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Published ahead of print on May 15, 2008, doi:10.1165/rcmb.2007-0328OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 522-529, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0328OC

Modulation of the Inflammatory Response to Streptococcus pneumoniae in a Model of Acute Lung Tissue Infection

Feng Xu1,2, Daniel Droemann1, Jan Rupp4, Huahao Shen2, Xiaohong Wu1,5, Torsten Goldmann6, Stefan Hippenstiel7, Peter Zabel1,3 and Klaus Dalhoff1

1 Medical Clinic III, and 4 Institute of Medical Microbiology and Hygiene, University of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany; 2 Department of Respiratory Medicine, Second Affiliated Hospital, and 5 Department of Pulmonary Medicine, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China; 3 Medical Clinic, and 6 Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany; and 7 Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-University, Berlin, Germany

Correspondence and requests for reprints should be addressed to Klaus Dalhoff, M.D., Medical Clinic III, Campus Lübeck, University of Schleswig-Holstein, 23538 Lübeck, Germany. E-mail: klaus.dalhoff{at}uni-luebeck.de


   Abstract
Top
 Abstract
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia and is a main cause of infectious deaths. However, little is known about host–pathogen interaction in human lung tissue. We tested the hypothesis that human alveolar macrophages (AMs) and alveolar epithelial cells (AECs) are important for initiating the host response against S. pneumoniae, and we evaluated the role of Toll-like receptor (TLR) 2, TLR4, and p38 mitogen-activated protein kinase (MAPK) signaling in the inflammatory response after pneumococcal infection. We established a novel model of acute S. pneumoniae infection using vital human lung specimens. In situ hybridization analysis showed that S. pneumoniae DNA was detected in 80 to 90% of AMs and 15 to 30% of AECs after in vitro infection accompanied by increased expression of inflammatory cytokines. Enhanced phosphorylation of p38 MAPK and increased TLR2 and 4 mRNA expression were observed in infected lung tissue. Thirty to fifty percent of AMs and 10 to 20% of AECs showed evidence of apoptosis 24 hours after pneumococcal infection. After macrophage deactivation with Clodronate/liposomes, infected lung tissue exhibited a significantly decreased release of inflammatory mediators. Inhibition of p38 MAPK signaling markedly reduced inflammatory cytokine release from human lungs, whereas TLR2 blockade revealed only minor effects. AMs are central resident immune cells during S. pneumoniae infection and are the main source of early proinflammatory cytokine release. p38 MAPK holds a major role in pathogen-induced pulmonary cytokine release and is a potential molecular target to modulate overwhelming lung inflammation.

Key Words: Streptococcus pneumoniae • pulmonary inflammatory response • alveolar macrophages • Toll-like receptor • mitogen-activated protein kinase

Despite the development of potent antimicrobial therapy, pneumonia remains one of the leading causes of death and the major cause of deaths from infectious diseases. Streptococcus pneumoniae is the most frequently isolated pathogen in community-acquired pneumonia and one of the most common causes of septic shock, bacterial meningitis, and adult respiratory distress syndrome (1). The rapid emergence of antibiotic resistance and the limited efficacy of the polysaccharide vaccine urge further efforts to understand the host response mechanisms involved in pneumococcal pneumonia.

Much of our knowledge about the pathogenesis of S. pneumoniae infection is derived from animal studies and cell culture experiments (2–7), whereas little is known about pathogen-cell-interaction within the human pulmonary compartment. Given the fact that humans are the natural hosts for S. pneumoniae, evaluation of the host–pathogen interaction in human lungs seems mandatory. As lung tissue specimens from patients with pneumococcal pneumonia are obviously not available, we established a model of acute S. pneumoniae infection in vital lung specimens from patients undergoing lung resection due to pulmonary nodules to analyze infection patterns and host cell response.

