Technical Comments

Role of Apoptosis in Pseudomonas aeruginosa Pneumonia

Science  30 Nov 2001:
Vol. 294, Issue 5548, pp. 1783
DOI: 10.1126/science.294.5548.1783a

Apoptosis plays a central role in the complex balance between invading pathogen and host defense. Depending on the pathogen, apoptosis of host cells may be beneficial or detrimental to survival. Studies using a clinically relevant intra-abdominal model of sepsis (peritonitis) have indicated that two types of cells, lymphocytes and gastrointestinal epithelial cells, undergo accelerated apoptosis (1, 2). Apoptosis appears to be confined predominantly to these two cell types, possibly because these cells normally die by apoptotic mechanisms. Furthermore, blocking lymphocyte apoptosis in peritonitis has been shown to improve survival (3–5). Consequently, some investigators have speculated that prevention of apoptosis may be efficacious in sepsis by preventing immune suppression (6, 7).

In striking contrast to the concept that apoptosis is detrimental in sepsis, Grassmé et al. (8) reported thatPseudomonas aeruginosa pneumonia resulted in bronchial cell apoptosis that was essential for survival. The survival benefit of apoptosis was demonstrated by showing that bronchial cell apoptosis did not occur in mice deficient in the cell death receptor Fas (CD95) and that mortality was greatly increased in Fas-deficient mice as well. Grassmé et al. (8) speculated that shedding of infected apoptotic bronchial cells in control mice (mice with normal Fas receptors) prevented bacterial dissemination and improved survival.

Although Grassmé et al. employed several methods to detect cell apoptosis in vitro, only the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) method was employed for in vivo determination of bronchial cell apoptosis. Because TUNEL may not reliably discriminate between apoptosis and necrosis and may yield false positives (9–11), however, their conclusions regarding in vivo effects of pneumonia must be interpreted cautiously.

Using the identical model of Grassmé et al.(8), we employed three methods in addition to TUNEL to determine if P. aeruginosa induced bronchial apoptosis (12). No bronchial apoptosis was detected by conventional light microscopy, electron microscopy (Fig. 1), or immunohistochemical staining for active caspase 3 (Fig. 2). Lymphocytes, occasional polymorphonuclear neutrophils, and, to a much lesser degree, capillary endothelial and alveolar cells demonstrated characteristic apoptotic changes of condensed and fragmented nuclei (Fig. 2). The incidence of apoptotic capillary endothelial and alveolar epithelial cell apoptosis was rare (less than one cell per five high powered fields; magnification, ×400). Although TUNEL was positive for bronchial cell apoptosis in mice with pneumonia, it was also positive in saline-treated (control) mice (Fig. 3). That TUNEL positive bronchial cells in both control and P. aeruginosa–treated mice were negative for active caspase 3 and had no apoptotic nuclear morphologic changes by conventional or electron microscopy strongly supports the contention that the TUNEL positive bronchial cells were falsely positive.

Figure 1

(A) Electron micrograph of ciliated bronchial epithelial cells from saline-treated mouse (sham), demonstrating normal morphology. Arrows identify nuclei with normal diffuse chromatin pattern. (B) Ciliated respiratory bronchial epithelial cells from P. aeruginosa–treated mouse show normal cell morphology. Nuclei are identified by arrows and have normal diffuse chromatin pattern without chromatin condensation or fragmentation. Bacteria (arrowheads) and inflammatory debris are in bronchial lumen in the right half of the image.

Figure 2

(A) Two capillary endothelial cells (arrows, upper left portion of photomicrograph) are positive for active caspase 3 and have secondary morphologic changes consistent with apoptosis. Two lymphocytes (remaining arrows) are also positive for active caspase 3. Magnification, ×600. It should be noted that bronchial cells (lower right margin of the image) are negative for active caspase 3. (B) Macrophages (arrows) have ingested apoptotic debris that is positive for active caspase 3. Magnification, ×600.

Figure 3

Lung tissue obtained 24 hours after intratracheal injection and evaluated by TUNEL method. TUNEL-positive bronchial epithelial cells have brown staining nuclei and are present in both (A) saline-treated (sham) lung and (B) bacterial-treated (pneumonia) lung. Magnification, ×400. It should be noted that the nuclei of the bronchial epithelial cells have normal morphology, without evidence of contraction or fragmentation.

The present study agrees with the work of Rajan et al. (13), who reported that airway epithelium is highly resistant to apoptosis in P. aeruginosa pneumonia and is also consistent with clinical studies in which no bronchial apoptosis was detected in lungs of patients who died of pneumonia (14,15). Interestingly, the TUNEL method did accurately demonstrate apoptosis in lymphocytes in mice with pneumonia (apoptosis confirmed by four methods), but not in controls. Recent studies have indicated that the accuracy of the TUNEL method may be related to cell-specific endogenous endonucleases and that TUNEL is falsely positive in cells with high endonuclease activity (11).

