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Am J Physiol Lung Cell Mol Physiol 290: L1131-L1138, 2006. First published January 13, 2006; doi:10.1152/ajplung.00383.2005
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Streptococcus pneumoniae induced p38 MAPK- and NF-{kappa}B-dependent COX-2 expression in human lung epithelium

Philippe Dje N’Guessan,1 Stefan Hippenstiel,1 Mirabelle O. Etouem,1 Janine Zahlten,1,2 Wiebke Beermann,1 David Lindner,1 Bastian Opitz,1 Martin Witzenrath,1 Simone Rosseau,1 Norbert Suttorp,1 and Bernd Schmeck1

1Department of Internal Medicine/Infectious Diseases and Respiratory Medicine, and 2Institute for Periodontology and Synoptic Dentistry, Charité Centrum 3 for Dental Medicine, Charité-Universitätsmedizin Berlin, Berlin, Germany

Submitted 6 September 2005 ; accepted in final form 9 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Streptococcus pneumoniae is a major cause of community-acquired pneumonia and death from infectious diseases in industrialized countries. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX)-derived prostaglandins, such as PGE2, are considered to be important regulators of lung function. Herein, we tested the hypothesis that pneumococci induced COX-2-dependent PGE2 production in pulmonary epithelial cells. Pneumococci-infected human pulmonary epithelial BEAS-2B cells released PGE2. Expression of COX-2 but not COX-1 was dose and time dependently increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with pneumococcal pneumonia. S. pneumoniae induced degradation of I{kappa}B{alpha} and DNA binding of NF-{kappa}B. A specific peptide inhibitor of the I{kappa}B{alpha} kinase complex blocked pneumococci-induced PGE2 release and COX-2 expression. In addition, we noted activation of p38 MAPK and JNK in pneumococci-infected BEAS-2B cells. PGE2 release and COX-2 expression were reduced by p38 MAPK inhibitor SB-202190 but not by JNK inhibitor SP-600125. We analyzed interaction of kinase pathways and NF-{kappa}B activation: dominant-negative mutants of p38 MAPK isoforms {alpha}, beta2, {gamma}, and {delta} blocked S. pneumoniae-induced NF-{kappa}B activation. In addition, recruitment of NF-{kappa}B subunit p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAPK but not on JNK activity. In summary, p38 MAPK- and NF-{kappa}B-controlled COX-2 expression and subsequent PGE2 release by lung epithelial cells may contribute significantly to the host response in pneumococcal pneumonia.

cyclooxygenase-2; p38 mitogen-activated protein kinase; nuclear factor-{kappa}B

PNEUMONIA IS THE MOST COMMON infectious disease leading to death in industrialized countries. (6). Over 40% of cases are due to infections with Streptococcus pneumoniae, and high mortality has been reported (7). Simultaneously, antibiotic-resistant strains have emerged (12). Although some pathogenic factors have been identified (40) and novel strategies for vaccination against pneumococci are under investigation (2), little is known about pneumococci-host interaction.

In pneumococcal pneumonia, massive leukocyte recruitment to the lung is observed (43). Recent studies pointed to the lung epithelium as an important sentinel and effector of innate immunity (4, 37). Detection of invading pneumococci by extracellular (19) and intracellular immune receptors (27) results in liberation of proinflammatory and chemotactic cytokines by lung epithelium contributing to leukocyte invasion (34, 37). Lipid metabolites of arachidonic acid, including prostaglandins and leukotrienes, have emerged as potent endogenous mediators and modulators of innate immunity in the lung (38, 41). PGE2 in particular was shown to modulate immune and inflammatory responses (3, 9, 38, 41) and is liberated by lung epithelial cells (16). It is produced at sites of inflammation, in which it demonstrates both pro- and anti-inflammatory effects (3, 9, 16, 38, 41). PGE2 is a product of the cyclooxygenase (COX)/prostaglandin H synthase pathway, which includes two distinct isoforms of COX, the constitutively expressed COX-1 and the (generally) inducible COX-2, as well as PGE synthase enzymes (5).

COX-2 gene expression is regulated at the level of transcription, as well as posttranscriptionally. The regulation of the cox2 promoter is subjected to a tight regulatory network involving NF-{kappa}B, CCAAT/enhancer binding protein (C/EBP), and the transcription factor activator protein-1 (AP-1) (5). The specific factors regulating COX-2 induction depend on the cell type and on the stimulus (5). These transcription factors can be activated by a complex kinase pathway centered around p38 MAPK and JNK (18, 22, 26, 34). Recently, four isoforms of p38 MAPK ({alpha}, beta2, {gamma}, {delta}) with either activating or inhibiting activity on gene transcription have been characterized (26), but their role in infectious disease pathogenesis remains to be clarified. JNKs are involved in the inflammatory response of genes, which are regulated by the transcription factors NF-{kappa}B and AP-1 (22). p38 MAPK- and JNK-stimulated pathways may lead to phosphorylation of p65/RelA, thereby regulating NF-{kappa}B-dependent gene transcription (26, 29, 42).