Alveolar macrophages (AMs) are central immune cells involved in the pulmonary host defense (8). However, the exact role of macrophages and other resident cells in the inflammatory response to pneumococcal infection has not been well characterized. Toll-like receptors (TLRs) are central molecules recognizing invading pathogens and activating signaling pathways of inflammation (9). TLR2 was identified as a key pattern recognition receptor in the immune response to cell wall components of gram-positive bacteria such as peptidoglycan and lipoteichoic acid (10). However, the role of TLR2 in the recognition of pathogens in human lung infections is less clear, although it is expressed on AMs and alveolar epithelial cells (11). TLR4 also has a role in the innate immune response to pneumolysin and intact pneumococci (12, 13). A central role of TLR-related activation of the immune system in pneumococcal pneumonia is furthermore evidenced by the demonstration of enhanced susceptibility of TLR9-deficient mice to pneumococcal infection (14). In addition, pneumococci are detected intracellularly by cytosolic receptors as nucleotide-binding oligomerization domain 2 (Nod2) (15). Both stimulation of TLRs and Nod2 culminates in the activation of nuclear factor (NF)-{kappa}B, as well as mitogen-activated protein kinases (MAPKs) (16). There is increasing evidence from in vitro cell stimulation experiments indicating that p38 MAPK is involved in the pneumococci-induced expression of inflammatory mediators in lung epithelium (17, 18).

The present study was undertaken to evaluate cellular and molecular mechanisms of the early immune response against S. pneumoniae in human lung tissue. We tested the hypothesis that human alveolar macrophages and alveolar epithelial cells are important for initiating the host response against S. pneumoniae, and we evaluated the role of TLR2, TLR4, and p38 MAPK signaling in the inflammatory response after pneumococcal infection.

Part of the results of this study has been previously reported in the form of an abstract (19).


   MATERIALS AND METHODS
Top
 Abstract
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Human Lung Tissue and Cell Infection Model
S. pneumoniae strain serotype 3 (ATCC 6303; Rockville, MD) was grown on sheep blood agar plates at 37°C and 5% CO2. Vital lung specimens at least 5 cm away from the tumor front were used in the experiments. The protocol was approved by the Ethical Committee of University of Lübeck, Germany. After 4 and 24 hours of infection, supernatants were harvested and lung specimens were fixed at 4°C in the Hepes–glutamic acid buffer mediated organic solvent protection effect (HOPE) solution (20). A549 cells and monocytes were infected with S. pneumoniae (multiplicity of infection [MOI]: 10 and 100).

In Situ Hybridization
In situ hybridization (ISH) was done as described previously (21). Hybridization targeting the CAP3 gene for uridine diphosphate glucose dehydrogenase of S. pneumoniae was performed in moist chambers.

RT-PCR
RT-PCR was performed using NucleoSpin RNA II kit (Macherey-Nagel, Dueren, Germany) and reverse transcribed into cDNA (First-Strand PCR kit; Roche, Mannheim, Germany), and PCR amplification was performed using LightCycler Detection System.

Western Blot Assay
Lung homogenates and cell pellets were lysed, subjected to 12% SDS-PAGE, and blotted on nitrocellulose membrane (Sartorius, Goettingen, Germany). Immunodetection of phosphorylated p38 MAPK was performed with specific antibodies (Cell Signaling Technology, Beverly, MA).

Macrophage Deactivation Experiments
For selective deactivation of macrophages from lung tissue, Clodronate/liposomes, kindly provided by Dr. Van Rooijen N (Vrije University, Amsterdam, The Netherlands), was used (22–25). Lung tissues were incubated with 1 ml of Clodronate/liposomes-RPMI 1640 (1:1; containing 2.5 mg Clodronate), or PBS/liposomes-RPMI 1640 (1:1) for 24 to 48 hours, then incubated with pneumococcal suspensions (107 colony-forming units [CFU]/ml) for 24 hours.

Lung Cell Apoptosis
Apoptosis in lung specimens was measured using the in situ Cell Death Detection TUNEL Kit AP (Roche) according to manufacturer's recommendations. Detection was performed with the same new-fuchsine substrate used in ISH.

Immunohistochemistry
Immunohistochemistry was performed as described previously (21). The anti-human CD11b and cleaved caspase-3 antibodies (BD Biosciences, Heidelberg, Germany) were applied in a dilution of 1/100. Detection was done by an alkaline phosphatase–labeled streptavidin-biotin technique but in higher dilution (Anti-mouse biotin 1/3, streptavidin-AP 1/3).

Inhibition Experiments
Lung specimens were pretreated for 1 hour with TLR antibodies (anti-TLR2: 10 µg/ml, anti-TLR4: 10 µg/ml; eBioscience, San Diego, CA) or p38 MAPK inhibitor (SB203580: 20 µM; Calbiochem, Darmstadt, Germany), then stimulated with S. pneumoniae (107 CFU/ml).

