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Am J Physiol Lung Cell Mol Physiol 292: L267-L277, 2007. First published September 29, 2006; doi:10.1152/ajplung.00100.2006
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Legionella pneumophila-induced PKC{alpha}-, MAPK-, and NF-{kappa}B-dependent COX-2 expression in human lung epithelium

Philippe Dje N'Guessan,1 Mirabelle O. Etouem,1 Bernd Schmeck,1 Andreas C. Hocke,1 Stefanie Scharf,1 Kremena Vardarova,1 Bastian Opitz,1 Antje Flieger,2 Norbert Suttorp,1 and Stefan Hippenstiel1

1Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité - Universitätsmedizin Berlin; and 2NG5 Pathogenesis of Legionella Infection, Robert Koch-Institut, Berlin, Germany

Submitted 20 March 2006 ; accepted in final form 26 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Legionella pneumophila causes community- and hospital-acquired pneumonia. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX) and microsomal PGE2 synthase-1 (mPGES-1)-derived prostaglandins like prostaglandin E2 (PGE2) are considered as important regulators of lung function. Herein we tested the hypothesis that L. pneumophila induced COX-2 and mPGES-1-dependent PGE2 production in pulmonary epithelial cells. Legionella induced the release of PGE2 in primary human small airway epithelial cells and A549 cells. This was accompanied by an increased expression of COX-2 and mPGES-1 as well as an increased PLA2 activity in infected cells. Deletion of the type IV secretion system Dot/Icm did not impair Legionella-related COX-2 expression or PGE2 release in A549 cells. L. pneumophila induced the degradation of I{kappa}B{alpha} and activated NF-{kappa}B. Inhibition of IKK blocked L. pneumophila-induced PGE2 release and COX-2 expression. We noted activation of p38 and p42/44 MAP kinase in Legionella-infected A549 cells. Moreover, membrane translocation and activation of PKC{alpha} was observed in infected cells. PKC{alpha} and p38 and p42/44 MAP kinase inhibitors reduced PGE2 release and COX-2 expression. In summary, PKC{alpha} and p38 and p42/44 MAP kinase controlled COX-2 expression and subsequent PGE2 release by Legionella-infected lung epithelial cells. These pathways may significantly contribute to the host response in Legionnaires' disease.

alveolar epithelium; protein kinase C; prostaglandin E2; cyclooxygenase-2; phospholipase A2; microsomal PGE2 synthase-1; mitogen-activated protein kinase

LEGIONELLA PNEUMOPHILA is an important causative agent of severe community-acquired pneumonia and the second most commonly detected pathogen in patients with pneumonia that are admitted to intensive care units in industrialized countries (48, 61). In addition, since Legionella spp. urine antigen detection is still not routinely used in some settings, worldwide the prevalence of Legionellosis is underestimated (47, 48). Approximately 15% of Legionellosis appears in community outbreaks. Although more than 40 Legionella species are known, the majority of human infections is caused by L. pneumophila serogroup 1 (57). L. pneumophila is a gram-negative, facultative intracellular pathogen of amoeba in natural and man-made aquatic environments. Infection of humans occurred after inhalation of contaminated water aerosol droplets.

L. pneumophila contain an array of important virulence factors including the DotA/Icm type IV secretion system, which is important for bacteria invasion and replication in the host cell (4). For L. pneumophila pathogenesis, essential results were obtained by analyzing infection of protozoans or immune cells like macrophages (4, 40). However, although Legionella replicate efficiently within lung epithelial cells, and recent studies pointed to the lung epithelium as an important sentinel and effector of innate immunity (6, 22, 52, 53, 56), little is known of the consequences of pulmonary epithelial cell infection with Legionella.

Lipid metabolites of arachidonic acid, including prostaglandins and leukotrienes, have emerged as potent endogenous mediators and modulators of innate immunity in the lung (59, 60).

The release of arachidonic acid from cell membrane phospholipids by phospholipase A2 (PLA2) enzymes impacts on the amount of eicosanoid production that occurs (7, 24). Whereas each class of PLA2 can release arachidonic acid from cell membrane phospholipids, cytosolic PLA2 (cPLA2) appears to have, as a primary function, the release of arachidonic acid for eicosanoid production, and cPLA2{alpha} is found in most cells and tissues (7, 24).

In particular, prostaglandin E2 (PGE2) produced at sites of infection was shown to modulate immune and inflammatory responses (18, 41, 60) and is liberated by lung epithelial cells (10, 26, 31, 35, 46). 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 [cytosolic PGES (cPGES) and microsomal PGES-1 (mPGES-1/-2)] (12, 44). cPGES is constitutively and ubiquitously expressed and is preferentially coupled with COX-1, promoting immediate production of PGE2. By contrast, mPGES-1 is markedly upregulated by proinflammatory stimuli and is functionally coupled with COX-2, promoting delayed PGE2 synthesis (44).

