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Basic Science Aspects

BOTH GRAM-NEGATIVE AND GRAM-POSITIVE EXPERIMENTAL PNEUMONIA INDUCE PROFOUND LYMPHOCYTE BUT NOT RESPIRATORY EPITHELIAL CELL APOPTOSIS

Schreiber, Torsten*†; Swanson, Paul E.; Chang, Katherine C.*; Davis, Christopher C.*; Dunne, W. Michael§; Karl, Irene E.; Reinhart, Konrad; Hotchkiss, Richard S.*

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doi: 10.1097/01.shk0000225856.32260.0d
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Abstract

INTRODUCTION

Apoptosis, or programmed cell death, is increasingly recognized as an important mechanism affecting the immune balance in the course of severe infections. Accelerated lymphocyte and gut epithelial cell apoptosis has been shown to occur in overwhelming infections in humans and in polymicrobial animal models of abdominal sepsis (1, 2). Experiments using a model of monomicrobial, gram-negative pneumonia caused by a clinically highly relevant organism (Pseudomonas aeruginosa) reported increased apoptotic cell death in spleen and thymus (3) and in the intestinal epithelium (4). The fact that prevention of gut epithelial cell apoptosis in this model was associated with improved survival (4), points to a functionally relevant role of apoptosis in critical illness.

Apoptosis can be induced by 2 major pathways, both of which ultimately lead to activation of caspase 3, a key enzyme that in turn affects many cellular proteins involved in DNA fragmentation and disintegration of the cell. The extrinsic pathway is initiated by ligand-binding to a cell membrane-bound death receptor (e.g., the Fas-receptor). This leads to downstream activation of caspase 8, which in turn activates caspase 3. The intrinsic pathway involves mitochondrial release of cytochrome c secondary to cellular stress, resulting in activation of caspase 9. Active caspase 9 then initiates activation of caspase 3.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and continues to be an important cause of death secondary to severe infection (5). A number of factors have been identified that contribute to the virulence of S. pneumoniae, including encapsulation and release of pneumolysin and hydrogen peroxide (6). It further has recently been demonstrated that pneumococcus infection may induce apoptosis in several cell types including monocytes, granulocytes, and lung epithelium (7-9).

Pneumonia caused by different bacteria involves diverse virulence factors resulting in unique immune responses (10). Thus, results obtained from gram-negative infection models may not necessarily be extrapolated to gram-positive pneumonia.

Although P. aeruginosa, a gram-negative organism, has been shown to induce systemic lymphocyte apoptosis (3), no studies have examined S. pneumoniae, a gram-positive organism. In addition, no studies have delineated the precise apoptotic pathways in pneumonia. Finally, there is debate whether respiratory epithelial cells undergo apoptosis in pneumonia (3, 11, 12).

Accordingly, the objectives of the present study were to determine if increased lymphocyte and bronchial epithelial cell apoptosis occurs in both gram-positive and gram-negative pneumonia and to identify if the external death receptor pathway or the intrinsic mitochondrial dependent pathway was involved in the process.

MATERIALS AND METHODS

A total of 30 male C57BL\6 mice were used for the experiments (Jackson Laboratories, Bar Harbor, Me). All studies were approved by the Washington University Animal Studies Committee and were in accordance with the National Institutes of Health guidelines.

Bacteria

Pseudomonas aeruginosa (strain: ATCC 27853) and S. pneumoniae (strain: 99.55, capsular type 6A) were grown overnight in trypticase soy broth to be used for the experiments on the following morning. For P. aeruginosa, density of the inoculum was adjusted to 0.3 A600 nm, corresponding to 5 × 108 to 1 × 109 CFU/mL as determined by serial dilution and colony counts. For S. pneumoniae, density of the inoculum was adjusted to 0.9 to 1.0 A600 nm, corresponding to a density approximately 1 × 109 CFU/mL. Previous studies had demonstrated bacterial dissemination into spleen and liver with similar doses of both organisms (4, 13).

