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Medical Research Service of the Veterans Affairs Puget Sound Health Care System, and Divisions of Pulmonary and Critical Care Medicine and Allergy and Infectious Diseases, Department of Medicine, University of Washington School of Medicine, Seattle
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana
Background.
The Fas/FasL system is both proapoptotic and proinflammatory. FasL is inhibited by decoy receptor3 (DcR3), a naturally occurring decoy receptor. We determined the effects of systemic blockade of the Fas/FasL system by a DcR3 analog (DcR3-a) in mice with pneumococcal pneumonia.
Methods.
Streptococcus pneumoniae (7.2 × 105 or 1.9 × 107 cfu/mL) was instilled intratracheally into untreated C57Bl/6 mice, C57Bl/6 mice treated with DcR3-a, or Fas-deficient lpr mice, and the mice were studied 48 h later.
Results.
After instillation of the lower bacterial dose, disruption of the Fas/FasL system by either DcR3-a or the lpr mutation resulted in improved clearance of bacteria in the lungs (mean ± SE, 4.6 ± 2.1 × 106 and 3.5 ± 1.6 × 106 cfu/lung, respectively, vs. 21.9 ± 9.3 × 106 cfu/lung in untreated C57Bl/6 mice; P < .05) and decreased percentage of polymorphonuclear neutrophils in bronchoalveolar lavage fluid (mean ± SE, 19.3% ± 9.5% and 20.2% ± 7.8%, respectively, vs. 55.0% ± 12.2% in untreated C57Bl/6 mice; P < .05). These changes were associated with decreased lung concentrations of the proinflammatory cytokines tumor necrosis factor and macrophage inflammatory protein2 and with a decrease in apoptotic cells in the alveolar walls.
Conclusion.
Blockade of the Fas/FasL system by DcR3-a in the lungs improves clearance of bacteria in mice with pneumococcal pneumonia.
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are important causes of morbidity and mortality in the United States [1]. The main histopathological characteristics of ALI and ARDS are destruction of the alveolar epithelium and severe neutrophilic alveolitis [2, 3]. The cause of these changes remains unknown. Recent evidence suggests that the membrane surface receptor Fas (CD95) and its natural ligand, FasL (CD178), may contribute to development of ALI by 2 separate and independent mechanisms: first, by inducing apoptosis of alveolar epithelial cells; and, second, by promoting inflammation via activation of NF-B. However, the precise role that the Fas/FasL system plays in the pathogenesis of ALI and the specific contribution of its proinflammatory and proapoptotic functions remain unclear. Clarifying that role could lead to the development of novel therapeutic strategies for ALI in humans.
The Fas/FasL pathway is an important biological system capable of triggering both apoptosis and activation of inflammatory responses [4, 5]. Binding of FasL to Fas results in activation of the caspase family of cysteine proteases, leading to DNA fragmentation and apoptosis [6]. Independently of its proapoptotic function, binding of FasL to Fas can also result in activation of NF-B, presumably via activation and recruitment of the caspase-8 inhibitor FLICE-inhibitory protein [7]. Monocyte-derived macrophages release proinflammatory cytokines after Fas ligation [8]. Both Fas and FasL are expressed in the lungs, and there is increasing evidence suggesting that activation of the Fas/FasL system is associated with epithelial damage in ALI [9].
Activation of the Fas/FasL system has been implicated in the pathogenesis of lung disease. Fas-mediated apoptosis of alveolar epithelial cells has been associated with pulmonary fibrosis and ARDS [911]. Biologically active soluble FasL (sFasL) is present in bronchoalveolar lavage fluid (BALF) from patients with ARDS and pulmonary fibrosis [9, 11]. In mice, the short-term activation of Fas leads to neutrophilic alveolitis and alveolar permeability changes, whereas repeated aerosol administration of a Fas-activating antibody results in pulmonary fibrosis [12, 13]. These observations suggest that activation of the Fas/FasL system in the lungs is associated with apoptosis of alveolar epithelial cells, increased alveolar permeability, and neutrophilic alveolitis, all of which are also features of ARDS. These findings suggest that a therapeutic strategy based on blockade of the Fas/FasL system could be useful for ALI.