Enzyme-Linked Immunosorbent Assay
Quantitative determination of IL-8, IL-6, and TNF-{alpha} was performed by following the manufacturer's recommendations (R&D Systems, Minneapolis, MN). The lower detection limits were 31.2 pg/ml for IL-8, 9.4 pg/ml for IL-6, and 15.6 pg/ml for TNF-{alpha}.

Statistical Analysis
Data are presented as the mean ± SEM. For independent samples, paired t test or one-way ANOVA with post hoc testing by the least significant difference (LSD) was used to analyze differences between groups as appropriate. A P value < 0.05 was considered statistically significant. Calculations were performed with SPSS for Windows software program 11.5 (SPSS, Chicago, IL).


   RESULTS
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Patients and Lung Tissues
The study population consisted of 26 patients who underwent lobar or atypical lung resections because of peripheral pulmonary nodules. Patients with clinical or laboratory signs of acute respiratory infection were excluded. The mean age of the study population was: 64.0 ± 1.9 years, 12 male and 14 female; lung function testing showed an FEV1/VC: 71.1 ± 3.0%, FEV1: 77.5 ± 3.8% predicted. Histologic diagnoses included 20 cases of lung cancer, 5 cases of metastases from extrapulmonary tumors, and 1 case of cystic echinococcosis.

Detection of S. pneumoniae in Human Lung Tissue
Twenty-four hours after infection, S. pneumoniae was predominantly detected in AMs, 80 to 90% of which stained positive for pneumococcal DNA (Figures 1A and 1B). In addition, 15 to 30% of alveolar epithelial cells (AECs) showing the morphologic characteristics of type II cells were S. pneumoniae positive (Figure 1C). In contrast, bronchial epithelial cells (BECs) were only sporadically infected (< 1%, not shown). Stimulation over 48 hours showed increasing infection rates of AECs.


Figure 1
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  		Figure 1. In situ hybridization analysis of Streptococcus pneumoniae DNA in human lung tissue specimens 24 hours after stimulation. The presence of S. pneumoniae DNA was observed to be mainly located in alveolar macrophages (AMs) (A, x200; B, x400; arrows) and alveolar epithelial cells (AECs) type II (C, x600; arrow) in infected lung tissue 24 hours after stimulation. Lung tissue without stimulation was used as a negative control (D, x400).

 
Lung Cell Apoptosis
After pneumococcal infection, a time-dependent increase of AM apoptosis was observed as compared with the control group (30–50% versus 4–8% [24 h], 40–70% versus 10–15% [48 h]; Figure 2A). In addition, AECs showed a higher proportion of apoptotic cells as compared with controls (10–20% versus 0–3% [24 h], Figure 2B), whereas no positive staining was found in noninfected lung tissues (Figure 2C). Apoptosis was associated with marked caspase-3 activation, as shown in Figure 2D.


Figure 2
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  		Figure 2. TUNEL and caspase-3 detection in human lung tissue 24 hours after S. pneumoniae infection in vitro. TUNEL staining was detected in AMs and AECs in infected lung specimens (A, x400; B, x600), whereas no positive staining was found in nonstimulated lung tissues (C, x400). Meanwhile, caspase-3–positive staining was detected in AMs and AECs of pneumococci-infected tissues (D, x400).

 
Role of AMs in Pneumococci-Induced Inflammation
To explore the relative contribution of AMs to the inflammatory response, lung tissue was pre-incubated with Clodronate/liposomes in vitro and followed by stimulation with S. pneumoniae. A decrease of 70 to 80% of CD11b-positive macrophages as compared with medium controls was demonstrated 24 hours after Clodronate/liposomes treatment (Figures 3A and 3B). This was accompanied by nearly complete ablation of TNF-{alpha} production and by a marked reduction of IL-6 and IL-8 levels in pneumococci-infected lung tissue (Figures 3C and 3D) underlining the central role of macrophages in the inflammatory response to S. pneumoniae. Clodronate/liposomes treatment was also associated with markedly enhanced TUNEL staining in AMs compared with PBS/liposomes control (45–75% versus 4–8% at 24 h), which gradually declined during further incubation in noninfected tissue (30–50% at 72 h). In contrast to apoptosis induced by pneumococcal infection, this effect was selective for macrophages and the rate of AEC apoptosis was not affected (Figure 3F). Furthermore, detection of activated caspase-3 indicated that Clodronate treatment induced caspase-3–dependent apoptosis of AMs in lung tissues (Figure 3G).


