Study On The Clinical And Pathologic Characterisitic And The Effects Of Lipopolysaccharide In Primary Biliary Cirrhosis

ObjectivePrimary biliary cirrhosis (PBC) is an autoimmune liver disease. Histopathology shows features of progressive destruction of intrahepatic bile ducts which ultimately leads to liver fibrosis and cirrhosis. Prevalence rates from west countries vary betwe- en 20 and 240 cases per million population. However, in China, PBC has traditionally been regarded as a rare disorder. However, with an increased awareness of PBC on clinic and the availability of diagnostic tests such as serological autoantibodies, espec -ially the measurement of AMA/AMA-M2, more PBC cases have been diagnosed in China. Furthermore, the incidence of PBC appears to be rising. The majority of invest -igators think the actual incidence rates for PBC are higher than other countries. So the effect of the disease on human health should be emphasized.The pathogenesis of PBC remains unknown up to date. In recent years, it has become clear that bacterial products play an important role in the pathogenesis of bile duct diseases. One or several candidate infectious agents have been postulated to trig -ger PBC in susceptible individuals through a mechanism known as molecular mim -icry. But the evidence is not sufficient. It is most likely not the type of infectious age-nt that determines the onset of autoimmunity, but rather the qualitative nature of innate immune responses by an individual that could potentially trigger an autoimmu -ne response. However, aberrant responses by monocyte to microbial infection have been hypothesized to initiate an autoimmune response. Inappropriate anti-infectious responses may lead to PBC. In our study, the clinical and pathologic characterisitic of PBC and the effects of LPS in the pathogenesis of the disease were studied systemati -cally in vivo and in vitro from the point of infection and immunology. Our aim is to study the features of patients with PBC including clinical symptoms, laboratory tests and pathology in order to improve recognition of the disease. Meanwhile, we try to explore lipopolysaccharide and its receptor impact on the initiation and progression of PBC and to expound the mechanism of immunologic injury from an entirely original points of infectious agents in order to provide a new possible treatment strategy and understanding for pathogenesis of PBC. MethodsAll patients were hospitalized and diagnosed as PBC with liver biopsy at 302 Hospital of PLA from 2001 to 2007. We reviewed and analysed the clinical and path -ological data including general status, clinical manifestations, laboratory findings and histological characteristics from one hundred cases of patients with PBC. Endotoxin level in sera was determined by limulus amebocyte lysate test.The serum levels of TNFα, IL-1β, IL-6, and IL-8 were detected by ELISA method.The expression of CD -14, TLR4, CD68 and NF-κB in liver tissues was examined by immunohistochemistry. In addition, we isolated peripheral blood mononuclear cells (PBMC) from PBC patients and healthy controls and stimulated the isolated and cultured PBMCs in vitro with LPS. The levels of TNFα, IL-1β, IL-6, and IL-8 in the supernatant fluids from the cultured PBMCs were analyzed with ELISA method. And the expression of monocyte cell surface molecules including TLR4, CD14 and CD83 was measured with flow cytometry. Western blot was used to detect TLR4 protein on cultured human biliary epithelial cells (hBEC) before and after being stimulated with LPS. Measurement of proinflammatory cytokines in supernatant fluids from cultured hBEC were performed using ELISA.The changes of immune cell populations in PBC liver tissues were studied by using methods of immunohistochemistry and double labeling immunohistochemistry.Results1. The analyzed clinical and pathological data show that the Chinese PBC cases presented some clinical and pathological characteristic, included a) that middle age females were popular in Chinese PBC population; b)the most frequent symptoms were fatigue, jaundice and pruritus; c)the levels ofγ-glutamyltransferase (r-GGT) , alkaline phosphatase (ALP) and bilirubin in the sera were markedly elevated in most patients,but decreased with PBC progression; d)70 % of patients were antimitocho -ndrial antibody (AMA) positive, and mean serum immunoglobulin M (lgM) level was significantly lower in AMA-negative group compared with AMA-positive PBC patients (P < 0.05). and e)the pathological features of PBC were as follows: in early stage, small bile ducts were destroyed and bile ductules were proliferated; with mono -nuclear cells aggregation or lymphocyte follicles around the damaged bile ducts, and in advanced stage, portal area is enlarged with fibrosis, lobular architectural distortion and finally liver cihrosis formation.2. The levels of endotoxin and proinflammatory cytokines in sera were increased significantly with PBC progression. Serum endotoxin levels in PBC patients were positively correlated with those biochemical parameters of ALP, TB, DB. TLR4, CD14, and NF-κBp65 antigen expression in PBC liver tissues are increased compared with normal liver tissues (P<0.05), especially TLR4 in biliary epithelial cells.3. Compared with healthy control, the CD14 positive cell frequency significantly increased in peripheral blood mononuclear cells from PBC patients, and the differences between both groups were significant. After stimulating with LPS, we found significantly higher amounts of TNFα, IL-1β, IL-6, and IL-8 production in PBC patients. And the levels of TLR4 and CD83 expression were significantly up -regulated on PBC monocytes as compared with healthy controls. Monocytes from patients with PBC appeared more sensitive to LPS.4. After cultured with LPS for 2hr, 8hr and 24hr, biliary epithelial cells originated from PBC patients presented significantly increased TLR4 expression in comparison with those from healthy controls(P<0.05). LPS-stimulated biliary epithelial cells from PBC patient were found to produce significantly higher amounts of TNFα, IL-6, and IL-8 after 8hr, 24hr culture than those from healthy controls(P<0.01). Biliary epithelial cells from PBC patients are more sensitive to LPS compared to those from healthy controls.5. In PBC liver tissues, immune cell populations were positive for CD3, CD4, CD8, CD20, CD83, CD57.The number of these cells increased compared with normal liver tissues. In early stage of PBC, the main immune cells in liver tissues were CD3+, CD4+, CD8+ lymphocytes, and CD8+ T cells were prodominant around the damaged interlobular bile duct. However, in the late stage of PBC, all of the cell number decreased.Conclusions1. According to our findings, we could conclude that PBC has some clinical character -istics. The most frequent symptoms in PBC patients are fatigue, jaundice and or pruritus. The presence of AMA in serum is the major hallmark of PBC. Serum ALP andγ-GGT levels were markedly elevated in all patients, while ALT and AST levels were mildly elevated. Liver biopsy is useful to confirm the diagnosis and differentiate the histopathologic stages with regard to atypical symptoms and AMA negative patients.2. LPS might play an important role in liver damage. LPS might directly contribute to the leisions of biliary epithelial cells as well as hepatocytes through its receptor way. On the other hand, proinflammatory cytokines produced by monocytes and biliary epithelial cells and other lymphocytes might be indirectly involved in the progression of liver inflammation, fibrosis and the small bile duct destruction. Therefore, considering the two ways of LPS induced liver damages, we infer this might be a new injury mechanism in PBC pathogenesis.3. The level of endotoxin in sera was increased significantly in PBC group when compared with healthy control.And the changes of endotoxin in PBC patients cor -related with pathologic stages.We could deduce the level of sera LPS might be a potential serological mark in clinical diagnosis, differentiation, progression estimation of PBC.4. According to our results, the changes of numbers, proportion and distribution of immune cells in PBC liver tissues showed the damage of intraheptic bile duct was mediated mainly by cell immunologic injury through Th1 way.The PBMCs from patients with PBC appear more sensitive to LPS signaling through TLR4 receptor and secretion of proinflammatory cytokines integral to the inflammatory response, which suggested that may be critical in the breakdown of immune balance in the process of PBC pathogenesis.