The most remarkable finding in the present study was the extensive lymphocyte apoptosis that occurred in lung, spleen, and thymus during pneumonia (Fig. 4). The percentage apoptosis increased from 3.3 ± 0.4% to 6.0 ± 0.8% (p < 0.05) in splenocytes and from 4.6 ± 1.0 to 20.0 ± 4.2 (p <0.05) in thymocytes. Fas receptor–deficient mice (MRL/MPJ = Faslpr) had no protection from lymphocyte apoptosis; percentage apoptosis was 3.7 ± 0.5% in control splenocytes versus 6.9 ± 0.4% in septic splenocytes and 2.8 ± 1.0% in control thymocytes versus 24.0 ± 4.3% in septic thymocytes (p <0.05). Although there was a trend toward improved survival in Fas receptor–deficient mice compared with control mice, the difference was not statistically significant (15). Lymphocyte apoptosis was totally inhibited in transgenic mice that overexpressed Bcl-2 in T cells; percent apoptosis was 2.5 ± 0.7% in control splenocytes versus 1.9 ± 0.4% in septic splenocytes and 2.62 ± 0.2% in control thymocytes versus 3.3 ± 0.4 in septic thymocytes in such mice. The results of the Fas and Bcl-2 transgenic mice are consistent with previous studies in sepsis that indicate that lymphocyte apoptosis occurs by a mitochondrial rather than a cell receptor death pathway (4,16).

Figure 4

Thymi obtained 24 hours after intratracheal injection in (A) saline-treated (sham) and (B) bacterial-treated (pneumonia) mice. Magnification, ×600. Cells positive for active caspase 3 (brown stain) show a marked increase in mice with pneumonia; nuclei are also compacted and fragmented.

The extensive apoptotic loss of lymphocytes in P. aeruginosapneumonia is comparable to that occurring in sepsis due to peritonitis (3, 4). Importantly, prevention of lymphocyte apoptosis by overexpression of Bcl-2 or administration of caspase inhibitors has been shown to improve survival in animal models of sepsis (4, 5, 17) and caused a non–statistically significant trend toward improved survival in pneumonia as well (15). Thus, extensive lymphocyte apoptosis may be a fundamental abnormality in bacterial sepsis irrespective of site of infection and may contribute to the accompanying immune suppression and mortality that characterize this highly lethal disorder. A recent clinical study in patients dying of sepsis showed profound loss of B and CD4+ T cells via apoptosis (18), a finding that highlights the potential importance of the current study.

REFERENCES AND NOTES

Role of Apoptosis in Pseudomonas aeruginosa Pneumonia

Response: TUNEL has been employed by many investigators to show apoptosis in the lung induced by a variety of stimuli [see, e.g. (1–11)]. In our study (12), using TUNEL, we showed that several P. aeruginosa strains induce apoptosis of lung epithelial cells by an up-regulation of the CD95/CD95 ligand system. Epithelial cells from lpr orgld mice lacking either CD95 or CD95 ligand were resistant to P. aeruginosa–triggered apoptosis. These mice, which were too young to suffer from lymph-adenoproliferative symptoms or reduction of body weight, were unable to control the infection and died by sepsis, while normal mice rapidly cleared pulmonary P. aeruginosa infections. Apoptosis as part of the host defense was recently also shown for Salmonella typhimurium infections ofCaenorhabditis elegans (13). In this response to the comments of Hotchkiss et al., we here confirm induction of apoptosis in lung epithelial cells by P. aeruginosa.

Transmission electron microscopy [TEM (14)] revealed marked chromatin condensation and fragmentation in nuclei of lung epithelial cells from infected mice, while the chromatin in nuclei from uninfected mice was homogenous (Fig. 1, A and B). The presence of cilia in apoptotic cells identifies them as lung epithelial cells (Fig. 1C). Apoptosis in lung epithelial cells was also confirmed by detection of single-stranded DNA (15), which was present only in nuclei of infected mice (Fig. 2). Single-stranded DNA is a typical marker of apoptosis and is absent in necrosis (16).

Figure 1

TEM photomicrographs (14) of lung tissue. (A) Lung section from uninfected mice show nuclei with homogeneous chromatin (magnification, ×6700). (B) Lung section from mice 12 hours after infection with ATCC 27853 reveals condensation and extensive fragmentation of nuclear chromatin (magnification, ×6700). (C) The presence of cilia indicates apoptosis in epithelial cells (magnification, ×21,000). Similar results were obtained in 5 independent infection experiments.