In this study, we tested the hypothesis that pneumococci induce COX-2 expression and subsequent PGE2 synthesis by combined stimulation of kinase pathways and NF-{kappa}B in lung epithelial cells. We report here that S. pneumoniae induced COX-2 expression and PGE2 release and activation of p38 MAPK, JNK, and NF-{kappa}B in human bronchial epithelial cells. PGE2 release and COX-2 expression depended on p38 MAPK and NF-{kappa}B activation, whereas JNK was not involved in this process. Pneumococci also induced lung COX-2 protein expression in vivo in mice. Subsequent studies demonstrated that pneumococci-related NF-{kappa}B activation depended on p38 MAPK isoforms {alpha}, beta2, {gamma}, and {delta}. Recruitment of NF-{kappa}B and RNA polymerase II to the cox2 promoter was blocked by inhibition of p38 MAPK but not of JNK. Thus pneumococci induced COX-2-dependent PGE2 liberation by lung epithelial cells may contribute significantly to the pathogenesis of pneumococcal pneumonia.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. SB-202190, SB-202474, SP-600125, indomethacin, SC-560, and NS-398 were purchased from Calbiochem (Merck, Darmstadt, Germany), TNF-{alpha} from R&D Systems (Wiesbaden, Germany), and IKK-NBD from Biomol (Plymouth Meeting, PA). All other chemicals used were of analytical grade and obtained from commercial sources.

Cell lines. Human bronchial epithelial BEAS-2B cells were a kind gift from C. Harris (National Institutes of Health, Bethesda, MD) (30). Human embryonic kidney cells (HEK-293) were purchased from American Type Culture Collection (Manassas, VA). BEAS-2B (1 x 106) cells were infected with 105, 106, and 107 cfu·ml bacteria–1·ml–1 and incubated in infection medium for a given time at 37°C and 5% CO2. The multiplicity of infection used was 0.1, 1, and 10.

Bacterial strains. Pneumonia in mice was induced by the encapsulated type 3 strain S. pneumoniae NCTC 7978 (27, 34). S. pneumoniae R6x is the unencapsulated derivative of type 2 strain D39 (39). Single colony isolates of R6x were maintained at 37°C with 5% CO2 on Columbia agar with 5% sheep blood. For cell culture stimulation studies, single colonies were expanded by resuspension in Todd-Hewitt broth supplemented with 0.5% yeast extract and incubation at 37°C for 3–4 h to midlog phase (absorption of 0.2–0.4 at 600-nm wavelength). Samples were harvested by centrifugation and resuspended in cell culture medium without antibiotics.

Plasmids and transient transfection procedures. HEK-293 cells were cultured in 12-well plates with DMEM supplemented with 10% FCS. Subconfluent cells were cotransfected by using the calcium phosphate precipitation method according to the manufacturer’s instructions (Clonetech, Palo Alto, CA) with 0.2 µg of NF-{kappa}B-dependent luciferase reporter (20), 0.2 µg of respiratory syncytial virus beta-galactosidase plasmid, 0.1 µg of human Toll-like receptor 2 (hTLR2; generously provided by Tularik, San Francisco, CA) (36), 0.1 µg of dominant-negative p38{alpha}/beta2/{gamma}/{delta}-AF, which cannot be phosphorylated (kind gift of Jiahuai Han, Scripps Research Institute, La Jolla, CA) (28), expression vectors or control vector, respectively. We used a luciferase reporter-gene assay (Promega, Mannheim, Germany) to measure luciferase activity, and results were normalized for transfection efficiency, with values obtained by respiratory syncytial virus-beta-galactosidase as described previously (34).

PGE2 ELISA. Confluent BEAS-2B cells were stimulated for 16 h as indicated in a humidified atmosphere. After incubation, supernatants were collected and processed for PGE2 quantification by immunoassay according to the manufacturer’s instructions (R&D Systems).

Western blot. For determination of COX-2 induction, I{kappa}B{alpha} degradation, p38 MAPK, and JNK phosphorylation in BEAS-2B cells, cells were stimulated as indicated, washed twice, and harvested. For analysis of COX-2 induction in vivo, pneumococci-infected mouse lungs were snap-frozen in fluid nitrogen and pulverized. BEAS-2B cells or lung homogenates were lysed in buffer containing Triton X-100, subjected to SDS-PAGE, and blotted on Hybond-ECL membrane (Amersham Biosciences, Freiburg, Germany). Immunodetection of target proteins was carried out with specific antibodies: COX-2 (Santa Cruz Biotechnologies, Santa Cruz, CA), COX-1 (Upstate Biotechnology, Lake Placid, NY), phospho-specific p38-MAPK antibody (Cell Signaling, Frankfurt, Germany), and phospho-specific JNK (Santa Cruz Biotechnologies) (25, 27). We analyzed degradation of I{kappa}B{alpha} in BEAS-2B cell lysates using a rabbit polyclonal antibody (Santa Cruz Biotechnologies) as described previously (13). In all experiments, actin (Santa Cruz Biotechnologies) or p38 and JNK (Santa Cruz Biotechnologies) were detected simultaneously to confirm equal protein load. Proteins were visualized by incubation with secondary IRDye800- or Cy5.5-labeled antibodies (Odyssey infrared imaging system, LI-COR) (14, 25).

