Abstract
Chlamydophila pneumoniae is an important respiratory pathogen. In this study we characterized C. pneumoniae strain TW183-mediated activation of human small airway epithelial cells (SAEC) and the bronchial epithelial cell line BEAS-2B and demonstrated time-dependent secretion of granulocyte macrophage colony-stimulating factor (GM-CSF) upon stimulation. TW183 activated p38 mitogen-activated protein kinase (MAPK) in epithelial cells. Kinase inhibition by SB202190 blocked Chlamydia-mediated GM-CSF release on mRNA and protein levels. In addition, the chemical inhibitor as well as dominant-negative mutants of p38 MAPK isoforms p38α, β2, and γ inhibited C. pneumoniae–related NF-κB activation. In contrast, blocking of MAPK ERK, c-Jun kinase/JNK, or PI-3 Kinase showed no effect on Chlamydia-related epithelial cell GM-CSF release. Ultraviolet-inactivated pathogens as compared with viable bacteria induced a smaller GM-CSF release, suggesting that viable Chlamydiae were only partly required for a full effect. Presence of an antichlamydial outer membrane protein–A (OmpA) antibody reduced and addition of recombinant heat-shock protein 60 from C. pneumoniae (cHsp60, GroEL-1)–enhanced GM-CSF release, suggesting a role of these proteins in epithelial cell activation. Our data demonstrate that C. pneumoniae triggers an early proinflammatory signaling cascade involving p38 MAPK–dependent NF-κB activation, resulting in subsequent GM-CSF release. C. pneumoniae–induced epithelial cytokine liberation may contribute significantly to inflammatory airway diseases like chronic obstructive pulmonary disease (COPD) or bronchial asthma.
Chlamydophila pneumoniae, a gram-negative obligate intracellular bacterium, is a widespread respiratory pathogen causing pneumonia, bronchitis, sinusitis, and pharyngitis (1, 2). The majority of C. pneumoniae infections are subclinical, but serious pulmonary infection and profound lymphocytic alveolitis have been observed. Moreover, chronic infection may be an important risk factor for adult-onset asthma, chronic obstructive pulmonary disease (COPD [3]), and development of vascular lesions (4–6). In airway infection bronchial epithelial cells are the first line of defense getting in contact with C. pneumoniae. However, little is known about Chlamydia–epithelial cell interaction.
Besides human airway epithelial cells monocytes, macrophages, smooth muscle cells, endothelial cells, and different cell lines (e.g., airway epithelial cell line BEAS-2B cells, HEp-2 cells, Hela-229 cells) have been shown to be susceptible for C. pneumoniae infection (7, 8). Chlamydia could reside and replicate in these cells and induce a profound proinflammatory activation including cytokine release as well as upregulation of adhesion molecules (9–11). A complex chlamydial growth cycle is initiated after an infectious elementary body (EB) attaches to a susceptible target cell, promoting entry into a host cell–derived phagocytic vesicle (12). Wuppermann and coworkers could identify heparan sulfate–like glycosaminoglycans (GAG) as possible receptors mediating initial attachment of C. pneumoniae (13). In addition, Chlamydia expresses a variety of potent virulence factors, including heat-shock protein 60 (cHsp60, GroEL-1), outer membrane protein–A (OmpA), or a type III secretion apparatus (2). However, the role of these factors for pathogen–host cell interaction is still unknown.
Several recent studies have suggested that in acute inflammatory reaction airway epithelium itself plays a prominent and active role by releasing pro- and anti-inflammatory mediators (14). Epithelial cells have been shown to liberate mediators such as granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, 15-hydroxyeicosatetraenoic acid (15-HETE), tumor necrosis factor- α (TNF-α), and nitric oxide (NO) (15–17). Among these mediators, GM-CSF seems to play an important role in several inflammatory pulmonary diseases. GM-CSF is a 23-kD glycoprotein with pleiotropic effects on myeloid cell function. It is released by airway epithelial cells upon stimulation with agents as diverse as cytokines (including TNF-α, IL-1β, IL-4, or IL-13 [15, 18]), viruses (e.g., respiratory syncytial virus), or histamine (19). Importantly, elevated levels of GM-CSF have been described in bronchoalveolar lavage, endobronchial biopsies, and sputum samples from humans suffering from bronchial asthma. GM-CSF may contribute to the disease in several ways: the cytokine is thought to be responsible for the increase in number of mononuclear cells as well as for eosinophil activation and survival (20). Moreover, GM-CSF activates the 5-lipoxygenase pathway, thereby contributing to airway hyperresponsiveness (21). Overall, production of GM-CSF by the epithelium seems to be critically involved in inflammatory airway disease.
GM-CSF transcription and expression is tightly controlled by complex signaling pathways. Ubiquitously expressed members of the mitogen-activated protein kinase (p38 MAPK, JNK, ERK1/2) family contribute to this regulation (22). Especially p38 MAPK is thought to be important for proinflammatory mediator expression under various conditions of infectious disease. p38 MAPK influences gene transcription by activation of transcription factors or by direct phosphorylation of nuclear transcription machinery constituents. In the case of various proinflammatory cytokines, including GM-CSF, activation of transcription factor nuclear factor κB (NF-κB) seems to be critical for gene expression. However, there is limited knowledge about the proinflammatory activation of human bronchial epithelial cells by C. pneumoniae.
