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* Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109;
Department of Microbiology, Toho University School of Medicine, Tokyo, Japan; and
Laboratory of Molecular Biology of Infectious Agents, Course of Emerging Infectious Diseases, Graduate School of Biomedical Science, Nagasaki University, Nagasaki, Japan
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Introduction |
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In lung tissue, bacteria multiply in several types of host cells, including macrophages, monocytes, and alveolar epithelial cells (7, 8, 9). Cytopathogenicity of L. pneumophila to host cells has been well demonstrated (10, 11, 12), although incompletely understood. Accumulating data indicate that L. pneumophila can induce apoptosis in macrophages and alveolar epithelial cells in vitro (13, 14, 15). Apoptosis is a highly regulated process of cell death that is required for development and homeostasis of multicellular organisms in physiological condition (16, 17). The apoptosis-inducing receptors (death receptors), such as TNFR (p55 and p75) and Fas, bind TNF-
and Fas ligand (death factors), respectively, which result in activation of downstream signaling molecules, including caspases and phospholipases. Fas-mediated signal appears to exclusively drive apoptosis, whereas TNFRs are capable of both activating cells, and eliminating cells by initiating apoptosis (18, 19). Several investigators have reported critical roles of death receptor-mediated apoptosis for modulation of host responses in certain infectious diseases (20, 21). A wide variety of pathogens modulate the host cell-death pathway by direct interaction with key components of the apoptosis machinery of the host (22, 23, 24). However, how apoptosis is involved in the pathogenesis of Legionella disease is still unknown.
Oxygen supplementation is commonly given to the patients with severe pneumonia, including Legionella disease. This is a critical supportive therapy, especially for the patients demonstrating severe hypoxemia. However, prolonged administration or even transient supplementation of oxygen can promote lung damage (25, 26, 27, 28). Cells at risk for hyperoxia-induced injury include alveolar epithelial cell and lung microvascular endothelial cells. Although mechanisms of oxygen toxicity to the lungs have not been carefully defined, it is likely that apoptosis plays a certain role in hyperoxia-associated lung injury (27, 29). Moreover, the effect of hyperoxia as a cofactor in the development of acute lung injury in bacterial pneumonia is unknown.
We examined the interaction between hyperoxia and L. pneumophila infection in a murine model of pneumonia. Our findings indicate that hyperoxia markedly increases mortality in mice with L. pneumophila pneumonia. The enhanced lethality is associated with acceleration of acute lung injury, but not increases in bacterial burden in the lungs. Interestingly, hyperoxia enhances apoptosis in the lungs of animals with Legionella pneumonia. It is likely that death receptor-mediated signaling is involved in the acceleration of apoptosis in hyperoxic conditions, because an increase in survival was observed in Fas-deficient mice with Legionella pneumonia in the setting of hyperoxia.
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Materials and Methods |
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Female specific pathogen-free 4- to 6-wk-old A/J, C57BL/6, and B6.MRL-Faslpr (Fas-deficient mice) were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in specific pathogen-free conditions within the animal care facility at the University of Michigan until the day of sacrifice.
Culture and inoculation of bacteria
We used a clinical isolate of L. pneumophila Suzuki strain (serogroup 1) for all experiments. N-(2-acetamido)-2-aminoethanesulfonic acid (Sigma-Aldrich, St. Louis, MO)-buffered yeast extract (BYE)3 broth supplemented with L-cysteine (0.4 mg/ml) and ferric nitrate (0.135 mg/ml) was used as liquid medium (BYE-broth). To prepare solid medium, activated charcoal (2 mg/ml) and agar (15 mg/ml) were added to liquid medium (buffered charcoal yeast extract-agar). L. pneumophila was incubated on buffered charcoal yeast extract-agar for 4 days at 37°C. A single colony was transferred to 3 ml of BYE-broth, and was then incubated overnight at 37°C with constant shaking. Bacterial suspension was again transferred to fresh BYE-broth, and incubated overnight in the same condition. According to a standard of absorbencies based on known CFU, the bacterial suspension was diluted to the desired concentration in saline. Animals were anesthetized i.p. with 6 and 100 mg/kg xylazine and ketamine, respectively. The trachea was exposed, and 30 µl of inoculum or saline was administered via a sterile 26-gauge needle. The skin incision was closed with surgical staples.
