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B- and MAPK-dependent cytokine release by lung epithelial cells
1 Dept of Internal Medicine/Infectious Diseases and Respiratory Medicine and 2 Institute for Periodontology and Synoptic Dentistry, Charité – Universitätsmedizin Berlin, and 3 Nachwuchsgruppe 5 Pathogenesis of Legionella Infection, Robert Koch Institute, Berlin, Germany.
CORRESPONDENCE: S. Hippenstiel, Dept of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Fax: 49 30450553906. E-mail: stefan.hippenstiel{at}charite.de
Keywords: Alveolar epithelium, bacteria, cytokines, signal transduction pathways
Received: November 30, 2005
Accepted August 7, 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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L. pneumophila infection induced liberation of interleukin (IL)-2, -4, -6, -8 and -17, monocyte chemoattractant protein-1, tumour necrosis factor-
, IL-1ß, interferon-
and granulocyte colony-stimulating factor, but not of IL-5, -7, -10, -12 (p70) or -13 or granulocyte-macrophage colony-stimulating factor. The present study focused on IL-8 and found induction by L. pneumophila strains 130b, Philadelphia 1, Corby and, to a lesser extent, JR32. Knockout of dotA, a central gene involved in type IVB secretion, did not alter IL-8 induction, whereas lack of flagellin significantly reduced IL-8 release by Legionella. Moreover, p38 mitogen-activated protein kinase (MAPK) was activated and kinase inhibition reduced secretion of induced cytokines, with the exception of IL-2 and granulocyte colony-stimulating factor. In contrast, inhibition of the MAPK kinase 1/extracellular signal-regulated kinase pathway only reduced the expression of a few cytokines. L. pneumophila also induced binding of nuclear factor-
B subunit RelA/p65 and RNA polymerase II to the il8 promoter, and a specific inhibitor of the inhibitor of nuclear factor-B complex dose-dependently lowered IL-8 expression.
Taken together, Legionella pneumophila activated p38 mitogen-activated protein kinase- and nuclear factor-B/RelA pathway-dependent expression of a complex pattern of cytokines by human alveolar epithelial cells, presumably contributing to the immune response in legionellosis.
An important causative agent of severe community-acquired pneumonia, Legionella pneumophila is the second most commonly detected pathogen in cases of pneumonia admitted to intensive care units in industrialised countries 1. Approximately 15% of legionellosis appears in community outbreaks. Although >40 Legionella species are known, the majority of human infections are caused by L. pneumophila serogroup 1 2. L. pneumophila is a Gram-negative facultative intracellular pathogen of amoeba in natural and man-made aquatic environments. Infection of humans occurs after inhalation of contaminated water aerosol droplets. L. pneumophila-containing phagosomes initially do not fuse with lysosomes and the bacteria induce remodelling of their membrane-bound compartment into an endoplasmic reticulum-like organelle 3. This remodelling depends on the defect in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion apparatus of L. pneumophila 4. Besides delivery of proteins by the type IVB secretion system, L. pneumophila contains a battery of additional virulence factors, including a type II secretion apparatus 3. Sequencing of L. pneumophila serogroup 1 genomes revealed
3,000 genes, among which are many genes with possible function in manipulation of host cell signalling 5. According to the Legionella–host interaction, recent studies demonstrated that L. pneumophila lipopolysaccharide was recognised by Toll-like receptor (TLR)2 and flagellin by TLR5 6, 7. A stop codon in the human TLR5 gene leads to increased susceptibility to legionellosis 7. Interestingly, although some studies have suggested a minor role of TLR4 in legionellosis 6, Hawn et al. 8 recently showed that TLR4 polymorphisms are associated with resistance to Legionnaires’ disease.