In the study presented we demonstrate that C. pneumoniae strongly activated GM-CSF liberation in human bronchial epithelial cells in a p38-MAPK dependent manner. Moreover, Chlamydia-related NF-κB activation was dependent on p38 MAPK. In addition, we show that chlamydial GroEL-1 protein sufficiently activated GM-CSF expression in epithelial cells.
Tissue culture plasticware was obtained from Becton Dickinson (Heidelberg, Germany). Keratinocyte serum-free medium (KSF-M), RPMI 1640, minimum essential medium (MEM, Eagle), PBS, trypsin-EDTA solution, and N-2-hydroxyethylpiperazine-N′-ethansulfonic acid (HEPES) were from Life Technologies (Karlsruhe, Germany). Small airway epithelial cell basal medium (SABM) was obtained from Cambrex (Baltimore, MD). Antibiotics were from Boehringer Mannheim (Mannheim, Germany). SB202190, U0126, SP600125, and LY294002 were from Calbiochem (San Diego, CA). Monoclonal antibody (mab) against chlamydial OmpA was from DAKO (Glostrup, Denmark), all other reagents from Sigma (Munich, Germany).
Primary human small airway epithelial cells (SAEC) were obtained from Cambrex (Clonetics Small Airway Epithelial Cell System; Cambrex, Baltimore, MD). The bronchial epithelial cell line BEAS-2B (CRL-9609), the human pulmonary mucoepidermoid carcinoma cell line NCI-H292 (CRL-1848), and the human embryonic kidney cell line HEK293 (CRL-1573) were obtained from American Type Culture Collection (Rockville, MD). All cells were grown to confluence in tissue culture flasks (80 cm2), and subsequently splitted into 6- or 24-well plates with 1.2 × 106 or 2.5 × 105 cells/well, respectively, using SABM (SAEC), KSF-M (BEAS-2B), RPMI 1640 (NCI-H292), or MEM-Eagle (HEK293) supplemented according to the supplier's instructions. Only confluent monolayers were used for experiments (23).
C. pneumoniae strain TW183 (ATCC) was cultured/purified as described by Maass and coworkers (4). Briefly, TW183 was grown to high titers in cycloheximide-treated HEp-2 cells. Infected monolayer were harvested from culture flasks and sonicated for 30 s. Cellular debris was removed by centrifugation at 500 × g for 10 min at 4°C. Aliquots diluted with an equal volume of sucrose-phosphate-glutamate (SPG)-buffer supplemented with 10% FCS were stored at −75°C until use. Titration in cycloheximide-treated HEp-2 cells was performed with a thawed aliquot (in triplicate). Epithelial cells/cell lines were inoculated with C. pneumoniae using a multiplicity of infection (MOI) as indicated in the figure legends. Chlamydia suspensions were thawed, diluted in the appropriate epithelial cell medium, and inoculated onto cells without centrifugation. After incubation at 37°C, plates were processed for further experiments at the times indicated in figure legends.
Purified recombinant GroEL-1 (heat-shock protein 60) from C. pneumoniae strain TW183 was a friendly gift of J. H. Hegemann (Institute of Microbiology, Heinrich-Heine-University, Düsseldorf, Germany). It was purified as described by Jantos and colleagues (24) with minor modifications; 20 μg / ml polymyxin B was used throughout the whole process of purification, and final dialysis was done using PBS without calcium or magnesium.
GM-CSF released by C. pneumoniae–infected SAEC or BEAS-2B cells was detected using a sandwich ELISA. Briefly, microtiterplates precoated overnight with a polyclonal goat anti-human GM-CSF mab (R&D Systems, Wiesbaden, Germany) were incubated with 1:10 diluted supernatants of control or stimulated epithelial cells. After 2 h, wells were washed three times and then incubated for another 2 h with a mouse anti-human GM-CSF mab (BD Biosciences, San Diego, CA). Wells were washed again three times and first incubated with a Biotin-SP–conjugated donkey anti-mouse mab (Dianova, Hamburg, Germany; 1 h, 37°C) and thereafter an additional hour with an avidin–horseradish peroxidase conjugate (DAKO, Hamburg, Germany). After additional washes, wells were exposed to 2,2′azino-di-(3-ethylbenzthiazoline sulfonsulfonate [6]) diammonium salt ABTS for 20 min. Optical density in the supernatant was determined at 405 nm in an ELISA reader (SLT-Reader400; SLT, Crailsheim, Germany).
HEK293 or NIC-H292 cells were cultured in 12-well plates with DMEM or RPMI 1640, respectively, each supplemented with 10% FCS. Subconfluent cells were co-transfected by using the calcium phosphate precipitation method according to the manufacturer's instructions (Clontech, Palo Alto, CA) with 0.2 μg of NF-κB–dependent luciferase reporter, 0.2 μg of RSV β-galactosidase plasmid, 0.1 μg of hTLR2 (generously provided by Tularik Inc., San Francisco, CA [25]), 0.1 μg of dominant negative p38α/β2/γ-AF, which cannot be phosphorylated (kind gift of Jiahuai Han, Scripps Research Institute, La Jolla, CA [26]) expression vectors or control vector, respectively. Cells were infected with C. pneumoniae strain TW183 on the following day. Luciferase activity was measured by using a luciferase reporter-gene assay (Promega, Mannheim, Germany), and results were normalized for transfection efficiency with values obtained by RSV–β-galactosidase.