Blockade of the Fas/FasL system can be achieved by use of decoy receptor3 (DcR3), a member of the tumor necrosis factor (TNF) receptor family, which binds and inactivates FasL [14]. DcR3 lacks a membrane domain and acts as a soluble receptor capable of blockade of Fas-mediated apoptosis of several cell lines in vitro, including Jurkat T cells and hepatocytes [15]. A DcR3 analog (DcR3-a) has been produced that antagonizes FasL but is more stable to proteolytic degradation in vitro and in vivo because of a change of an arginine to a glutamine residue at position 218 [16]. DcR3-a attenuates FasL-induced lung inflammation in mice [17]. The potential therapeutic role of DcR3 has been shown in experiments demonstrating that administration of DcR3 significantly improves mortality in mice with Fas-induced hepatitis [15]. However, blockade of the Fas/FasL system could render the host more susceptible to infection, by suppressing the proinflammatory function of the Fas/FasL system.
Information regarding the role that the Fas/FasL system plays in pulmonary host defenses is conflicting. We have previously reported that mouse pulmonary host defenses are not impaired by Fas deficiency and that Fas deficiency appears to be protective against permeability changes in mice with diffuse pneumonia induced by administration of aerosolized Escherichia coli, Streptococcus pneumoniae, or Staphylococcus aureus [18]. However, using a mouse model of focal pneumonia with higher doses of Pseudomonas aeruginosa, Grassme et al. discovered that mice deficient in Fas or FasL are prone to systemic dissemination of bacteria and have increased mortality [19]. Grassme et al. postulated that apoptosis of lung epithelial cells could act as a protective mechanism against dissemination of bacteria from the lungs. These findings raise the potential that blockade of the Fas/FasL system as a treatment of ALI/ARDS could increase the risk of disseminated infections, because bacteria are frequently found in the airways and lungs of patients with ALI/ARDS [20, 21]. The goal of the present study was to determine the effect of blockade of the Fas/FasL system by either DcR3-a or the lpr mutation on the pulmonary host defenses of mice with pneumonia due to S. pneumoniae, which is the most common cause of community acquired pneumonia in adults [22].
MATERIALS AND METHODS
Reagents
DcR3-a was produced by changing an arginine to a glutamine residue at position 218, as described elsewhere [16]. Reagents used for immunohistochemical analysis included purified antiactive caspase-3 monoclonal antibody clone c92-605 (PharMingen), biotinylated goat antirabbit IgG (Vector), and normal rabbit IgG (Jackson Laboratories).
Animal Protocol
The animal protocol was approved by the Animal Care Committee of the Veterans Affairs Puget Sound Health Care System. Two strains of mice were used: C57Bl/6 and lpr (C57Bl/6 background). All mice were male, weighed 2030 g, and were obtained from Jackson Laboratories. The lpr mice carry a naturally occurring mutation that renders them deficient in membrane Fas [23].
The mice were anesthetized by intraperitoneal (ip) administration of ketamine/xylazine and placed on an inclined surface (70°80°). The mouth was held open with a rubber band, and the tongue was displaced by use of thumb forceps. A 22-gauge cannula was inserted directly into the trachea, and 3.3 L of bacterial slurry/g of body weight was instilled into each mouse. Colloidal carbon was added to the slurry, to allow macroscopic and microscopic verification of the distribution of the inoculum. The cannula was removed, and the mouse was allowed to recover from anesthesia and was allowed free access to water and food for the duration of the experiment. The mice were euthanized 48 h after instillation of bacteria. Mice were euthanized earlier if they exhibited loss of >15% of original body weight [1] or 3 of the following: dehydration (evaluated by skin tenting test), lethargy and decreased movement, abnormal posture (such as hunching), ruffled fur, pale eyes, or loose feces [2].
