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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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Inhibition of NF-κB activation by TPCK blocks  the induction of cytokine genes  in Ngo-infected epithelial cells.  (A) Nuclear extracts from HeLa  cells were prepared at different  time points after infection (Ngo  P+ strain), incubated with a 32P-labeled oligonucleotide containing the NF-κB H-2K DNA-binding site, and analyzed for  NF-κB activation in an EMSA  (lanes 1–5). Additionally, cells  were treated with the serine protease inhibitor TPCK 30 min before the infection (lanes 6–10).  Only a section of the autoradiogram containing the protein– DNA complexes is shown. The  position of the NF-κB–DNA  complexes is indicated with an  arrow. (B) Shown is an analysis of  cytokine mRNA levels in HeLa  cells in response to Ngo P+ strain  infection by duplex RT-PCR  either in the absence or presence  of the protease inhibitor TPCK.  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.
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Figure 6: Inhibition of NF-κB activation by TPCK blocks the induction of cytokine genes in Ngo-infected epithelial cells. (A) Nuclear extracts from HeLa cells were prepared at different time points after infection (Ngo P+ strain), incubated with a 32P-labeled oligonucleotide containing the NF-κB H-2K DNA-binding site, and analyzed for NF-κB activation in an EMSA (lanes 1–5). Additionally, cells were treated with the serine protease inhibitor TPCK 30 min before the infection (lanes 6–10). Only a section of the autoradiogram containing the protein– DNA complexes is shown. The position of the NF-κB–DNA complexes is indicated with an arrow. (B) Shown is an analysis of cytokine mRNA levels in HeLa cells in response to Ngo P+ strain infection by duplex RT-PCR either in the absence or presence of the protease inhibitor TPCK. 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.

Mentions: From the experiments described above, we suggest that the activation of the transcription factor NF-κB represents a critical event in Ngo infection of HeLa cells. To study the importance of NF-κB in the downstream signaling after Ngo infection, we asked whether the blockage of NF-κB activation by the addition of the serine protease inhibitor TPCK could cause a block of cytokine mRNA upregulation. As shown in Fig. 6 A, TPCK effectively blocked IκBα degradation and NF-κB activation in response to P+ Ngo infection as determined in the gel retardation assay. The TPCK inhibitor does not generally or nonspecifically affect DNA-binding proteins since, for example, Oct-1 DNA-binding activity remains unaffected (data not shown). To determine if cytokine mRNA upregulation is affected by Ngo infection of TPCK pretreated cells, we investigated a panel of cytokine genes as shown in Fig. 1 A. TPCK pretreatment of HeLa cells led to a strong inhibition of GM-CSF, TNF-α, IL-8, IL-6, and MCP-1 cytokines mRNAs synthesis upon infection with P+ Ngo (Fig. 6 B). Marginal or no blockage of mRNA synthesis was observed for the cytokines IL-1α, IL-1β, and TGF-β. Thus, in most of the cases studied, the activation of the immediate early factor NF-κB is sufficient for the transcriptional activation of cytokine genes in response to infection with Ngo.


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)

Inhibition of NF-κB activation by TPCK blocks  the induction of cytokine genes  in Ngo-infected epithelial cells.  (A) Nuclear extracts from HeLa  cells were prepared at different  time points after infection (Ngo  P+ strain), incubated with a 32P-labeled oligonucleotide containing the NF-κB H-2K DNA-binding site, and analyzed for  NF-κB activation in an EMSA  (lanes 1–5). Additionally, cells  were treated with the serine protease inhibitor TPCK 30 min before the infection (lanes 6–10).  Only a section of the autoradiogram containing the protein– DNA complexes is shown. The  position of the NF-κB–DNA  complexes is indicated with an  arrow. (B) Shown is an analysis of  cytokine mRNA levels in HeLa  cells in response to Ngo P+ strain  infection by duplex RT-PCR  either in the absence or presence  of the protease inhibitor TPCK.  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.
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Figure 6: Inhibition of NF-κB activation by TPCK blocks the induction of cytokine genes in Ngo-infected epithelial cells. (A) Nuclear extracts from HeLa cells were prepared at different time points after infection (Ngo P+ strain), incubated with a 32P-labeled oligonucleotide containing the NF-κB H-2K DNA-binding site, and analyzed for NF-κB activation in an EMSA (lanes 1–5). Additionally, cells were treated with the serine protease inhibitor TPCK 30 min before the infection (lanes 6–10). Only a section of the autoradiogram containing the protein– DNA complexes is shown. The position of the NF-κB–DNA complexes is indicated with an arrow. (B) Shown is an analysis of cytokine mRNA levels in HeLa cells in response to Ngo P+ strain infection by duplex RT-PCR either in the absence or presence of the protease inhibitor TPCK. 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.
Mentions: From the experiments described above, we suggest that the activation of the transcription factor NF-κB represents a critical event in Ngo infection of HeLa cells. To study the importance of NF-κB in the downstream signaling after Ngo infection, we asked whether the blockage of NF-κB activation by the addition of the serine protease inhibitor TPCK could cause a block of cytokine mRNA upregulation. As shown in Fig. 6 A, TPCK effectively blocked IκBα degradation and NF-κB activation in response to P+ Ngo infection as determined in the gel retardation assay. The TPCK inhibitor does not generally or nonspecifically affect DNA-binding proteins since, for example, Oct-1 DNA-binding activity remains unaffected (data not shown). To determine if cytokine mRNA upregulation is affected by Ngo infection of TPCK pretreated cells, we investigated a panel of cytokine genes as shown in Fig. 1 A. TPCK pretreatment of HeLa cells led to a strong inhibition of GM-CSF, TNF-α, IL-8, IL-6, and MCP-1 cytokines mRNAs synthesis upon infection with P+ Ngo (Fig. 6 B). Marginal or no blockage of mRNA synthesis was observed for the cytokines IL-1α, IL-1β, and TGF-β. Thus, in most of the cases studied, the activation of the immediate early factor NF-κB is sufficient for the transcriptional activation of cytokine genes in response to infection with Ngo.

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