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Expression of cytokine and chemokine mRNA and secretion of tumor necrosis factor-alpha by gallbladder epithelial cells: response to bacterial lipopolysaccharides.

Savard CE, Blinman TA, Choi HS, Lee SK, Pandol SJ, Lee SP - BMC Gastroenterol (2002)

Bottom Line: TNF-alpha protein was measured by immunoassays.This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFalpha protein.This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Washington and VA Puget Sound Health Care System, Seattle, Washington, USA.

ABSTRACT

Background: In addition to immune cells, many other cell types are known to produce cytokines. Cultured normal mouse gallbladder epithelial cells, used as a model system for gallbladder epithelium, were examined for their ability to express the mRNA of various cytokines and chemokines in response to bacterial lipopolysaccharide. The synthesis and secretion of the tumor necrosis factor-alpha (TNF-alpha) protein by these cells was also measured.

Results: Untreated mouse gallbladder cells expressed mRNA for TNF-alpha, RANTES, and macrophage inflammatory protein-2 (MIP-2). Upon treatment with lipopolysaccharide, these cells now produced mRNA for Interleukin-1beta (IL-1beta), IL-6, monocyte chemoattractant protein-1 (MCP-1), and showed increased expression of TNF-alpha and MIP-2 mRNA. Untreated mouse gallbladder cells did not synthesize TNF-alpha protein; however, they did synthesize and secrete TNF-alpha upon treatment with lipopolysaccharide.

Methods: Cells were treated with lipopolysaccharides from 3 strains of bacteria. Qualitative and semi-quantitative RT-PCR, using cytokine or chemokine-specific primers, was used to measure mRNA levels of TNFalpha, IL-1beta, IL-6, IL-10, KC, RANTES, MCP-1, and MIP-2. TNF-alpha protein was measured by immunoassays.

Conclusion: This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFalpha protein. This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis.

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Semi-quantitative RT-PCR Semi-quantitative RT-PCR products from cytokine mRNA using MIP-2, TNF-α, and IL-1β specific primers. C is untreated control cells. Treated cells were exposed to 100 μg/mL LPS from either E. coli or P. aeruginosa on the apical surface for two hours. ARP was used as an internal control to standardize starting mRNA quantity. PCR cycle number used was between 22 for ARP and 31 for TNF-α.
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Figure 2: Semi-quantitative RT-PCR Semi-quantitative RT-PCR products from cytokine mRNA using MIP-2, TNF-α, and IL-1β specific primers. C is untreated control cells. Treated cells were exposed to 100 μg/mL LPS from either E. coli or P. aeruginosa on the apical surface for two hours. ARP was used as an internal control to standardize starting mRNA quantity. PCR cycle number used was between 22 for ARP and 31 for TNF-α.

Mentions: Using cytokine/chemokine-specific primers with RT-PCR, untreated MGBE showed distinct bands representing mRNA of TNF-α, RANTES (Regulated on activation, normal T cell expressed and secreted) (CCL5), and macrophage inflammatory protein-2 (MIP-2/CXCL2) (Figure 1). Upon apical treatment with LPS from E. coli for two hours, these cells additionally produced mRNA for interleukin-1β (IL-1β), IL-6, and monocyte chemoattractant protein-1 (MCP-1/CCL2). There was no detectable expression of IL-10 or KC (murine GRO-α/CXCL1) mRNA before or after LPS treatment. Semi-quantitative RT-PCR using specific primers for TNF-α, MIP-2 (CXCL2) and IL-1β showed that LPS treatment caused the amount of TNF-α mRNA to slightly increase compared to the already abundant levels found in untreated cells (figure 2 and 3). In contrast, there was a substantial increase in the expression of MIP-2 (CXCL2) mRNA and IL-1β mRNA following LPS treatment compared to control. Similar responses were seen when the cells were treated with LPS from P. aeruginosa (Figure 2 and 3). All visualized PCR product bands were of the expected size and nucleotide sequence confirming proper identification. Although the RANTES (CCL5) sample had extra bands of unknown origin, the band at the expected size of 205 bp had a nucleotide sequence that matched RANTES.


