Streptococcus pneumoniae-induced p38 MAPK-dependent Phosphorylation of RelA at the Interleukin-8 Promotor
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m313702200
ISSN1083-351X
AutoresBernd Schmeck, Janine Zahlten, Kerstin Moog, Vincent van Laak, S Huber, Andreas C. Hocke, Bastian Opitz, Elke Hoffmann, Michael Kracht, Jens Zerrahn, Sven Hammerschmidt, Simone Rosseau, Norbert Suttorp, Stefan Hippenstiel,
Tópico(s)Pneumonia and Respiratory Infections
ResumoStreptococcus pneumoniae is the major cause of community-acquired pneumonia and one of the most common causes of death by infectious disease in industrialized countries. Little is known concerning the mechanisms of target cell activation in this disease. The present study shows that NF-κB and p38 MAPK signaling pathways contribute to chemokine synthesis by lung epithelial cells in response to pneumococci. In infected lungs of mice pneumococci stimulate expression of the interleukin (IL)-8 homolog keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor, as well as activate p38 MAPK. Human bronchial epithelium was chosen as a cellular model, because it establishes the first barrier against pathogens, and little is known about its function in innate immunity. Pneumococci infection induces expression of IL-8 and granulocyte-macrophage colony-stimulating factor as well as activation of p38 MAPK in human bronchial epithelial cells (BEAS-2B). Inhibition of p38 MAPK activity by SB202190 and SB203580 blocks pneumococci-induced cytokine release. In mouse lungs in vivo as well as in cultured cells, pneumococci activate NF-κBinanIκB kinase-dependent manner. Inhibition of p38 MAPK by chemical inhibitors or by RNA interference targeting p38α reduces pneumococci-induced NF-κB-dependent gene transcription. Blockade of p38 activity did not affect inducible nuclear translocation and recruitment of NF-κB/RelA to the IL-8 promotor but did reduce the level of phosphorylated RelA (serine 536) at IL-8 promotor and inhibited pneumococci-mediated recruitment of RNA polymerase II to IL-8 promotor. Thus, p38 MAPK contributes to pneumococci-induced chemokine transcription by modulating p65 NF-κB-mediated transactivation. Streptococcus pneumoniae is the major cause of community-acquired pneumonia and one of the most common causes of death by infectious disease in industrialized countries. Little is known concerning the mechanisms of target cell activation in this disease. The present study shows that NF-κB and p38 MAPK signaling pathways contribute to chemokine synthesis by lung epithelial cells in response to pneumococci. In infected lungs of mice pneumococci stimulate expression of the interleukin (IL)-8 homolog keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor, as well as activate p38 MAPK. Human bronchial epithelium was chosen as a cellular model, because it establishes the first barrier against pathogens, and little is known about its function in innate immunity. Pneumococci infection induces expression of IL-8 and granulocyte-macrophage colony-stimulating factor as well as activation of p38 MAPK in human bronchial epithelial cells (BEAS-2B). Inhibition of p38 MAPK activity by SB202190 and SB203580 blocks pneumococci-induced cytokine release. In mouse lungs in vivo as well as in cultured cells, pneumococci activate NF-κBinanIκB kinase-dependent manner. Inhibition of p38 MAPK by chemical inhibitors or by RNA interference targeting p38α reduces pneumococci-induced NF-κB-dependent gene transcription. Blockade of p38 activity did not affect inducible nuclear translocation and recruitment of NF-κB/RelA to the IL-8 promotor but did reduce the level of phosphorylated RelA (serine 536) at IL-8 promotor and inhibited pneumococci-mediated recruitment of RNA polymerase II to IL-8 promotor. Thus, p38 MAPK contributes to pneumococci-induced chemokine transcription by modulating p65 NF-κB-mediated transactivation. Pneumonia is the most common infectious disease leading to death in industrialized countries (1Garibaldi R.A. Am. J. Med. 1985; 78: 32-37Abstract Full Text PDF PubMed Scopus (377) Google Scholar). Over 40% of cases are due to infections with Streptococcus pneumoniae, and high mortality has been reported (2Finch R. Clin. Microbiol. Infect. 