0 pg/ml, P < 0.001). Similar results were obtained with A549 cells (Fig. 5B). The addition of PA to A549 cells increased expression by 69% compared with RA control (from 22.1 to 37.5 pg/ml, P < 0.001). Exposure to hyperoxia before PA exposure resulted in a 134% (51.9 pg/ml) increase in IL-8 production relative to RA controls (P < 0.0001) and corresponding to an additional 72.5% (16.1 pg/ml) relative to cells exposed to hyperoxia alone (P < 0.001). The induction of IL-8 by exposure to hyperoxia in 16HBE cells was more prominent than in A549 cells, supporting the observation that bronchial epithelial cells were more susceptible to hyperoxia and that IL-8 serves as a marker for cell stress.
The effects of MnSOD overexpression on IL-8 production were then examined. MnSOD-overexpressing cell lines demonstrated reductions in both basal and induced levels of expression (P < 0.0001 relative to pWE4#5, Fig. 6A). In MnSOD-overexpressing cells, the stimulated levels of IL-8 were reduced to 41.8% with PA treatment, to 19.6% with hyperoxia exposure, and to 58.7% with both hyperoxia and PA treatment. Although the reduction of IL-8 levels was evident, IL-8 expression was still induced in a similar manner by exposure to either PA or hyperoxia. The patterns were similar in both MnSOD cell lines. However, to ensure that this was not a cloning anomaly, we tested the effect of transiently expressing MnSOD. As illustrated in Fig. 6B, an identical pattern was seen with MnSOD reducing the basal and stimulated levels of IL-8 in transiently induced cells (P < 0.0001 relative to Ad.CBlacZ, Fig. 6B). A reduced basal level of expression indicates that ROS signaling is involved in regulation of IL-8 expression, a response that is moderated by MnSOD. The induction by all stimuli was observed in the MnSOD overexpressors, but levels of IL-8 were well below RA-exposed control cells (pWE4#5 and Ad.CBLacZ). These data indicate that both hyperoxia and bacterial infection increase inflammatory cytokine production in lung epithelial cells in an additive fashion, with increased MnSOD activity dampening the overall response.

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Fig. 6. MnSOD reduces the hyperoxia-induced IL-8 production. A: empty vector control cells (pWE4#5) and SOD2 A549 cells. B: Ad.CMVLacZ-transduced and Ad.RSVMnSOD-transduced A549 cells. Cells were exposed to either RA or to 95% O2 for 24 h. A subset of cultures were then exposed to PAO-1 for 1 h, and IL-8 levels were determined by ELISA. *P < 0.01, **P < 0.02, and #P < 0.0001, relative to controls (Fisher's PLSD).
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DISCUSSION
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Data indicate that hyperoxia increases PA adherence in lung epithelial cells, suggesting an upregulation of specific receptors responsible for the increase in PA adherence. Indeed, several adhesin-receptor interactions have been identified for PA (12, 22, 25, 26, 30). The increases in SA adherence, although statistically significant in A549 cells, were moderate in comparison. Because both SA and PA strains can bind to asialo-GM1 (22) and their adherence levels differed in hyperoxia, it is unlikely that the increased adherence was due to an increase in this receptor, and further characterization is needed to identify the receptor(s) responsible. Cells exposed to hyperoxia followed by incubation with SA demonstrated a greater increase in PA adherence than those exposed to hyperoxia alone. This suggests that SA infection (or colonization) might further contribute to secondary PA infection in ventilated patients. Although this progression of infection has been established in patients with cystic fibrosis, our data together with microbiological studies (33) would indicate that ventilated patients are also susceptible to this type of progression.
Hyperoxia generates ROS that cause oxidation of macromolecules, including proteins (34), lipids (35), and DNA (14). The decrease in MTT activity found in cells exposed to hyperoxia is not surprising, considering that this assay measures mitochondrial dehydrogenase activity and hyperoxia is known to inhibit key metabolic enzymes (13, 34). Although this forces cells to utilize glycolysis to generate ATP (34), ATP is not depleted in these cells after 24 h of hyperoxia and is not a limiting factor (data not shown). As shown in electron micrographs, mitochondria were swollen under these exposure conditions in both airway and alveolar cells. It is unclear whether inhibiting these metabolic enzymes directly affects the ability of epithelial cells to clear bacteria.