RT-PCR. To detect the presence of mRNA encoding COX-2 and GAPDH, RT-PCR analyses were performed as previously described (34). Briefly, total RNA was isolated from BEAS-2B cells with RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse transcribed with AMV reverse transcriptase (Promega). We amplified generated cDNA by PCR using specific intron-spanning-specific primers for COX-2 and GAPDH. All primers were purchased from TIB MOLBIOL (Berlin, Germany). After 35 amplification cycles, PCR products were analyzed on 1.5% agarose gels, stained with ethidium bromide, and subsequently visualized. To confirm use of equal amounts of RNA in each experiment, all samples were checked for GAPDH mRNA expression. The following primers were used: for COX-2, forward 5'-CTTCAAAATAAGCTTGAATTCAGGATTGTAATG-3' and reverse 5'-CTTTTTGATAATTTAATAATTTCAATCTTCTGTTTC-3'; for GAPDH, forward 5'-CCACCCATGGCAAATTCCATGGCA-3' and reverse 5'-TCTAGACGGCAGGTCAGGTCCACC-3'.

EMSA. After stimulation of BEAS-2B cells, nuclear protein was isolated and analyzed by EMSA as described previously (33). IRDye800-labeled consensus NF-{kappa}B oligonucleotides were purchased from Metabion (Planegg-Martinsried, Germany). Briefly, EMSA binding reactions were performed by incubating 2 µg of nuclear extract with the annealed oligos according to the manufacturer’s instructions. The reaction mixture was subjected to electrophoresis on a 5% native gel and analyzed by Odyssey infrared imaging system (LI-COR).

Chromatin immunoprecipitation. BEAS-2B cells were stimulated, culture medium was removed, and 1% formaldehyde was added. After 1 min, cells were washed in ice-cold 0.125 M glycin in PBS and then rapidly collected in ice-cold PBS, centrifuged, and washed twice with ice-cold PBS. Cells were lysed in RIPA buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 1% desoxycholic acid, 0.1% SDS, 1 mM EDTA, 1% aprotinin], and the chromatin was sheared by sonication. Lysates were cleared by centrifugation, and supernatants were stored in aliquots at –80°C until further use. Antibodies were purchased from Santa Cruz Biotechnology (p65/RelA and polymerase II). Immunoprecipitations from soluble chromatin were carried out overnight at 4°C. Immune complexes were collected with protein A/G agarose for 60 min and washed twice with RIPA buffer and once with high-salt buffer [2 M NaCl, 10 mM Tris (pH 7.5), 1% NP-40, 0.5% desoxycholic acid, 1 mM EDTA], which was followed by another wash in RIPA buffer and one wash with TE buffer [10 mM Tris (pH 7.5), 1 mM EDTA]. Immune complexes were extracted in elution buffer (1 TE buffer containing 1% SDS) by shaking the lysates for 15 min at 1,200 rpm (30°C). They were then digested with RNase (1 µg/20 µl) for 30 min at 37°C. After proteinase K digestion (1 µg/8 µl for 6 h at 37°C and 6 h at 65°C), we used a PCR purification kit (Qiagen) to extract DNA. COX-2 promoter DNA was amplified by PCR using Hotstart Taq (Qiagen) polymerase. PCR products were separated by agarose gel electrophoresis, which was detected by ethidium bromide staining of gels. Equal amounts of input DNA was controlled by gel electrophoresis. The following promoter-specific primers for COX-2 (44) were used: sense 5'-CTTCAAAATAAGCTTGAATTCAGGATTGTAATG-3' and antisense 5'-CTTTTTGATAATTTAATAATTTCAATCTTCTGTTTC-3'.

Mouse pneumonia model. All animal procedures were approved by the local authorities. Pathogen-free, female C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany). Animals were housed under a 12:12-h light-dark cycle and were given free access to food and water. Mice were infected at 10 ± 1 wk of age and 18–21 g of weight. Pneumonia was induced by use of encapsulated S. pneumoniae type 3 (NCTC 7978) as described (34). Bacteria were grown as described above and resuspended in sterile PBS, at ~2.5 x 108 cfu/ml. Mice were lightly anesthetized by intraperitoneal injection of ketamine and xylazine and inoculated intranasally with 20 µl of bacterial suspension (5 x 106 cfu). Control mice were challenged with 20 µl of sterile PBS. Groups of three mice were killed 6, 12, 24, or 48 h postinfection, and lungs were removed aseptically and immediately frozen in liquid nitrogen. Control mice were killed 6 h postinfection (n = 3).