2001; 7: 30-38Abstract Full Text Full Text PDF PubMed Google Scholar). Simultaneously, antibiotic-resistant strains have emerged (3Heffelfinger J.D. Dowell S.F. Jorgensen J.H. Klugman K.P. Mabry L.R. Musher D.M. Plouffe J.F. Rakowsky A. Schuchat A. Whitney C.G. Arch. Intern. Med. 2000; 160: 1399-1408Crossref PubMed Scopus (605) Google Scholar). Considering the medical relevance of these perilous bacteria, an increase in the knowledge and insights into the pathophysiological mechanisms of pneumococci-host interaction appears expedient. Although some pathogenic factors have been identified (4Tuomanen E.I. Austrian R. Masure H.R. N. Engl. J. Med. 1995; 332: 1280-1284Crossref PubMed Scopus (319) Google Scholar) and new strategies for pneumococcal vaccination (5Austrian R. J Infect. Dis. 1999; 179: S338-S341Crossref PubMed Scopus (57) Google Scholar) are under investigation, little is known about the activation of signaling cascades in target cells. In pneumococcal pneumonia massive leukocyte recruitment to the lung is observed (6Wang E. Ouellet N. Simard M. Fillion I. Bergeron Y. Beauchamp D. Bergeron M.G. Infect. Immun. 2001; 69: 5294-5304Crossref PubMed Scopus (37) Google Scholar). Liberation of pro-inflammatory and chemotactic cytokines by lung epithelium and cells of innate immune response such as the alveolar macrophages contributed significantly to leukocyte invasion (7Strieter R.M. Belperio J.A. Keane M.P. Curr. Opin. Infect. Dis. 2003; 16: 193-198Crossref PubMed Scopus (105) Google Scholar). IL-8/CXCL8 1The abbreviations used are: IL, interleukin; IκBα, inhibitor of κBα; IKK, IκBα kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor; NF-κB, nuclear factor-κB; MAPK, mitogen-activated protein kinase; TLR2, toll-like receptor 2; TNF, tumor necrosis factor; siRNA, small interference RNA; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline; cfu, colony-forming unit; KC, keratinocyte-derived chemokine. is an important chemotactic cytokine and a key mediator in neutrophil-mediated inflammation in the lung (8Strieter R.M. Am. J. Physiol. 2002; 283: L688-L689Crossref PubMed Scopus (48) Google Scholar). In addition, GM-CSF is produced in inflammation by tissue cells, including lung epithelium (9Chung K.F. Barnes P.J. Thorax. 1999; 54: 825-857Crossref PubMed Scopus (514) Google Scholar). GM-CSF participates in the inflammatory response by direct activation of leukocytes at the local site of infection as well as by activation of immature precursor cells (9Chung K.F. Barnes P.J. Thorax. 1999; 54: 825-857Crossref PubMed Scopus (514) Google Scholar). Both cytokines are subjected to a tight regulatory network involving the transcription factors activator protein-1 and NF-κB (10Zhou L. Tan A. Iasvovskaia S. Li J. Lin A. Hershenson M.B. Am. J. Respir. Cell Mol. Biol. 2003; 28: 762-769Crossref PubMed Scopus (58) Google Scholar). Under certain conditions, these transcription factors may be activated by a complex kinase pathway centered around p38 MAPK (11Koch A. Giembycz M. Ito K. Lim S. Jazrawi E. Barnes P.J. Adcock I. Erdmann E. Chung K.F. Am. J. Respir. Cell Mol. Biol. 2003; 30: 342-349Crossref PubMed Scopus (48) Google Scholar). Recently, four isoforms of p38 MAPK (α, β, γ, and δ) with either activating or inhibiting activity on gene transcription have been characterized (12Pramanik R. Qi X. Borowicz S. Choubey D. Schultz R.M. Han J. Chen G. J. Biol. Chem. 2003; 278: 4831-4839Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). However, their distinct roles in infectious disease pathogenesis remain to be clarified. Increasing evidence pointed to a critical role of transcription factor modification for the regulation of gene transcription. p38 MAPK-stimulated pathways may phosphorylate p65/RelA, thereby regulating NF-κB-dependent gene transcription (14Vermeulen L. De Wilde G. Van Damme P. Vanden Berghe W. Haegeman G. EMBO J. 2003; 22: 1313-1324Crossref PubMed Scopus (644) Google Scholar, 15Rahman A. Anwar K.N. Minhajuddin M. Bijli K.M. Javaid K. True A.L. Malik A.B. Am. J. Physiol. 