One hour before being euthanized, each mouse received an ip injection of 200 L of 5% human serum albumin (HSA) in PBS (Baxter). Euthanasia was performed by ip administration of ketamine/xylazine. After the mouse was euthanized, the thorax was rapidly opened, and the mouse was exsanguinated by direct cardiac puncture. The trachea was cannulated by use of a 20-gauge catheter, the left hilum was clamped, and the left lung was removed for homogenization. The right lung was lavaged with an initial 0.6-mL aliquot of 0.9% NaCl plus 0.6 mmol/L EDTA (NaCl/EDTA) at 37°C, followed by 3 separate 0.5-mL aliquots of NaCl/EDTA, and then was fixed by intratracheal (int) instillation of 4% paraformaldehyde at 15 cm of H2O transpulmonary pressure.
The BALF aliquots from each mouse were pooled. An aliquot was processed immediately for total and differential cell counts by use of a hemacytometer. The remainder of the BALF was spun at 200 g to pellet cells, and the supernatants were stored in individual aliquots at -70°C for determination of total proteins and cytokines.
The left lung was weighed and then homogenized in 1.0 mL of sterile distilled H2O by use of a handheld homogenizer. An aliquot was used for quantitative bacterial cultures. The remainder of the homogenate was divided into aliquots and stored at -70°C for determination of cytokines and myeloperoxidase (MPO). For determination of cytokines, the homogenate aliquot was vigorously mixed with a buffer containing 0.5% Triton X-100, 150 mmol/L NaCl, 15 mmol/L Tris, 1 mmol/L CaCl2, and 1 mmol/L MgCl (pH 7.4), was incubated for 30 min at 4°C, and was spun at 10,000 g for 20 min. The supernatants were stored at -70°C. For determination of MPO, the homogenate was vigorously mixed with 50 mmol/L potassium phosphate (pH 6.0), with 5% hexadecyltrimethyl ammonium bromide (Sigma) and 5 mmol/L EDTA. The mixture was sonicated and spun at 12,000 g for 15 min at 25°C, and the supernatants were stored at -70°C.
Experimental Protocol
The mice were divided into 3 treatment groups. The DcR3-a group received DcR3-a (400 g/mouse) administered subcutaneously (sc) every 12 h, beginning immediately before instillation of bacteria. The C57Bl/6 and the lpr groups received bovine serum albumin (BSA) sc every 12 h. The lpr mice were used as a genetic negative comparison group.
Mice from each of these groups received S. pneumoniae (serotype 3; ATCC 6303) at either 7.2 × 105 or 1.9 × 107 cfu/mL by int instillation, as described above. The times and doses were derived from pilot experiments that determined the clearance rates and optimal inocula in the mouse strains used. The pneumococci were prepared and grown as described elsewhere [18].
Measurements
Bacterial cultures.
Quantitative cultures of lung homogenates were performed by spreading serial 10-fold dilutions on 5% sheep-blood agar plates and counting colonies after 24 h of incubation at 37°C in 5% CO2.
Lung alveolar permeability.
Lung alveolar permeability was measured by determining the concentration of HSA in BALF, by immunoassay, by use of rabbit anti-HSA IgG (Dako) as capture antibody, horseradish peroxidase (HRP)labeled rabbit anti-HSA IgG P0356 (Dako) as detecting antibody, and HSA (Albuminar-25; Centeon) as the standard. Briefly, a 96-well flat-bottom high-binding plate (Costar) was incubated overnight at 4°C with 0.14 g/mL rabbit anti-HSA IgG in 25 mmol/L Na2CO3 buffer (pH 9.5). After incubation, the plate was washed once with PBS containing 0.05% Tween-20 (PBS-T) (Sigma) and blocked for 1 h at 37°C with 0.2% I-Block (Tropix) and PBS-T (pH 7.2). The plate was washed once with PBS-T, and the standards and samples were added to the plate and incubated for 1 h at 37°C. The plate was washed 3 times with PBS-T, and then HRP-labeled rabbit antiHSA P0356 was added at 0.26 g/mL. After incubation for 1 h at 37°C, the plate was washed 3 times with PBS-T and developed with 3,35,5-tetramethylbenzidine/peroxide substrate solution (KPL). After 20 min at 37°C, the reaction was stopped with 1.0 mol/L phosphoric acid, and the optical density (450 nm) in each well was read in a microtiter plate reader (Dynatech). The antibodies were diluted in 0.2% I-Block. This assay is insensitive to mouse albumin, and no reactivity with mouse serum was detected.
