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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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Related in: MedlinePlus

Intracellular TNF-α following LPS treatment Intracellular TNF-α concentration of MGBE over time following apical exposure to 100 μg/mL E. coli LPS was measured by immunoassay. Values are the means ± standard error of results from three to four assays.
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Figure 4: Intracellular TNF-α following LPS treatment Intracellular TNF-α concentration of MGBE over time following apical exposure to 100 μg/mL E. coli LPS was measured by immunoassay. Values are the means ± standard error of results from three to four assays.

Mentions: The immunoassay experiments showed no detectable TNF-α in untreated MGBE or their surrounding media after 5 hours of incubation (limit of detection = 5.1 pg/mL). In contrast, following treatment with 100 μg/mL E. coli LPS the concentration of TNF-α protein in the cells was 33.6 pg per 106 cells in one hour (Figure 4). Intracellular TNF-α concentration reached a peak two hours after LPS treatment with a subsequent decrease by five hours. After two and five hours of LPS treatment, there was measurable release of TNF-α into the media of both the apical and basolateral compartments (figure 5). Three different species of LPS (E. coli, P. aeruginosa, and Klebsiella pneumoniae) stimulated TNF-α release in a similar fashion (figure 6). Treatment with 2 μg/mL E. coli LPS showed comparable results. None of the treatments increased lactate dehydrogenase in the medium compared with controls showing that the LPS treatments did not alter epithelial cell integrity (data not shown). LPS treatment also had no effect on the transepithelial permeability of these cells for up to 24 hours as measured by 3H-mannitol diffusion experiments when compared to controls (data not shown).


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)

Intracellular TNF-α following LPS treatment Intracellular TNF-α concentration of MGBE over time following apical exposure to 100 μg/mL E. coli LPS was measured by immunoassay. Values are the means ± standard error of results from three to four assays.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Intracellular TNF-α following LPS treatment Intracellular TNF-α concentration of MGBE over time following apical exposure to 100 μg/mL E. coli LPS was measured by immunoassay. Values are the means ± standard error of results from three to four assays.
Mentions: The immunoassay experiments showed no detectable TNF-α in untreated MGBE or their surrounding media after 5 hours of incubation (limit of detection = 5.1 pg/mL). In contrast, following treatment with 100 μg/mL E. coli LPS the concentration of TNF-α protein in the cells was 33.6 pg per 106 cells in one hour (Figure 4). Intracellular TNF-α concentration reached a peak two hours after LPS treatment with a subsequent decrease by five hours. After two and five hours of LPS treatment, there was measurable release of TNF-α into the media of both the apical and basolateral compartments (figure 5). Three different species of LPS (E. coli, P. aeruginosa, and Klebsiella pneumoniae) stimulated TNF-α release in a similar fashion (figure 6). Treatment with 2 μg/mL E. coli LPS showed comparable results. None of the treatments increased lactate dehydrogenase in the medium compared with controls showing that the LPS treatments did not alter epithelial cell integrity (data not shown). LPS treatment also had no effect on the transepithelial permeability of these cells for up to 24 hours as measured by 3H-mannitol diffusion experiments when compared to controls (data not shown).

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