2004; 287: 1017-1024Crossref PubMed Scopus (59) Google Scholar). However, there are no reports addressing the role of these important regulation pathways during infection by intact bacteria. In this study we tested the hypothesis that pneumococci directly activate gene expression of lung epithelial cells by combined stimulation of p38 MAPK and of NF-κB. We report here that transcriptome analysis of isolated lungs obtained from a mouse model of pneumococcal pneumonia by DNA microarray revealed elevated mRNA levels of two cytokines KC, a functional murine IL-8 homolog (16Nick J.A. Young S.K. Arndt P.G. Lieber J.G. Suratt B.T. Poch K.R. Avdi N.J. Malcolm K.C. Taube C. Henson P.M. Worthen G.S. J. Immunol. 2002; 169: 5260-5269Crossref PubMed Scopus (102) Google Scholar), and GM-CSF. In addition, activation of p38 MAPK and of NF-κB pathway was noted in infected lungs. Pneumococci also induced IL-8 and GM-CSF gene expression in human bronchial epithelial cells in vitro. Blocking of both the p38 MAPK signaling pathway and the canonical NF-κB pathway reduced pneumococci-dependent cytokine secretion. Subsequent studies demonstrated that pneumococci activated NF-κB in a p38 MAPK-independent manner, whereby p38 apparently regulates IL-8 transcription at the promotor level. Actually, both p38 MAPK and NF-κB are required to modulate the host response at the transcriptional level in response to pneumococci infection. Materials—SB202190, SB203580, and SB202474 were purchased from Calbiochem (Merck), TNFα from R & D Systems (Wiesbaden, Germany), IKK-NBD from Biomol (Plymouth Meeting, PA), and Pam 3-Cys from Invitrogen. Peptidoglycan was a kind gift from R. R. Schumann (Berlin, Germany). All other chemicals used were of analytical grade and obtained from commercial sources. Cell Lines—Human bronchial epithelial BEAS-2B cells were a kindly gift of C. Harris (National Institutes of Health, Bethesda, MD) (17Reddel R.R. Ke Y. Gerwin B.I. McMenamin M.G. Lechner J.F. Su R.T. Brash D.E. Park J.B. Rhim J.S. Harris C.C. Cancer Res. 1988; 48: 1904-1909PubMed Google Scholar). Human embryonic kidney cells (HEK293) were purchased from ATCC (Manassas, VA). Bacterial Strains—Pneumonia in mice was induced by the encapsulated S. pneumoniae type 3 (NCTC 7978). S. pneumoniae R6x is the unencapsulated derivative of type 2 strain D39 (18Tiraby J.G. Fox M.S. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 3541-3545Crossref PubMed Scopus (126) Google Scholar). A pneumolysin-negative mutant of R6x (R6xply–) was generated by insertion-duplication mutagenesis using the pJDC9::ply construct (19Zysk G. Schneider-Wald B.K. Hwang J.H. Bejo L. Kim K.S. Mitchell T.J. Hakenbeck R. Heinz H.P. Infect. Immun. 2001; 69: 845-852Crossref PubMed Scopus (125) Google Scholar). After transformation of the plasmid into S. pneumoniae strain R6x, erythromycin-resistant transformants were selected. Insertion of the plasmid into pneumolysin encoding gene ply was confirmed by PCR analysis and DNA sequencing. Loss of function was analyzed using the hemolysis assay as described previously (20Benton K.A. Paton J.C. Briles D.E. Microb. Pathog. 1997; 23: 201-209Crossref PubMed Scopus (45) Google Scholar). Single colony isolates of R6x and its corresponding mutant R6xply– were maintained at 37 °C with 5% CO2 on Columbia agar with 5% sheep blood. For cell culture stimulation studies, single colonies were expanded by resuspension in Todd-Hewitt broth supplemented with 0.5% yeast extract and incubation at 37 °C for 3–4 h to midlog phase (A600 0.2–0.4), harvested by centrifugation, and resuspended in cell culture medium without antibiotics. Plasmids, RNA Interference, and Transient Transfection Procedures—HEK293 cells were cultured in 12-well plates with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. 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 (21Krull M. Klucken A.C. Wuppermann F.N. Fuhrmann O. Magerl C. Seybold J. Hippenstiel S. Hegemann J.H. Jantos C.A. Suttorp N. J. Immunol. 