Statistical methods. A one-way ANOVA was used for data of Figs. 1, 3, 5, 6D, and 7, and data are shown as means ± SE of 4 separate experiments. Main effects were then compared by an F probability test. P < 0.05 was considered to be significant and indicated by asterisks (if not indicated otherwise, test was performed vs. control).


Figure 1
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  		Fig. 1. Increased PGE2 expression in pneumococci-infected human bronchial epithelial cells. BEAS-2B cells were infected for 16 h with indicated concentrations of Streptococcus pneumoniae R6x and TNF-{alpha} (50 ng/ml)/IL-1beta (10 ng/ml). PGE2 release was detected by ELISA. Data presented are means ± SE of 4 separate experiments. *P < 0.05 vs. unstimulated control.

 

Figure 3
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  		Fig. 3. Inhibition of COX-2 but not COX-1 strongly reduced pneumococci-induced PGE2 release. BEAS-2B cells were pretreated with nonselective COX inhibitors indomethacin (INDO) (0.1 and 1 µM), selective COX-1 (SC-560; 0.1 and 1 µM), and COX-2 inhibitor (NS-398; 0.1 and 1 µM) for 30 min and then infected with R6x (107 cfu/ml) for 16 h. PGE2 release was measured using ELISA. Data are means ± SE of 4 separate experiments. *P < 0.05 vs. unstimulated control. #P < 0.05 with or without inhibitor.

 

Figure 5
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  		Fig. 5. Pneumococci-induced PGE2 release depended on p38 MAPK. BEAS-2B cells were pretreated with specific p38 inhibitor SB-202190 (10 µM), nonactive control compound SB-202474 (10 µM) for 60 min, and then infected with R6x (107 cfu/ml) for 16 h. PGE2 release was assessed by ELISA. Data are means ± SE of 4 separate experiments. *P < 0.05 vs. unstimulated control. #P < 0.05 with or without inhibitor.

 

Figure 6
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  		Fig. 6. Pneumococci-induced PGE2 release and COX-2 expression depended on NF-{kappa}B activation. BEAS-2B cells were infected with 107 cfu/ml R6x for the indicated time periods. A: I{kappa}B{alpha} protein levels were detected with a specific antibody in Western blot and simultaneous detection of actin demonstrated equal protein load. B: increased DNA binding of NF-{kappa}B in nuclear cell extracts in pneumococci-exposed cells was shown by EMSA. C: BEAS-2B cells were pretreated with a specific I{kappa}B{alpha} kinase inhibitor IKK-NBD (0.1, 1, 10 µM) for 60 min and infected with R6x for 8 h. Induction of COX-2 was assessed by Western blot. D: PGE2 release was measured with ELISA in presence or absence of IKK-NBD. Representative blots or gels out of 3 are shown in A, B, and C. Data presented in D are means ± SE of 4 separate experiments. *P < 0.05 vs. unstimulated control. #P < 0.05 with or without inhibitor.

 

Figure 7
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  		Fig. 7. Inhibition of p38 MAPK-inhibited pneumococci induced NF-{kappa}B activation. HEK-293 cells were cotransfected with human Toll-like receptor 2 (hTLR2), an NF-{kappa}B-dependent luciferase reporter plasmid, dominant-negative (dn) mutants of MAPK isoforms p38{alpha}, beta2, {gamma}, and {delta}, and a beta-galactosidase (beta-Gal) construct. Cells pretreated with SB-202474 (10 µM) or SB-202190 (10 µM) were stimulated for 6 h with pneumococci as indicated, and luciferase and beta-Gal activities were determined and normalized. Data are means ± SE of 4 separate experiments. *P < 0.05 vs. unstimulated control. #P < 0.05 with or without dn mutant.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Pneumococci induced expression of PGE2 in human bronchial epithelial cells in vitro. Human bronchial epithelial BEAS-2B cells were stimulated with S. pneumoniae R6x (105–107 cfu/ml) or TNF-{alpha} (50 ng/ml) and IL-1beta (10 ng/ml) for 16 h, and PGE2 release was analyzed. Pneumococci infection of BEAS-2B cells dose dependently induced the release of PGE2 (Fig. 1) by these cells.

Pneumococci induced time-dependent expression of COX-2 in human bronchial epithelial cells in vitro and in mouse lungs. BEAS-2B cells were stimulated with S. pneumoniae R6x in a concentration between 105 and 107 cfu/ml for 2 and 4 h (mRNA, protein) and 8 h (protein). Pneumococci infection of BEAS-2B cells time dependently induced COX-2 mRNA (Fig. 2, A and B) or protein (Fig. 2C) expression in these cells. In contrast, there was no effect on COX-1 expression (Fig. 2C).