Essential results according L. pneumophila pathogenesis were obtained by analysing infection of protozoans or immune cells such as macrophages. However, lung epithelial cells constitute a first mechanical and immunological barrier against airborne pathogens and are important sources of cytokines in the lung 9, 10. Activation of pro-inflammatory signalling pathways in lung epithelial cells, including p38 mitogen-activated protein kinase (MAPK)- and nuclear factor-B (NF-B)-dependent gene transcription, by bacterial infection, contribute significantly to cytokine release 10, 11. Although Legionella efficiently infects and stimulates lung epithelial cells 7, 12, mechanisms of L. pneumophila-induced activation of and cytokine release in lung epithelial cells are widely unknown. Therefore, the pro-inflammatory activation of lung epithelial cells by Legionella infection was analysed in detail.
In the present study, it was shown that L. pneumophila induced the release of several important cytokines in human alveolar epithelial A549 cells, e.g. interleukin (IL)-2, -4, -6, -8 and -17, monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-, IL-1ß, interferon (IFN)- and granulocyte colony-stimulating factor (G-CSF), as well as activating the p38 MAPK, extracellular signal-regulated kinase (ERK) and NF-B pathways. Blocking p38 MAPK reduced secretion of all cytokines in Legionella-infected cells, with the exception of IL-2 and G-CSF, whereas blocking the ERK pathway diminished only release of IFN-, IL-1ß, IL-6 and TNF-. By addressing expression of IL-8 as a model cytokine in more detail, the important role of p38 MAPK- and NF-B/RelA-dependent gene transcription in the activation of L. pneumophila-infected epithelial cells was verified. Moreover, activation of IL-8 expression was reduced by a flagellin deletion mutant, implying a role for TLR5 or possible intracellular receptors in Legionella sensing by A549 cells. However, IL-8 expression in A549 cells was not affected by a dotA-knockout mutant, suggesting that the type IVB Dot/Icm secretion system and intracellular replication are not needed for IL-8 expression in A549 cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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B-dependent reporter cell line, A549 6Btkluc, was a kind gift of R. Newton (Dept of Biological Sciences, University of Warwick, Coventry, UK). These cells contain a stably integrated plasmid with three tandem repeats of the sequence 5’-AGCTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGA-3’, which contains two copies of the decameric NF-B binding site upstream of a minimal thymidine kinase promoter (-105–51) driving a luciferase gene.
Infection with bacterial strains and isogenic mutants
L. pneumophila sg1 strains 130b (ATCC BAA-74, kindly provided by N.P. Cianciotto, Northwestern University Medical School, Chicago, IL, USA 13), Philadelphia 1 (ATCC 33152, kindly provided by B. Neumeister, Tübingen University, Tübingen, Germany 14), JR32 wildtype 15 and JR32 dotA mutant (LELA 3118, both kindly provided by H. Shuman, Columbia University, New York, NY, USA 16), and Corby wildtype and a Corby flaA, defective in flagellin, mutant (both kindly provided by K. Heuner, Würzburg University, Würzburg, Germany) were routinely grown on buffered charcoal–yeast extract (BCYE) agar for 2–3 days at 37°C 17 and subsequently inoculated into plain RPMI medium at an optical density at 660 nm of 0.2–0.4. A549 cells (1x105 cells·mL-1) were infected with 1x105–1x108 colony-forming units (cfu)·mL-1 L. pneumophila, i.e. at a multiplicity of infection of 1:1–1:1,000, in 1 mL epithelial cell growth medium for the indicated times. Extracellular bacteria were not routinely killed with antibiotics. L. pneumophila strains did not grow significantly in epithelial cell growth medium, as controlled by serial dilutions plated on BCYE agar.
In order to verify intracellular infection, A549 cells were incubated with the added bacteria for 2 h with or without kinase inhibitors, gentamicin (100 µg·mL-1) was added for a further 2 h and then the cells were washed three times with plain medium, to remove unbound bacteria, and treated with 10% (weight/volume) saponin (Sigma Chemical Company, Munich, Germany) to lyse the host cells. Serial dilutions were plated on BCYE agar.