Figure 3
Figure 3
Figure 3
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  		Figure 3. Immunohistochemistry analysis of CD11b showed positive staining of AMs in experiments without Clodronate (A, x600), whereas 24 hours of Clodronate treatment induced a marked reduction of CD11b expression (B, x600). Lung specimens were pretreated with PBS/liposomes or Clodronate/liposomes for 24 hours (C, n = 6) and 48 hours (D, n = 5), respectively, before S. pneumoniae infection. Clodronate significantly inhibited the production of TNF-{alpha}, IL-8, and IL-6 in infected lung tissue in a time-dependent manner. A marked reduction of IL-6 and IL-8 levels was also seen in noninfected samples (E, n = 3). The detection of TUNEL (F, x400) and caspase-3 (G, x600) indicated that Clodronate treatment induced caspase-3–dependent apoptosis of AMs in lung tissues. Spn, S. pneumoniae; ND, not detected; *P < 0.05 versus PBS/liposomes-treated tissues.

 
TLR2 and TLR4 mRNA Expression
We next determined the regulation of TLR expression in the human lung after pneumococcal infection. Compared with noninfected lung specimens, S. pneumoniae stimulation resulted in a 2.9-fold increase in TLR2 mRNA expression 24 hours after infection. Similarly, there was a 2.7-fold increase in the expression of TLR4 mRNA in response to pneumococcal stimulation (Figure 4). These data indicate that TLR2 and TLR4 are both up-regulated in the course of the pulmonary immune response during pneumococcal infection.


Figure 4
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  		Figure 4. Significantly enhanced Toll-like receptor (TLR)2 and TLR4 mRNA expression was detected by RT-PCR in lung tissue 24 hours after pneumococcal infection (n = 11). Spn, S. pneumoniae; *P < 0.05 versus medium-treated group.

 
p38 MAPK Activation
Activation of p38 MAPK is considered to be an important step in the regulation of pathogen-induced cytokine expression. Enhanced phosphorylation of p38 MAPK 4 to 24 hours after infection with pneumococci was observed in lung tissue (Figure 5). Similarly, phosphorylation of p38 MAPK was induced within 15 to 30 minutes in A549 cells and monocytes after pneumococcal stimulation, confirming that p38 MAPK signaling is involved in the host cell activation upon pneumococcal infection.


Figure 5
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  		Figure 5. Immunoblot analysis of p38 MAPK in the lung tissue and cells. (A) Enhanced phosphorylation of p38 MAPK 4 to 24 hours after infection with pneumococci was observed in the lung tissue model. Phosphorylation of p38 MAPK was rapidly induced by S. pneumoniae 15 to 30 minutes after infection in (B) A549 cells and (C) blood monocytes, indicating that p38 MAPK is involved in host cell activation upon pneumococcal stimulation. Representative gels from each three independent experiments are shown. Spn, S. pneumoniae.

 
Roles of TLRs and p38 MAPK in the S. pneumoniae–Induced Immune Response
S. pneumoniae (107 CFU/ml) induced a time-dependent innate response in the lung tissue model (24 h versus 4 h: 525.8 ± 119.0 ng/ml versus 30.6 ± 6.4 ng/ml [IL-8]; 1,167.8 ± 446.4 pg/ml versus 737.6 ± 212.7 pg/ml [TNF-{alpha}]; 1,452.5 ± 242.2 ng/ml versus 146.4 ± 27.0 ng/ml [IL-6]; n = 8). Using increasing pneumococcal inocula a dose-dependent IL-8 release from lung tissue was demonstrated 24 hours after infection: 184 ± 53 ng/ml (106 CFU/ml) versus 222 ± 40 ng/ml (107 CFU/ml) versus 314 ± 51 ng/ml (108 CFU/ml); n = 3. To further investigate the roles of TLR2, TLR4, and the p38 MAPK pathway, neutralizing TLR2 and TLR4 antibodies and the p38 inhibitor SB203580 were adopted to block inflammatory cytokine production in human lung tissue. TLR2 blockade reduced IL-8, TNF-{alpha}, and IL-6 production by 15.5%, 22.3%, and 13.8%, respectively, without reaching statistical significance. Blocking of TLR4 had no effect on the pulmonary inflammatory response in this model. Combined blockade of TLR2 and 4 Abs revealed no additive effect (Figure 6). In contrast, inhibition of p38 MAPK signaling by SB203580 significantly decreased cytokine release from infected lung tissues. Comparable changes were found when isolated blood monocytes were infected in vitro; infection of A549 epithelial cells resulted in enhanced IL-8 levels, but not in a detectable TNF-{alpha} protein release (Figure 7).