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) (12). These transcription factors can be activated by complex kinase pathways centered around PKC (protein kinase C) (12, 36) and p38 and p42/44 mitogen-activated protein kinase (MAPK) (12, 30, 50).

The PKC family of proteins phosphorylates serine or threonine residues on multiple protein substrates. These kinases participate, e.g., in the regulation of the immune response by the regulation of gene transcription (45, 58). The PKC enzyme family includes multiple isoforms that display different activities in the presence or absence of cofactors, including calcium, diacylglycerols (DAG), and phospholipids (58). The PKC isoforms can be categorized into three classes based on these differences: the conventional isoforms ({alpha}, beta1, beta2, and {gamma}) are dependent on both Ca2+ and DAG, the novel isoforms ({delta}, {epsilon}, {eta}, µ, and {theta}) are Ca2+ independent but DAG dependent, and the atypical isoforms ({lambda} and {zeta}) are neither Ca2+ nor DAG dependent (58).

The MAPK family is involved in multiple cell functions, including inflammation, proliferation, and apoptosis (30, 38, 42, 53). Five distinguishable MAPK subfamilies have been identified in mammalian systems; the best described of these are the ERK (p42/p44), p38, and c-Jun NH2-terminal kinase pathways (27).

Activation of proinflammatory signaling pathways in lung epithelial cells, including the PKC{alpha}, p38 and p42/p44-MAP kinase, and NF-{kappa}B pathways, by bacterial infection are suggested to contribute significantly to disease process in pneumonia (13, 28, 36, 53). Although Legionella efficiently infected and stimulated lung epithelial cells (20, 33), mechanisms of L. pneumophila-induced activation of COX-2 and PGE2 release in lung epithelial cells are widely unknown.

In this study, we tested the hypothesis that L. pneumophila induces 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 Legionella induced COX-2 expression, mPGES-1 transcription, and PGE2 release as well as activation of p38 MAP kinase, p42/44, PKC{alpha}, and NF-{kappa}B in human alveolar epithelial cells. PGE2 release and COX-2 expression depended on p38 and p42/44 MAP kinase, PKC{alpha}, and NF-{kappa}B activation. Inactivation of the Icm/DotA system, a major virulence factor of L. pneumophila, did not reduce COX-2 expression and PGE2 release in epithelial cells. Thus Legionella-induced PGE2 liberation by lung epithelial cells may contribute significantly to the pathogenesis of Legionnaires' disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DMEM, FCS, trypsin-EDTA solution, CA-650, and antibiotics were obtained from Life Technologies (Karlsruhe, Germany). Protease inhibitors, Triton X-100, and Tween 20 were purchased from Sigma Chemical (Munich, Germany); TNF-{alpha} was from R&D Systems (Wiesbaden, Germany); SB-202190, Gö-6976, PKC-I20–28, calphostin C, indomethacin, SC-560, NS-398, and PMA (phorbol 12-myristate 13-acetate) were from Calbiochem-Merck (Darmstadt, Germany); and IKK-NBD was from Biomol (Plymouth Meeting, PA). All other chemicals used were of analytical grade and obtained from commercial sources.

Cell lines. Alveolar epithelial cell line A549 was purchased from American Type Culture Collection (ATCC; Rockville, MD) and cultured in Ham's F-12 with L-glutamine and 10% FCS without antibiotics. Primary human small airway epithelial cells (SAEC) were obtained from Cambrex (Clonetics Small Airway Epithelial Cell System; Cambrex, Baltimore, MD) and cultured according to the supplier's instructions.

Infection with bacterial strains. L. pneumophila sg1 130b [ATCC BAA-74, kindly provided by Nicholas P. Cianciotto, Northwestern Univ. Medical School, Chicago, IL (16)] and JR32 and JR32 DotA mutant [LELA 3118 (JR32 DotA–/–), both kindly provided by Howard Shuman, Columbia Univ., New York, NY (49)] were routinely grown on buffered charcoal-yeast extract agar for 2 or 3 days at 37°C (15) and subsequently resuspended in epithelial cell medium at 37°C with shaking at 350 rpm (3). Bacterial density was checked by determining the optical density at 660 nm (OD660) with a Beckman spectrophotometer DU520 (Beckman Coulter, Unterschleissheim, Germany). A549 (1 x 106) cells were infected with 105, 106, and 107 colony-forming units (cfu) bacteria per milliliter and incubated in infection medium (Ham's F-12 with L-glutamine without antibiotics) for a given time at 37°C and 5% CO2. The multiplicity of infection used was 0.1, 1, and 10.

PGE2 ELISA. Confluent A549 cells were infected 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) (37).

