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Mycoplasma genitalium lipoproteins induce human monocytic cell expression of proinflammatory cytokines and apoptosis by activating nuclear factor kappaB.

Wu Y, Qiu H, Zeng Y, You X, Deng Z, Yu M, Zhu C - Mediators Inflamm. (2008)

Bottom Line: LPs were also found to increase the DNA-binding activity of NF-kappaB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis.These effects were abrogated by PDTC, an inhibitor of NF-kappaB.Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappaB.

View Article: PubMed Central - PubMed

Affiliation: Pathogenic Biology Institute, University of South China, 421001 Hengyang, China. yimouwu@sina.com

ABSTRACT
This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins (LPs) prepared from Mycoplasma genitalium. Cultured cells were stimulated with M. genitalium LP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide (PI) staining and acridine orange (AO)-ethidium bromide (EB) staining. The DNA-binding activity of nuclear factor-kappaB (NF-kappaB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-kappaB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-kappaB. Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappaB.

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Cell apoptosis of different groups wasdetected by Annexin-V-propidium iodide staining. THP-1 cells were stimulated with 3 μg/mL of LP, 3 μg/mL of LP incombination with 25 μM PDTC, or 0.1 μg/mL LPS for 12 hours, stained withAnnexin-V-FITC-PI and analyzed by FACS. Double negative staining represents livingcells (c), positive staining for Annexin-V-FITC, and negative stainingfor PI represent the early apoptotic stage (e), and double-positivestaining represents the late apoptotic stage (b).
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fig5: Cell apoptosis of different groups wasdetected by Annexin-V-propidium iodide staining. THP-1 cells were stimulated with 3 μg/mL of LP, 3 μg/mL of LP incombination with 25 μM PDTC, or 0.1 μg/mL LPS for 12 hours, stained withAnnexin-V-FITC-PI and analyzed by FACS. Double negative staining represents livingcells (c), positive staining for Annexin-V-FITC, and negative stainingfor PI represent the early apoptotic stage (e), and double-positivestaining represents the late apoptotic stage (b).

Mentions: The percentage of apoptotic cells was examinedby two techniques as follows. (1) a typical experiment of Annexin-V-FITC-PIstaining was shown in Figure 5. By this technique, necrotic and late apoptosiscells could not be distinguished from each other, so we concluded that the double-positivecells (for Annexin-V-FITC and PI) represented the late apoptotic cells and notnecrotic ones. The results obtained in the three independentexperiments showed approximately 14.23 ± 1.56% reduction in the percentage of theapoptotic cells induced by M. genitalium LP, in comparison with LPS control (16.53 ± 1.68%) and LP in combination with 25 μMPDTC-induced cells (2.79 ± 0.46%). (2) AO-EB staining,which distinguishes between apoptotic cells by the morphological changes in thenucleus (lack of DNA condensation in necrotic cells as supposed to apoptotic cells), was used to determine whether M. genitalium LP induced apoptosis ornecrosis. The apoptosis was reduced by about 61%in THP-1 cells infected with M.genitalium LP in combination with 25 μM PDTC (15.34 ± 3.94%), in comparison with M. genitalium LP-infected cells (38.50 ± 4.46%) and LPS-treated cells (39.60 ± 4.45%) (Table 1).


Mycoplasma genitalium lipoproteins induce human monocytic cell expression of proinflammatory cytokines and apoptosis by activating nuclear factor kappaB.

Wu Y, Qiu H, Zeng Y, You X, Deng Z, Yu M, Zhu C - Mediators Inflamm. (2008)

Cell apoptosis of different groups wasdetected by Annexin-V-propidium iodide staining. THP-1 cells were stimulated with 3 μg/mL of LP, 3 μg/mL of LP incombination with 25 μM PDTC, or 0.1 μg/mL LPS for 12 hours, stained withAnnexin-V-FITC-PI and analyzed by FACS. Double negative staining represents livingcells (c), positive staining for Annexin-V-FITC, and negative stainingfor PI represent the early apoptotic stage (e), and double-positivestaining represents the late apoptotic stage (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2366083&req=5

fig5: Cell apoptosis of different groups wasdetected by Annexin-V-propidium iodide staining. THP-1 cells were stimulated with 3 μg/mL of LP, 3 μg/mL of LP incombination with 25 μM PDTC, or 0.1 μg/mL LPS for 12 hours, stained withAnnexin-V-FITC-PI and analyzed by FACS. Double negative staining represents livingcells (c), positive staining for Annexin-V-FITC, and negative stainingfor PI represent the early apoptotic stage (e), and double-positivestaining represents the late apoptotic stage (b).
Mentions: The percentage of apoptotic cells was examinedby two techniques as follows. (1) a typical experiment of Annexin-V-FITC-PIstaining was shown in Figure 5. By this technique, necrotic and late apoptosiscells could not be distinguished from each other, so we concluded that the double-positivecells (for Annexin-V-FITC and PI) represented the late apoptotic cells and notnecrotic ones. The results obtained in the three independentexperiments showed approximately 14.23 ± 1.56% reduction in the percentage of theapoptotic cells induced by M. genitalium LP, in comparison with LPS control (16.53 ± 1.68%) and LP in combination with 25 μMPDTC-induced cells (2.79 ± 0.46%). (2) AO-EB staining,which distinguishes between apoptotic cells by the morphological changes in thenucleus (lack of DNA condensation in necrotic cells as supposed to apoptotic cells), was used to determine whether M. genitalium LP induced apoptosis ornecrosis. The apoptosis was reduced by about 61%in THP-1 cells infected with M.genitalium LP in combination with 25 μM PDTC (15.34 ± 3.94%), in comparison with M. genitalium LP-infected cells (38.50 ± 4.46%) and LPS-treated cells (39.60 ± 4.45%) (Table 1).

Bottom Line: LPs were also found to increase the DNA-binding activity of NF-kappaB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis.These effects were abrogated by PDTC, an inhibitor of NF-kappaB.Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappaB.

View Article: PubMed Central - PubMed

Affiliation: Pathogenic Biology Institute, University of South China, 421001 Hengyang, China. yimouwu@sina.com

ABSTRACT
This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins (LPs) prepared from Mycoplasma genitalium. Cultured cells were stimulated with M. genitalium LP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide (PI) staining and acridine orange (AO)-ethidium bromide (EB) staining. The DNA-binding activity of nuclear factor-kappaB (NF-kappaB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-kappaB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-kappaB. Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappaB.

Show MeSH
Related in: MedlinePlus