1999; 162: 4834-4841PubMed Google Scholar), 0.2 μg of Rous sarcoma virus β-galactosidase plasmid, 0.1 μg of hTLR2 (generously provided by Tularik Inc., San Francisco, CA (22Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (506) Google Scholar)) expression vectors, or control vector, respectively. The cells were incubated with R6xply– for 6 h, because pneumolysin is cytotoxic for Hek293 cells. The cells were lysed, and luciferase activity was measured by using a luciferase reporter gene assay (Promega, Mannheim, Germany), and the results were normalized for transfection efficiency with values obtained by Rous sarcoma virus β-galactosidase (Roche Applied Science). For RNA interference experiments, the cells were transfected with p38α-specific Smartpool® or nonsense siRNA (both from Upstate Biotechnology, Inc., Lake Placid, NY) using RNAiFect® (Qiagen) following the manufacturer's instructions for 96 h and then processed as described above. IL-8 and GM-CSF ELISA—Confluent BEAS-2B cells were stimulated for 15 h as indicated in a humidified atmosphere. After the incubation, the supernatants were collected and processed for IL-8 or GM-CSF quantification by sandwich ELISA as described previously (23Hippenstiel S. Soeth S. Kellas B. Fuhrmann O. Seybold J. Krull M. Eichel-Streiber C. Goebeler M. Ludwig S. Suttorp N. Blood. 2000; 95: 3044-3051Crossref PubMed Google Scholar). Western Blot—For determination of p38α expression or p38 MAPK phosphorylation in BEAS-2B cells, the cells were stimulated as indicated, washed twice, and harvested. For analysis of p38 phosphorylation pneumococci-infected mice, lungs were snap-frozen in fluid nitrogen and pulverized. BEAS-2B cells or lung homogenates were lysed in buffer containing Triton X-100, subjected to SDS-PAGE, and blotted on Hybond-ECL membrane (Amersham Biosciences). Immunodetection of p38α or phosphorylated p38 MAPK was carried out with p38α-specific antibody (Upstate Biotechnology, Inc.) or phospho-specific p38 MAPK antibody (Cell Signaling, Frankfurt, Germany) respectively (23Hippenstiel S. Soeth S. Kellas B. Fuhrmann O. Seybold J. Krull M. Eichel-Streiber C. Goebeler M. Ludwig S. Suttorp N. Blood. 2000; 95: 3044-3051Crossref PubMed Google Scholar). Degradation of IκBα was analyzed in BEAS-2B cell lysates or lung homogenates using a rabbit polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) as described previously (24Hippenstiel S. Schmeck B. Seybold J. Krull M. Eichel-Streiber C. Suttorp N. Biochem. Pharmacol. 2002; 64: 971-977Crossref PubMed Scopus (46) Google Scholar). In all of the experiments, ERK2 kinase (Santa Cruz Biotechnologies) was detected simultaneously to confirm equal protein load. The proteins were visualized by incubation with secondary IRDye 800- or Cy5.5-labeled antibodies, respectively (Odyssey infrared imaging system, LI-COR Inc.) (24Hippenstiel S. Schmeck B. Seybold J. Krull M. Eichel-Streiber C. Suttorp N. Biochem. Pharmacol. 2002; 64: 971-977Crossref PubMed Scopus (46) Google Scholar, 25Hippenstiel S. Witzenrath M. Schmeck B. Hocke A. Krisp M. Krull M. Seybold J. Seeger W. Rascher W. Schutte H. Suttorp N. Circ. Res. 2002; 91: 618-625Crossref PubMed Scopus (150) Google Scholar). RT-PCR—For analysis of IL-8, GM-CSF, and GAPDH gene expression in BEAS-2B cells, total RNA was isolated with RNEasy Mini kit (Qiagen) and reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega). Generated cDNA was amplified by PCR using specific intron-spanning specific primers for IL-8, GM-CSF, and GAPDH. In isolated mice lungs, KC, GM-CSF, and GAPDH were analyzed after isolation of total cellular RNA. Frozen lung tissue was crushed using a sterile, nitrogen-cooled plate homogenizer. Total RNA was extracted using a RNEasy mini isolation kit. All of the primers were purchased from TIB MOLBIOL (Berlin, Germany). After 35 amplification cycles, the 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 of the samples were checked for GAPDH mRNA expression. Electrophoretic Mobility Shift Assay (EMSA)—After stimulation of BEAS-2B cells, nuclear protein was isolated and analyzed by EMSA as described previously (21Krull M. Klucken A.C. Wuppermann F.N. Fuhrmann O. Magerl C. Seybold J. Hippenstiel S. Hegemann J.H. Jantos C.A. Suttorp N. J. Immunol. 