Oxygen exposure
Two hours after intratracheal (i.t.) administration of bacteria, one group of mice was kept in hyperoxic conditions in a 50 x 30 x 30 cm airtight chamber for 60 h, whereas another group was placed in room air conditions. For hyperoxic exposure, the oxygen concentration in the chamber was kept between 90 and 94% by a constant flow of gas, which was monitored with an in-line oxygen analyzer (model D2; Beckman, Fullerton, CA). Carbon dioxide levels in the chamber were maintained at 
0.03–0.04% during the course of experiments. Both groups of mice were fed food and water ad lib and kept on a 12-h dark-light cycle at room temperature.
Lung harvesting for analysis
At designated time points, mice were sacrificed by CO2 asphyxia. Before lung removal, the pulmonary vasculature was perfused with 1 ml of PBS containing 5 mM EDTA, via the right ventricle. Whole lungs were then harvested for assessment of bacterial number, cytokine protein expression, and apoptosis. After removal, whole lungs were homogenized in 1.0 ml of PBS with protease inhibitor (Boehringer Mannheim, Indianapolis, IN) using a tissue homogenizer (Biospec Products, Bartlesville, OK) under a vented hood. Portions of homogenates (10 µl) were inoculated on agar after serial 1/10 dilutions with PBS. The remaining homogenates were incubated on ice for 30 min, then centrifuged at 2500 rpm for 10 min. Supernatants were collected, passed through a 0.45-µm filter (Gelman Sciences, Ann Arbor, MI), and then stored at -20°C for further analysis.
Bronchoalveolar lavage (BAL)
Mice were sacrificed 2 days after bacterial challenge, and BAL was performed. The trachea was exposed and intubated using a 1.7-mm outer diameter polyethylene catheter. BAL was performed by instilling PBS containing 5 mM EDTA in 1-ml aliquots. Approximately 5 ml of lavage fluid was retrieved per mouse. Cytospins were subsequently prepared from BAL cells for cell differentiation. In some experiments, albumin concentrations in BAL fluid were determined by mouse albumin ELISA quantification kit (Bethyl Laboratories, Montgomery, TX).
TUNEL staining
The TUNEL assay was performed on paraffin-embedded lung tissue sections using a commercially available kit (FragEL DNA fragmentation detection kit; Oncogene Research Products, Boston, MA).
Murine alveolar epithelial cell isolation and culture
Murine type II alveolar epithelial cells were isolated using the method developed by Corti et al. (30). Briefly, following pentobarbital anesthesia and heparin administration, C57B/6 mice were exsanguinated, and the pulmonary vasculature was perfused via the right ventricle with 0.9% NaCl. The lungs were then filled via the trachea with 1–2 ml of dispase (Worthington, Lakewood, NJ). Subsequently, 0.45 ml of low melting point agarose was infused via the trachea, and the lungs were placed in ice for 2 min to harden the agarose. The lungs were then submerged in dispase for 45 min at 24°C before the lung tissue was teased from the airways and minced in DMEM with 0.01% DNase. After swirling for 10 min followed by passage through a series of nylon filters, the cell suspension was collected by centrifugation and incubated with biotinylated Abs (anti-CD32 and anti-CD45; BD PharMingen, San Diego, CA). After incubation with streptavidin-coated magnetic particles, bone marrow-derived cells were removed with a magnetic tube separator. Mesenchymal cells were removed by overnight adherence in a petri dish. The nonadherent cells after this initial plating were plated at a density of 2 x 105–2 x 106/cm2 on plastic dishes coated with fibronectin and were maintained in DMEM with penicillin/streptomycin and 10% FCS. Cells were washed with PBS 1 h after plating. This technique routinely generates 5–6 x 106 cells/mouse, with a final adherent population that includes only 4% nonepithelial cells (by intermediate filament staining). Culture cells on day 3 were placed in a glass chamber and exposed to either normoxic or hyperoxic conditions (95% O2) in the presence or absence of L. pneumophila (15 to 1 bacteria to cell ratio). After inoculation of 106 CFU of bacteria, no increase of bacterial number in each well was observed during the course of experiments. The 3 day time point was chosen because isolated alveolar epithelial cells by this time in culture are relatively pure (>90% alveolar epithelial cells) and have begun a transition to a more type I-like phenotype. At indicated time points, histone-associated DNA fragments and transepithelial voltage were determined as described in Materials and Methods.