Experimental protocol

Under halothane anesthesia, the trachea of the animals was surgically exposed and an intratracheal injection of either 50 μL of the bacterial solution or isotonic saline (sham group) was performed. Animals were held upright for 10 s to facilitate delivery of the solution to the lungs. The neck incision was closed, and after injection of 1 mL of 0.9% saline s.c., the mice were transferred to their cages, where they had free access to food and water. No antibiotics were given. With these doses, lethality within 10 days was more than 70% for S. pneumoniae (own unpublished data) and more than 90% for P. aeruginosa infection (4).

Animals injected with P. aeruginosa were killed at 24 (±2) h, and animals injected with S. pneumoniae were killed at 32 (±2) or 48 (±2) h. Although P. aeruginosa infected mice commonly present with severe illness at 20 to 24 h, the onset of clinical symptoms in S. pneumoniae infected mice is protracted, and animals exhibit a more gradual deterioration. Hence, these time points reflect the clinical course of illness.

At each time point (24, 32, 48 h), bacteria-instilled and sham-treated animals were killed (n = 5 per group) by cervical dislocation and spleen; thymus and lungs were removed immediately. Lungs were evaluated via gross visual examination for presence of consolidation and hemorrhage (lung injury was further assessed by light microscopy in hematoxylin and eosin (H&E)-stained, longitudinal sections of the whole lung).

The whole lungs and portions of spleen and thymus were immersed and fixed in 10% buffered formalin overnight for histological examination. The other portion of thymus and spleen was used for flow cytometry.

Flow cytometry

For flow cytometry, tissue sections from spleen and thymi were gently ground to dissociate cells. After hypotonic lysis of red blood cells, cells were incubated with FITC-labeled antimouse mAbs (CD3) specific for mouse T-lymphocytes for 30 min at room temperature. For staining of active caspases 3, 8, and 9, paraformaldehyde fixed cells were incubated with their respective primary antibodies raised in rabbit and stained with phycoerythrin-conjugated donkey antirabbit Ab. The primary Ab (1/200 concentration) for active caspase 3 was purchased from Cell Signaling Technology (Beverly, Mass). This Ab is specific for cleaved fragment of caspase 3 and does not recognize the procaspase 3 form. The primary antibodies for active caspases 8 and 9 were generously provided by Merck Frosst. Flow cytometric analysis (50,000 events/sample) was performed on FACScan (Becton Dickinson, Biosciences, San Jose, CA).

Histology and immunohistochemistry

Three techniques were used for detection of apoptosis in tissue sections: (1) conventional microscopy of H&E-stained tissue sections, (2) immunohistochemical (IHC) staining for active caspase 3 (mAB to active caspase 3, Cell Signaling Technology), and (3) DNA strand breaks. For the latter technique terminal de-oxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL; Kit: Roche Molecular Laboratories) was performed in spleens and thymi. Because of the high false positive rate of apoptosis in lung tissues examined by conventional TUNEL, (1, 14), a novel in situ oligo ligation (ISOL) method (Kit: ApopTag, Chemicon Int, Temecula, Calif) preferentially labeling the DNA strand breaks occurring in apoptosis (predominantly blunt-ends or 3′single-base overhangs) was used for labeling apoptotic cells in lung tissue sections. All kits were used according to manufacturer guidelines.

For evaluation of H&E-stained organ sections, cells were graded as apoptotic if they exhibited characteristic changes of nuclear condensation (pyknosis) and/or nuclear fragmentation (karyorrhexis). For IHC staining of active caspase 3 or TUNEL/ISOL, cells were judged to be apoptotic if they displayed a brown precipitate on examination by brightfield microscopy. Lung tissue sections were examined and evaluated for apoptosis by the author (P.E.S.), who was blinded to sample identity.

Statistical analysis

Data are shown as mean ± SEM. T test for unpaired samples was used to compare flow cytometry data between groups. A P value less than 0.05 was considered significant. The statistics software SPSS Version 11 (SPSS Inc., Cary, NC) was used for data analysis.