1999; 162: 4834-4841PubMed Google Scholar, 24Hippenstiel S. Schmeck B. Seybold J. Krull M. Eichel-Streiber C. Suttorp N. Biochem. Pharmacol. 2002; 64: 971-977Crossref PubMed Scopus (46) Google Scholar, 26Schmeck B. Brunsch M. Seybold J. Krull M. Eichel-Streiber C. Suttorp N. Hippenstiel S. Inflammation. 2003; 27: 89-95Crossref PubMed Scopus (30) Google Scholar). IRDye800-labeled consensus NF-κB oligonucleotides were purchased from Metabion (Planegg-Martinsried, Germany). Briefly, EMSA binding reactions were performed by incubating 2 μg of nuclear extract with the annealed oligonucleotides according to the manufacturer's instructions. The reaction mixture was subjected to electrophoresis on a 5% native gel and analyzed by an Odyssey infrared imaging system (LI-COR Inc.). NF-κB Transcription Factor Assay Kit (TransAM™)—The NF-κB TransAM™ assay (Active Motif, Carlsbad, CA) was used to detect DNA binding of p65/RelA containing NF-κB dimers according to the manufacturer's instructions. Briefly, BEAS-2B cells were stimulated, and 10 μg of nuclear cell extract (containing activated transcription factor) were given in oligonucleotide-coated wells. After 20 min of incubation at room temperature with mild agitation, the plate was washed, and 100 μl/well of the diluted p65/RelA antibody (1:1000) was incubated for 1 h. The plate was washed three times and 100 μl of horseradish peroxidase-conjugated antibody (1:1000) was added for 1 h. Developing solution was incubated for 10 min. The reaction was stopped, and the absorbance was read at 450 nm. Chromatin Immunoprecipitation—BEAS-2B cells were stimulated, culture medium was removed, and 1% formaldehyde was added. After 1 min, the cells were washed in ice-cold 0.125 m glycin in PBS, then rapidly collected in ice-cold PBS, centrifuged, and washed twice with ice-cold PBS. The cells were lysed in radioimmune precipitation assay buffer (10 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 1% desoxycholic acid, 0.1% SDS, 1 mm EDTA, 1% aprotinin), and the chromatin was sheared by sonication. The lysates were cleared by centrifugation, and the supernatants were stored in aliquots at –80 °C until further use. Antibodies were purchased from Cell Signaling (Beverly, MA) (p65-Pser536) and Santa Cruz Biotechnology (p65/RelA and polymerase II). Immunoprecipitations from soluble chromatin were carried out overnight at 4 °C. Immune complexes were collected with protein A/G-agarose for 60 min and washed twice with radioimmune precipitation assay buffer and once with high salt buffer (2 m NaCl, 10 mm Tris, pH 7.5, 1% Nonidet P-40, 0.5% desoxycholic acid, 1 mm EDTA) followed by another wash in radioimmune precipitation assay buffer and one wash with TE buffer (10 mm Tris, pH 7.5, 1 mm EDTA). Immune complexes were extracted in elution buffer (1 TE buffer containing 1% SDS) by shaking the lysates for 15 min at 1200 rpm, 30 °C. They were then digested with RNase (1 μg/20 μl) for 30 min at 37 °C. After proteinase K digestion (1 μg/8 μl for 6 h at 37 °C and 6 h at 65 °C) DNA was extracted using a PCR purification kit (Qiagen). IL-8 promotor DNA was amplified by PCR using Hotstart Taq (Qiagen) polymerase. The PCR conditions were 95 °C for 15 min and 33–35 cycles of 94 °C for 20 s, 60 °C for 20 s, 72 °C for 20 s. PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining of gels. Equal amounts of input DNA was controlled by gel electrophoresis. The following promotor-specific primers for IL-8 were used: sense, 5′-AAG AAA ACT TTC GTC ATA CTC CG-3′; antisense, 5′-TGG CTT TTT ATA TCA TCA CCC TAC-3′. Mouse Pneumonia Model—Pathogen-free, female C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany). The animals were kept with a 12-h light/dark cycle and were given free access to food and water. The study was approved by the Institutional Review Board for the care of animal subjects. The mice were challenged at 10 weeks of age and 19–20 grams of weight. Pneumonia was induced by use of encapsulated S. pneumoniae type 3 (NCTC 7978). The bacteria were grown as described above and resuspended in sterile PBS at ∼2.5 × 108 cfu/ml. The mice were lightly anesthetized by intraperitoneal injection of ketamine and xylazine and inoculated