Figure 2
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  		Fig. 2. Time- and dose-dependent induction of cyclooxygenase-2 (COX-2) by pneumococci in human bronchial epithelial cells. BEAS-2B cells were treated with indicated doses of bacteria for 4 h (A) or infected with 107 cfu/ml S. pneumoniae R6x for the indicated time periods (B and C). COX-2 mRNA was detected by RT-PCR (A and B) and COX-1 or COX-2 protein (C) by using specific antibodies in Western blot. TNF-{alpha} (50 ng/ml) was used as positive control. Furthermore, lungs of mice infected with NCTC 7978 pneumococci for 6, 12, 24, and 48 h were explanted, soluble proteins were subjected to SDS-PAGE, and COX-2 was detected with a specific antibody (D). Representative blots or gels out of 3 are shown.

 
C57BL/6 mice were infected with S. pneumoniae NCTC 7978 (5 x 106 cfu/ml) by intranasal infection, thereby inducing pneumonia as described previously (34). In Western blot analysis of infected lungs, we found an increase of the very low basal level of COX-2 protein expression at 6 and 12 h and an even stronger expression after 24 h (Fig. 2D).

Pneumococci-induced expression of PGE2 in human bronchial epithelial cells is largely dependent on COX-2. To test the role played by COX-1 and COX-2 in pneumococci-induced PGE2 synthesis in lung epithelium, cells were infected in the absence or presence of the nonselective COX inhibitor indomethacin (0.1 µM, 1 µM), the selective COX-2 blocker NS 398 (0.1 or 1 µM), or the selective COX-1 inhibitor SC 560 (0.1 or 1 µM). These drugs were incubated with the cells 30 min beforehand. Inhibition of COX-2 (NS-398) in pneumococci-infected BEAS-2B cells blocked PGE2 release. The nonselective cyclooxygenase inhibitor indomethacin (COX-1 and COX-2) also strongly reduced PGE2 secretion (Fig. 3). A small but significant decrease in pneumococci-related PGE2 liberation was observed by using the COX-1 inhibitor.

Inhibition of p38 MAPK, but not of JNK, blocked pneumococci-induced expression of COX-2 in human bronchial epithelial cells. Activation of p38 MAPK and JNK is considered to participate in the regulation of inflammatory processes in bronchial epithelial cells (34). Phosphorylation of p38 MAPK by pneumococcal infection of epithelial cells was detected 60 min after infection and increased up to 120 min (Fig. 4A). In addition, a persistent phosphorylation of JNK in the same cells was noted within the observed time frame (30–240 min).


Figure 4
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  		Fig. 4. Inhibition of p38 MAPK but not JNK strongly reduced pneumococci-related COX-2 expression. BEAS-2B cells were incubated with R6x (107 cfu/ml) for the indicated times and phosphorylated (P) or total p38 MAPK and JNK were detected by Western blot (A). Expression of p38 or JNK was performed simultaneously to confirm equal protein load. BEAS-2B cells were pretreated with specific p38 inhibitor SB-202190 (10 µM), nonactive control compound SB-202474 (10 µM), or specific JNK inhibitor SP-600125 (10 µM) for 60 min and then infected with R6x (107 cfu/ml) for 4 h (B) and 8 h (C and D), and induction of COX-2 was assessed by RT-PCR (B) or Western blot (C and D). Representative gels or blots out of 3 are shown.

 
To test the role of p38 MAPK and JNK activation for COX-2 expression in pneumococci-infected epithelium, we made use of specific chemical inhibitors SB-202190 (p38 MAPK), its nonfunctional homolog SB-202474 (p38 MAPK), and SP-600125 (JNK). Inhibition of p38 MAPK in BEAS-2B cells by preincubation of cells with SB-202190 60 min before pneumococcal infection blocked induction of COX-2 mRNA (Fig. 4B) and protein (Fig. 4C), whereas the nonfunctional homolog SB-202474 or solvent alone had no effect. A preincubation of pneumococci-stimulated BEAS-2B cells for 60 min with the JNK inhibitor SP-600125 had no effect on COX-2 protein expression (Fig. 4D). Therefore, pneumococci-dependent p38 MAPK activation seems to be important for COX-2 expression. The concentrations of SB-202190 and SP-600125 used in this study did not alter bacterial growth within the time frame tested (data not shown).

Because S. pneumoniae induces the release of PGE2 from bronchial epithelial cells (Fig. 1) and expression of COX-2 in a p38 MAPK-dependent manner (Fig. 4, B and C), we postulated that this kinase participated in the regulation of PGE2 release. Therefore, we preincubated BEAS-2B cells for 60 min with specific p38 MAP kinase-inhibitor SB-202190 before infection of the cells with pneumococci for 16 h. We observed a strong reduction of PGE2 release in these cells through p38 MAPK inhibition (Fig. 5), indicating that pneumococci-dependent p38 MAPK activation seems to be important for PGE2 release.