Western blot. For determination of COX-2 induction, I{kappa}B{alpha} degradation, PKC{alpha} translocation, and p38 MAP and ERK kinase phosphorylation, A549 cells were infected as indicated, washed twice, and harvested. Cells 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 MAP kinase antibody (38, 42, 53) (Cell Signaling, Frankfurt, Germany), phospho-specific p42/44 (38, 51) and PKC{alpha} (Santa Cruz Biotechnologies), and I{kappa}B{alpha} (37, 53) (Santa Cruz Biotechnologies). In all experiments, actin (Santa Cruz Biotechnologies) or p38 and p42 (Santa Cruz Biotechnologies) were detected simultaneously to confirm equal protein load (37, 38, 52).

RT-PCR. For analysis of COX-2, mPGES-1, and GAPDH gene expression in A549 cells, total RNA was isolated with the RNeasy Mini kit (Qiagen, Hilden, Germany) and reversely transcribed using avian myeloblastosis virus reverse transcriptase (Promega, Heidelberg, Germany) (37, 43, 52). Generated cDNA was amplified by PCR using specific intron-spanning specific primers for COX-2 (forward: 5'-TGCTGTGGAGCTGTATCC-3', reverse: 5'-GACTCCTTTCTCCGCAAC-3'), mPGES-1 (forward: 5'-CCAAGTGAGGCTGCGGAAGAA-3', reverse: 5'-GCTTCCCAGAGGATCTGCAGA-3'), 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 (37, 43, 52, 54).

cPLA2 assay. A cPLA2 assay kit (Cayman Chemical, Ann Arbor, MI) was used to detect cPLA2 activity. A549 cells were infected with L. pneumophila, and cell extract was collected and processed for cPLA2 activity according to the manufacturer's instructions.

PKC assay. The PKC assay StressXpress (Stressgen Bioreagents, Victoria, Canada) was used to detect PKC activity. A549 cells were stimulated with L. pneumophila. A cell extract containing activated PKC was collected and processed for PKC activity by ELISA according to the manufacturer's instructions.

Immunofluorescence. Cells (1 x 105) grown on glass coverslips were infected as indicated, washed twice, and incubated in a humidified atmosphere. Briefly, cells were fixed in freshly prepared paraformaldehyde (3% in PBS, pH 7.6; Sigma-Aldrich, Munich, Germany), permeabilized, washed, and incubated with specific antibody against PKC{alpha} (Santa Cruz Biotechnologies). The signal intensity was enhanced by application of Alexa 488 anti-FITC antibody (1:1,000, 1 h at 37°C) and analyzed by using a Zeiss Pascal 5 confocal microscope. F-actin was visualized by being marked with Alexa 546-labeled phalloidin (1:200, 30 min) as described previously (23, 38, 55).

EMSA. After stimulation of A549 cells, nuclear protein was isolated and analyzed by EMSA as described previously (53). 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 native gel PAGE and analyzed by Odyssey infrared imaging system (LI-COR).

Statistical methods. Data are shown as means ± SE of at least three independent experiments. A one-way ANOVA was used for data of Figs. 1A–C, Go3, 4A, 5B, 6A, 7B, 8C, and 9D. The main effects were then compared by a Newman-Keuls posttest. P < 0.05 was considered to be significant and is indicated by asterisks (if not indicated otherwise, test was performed vs. control).


Figure 1
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  		Fig. 1. Increased PGE2 expression in Legionella pneumophila (L.p.)-infected human alveolar A549 epithelial cells (A and B) and small airway epithelial cells (SAEC; C). A549 cells were infected for 12 h with the indicated concentrations of Legionella (cfu/ml) or TNF-{alpha} (50 ng/ml) for 4, 8, and 12 h with a bacteria concentration of 107 cfu/ml. SAEC were also infected in the same condition for also 4, 8, and 12 h. PGE2 release was detected by ELISA. The experiments in A and B were conducted simultaneously. Data presented are means ± SE of 4 separate experiments. *P < 0.05 vs. infected control.

 

Figure 2
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  		Fig. 2. Time- and dose-dependent induction of cyclooxygenase (COX)-2 by L. pneumophila in human alveolar epithelial cells. A549 cells and SAEC were treated with the indicated doses of bacteria for 1 (A) and 4 h (B) or infected with 107 cfu/ml Legionella 130b for the indicated time periods (C and D). COX-2 mRNA was detected by RT-PCR (A) and COX-1 or -2 protein (B and C) by using specific antibodies in Western blot. TNF-{alpha} (50 ng/ml) was used as a positive control. Representative blots or gels out of 3 separate experiments are shown.