Figure 6
Figure 6
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  		Figure 6. The effect of anti-TLR2 and anti-TLR4 antibodies, and p38 MAPK inhibitor, on inflammatory response in the lung tissue model. S. pneumoniae infection enhanced the release of inflammatory mediators such as IL-8, TNF-{alpha}, and IL-6 from lung tissue after 24 hours of stimulation. Blockade of TLR2 generated a modest inhibitory effect on the production of IL-8, TNF-{alpha}, and IL-6 without reaching statistical significance, whereas TLR4 blockade had no influence on pulmonary inflammation. Furthermore, combined blockade of TLR2 and TLR4 antibodies revealed no additive effect (A, n = 4). The p38 MAPK inhibitor SB203580 markedly reduced the production of IL-8, TNF-{alpha}, and IL-6 in infected lung tissue (B, n = 11). Spn, S. pneumoniae; *P < 0.05 versus medium-treated tissue, **P < 0.05 versus Spn-treated tissue.

 

Figure 7
Figure 7
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  		Figure 7. The effect of p38 MAPK inhibitor on inflammatory response in host cells. SB203580 significantly inhibits IL-8 release from A549 cells infected with multiplicity of infection (MOI) 10 of S. pneumoniae (A, n = 7) and markedly suppresses the production of IL-8, IL-6, and TNF-{alpha} in blood monocytes stimulated with MOI 100 of S. pneumoniae (B, n = 7). Spn, S. pneumoniae; *P < 0.05 versus medium-treated cells, **P < 0.05 versus Spn-treated cells.

 

   DISCUSSION
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study, we explored interactions between S. pneumoniae and the human pulmonary immune system during the early phase of infection using a new ex vivo stimulation model of human lung tissue culture. The main findings are that AMs are central resident immune cells after pneumococcal infection which orchestrate a marked innate immune response, but are undergoing pathogen induced apoptosis after a period of 24 hours. Deactivation of AMs with Clodronate/liposomes led to a significantly reduced cytokine release from lung tissue. Concerning signaling pathways, inhibition of p38 MAPK signaling markedly reduced the inflammatory response, while blockade of TLR2 and TLR4 had no significant effects on the expression of analyzed cytokines.

Although pneumococcal pneumonia is one of the leading causes of death from infectious diseases, studies on the host–pathogen interaction in the human lung are scarce. Data from animal models cannot be easily generalized because of important interspecies differences in the expression, function, and localization of immune molecules (e.g., receptors, signaling intermediates, response molecules). Therefore, we developed a new human lung tissue infection model, which has already been successfully used for evaluation of intracellular infections (21, 26). Although this model has some inherent limitations with regard to the period of viability and the lack of recruitment of cells from the circulation, it allows for the investigation of the early innate immune response in the human lung.

It is not clear what are the most relevant target cells within the human lung after pneumococcal infection. In cell culture models, pharyngeal epithelial cells, BECs, AECs type II, monocyte-derived macrophages, erythrocytes, and vascular endothelial cells have been identified as target cells for S. pneumoniae (27–32). In contrast to transformed cell lines, the infection pattern in primary lung tissue more closely reflects the interactions between pneumococci and host cells in vivo. ISH analysis showed that S. pneumoniae was located mainly in AMs and to a minor degree in AECs type II. The infection of macrophages and the epithelial cell line A549 by S. pneumoniae was described previously in cell culture models (31, 33). The uptake by AECs may be helpful for short-term survival of pneumococci because intracellular bacteria in epithelial cells are partly protected from professional phagocytes and shielded from antibiotics. However, only few BECs were infected in the tissue model. The different infection rates of AECs and BECs may be due to their distinct characteristics with respect to pneumococcal adherence and invasion. Indeed, the primary site of colonization for pneumococci is the nasopharynx, while bronchial cells may serve as transient sites for pneumococcal attachment when the pathogens pass from the nasopharynx to the lower respiratory tract (30). Unlike other respiratory pathogens such as Hemophilus influenzae, which initiate infection in the nasopharynx and descend along the tracheobronchial tree causing bronchitis, pneumococci do not typically cause bronchial infections, although they have been associated with exacerbations of chronic bronchitis (34).