RESULTS

Assessment of illness and lung injury

All bacteria-inoculated animals were sick at the time they were killed, as indicated by reduced locomotion, lethargy, and piloerection. Macroscopically, their lungs revealed areas of consolidation and appeared in some cases hemorrhagic. In all bacteria-instilled animals, H&E-stained lung tissue sections demonstrated extensive areas of leukocyte infiltration (predominantly polymorphonuclear and mononuclear cells) and loss of normal alveolar structure in more severely affected foci, findings typical of bacterial pneumonia (Fig. 1). Infection with P. aeruginosa caused a more diffuse involvement of the lung, whereas S. pneumoniae caused a more lobar involvement. Saline-treated animals appeared to be unaffected, and their lungs looked macroscopically and histologically normal.

Fig. 1
Fig. 1:
Typical H&E-stained lung tissue sections obtained 24 h after intratracheal injection of (A) normal saline or (B)P.aeruginosa and (C) 30 h after intratreacheal injection ofS. pneumoniae. Magnification is ×100. Normal pulmonary architecture is preserved after saline instillation. Note the loss of alveolar integrity and the extensive inflammatory infiltrations consistent with pneumonia, which are present in P. aeruginosa and S. pneumoniae inoculated lungs.

Assessment of apoptosis

Flow cytometry-Compared with control animals, percentage of T lymphocytes staining positive for active caspase 3 was significantly increased in spleen and thymus in both gram-positive and gram-negative pneumonia (Fig. 2A). Examination of key markers for the intrinsic and extrinsic pathway of apoptosis showed that cells staining positive for active caspase 8 and 9 were significantly increased in thymus and spleen in both pneumonia models (Fig. 2B). The single exception of this finding was that in pneumoccus-infected animals at 30 h, the percentage of cells positive for active caspase 8 in thymocytes was not increased.

Fig. 2
Fig. 2:
A, Percentage of T lymphocytes, which were stained positive for active caspase 3 in spleen and thymus. B, Percentage of lymphocytes in spleen and thymus, which were stained positive for active caspase 8 (upper panel) or active caspase 9 (lower panel), respectively. Black bars represent bacteria-instilled animals (n = 5/group). Gray bars represent corresponding saline-instilled control animals (n = 5/group). Please note the different scales on the x axes. PNEUMO, animals infected with S. pneumoniae; PSEUDO, animals infected with P. aeruginosa. *P < 0.05 vs. corresponding control group.

Histology

Spleen and thymus-Hematoxylin and eosin: Hematoxylin and eosin-stained tissue sections revealed extensive apoptosis in spleen and thymus (Fig. 3) in both pneumonia models. In thymi, apoptosis was more extensive in the cortex than in the medulla. In spleens, apoptosis was more extensive in white than in red pulp.

Fig. 3
Fig. 3:
Representative H&E-stained tissue sections from thymi obtained from (A) a sham animal and (B) a mouse infected withS.pneumoniae (magnification ×400). Note the extensive degree of apoptotic cellular changes (nuclear condensation and nuclear fragmentation) in (B).

Immunohistochemistry: Compared with control animals, grossly increased numbers of cells staining positive for active caspase 3 were present in spleen and thymus in both gram-positive and gram-negative pneumonia (Fig. 4). Similarly, the TUNEL method revealed numerous areas of TUNEL-positive cells in both types of pneumonia. In thymic cortex, greater than 75% of cells exhibited morphological changes classic for apoptosis. In splenic follicles, focal areas were present, in which more than 25% cells were apoptotic. Only occasional thymocytes or splenocytes (<2%-3%) were positive for active caspase 3 or TUNEL in sham-treated mice.

Fig. 4
Fig. 4:
Representative immunohistochemically stained tissue sections (active caspase 3) from spleen and thymi obtained from controls (A) or mice infected withS.pneumoniae (B) orP. aeruginosa(C). Cells positive for active caspase 3 are labeled brown by diaminobenzidine activation system. In conjunction with typical morphological cellular changes (cell shrinkage, condensed nuclei), this indicates apoptotic cell death. These findings are rare in control animals but numerous in thymi and spleens in both types of pneumonia. Note that thymi appear to be much more affected than spleens.