 

Figure 3
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  		Fig. 3. Inhibition of COX-2, but not COX-1, strongly reduced L. pneumophila-induced PGE2 release. A549 cells were pretreated with nonselective COX inhibitors indomethacin (INDO; 0.1 µM, 1 µM), selective COX-1 (SC-560; 0.1 µM, 1 µM), and COX-2 inhibitor (NS-398; 0.1 µM, 1 µM) for 30 min and then infected with L. pneumophila (107 cfu/ml) for 12 h. PGE2 release was measured using ELISA. Data presented are means ± SE of 4 separate experiments. *P < 0.05 vs. unstimulated control; #P < 0.05 with or without inhibitors.

 

Figure 4
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  		Fig. 4. Increased cytosolic PLA2 (cPLA2) activity and microsomal PGE2 synthase-1 (mPGES-1) transcription in L. pneumophila-infected human alveolar A549 epithelial cells. A549 cells were infected for 4, 8, and 12 h or 1, 2, 4, and 8 h with Legionella (107 cfu/ml). cPLA2 activity was accessed with an activity assay and mPGES-1 transcription by RT-PCR. TNF-{alpha} (50 ng/ml) was used as positive control for cPLA2 activity (12 h) and mPGES-1 transcription (8 h). Data in A are presented as fold of unstimulated control cells and are means ± SE of 4 separate experiments. *P < 0.05 vs. infected control. Representatives of 3 independent gels with similar results are shown B.

 

Figure 5
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  		Fig. 5. A and B: induction of COX-2 and PGE2 release by different strains of L. pneumophila. A549 were incubated for 4 h (COX-2) and 12 h (PGE2) with 107 cfu/ml of L. pneumophila 130b, JR32, and JR32 DotA defective mutant (JR32–/– DotA). COX-2 expression was analyzed by Western blot and PGE2 release by ELISA.

 

Figure 6
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  		Fig. 6. L. pneumophila induced activation of PKC. A549 cells were incubated with L. pneumophila 130b (107 cfu/ml) for 30, 60, and 120 min, and PKC activity was detected by PKC activity assay (A). Data presented are means ± SE of 4 separate experiments. *P < 0.05. Phorbol 12-myristate 13-acetate (PMA; 160 nM, 60 min) and recombinant active PKC (10 ng) were used as positive control. To assess PKC{alpha} translocation by immunofluorescence (B) or Western blot (C), lung alveolar epithelial cells were infected with L. pneumophila 130b (107 cfu/ml) for 60 min (B) or 30, 60, and 120 min (C). In the immunofluorescence images, translocated PKC{alpha} is shown with white arrow. PMA (160 nM, 60 min) was used as positive control. Representatives of 3 independent experiments with similar results are shown.

 

Figure 7
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  		Fig. 7. L. pneumophila-induced COX-2 expression and PGE2 release depended on PKC{alpha} activity. A549 cells were preincubated (60 min) with 10 µM PKC{alpha} inhibitor Gö-6976 and PKC-I20–28 or pan-PKC inhibitor calphostin C and infected with L. pneumophila 130b (107 cfu/ml) for 12 h. Induction of COX-2 was assessed by Western blot (A) and PGE2 release by ELISA (B). Representatives of 3 independent blots with similar results are shown. ELISA data presented are means ± SE of 4 separate experiments. *P < 0.05 vs. unstimulated control; #P < 0.05 with or without inhibitor.

 

Figure 8
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  		Fig. 8. Inhibition of p38 MAP kinase and p42/44 strongly reduced L. pneumophila-related COX-2 expression and PGE2 release. A549 cells were incubated with L. pneumophila 130b (107 cfu/ml) for 30, 60, and 120 min or with TNF-{alpha} for 30 min. Phosphorylated or total p38 and p42/44 MAP kinase were detected by Western blot (A). Expression of p38 or p42 was performed simultaneously to confirm equal protein load. A549 cells were pretreated with specific p38 inhibitor SB-202190 (10 µM), specific p42/44 inhibitor U0126 (10 µM), or Gö-6976 (10 µM) for 60 min and then infected with Legionella (107 cfu/ml) for 4 (B) and 12 h (C). A combination of the above-mentioned inhibitors was also used in the same condition (C). Induction of COX-2 was assessed by Western blot (B) and PGE2 release by ELISA (C). Representatives of 3 independent blots with similar results are shown. ELISA data presented are means ± SE of 4 separate experiments. *P < 0.05 vs. unstimulated control; #P < 0.05 with or without inhibitor.

