Subscribe to RSS
DOI: 10.1160/TH04-12-0822
Streptococcus pneumoniae R6x induced p38 MAPK JNK-mediated Caspase-dependent apoptosis in human endothelial cells
Grant support: This work was supported in part by tBundesministerium he für Bildung und Forschung to B. Schmeck (BMBF-CAPNETZ C15), S. Hippenstiel (BMBF-NBL-III 3.3.1.A, BMBFCAPNETZ C15), S. Hammerschmidt (BMBF-CAPNETZ C8), N. Suttorp and S. Rosseau (BMBF-CAPNETZ C4) and Deutsche Forschungsgemeinschaft to N. Suttorp (DFGSPP1130).Publication History
Received: 20 December 2004
Accepted after major revision: 13 April 2005
Publication Date:
05 December 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/200502/lookinside/thumbnails/10-1160-th04-12-0822-1.jpg)
Summary
Streptococcus pneumoniae is the major pathogen of communipnetyacquired umonia and a common cause of otitis, meningitis and sepsis. During pneumococci infection accompanied with bacterial invasion and hematogenous spreading, the endothelium is directly targeted by pneumococci and their virulence factors. Therefore, we tested the hypothesis that pneumococci induced endothelial apoptosis. Unencapsulated R6x pneumococci strongly induced apoptosis of human endothelial cells both from lung microvasculature and umbilical vein, whereas an encapsulated strain D39 mainly led to necrotic cell death. Deletion of the gene coding for pneumolysin reduced pneumococci-induced apoptosis in HUVEC. Furthermore, N-acetyl-L-cysteine, an antioxidant thiol, significantly reduced apoptosis caused by R6x, and LDH release induced by D39, pointing to a role for reactive oxygen species in the pathogenesis. Apoptotic cells showed increased cleavage and activity of caspases 6 and 9 but only late activation of caspase 3. Programmed cell death could be strongly reduced by pan-caspase inhibitor zVAD. Reduced levels of Bcl2 and cytosolic increase of apoptosis-inducing factor in pneumococci-infected cells implicated involvement of mitochondrial death pathways. Caspase activation and apoptosis were abolished by cAMP elevation. Moreover, p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase were activated in pneumococci-infected cells and inhibitors of both kinases strongly reduced pneumococci-induced caspase activation and apoptosis. Hence,kinase- and caspase-dependence of pneumococci-induced endothelial apoptosis may bear relevance to novel therapeutic approaches to pneumococci-related disease.
* These authors contributed equally to the manuscript.
-
References
- 1 Musher DM. Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clin Infect Dis 1992; 14: 801-7.
- 2 Braun JS, Sublett JE, Freyer D. et al. Pneumococcal pneumolysin and H(2)O(2) mediate brain cell apoptosis during meningitis. J Clin Invest 2002; 109: 19-27.
- 3 Hippenstiel S, Suttorp N. Interaction of pathogens with the endothelium. Thromb Haemost 2003; 89: 18-24.
- 4 Braun JS, Novak R, Murray PJ. et al. Apoptosis-inducing factor mediates microglial and neuronal apoptosis caused by pneumococcus. J Infect Dis 2001; 184: 1300-9.
- 5 Zysk G, Bejo L, Schneider-Wald BK. et al. Induction of necrosis and apoptosis of neutrophil granulocytes by Streptococcus pneumoniae. Clin Exp Immunol 2000; 122: 61-6.
- 6 Schmeck B, Gross R, N’Guessan PD. et al. Streptococcus pneumoniae-induced caspase 6-dependent apoptosis in lung epithelium. Infect Immun 2004; 72: 4940-7.
- 7 Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407: 770-6.
- 8 Desagher S, Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol 2000; 10: 369-77.
- 9 Su B, Karin M. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol 1996; 8: 402-11.
- 10 Aleman M, Schierloh P, dela Barrera SS. et al. Mycobacterium tuberculosis triggers apoptosis in periph eral neutrophils involving toll-like receptor 2 and p38 mitogen protein kinase in tuberculosis patients. Infect Immun 2004; 72: 5150-8.
- 11 Esen M, Schreiner B, Jendrossek V. et al. Mechanisms of Staphylococcus aureus induced apoptosis of human endothelial cells. Apoptosis 2001; 6: 431-9.
- 12 Krump-Konvalinkova V, Bittinger F, Unger RE. et al. Generation of human pulmonary microvascular endothelial cell lines. Lab Invest 2001; 81: 1717-27.
- 13 Unger RE, Krump-Konvalinkova V, Peters K. et al. In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines, including the novel cell line HPMEC-ST1.6R. Microvasc Res 2002; 64: 384-397.