Pneumococci-induced expression of COX-2 and PGE2 depended on NF-{kappa}B activation. Expression of COX-2 and subsequent PGE2 release in cells is considered to be regulated by NF-{kappa}B, which is released of its cytosolic sequestration because of phosphorylation of its inhibitor I{kappa}B{alpha} by IKKbeta and subsequent proteolytic degradation (5).

To assess I{kappa}B{alpha} degradation, BEAS-2B cells were infected with S. pneumoniae for different time periods. As shown in Fig. 6A, 107 cfu/ml pneumococci induced degradation of I{kappa}B{alpha} within 60 min. We verified pneumococci induced NF-{kappa}B activation by EMSA. Pneumococci (107 cfu/ml) induced DNA binding of NF-{kappa}B within 60 min to the same extent as 50 ng/ml TNF-{alpha} (Fig. 6B). In the next step, the role of the central kinase complex of the canonical NF-{kappa}B pathway, I{kappa}B{alpha} kinase, for pneumococci-dependent COX-2 induction and PGE2 release was analyzed. The I{kappa}B kinase complex was blocked by preincubation of BEAS-2B cells with the cell-permeable peptide inhibitor IKK-NBD (23). IKK-NBD significantly reduced COX-2 protein expression (Fig. 6C) and release of PGE2 (Fig. 6D) in pneumoncocci-infected BEAS-2B cells. Overall, these data demonstrate that activation of the canonical NF-{kappa}B signaling pathway by pneumococci was necessary for expression of COX-2 and PGE2 release in lung epithelium. IKK-NBD did not alter bacterial growth within the concentration and time tested (data not shown).

Pneumococci activated NF-{kappa}B-dependent gene expression via p38 MAPK. Our data suggest that activation of both p38 MAPK and NF-{kappa}B-dependent signaling contributed to pneumococci-related expression of COX-2 and subsequent PGE2 release in BEAS-2B cells. We hypothesized that p38 MAPK activity is necessary for NF-{kappa}B-dependent gene transcription in pneumococci-infected cells. p38 MAPK {alpha}/beta inhibitor SB-202190 (10 µM) but not the inactive compound SB-202474 blocked pneumococci-induced reporter gene expression in HEK-293 cells cotransfected with hTLR2 and a NF-{kappa}B-dependent luciferase reporter plasmid (Fig. 7). Because several isoforms of p38 MAPK have been described that may induce differentiated activation of transcription factors, we performed transfection experiments using dominant-negative mutants of MAPK p38{alpha}, beta2, {gamma}, and {delta}, which cannot be phosphorylated and thereby activated (28). As shown in Fig. 7, pneumococci-induced expression of the NF-{kappa}B-dependent reporter gene was reduced to a great extent by dominant-negative isoforms {alpha}, beta2, {gamma}, and {delta} of p38 MAPK.

Inhibition of p38 MAPK but not JNK-blocked polymerase II binding on the cox2 promoter. To further characterize the mechanism by which p38 MAPK contributes to pneumococci-mediated NF-{kappa}B activation and COX-2 transcription, association of NF-{kappa}B subunit p65 and polymerase II with the COX-2 promoter was evaluated by ChIP assay (Fig. 8A). After infection of human lung epithelial BEAS-2B cells with S. pneumoniae, p65 NF-{kappa}B and polymerase II immunoprecipitates showed an enrichment of cox2 promoter DNA (Fig. 8A). To investigate the role of p38 MAPK and JNK in this process, BEAS-2B cells were preincubated with the specific inhibitors SB-202190 (60 min) for p38 MAPK or SP-600125 (60 min) for JNK before infection with R6x pneumococci (107 cfu/ml) for 60 min. We observed a p38-dependent but JNK-independent binding of polymerase II the cox2 promoter induced by S. pneumoniae (Fig. 8B). Inhibition of p38 MAPK before infection with pneumococci R6x reduced the binding of p65 and polymerase II to the cox2 promoter, whereas JNK inhibition had no effect on polymerase II and a slight influence on p65 association with the cox2 promoter. In conjunction with the data shown in Fig. 7, these experiments suggest that p38 MAPK controlled the p65 and polymerase II recruitment in pneumococci-driven COX-2 transcription.