 

Figure 9
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  		Fig. 9. L. pneumophila-induced COX-2 expression and PGE2 release depended on NF-{kappa}B activation in alveolar epithelial cells. A549 cells were infected with 107 cfu/ml L. pneumophila 130b for the indicated time periods (A). I{kappa}B{alpha} protein levels were detected using a specific antibody in Western blot. Simultaneous detection of actin demonstrated equal protein load (A). B: increased DNA binding of NF-{kappa}B in nuclear cell extracts of L. pneumophila 130b-exposed cells was shown by EMSA. C: A549 cells were pretreated with a specific I{kappa}B{alpha} kinase inhibitor, IKK-NBD (10 µM), for 60 min and infected with L. pneumophila 130b for 4 h. Induction of COX-2 was assessed by Western blot. D: PGE2 release was measured using ELISA in the presence or absence of IKK-NBD after infection of A549 cells with Legionella. Representative blots or gels out of 3 are shown in A–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.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
L. pneumophila induced COX-2-dependent release of PGE2 in human alveolar epithelial cells. Human alveolar epithelial A549 cells were infected with L. pneumophila strain 130b (105 to 107 cfu/ml) or exposed to TNF-{alpha} (50 ng/ml) for 12 h (Fig. 1A). In addition, cells were infected with 107 cfu/ml L. pneumophila strain 130b for 4, 8, and 12 h, and PGE2 release was analyzed by ELISA (Fig. 1B). Legionella infection of A549 cells time and dose dependently induced the release of PGE2 by these cells. Furthermore, we noticed a time-dependent release of PGE2 in human primary SAEC, suggesting that Legionella-driven induction of PGE2 is not restricted to the A549 cell line (Fig. 1C).

PGE2 release is dependent on the activation of COX-1 and/or COX-2. COX-2 expression may be increased after proinflammatory stimulation of cells. Therefore, we analyzed the expression of both isoenzymes in Legionella-infected lung epithelium. As shown in Fig. 2A, Legionella (105 to 107 cfu/ml) induced the expression of COX-2 mRNA after 1 h of infection. Moreover, we noted a dose (105 to 107 cfu/ml)- and time (1–10 h)-dependent increase in the expression of COX-2 protein, but not of COX-1 protein (Fig. 2, B and C, respectively), in Legionella-infected A549 cells. Furthermore, Legionella increased COX-2 protein expression in SAEC (Fig. 2D).

To test the role played by COX-1 and COX-2 in Legionella-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 µM, 1 µM), or the selective COX-1 inhibitor SC-560 (0.1 µM, 1 µM). These drugs were incubated with the cells 30 min beforehand. Inhibition of COX-2, but not COX-1, in Legionella-infected A549 cells blocked PGE2 release. The nonselective COX inhibitor indomethacin also strongly reduced PGE2 secretion (Fig. 3). Thus L. pneumophila induced COX-2-dependent release of PGE2 secretion by cultured lung epithelial cells. The concentration of indomethacin, NS-398, and SC-560 used in this study did not alter bacteria growth within the time frame tested (data not shown). The inhibitors did not reduce epithelial cell numbers or induce morphological signs of cytotoxicity (data not shown).

L. pneumophila increased cPLA2 activity and mPGES-1 transcription in human alveolar epithelial cells. PLA2 and mPGES-1 were known to contribute to COX-related mediator formation by the release of arachidonic acid (7, 17, 44). Therefore, we analyzed the activation of cPLA2 and the transcription of mPGES-1 in Legionella-infected lung epithelium.

A549 cells were infected with L. pneumophila strain 130b (4, 8, 12 h) or exposed to TNF-{alpha} (50 ng/ml, 12 h), and cPLA2 activity was analyzed by a specific activity assay (Fig. 4A). Legionella time dependently increased activity of cPLA2. Legionella-related cPLA2 activity was threefold higher than that induced by TNF-{alpha}, a potent inductor of cPLA2 activity (32). In addition, cells were infected with 107 cfu/ml L. pneumophila strain 130b for 1, 2, 4, and 8 h or TNF-{alpha} (50 ng/ml, 8 h), and mPGES-1 transcription was quantified by RT-PCR (Fig. 4B). Legionella infection of A549 cells time dependently induced the transcription of mPGES-1 to the same extent as TNF-{alpha}.

L. pneumophila-induced COX-2 expression and PGE2 release in alveolar epithelial cells is not controlled by the Icm/DotA system. The Icm/dotA system is known as an important virulence system within Legionella (49). Infection of A549 cells with L. pneumophila strain 130b and JR32 induced comparable COX-2 expression (107 cfu/ml, 2 h) (Fig. 5A) and PGE2 release (107 cfu/ml, 12 h) (Fig. 5B). However, there was no significant difference among the effects of JR32 DotA deletion mutant or wild-type strains, with respect to COX-2 expression or PGE2 liberation.