Interleukin-8 ELISA
Confluent A549 cells were infected as indicated in a humidified atmosphere for 15 h. After incubation, supernatants were collected and processed for IL-8-quantification by sandwich ELISA, as described previously 11, 18. In some experiments, medium was changed after certain time periods (1, 2 or 4 h) with or without gentamicin (100 µg·mL-1) and then the cells were incubated for the remaining time in the same medium before IL-8 was analysed in the supernatant.
Bioplex protein array system
Confluent A549 cells were infected as indicated in a humidified atmosphere for 15 h. After incubation, supernatants were collected and cytokine release was analysed using the Bioplex Protein Array system (BioRad, Hercules, CA, USA) and beads specific for IL-2, -4, -5, -6, -7, -8, -10, -12 (p70), -13 and -17, MCP-1, TNF-, IL-1ß, IFN-, granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF, according to the manufacturer’s instructions 19, 20.
Western blotting
For determination of p38 MAPK and ERK phosphorylation, A549 cells were infected as indicated, washed twice and harvested. Cells were lysed in buffer containing Triton X-100 (Sigma Chemical Company), subjected to sodium dodecylsulphate-polyacrylamide gel electrophoresis and blotted on to Hybond ECL membrane (Amersham Biosciences, Freiburg, Germany). Immunodetection of phosphorylated MAPK was carried out using phospho-specific p38 MAPK or ERK antibodies (Cell Signaling, Frankfurt, Germany) 11. Degradation of inhibitor of NF-B (IB) was analysed using a rabbit polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as described previously 11. In all experiments, unphosphorylated ERK2 and p38 MAPK (Santa Cruz Biotechnologies) were detected simultaneously in order to confirm equal protein load. Proteins were visualised by incubation with secondary IRDye 800- or Cy5.5-labelled antibodies, respectively (Odyssey infrared imaging system; LI-COR Inc., Bad Homburg, Germany) 11, 21. All primary antibodies were used at a dilution of 1:200 and all secondary antibodies at a dilution of 1:2,000.
RT-PCR
For analysis of IL-8 and reduced glyceraldehye-3-phosphate dehydrogenase (GAPDH) gene expression in A549 cells, total RNA was isolated by means of the RNEasy Mini kit (Qiagen, Hilden, Germany) and reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega, Heidelberg, Germany). The complementary DNA generated was amplified by PCR using specific intron-spanning IL-8 and GAPDH primers 11. All primers were purchased from TIB MOLBIOL (Berlin, Germany). After 35 amplification cycles, PCR products were analysed on 1.5% agarose gels, stained with ethidium bromide and subsequently visualised. In order to confirm use of equal amounts of RNA in each experiment, all samples were checked for GAPDH mRNA expression.
Reporter gene assay
A549 cells stably transfected with a NF-B-dependent luciferase reporter plasmid 22 were cultured in 12-well plates in Dulbecco’s modified Eagle medium (Life Technologies). Cells were incubated with Legionella for 15 h and lysed, and then luciferase activity was measured using a luciferase reporter gene assay (Promega, Mannheim, Germany).
Chromatin immunoprecipitation
A549 cells in 75-cm2 culture flasks were infected with L. pneumophila 130b as indicated and then subjected to a chromatin immunoprecipitation assay, as previously described, using anti-p65 or anti-RNA polymerase II antibodies (both Santa Cruz Biotechnologies) 11, 20. The il8 promoter was amplified by PCR using HotstarTaq polymerase (Qiagen) and specific primers as follows: 5’-AAGAAAACTTTCGTCATACTCCG–3’ (sense); and 5’–TGGCTTTTTATATCATCACCCTAC-3’ (antisense). PCR amplification of the total input DNA in each sample was performed as a control 18, 19.
Statistical methods
Data are presented as mean±SEM of at least three independent experiments. One-way ANOVA was used for numerical data shown in the figures. Main effects were then compared using Newman–Keuls post-test. A p-value of <0.01 was considered significant.
