Bacteria are found in the bile of the gallbladder and bile ducts [15, 16], the presence of which may have relevance to clinical diseases such as stone formation [19–21], primary sclerosing cholangitis , and cholestasis [23, 24]. Direct exposure of sub-epithelial tissue to LPS from gram-negative bacteria causes release of cytokines by resident leukocytes and activation of macrophages . The biliary fluid of patients with cholangitis has increased concentrations of the inflammatory cytokines TNF-α and IL-6 suggesting that bacterial infection leads to an increase in cytokines released into the bile . Adding LPS to the bile of guinea pig gallbladders in vivo led to increased IL-1 concentration in the bile . What role biliary epithelial cells play in the response to bacteria in bile is not known.
While most cytokine research has focused on cells of the immune system, other cell types produce cytokines including the epithelial cells of the lung [28, 29], retina , and intestines [31, 32]. Among the few studies involving the biliary system, it was demonstrated that cultured human intrahepatic duct epithelial cells produce functional IL-8 and MCP-1 (CCL2) [14, 33]. Isolated human intrahepatic epithelial ducts expressed immunoreactive IL-6 when treated with IL-1β or phorbal myristate acetate . Intracellular TNF-α mRNA and protein was detected in intrahepatic biliary duct cells in tissue sections , but not in cultured intrahepatic epithelial cells; therefore, there was no clear demonstration that biliary epithelium secretes TNF-α. Little research has focused on the ability of the epithelial cells of the extrahepatic biliary system to produce cytokines or chemokines.
As described in this paper, MGBE were used as an in vitro model system of gallbladder epithelium to measure the endogenous production of cytokines and chemokines by extrahepatic biliary epithelial cells. These cultured cells consist exclusively of normal, non-neoplastic epithelial cells , without other cell types such as leukocytes ordinarily found in biliary tissues. The experiments showed that unstimulated MGBE express mRNA for TNF-α, MIP-2, and RANTES (CCL5). After treating the apical surface of the cells with LPS, the mRNA levels for TNF-α and MIP-2 (CXCL2) increased, while mRNA for IL-1β, IL-6, and MCP-1 (CCL2) appeared for the first time. Therefore, cytokines (TNF-α, IL-1β, and IL-6), a C-C type chemokine (MCP-1/CCL2), and a C-X-C type chemokine (MIP-2/CXCL2) are all up regulated at the RNA level by bacterial product interaction with the epithelial cells. The effects of LPS treatment for two hours on cytokine and chemokine mRNA expression clearly suggests that biliary epithelial cells can modify their own cytokine and chemokine production after bacterial challenge at the mRNA level. As with all in vitro cell systems there is the possibility that the cells in culture may have phenotypic changes to their cytokine profile compared to in vivo cells. In other parameters tested (i.e., morphology, mucin secretion, etc.), these cells retain the normal phenotype of cells in vivo.
Measurements of TNF-α protein showed that MGBE synthesize and secrete TNF-α when exposed to LPS, while untreated cells do not. This demonstrates that gallbladder epithelial cells themselves secrete a cytokine in response to bacterial products. The results from RT-PCR (showing a relatively high TNF-α mRNA expression levels in untreated cells which is only slightly increased by LPS treatment) along with the immunoassay results suggest that TNF-α mRNA is constantly present in these cells, but is not translated into protein until certain triggers are provided. This is consistent with the post-transcriptional regulation of TNF-α observed in other cells . In contrast, the mRNA of other factors, such as IL-1β, IL-6, and MCP-1 (CCL2) are not synthesized until the cells are stimulated, suggesting that their production is under transcriptional control. The protein expression of these other cytokines and chemokines along with the precise nature of the interaction between LPS and cell surface receptors warrants further study.
While the exact role that gallbladder epithelial cells play in inflammatory and infectious diseases has yet to be determined, our research supports the hypothesis that gallbladder epithelium have some innate cytokine-producing capabilities in response to the presence of bacteria on their apical surface. The accumulation of TNF-α in the gallbladder bile of patients with cholangitis may be due in part to the apical release of TNF-α by biliary epithelial cells. In contrast, basolateral secretion of TNF-α could lead to a focal accumulation of TNF-α in the sub-mucosa of biliary tissue. Sufficient cytokine secretion could then stimulate the leukocytes in the sub-epithelial tissue, leading to inflammation of the tissue, without the need for the bacteria to pass through the epithelial barrier. The production of cytokines by gallbladder epithelial cells following LPS exposure demonstrates that biliary epithelial cells may be an active partner with the immune system in the detection of and defense against bacteria in bile.
During sepsis, biliary epithelial cells could mediate biliary secretory function through cytokine and chemokine production. Dog gallbladder epithelial cells demonstrated increased mucin secretion following TNF-α treatment . In addition, exogenous TNF-α altered the absorptive properties of cultured human gallbladder epithelial cells . It is therefore reasonable to suggest that TNF-α produced by gallbladder epithelial cells in response to LPS could affect cellular function in an autocrine manner. Since mucin secretion and gallbladder transport properties play vital roles in cholelithiasis, changes in these parameters caused by endogenous cytokines and chemokines may promote stone formation. Epithelial-derived cytokines and chemokines also may alter biliary function leading to cholestasis.