L. pneumophila-induced COX-2 expression and PGE2 release was dependent on PKC{alpha} activation in alveolar epithelial cells. PKC signaling pathways have been shown to play important roles in inflammation (12, 13, 45, 58). Therefore, we hypothesized that PKC{alpha} may contribute to Legionella pathologies in lung epithelium. Legionella infection increased total PKC activity in A549 cells, as shown by PKC activity assay (Fig. 6A). PKC activity was strongly induced 60 and 120 min after Legionella exposure. Stimulation of PKC activity observed in Legionella-infected cells was comparable to stimulation observed by the positive controls used (160 nM PMA for 30 min, 10 ng recombinant PKC) (Fig. 6A).

PKC{alpha} translocation from the cytosol to the cell membrane is associated with activation of PKC{alpha} (21, 28). To determine whether PKC{alpha} activation participated in L. pneumophila-induced PKC activity, A549 cells were infected with Legionella 130b. As shown in Fig. 6, B and C, PKC{alpha} strongly translocates from the cytosol to the cell membrane after cell infection. The effect observed was similar to PKC{alpha} translocation induced by the strong PKC{alpha} activator PMA (160 nM, 30 min) (Fig. 6B).

Next, we analyzed the impact of L. pneumophila-induced activation of the PKC{alpha} pathway on COX-2 expression and PGE2 release. A549 cells were preincubated with PKC{alpha} inhibitors [Gö-6976, PKC-I20–28 (10), and pan-PKC inhibitor calphostin C (13, 28)] before infection with L. pneumophila 130b. Activation of COX-2 expression (see Fig. 8A) and release of PGE2 (Fig. 7B) were assessed by Western blot or ELISA, respectively. Blocking of PKC{alpha} with Gö-6976 and PKC-I20–28 reduced Legionella-induced COX-2 expression (Fig. 7A) and PGE2 liberation (Fig. 7B), as did the pan-PKC inhibitor calphostin C. Neither inhibitors nor control compounds (Gö-6976, PKC-I20–28, and calphostin C) reduced epithelial cell numbers or induced morphological signs of cytotoxicity (data not shown). The inhibitors did not alter bacteria growth within the time frame tested (data not shown).

Inhibition of p38 and p42/44 MAP kinase blocked Legionella-induced expression of COX-2 in human alveolar epithelial cells. Activation of p38 MAP kinase and p42/44 is considered to participate in the regulation of inflammatory processes in alveolar epithelial cells (30). Phosphorylation of p38 and p42/44 MAP kinase by Legionella infection of epithelial cells was detected 30 min after infection, increased up to 60 min, and decreased at 120 min (Fig. 8A).

To test the role of p38 MAP kinase and p42/44 activation for COX-2 expression in Legionella-infected epithelium, we used the specific chemical inhibitors SB-202190 (p38 MAP kinase) and U0126 (MEK1 and p42/44 MAP kinase). Inhibition of p38 and p42/44 MAP kinase in A549 cells by preincubation of cells with SB-202190 and U0126 60 min before Legionella infection reduced COX-2 expression in infected cells (Fig. 8B). Therefore, Legionella-dependent p38 and p42/44 MAP kinase activation seems to be important for COX-2 expression.

Moreover, preincubation of A549 cells for 60 min with specific MAP kinase inhibitors SB-202190 (p38) and U0126 (p42/44) before infection of the cells with Legionella for 12 h reduced PGE2 release (Fig. 8C) indicating that Legionella-dependent p38 and p42/44 MAP kinase activation seems to be important for PGE2 release.

However, there is evidence of complex interconnections between these pathways suggesting they may act together to stimulate maximal Legionella-induced cell activation, including PGE2 release (7, 12, 44). Whereas the combination of SB-202190 and U0126 completely abolished PGE2 release, addition of Gö-6976 to U0126 before cell infection affected PGE2 to the same extent observed after incubation with one inhibitor alone (Fig. 8C). Combining SB-202190 and Gö-6976 also completely abolished PGE2 expression (Fig. 8C). The concentration of SB-202190, U0126, and Gö-6976 (or the inhibitor combinations) used in this study neither altered bacteria growth nor reduced epithelial cell numbers, or did not induce morphological signs of cytotoxicity within the time frame tested (data not shown).

L. pneumophila-induced COX-2 expression and PGE2 release depended on NF-{kappa}B activation in alveolar epithelial cells. 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 by I{kappa}B due to phosphorylation of its inhibitor I{kappa}B{alpha} by IKKbeta and subsequent proteolytic degradation (12).

