フラクタルカインとCX3CR1は肝内胆管の胆管上皮膚内リンパ球の遊走に関連する

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第1678号, 学位授与年月日 : 平成17年3月22日, 学位授与大学 : 金沢大

Endotoxin tolerance in human intrahepatic biliary epithelial cells is induced by upregulation of IRAK-M

金沢大学大学院医学系研究科がん細胞学Background/Aim: Biliary epithelial cells possess an innate immune system consisting of Toll-like receptors (TLRs). Although the human bile contains lipopolysaccharide (LPS) in normal as well as diseased livers, LPS physiologically does not elicit an inflammatory response in the biliary tree. This absence of a response to LPS could be due to the \u27endotoxin tolerance\u27speculated to maintain innate immune homeostasis in organs. Our aim here is to clarify the presence and molecular mechanisms of endotoxin tolerance of biliary epithelium. Methods and Results: In nuclear factor-κB (NF-κB)-DNA binding assays using three-cultured human intrahepatic biliary epithelial cell (HIBEC) lines, all the cells responded to LPS (TLR4 ligand) by activating NF-κB, but pretreatment with LPS for 24h effectively induced tolerance against any subsequent stimulation with LPS (endotoxin tolerance). This tolerance was also induced by pretreatment with Pam3Cys-Ser-(Lys)4 trihydrochloride (Pam3CKS4, TLR1/2 ligand). Then, real-time polymerase chain treaction and Western blotting revealed that LPS treatment upregulated the expression of IRAK-M (a negative regulator of TLR signaling), but did not affect interleukin-1 receptor-associated kinase-1 (IRAK-1, an essential molecule of TLR signaling), in HIBECs. Moreover, immunohistochemistry revealed that IRAK-M was diffusely expressed in intrahepatic bile ducts. Conclusions: This study showed that the mechanism of endotoxin tolerance exists in the intrahepatic biliary tree and is possibly induced by the expression of IRAK-M in the intrahepatic biliary epithelium, suggesting that the endotoxin tolerance is important in maintaining innate immune biliary homeostasis. © 2006 Blackwell Munksgaard

CD39 deficiency in murine liver allografts promotes inflammatory injury and immune-mediated rejection

Adenosine triphosphate (ATP), an essential metabolic energy source, is released following cell apoptosis or necrosis. It acts as a damage-associated molecule pattern to stimulate innate immune cells. The ectonucleotidase CD39 regulates immune activation by hydrolysis of extracellular ATP. We have shown previously that CD39 expression by donor livers helps protect syngeneic grafts with extended (24 hr) cold preservation time from ischemia reperfusion injury. Given its immune regulatory properties, we hypothesized that CD39 expression in donor livers might modulate transplant tolerance that occurs following mouse allogeneic liver transplantation (LTx). Livers from C57BL/6 (B6) wild-type (WT) or CD39 KO mice were transplanted into normal C3H recipients with minimal (approximately 1 hr) cold ischemia. Serum alanine aminotransferase levels at day 4 post LTx were significantly higher in animals given CD39KO compared with WT livers. Moreover, IFN-γ production by liver-infiltrating CD8+ T cells at day 4 was significantly higher in CD39KO than in WT grafts. Furthermore, splenic T cells from CD39KO liver recipients exhibited greater proliferative responses to donor alloantigens than those from mice given WT grafts. By contrast, there was a concomitant significant reduction in the frequency of regulatory T cells (Treg) in CD39KO than in WT livers. Whereas WT liver allografts survived > 100 days, no CD39KO grafts survived beyond 40 days (median survival time [MST]: WT: >100 days vs CD39KO: 8 days; p<0.01). In addition, soluble CD39 administration significantly prolonged CD39KO liver allograft survival (MST: 27.5 days). These novel data suggest that CD39 expression in liver allografts modulates tissue injury, inflammation, anti-donor effector T cell responses and Treg infiltration and can suppress transplant rejection

Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics

Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype

Histologic graft assessment after clinical islet transplantation

BACKGROUND: An accurate monitoring would help understanding the fate of islet grafts after transplantation. METHODS: This work assessed the feasibility of needle biopsy monitoring after intraportal islet transplantation (n=16), and islet graft morphology was studied with the addition of autopsy samples (n=2). Pancreas autopsy samples from two nondiabetic individuals were used as control. RESULTS: Islet tissue was found in five needle samples (31%). Sampling success was related to size (100% sampling for the four biopsies of 1.8 cm in length or higher, P ≤ 0.01). Mild liver abnormalities included localized steatosis (n=8), mild nodular regenerative hyperplasia and mild portal venopathy (n=3), and hepatocyte swelling (n=2). Endocrine cell composition and distribution were similar between islet grafts and normal islets within the native pancreas. There was no or minimal immune cell infiltrate in patients on and off exogenous insulin, including two patients with ongoing negative metabolic events (increasing HbA1c or insulin requirement). The infiltrate was mainly composed of CD4- and CD8-positive cells. CONCLUSION: This study demonstrates that needle biopsy is feasible after clinical islet transplantation but with a limited practical value because of its low islet sampling rate using current sampling and analysis methods. Both biopsy and autopsy samples demonstrated the well-preserved islet endocrine composition after transplantation and the presence of focal areas of steatosis. Islet grafts showed no or minimal immune cell infiltration, even in the case of ongoing islet loss. On the basis of the findings, possible reasons for allograft islet loss are discussed