For determination of p38 MAP kinase phosphorylation in BEAS-2B cells, cells were stimulated as indicated, washed twice in ice-cold HEPES buffer (pH 7.4) containing 100 mM sodium fluoride, 2 mM sodium vanadate, and 15 mM sodium pyrophosphate. Cells were then harvested on ice by scraping with lysis buffer containing EDTA 1 mM, Triton X-100 1%, PMSF 1 mM, leupeptin, pepstatin, and antipain (2 μg/ml each). After removal of the cell debris by centrifugation, cell lysates were subjected to 12.5% SDS-PAGE and blotted on Hybond-ECL membranes (Amersham Biosciences, Freiburg, Germany). Each lane contained 80 μg protein (ascertained by Bradford assay). Immunodetection of phosphorylated p38 MAP kinase was performed with phospho-specific p38 MAP kinase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). In all experiments, nonphosphorylated p38 MAP kinase (Santa Cruz Biotechnology) and β-actin (Sigma) was detected simultaneously to confirm that changes in phosphorylated p38-MAPK are not attributed to changes in total p38 expression and to confirm equal protein loads. Proteins were visualized by incubation with secondary IRDye 800– or Cy5.5-labeled antibodies (27), respectively, using an Odyssey infrared imaging system (LICOR Inc., Bad Homburg, Germany).
Expression of GM-CSF-mRNA in SAEAC or BEAS-2B was analyzed semiquantitatively by RT-PCR as described previously (28). Briefly, total RNA was extracted using RNeasy-Midi-kit (Qiagen, Hilden, Germany). One microgram of RNA was reverse transcribed in a total volume of 20 μl in the presence of 8 units of reverse transcriptase, 30 units of RNAse inhibitor, 0.2 μg of random hexamers (Pharmacia, Uppsala, Sweden), and 1 mM deoxynucleotides. RT-generated cDNA encoding the GM-CSF gene was amplified by PCR using specific primers designed from the reported primary sequences deposited with the GenBank database (GM-CSF: forward primer, 5′-AGCATGTGAATGCCATCCAG-3′; reverse primer, 5′-AGGGGATGACAAGCAGAAAG-3′; base pairs 100–119 and 438–419, respectively [GenBank NM000758]). To confirm the integrity of RNA and equal loading of sample, RT-PCR analysis of the Glyceraldehyd-3-phosphate dehydrogenase (GAPDH) gene was routinely performed (GAPDH: forward primer, 5′-CCACC CATGGCAAATTCCATGGCA-3′; reverse primer, 5′-TCTAGACG GCAGGTCAGGTCC-3′; base pairs 212–235 and 809–789, respectively [GenBank M33197.1]). PCR products were subsequently size-fractionated on 1.5% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. To guard against contamination by PCR products, water blanks were subjected to PCR in parallel with test samples.
Depending on the number of groups and the number of different time points studied, data of Figures 1A and 1C were analyzed by a two-way ANOVA. A one-way ANOVA was used for data of Figures 1B, 1D, 4A, 4B, 6, and 7. P ⩽ 0.05 was considered to be significant and indicated by asterisks (if not indicated otherwise, test was performed versus control).
Figure 1. C. pneumoniae–mediated GM-CSF release. Epithelial GM-CSF release was time- (A, BEAS-2B; C, SAEC) and dose- (B, BEAS-2B; D, SAEC) dependently increased in the supernatant of C. pneumoniae strain TW183-stimulated BEAS-2B cells or SAEC. Note that GM-CSF secretion increased as early as 2 h after infection and reached a maximum level after 24 h in both cell types (A, BEAS-2B; C, SAEC). There was no further significant increase after stimulation for more than 24 h. The total amount of secreted GM-CSF was markedly higher in SAEC than in BEAS-2B cells. Stimulation with TNF-α (10 ng/ml for 4 h) was used as a positive control for both cell types (B, BEAS-2B; D, SAEC). Data presented are mean ± SEM of five separate experiments. *P ⩽ 0.05 versus control (“ctrl.”). Circles, control; inverted triangles, TW183, MOI = 0.5; triangles, TW183, MOI = 5.
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Figure 4. Epithelial GM-CSF secretion is dependent on p38 MAPK activation. BEAS-B2 (A) and SAEC (B) were pretreated with inhibitors of epithelial p38 MAPK (SB 202190, 10 μM, 15 min), ERK1/2 (U0126, 1 μM, 15 min), JNK (SP600125, 1 μM, 15 min) to block epithelial MAP kinases or with LY294002 (10 μM, 15 min) to block PI-3 kinase. Afterwards, cells were infected with an MOI = 5 of C. pneumoniae, strain TW183. As demonstrated by ELISA, SB202190 reduced GM-CSF-expression 24 h after infection by more than 60% in BEAS-2B cells (A) and almost 50% in SAEC (B), respectively. Solvent controls for each inhibitor (ethanol and DMSO, respectively) were without effects (data not shown). Data presented are mean ± SEM of four separate experiments. #P ⩽ 0.05 versus control; *P ⩽ 0.05 versus untreated; n.s. = not significant.