Cytokine and FasL assays.
FasL, monocyte chemotactic protein (MCP)1, macrophage inflammatory protein (MIP)2, and TNF- were measured in lung homogenates by use of mouse-specific ELISA-based immunoassays (R&D Systems). The lower limits of sensitivity were 4.2 pg/mL for FasL, 39.06 pg/mL for MCP-1, 7.81 pg/mL for MIP-2, and 19.5 pg/mL for TNF-.
MPO.
MPO was measured in lung homogenates by use of the Amplex Red fluorometric assay, in accordance with the manufacturer's instructions (Molecular Probes).
Histopathological Analysis
Caspase-3 immunohistochemical analysis.
Immunohistochemical analysis was performed by use of the Vector Elite ABC-HP kit (Vector). Briefly, the slides were deparaffinized by heating for 60 min at 57°C and were rehydrated by washing twice in Clear Rite (Richard Allan Scientific) for 5 min, twice in 100% ethanol for 3 min, twice in 95% ethanol for 3 min, and once in distilled H2O for 5 min. The slides were rinsed twice with PBS for 5 min, and the samples were digested with citric buffer (Vector) in a microwave for 15 min at medium setting. After digestion, the slides were cooled for 10 min to room temperature, rinsed twice with PBS for 5 min, and blocked with 3% normal goat serum in nonfat milk for 60 min at room temperature. The samples were labeled with rabbit antiactive caspase-3 (p17) in a moist chamber overnight at 4°C. Next, the slides were rinsed twice with PBS and labeled with goat antirabbit biotinylated antibody for 2 h at room temperature. The slides were rinsed twice with PBS, incubated with 0.3% H2O2 in MeOH for 60 min (to block endogenous peroxidases), and then rinsed twice with PBS. The samples were labeled with ABC-HP and incubated in a moist chamber for 90 min at room temperature, rinsed twice with PBS, and developed in a moist chamber with diaminobenzidine (DAB) substrate (Sigma) in the dark for 12 min at room temperature. The slides were rinsed with running deionized H2O for 5 min and counterstained with 1% methyl green for 6 min. The slides were dehydrated with ethanol, incubated in xylene for 5 min, and mounted with Permount.
Active caspase-3 index.
Cells staining with the brown DAB reaction product were counted in randomly generated high-power fields (×400) on each slide. A minimum of 40 fields were counted in each mouse lung specimen. Results are shown as the caspase-3 index, which is the total number of positive cells for a mouse lung divided by the number of fields counted in that lung and multiplied by 100, to yield the number of positive cells per 100 fields.
In Vitro Experiments
Isolation of bone marrowderived (BMD) macrophages.
Mouse BMD macrophages from C57Bl/6 and lpr mice were prepared by cultivation in L929 cellconditioned medium, as described elsewhere [24]. Briefly, the femurs were dissected free of tissue, and the bone marrow cells were flushed with sterile Dulbecco's modified Eagle medium (BioWhittaker) and cultured on plastic petri dishes (100 × 30 mm; Nalge Nunc International) for 7 days, in medium consisting of a 1 : 1 mixture of RF-20 (RPMI 1640 medium supplemented with 20% fetal bovine serum, 1% HEPES, 1% L-glutamine, and 0.5% penicillin-streptomycin) and L929 cellconditioned medium, prepared as described elsewhere [24].
Assessment of phagocytosis by flow cytometry.
Heat-inactivated S. pneumoniae were labeled with the red fluorophore DyLight 547 (Pierce), in accordance with the manufacturer's instructions. The pneumococci were then opsonized by incubation with normal mouse serum for 30 min at 37°C, followed by 2 washes with PBS. The labeled, opsonized pneumococci were then incubated with BMD macrophages at a ratio of 10 : 1 for 2 h in 5-mL polypropylene round-bottom tubes (final concentration of macrophages, 1 × 106 cells/mL), in RF-10 (RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% HEPES, 1% L-glutamine, and 0.5% penicillin-streptomycin). After incubation, the tubes were placed on ice to terminate the reaction, the cells were washed with 2% BSA in PBS (FACS buffer), and the cell pellet was resuspended in 0.1% trypan blue for 1 min. Cells were then washed twice, resuspended in FACS buffer, and analyzed by use of the FL3 channel of a FACScan (Beckton Dickinson) cytometer, while gates were drawn to exclude debris.