To assess I{kappa}B{alpha} degradation, A549 cells were infected with L. pneumophila for different time periods. As shown in Fig. 9A, 107 cfu/ml Legionella induced degradation of I{kappa}B{alpha} within 30 min. We verified L. pneumophila induced NF-{kappa}B activation by EMSA. L. pneumophila (107 cfu/ml) induced strong DNA binding of NF-{kappa}B within 30 min, comparable or even stronger to TNF-{alpha}-related (50 ng/ml) activation (Fig. 9B). In the next step, the role of the central kinase complex of the canonical NF-{kappa}B pathway, I{kappa}B kinase (34), for Legionella-dependent COX-2 induction and PGE2 release was analyzed. The I{kappa}B kinase complex was blocked by preincubation of A549 cells with the cell-permeable peptide inhibitor IKK-NBD (34). IKK-NBD significantly reduced COX-2 protein expression (Fig. 9C) and release of PGE2 (Fig. 9D) in Legionella-infected A549 cells. Overall, the data demonstrate that activation of the canonical NF-{kappa}B signaling pathway by L. pneumophila was necessary for expression of COX-2 and PGE2 release in lung epithelium. IKK-NBD did not alter bacteria growth within the concentration and time frame tested (data not shown).


   DISCUSSION
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In the present study, we found that L. pneumophila induced cPLA2 activation, COX-2 expression, mPGES-1, and subsequent PGE2 release in human alveolar epithelial cells in vitro. Detailed analysis of signal transduction pathways provides evidence that this process depended on activation of PKC{alpha}, p38 and p42/44 MAP kinase, and the canonical NF-{kappa}B pathway.

During airborne lung infections, the airway and lung epithelium comprise the first host barrier against invading pathogens and is thereby directly exposed to pathogens and their products. Besides acting as a mechanical barrier, liberation of agents regulating inflammation by the epithelium may contribute significantly to the initiation and regulation of host innate immune response (22). L. pneumophila is known as one of the most important pathogens causing pneumonia in man (61) and is shown to activate lung tissue cells (8, 9, 33). Although lipid metabolites produced by PLA2, COX, and mPGES are known as important mediators of pulmonary (patho-) physiology (19, 60), little is known about Legionella-related cPLA2, COX, and mPGES-1 activation in lung cells. Therefore, we tested the hypothesis that L. pneumophila induced the activation of cPLA2, COX, and mPGES-1 in human lung epithelial cells.

Herein, we showed that Legionella infection resulted in increased expression of COX-2, but not COX-1, in A549 cells and primary lung SAEC. Subsequently, increased COX-2 protein expression was accompanied by PGE2 liberation, which is known to be the major COX product released by pulmonary epithelial cells as shown in previous studies (26, 37). Since two serotypes of L. pneumophila (130b and JR32) induced COX-2 expression and PGE2 release, induction of this cell pathway may be a common phenomenon in Legionella-related epithelial cell activation.

Increased PGE2 in the lung has been shown to stimulate the secretion of surfactant by alveolar type II cells and wound closure in airway epithelium (60). It has also been reported that PGE2 downregulates the production of important inflammatory cytokines such as IL-8, IL-12, monocyte chemoattractant protein-1, and granulocyte/macrophage colony-stimulating factor, which are essential for leukocyte migration (60). In addition, Aronoff et al. (2) demonstrated that PGE2 also inhibits alveolar macrophage phagocytosis through the increase of intracellular cAMP. Apart from lung epithelium, a recent report by Neild et al. (41) demonstrates an increased COX-2 expression and PGE2 production in Legionella-infected macrophages. They also demonstrate that macrophage-derived PGE2 inhibits T cells. Since an effective immune response that results in the clearance and growth control of intracellular pathogens often requires the activation of T cells (29), the Legionella-induced COX-2 expression and PGE2 release from alveolar epithelium may interfere with the host response in Legionnaires' disease.

The observation that Chlamydia pneumoniae (26), pneumococci (37), respiratory syncytial virus (46), as well as influenza virus (35), also induced COX-2 expression in pulmonary epithelium underlines the potential role of COX-2 in lung infection.

However, we noted that L. pneumophila provoked PGE2 production to a greater extent than TNF-{alpha} (Fig. 1A), although TNF-{alpha} stimulated COX-2 expression to a greater degree than L. pneumophila (Fig. 2C). Indeed, we demonstrated that Legionella activated cPLA2, a major enzyme of arachidonic acid production stronger than TNF-{alpha} in lung epithelial cells. Since we noticed no difference between L. pneumophila and TNF-{alpha} with regard to mPGES-1 transcription in the same cells, the data provided here might explain the phenomenon described above. Thus the Legionella-induced PGE2 release seems to be critically controlled by cPLA2. Further studies are needed to test whether phospholipases expressed by Legionella also contribute to cPLA2-related PGE2 release in human lung epithelial cells (3, 5).