Figure 8
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  		Fig. 8. Inhibition of p38 MAPK- but not of JNK-blocked pneumococci induced recruitment of RNA polymerase II (POL II) to the cox2 promoter. BEAS-2B cells were stimulated with R6x (107 cfu/ml) for 30 and 60 min (A) or pretreated with SB-202190 (10 µM, 60 min) or SP-600125 (10 µM, 60 min) (B) before infection with pneumococci. Representative experiments out of 3 are shown.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present report, we provide evidence that S. pneumoniae-induced p38 MAPK and NF-{kappa}B activation impacts on the cox2 promoter of infected human lung epithelial BEAS-2B cells. This process is important for pneumococci-induced COX-2 expression and PGE2 synthesis in lung epithelium. In addition, expression of COX-2 protein was increased in a mouse model of pneumococcal pneumonia. Pneumococci infection led to NF-{kappa}B and p38 MAPK and JNK activation in BEAS-2B cells. Whereas chemical inhibition of p38 MAPK activity blocked pneumococci-induced PGE2 release, COX-2 expression, and recruitment of RNA polymerase II and of NF-{kappa}B/RelA to the cox2 promoter, blockade of JNK activity had no effect on COX-2 expression and promoter activity in infected lung epithelial cells.

During airborne lung infections, the airway and lung epithelia comprise the first host barrier against invading pathogens and are thereby directly exposed to pathogens and their products. Besides acting as a mechanical barrier, liberation of proinflammatory agents by the epithelium may contribute significantly to the initiation of host innate immune response. Pneumococci are known as the most important pathogen causing pneumonia in humans (6) and are shown to activate pulmonary tissue cells (17, 27, 34). For example, lung epithelial cells were able to detect invading pneumococci by specific receptors (27), resulting in proinflammatory cytokine release (27, 34). Although lipid metabolites produced by COX are known as important mediators of pulmonary physiology (10, 41), little is known about pneumococci-related COX activation in lung cells. Therefore, we tested the hypothesis that pneumococci induced COX-2 expression in human epithelial BEAS-2B cells and in a mouse pneumonia model.

Herein, we showed that pneumococci infection resulted in increased expression of COX-2, but not of COX-1, in BEAS-2B cells in vitro as well as in lung homogenates of pneumococci-infected mice in vivo. In addition, increased COX-2 protein expression was accompanied by subsequent PGE2 liberation in vitro, which is known to be the major cyclooxygenase product liberated by pulmonary epithelial cells as shown in a previous study (16). The slight decrease of PGE2 through the COX-1 inhibitor suggests that this enzyme may also be involved in pneumococci-induced PGE2 production, presumably because of higher amounts of arachidonic acid as substrate in proinflammatory activated cells. Our data are in line with the recent report of Seki et al. (35) of increased COX-2 expression and PGE2 production in pneumococci infected mice lungs. In addition, it was shown that Chlamydia pneumoniae (15), respiratory syncytial virus (31), and influenza virus (24) induced COX-2 expression in pulmonary epithelium, underlining the potential role of COX-2 in lung infection caused by different agents.

In contrast to the constitutively expressed COX-1, a complex signaling network regulates the expression of inducible COX-2 (5). In pneumococci-infected lung epithelial cells, we noted activation of p38 MAPK and JNK. Both kinases were considered as important regulators of proinflammatory signaling pathways (5). Inhibition of p38 MAPK but not of JNK reduced pneumococci-related expression of COX-2 and PGE2 liberation. This is in line with previous observations, which demonstrated a pivotal role of the p38 MAPK pathway in the regulation of COX-2 expression (32). PGE2 liberation is even stronger impaired by p38 MAPK inhibition than COX-2 expression. Presumably, other steps of COX-2 synthesis were additionally affected by p38 MAPK inhibition and acted synergistically on PGE2 liberation. However, in, e.g., influenza virus-infected bronchial epithelium, COX-2 expression was dominantly mediated by ERK and JNK but not by p38 MAPK (24), suggesting a pathogen-specific regulation of COX-2 expression in lung tissue cells. Besides regulating pneumococci-dependent COX-2 expression, activation of p38 MAPK was also critically involved in the expression of proinflammatory cytokines like IL-8 and GM-CSF in pneumococci-infected lung epithelium (34). Overall, p38 MAPK dependent signaling seems to be an important step in pneumococci-dependent activation of pulmonary epithelial cells.

Stimulation of the transcription factor NF-{kappa}B was considered to contribute significantly to COX-2 expression. In resting cells, I{kappa}B molecules sequester NF-{kappa}B in the cytosol. After cell activation, a complex signaling cascade containing IKK kinase results in degradation of I{kappa}B{alpha}, thus allowing NF-{kappa}B transfer into the nucleus (11). We found degradation of I{kappa}B{alpha} in the cytosol and increased binding of NF-{kappa}B to the DNA in pneumococci-infected epithelium. Moreover, a highly specific cell-permeable inhibitor of IKK, IKK-NBD (1, 23), abolished pneumococci-related COX-2 protein expression and subsequent PGE2 release. Finally, pneumococci infection activated NF-{kappa}B-dependent reporter-gene expression in TLR2-overexpressing HEK-293 cells. These data implicate a crucial involvement of NF-{kappa}B in pneumococci-caused COX-2 induction.