Assessment of apoptosis by flow cytometry.
BMD macrophages were plated onto 12-well tissue-culture plates, in RF-10, and were incubated, for 18 h at 37°C in 5% CO2, with heat-inactivated S. pneumoniae at a 10 : 1 ratio (final concentration of macrophages, 1 × 106 cells/mL). Staurosporine (0.5 mol/L; Sigma) was added to some of the wells to serve as a "dead cell" control. After incubation, the cells were washed with PBS and incubated with fluorescein isothiocyanatelabeled Annexin V and propidium iodide, in accordance with the manufacturer's instructions (apoptosis detection kit; Trevigen). The percentage of cells binding to Annexin V and/or propidium iodide was determined by 2-color fluorescence analysis by use of the FL1 and FL2 channels of a FACScan cytometer, after gates were drawn to exclude debris.
Assessment of phagocytosis and apoptosis by confocal microscopy.
BMD macrophages and heat-inactivated, opsonized S. pneumoniae labeled with DyLight 577 were incubated, as described above, for 4 h in 8-well glass-chamber slides (LabTek; Nalge Nunc). After incubation, the glass-chamber slides were spun at 1200 g, the supernatant was removed, and the cells were labeled with Alexa Fluor 488conjugated Annexin V, in accordance with the manufacturer's instructions (Molecular Probes), and the nuclei were counterstained with TOpro1 (Molecular Probes). After labeling and washing, in accordance with the manufacturer's instructions, the slides were covered with a thin (<1.5-mm) coverslip and mounted with aqueous media. The cells were visualized by use of a Leica confocal microscope with a 488-nm argon laser (fluorescein/rhodamine).
Statistical Analysis
When the data followed a normal distribution, comparisons between continuous variables were performed by use of Fischer's exact test, and comparisons between multiple groups were performed by use of factorial analysis of variance followed by Fischer's post hoc test. When the data deviated from a normal distribution, comparisons between multiple groups were performed by use of Kruskall-Wallis nonparametric analysis, and comparisons between 2 groups were performed by use of the Mann-Whitney U test. P < .05 was considered to be significant.
RESULTS
FasL Concentrations in the Lungs of Mice with Experimental Pneumococcal Pneumonia
Lung homogenates from mice euthanized 48 h after int instillation of S. pneumoniae at either 7.2 × 105 (high dose) or 1.9 × 107 (low dose) cfu/mL contained FasL at mean ± SE concentrations of 32.3 ± 6 and 78.7 ± 11.7 pg/mL, respectively (P < .05) (figure 1). FasL was undetectable in the lungs of mice without pneumonia.
Effects of Systemic Blockade of the Fas/FasL System by DcR3-a on Mice with Pneumococcal Pneumonia
Mortality.
After instillation of low-dose S. pneumoniae, 1 of 10 mice in each of the lpr and C57Bl/6 groups died before the end of the 48-h observation period. After instillation of high-dose S. pneumoniae, 1 of 10 mice in the C57Bl/6 group died prematurely, whereas all of the mice in the DcR3-a and lpr groups survived.
Clearance of bacteria.
Forty-eight hours after instillation of low-dose S. pneumoniae, the mean ± SE lung bacterial burdens in the DcR3-a and lpr mice were significantly lower than that in the C57Bl/6 mice (4.6 ± 2.1 × 106 and 3.5 ± 1.6 × 106 vs. 21.9 ± 9.3 × 106 cfu/lung, respectively; P < .05) (figure 2). However, the effect of DcR3-a and Fas-deficiency on clearance of bacteria was lost at higher doses of bacteria (figure 2). The clinical responses of the mice, as measured by the percentage of weight loss throughout the experiment, mirrored the recovery of bacteria in the lungs. At the lower dose of bacteria, the C57Bl/6 mice lost significantly more weight (mean ± SE, 12.0% ± 1.6%) than did the DcR3-a (mean ± SE, 6.5% ± 1.7%) (P < .05) or lpr (mean ± SE, 5.8% ± 1.8%) (P < .05) mice (figure 3), whereas, at the higher dose of bacteria, all of the mice lost similar amounts of weight.