Lung-Hematoxylin and eosin: In bacteria-instilled animals, H&E-stained slides of the lung demonstrated dense inflammatory infiltrate with mononuclear cells; many of which had compacted and fragmented nuclei-characteristic findings of apoptosis. Although it was frequently impossible to identify specific cell types, many of the cells were clearly lymphocytes and neutrophils. In addition, we frequently found neutrophils adherent to the epithelium or within the lumen of respiratory bronchioles, and many of these cells had the characteristic cellular findings of apoptosis. In contrast to the lymphocytes and neutrophils, we only rarely were able to identify alveolar epithelial and capillary endothelial cells exhibiting morphological changes consistent with apoptosis (<1 apoptotic cells per 4 high-powered fields; magnification ×400). In addition, no apoptosis was detected in the epithelium of respiratory bronchioles and larger airways.

Immunohistochemistry: A series of representative photomicrographs obtained from bacteria-infected animals are shown in Figure 5.

Fig. 5
Fig. 5:
Representative immunohistochemically stained lung tissue sections (magnification ×200-400) obtained from mice infected withS.pneumoniae (left) orP.aeruginosa (right). All sections are stained for active caspase 3, except section D, which is stained using the TUNEL method. A, Interstitial inflammatory infiltrate consisting mostly of polymorphonuclear neutrophils and mononuclear cells. Apoptotic cell death is present in the inflamed areas, but only rarely noted in the preserved airspaces (arrow). Note that the respiratory epithelium in the adjacent bronchiole does not exhibit signs of apoptosis. B, No signs of apoptosis were present in many other areas of inflammation as well as in the adjacent airspaces. C, Apoptotic cell death in interstitial tissue and in the wall of airspaces (arrows). Because of the location of the cells, the latter finding could represent dying epithelial cells. D, Overview on inflammatory infiltrate (center), adjacent airspaces (upper part), and a segment of airway epithelium (bottom part) showing apoptotic cell death in the lung interstitium but not in epithelial cells. E, Section of a large airway subtotally filled with cellular debris, nuclear fragments, and sloughed cells, most likely neutrophils. Note that the respiratory epithelium appears to be intact and does not exhibit signs of apoptotic cell death. F, Small blood vessel (within the inflammatory infiltrate) with possible endothelial cell apoptosis (arrow). This is a rare finding. G, Apoptotic cell death (brown spots) in the inflammatory infiltrate. H, Apoptotic cells, most likely neutrophils, within preserved alveoli. Br indicates bronchiole.

Cells stained positive for active caspase 3 were seen predominantly within areas of extensive inflammatory infiltrate, and similar to the H&E stains, many of these apoptotic cells had morphology typical of neutrophils and lymphocytes. Because of the disintegration of normal alveolar architecture and the density of the cellular inflammatory infiltrate, it was frequently not possible to determine if some or how many labeled cells originally were residential cells of the lung. We also found caspase 3-positive cells, most likely representing apoptotic neutrophil granulocytes, superposed the respiratory epithelium or shed into the airway lumen. Similar to the H&E-stained sections, we only very occasionally were able to identify alveolar epithelial, respiratory epithelial or endothelial cells, which were labeled for active caspase 3 in the less inflamed lung areas. The number of these cells was low in all groups and not apparently different between groups. Similar results were obtained with the ISOL technique used in the lung.

With both IHC techniques occasional labeling of residential cells of the lung (airway and alveolar epithelial cells, and endothelial cells) was also seen in saline-treated animals (Fig. 6).

Fig. 6
Fig. 6:
Typical aspect of an immunohistochemically (active caspase 3) stained lung tissue section obtained from a saline-instilled (control) animal. The specimen (magnification ×100) shows intact alveolar structures and sections of 2 larger airways. Apoptotic cell death is present in the respiratory epithelium (arrow), but it is difficult to identify the type of the dying cells.

DISCUSSION

In this study, using clinically relevant bacteria, we demonstrate by conventional light microscopy, immunohistochemistry, and flow cytometry that both gram-positive and gram-negative pneumonia caused a pronounced increase in lymphocyte apoptosis in spleen and thymus. Furthermore, our finding of increased levels of active caspase 8 and 9 in splenocytes and lymphocytes in both models suggests that both the extrinsic and intrinsic pathway is involved in the process of apoptotic cell death. However, we found only a small number of cells (<1 cell per 4 high-powered fields; magnification ×400) with typical changes indicative of apoptosis in the alveoli or respiratory epithelium, suggesting that alveolar and bronchial cell apoptosis is not a major effect of pseudomonas or pneumococcus infection.