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Figure 6. HEK293 or NCI-H292 cells were co-transfected with hTLR2, an NF-κB–dependent luciferase reporter plasmid, a β-Gal construct and a control vector (“mock,” A), or additionally with dominant-negative mutants of p38 isoforms α/β2/γ, respectively (B). A quantity of 10 μM SB202190 was used to block p38 MAP kinase (B). Cells were stimulated the day after transfection with C. pneumoniae strain TW183 with the indicated concentration. After 6 h cells were harvested and luciferase- and β-Gal-activity were determined and normalized. TW183 induced dose-dependent NF-κB activation in HEK293 and NCI-H292 cells with maximal effects after 6 h, using an MOI = 5 (A). A quantity of 10 ng/ml of TNF-α was used as positive control. Data presented are mean ± SEM of three separate experiments. (A) *P ⩽ 0.05 of TW-infected cells versus uninfected cells with control vector alone (“mock”). (B) *P ⩽ 0.05 of TW-infected cells versus uninfected cells with control vector alone (“mock”); #P ⩽ 0.05 TW-infected cells with dn-vector versus TW183-infected cells with control vector alone (“mock”), but no significance between different p 38 MAP kinase isoforms.
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Figure 7. Importance of different chlamydial virulence factors for GM-CSF secretion. Chlamydia were either heat-inactivated (90°C, 30 min), ultraviolet-inactivated, or pretreated with antibodies against outer membrane protein-1 (OmpA, 1:50, 30 min) or polymyxin B (100 μM, 15 min). Afterward BEAS-2B cells were stimulated with native (“untreated”) or pretreated C. pneumoniae, strain TW183 (MOI = 5, A). GM-CSF secretion in the supernatant was determined by ELISA 24 h after infection. To evaluated importance of purified GroEL-1, BEAS-2B cells were stimulated for 24 h with increasing concentrations of GroEL-1 (B). Our results could be confirmed using SAEC. Again, GroEl-1 (10 μg/ml, 24 h) was almost as effective as whole viable TW183 to induce a significant increase of GM-CSF from SAEC (C). Stimulation with TNF-α (10 ng/ml) was used as a positive control in all settings. Data presented are mean ± SEM of three separate experiments. (A) #P ⩽ 0.05 versus control; *P ⩽ 0.05 versus untreated. (B) *P ⩽ 0.05 versus unstimulated (“control”). (C) *P ⩽ 0.05 versus unstimulated (“control”).
[More] [Minimize]We have recently demonstrated that C. pneumoniae is able to infect human bronchial epithelial cells (freshly isolated human airway epithelial cell [HAEC] and the bronchial epithelial cell line BEAS-2B), followed by subsequent replication (23). In this study we were able to demonstrate that infection of BEAS-2B cells with C. pneumoniae strain TW183 time- (Figure 1A) and dose-dependently (Figure 1B) increased secretion of epithelial GM-CSF as shown by ELISA. GM-CSF in the supernatant of TW1983-infected epithelial cells continuously increased starting 2 h after infection, reached maximal levels after ∼ 24 h (Figure 1A), and remained elevated for more than 72 h (data not shown). Maximal effects occurred in the presence of an MOI of 5 (Figure 1B). These results could be confirmed using primary human epithelial cells from small airways (SAEC). Infection of SAEC with TW183 time- (Figure 1C) and dose-dependently (Figure 1D) increased secretion of epithelial GM-CSF with a kinetic similar to that of BEAS-2B. Again, maximal effects were seen using a MOI of 5 (Figure 1D). The total amount of secreted GM-CSF was markedly higher in primary epithelial cells than in BEAS-2B cells (up to 4-fold). Stimulation with TNF-α (10 ng/ml, 4 h) was used as a positive control for both cell types (Figure 1B, BEAS-2B; Figure 1D, SAEC).
PCR analysis was performed to analyze C. pneumoniae–mediated mRNA expression for GM-CSF. GM-CSF mRNA in TW183-infected (MOI = 5) epithelial cells was increased 30 min after infection in BEAS-2B cells (Figure 2A) and in SAEC cells (Figure 2B). The level of GM-CSF mRNA in these cells continued to increase over time, reaching maximal levels at 2 h. GM-CSF-RNA decreased to baseline (undetectable) after 24 h. TNF-α (10 ng/ml) was used as a positive control.
Figure 2. C. pneumoniae–induced GM-CSF mRNA expression in epithelial cells. Semiquantitative RT-PCR demonstrated a time-dependent mRNA expression in TW183-stimulated epithelial cells (A, BEAS-2B; B, SAEC). Epithelial cells were incubated with an MOI = 5 of TW183 for 0.5, 1, 2, 8, and 24 h. Unstimulated control cells were lysed after 2 h. TNF-α (10 ng / ml) was used as a positive control and maximal effects were seen after at 2 h. Expression of GAPDH was used to confirm equal loading. Representative gels (out of three) are demonstrated.
[More] [Minimize]Western blot analysis demonstrated that preincubation of BEAS-2B with C. pneumoniae strain TW183 (MOI = 0.05–5) time- and dose-dependently increased phosphorylation of p38 MAPK (Figure 3). Phosphorylation increased as early as 5–10 min after contact of Chlamydiae with epithelial cells, peaked at 15–30 min, and decreased again to almost baseline after 60–120 min (Figure 3A). Dose dependency (Figure 3B) was demonstrated after 15 min of incubation. Nonphosphorylated p38 MAPK was detected simultaneously to confirm that changes in phosphorylated p38 MAPK are not attributed to changes in total p38 expression. Equal protein load was confirmed by simultaneous detection of β-actin.