Our results suggest that the increased susceptibility of the lower respiratory tract to gram-negative bacterial colonization is an early event (with airway cells significantly more susceptible to bacterial adherence and colonization than alveolar cells in both RA and in 95% O2). The deleterious effects of hyperoxia have been observed from various experimental models that demonstrate that hyperoxia can potentiate Ureaplasma urealyticum pneumonia in newborn mice and impair pulmonary clearance of PA in adult mice (4, 10). Johanson et al. (19) demonstrated an increased mortality from acute PA infection in hamsters, when infected hamsters were subsequently exposed to hyperoxia. If the increased mortality was due to damage, one would predict that preexposure would increase the mortality rate. However, a 4-day preexposure to hyperoxia did not result in a further increase of mortality compared with those exposed concurrently. Our data demonstrating that hyperoxia increases susceptibility to infection by increasing bacterial adherence without loss of tight junction integrity support the notion that the increase is pathway driven and not merely a function of oxidant damage to the epithelial layer. This initial trigger could lead to an inflammatory cascade, resulting in increased damage to the epithelium followed by colonization and subsequent infection.
In this report, an additive induction with the combination of hyperoxia and PA adherence was found. Exposure to hyperoxia induces signal transduction pathways, resulting in the rapid activation of the NF-
B (24) and a biphasic activation of AP-1 transcription factors (23). Given the role of these transcription factors in oxidative stress and the ability of MnSOD to reduce their activation (40), it is likely that these factors play a central role in the regulation of infection and inflammation in the lung. Studies to delineate the molecular pathways involved in their regulation are underway.
A moderate increase in MnSOD activity significantly reduced bacterial adherence and IL-8 expression in response to hyperoxia. The mechanism of this protection is unclear, but inactivation of mitochondrial enzymes was evident during O2 exposures and overexpression of MnSOD protects mitochondria, suggesting that protection of the mitochondria ameliorated cell stress and reduced subsequent bacterial adherence. We tested this notion by studying cells with increased expression of the CuZnSOD, which also demonstrated a similar but slightly less significant reduction of bacterial adherence than that of MnSOD (data not shown). It should be noted that transduction with Ad.CMVMnSOD increases both mitochondrial and cytosolic SOD levels; therefore, it is difficult to address the specific contributions of mitochondrial protection in these experiments. These results suggest that the reduction of bacterial adherence could be due to mitochondrial protection from oxidative damage or through moderation of the ROS-mediated signaling. H2O2 is a potent second messenger (18) that can affect transcriptional activation and cell cycle regulation. Because SOD catalyzes the conversion of superoxide to H2O2, scavenging H2O2 by the coexpression of other antioxidants (e.g., catalase or glutathione peroxidase) may further affect the response. Whether the source of the ROS is from normal respiration or from other ROS-generating enzyme systems (e.g., NADPH oxidase and xanthine oxidase), the scavenging of other ROS may also suppress the induced expression of PA receptors. These hypotheses will require further testing. Taken together, our data suggest a potential role of antioxidant enzymes in prevention of not only hyperoxic lung injury but also subsequent bacterial infection. Animal models of hyperoxic injury and human trials in premature infants treated with hyperoxia and mechanical ventilation have consistently demonstrated less lung injury and improved outcome with the prophylactic use of recombinant human CuZnSOD (5, 6). These studies, together with our results, demonstrate that reducing oxidant stress will reduce adherence and therefore may prevent colonization of the airways and subsequent bacterial infection.
In summary, cell culture models of airway epithelium were used to examine the role of hyperoxia on bacterial adhesion, IL-8 expression, and the response of these events to antioxidants. Hyperoxia increased the susceptibility of epithelial cells to bacterial adherence and resulted in an additive induction of IL-8 when bacteria (PA) were present. MnSOD was able to significantly reduce hyperoxia-induced increases in bacterial adherence and IL-8 expression and minimize mitochondrial damage. This has important implications in the development of therapeutic interventions using antioxidants to prevent bacterial lung infection in patients receiving supplemental oxygen.
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GRANTS
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This work was supported by grants from the American Lung Association (Y. Arita), the Cystic Fibrosis Foundation (J. A. Kazzaz), and the National Heart, Lung, and Blood Institute (J. M. Davis, Grant HL-64158).