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RESULTS |
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, IL-1ß, IFN- and G-CSF, but not of IL-5, -7, -10, -12 (p70) or -13 or GM-CSF was observed (fig. 1
). Pre-incubation of A549 cells with the specific p38 MAPK inhibitor SB202190 (Calbiochem-Merck, Darmstadt, Germany) reduced levels of IL-4, -6, -8 and -17, MCP-1, TNF-, IL-1ß and IFN-, but not of IL-2 or G-CSF (TNF- was obtained from R&D Systems, Wiesbaden, Germany). Inhibition of MAPK kinase 1 (MEK1) by U0126 reduced release of only IFN-, IL-1ß, IL-6 and TNF- (fig. 1). Incubation with inhibitors alone showed no cytotoxicity and did not alter cytokine expression or infection of epithelial cells (data not shown).
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). L. pneumophila 130b, Philadelphia 1 and Corby similarly induced dose-dependent IL-8 release (fig. 2a and b), whereas JR32-provoked cytokine secretion was lower (fig. 2c). L. pneumophila 130b also induced dose- (fig. 2d) and time-dependent (fig. 2e) expression of IL-8 mRNA. Gene expression started as early as 60 min after infection. A flaA knockout mutant, defective in flagellin production, resulted in strongly reduced IL-8 release by L. pneumophila Corby-infected A549 cells over 15 h (fig. 2b). In contrast, a dotA knockout mutant, defective in a gene essential for the establishment of a functional L. pneumophila type IVB secretion apparatus 3, did not alter IL-8 release by L. pneumophila JR32-infected A549 cells over 15 h (fig. 2c) or the time course of IL-8 mRNA induction up to 4 h (fig. 2f).
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) and ERK2 (fig. 3b) was assessed by Western blot analysis. L. pneumophila infection induced phosphorylation of both kinases within 60–120 min. Blocking p38 MAPK with SB202190 reduced IL-8 mRNA accumulation (fig. 3d). Moreover, p38 MAPK inhibitors SB202190 and SB203580 (Calbiochem-Merck) dose-dependently reduced L. pneumophila-induced IL-8 release (fig. 3c). Control compound SB202474 (Calbiochem-Merck) had no effect on cytokine release (fig. 3c). Neither inhibitors nor control compound reduced cell number or induced morphological signs of cytotoxicity.
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B activationB. IB kinase complex was blocked using the specific peptide inhibitor, IB kinase NF-B essential modulator-binding domain (IKK-NBD; Biomol, Plymouth Meeting, PA, USA), and a dose-dependent reduction in IL-8 secretion by L. pneumophila 130b-infected A549 cells observed (fig. 4a). In A549 cells, transfected with a NF-B-dependent reporter gene construct, dose-dependent induction of reporter gene expression was found (fig. 4b). IKK-NBD displayed no cytotoxicity and did not alter infection of A549 by Legionella strain 130b or basal cytokine expression (data not shown). Western Blot analysis revealed degradation of cytosolic NF-B inhibitor IB starting 60 min after stimulation of A549 cells with L. pneumophila 130b (fig. 4c), and chromatin immunoprecipitation showed recruitment of NF-B/p65 and RNA polymerase II to the il8 promoter (fig 4d). IKK-NBD did not reduce cell number or induce morphological signs of cytotoxicity. These data indicate that L. pneumophila induces IL-8 expression by activation of the canonical NF-B pathway.
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). Removal of extracellular bacteria early in infection resulted in significantly reduced IL-8 release in comparison with late removal after 4 h of infection. Moreover, killing of extracellular bacteria with gentamicin further reduced IL-8 release.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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B/RelA-pathway, and, to a lesser extent, the MEK1–ERK1/2 pathway (fig. 6).
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Since chemokine synthesis is important for orchestration of the innate and adaptive immune response, the chemokine pattern released by L. pneumophila-infected alveolar epithelial cells was analysed first. For this purpose, the well-established human alveolar epithelial cell line A549, which Legionella infects efficiently, as shown in several studies 12, 23, was used.