Figure 3. C. pneumoniae–mediated phosphorylation of p38 MAPK. C. pneumoniae induced a time- (A) and dose- (B) dependent phosphorylation of p38 MAPK in BEAS-2B cells as demonstrated by Western blot. Cells were incubated with an MOI = 5 of strain TW183 for 5, 15, 30, 60, 120, and 240 min; control cells were lysed after 15 min (A). Dose dependency (B) was demonstrated after an incubation time of 15 min. Proteins were separated using a 12.5% SDS-polyacrylamide gel; equal gel loading (80 μg protein/lane) was confirmed by detection of β-actin on the same blot. Nonphosphorylated p38 MAPK was detected simultaneously to confirm that changes in phosphorylated p38 MAPK are not attributed to changes in total p38 expression. Representative gels (out of three for all figures) are demonstrated.
[More] [Minimize]Using specific inhibitors against MAPK, we demonstrated the contribution of p38 MAPK for a C. pneumoniae–mediated GM-CSF-expression on the level of (1) protein expression, (2) mRNA–expression, and (3) transcription-factor activation.
Inhibition of epithelial p38 MAPK by SB202190 (10 μM, 15 min) reduced C. pneumoniae–mediated GM-CSF secretion of BEAS-2B cells into the supernatant by almost 60% as demonstrated by ELISA (Figure 4A). In contrast, blockade of MAPK ERK1/2 (U0126, 1 μM) or JNK (SP600125, 1 μM) failed to have an effect on GM-CSF expression. Solvent controls for each inhibitor (ethanol and DMSO, respectively) as well as SB202474 (a negative control for SB202190) were without effect (data not shown). In addition, Chlamydia-related GM-CSF expression was not affected by a PI-3K inhibitor (LY294002, 10 μM). These results could be confirmed using SAEC. Inhibition of p38 MAPK by SB202190 reduced C. pneumoniae–mediated GM-CSF secretion of SAEC by almost 50%.
Epithelial preincubation with SB202190 reduced TW183-mediated GM-CSF mRNA expression in a concentration- dependent manner (Figure 5). Maximal effects were seen using 10 μM of the inhibitor. Again, blockade of either ERK1/2 or JNK did not have any effects on Chlamydiae-induced GM-CSF mRNA-expression (data not shown).
Figure 5. C. pneumoniae–induced GM-CSF-mRNA expression is dependent on p38 MAPK-activation. Epithelial preincubation with SB202190 (0.1–10 μM, 15 min) reduced TW183-mediated GM-CSF expression dose-dependently. Total RNA was extracted 2 h after infection with C. pneumoniae (MOI = 5). Epithelial stimulation with TNF-α (10 ng/ml, 2 h) was used as a positive control. Expression of GAPDH was used to confirm equal loading. Representative gels (out of three) are demonstrated.
[More] [Minimize]BEAS-2B cells could not be transfected with a satisfactory efficiency. To further study importance of p38 MAPK for C. pneumoniae–mediated signal transduction, we therefore made use of HEK293 cells transiently transfected with a NF-κB–dependent luciferase reporter. Transfected cells were infected with increasing doses of C. pneumoniae (MOI 0.05–5) or TNFα (10 ng/ml). After incubation (6 h), luciferase activity was assessed. Chlamydia dose-dependently induced transcription of the NF-κB–dependent reporter gene. An MOI of 5 was as effective as TNF-α with respect to NF-κB activation (Figure 6A). Our results from the HEK293 cell system could be confirmed using the human pulmonary mucoepidermoid carcinoma cell line NCI-H292 (a pulmonary epithelial cell line). We were also able to transiently transfect the NCI-H292 cells with the NF-κB–dependent luciferase reporter plasmide. Although transfection efficiency was lower than in the HEK293 cell system, we could demonstrate that TW183 dose-dependently induced transcription of the NF-κB–dependent reportergene (Figure 6A).
In previous studies we and others showed a Chlamydia-dependent NF-κB–activation, and NF-κB is known to be critical for sufficient GM-CSF expression. We hypothesized that p38 MAPK activated NF-κB, thereby causing cytokine liberation. HEK293 or NCI-H292 cells, transiently transfected with an NF-κB–dependent luciferase reporter plasmid and preincubated with SB202190 (10 μM), did not respond with reporter gene expression upon infection with TW183 (Figure 6B).
Several isoforms of p38 MAPK induce differentiated activation of transcription factors; therefore, we performed transfection experiments using dominant-negative mutants of MAPK p38α, β2, and γ, which cannot be phosphorylated and thereby activated (26). HEK293 and NCI-H292 cells were co-transfected as described. As shown in Figure 6B, C. pneumoniae–induced expression of NF-κB–dependent reporter gene was markedly blocked by dominant-negative isoforms α, β2, and γ of p38 MAPK. Again, transfection efficiency in the NCI-H292 cell system was lower than in the HEK293 cell system; overall results, however, were comparable between both cell types. These experiments provide evidence that Chlamydia activated NF-κB–dependent gene transcription in a p38 MAPK–dependent manner.