Assessment of transepithelial voltage differences
To assess alveolar epithelial cell barrier function in vitro, type II cells were cultured in the upper chamber of Transwell culture dishes on tissue culture-treated polycarbonate membranes (0.4-µm pore size, 6.5 mm diameter; Corning Costar, Cambridge, MA). The resistance across the monolayer was determined at various time points using a Millicell device (Millipore, Bedford, MA) with one sterile electrode in the medium in the upper chamber and one sterile electrode in the medium in the lower chamber (31).
Determination of histone-associated DNA fragments and caspase-3 activity
To evaluate induction of apoptosis, levels of histone-associated DNA fragments and caspase-3 activity were determined in lung homogenates. DNA fragmentation was quantified by measuring histone-associated DNA fragments using an ELISA kit (Cell Death Detection ELISAplus, Roche Diagnostics, Mannheim, Germany). Because L. pneumophila is reported to induce apoptosis in vitro in a caspase-3-dependent manner (14), caspase-3 activity was determined by a colorimetric assay (R&D Systems, Minneapolis, MN), in which caspase-specific peptide conjugated to the color reporter molecule p-nitroanilide was used. The data are expressed as a fold increase, comparing to those of control mice (n = 5).
Murine TNF- ELISA
Murine TNF- was quantitated using a modification of a double ligand method as previously described (32). Standards were 2-fold dilutions of murine recombinant cytokine from 1 pg/ml to 100 ng/ml. This ELISA method consistently detected murine TNF- concentration >20–50 pg/ml. The ELISA did not cross-react with other cytokines, such as IL-1,IL-2, IL-6, IL-12, and IFN-
. In addition, the ELISA did not cross-react with members of the murine chemokine family, including murine KC, macrophage-inflammatory protein-2 and -1, monocyte chemoattractant protein-1, or RANTES.
Neutralization of TNF- by specific antiserum
To neutralize TNF- biologic activity, rabbit anti-murine TNF- antiserum was i.p. injected into mice 2 h (0.5 ml) before and 48 h (0.2 ml) after bacterial challenge. Normal rabbit preimmune serum was used as a control. This Ab has been shown to neutralize TNF- both in vitro and in vivo (32).
Statistical analysis
Statistical significance was determined using the unpaired, two-tailed alternate Welch t test and the nonparametric Mann-Whitney test. Calculations were performed using InStat for Macintosh (GraphPad, San Diego, CA). Statistical analyses of survival curves were performed by the log rank test using the Prism software program (GraphPad).
