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

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.

Figure 1

RT-PCR for cytokine mRNAs Treated MGBE were exposed to 100 μg/mL E. coli LPS on the apical surface for two hours. GAPDH was used as an internal control to standardize starting mRNA quantity. This experiment was repeated twice with equivalent results.

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.

Figure 3

Densitometry analysis of semi-quantitative RT-PCR results Densitometry of PCR bands for MIP-2, TNF-α, and IL-1β (from figure 2) based on % of ARP control bands using ImageJ Program (Version 1.20s).

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.

Figure 5

TNF-α secretion into medium Release of TNF-α into the apical and basolateral medium by cultured MGBE exposed to 100 μg/mL E. coli LPS over time. TNF-α secretion was calculated as the total pg amount of TNF-α present in the apical or basolateral medium. Values are the means of triplicates ± standard error from a single assay. This assay was repeated twice more with similar results. Statistical differences between the concentration of TNF-α in the basal and apical medium were measured by paired Student's t-test (* = p < 0.01).

Figure 6

Effects of LPS from different bacteria on TNF-α secretion Release of TNF-α into the apical and basolateral medium by cultured MGBE exposed to 100 μg/mL LPS from various bacteria for two hours. TNF-α was measured by immunoassay and calculated as the total pg present in the apical and basolateral medium. Each treatment was done in triplicate and assay repeated twice more with E. coli LPS and once more with P. aeruginosa and K. pneumoniae LPS. Values represent the mean of each treatment (n = 9 for E. coli LPS and n = 6 for P. aeruginosa and K. pneumoniae LPS) ± standard error. There was a statistical difference between the amount of TNF-α in the basal versus apical medium as measured by paired Student's t-test (* = p < 0.01).