L. pneumophila-infected A549 cells released the chemoattractants IL-8 (for polymorphonuclear neutrophils (PMNs)) and MCP-1 (for monocytes), the T-helper cell (Th) type 1 cytokines IL-2, TNF- and IFN-, and the Th2 cytokines IL-4 and IL-6 within 15 h. In addition, the pro-inflammatory cytokines IL-1ß and IL-17 and the myeloid growth factor G-CSF were secreted (fig. 6
). In accordance with the present findings, Chang et al. 23 described IL-6 and IL-8, as well as TNF-, expression in Legionella-infected A549 cells.
In humans, Legionella infection increases serum levels of, for example, IFN- and IL-6, -12 and -10 25, 26. In experimental studies using murine models or isolated macrophages, mainly cytokines attracting and activating PMNs and moncytes/macrophages have been analysed. In experimental Legionella pneumonia in A/J mice, Tateda et al. 27 found induction of the chemotactic cytokines chemokine (CXC motif) ligand 1 (KC), macrophage inflammatory protein (MIP)-2 and chemokine (CXC motif) ligand 5 (LIX), recruiting PMNs into the lung. Accordingly, attachment of L. pneumophila to cultured mouse peritoneal macrophages increased steady-state levels of cellular mRNAs encoding the cytokines IL-1ß, IL-6 and GM-CSF, and the chemokines MIP-1ß, MIP-2 and KC 28. Since PMN recruitment was seen in Legionella pneumonia in humans 29 and mice 30, and its blockage increased mortality in the A/J mouse model 27, alveolar epithelium may play an important role in orchestrating the immune response against Legionella.
Recruited monocytes were activated by the Th1 cytokines IFN- and TNF-; IFN- promotes Legionella clearance in macrophages 31, and transgenic overexpression of IFN- in A/J mice reduces the bacterial burden 32. Since IFN- was also found to be crucial to immune defence against Listeria monocytogenes in mice 33 and humans 34, an important role of IFN- in host defence against intracellular bacteria, including Legionella, has to be considered. Similarly, TNF- promoted L. pneumophila clearance in human monocytes and proved to be protective in mice 35. As release of both cytokines was reduced by inhibition of p38 MAPK, this pathway might be crucial to an effective immune response in L. pneumophila infection.
L. pneumophila-infected A549 cells did not, however, release Th1 cytokine IL-12 (p70), Th2 cytokines IL-5, -10 or -13, or the lymphoid and myeloid growth factors IL-7 and GM-CSF. Interestingly, high IL-12 (p70) levels accompanied decreased mortality in A/J mice with L. pneumophila infection 27. Expression was also found in human L. pneumophila pneumonia 25, and it could be produced by Legionella-exposed dendritic cells 36, but alveolar epithelium does not seem to be a source of this cytokine. In contrast, IL-10 reversed the Legionella-protective effects of IFN- 37. Taken together, L. pneumophila-infected human alveolar epithelial cells secreted chemotactic CC and CXC chemokines, as well as Th1 and Th2 chemokines (fig. 6). Of these, TNF-, IL-1ß, -6 and -8 and G-CSF were considered as uniform inflammatory reaction factors, e.g. induced by TLR2, whereas IFN-, IL-2, -4 and -17 and MCP-1 seem to be part of a pathogen-specific reaction 38. Thus, Legionella-infected alveolar epithelial cells may potently and specifically contribute to the regulation of the host immune response in legionellosis.