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Results |
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Macrophages from A/J mice are permissive for growth of Legionella organism, whereas those from other strains of mice, including C57BL/6, are nonpermissive (33, 34). However, recent data showed that this organism is capable of inducing apoptosis in host cells without intracellular bacterial replication in vitro (13, 14). Therefore, we first examined effects of hyperoxia on survival in permissive A/J and nonpermissive C57BL/6 mice with Legionella pneumonia (Fig. 1). After i.t. administration of bacteria, one group of animals was kept in hyperoxic conditions for 60 h, while another group was placed in room air during the observation period. Interestingly, in both strains of mice, drastic decreases in survival were observed in the group of animals exposed to hyperoxic conditions. Specifically, in C57BL/6 mice, only 10% of control mice died, whereas >90% of mice exposed to hyperoxia died by 7 days post-infectious challenge. A similar increase in lethality was observed in infected A/J mice under hyperoxic condition, as compared with infected animals breathing room air. Importantly, hyperoxia treatment alone (90–94% oxygen for 60 h), without infection, induced no death of mice in either strain (data not shown). To determine whether the hyperoxia-associated increase in lethality was specific for Legionella pulmonary infection, we assessed survival of mice with Klebsiella pneumoniae pneumonia in normal and hyperoxic conditions. In contrast to Legionella pneumonia, we did not observe any detrimental effects of hyperoxia on survival of animals with Klebsiella pneumonia (data not shown). These data clearly demonstrate that hyperoxia increases the lethality of L. pneumophila pneumonia, but not K. pneumoniae pneumonia. Moreover, increase of lethality was observed in both permissive A/J and nonpermissive C57BL/6 mice, suggesting that intracellular growth of Legionella is not required for hyperoxia-induced lethality.
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To determine the cause of increased lethality under hyperoxic conditions, we next examined bacterial number in the lungs of C57BL/6 mice on days 2 and 4 after challenge with L. pneumophila (6.7 x 106 CFU). In the room air and hyperoxia groups, bacterial numbers on day 2 were 6.6 x 105 and 7.0 x 105 CFU/lungs, which decreased to 3.0 x 104 and 5.2 x 104 CFU/lungs on day 4, respectively. Importantly, we did not observe any differences in pulmonary bacterial number between these two groups. In addition, there was no evidence of dissemination of bacteria to extrapulmonary organs, including spleen and blood (data not shown). Similarly, exposure to hyperoxia did not alter bacterial counts in the lungs of A/J mice challenged with L. pneumophila (data not shown). Next, we examined cell numbers and differential in BAL fluid of mice on day 2 after Legionella challenge. Hyperoxia alone did not induce any changes in total BAL cell number. A >10-fold increase in total cell number was demonstrated in the BAL fluid of infected mice; however, no difference between mice exposed to hyperoxia or room air was observed. Cell differential of the infected mice indicated that 80% of cells were neutrophils in both hyperoxia and room air groups, and again there was no difference between these two groups (data not shown). These data indicate that hyperoxia-induced lethality is not due to increased bacterial burden in the lungs nor bacterial dissemination to extrapulmonary organs. Moreover, inflammatory cell numbers and cell types recruited into the lung did not account for the increased lethal sensitivity to Legionella pneumonia after exposure to hyperoxia.
Effect of hyperoxia on the development of lung injury in mice with Legionella pneumonia
To evaluate the magnitude of lung injury in Legionella-infected mice exposed to room air and hyperoxic conditions, we compared total lung weights and albumin concentrations in BAL fluid on day 2 after Legionella challenge (Fig. 2). Hyperoxic conditions used in the present study (90–95% oxygen for 60 h) failed to induce changes in lung weights, as compared with that observed in control uninfected mice. In contrast, Legionella infection induced a clear increase in total lung weights, with a 1.5-fold increase in infected mice kept in room air and a nearly 2-fold increase of total lung weight in Legionella-infected animals exposed to hyperoxic conditions (p < 0.05). Albumin concentration in BAL fluid correlated well with changes in lung weights. Specifically, Legionella challenge resulted in significantly higher BAL albumin concentrations, as compared with uninfected animals. Importantly, a >2-fold increase in BAL albumin levels were noted in infected mice exposed to hyperoxia compared with those observed in infected mice kept in normoxic conditions (p < 0.05). These data demonstrate that Legionella infection significantly increases lung permeability and that lung leak is exacerbated by exposure to hyperoxia independent of changes in lung bacterial burden.