Systemic dissemination of the infection.
To evaluate systemic dissemination of the infection, we measured recovery of bacteria in the spleens of the mice. With the exception of 1 mouse in the lpr group, mean ± SE bacterial cultures in the spleens of the C57Bl/6, DcR3-a, and lpr mice were similar (5.6 ± 4.6 × 103, 3.2 ± 1.9 × 103, and 3.6 ± 2.2 × 103 cfu/mL, respectively). The 1 outlier in the lpr group had a spleen bacterial count of 7.0 × 106 cfu/spleen and was the sickest mouse of its group. Treatment with DcR3-a had no effect on recovery of bacteria in the spleens. After instillation of high-dose S. pneumoniae, the bacterial counts in spleens were similar in each of the 3 groups of mice.
Inflammatory response.
After instillation of low-dose S. pneumoniae, the mean ± SE percentage of polymorphonuclear neutrophils (PMNs) in BALF from the DcR3-a and lpr mice was significantly lower than that in BALF from the C57Bl/6 mice (19.3% ± 9.5% and 19.3% ± 9.5% vs. 55.0% ± 12.2%, respectively; P = .02 and P = .03, respectively) (figure 4A). The decrease in the percentage of PMNs in BALF was due to a decrease in the number of total PMNs in BALF relative to the number of total macrophages in BALF (figure 4B). The changes in numbers of PMNs were limited to the airspaces because MPO activity in whole-lung homogenates, which reflects numbers of total intravascular and extravascular PMNs, was similar in the 3 groups (figure 4C). The lower percentage of PMNs in BALF from the DcR3-a and lpr mice was associated with significantly lower concentrations of TNF- and MIP-2 in lung homogenates, compared with those in the C57Bl/6 mice (table 1). The number of total cells in BALF from mice instilled with low-dose S. pneumoniae was similar in the 3 groups (data not shown).
Alveolar epithelial and tissue responses.
Alveolar permeability was assessed by determining the concentration of HSA in BALF (table 2). There was no significant difference in the concentration of HSA in BALF between C57Bl/6 mice, lpr mice, and DcR3-a mice.
In Vitro Determination of Macrophage Phagocytosis of Bacteria, Apoptosis, and Release of Cytokines
To determine whether the improved clearance of pneumococci seen in lpr mice was due to either improved survival of macrophages or enhanced phagocytosis of pneumococci, we measured phagocytosis and apoptosis of BMD macrophages from C57Bl/6 and lpr mice after incubation with heat-inactivated, opsonized S. pneumoniae. After 4 h of incubation, BMD macrophages from C57Bl/6 and lpr mice showed similar phagocytic activity toward heat-inactivated S. pneumoniae (data not shown) and released similar amounts of cytokines (table 3). After 18 h of incubation with heat-inactivated, opsonized S. pneumoniae, the mean ± SE rates of apoptosis of BMD macrophages from C57Bl/6 and lpr mice remained similar (28.0% ± 8.2% and 30.6% ± 8.8% apoptotic cells, respectively).
DISCUSSION
The main goal of the present study was to determine whether blockade of the Fas/FasL system affects host defenses in a mouse model of S. pneumoniae pulmonary infection. The primary finding was that disruption of the Fas/FasL system, either by systemic administration of DcR3-a or as a result of the mutation encoded by the lpr gene, reduced the bacterial load in the lungs 48 h after instillation of bacteria. This was associated with a decreased percentage of PMNs in BALF and a lower concentration of TNF- and MIP-2 in lung homogenates. Thus, in this mouse model of infection, blockade of the Fas/FasL system improved clearance of bacteria and reduced inflammation in the lungs.