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ACKNOWLEDGMENTS
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The authors thank Dr. Dieter Gruenert (University of Vermont) for providing the 16HBE cell line, Dr. Marlene Strayer (Thomas Jefferson University Medical College) for assistance and suggestions with the adenovirus experiments, Dr. Lin Mantell (North Shore University Hospital) for insight and suggestions throughout the course of this project, Dr. Scott Schroeder (Winthrop-University Hospital) for critical evaluation of this manuscript, and E. M. Gurzenda for technical help.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. A. Kazzaz, CardioPulmonary Research Institute, 222 Station Plaza North, Suite 604, Mineola, NY 11501 (E-mail: jkazzaz{at}winthrop.org)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
|
---|
- Allen C and White C. Glucose modulates cell death due to normobaric hyperoxia by maintaining cellular ATP. Am J Physiol Lung Cell Mol Physiol 274: L159L174, 1998.[Abstract/Free Full Text]
- Chastre J, Trouillet JL, Vuagnat A, Joly-Guillou ML, Clavier H, Dombret MC, and Gibert C. Nosocomial pneumonia in patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 157: 11651172, 1998.[Abstract/Free Full Text]
- Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, and Gruenert DC. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10: 3847, 1994.[Abstract]
- Crouse DT, Cassell GH, Waites KB, Foster JM, and Cassady G. Hyperoxia potentiates Ureaplasma urealyticum pneumonia in newborn mice. Infect Immun 58: 34873493, 1990.[ISI][Medline]
- Davis JM, Rosenfeld WN, Richter SE, Parad R, Gewolb IH, Spitzer AR, Carlo WA, Couser RJ, Price A, Flaster E, Kassem N, Edwards L, Tierney J, and Horowitz S. Safety and pharmacokinetics of multiple doses of recombinant human CuZn superoxide dismutase administered intratracheally to premature neonates with respiratory distress syndrome. Pediatrics 100: 2430, 1997.[Abstract/Free Full Text]
- Davis JM, Rosenfeld WN, Sanders RJ, and Gonenne A. Prophylactic effects of recombinant human superoxide dismutase in neonatal lung injury. J Appl Physiol 74: 22342241, 1993.[Abstract]
- De Bentzmann S, Roger P, and Puchelle E. Pseudomonas aeruginosa adherence to remodelling respiratory epithelium. Eur Respir J 9: 21452150, 1996.[Abstract/Free Full Text]
- Delclaux C, Roupie E, Blot F, Brochard L, Lemaire F, and Brun-Buisson C. Lower respiratory tract colonization and infection during severe acute respiratory distress syndrome: incidence and diagnosis. Am J Respir Crit Care Med 156: 10921098, 1997.[Abstract/Free Full Text]
- DiMango E, Zar HJ, Bryan R, and Prince A. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest 96: 22042210, 1995.[ISI][Medline]
- Dunn MM and Smith LJ. The effects of hyperoxia on pulmonary clearance of Pseudomonas aeruginosa. J Infect Dis 153: 676681, 1986.[ISI][Medline]
- Engelhardt JF, Yang Y, Stratford-Perricaudet LD, Allen ED, Kozarsky K, Perricaudet M, Yankaskas JR, and Wilson JM. Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses. Nat Genet 4: 2734, 1993.[ISI][Medline]
- Feldman M, Bryan R, Rajan S, Scheffler L, Brunnert S, Tang H, and Prince A. Role of flagella in pathogenesis of Pseudomonas aeruginosa pulmonary infection. Infect Immun 66: 4351, 1998.[Abstract/Free Full Text]
- Gardner PR, Nguyen DD, and White CW. Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs. Proc Natl Acad Sci USA 91: 1224812252, 1994.[Abstract/Free Full Text]
- Gille JJ, van BCG, Mullaart E, Vijg J, and Joenje H. Effects of lethal exposure to hyperoxia and to hydrogen peroxide on NAD(H) and ATP pools in Chinese hamster ovary cells. Mutat Res 214: 8996, 1989.[CrossRef][ISI][Medline]
- Grant MM, Niederman MS, Poehlman MA, and Fein AM. Characterization of Pseudomonas aeruginosa adherence to cultured hamster tracheal epithelial cells. Am J Respir Cell Mol Biol 5: 563570, 1991.[ISI][Medline]
- Ilizarov AM, Koo HC, Kazzaz JA, Mantell LL, Li Y, Bhapat R, Pollack S, Horowitz S, and Davis JM. Overexpression of manganese superoxide dismutase protects lung epithelial cells against oxidant injury. Am J Respir Cell Mol Biol 24: 436441, 2001.[Abstract/Free Full Text]
- Jacobson JM, Michael JR, Jafri MH Jr, and Gurtner GH. Antioxidants and antioxidant enzymes protect against pulmonary oxygen toxicity in the rabbit. J Appl Physiol 68: 12521259, 1990.[Abstract/Free Full Text]
- Janssen YM, Matalon S, and Mossman BT. Differential induction of c-fos, c-jun, and apoptosis in lung epithelial cells exposed to ROS or RNS. Am J Physiol Lung Cell Mol Physiol 273: L789L796, 1997.[Abstract/Free Full Text]
- Johanson WG Jr, Higuchi JH, Woods DE, Gomez P, and Coalson JJ. Dissemination of Pseudomonas aeruginosa during lung infection in hamsters. Role of oxygen-induced lung injury. Am Rev Respir Dis 132: 358361, 1985.[ISI][Medline]
- Kazzaz JA, Xu J, Palaia TA, Mantell L, Fein AM, and Horowitz S. Cellular oxygen toxicityoxidant injury without apoptosis. J Biol Chem 271: 1518215186, 1996.[Abstract/Free Full Text]
- Kollef MH. Epidemiology and risk factors for nosocomial pneumonia. Emphasis on prevention. Clin Chest Med 20: 653670, 1999.[ISI][Medline]
- Krivan HC, Roberts DD, and Ginsburg V. Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAc
14Gal found in some glycolipids. Proc Natl Acad Sci USA 85: 61576161, 1988.[Abstract]
- Li Y, Arita Y, Koo HC, Davis JM, and Kazzaz JA. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol 29: 779783, 2003.[Abstract/Free Full Text]
- Li Y, Zhang W, Mantell L, Kazzaz J, Fein A, and Horowitz S. Nuclear factor-
B is activated by hyperoxia but does not protect from cell death. J Biol Chem 272: 2064620649, 1997.[Abstract/Free Full Text]
- Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol 280: L181L187, 2001.[Abstract/Free Full Text]
- Lillehoj EP and Kim KC. Pseudomonas aeruginosa flagellin is an adhesin for Muc1 mucin and stimulates its phosphorylation (Abstract). Am J Respir Crit Care Med 163: A674, 2001.