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To investigate the cause of accelerated lung injury in hyperoxia, we examined quantitative markers of apoptosis, including histone-associated DNA fragments and caspase-3, in the lungs of mice 2 days after Legionella challenge (Fig. 3). Histone-associated DNA fragments are a marker for DNA fragmentation, one of the main characteristics of apoptosis, whereas caspase-3 is an essential protease mediating apoptosis. Hyperoxia alone did not induce evidence of apoptosis. However, Legionella infection induced a clear increase in the presence of both markers within the lungs. The increase in DNA fragments and caspase-3 activity was most marked in the lungs of infected animals exposed to hyperoxia, with 22- and 1.8-fold increases, respectively, compared with control uninfected animals (p < 0.05). In addition, evidence of DNA laddering was observed in agarose gel analysis after extraction of chromosomal DNA from infected lungs, which was more pronounced in the lungs of hyperoxic animals (data not shown). These studies indicate that hyperoxia treatment exaggerates apoptosis in the lungs of mice infected with L. pneumophila. In contrast, we did not observe an increase of histone-associated DNA levels in K. pneumoniae-infected lungs in the setting of hyperoxia (data not shown), which closely correlated with the lack of oxygen effects on survival in Klebsiella-infected mice.
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To more directly define the effect of hyperoxia on Legionella-induced alveolar epithelial cell apoptosis and barrier dysfunction, we established an in vitro system to model in vivo oxygen exposure. As shown in Fig. 5A, exposure to normoxia for 24 h in the presence or absence of L. pneumophila resulted in minimal alveolar epithelial cell apoptosis. In contrast, exposure to hyperoxia alone resulted in a modest elevation of histone-DNA complexes, which was significantly increased in the presence of L. pneumophila (p < 0.01, comparing to room air plus Legionella infection). To assess the effects of hyperoxia on barrier function, transepithelial voltage differences were determined across the epithelial monolayer (Fig. 5B). No differences in initial transepithelial voltage were noted in infected or uninfected alveolar epithelial cells cultured for 24 h at various oxygen tensions. However, by 48 h of exposure to hyperoxia, there was a significant decrease in transepithelial voltage (indicative of increased lung permeability) in Legionella-infected cells, as compared with infected or uninfected cells cultured at room air, or uninfected cells cultured in hyperoxia. Taken together, these data indicate that hyperoxia can potentiate Legionella-induced alveolar epithelial cell apoptosis and epithelial permeability.
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To examine the role of apoptosis in the survival of mice with Legionella pneumonia exposed to hyperoxia, we infected Fas-deficient and control C57BL/6 mice with L. pneumophila, and then placed animals in hyperoxic conditions for 60 h (Fig. 6). This dose of bacteria was sublethal for control mice in normoxic conditions. However, C57BL/6 mice exposed to hyperoxia started to die by day 3 after bacterial challenge, with survival of only 33% by the end of observation. In contrast, Fas-deficient mice demonstrated partial resistance to the lethal effects of Legionella after hyperoxic exposure, with no death observed until day 5 after bacterial challenge, and 67% of Fas-deficient mice surviving to the end of observation (p < 0.05). Next, we examined histone-associated DNA fragments in the lungs of these control C57BL/6 and Fas-deficient mice (Fig. 7). Consistent with survival data, the hyperoxia-induced increase in histone-associated DNA fragments observed in infection was significantly attenuated in Fas-deficient mice (p < 0.05). These results strongly suggest that Fas-mediated apoptosis partially mediates hyperoxia-associated lethality in mice with Legionella pneumonia.