Figure 2

Monoclonal antibody test for single-stranded DNA (15) in (A) cells from uninfected mice, (B) cells from mice infected with early P. aeruginosa grown to early mid-logarithmic phase, and (C) cells from mice infected with plateau phase–grown bacteria. Single-stranded DNA is detected in (B) but is absent in (A) and (C).

Hotchkiss et al. used P. aeruginosa in the plateau growth phase, whereas we employed bacteria cultured until the early mid-logarithmic growth phase (17). Because growth and infection conditions have been shown to be crucial for biological effects of many bacteria, such as Salmonella typhimurium,Yersinia enterocolitica, Yersinia pseudotuberculosis, Escherichia coli, and P. aeruginosa (18, 19), we tested the effect of different P. aeruginosa growth conditions on apoptosis. Our experiments revealed that P. aeruginosa cultured until early mid-logarithmic growth phase, but not bacteria in plateau growth phase or taken directly from the agar plate, induced apoptosis in lung epithelial cells in vivo (Fig. 2) or in vitro [Fig. 3 (20)]. The very efficient induction of apoptosis byP. aeruginosa ATCC 27853 is consistent with our previous data (12).

Figure 3

FACS and fluorescence microscopy (20) confirm that only logarithmic-grown P. aeruginosa trigger apoptosis of more than 90% of WI-38 cells after 60 min of infection. Apoptosis was detected by FITC-Annexin labeling; simultaneous staining with PI excluded necrosis. Data are representative for five experiments. (A) Histogram of FITC-Annexin fluorescence 1 hour after infection. Blue line, uninfected cells; red line, cells infected with early mid-logarithmic growth bacteria; yellow line, cells infected with plateau phase bacteria; pink line, cells infected with plate-grown bacteria. (B) Dot blots for logarithmic-grown bacteria 1 hour after infection, comparing fluorescence for PI against fluorescence for FITC-Annexin for uninfected and infected cells. (C) Fluorescence microscopy for uninfected cells (top) and for cells 1 hour after infection with logarithmic-grown bacteria.

Likewise, only early mid-logarithmic P. aeruginosawere internalized by mammalian cells in vitro and in vivo [Fig. 4 (21)]. P. aeruginosa grown to plateau phase or directly taken from agar plates were poorly internalized. Plateau phase P. aeruginosa, but not early mid-logarithmic grown P. aeruginosa, seemed to develop capsules (Fig. 4, A to C), which might interfere with the infection of mammalian cells.

Figure 4

Internalization of P. aeruginosa by epithelial cells depends on bacterial growth conditions (21). Left-hand panels show representative results of crystal violet stainings of WI-38 cells in vitro, for (A) uninfected cells, (B) cells infected with early mid- logarithmic–grown P. aeruginosa ATCC 27853, and (C) cells infected with plateau phase bacteria. (D) Invasion of WI-38 cells by P. aeruginosa in vitro, 15 min and 30 min after infection. Results show mean ± SD for 600 cells. Gray bars, early mid-logarithmic; black bars, plateau phase; white bars, plate growth. (E) Invasion of murine lung cells by P. aeruginosaATCC 27853 in vivo. Results show mean ± SD for five independent infections. Gray bar, early mid-logarithmic; black bar, plateau phase; white bar, plate growth.

Our TEM, single-stranded DNA, and FITC-Annexin/PI labeling studies confirm the previously described TUNEL assays (12) and demonstrate apoptosis of lung epithelial cells upon P. aeruginosa ATCC 27853 infection. In our previous studies, we have observed positive TUNEL exclusively in epithelial cells from infected normal mice. No signal was detected in lungs from uninfected normal or infected lpr or gld mice, respectively, which indicates the specificity of TUNEL, consistent with many previous studies (1–11) that have employed TUNEL to detect apoptosis in the lung under different conditions. None of those studies have reported unspecific TUNEL staining of naive lungs (1–11), in contrast to the data presented by Hotchkiss et al. In addition, because apoptosis may involve a variety of different caspases and even caspase-independent apoptosis has been observed, absence of active caspase 3 immunoreactivity does not rule out apoptosis.

Our data indicate the importance of P. aeruginosagrowth conditions for triggering apoptosis and invasion of epithelial cells. However, early mid-logarithmic growth conditions of P. aeruginosa upon intranasal infections might be most appropriate for mimicking the clinical situation of an early pulmonary P. aeruginosa infection. Although CD95 stimulation and apoptosis of lung epithelial cells seem to be beneficial in acute pulmonaryP. aeruginosa infections, apoptosis of lymphocytes inP. aeruginosa peritonitis or sepsis might be detrimental. This suggests that induction of apoptosis has specific roles depending on the conditions of the bacterial infection.

REFERENCES AND NOTES

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