Our data on lymphocyte apoptosis in P. aeruginosa pneumonia are in accordance with previous findings in P. aeruginosa pneumonia, demonstrating increased apoptosis in splenocytes and in thymocytes using the TUNEL method (3). No data have been reported so far on apoptotic cell death in spleens and thymi of animals infected with S. pneumoniae. The extensive lymphocyte apoptosis demonstrated in the present study is consistent with the striking depletion of lymphocytes reported in adult (1), pediatric (15), and neonatal sepsis (16). This loss of lymphocytes has highly negative consequences on host defenses. Prevention of lymphocyte death in sepsis has been shown to decrease mortality (17).

It is important to determine if lymphocyte apoptosis in sepsis occurs by the death receptor or mitochondrial mediated pathway because this information provides insight into potential mediators of cell death and possible therapeutic approaches. The pronounced increase in both active caspase 8 and 9 in T lymphocytes in both pneumonia models in our study is consistent with activation of both the death-receptor and mitochondrial mediated pathway of apoptosis. The present results are in accordance with previous work from our group, which has shown that E. coli sepsis in primates activates both the intrinsic and extrinsic pathways (18). We speculate that pneumonia-induced sepsis induces a myriad of noxious stimuli, many of which can activate the cell death programs by initiating death receptor and mitochondrial mediated pathways.

Although the present study suggests that pneumonia activates both pathways of cell death, it is important to note that recent work demonstrates possible crosstalk between the extrinsic and intrinsic death pathways (19). Despite this limitation, there are several reasons why we speculate that both pathways directly caused cell death in sepsis. Transgenic and knockout mouse mutations that target the death receptor or mitochondrial mediated pathways have both independently shown the ability to block sepsis-induced apoptosis and to improve survival (20). Furthermore, studies in mice with knockout of Bid, a key Bcl-2 family member that links the death receptor and mitochondrial mediated pathway, showed only a small decrease in sepsis-induced apoptosis (own unpublished observations).

A second possible limitation of the present study relates to the fact that caspase activation is not synonymous with cell death. Caspases are also involved in lymphocyte activation and proliferation (21, 22). In our study however, given the fact that there is extensive collaborating evidence of lymphocyte apoptosis, that is, compacted and fragmented nuclei (Fig. 3) and positive DNA strand breaks, the finding of active caspase 3 most likely reflects cell death.

Our observations of a low degree of apoptosis in the respiratory epithelium might reflect the low rate of cell death occurring during the slow normal turnover of residential cells in the lung (23). Our data do not support the hypothesis that either gram-positive or gram-negative pneumonia significantly increases apoptosis in alveolar, endothelial, or respiratory epithelial cells.

Of note, we did see increased numbers of apoptotic cells in the inflamed areas of the lung. According to cell morphology, these in most cases were leukocytes, such as polymorphonuclear neutrophils and lymphocytes that had migrated to the lung in response to the bacterial insult but not residential cells of the lung. Apoptosis is an important pathway of eliminating neutrophils that have migrated into the lung after various stimuli (24) and has been demonstrated to occur in bacterial pneumonia (25).

Our findings on apoptosis in the lung in pseudomonas pneumonia do not confirm findings by Grassme et al. (11), reporting increased respiratory epithelial cell apoptosis using the TUNEL technique in mice infected with P. aeruginosa but are consistent with results from a previous study by our group which failed to show increased pulmonary cell apoptosis with this model (3) and are in accordance with data reported by Rajan et al. (26), indicating that airway epithelium is highly resistant to apoptosis in pseudomonas infection.

In conclusion, extensive systemic lymphocyte apoptosis occurs in both gram-positive and gram-negative pneumonia, and it is likely that pneumonia activates both the death receptor and mitochondrial mediated pathways of apoptosis. Neither P. aeruginosa nor S. pneumoniae pneumonia induced a significant increase in respiratory epithelial or endothelial cell apoptosis. Strategies to prevent lymphocyte apoptosis in severe infection may improve immune function and increase survival.

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Keywords:

Sepsis; caspases; flow cytometry; immunohistochemistry

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