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on hyperoxia-associated apoptosis
To further define potential mechanisms of hyperoxia-associated apoptosis in Legionella pneumonia, we examined pulmonary TNF- levels 2 days after bacterial challenge (Fig. 8). TNF- was targeted because this cytokine has been shown to be up-regulated in the setting of oxidant stress (35, 36), and can induce apoptosis in certain settings (16, 19). Hyperoxia alone failed to induce TNF- production in lungs. However, i.t. challenge with L. pneumophila substantially increased lung TNF- levels. This was particularly evident in animals exposed to hyperoxia, with TNF- levels being 2.2-fold greater than those observed in infected animals under room air conditions (p < 0.05). To examine the role of TNF- in the induction of apoptosis, mice were treated with anti-TNF- antiserum, and then apoptosis in the lungs was examined on day 2 after bacterial challenge (Fig. 9). TNF- blockade attenuated apoptosis, as indicated by a decrease in histone-associated DNA fragments and caspase-3 levels in both mice kept in hyperoxia and those kept in room air conditions. However, TNF blockade did not provide survival benefit to hyperoxic mice (data not shown). These results suggest that TNF- plays a role in the induction of apoptosis observed in Legionella-infected mice, although in contrast to Fas-mediated apoptosis, TNF-mediated apoptosis was not appreciably enhanced in the setting of hyperoxia.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of apoptosis in the lungs of L. pneumophila pneumonia was demonstrated using multiple approaches, including quantification of histone-associated DNA fragments, caspase-3 activity, DNA laddering, and TUNEL staining. We observed acceleration of apoptosis in the infected lungs of mice exposed to hyperoxia, which was well correlated with enhanced lethal sensitivity. L. pneumophila has been reported to induce apoptosis in macrophages and alveolar epithelial cells in vitro in a caspase-3-dependent fashion within a few hours of infection (14). Our data are consistent with these previous results and further support a critical role of caspase-3-mediated apoptosis in the pathogenesis of Legionella pneumonia. Because Legionella-associated apoptosis was demonstrated in nonreplicative C57BL/6 mice, intracellular multiplication may not be prerequisite for induction of apoptosis. Moreover, it is likely that exaggeration of apoptosis in the setting of hyperoxia is detrimental to outcome, in part through acceleration of acute lung injury.
TUNEL staining of the infected lung sections demonstrates that macrophages (alveolar and interstitial) and alveolar epithelial cells are the predominant cells undergoing apoptosis. In particular, alveolar epithelial cell apoptosis may be crucial for initiation of enhanced pulmonary permeability because these cells cover >95% of alveolar air space and are paramount in maintaining the integrity of the alveolar-capillary barrier (37). In addition, because Legionella has a strong predilection for invasion of alveolar epithelial cells (9, 13, 14), bacterial cytotoxicity, in combination with host factors (e.g., TNF-), may be pivotal to the development of epithelial injury, acute lung damage, and increased mortality in Legionella pneumonia. Our in vitro experiments mimicking in vivo hyperoxia support a direct effect of hyperoxia as a mediator of alveolar epithelial cell apoptosis. In contrast, the role of macrophage apoptosis in Legionella pneumonia is still poorly understood. It has been shown in vitro that L. pneumophila kills macrophages by activating the host apoptosis cascade upon contact with these cells (13, 14), although whether apoptosis of infected macrophages benefits the host or the bacteria is still unclear. For example, apoptosis, but not necrosis of human monocytes, is shown to limit the growth of other intracellular pathogens, including Mycobacterium bovis (38) and Mycobacterium avium (39). An area of interest in this regard is how macrophage apoptosis affects the viability of intracellular bacteria in replicative A/J mice model of pneumonia, which is an area of ongoing investigation.
Several investigators have reported crucial roles of death receptor-mediated apoptosis in certain infectious diseases. Kanaly et al. (21) have reported that the TNF-receptor is important for successful regression through induction of lymphocyte apoptosis in Leishmania major or Rhodococcus equi infections, once pathogens are eliminated. In murine CMV infection, Fas-mediated apoptosis is required for the down-modulation of the immune response to virus, which prevented detrimental chronic inflammatory responses (20). Our data suggest that apoptosis occurs in Legionella infection, especially in the setting of hyperoxia, and that both TNF and Fas are relevant mediators. Specifically, we observed a >2-fold increase in TNF- in the infected lungs of mice exposed to hyperoxic conditions. In addition, TNF- blockade resulted in attenuation of apoptosis in both infected mice kept in hyperoxia and those kept in room air. Furthermore, the observation obtained in Fas-deficient mice suggests that Fas mediates the acceleration of apoptosis in hyperoxic conditions, and that Fas-mediated apoptosis may be detrimental in Legionella pneumonia in the setting of hyperoxia. The fact that Fas deficiency only partially, not totally, restored resistance to the lethal effects of Legionella after hyperoxic exposure suggests involvement of Fas-independent pathway(s) (e.g., TNFRI, -II) in the pathogenesis of exaggerated lethal sensitivity of these mice. However, it should be noted that Fas knockout mice are not completely Fas-deficient, which may have resulted in an underestimation of Fas-dependent events (40).