Viable bacteria were required to induce a maximal GM-CSF secretion of BEAS-2B cells (Figure 7A). Heat inactivation (90°C, 30 min) reduced GM-CSF-expression almost completely. Ultarviolet-inactivated bacteria were still able to induce a marked release of GM-CSF from BEAS-2B cells. Pretreatment of C. pneumoniae with an antibody against chlamydial OmpA (1:50, 30 min) resulted in a 33% reduction of the pathogen-mediated GM-CSF release. Chlamydial preincubation with polymyxin B (100 μM, 15 min) to block chlamydial LPS did not have significant effects on TW183-mediated GM-CSF release by BEAS-2B cells. Purified recombinant C. pneumoniae GroEL-1 was almost as effective as viable bacteriae and induced a time- (data not shown) and dose-dependent GM-CSF expression (Figure 7B). Maximal release was seen using a concentration of 20 μg GroEL-1/ml; higher concentrations did not increase these effects (data not shown). These results could be confirmed using SAEC (Figure 7C). Heat inactivation of GroEL-1 (90°C, 60 min) abolished GM-CSF almost completely (data not shown).
C. pneumoniae, an obligate intracellular bacterium, is a widespread respiratory pathogen causing serious airway infection (bronchitis, pneumonia, etc.) with subsequent persistent or recurrent severe immunopathologic effects (1–3, 29). Respiratory epithelium has been identified as a primary target of C. pneumoniae infection (2, 30). C. pneumoniae strain TW183, used in our study, was shown to induce a marked release of the proinflammatory cytokine GM-CSF from SAEC and the bronchoepithelial cell line BEAS-2B. GM-CSF expression observed was dependent on heat labile factors expressed by Chlamydiae. Effects in both cell types were mediated via epithelial p38-MAPK but not ERK1/2, JNK, or PI-3K activity. Interestingly Chlamydia-induced p38 MAPK activity seemed to be critical for NF-κB stimulation. These observations add important new properties to this bacterium—i.e., its capacity to induce a cascade of events leading to a profound inflammatory activation of airway epithelial cells.
Members of the MAPK family are ubiquitously expressed and activated in response to a variety of stimuli. Recently we identified the importance of ERK1/2 in C. pneumoniae–mediated endothelial cell activation (9). In addition, Coombes and Mahony demonstrated that activation of the MEK-ERK1/2 pathway is important for C. pneumoniae invasion of epithelial cells (31). In the present study we analyzed the impact of p38 MAPK in Chlamydia-mediated epithelial cell activation and demonstrated that TW183 induced a distinct activation of p38 MAPK in BEAS-2B cells. Moreover, inhibition of p38 MAPK by SB202190 markedly reduced expression of a NF-κB reporter gene as well as GM-CSF expression on protein and mRNA level, indicating that both p38 MAP kinase activation and NF-κB–dependent gene transcription seem to be involved in C. pneumoniae–induced expression of proinflammatory genes. Four different isoforms of p38 MAP kinase have been identified (32). p38α and β2 were found to activate AP1-related gene expression, while γ and δ had inhibitory effects on gene expression (26). However, the function of the different isoforms in bacterial-induced cell activation is still unknown. Using dominant-negative mutants of p38 MAP kinase α, β2, and γ we could show that all isoforms significantly reduced Chlamydia-dependent NF-κB activation. Although our data emphasize the central role of p38 MAP kinase in C. pneumoniae–mediated epithelial cell infection, further studies investigating additional signaling pathways as well as transcription factors are needed to clarify the role of these isoforms during target cell activation. Our studies, aimed to identify possible intracellular signaling steps involved during (initial) contact to and infection of epithelium, indicated that p38 MAPK phosphorylation occurred within 15 min after the addition of TW183 to epithelial cells. Subsequent p38 MAPK–dependent activation of NF-κB and increased expression of GM-CSF mRNA and protein was noted. Viable bacteria were only in part required for a full effect on epithelial cell activation (GM-CSF mRNA expression and translation as well as GM-CSF release into the supernatant).
Almost nothing is known about the importance of different chlamydial virulence factors for activation of signal transduction pathways in target cell infection. After initial attachment to heparan sulfate–like glycosaminoglycans (13) Chlamydiae are internalized; several studies have demonstrated that Chlamydia-mediated target cell activation is mediated via Toll-like receptor (TLR)-2 and -4, as well as by the recently identified (intracellular) nucleotide-binding oligomerization domain (Nod) proteins (11, 33–35). After internalization, elementary bodies dissociate from the endocytotic pathway by actively modifying the vacuole to become fusogenic with exocytic vesicles (12). Interaction with this secretory pathway appears to provide a pathogenic mechanism that allows Chlamydiae to establish themselves in a site that is not destined to fuse with lysosomes.