Activation of Fas can trigger apoptosis of target cells. In vitro studies have shown that rabbit primary alveolar type II cells, distal lung epithelial cells, and the neoplastic bronchoalveolar cell line A549 express Fas on their surface and become apoptotic in response to Fas ligation [9, 25, 26]. In vivo studies have shown that Fas ligation with recombinant sFasL or with an agonistic anti-Fas antibody (Jo-2) is followed by increased alveolar permeability and apoptosis of alveolar epithelial cells and that chronic stimulation of Fas with Jo-2 leads to pulmonary fibrosis in mice [12, 27]. sFasL has been detected in BALF from patients with ARDS, and higher concentrations of FasL are associated with increased mortality [9]. On the basis of these findings, we and others have proposed that the permeability changes seen during noninfectious lung inflammatory diseases such as ARDS could be associated with Fas-mediated apoptosis of alveolar epithelial cells, raising the possibility that blockade of the Fas/FasL system could be a potential therapeutic strategy for ALI.
A potential limitation of a therapeutic strategy of blockade of the Fas/FasL system is that it might increase host susceptibility to infections by disrupting release of cytokines. Independently of its proapoptotic effects, activation of Fas can also lead to NF-B translocation and the release of proinflammatory cytokines. Distal lung epithelial cells can release IL-8 after exposure to an activating anti-Fas antibody in vitro [28], and the intrabronchial administration of recombinant human FasL to rabbits resulted in lung injury and increased expression of IL-8 by alveolar macrophages in vivo [25]. Although inhibition of release of cytokines could be of some benefit in certain inflammatory diseases (such as ARDS), it could also increase the susceptibility to secondary pulmonary infections.
Because of this concern, in a previous study, we investigated the antibacterial responses of normal mice and mice deficient in Fas 6 and 12 h after administration of aerosolized E. coli, S. aureus, or S. pneumoniae [12]. We found that clearance of bacteria was not impaired by Fas deficiency in response to any of the organisms tested, and, surprisingly, the lpr mice had improved clearance of S. aureus at 6 h. At 12 h, neither the wild-type (wt) nor the lpr mice were able to clear S. pneumoniae, but S. pneumoniae proliferated faster in the wt than in the lpr mice, and this was associated with lower numbers of total PMNs in BALF. These findings unexpectedly suggested that Fas deficiency might be associated with improved host responses to gram-positive bacteria.
In the present study, we have evaluated the role that the Fas/FasL system plays in pulmonary responses to int instillation of S. pneumoniae. The study was performed over the course of 48 h, a longer period of time than that in our previous study, and the int technique of administration allowed us to investigate different doses of bacteria. We chose pneumococcus because it is the most common cause of community-acquired pneumonia requiring care in an intensive-care unit in adults and also because humans with acute pneumococcal pneumonia have elevated concentrations of sFasL in plasma [22, 29]. Two different strategies to disrupt Fas/FasL responses were used: the use of mice naturally deficient in membrane Fas (lpr mice) and the administration of DcR3-a, which neutralizes sFasL with the same efficiency as does DcR3 but is more resistant to proteolytic degradation in vitro and in vivo [16]. DcR3-a prevents lung injury induced by activation of Fas in mice [15, 17].
The concentration of sFasL in mouse lung homogenates increased in a dose-dependent manner in response to int instillation of S. pneumoniae. Blockade of the Fas/FasL system improved clearance of bacteria in the lungs of mice 48 h after instillation of S. pneumoniae. This improvement in clearance of bacteria was associated with decreased weight loss, indicating a better clinical status. The finding that blockade of Fas did not worsen pneumonia confirms our previously reported findings, suggesting that therapeutic blockade of Fas would not necessarily place a critically ill patient at a higher risk for gram-positive infectious complications in the lungs. Interestingly, systemic dissemination of bacteria was not affected by the improvement in local clearance of bacteria. Moreover, despite the more rapid clearance of bacteria observed in the lungs of the lpr and DcR3-a mice, the bacterial content of the spleens was similar in all 3 groups of mice. One possible explanation is that dissemination of bacteria results from early bacteremia, which occurs shortly after the instillation of bacteria and is probably associated with early bacterial growth in the lungs. Previous studies have shown that, within the first 12 h after experimental pneumococcal infection in the lungs, there is local proliferation of bacteria in the lungs of both wt and lpr mice, although proliferation is slower in the lpr mice [18]. It is quite possible that dissemination of bacteria occurs and that the spleens are seeded during this initial phase of proliferation. Local differences in the role that the Fas/FasL system plays in clearance of bacteria in these separate organs may reflect differences in specific resident immune effector cells, such as resident tissue macrophages [30, 31].
