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Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

Naumann M, Wessler S, Bartsch C, Wieland B, Meyer TF - J. Exp. Med. (1997)

Bottom Line: In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer.Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation.Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, 10117 Berlin. naumann@mpiib-berlin.mpg.de

ABSTRACT
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

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Ngo infection induces proinflammatory cytokines  in epithelial cells. (A) Shown is  an analysis of cytokine mRNA  levels in ME180 cells in response  to Ngo infection (the invasive  Opa+ strain, the piliated, noninvasive P+ strain, and the P−  Opa− control strain) by duplex  RT-PCR. Total RNA was isolated at the indicated time points  after infection and reverse transcribed into cDNA, and cytokine mRNAs as well as β-actin  mRNA were semiquantitated  by several cycles of PCR using  cytokine-specific primers so that  products were below the saturation stage of amplification. Equal  RNA was amplified for each  sample within an infection kinetic as indicated by the internal  β-actin amount. DNA products  were separated by electrophoresis  on a agarose gel and visualized  with ethidium bromide. Shown is  an experiment representative of  at least three. *, β-actin. Not  shown: IL-2, IL-3, IL-4, IL-5,  IL-10, IL-13, I-309, and IFN-γ  RT-PCR reactions that recognized no transcripts. (B) The secretion of the cytokines TNF-α,  GM-CSF, and IL-8 was assayed  in ELISA systems 3 h and 6 h after infection (p.i.). Values are  means, the standard errors of the  means are representative for  three experiments.
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Figure 1: Ngo infection induces proinflammatory cytokines in epithelial cells. (A) Shown is an analysis of cytokine mRNA levels in ME180 cells in response to Ngo infection (the invasive Opa+ strain, the piliated, noninvasive P+ strain, and the P− Opa− control strain) by duplex RT-PCR. Total RNA was isolated at the indicated time points after infection and reverse transcribed into cDNA, and cytokine mRNAs as well as β-actin mRNA were semiquantitated by several cycles of PCR using cytokine-specific primers so that products were below the saturation stage of amplification. Equal RNA was amplified for each sample within an infection kinetic as indicated by the internal β-actin amount. DNA products were separated by electrophoresis on a agarose gel and visualized with ethidium bromide. Shown is an experiment representative of at least three. *, β-actin. Not shown: IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, I-309, and IFN-γ RT-PCR reactions that recognized no transcripts. (B) The secretion of the cytokines TNF-α, GM-CSF, and IL-8 was assayed in ELISA systems 3 h and 6 h after infection (p.i.). Values are means, the standard errors of the means are representative for three experiments.

Mentions: Each of the three studied epithelial cell lines expressed inflammatory cytokines in response to Ngo infection. As shown in Fig. 1 A, infection of ME180 cells with Ngo led to an increased synthesis of several cytokines, i.e., GM-CSF, TNF-α, IL-8, MCP-1, TGF-β, and IL-1β as soon as 15 min after infection. Maximum expression was achieved 2 h after infection. Furthermore, the cytokine IL-6 was induced within 45 min after infection and the cytokines IL-1α and IL-12 showed constitutive mRNA expression with a slight increase upon infection. The chemokine RANTES (regulated on activation, normal T cell expressed and secreted) was constitutively expressed without change in infected cells. Both the adherent (P+) and the invasive (Opa+) gonococci were efficient in cytokine upregulation, whereas the P− Opa− gonococci did not significantly increase cytokine mRNA levels. LPS, a major constituent of the gram-negative bacterial outer membrane and a potent inducer of cytokine mRNA upregulation in CD14 positive cells, did not effect cytokine expression (data not shown). None of the tested cell lines induced detectable levels of mRNA coding for IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, intercrine 309 (I-309), or IFN-γ (data not shown).


Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

Naumann M, Wessler S, Bartsch C, Wieland B, Meyer TF - J. Exp. Med. (1997)

Ngo infection induces proinflammatory cytokines  in epithelial cells. (A) Shown is  an analysis of cytokine mRNA  levels in ME180 cells in response  to Ngo infection (the invasive  Opa+ strain, the piliated, noninvasive P+ strain, and the P−  Opa− control strain) by duplex  RT-PCR. Total RNA was isolated at the indicated time points  after infection and reverse transcribed into cDNA, and cytokine mRNAs as well as β-actin  mRNA were semiquantitated  by several cycles of PCR using  cytokine-specific primers so that  products were below the saturation stage of amplification. Equal  RNA was amplified for each  sample within an infection kinetic as indicated by the internal  β-actin amount. DNA products  were separated by electrophoresis  on a agarose gel and visualized  with ethidium bromide. Shown is  an experiment representative of  at least three. *, β-actin. Not  shown: IL-2, IL-3, IL-4, IL-5,  IL-10, IL-13, I-309, and IFN-γ  RT-PCR reactions that recognized no transcripts. (B) The secretion of the cytokines TNF-α,  GM-CSF, and IL-8 was assayed  in ELISA systems 3 h and 6 h after infection (p.i.). Values are  means, the standard errors of the  means are representative for  three experiments.
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Related In: Results  -  Collection

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Figure 1: Ngo infection induces proinflammatory cytokines in epithelial cells. (A) Shown is an analysis of cytokine mRNA levels in ME180 cells in response to Ngo infection (the invasive Opa+ strain, the piliated, noninvasive P+ strain, and the P− Opa− control strain) by duplex RT-PCR. Total RNA was isolated at the indicated time points after infection and reverse transcribed into cDNA, and cytokine mRNAs as well as β-actin mRNA were semiquantitated by several cycles of PCR using cytokine-specific primers so that products were below the saturation stage of amplification. Equal RNA was amplified for each sample within an infection kinetic as indicated by the internal β-actin amount. DNA products were separated by electrophoresis on a agarose gel and visualized with ethidium bromide. Shown is an experiment representative of at least three. *, β-actin. Not shown: IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, I-309, and IFN-γ RT-PCR reactions that recognized no transcripts. (B) The secretion of the cytokines TNF-α, GM-CSF, and IL-8 was assayed in ELISA systems 3 h and 6 h after infection (p.i.). Values are means, the standard errors of the means are representative for three experiments.
Mentions: Each of the three studied epithelial cell lines expressed inflammatory cytokines in response to Ngo infection. As shown in Fig. 1 A, infection of ME180 cells with Ngo led to an increased synthesis of several cytokines, i.e., GM-CSF, TNF-α, IL-8, MCP-1, TGF-β, and IL-1β as soon as 15 min after infection. Maximum expression was achieved 2 h after infection. Furthermore, the cytokine IL-6 was induced within 45 min after infection and the cytokines IL-1α and IL-12 showed constitutive mRNA expression with a slight increase upon infection. The chemokine RANTES (regulated on activation, normal T cell expressed and secreted) was constitutively expressed without change in infected cells. Both the adherent (P+) and the invasive (Opa+) gonococci were efficient in cytokine upregulation, whereas the P− Opa− gonococci did not significantly increase cytokine mRNA levels. LPS, a major constituent of the gram-negative bacterial outer membrane and a potent inducer of cytokine mRNA upregulation in CD14 positive cells, did not effect cytokine expression (data not shown). None of the tested cell lines induced detectable levels of mRNA coding for IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, intercrine 309 (I-309), or IFN-γ (data not shown).

Bottom Line: In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer.Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation.Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, 10117 Berlin. naumann@mpiib-berlin.mpg.de

ABSTRACT
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

Show MeSH
Related in: MedlinePlus