In contrast to the constitutively expressed COX-1, a complex signaling network regulates the expression of inducible COX-2 (12). In Legionella-infected lung epithelial cells, we noted activation of p38 and p42/44 MAPK as well as of PKC{alpha}. These kinases were considered as important regulators of COX-2 and proinflammatory signaling pathways (10, 12, 28, 36, 58). We demonstrate that inhibition of p38 and p42/44 MAP kinase and PKC{alpha} reduced Legionella-related expression of COX-2 and PGE2 liberation. For example, since in influenza virus-infected lung epithelium, COX-2 expression was dominantly mediated by ERK and JNK, but not by p38 MAPK (35), a pathogen-specific regulation of COX-2 expression in lung tissue cells has to be considered. Activation of PKC{alpha} was shown to contribute to bacteria-related expression of COX-2 in Helicobacter pylori- or Staphylococcus aureus-infected cells (11, 31). We now demonstrate that L. pneumophila induced the activation of PKC in lung epithelium. In the case of macrophages and monocytes, it was shown that L. pneumophila heat shock protein induced the increase of IL-1 through PKC (45). Furthermore, Coxon et al. (13) found that PKC{alpha} was activated early during the uptake of L. pneumophila by macrophages.

Inhibition of both p42/44 MAPK and p38 MAPK, as well as inhibition of p38 MAPK and PKC, synergistically reduced Legionella-related PGE2 release. For example, since Chang et al. (10) found that PKC controlled p42/44 MAPK activity in A549 cells stimulated with PMA, and in microglia, PKC was found to control p38 activity (39), these pathways may be interconnected in a complex way in Legionella-infected epithelial cells. Overall, p38 and p42/44 MAP kinase, as well as PKC{alpha}-dependent signaling, seems to be an important step in Legionella-dependent activation of alveolar epithelial cells.

Stimulation of the transcription factor NF-{kappa}B is considered to contribute significantly to COX-2 expression (12). 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 (12, 34). We found degradation of I{kappa}B in the cytosol and increased binding of NF-{kappa}B to the DNA in Legionella-infected epithelium. Moreover, a highly specific cell-permeable inhibitor of IKK, IKK-NBD (34), abolished Legionella-related COX-2 protein expression and subsequent PGE2 release. The data implicates a crucial involvement of NF-{kappa}B in L. pneumophila-caused COX-2 induction.

Pulmonary epithelial cells may detect Legionella by transmembranous Toll-like receptors (TLR) or cytosolic pathogen pattern recognition receptors. For example, in recent studies, TLR2 (1) and TLR5 (20) were implicated in the host response against Legionella. In addition, cytosolic receptors of the NACHT (domain present in NAIP, CIITA, HET-E, TP-1)-LRR (leucine-rich repeats) (NLR) family are suspected to contribute to the host response against intracellular bacteria (25). In particular, it was demonstrated that A/J mice showed an increased susceptibility to Legionella infection due to a defect in the expression of the NLR protein Naip5 (Birc1e) (14, 62). However, the extent to which these receptors contribute to the activation of MAPK and PKC in Legionella-infected lung epithelium has to be established in further studies. Legionella express a variety of effector transport systems (e.g., PilD-dependent Lsp type II secretion pathway, type IV secretion system encoded by the DotA/Icm genes). The type IV secretion system, for instance, allows the bacterium to release pathogenic factors directly into the host cell cytosol (4). Interestingly, COX-2 expression and PGE2 liberation was not reduced in infections with a dotA mutant strain, suggesting that other, not type IV secretion system-dependent factors of Legionella, are involved in the described pathways. Nevertheless, further studies are needed to address, in depth, the role of different Legionella virulence factors for the activation of host cell pattern recognition receptors and signaling pathways like p42/44 MAPK, p38 MAPK, or PKC.

In conclusion, we found that L. pneumophila-induced PGE2 release in human alveolar epithelial A549 cells was dependent on COX-2 induction. It required p38, p42/44 MAP kinase, PKC{alpha}-, and NF-{kappa}B activation. Since control of the immune response is crucial to assure bacteria clearance and prevent excessive tissue damage in pneumonia, the mechanism described above could be important in Legionnaires' disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported with grants from the Bundesministerium für Bildung und Forschung to B. Schmeck (BMBF-Competence Network CAPNETZ C15), S. Hippenstiel (BMBF-Competence Network CAPNETZ C15), and N. Suttorp (BMBF-Competence Network CAPNETZ C4).


   ACKNOWLEDGMENTS
 
The excellent technical assistance of Sylwia Schapke, Frauke Schreiber, Jacqueline Hellwig (Dept. of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité - Universitätsmedizin Berlin, Berlin), and Kerstin Rydzewski (Robert Koch-Institut, Berlin) is greatly appreciated. Particular thanks goes to Ulrike Reichelt, a trainee in our department, for assistance. Part of this work will be included in the doctoral thesis of M. O. Etouem.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Hippenstiel, Dept. of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany (e-mail: stefan.hippenstiel{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.


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