- Mantell LL, Kazzaz JA, Xu J, Palaia TA, Piedboeuf B, Hall S, Rhodes G, Niu G, Fein AM, and Horowitz S. Unscheduled apoptosis during acute inflammatory lung injury. Cell Death Differ 4: 604607, 1997.
- Marquette CH, Copin MC, Wallet F, Neviere R, Saulnier F, Mathieu D, Durocher A, Ramon P, and Tonnel AB. Diagnostic tests for pneumonia in ventilated patients: prospective evaluation of diagnostic accuracy using histology as a diagnostic gold standard. Am J Respir Crit Care Med 151: 18781888, 1995.[Abstract]
- Meduri GU, Reddy RC, Stanley T, and El-Zeky F. Pneumonia in acute respiratory distress syndrome. A prospective evaluation of bilateral bronchoscopic sampling. Am J Respir Crit Care Med 158: 870875, 1998.[Abstract/Free Full Text]
- Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, and Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 20852088, 1998.[Abstract/Free Full Text]
- Rahman I. Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. J Biochem Molec Biol 36: 95109, 2003.[ISI]
- Rajan S, Cacalano G, Bryan R, Ratner AJ, Sontich CU, van Heerckeren A, Davis P, and Prince A. Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells. Analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors. Am J Respir Cell Mol Biol 23: 304312, 2000.[Abstract/Free Full Text]
- Rello J and Torres A. Microbial causes of ventilator-associated pneumonia. Semin Respir Infect 11: 2431, 1996.[Medline]
- Schoonen WG, Wanamarta AH, Van der Klei-Van Moorsel JM, Jakobs C, and Joenje H. Hyperoxia-induced clonogenic killing of HeLa cells associated with respiratory failure and selective inactivation of Krebs cycle enzymes. Mutat Res 237: 173181, 1990.[CrossRef][ISI][Medline]
- Sullivan SJ, Roberts RJ, and Spitz DR. Replacement of media in cell culture alters oxygen toxicity: possible role of lipid aldehydes and glutathione transferase in oxygen toxicity. J Cell Physiol 147: 427433, 1991.[ISI][Medline]
- Tanswell AK and Freeman BA. Liposome-entrapped antioxidant enzymes prevent lethal O2 toxicity in the newborn rat. J Appl Physiol 63: 34752, 1987.[Abstract/Free Full Text]
- Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A, and Boyd MR. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res 51: 25152520, 1991.[Abstract]
- Wispe JR, Warner BB, Clark JC, Dey CR, Neuman J, Glasser SW, Crapo JD, Chang LY, and Whitsett JA. Human Mn-superoxide dismutase in pulmonary epithelial cells of transgenic mice confers protection from oxygen injury. J Biol Chem 267: 2393723941, 1992.[Abstract/Free Full Text]
- Wu CL, Domenico P, Hassett DJ, Beveridge TJ, Hauser AR, and Kazzaz JA. Subinhibitory bismuth-thiols reduce virulence of Pseudomonas aeruginosa. Am J Respir Cell Mol Biol 26: 731738, 2002.[Abstract/Free Full Text]
- Zwacka RM, Zhou W, Zhang Y, Darby CJ, Dudus L, Halldorson J, Oberley L, and Engelhardt JF. Redox gene therapy for ischemia/reperfusion injury of the liver reduces AP1 and NF-
B activation. Nat Med 4: 698704, 1998.[ISI][Medline]
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