intranasally with 20 μl of bacterial suspension (5 × 106 cfu). The control mice were challenged with 20 μl of sterile PBS. Groups of three mice were sacrificed 6, 12, 24, or 48 h postinfection, and the lungs were removed aseptically and immediately frozen in liquid nitrogen. The control mice were sacrificed 6 h postinfection (n = 3). Microarray Transcriptome Analysis—Microarray experiments were done as two-color hybridizations. Total RNA was extracted from whole lungs of at least three C57BL/6 mice at 12, 24, and 48 h after infection and from at least three noninfected C57BL/6 control mice by lysis in 1 ml of TRIzol (Invitrogen) per lung using an Ultraturrax T8 (IKA) homogenisator. Homogenates from each group were pooled, shock-frozen, and stored at –70 °C. An amount of 4 μg of total RNA was reverse transcribed with an oligo(dT)-T7-promotor primer by a fluorescent linear amplification reaction (Agilent Technologies, Palo Alto, CA), and cDNA was labeled either with cyanine 3-CTP or cyanine 5-CTP (New England Biolabs Life Science Products, Beverly, MA). To compensate specific effects of dyes, e.g. incorporation, and to ensure statistically relevant data analysis, a color swap was performed. The RNA samples were labeled vice versa with the two fluorescent dyes (fluorescence reversal). After precipitation, purification, and quantification, 1.25 μg of each labeled cRNA was mixed, fragmented, and hybridized to the 8.4 K custom in situ mouse array according to the supplier's protocol (Agilent Technologies). Scanning of microarrays was performed with 5-μm resolution using a DNA microarray laser scanner (Agilent Technologies). The features were extracted with an image analysis tool (version A4.045) from Agilent Technologies using default settings. The data analysis was carried out on the Rosetta Inpharmatics platform Resolver Built 3.0.0.3.22. Statistical Methods—A one-way analysis of variance was used for data of Figs. 1 (B and D), 3(A, C, and D), 5, 6 (A and B), and 7A, and the data are shown as the means ± S.E. for one representative experiment of three. The main effects were then compared by an F probability test. p < 0.05 was considered to be significant and indicated by asterisks (if not indicated otherwise, the test was performed versus control).Fig. 3p38 MAPK inhibition blocked pneumococci-induced IL-8 and GM-CSF expression. BEAS-2B cells were pretreated with indicated doses of specific p38 inhibitor SB202190, SB203580, or non-active control compound SB202474 for 20 min and then infected with R6x or stimulated with TNFα/IL-1β for 15 h, and release of IL-8 (A) and GM-CSF (C) was assessed by ELISA. B, BEAS-2B were pretreated with 10 μm SB202190 for 20 min and then infected with 107 cfu/ml R6x for 3 h, and IL-8 mRNA was detected by RT-PCR. One representative gel of three is shown. D, BEAS-2B cells were pretreated with indicated doses of specific p38 inhibitor SB202190, SB203580, or nonactive control compound SB202474 for 20 min and then stimulated with phorbol 12-myristate 13-acetate (0.5 μg/ml) for 15 h, and release of IL-8 was measured by ELISA. S. p., S. pneumoniae.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Dose-dependent induction of NF-κB transcriptional activity by pneumococci. HEK293 cells were transfected with hTLR2, a NF-κB-dependent luciferase reporter plasmid, and a β-galactosidase construct and stimulated with the indicated doses of R6xply– or peptidoglycan (PGN), Pam-3-Cys (P-3-C), or TNFα. After 6 h the cells were harvested, and luciferase and β-galactosidase activity were determined and normalized. S. p., S. pneumoniae.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Pneumococci-induced pro-inflammatory cell activation was IKK-dependent. BEAS-2B cells were pretreated with indicated doses of specific IKK inhibitor IKK-NBD for 20 min and infected with R6x or stimulated with TNFα/IL-1β (T/I) for 15 h, and release of IL-8 (A) and GM-CSF (B) was assessed by ELISA. S. p., S. pneumoniae.