The improved clearance of S. pneumoniae resulting from disruption of the Fas/FasL system could result from several possible cellular mechanisms. One possibility is that the ability of macrophages to phagocytose S. pneumoniae is improved by Fas deficiency. However, we found that BMD macrophages from wt and lpr mice showed the same phagocytic ability, released similar amounts of cytokines, and demonstrated similar rates of apoptosis in response to S. pneumoniae in vitro. Furthermore, apoptosis of macrophages has been associated with improved phagocytosis and killing of S. pneumoniae, but the mechanism does not depend on membrane Fas [32]. It is also unlikely that decreased apoptosis of PMNs played a role in the improved response to S. pneumoniae, because, if this were the case, one would expect to see higher numbers of PMNs in lpr mice infected with S. pneumoniae. However, in both of our studies, we failed to see an enhancement of the neutrophilic response to pneumococcus in the lungs. Thus, it appears unlikely that the effects of the Fas/FasL system on host responses to S. pneumoniae are mediated by macrophages or neutrophils.
A second possibility explaining the protective effect of disruption of the Fas/FasL system on host defenses to S. pneumoniae is that protection of the epithelium from Fas-mediated apoptosis improves the ability of the host to respond to pneumococci by preserving the integrity of the alveolar-capillary barrier and, thus, facilitating localization of the infection to the airspaces. In support of this possibility, in the present study, pneumococcal infection was associated with apoptosis of cells of the alveolar walls, which was decreased both by Fas deficiency and by blockade of Fas. However, we did not see clear evidence that disruption of the Fas/FasL system protected the functional integrity of the alveolar-capillary barrier. The role that the epithelium plays in host defenses to pneumonia remains controversial. Grassme et al. have suggested that Fas-mediated apoptosis of bronchial epithelial cells is protective in a mouse model of P. aeruginosa pneumonia, but these findings have not been reproduced [19, 33]. Further studies are needed to clarify the role that apoptosis of lung epithelial cells plays in host responses to bacterial pneumonia.
A third possibility is that the protective effect of disruption of the Fas/FasL system on the host response to S. pneumoniae experimental pneumonia was due to decreased apoptosis of lymphocytes. Several studies have shown that experimental sepsis and pneumonia are associated with apoptotic depletion of lymphocytes [34]. Apoptotic depletion of lymphocytes has also been observed in humans during the early phases of pneumococcal pneumonia [29]. In mouse models of sepsis, prevention of apoptosis of lymphocytes improves survival, whereas adoptive transfer of apoptotic splenocytes increases mortality [3537]. The deleterious effect of apoptosis of lymphocytes in these models of sepsis appears to be mediated, at least in part, by a decrease in the production of interferon (IFN) [35]. In agreement with these findings, we have previously reported that the expression of IFN- is down-regulated in Fas-mediated ALI in mice [12]. The present study was not designed to evaluate apoptosis of lymphocytes, and further studies will be necessary to clarify the role that lymphocytes play in the host response to S. pneumoniae lung infections.
In summary, the concentration of sFasL increases in the lungs of mice with pneumococcal pneumonia. Disruption of the Fas/FasL system results in decreased bacterial loads and a lower concentration of proinflammatory cytokines in response to pneumococcal pneumonia. Systemic blockade of the Fas/FasL system by DcR3-a results in faster clearance of bacteria in the lungs of mice with pneumococcal pneumonia but does not improve clearance of bacteria in the spleens. Partial or complete inhibition of the Fas/FasL system by systemic inhibitors, such as DcR3-a, may play a role in some intrapulmonary bacterial infections.
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