In order to gain more insight into alveolar epithelial cell activation by L. pneumophila, expression of the important chemotatic cytokine IL-8 was analysed in more detail. L. pneumophila serogroup 1 strains 130b, Philadelphia 1, JR32 and Corby induced IL-8 secretion by infected A549 cells. Philadelphia 1-derived strain JR32 induced IL-8 expression to a lesser extent than the other strains in lung epithelial cells, underlining existing differences between these strains. In accordance with the recent findings of Hawn et al. 7, Ren et al. 39 and Molofsky et al. 40, experiments using a Corby flaA knockout mutant strain indicated that recognition of flagellin by TLR5 or other possibly intracellular receptors seems to be essential to the early induction of IL-8 release in alveolar epithelial cells in vitro. Moreover, removal or killing (gentamicin) of extracellular bacteria reduced IL-8 release by A549 cells significantly.
In addition, data obtained using a JR32 dotA knockout mutant indicated that type IVB-secreted effectors do not seem to be essential to the early induction of IL-8 release in alveolar epithelial cells in vitro. Furthermore, this implies that bacterial replication is not necessary for induction of IL-8 release. However, Chang et al. 23 have found that knockout of dotG/icmE in L. pneumophila strain 80-045 reduced cytokine expression at later time points 23. L. pneumophila genomes showed marked plasticity and diversity, as recently demonstrated, for example, for the strains Paris and Lens 5, and showed different expression patterns of pathogenetic factors. The physiological importance of such differences is furthermore highlighted by, for example, the observation that Philadelphia 1-derived strain JR32 induced IL-8 expression to a lesser extent in lung epithelial cells. Thus, it cannot be ruled out that the importance of a particular virulence factor may vary between infections with different L. pneumophila strains. Moreover, different genes within the icm/dot loci were manipulated by mutagenesis. In accordance with Chang et al. 23, the time course of IL-8 mRNA induction by the JR32 strain or dotA knockout mutant did not differ up to 4 h. Overall, it seems reasonable that recognition of extracellular Legionella by TLRs initially contributes to alveolar epithelial activation 6, 7.
Activation of p38 MAPK has been shown to contribute to bacteria-related expression of IL-8 in infected lung epithelial cells 11, and Welsh et al. 41 found that p38 MAPK and Janus kinase were activated early during the uptake of L. pneumophila by macrophages. In lung epithelial cells, p38 MAPK activation 60 min after infection was critical to the release of all induced cytokines, with the exception of IL-2 and G-CSF, but seems not to be necessary for invasion of the cells (data not shown). Thus, cell-specific effects should be considered concerning replication of L. pneumophila. Interestingly, although ERK2 kinase was activated in infected epithelium, ERK kinase activity contributed to a lesser extent to epithelial cell activation with respect to cytokine and chemokine release. Besides p38 MAPK activity, stimulation of IL-8 expression was dependent upon activation of the transcription factor NF-B in L. pneumophila-infected epithelium, as was shown for Streptococcus pneumoniae in lung epithelial cells 11. Further experiments addressing the role of the different pathogenic factors of Legionella, such as lipopolysaccharide, flagella or hydrolysing enzymes, for the activation of these important pro-inflammatory pathways are needed in order to gain more insight into the molecular mechanisms involved.
In summary, it is shown here that Legionella pneumophila infection induced strong chemokine and cytokine release in human alveolar epithelial A549 cells. Expression of these molecules depends predominantly upon activation of the p38 mitogen-activated protein kinase pathway and nuclear factor-B-dependent gene transcription in A549 cells. Overall, activation of alveolar epithelium seems to contribute significantly to the orchestration of the immune response in legionellosis.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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[Order article via Infotrieve]: differential responses of blood monocytes and alveolar macrophages. J Immunol 1996;157:2528–2538.[Abstract]This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 5 µM SB202190 (p38 MAPK inhibitor; 
) or U0126 (MAPK kinase 1 inhibitor; 
) and infected with L. pneumophila 130b (1x107 colony-forming units·mL-1; uninfected control (C; 
)). Cytokine release in the supernatant was measured by Bioplex assay. Data are presented as mean±SEM. **: p<0.01 versus C; ##: p<0.01 versus infected cells without pre-incubation with inhibitors (in at least three independent experiments).
flaA knockout mutant (