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Inhibition of p38 MAPK reduced pneumococci-induced NF-κB activation. HEK293 cells were co-transfected with hTLR2, a NF-κB-dependent luciferase reporter plasmid, and a β-galactosidase construct. Cells pretreated with p38α-specific or nonsense (ns) siRNA or SB202190 (SB) were stimulated for 6 h with pneumococci as indicated, and luciferase and β-Gal activity were determined and normalized (A). # indicates significance (p < 0.05) versus infected cells without siRNA or inhibitor. B, HEK293 cells were transfected with p38α-specific or non-sense siRNA, and reduction of p38α MAPK expression was verified by Western blot. S. p., S. pneumoniae.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pneumococci-induced Expression of Pro-inflammatory Cytokines KC/IL-8 and GM-CSF in Mice Lungs and Human Bronchial Epithelial Cells in Vitro—C57BL/6 mice were infected with S. pneumoniae NCTC 7978 by intranasal infection, thereby inducing pneumonia, characterized by massive leukocyte influx. In transcriptome analysis of infected lungs, we found a time-dependent increase in mRNA expression of the pro-inflammatory chemokines KC (12 h, 13.79-fold; 24 h, 10.39-fold; and 48 h, 10.72-fold) and GM-CSF (12 h, 2.13-fold; 24 h, 1.35-fold; and 48 h, 1.16-fold) with p values smaller than 0.01. Using RT-PCR we verified increased mRNA expression of KC and GM-CSF in pneumococcal-infected lungs and noted strongly elevated KC and GM-CSF mRNA levels 6 and 12h post infection (Fig. 1A). These data cannot be assigned to one single cell type. We have picked human bronchial epithelium as an interesting cellular model, because it establishes the first barrier against pathogens, including pneumococci entering the lung by the airways. Moreover, little is known about its function in innate immunity. Pneumococci-stimulated expression of these pro-inflammatory cytokines by tracheobronchial epithelium may contribute significantly to the prominent leukocyte recruitment observed in pneumococci-pneumonia. S. pneumoniae NCTC 7978 revealed massive cytotoxic effects in vitro (data not shown). Therefore, we stimulated human bronchial epithelial BEAS-2B cells with S. pneumoniae R6x and analyzed IL-8 (the functional human KC homolog (16Nick J.A. Young S.K. Arndt P.G. Lieber J.G. Suratt B.T. Poch K.R. Avdi N.J. Malcolm K.C. Taube C. Henson P.M. Worthen G.S. J. Immunol. 2002; 169: 5260-5269Crossref PubMed Scopus (102) Google Scholar)) and GM-CSF expression. Pneumococci infection of BEAS-2B cells dose-dependently induced the release of IL-8 (Fig. 1B) and GM-CSF (Fig. 1D) by these cells. Onset of IL-8 mRNA expression was noted as early as 2 h post-infection (Fig. 1C). Pneumococcal infection was at least as effective as cell stimulation of BEAS-2B cells by TNFα/IL-1β. Inhibition of p38 MAPK Blocked Pneumococci-induced Expression of IL-8 and GM-CSF in Human Bronchial Epithelial Cells—Activation of p38 MAPK is considered to participate in the regulation of pro-inflammatory cytokine expression. We noted phosphorylation of p38 MAPK in mice lungs 6–48 h post infection with pneumococci, indicating kinase activation (Fig. 2A). Moreover, exposure of BEAS-2B cells to 106 cfu/ml of S. pneumoniae for 2 h strongly induced p38 MAPK phosphorylation in vitro (Fig. 2B). Phosphorylation of p38 MAPK by pneumococcal infection of epithelial cells was detected after 1 h of cell infection and increased up to 3 h (Fig. 2C). To test the role of p38 MAPK activation for IL-8 and GM-CSF expression, we made use of specific chemical inhibitors SB202190 and SB203580 and its nonfunctional homolog SB202474. Inhibition of p38 MAPK in BEAS-2B cells by pre-incubation of cells with SB202190 or SB202580 20 min before pneumococcal infection dose-dependently blocked release of IL-8 (Fig. 3A) and significantly decreased liberation of GM-CSF, whereas the nonfunctional homolog SB202474 or solvent alone had no effect (Fig. 3, A and C). All three compounds or their solvents had no effect on spontaneous or phorbol 12-myristate 13-acetate-stimulated IL-8 release in BEAS-2B cells (Fig. 3D). Moreover, inhibition of p38 MAPK reduced expression of IL-8 mRNA by BEAS-2B cells after 3 h of incubation with 107 cfu/ml S. pneumoniae (Fig. 3B). Therefore, pneumococci-dependent p38
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