Because both pathways, p38 MAPK and NF-{kappa}B activation, seem to be essentially involved in COX-2 expression in pneumococci-infected lung epithelium, we analyzed the impact of p38 MAPK on NF-{kappa}B activation in more detail.

However, because BEAS-2B cells could poorly be transfected, we made use of TLR2-overexpressing HEK-293 epithelial cells as a model that has been applied successfully in earlier studies investigating pneumococci-related cell activation (27, 34). A chemical inhibitor of p38 MAPK, but not an inactive chemical control compound, blocked pneumococci-driven NF-{kappa}B-dependent reporter-gene expression in these cells. Four different isoforms of p38 MAPK have been described (8). Dominant-negative mutants of MAPK p38{alpha}, beta2, {gamma}, and {delta} blocked to a comparable extent pneumococci-driven NF-{kappa}B-dependent reporter-gene expression. This is in line with a recent study demonstrating that gene silencing of p38 MAPK p38{alpha} by siRNA also reduced pneumococci-related expression of a NF-{kappa}B-dependent reporter gene (34). In contrast to NF-{kappa}B, AP-1-dependent gene expression in arsenite-stimulated human breast cancer cells was mediated through p38beta and inhibited through p38{gamma} and {delta} (28).

It has been shown in a recent study that p38 MAPK inhibition did not block I{kappa}B{alpha} degradation or nuclear translocation of NF-{kappa}B in BEAS-2B cells (34). To analyze more directly the influence of p38 MAPK on NF-{kappa}B dependent COX-2 transcription, we performed chromatin immunoprecipitation assays. Pneumococci infection of BEAS-2B cells increased recruitment of the NF-{kappa}B subunit p65/RelA as well as of RNA polymerase II (as a measure of overall promoter activity) to the promoter. Blocking of p38 MAPK, abolished binding of p65/RelA and of polymerase II to the cox2 promoter. Inhibition of JNK slightly reduced p65/RelA binding but did not affect polymerase II recruitment or COX-2 gene transcription. Interestingly, in contrast to the cox2 promoter, at the il8 gene, p38 MAPK inhibition did not suppress p65/RelA recruitment but also blocked RNA polymerase II binding in pneumococci-infected BEAS-2B cells (34). This points to a promoter-specific interaction of p38 MAPK pathway and NF-{kappa}B and highlights the complex nature of gene transcription in (pneumococci) infected pulmonary epithelial cells.

Recent studies demonstrated an impact of p38 MAPK-dependent signals on NF-{kappa}B by, e.g., phosphorylation of the subunit p65 after IL-1beta (21), thrombin (29) stimulation, or bacterial infection (34). However, presently, it remains unclear whether p38 MAPK directly phosphorylated NF-{kappa}B/RelA in pneumococci infection and which phosphorylation sites were affected.

Although JNK was activated by S. pneumoniae in our model and has been shown to be involved in NF-{kappa}B signaling (26), we could not assign any importance to this kinase in pneumococci-induced COX-2 expression. Taken as a whole, the data presented implicate that the p38 MAPK pathway is crucial for pneumococci-induced NF-{kappa}B/RelA activity at the cox2 promoter rather than the JNK pathway.

Even though increased COX-2 expression as well as increased PGE2 production (35) was noted in infected mice lungs, further investigations in vivo are needed to expand our knowledge about the role of COX-2 in pneumonia. For example, invading immune cells as well as stimulated lung epithelium may also contribute to increased lung PGE2 liberation. In addition, studies with specific COX-2 inhibitors or COX-2-deficient mice would help to understand the role of COX-2 in pneumococcal pneumonia. Because there is evidence provided for a central role of p38 MAPK-related gene expression in pneumococci-infected lung cells (this study) (34), in vivo studies should address its role in pneumococcal pneumonia. Overall, by expression of p38 MAPK- and NF-{kappa}B-dependent proinflammatory gene products, the pulmonary epithelium may contribute significantly to the initiation of the host response in pneumococcal pneumonia.

In conclusion, we found that pneumococci induced PGE2 release dependent on COX-2 induction in human bronchial epithelial cells. It required p38 MAPK-dependent recruitment of NF-{kappa}B and RNA polymerase II to the cox2 promoter and might be beneficial for the host immunoreactions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the Bundesministerium für Bildung und Forschung to B. Schmeck (BMBF-competence network CAPNETZ C15), S. Hippenstiel (BMBF-competence network CAPNETZ C15), and N. Suttorp and S. Rosseau (BMBF-competence network CAPNETZ C4) and Deutsche Gesellschaft für Pneumologie to J. Zahlten.


   ACKNOWLEDGMENTS
 
The excellent technical assistance of Kerstin Möhr and Sylwia Schapke is greatly appreciated. Part of this work will be included in the doctoral thesis of M. O. Etouem.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Schmeck, Dept. of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany (e-mail: bernd.schmeck{at}charite.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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