Expression of cytokine and chemokine mRNA and secretion of tumor necrosis factor-alpha by gallbladder epithelial cells: response to bacterial lipopolysaccharides.

Savard CE, Blinman TA, Choi HS, Lee SK, Pandol SJ, Lee SP - BMC Gastroenterol (2002)

Semi-quantitative RT-PCR Semi-quantitative RT-PCR products from cytokine mRNA using MIP-2, TNF-α, and IL-1β specific primers. C is untreated control cells. Treated cells were exposed to 100 μg/mL LPS from either E. coli or P. aeruginosa on the apical surface for two hours. ARP was used as an internal control to standardize starting mRNA quantity. PCR cycle number used was between 22 for ARP and 31 for TNF-α.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC130965&req=5

Figure 2: Semi-quantitative RT-PCR Semi-quantitative RT-PCR products from cytokine mRNA using MIP-2, TNF-α, and IL-1β specific primers. C is untreated control cells. Treated cells were exposed to 100 μg/mL LPS from either E. coli or P. aeruginosa on the apical surface for two hours. ARP was used as an internal control to standardize starting mRNA quantity. PCR cycle number used was between 22 for ARP and 31 for TNF-α.
Mentions: Using cytokine/chemokine-specific primers with RT-PCR, untreated MGBE showed distinct bands representing mRNA of TNF-α, RANTES (Regulated on activation, normal T cell expressed and secreted) (CCL5), and macrophage inflammatory protein-2 (MIP-2/CXCL2) (Figure 1). Upon apical treatment with LPS from E. coli for two hours, these cells additionally produced mRNA for interleukin-1β (IL-1β), IL-6, and monocyte chemoattractant protein-1 (MCP-1/CCL2). There was no detectable expression of IL-10 or KC (murine GRO-α/CXCL1) mRNA before or after LPS treatment. Semi-quantitative RT-PCR using specific primers for TNF-α, MIP-2 (CXCL2) and IL-1β showed that LPS treatment caused the amount of TNF-α mRNA to slightly increase compared to the already abundant levels found in untreated cells (figure 2 and 3). In contrast, there was a substantial increase in the expression of MIP-2 (CXCL2) mRNA and IL-1β mRNA following LPS treatment compared to control. Similar responses were seen when the cells were treated with LPS from P. aeruginosa (Figure 2 and 3). All visualized PCR product bands were of the expected size and nucleotide sequence confirming proper identification. Although the RANTES (CCL5) sample had extra bands of unknown origin, the band at the expected size of 205 bp had a nucleotide sequence that matched RANTES.

Bottom Line: TNF-alpha protein was measured by immunoassays.This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFalpha protein.This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Washington and VA Puget Sound Health Care System, Seattle, Washington, USA.

ABSTRACT

Background: In addition to immune cells, many other cell types are known to produce cytokines. Cultured normal mouse gallbladder epithelial cells, used as a model system for gallbladder epithelium, were examined for their ability to express the mRNA of various cytokines and chemokines in response to bacterial lipopolysaccharide. The synthesis and secretion of the tumor necrosis factor-alpha (TNF-alpha) protein by these cells was also measured.

Results: Untreated mouse gallbladder cells expressed mRNA for TNF-alpha, RANTES, and macrophage inflammatory protein-2 (MIP-2). Upon treatment with lipopolysaccharide, these cells now produced mRNA for Interleukin-1beta (IL-1beta), IL-6, monocyte chemoattractant protein-1 (MCP-1), and showed increased expression of TNF-alpha and MIP-2 mRNA. Untreated mouse gallbladder cells did not synthesize TNF-alpha protein; however, they did synthesize and secrete TNF-alpha upon treatment with lipopolysaccharide.

Methods: Cells were treated with lipopolysaccharides from 3 strains of bacteria. Qualitative and semi-quantitative RT-PCR, using cytokine or chemokine-specific primers, was used to measure mRNA levels of TNFalpha, IL-1beta, IL-6, IL-10, KC, RANTES, MCP-1, and MIP-2. TNF-alpha protein was measured by immunoassays.

Conclusion: This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFalpha protein. This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis.

Show MeSH
Related in: MedlinePlus