TNF- blockade failed to provide a survival benefit in hyperoxic mice infected with Legionella. It has been shown that TNF- contributes not only to apoptosis but also to antimicrobial immunity against a variety of intracellular and extracellular pathogens, including Legionella (41, 42). Therefore, the lack of beneficial effects of TNF blockade on survival of mice in hyperoxic conditions may reflect multipotential activity of TNF- on host biological and immunological systems.
How hyperoxia accelerates Legionella-associated apoptosis in the lung is not clearly defined, although death receptor-mediated signaling appears to play a certain role. We speculate on at least two possibilities in which hyperoxia affects apoptosis in Legionella pneumonia. First, hyperoxia or higher oxygen concentration may affect the expression of virulence factors produced by L. pneumophila, which may amplify host responses, including TNF- production. Alternatively, these virulence factors may directly induce apoptosis through death receptor-dependent and/or -independent mechanisms. Although apoptosis-inducing factor(s) in Legionella pneumonia have not yet been defined, dot/icm (14) and pore-forming toxins (11) may be potential candidates. It is of great interest to investigate whether the virulence-deficient mutants are capable of accelerating apoptosis in the setting of hyperoxia. A second possibility is that hyperoxia sensitizes or primes host cells to apoptosis-triggering stimuli that are elaborated by Legionella infection. Hyperoxia may affect a variety of host biological systems, such as antioxidant enzymes (43) and cytokine production (28), through excessive production of reactive oxygen species. Several investigators have reported that hyperoxia up-regulates TNF- gene and protein expression, especially after triggering stimuli (35, 36, 44, 45). In addition, hyperoxia has been reported to induce a marked increase in message and protein levels of Fas in lungs, although Fas-deficient mice are not resistant to hyperoxia-induced lung damage itself (29). These suggest that both Fas and bacteria/bacterial products may cooperatively mediate Legionella-induced lethality in hyperoxic conditions. Modulation of host cell sensitivity to apoptosis-triggering stimuli, both host and bacterial factors, in addition to expression of death receptors on responsible cells, remained to be investigated in this model.
In conclusion, the present results suggest that hyperoxia serves as an important cofactor for the development of acute lung injury and lethality in L. pneumophila pneumonia. Exaggerated apoptosis, in part through the death receptor-mediated signals, accelerates hyperoxia-induced acute lung injury in Legionella pneumonia. Further investigations into the roles of death receptors and death factors in hyperoxia-associated apoptosis in Legionella pneumonia, and identification of related virulence factor(s), are warranted. Given the large number of patients with pneumonia exposed to high oxygen tensions, the observations made in this study are of greater clinical relevance and similar studies should be pursued in patient populations at risk.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kazuhiro Tateda, Department of Microbiology, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ohtaku, Tokyo 143-8540, Japan. E-mail address: kazu{at}med.toho-u.ac.jp
3 Abbreviations used in this paper: BYE, buffered yeast extract; i.t., intratracheal(ly); BAL, bronchoalveolar lavage.
Received for publication August 19, 2002. Accepted for publication February 6, 2003.
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) was kept in room air, whereas another group (•) was placed in hyperoxic condition for 60 h, and then survival was observed 7–8 days after bacterial challenge (n = 14–19). *, p < 0.05, compared with C57BL/6 control mice.
, Normoxic condition; 
, normoxic condition plus Legionella; 