Further studies are required to determine the relationship between these distinct steps of this chlamydial development cycle and initiation of host cell signaling pathways. To characterize impact of potential chlamydial virulence factors for initial target cell activation, bacteriae were either heat- or ultraviolet-treated. Ultraviolet-inactivated Chlamydiae were still able to induce GM-CSF mRNA expression (data not shown) and GM-CSF release into the supernatant. Heat-killed pathogens were substantially less effective; GM-CSF release was almost completely abolished. This suggests that heat-labile chlamydial membrane compounds like outer membrane proteins or heat shock proteins, or other yet unidentified virulence factors, are possible candidates for initiation of target cell activation; heat-resistant structures like chlamydial LPS (cLPS) are not very likely. Several recent studies have suggested that chlamydial heat shock protein-60 (cHsp60, GroEL-1) may act as a possible mediator of target cell activation (11, 34, 35). Using recombinant GroEL-1 from C. pneumoniae, we now could demonstrate that the purified protein dose-dependently induced expression of GM-CSF in epithelial cells. The protein was almost as effective as viable or ultraviolet-inactivated whole bacteria. Heat inactivation of GroEL-1 reduced GM-CSF expression almost completely. Additional studies using a monoclonal antibody against GroEL-1 are necessary to specify the importance of this protein during target cell activation by whole bacteria. In addition, using a commercially available antibody against chlamydial OmpA we were able to demonstrate that this membrane component may also have some effects for target cell activation; GM-CSF expression was significantly reduced in cells incubated with pretreated Chlamydia. However, if triggering GroEL-1 is on the surface of the elementary bodies, use of antibodies against OmpA might be able to partly cover the heat shock proteins and therefore modify target cell activation. TW183 preincubation with polymyxin B to inactivate chlamydial LPS had no effects on epithelial MAPK phosphorylation or GM-CSF expression. Heated bacteria did not induce a GM-CSF expression. Since LPS is thermostable, this also speaks against the role of LPS in this response. Additional studies, however, using purified OmpA or LPS from C. pneumoniae as well as monoclonal antibodies against chlamydial LPS, may help to clarify the role of these virulence factors during target cell activation.
Most data of our study were obtained using the bronchoepithelial cell line BEAS-2B. However, key experiments using SAEC reproduced the findings. Interestingly, the total amount of secreted GM-CSF was markedly higher in SAEC than in BEAS-2B cells (up to 4-fold). In addition, we were able to confirm our data from the system of transiently transfected HEK293 cells using the respiratory cell line NCI-H292. For an exact analysis of epithelial function in inflammatory airway disease in terms of C. pneumoniae–mediated clinical disorders (pneumonia, asthma, etc.), in vivo studies are required to determine the relationship between distinct steps of the chlamydial development cycle, the importance of different chlamydial virulence factors, and the initiation of host cell signaling pathways.
In conclusion, we present evidence that C. pneumoniae can induce a sustained proinflammatory phenotype in the human epithelial cells. GM-CSF expression induced by C. pneumoniae is mediated via a p38 MAPK–dependent activation of NF-κB–related gene transcription. Chlamydial OmpA and especially heat shock protein-60, GroEL-1, could be identified as an important virulence factors for target cell activation. The data demonstrated that during an acute infection with C. pneumoniae the airway epithelium itself plays a prominent and active role. This information may improve our understanding of the pathogenesis of C. pneumoniae–mediated inflammatory airway diseases and may help in the establishment of innovative therapeutic strategies.
The technical assistance of K. Möhr is greatly appreciated. Parts of this work will be included in the M.D. thesis of P. Bockstaller.
| 1. | Grayston JT, Kuo CC, Wang SP, Altman J. A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. N Engl J Med 1986;315:161–168. |
| 2. | Kuo CC, Jackson LA, Campbell LA, Grayston JT. Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 1995;8:451–461. |
| 3. | Hahn DL. Chlamydia pneumoniae, asthma, and COPD: what is the evidence? Ann. Allergy Asthma Immunol 1999;83:271–288, 291. |
| 4. | Maass M, Bartels C, Engel PM, Mamat U, Sievers HH. Endovascular presence of viable Chlamydia pneumoniae is a common phenomenon in coronary artery disease. J Am Coll Cardiol 1998;31:827–832. |
| 5. | Saikku P, Leinonen M, Tenkanen L, Linnanmaki E, Ekman MR, Manninen V, Manttari M, Frick MH, Huttunen JK. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Intern Med 1992;116:273–278. |
| 6. | Shor A, Kuo CC, Patton DL. Detection of Chlamydia pneumoniae in coronary arterial fatty streaks and atheromatous plaques. S Afr Med J 1992;82:158–161. |
| 7. | Gaydos CA, Summersgill JT, Sahney NN, Ramirez JA, Quinn TC. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells. Infect Immun 1996;64:1614–1620. |
| 8. | Roblin PM, Dumornay W, Hammerschlag MR. Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae. J Clin Microbiol 1992;30:1968–1971. |