Macrophages play an essential role in pulmonary host defense by generating antimicrobial mediators, presenting antigens, and phagocytosing foreign pathogens. However, the exact role of AMs in the host defense against pneumococci is poorly understood (25). Much of our knowledge about the role of AMs in the pathogenesis of pneumonia derives from animal studies. Pulmonary macrophages, but not epithelial cells and granulocytes, have been shown to be the main cellular source of TNF-{alpha} in a mouse model of pneumococcal infection (35). Accordingly, TNF-{alpha} release from A549 cells stimulated with S. pneumoniae was negligible in our study compared with monocytes/macrophages. Furthermore, macrophage deactivation experiments resulted in a significantly decreased release of TNF-{alpha} and other inflammatory cytokines from the lung, indicating that macrophages, but not epithelial cells, are the main source of pro-inflammatory cytokines after pneumococcal infection. The residual cytokine release after macrophage deactivation is likely due to production by alveolar epithelial cells, which are not affected by Clodronate treatment and have been shown to respond to pneumococcal infection with production of IL-6 and IL-8 by previous authors (17) and in this study. Interestingly, Knapp and coworkers found that AMs have a protective anti-inflammatory role in a mouse model of pneumococcal infection by eliminating apoptotic polymorphonuclear neutrophils (24). This apparent discrepancy is likely due to the different experimental conditions, particularly the involvement of neutrophils in the mouse model and the different phases of infection addressed.

Macrophage apoptosis is a feature of several respiratory infections such as pulmonary tuberculosis. Using a low-dose pneumococcal infection model characterized by the clearance of bacteria without neutrophil recruitment, Dockrell and colleagues showed that apoptosis of alveolar macrophages contributes to the host defense against pneumococci (25). Pneumococcal infection causes apoptosis in different target cells, including neuronal cells (36), monocyte-derived macrophages (31), and A549 cells (37). Our study confirms and extends these findings by showing that AM apoptosis is a prominent feature of pneumococcal infection in the human lung tissue. The comparably high rate of apoptosis in our study may be due to the fact that apoptotic cells are not removed from the tissues in this model. Regarding the mechanism of apoptosis, Marriott and coworkers demonstrated that nitric oxide has a major role in regulating the initiation of macrophage apoptosis by S. pneumoniae, which is associated with bacterial killing (38). In addition, pneumolysin challenge leads to a depletion of AMs due to its pore-forming activity (39). We cannot totally exclude the possibility that apoptotic cells resulting from Clodronate/liposomes treatment are mediating some of the anti-inflammatory effects observed by macrophage deactivation. However, this appears unlikely, since pneumococcal infection by itself elicited comparable rates of apoptosis and caspase activation without inhibiting cytokine responses. Previous studies suggested that the immunoregulatory effects of apoptosis depend on the microenvironment in which the phagocytosis of apoptotic cells is taking place: in the presence of ongoing microbial stimulation proinflammatory effects can be observed, whereas immune deactivation is induced in the absence of such stimuli (40). In contrast, the effects of Clodronate treatment were independent of concomitant microbial stimulation in our study.