- 14 Hippenstiel S, Witzenrath M, Schmeck B. et al. Adrenomedullin reduces endothelial hyperpermeability. Circ Res 2002; 91: 618-25.
- 15 Schmeck B, Brunsch M, Seybold J. et al. Rho protein inhibition blocks cyclooxygenase-2 expression by proinflammatory mediators in endothelial cells. Inflammation 2003; 27: 89-95.
- 16 Hippenstiel S, Soeth S, Kellas B. et al. Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells. Blood 2000; 95: 3044-51.
- 17 Hippenstiel S, Schmeck B, N’Guessan PD. et al. Rho protein inactivation induced apoptosis of cultured human endothelial cells. Am J Physiol Lung Cell Mol Physiol 2002; 283: L830-L838.
- 18 Tiraby JG, Fox MS. Marker discrimination in transformation and mutation of pneumococcus. Proc Natl Acad Sci U S A 1973; 70: 3541-5.
- 19 Zysk G, Schneider-Wald BK, Hwang JH. et al. Pneumolysin is the main inducer of cytotoxicity to brain microvascular endothelial cells caused by Streptococcus pneumoniae. Infect Immun 2001; 69: 845-52.
- 20 Benton KA, Paton JC, Briles DE. The hemolytic and complement-activating properties of pneumolysin do not contribute individually to virulence in a pneumococcal bacteremia model. Microb Pathog 1997; 23: 201-9.
- 21 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
- 22 Opitz B, Puschel A, Schmeck B. et al. Nucleotidebinding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae. J Biol Chem 2004; 279: 36426-32.
- 23 Schmeck B, Zahlten J, Moog K. et al. Streptococcus pneumoniae-induced p38 MAPK-dependent phosphorylation of RelA at the interleukin-8 promotor. J Biol Chem 2004; 279: 53241-7.
- 24 Sedger LM, Shows DM, Blanton RA. et al. IFNgamma mediates a novel antiviral activity through dynamic modulation of TRAIL and TRAIL receptor expression. J Immunol 1999; 163: 920-6.
- 25 Duane PG, Rubins JB, Weisel HR. et al. Identification of hydrogen peroxide as a Streptococcus pneumoniae toxin for rat alveolar epithelial cells. Infect Immun 1993; 61: 4392-7.
- 26 Hoskins J, Alborn WE, Jr. Arnold J. et al. Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 2001; 183: 5709-17.
- 27 Suttorp N, Weber U, Welsch T. et al. Role of phosphodiesterases in the regulation of endothelial permeability in vitro. J Clin Invest 1993; 91: 1421-8.
- 28 Wada T, Penninger JM. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 2004; 23: 2838-49.
- 29 Chen M, Bao W, Aizman R. et al. Activation of extracellular signal-regulated kinase mediates apoptosis induced by uropathogenic Escherichia coli toxins via nitric oxide synthase: protective role of heme oxygenase-1. J Infect Dis 2004; 190: 127-35.
- 30 Menzies BE, Kourteva I. Internalization of Staphylococcus aureus by endothelial cells induces apoptosis. Infect Immun 1998; 66: 5994-8.
- 31 Geelen S, Bhattacharyya C, Tuomanen E. The cell wall mediates pneumococcal attachment to and cytopathology in human endothelial cells. Infect Immun 1993; 61: 1538-43.
- 32 Rubins JB, Duane PG, Charboneau D. et al. Toxicity of pneumolysin to pulmonary endothelial cells in vitro. Infect Immun 1992; 60: 1740-6.
- 33 Malley R, Henneke P, Morse SC. et al. Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection. Proc Natl Acad Sci U S A 2003; 100: 1966-71.
- 34 Martens P, Worm SW, Lundgren B. et al. Serotypespecific mortality from invasive Streptococcus pneumoniae disease revisited. BMC Infect Dis 2004; 4: 21.
- 35 Tauber MG, Burroughs M, Niemoller UM. et al. Differences of pathophysiology in experimental meningitis caused by three strains of Streptococcus pneumoniae. J Infect Dis 1991; 163: 806-11.
- 36 Engelhard D, Pomeranz S, Gallily R. et al. Serotyperelated differences in inflammatory response to Streptococcus pneumoniae in experimental meningitis. J Infect Dis 1997; 175: 979-82.
- 37 Ali F, Lee ME, Iannelli F. et al. Streptococcus pneumoniae- associated human macrophage apoptosis after bacterial internalization via complement and Fcgamma receptors correlates with intracellular bacterial load. J Infect Dis 2003; 188: 1119-31.