| 9. | Krull M, Klucken AC, Wuppermann FN, Fuhrmann O, Magerl C, Seybold J, Hippenstiel S, Hegemann JH, Jantos CA, Suttorp N. Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae. J Immunol 1999;162:4834–4841. |
| 10. | Kaukoranta-Tolvanen SS, Teppo AM, Laitinen K, Saikku P, Linnavuori K, Leinonen M. Growth of Chlamydia pneumoniae in cultured human peripheral blood mononuclear cells and induction of a cytokine response. Microb Pathog 1996;21:215–221. |
| 11. | Kol A, Bourcier T, Lichtman AH, Libby P. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages. J Clin Invest 1999;103:571–577. |
| 12. | Moulder JW. Interaction of chlamydiae and host cells in vitro. Microbiol Rev 1991;55:143–190. |
| 13. | Wuppermann FN, Hegemann JH, Jantos CA. Heparan sulfate-like glycosaminoglycan is a cellular receptor for Chlamydia pneumoniae. J Infect Dis 2001;184:181–187. |
| 14. | Barnes PJ. Cytokines as mediators of chronic asthma. Am J Respir Crit Care Med 1994;150:S42–S49. |
| 15. | Cromwell O, Hamid Q, Corrigan CJ, Barkans J, Meng Q, Collins PD, Kay AB. Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1 beta and tumour necrosis factor-alpha. Immunology 1992;77:330–337. |
| 16. | Mitchell JA, Belvisi MG, Akarasereenont P, Robbins RA, Kwon OJ, Croxtall J, Barnes PJ, Vane JR. Induction of cyclo-oxygenase-2 by cytokines in human pulmonary epithelial cells: regulation by dexamethasone. Br J Pharmacol 1994;113:1008–1014. |
| 17. | Widdicombe JH, Ueki IF, Emery D, Margolskee D, Yergey J, Nadel JA. Release of cyclooxygenase products from primary cultures of tracheal epithelia of dog and human. Am J Physiol 1989;257:L361–L365. |
| 18. | Nakamura Y, Azuma M, Okano Y, Sano T, Takahashi T, Ohmoto Y, Sone S. Upregulatory effects of interleukin-4 and interleukin-13 but not interleukin-10 on granulocyte/macrophage colony-stimulating factor production by human bronchial epithelial cells. Am J Respir Cell Mol Biol 1996;15:680–687. |
| 19. | Noah TL, Becker S. Respiratory syncytial virus-induced cytokine production by a human bronchial epithelial cell line. Am J Physiol 1993;265:L472–L478. |
| 20. | Cox G, Ohtoshi T, Vancheri C, Denburg JA, Dolovich J, Gauldie J, Jordana M. Promotion of eosinophil survival by human bronchial epithelial cells and its modulation by steroids. Am J Respir Cell Mol Biol 1991;4:525–531. |
| 21. | Stampfli MR, Wiley RE, Neigh GS, Gajewska BU, Lei XF, Snider DP, Xing Z, Jordana M. GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice. J Clin Invest 1998;102:1704–1714. |
| 22. | Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature 2001;410:37–40. |
| 23. | Jahn HU, Krull M, Wuppermann FN, Klucken AC, Rosseau S, Seybold J, Hegemann JH, Jantos CA, Suttorp N. Infection and activation of airway epithelial cells by Chlamydia pneumoniae. J Infect Dis 2000;182:1678–1687. |
| 24. | Jantos CA, Krombach C, Wuppermann FN, Gardemann A, Bepler S, Asslan H, Hegemann JH, Haberbosch W. Antibody response to the 60-kDa heat-shock protein of Chlamydia pneumoniae in patients with coronary artery disease. J Infect Dis 2000;181:1700–1705. |
| 25. | Song HY, Regnier CH, Kirschning CJ, Goeddel DV, Rothe M. Tumor necrosis factor (TNF)-mediated kinase cascades: Bifurcation of nuclear factor-kappa B and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2. Proc Natl Acad Sci USA 1997;94:9792–9796. |
| 26. | Pramanik R, Qi XM, Borowicz S, Choubey D, Schultz RM, Han JH, Chen G. p38 isoforms have opposite effects on AP-1-dependent transcription through regulation of c-Jun - The determinant role of the isoforms in the p38 MAPK signal specificity. J Biol Chem 2003;278:4831–4839. |
| 27. | Hippenstiel S, Witzenrath M, Schmeck B, Hocke A, Krisp M, Krull M, Seybold J, Seeger W, Rascher W, Schutte H, et al. Adrenomedullin reduces endothelial hyperpermeability. Circ Res 2002;91:618–625. |
| 28. | Seybold J, Newton R, Wright L, Finney PA, Suttorp N, Barnes PJ, Adcock IM, Giembycz MA. Induction of phosphodiesterases 3B, 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in human peripheral blood T-lymphocytes by 8-bromo-cAMP and Gs-coupled receptor agonists: potential role in beta2-adrenoreceptor desensitization. J Biol Chem 1998;273:20575–20588. |
| 29. | Dalhoff K, Maass M. Chlamydia pneumoniae pneumonia in hospitalized patients: clinical characteristics and diagnostic value of polymerase chain reaction detection in BAL. Chest 1996;110:351–356. |
| 30. | Wong KH, Skelton SK, Chan YK. Efficient culture of Chlamydia pneumoniae with cell lines derived from the human respiratory tract. J Clin Microbiol 1992;30:1625–1630. |
| 31. | Coombes BK, Mahony JB. Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells. Cell Microbiol 2002;4:447–460. |
| 32. | Hale KK, Trollinger D, Rihanek M, Manthey CL. Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages. J Immunol 1999;162:4246–4252. |
| 33. | Opitz B, Forster S, Hocke AC, Maass M, Schmeck B, Hippenstiel S, Suttorp N, Krull M. Nod1-mediated endothelial cell activation by Chlamydophila pneumoniae. Circ Res 2005;96:319–326. |
| 34. | Bulut Y, Faure E, Thomas L, Karahashi H, Michelsen KS, Equils O, Morrison SG, Morrison RP, Arditi M. Chlamydial heat shock protein 60 activates macrophages and endothelial cells through Toll-like receptor 4 and MD2 in a MyD88-dependent pathway. J Immunol 2002;168:1435–1440. |
| 35. | Sasu S, LaVerda D, Qureshi N, Golenbock DT, Beasley D. Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation. Circ Res 2001;89:244–250. |