The identification of the TLR family and their signaling pathways provides insights into the pathogen recognition system of the airways. Functional TLR2 and TLR4 are constitutively expressed on AMs and airway epithelia, indicating important roles in the pulmonary immune response (11, 41). In the present study, we determined the role of TLR2 and TLR4 in the inflammatory response to S. pneumoniae in human lung tissue. We found an up-regulated expression of TLR2 and TLR4 mRNA, followed by an increased release of inflammatory mediators. These findings extend data from studies observing expression of human TLR2 but not TLR4 in Chinese hamster ovary fibroblasts in response to heat-killed S. pneumoniae (42) and up-regulation of TLR1 and TLR2 in infected lung epithelial cells (43). A recent report demonstrated that isolated AMs from TLR2–/– mice failed to release TNF-{alpha} and keratinocyte chemoattractant (murine analog of IL-8) upon stimulation with heat-killed S. pneumoniae compared with wild-type AMs, suggesting that TLR2 is indispensable for alveolar macrophage responsiveness toward pneumococci (44). To delineate the roles of TLR2 and TLR4 in human lung tissue, we performed blocking experiments using monoclonal antibodies. TLR2 blockade led to a moderate, nonsignificant inhibition of cytokine release, whereas TLR4 ligation had no effect. Furthermore, combined blockade of both TLRs did not reveal additive effects in our model. This suggests that the inflammatory response to pneumococcal challenge is only partly mediated via TLR2 and that alternative signaling pathways may provide redundancy. Indeed, MyD88–/–, but not TLR2–/–, mice were markedly defective in their expression of pro-inflammatory cytokines and chemokines after infection with heat-killed S. pneumoniae (45). However, the precise role of different pattern recognition receptors in pneumococcal pneumonia requires further in-depth investigation.

MAPK signaling is a key element in the inflammatory response by regulating the production of pro-inflammatory cytokines, and modulating intracellular enzymes and adhesion molecules (46). Recent data indicate that the p38 pathway has a role in S. pneumoniae–induced host cell responses. Monier and colleagues showed that p38 MAPK is a key signaling pathway in the up-regulation of TNF expression in murine macrophages stimulated with antibiotic-killed pneumococci and pneumococcal cell wall preparations (47). Schmeck and coworkers reported that p38 MAPK was rapidly activated and phosphorylated in the lung of mice and human bronchial epithelial cell line BEAS-2B when exposed to S. pneumoniae. Pneumococcal stimulation activated NF-{kappa}B–dependent gene transcription in HEK293 cells and induced the expression of IL-8 and granulocyte-macrophage colony-stimulating factor in BEAS-2B cells via p38 MAPK signaling pathway (17). A recent study found that pneumococci induced cyclooxygenase-2 expression and subsequent prostaglandin E2 synthesis via p38 MAPK and NF-{kappa}B in lung epithelial cells. Furthermore, the recruitment of NF-{kappa}B subunit p65 to the cyclooxygenase-2 promoter depended on p38 activation (18). Our data show phosphorylation of p38 MAPK in human lung tissue 4 hours after pneumococcal infection, increasing up to 24 hours in contrast to nonstimulated tissue. We also found that p38 MAPK was rapidly activated in A549 cells and monocytes upon stimulation, further indicating that the p38 signaling pathway is implicated in pneumococci-related lung cell activation. The blockade of p38 MAPK using SB203580 markedly inhibited the release of proinflammatory cytokines such as IL-8, TNF-{alpha}, and IL-6 from lung tissue as well as from A549 cells and monocytes. This indicates that the inflammatory response after pneumococcal infection of human lung tissue is, at least in part, p38 MAPK dependent. These results are in line with the finding from Schmeck and colleagues that p38 MAPK plays an important role in pneumococci-induced inflammatory cytokines transcription by modulating p65 NF-{kappa}B–mediated transactivation in epithelial cell line BEAS-2B (17). The fact that p38 MAPK inhibition, but not TLR blockade, is able to down-regulate proinflammatory cytokines suggests that modification of downstream signal molecules may have more profound effects on the pathogen-induced innate response than targeting single pattern recognition receptors, multiple of which are activated by the pneumococcus during respiratory infection. In vivo experiments are needed to test the efficacy of p38 inhibition as a novel therapeutic strategy to attenuate the pathology induced by pneumococcal infection.

In conclusion, we have found for the first time using a human lung tissue model that AMs are pivotal host defense cells after pneumococcal infection. AMs are the main source of proinflammatory cytokine release, but undergo infection-induced apoptosis, which may contribute to the resolution of inflammation in later stages of disease. AECs are also involved in the host defense against pneumococcal infection and contribute to lung inflammation. TLR2 appears to be implicated in cell activation, although blockade of TLR2 elicits only a slight decrease of inflammatory response and other recognition receptors may be equally important. p38 MAPK holds a major role in the pneumococci-induced pulmonary inflammation and is a potential molecular target to modulate pathogen-induced lung inflammation.


   Footnotes
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0328OC on May 15, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 6, 2007

Accepted in final form March 30, 2008


   References
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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