- 38 Elm C, Braathen R, Bergmann S. et al. Ectodomains 3 and 4 of human polymeric Immunoglobulin receptor (hpIgR) mediate invasion of Streptococcus pneu moniae into the epithelium. J Biol Chem 2004; 279: 6296-304.
- 39 Novak R, Charpentier E, Braun JS. et al. Signal transduction by a death signal peptide: uncovering the mechanism of bacterial killing by penicillin. Mol Cell 2000; 5: 49-57.
- 40 Ring A, Weiser JN, Tuomanen EI. Pneumococcal trafficking across the blood-brain barrier. Molecular analysis of a novel bidirectional pathway. J Clin Invest 1998; 102: 347-60.
- 41 Zhang JR, Mostov KE, Lamm ME. et al. The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells. Cell 2000; 102: 827-37.
- 42 Li JM, Shah AM. Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology. Am J Physiol Regul Integr Comp Physiol 2004; 287: R1014-R1030.
- 43 von Mering M, Wellmer A, Michel U. et al. Transcriptional regulation of caspases in experimental pneumococcal meningitis. Brain Pathol 2001; 11: 282-95.
- 44 Rouaux C, Jokic N, Mbebi C. et al. Critical loss of CBP/p300 histone acetylase activity by caspase-6 during neurodegeneration. EMBO J 2003; 22: 6537-49.
- 45 Neuzil J, Schroder A, von Hundelshausen P. et al. Inhibition of inflammatory endothelial responses by a pathway involving caspase activation and p65 cleavage. Biochemistry 2001; 40: 4686-92.
- 46 Ruchaud S, Korfali N, Villa P. et al. Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation. EMBO J 2002; 21: 1967-77.
- 47 Cannon CL, Kowalski MP, Stopak KS. et al. Pseudomonas aeruginosa-induced apoptosis is defective in respiratory epithelial cells expressing mutant cystic fibrosis transmembrane conductance regulator. Am J Respir Cell Mol Biol 2003; 29: 188-97.
- 48 Potthoff A, Ledig S, Martin J. et al. Significance of the caspase family in Helicobacter pylori induced gastric epithelial apoptosis. Helicobacter 2002; 7: 367-77.
- 49 Allsopp TE, McLuckie J, Kerr LE. et al. Caspase 6 activity initiates caspase 3 activation in cerebellar granule cell apoptosis. Cell Death Differ 2000; 7: 984-93.
- 50 Braun JS, Novak R, Herzog KH. et al. Neuroprotection by a caspase inhibitor in acute bacterial meningitis. Nat Med 1999; 5: 298-302.
- 51 Mogga SJ, Mustafa T, Sviland L. et al. Increased Bcl-2 and reduced Bax expression in infected macrophages in slowly progressive primary murine Mycobacterium tuberculosis infection. Scand J Immunol 2002; 56: 383-91.
- 52 Lassus P, Opitz-Araya X, Lazebnik Y. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science 2002; 297: 1352-4.
- 53 Zamzami N, El Hamel C, Maisse C. et al. Bid acts on the permeability transition pore complex to induce apoptosis. Oncogene 2000; 19: 6342-50.
- 54 Robertson JD, Enoksson M, Suomela M. et al. Caspase- 2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J Biol Chem 2002; 277: 29803-9.
- 55 Arnoult D, Tatischeff I, Estaquier J. et al. On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death. Mol Biol Cell 2001; 12: 3016-30.
- 56 Virdee K, Parone PA, Tolkovsky AM. Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr Biol 2000; 10: R883.
- 57 Monier RM, Orman KL, Meals EA. et al. Differential effects of p38– and extracellular signal-regulated kinase mitogen-activated protein kinase inhibitors on inducible nitric oxide synthase and tumor necrosis factor production in murine macrophages stimulated with Streptococcus pneumoniae. J Infect Dis 2002; 185: 921-6.
- 58 Liberto MC, Matera G, Lamberti AG. et al. Bartonella quintana-induced apoptosis inhibition of human endothelial cells is associated with p38 and SAPK/JNK modulation and with stimulation of mitosis. Diagn Microbiol Infect Dis 2004; 50: 159-66.
- 59 Kaiser RA, Bueno OF, Lips DJ. et al. Targeted inhibition of p38 mitogen-activated protein kinase antagonizes cardiac injury and cell death following ischemia- reperfusion in vivo. J Biol Chem 2004; 279: 15524-30.
- 60 Choi IJ, Kim JS, Kim JM. et al. Effect of inhibition of extracellular signal-regulated kinase 1 and 2 pathway on apoptosis and bcl-2 expression in Helicobacter pylori-infected AGS cells. Infect Immun 2003; 71: 830-7.