disease Kupffer cell uptake function in non-alcoholic fatty liver Super paramagnetic iron oxide MRI shows defective

Background The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is incompletely understood. Kupffer cells (KCs), phagocytic liver-resident macrophages, provide a protective barrier against egress of endotoxin from the portal to the systemic circulation. It is not known if KC phagocytic function is impaired in NAFLD. Super-paramagnetic iron oxide (SPIO) magnetic resonance imaging is a comparative technology dependent on KC phagocytic function. Objective To evaluate KC uptake function, in patients and experimental animals with NAFLD, using SPIO. Methods Abdominal CT and histological examination of liver biopsy specimens were used to estimate the degree of steatosis in patients with NAFLD and controls with chronic hepatitis C. SPIO-MRI was then performed in all patients. Normal rats fed a methionine-choline-deficient diet to induce non-alcoholic steatohepatitis (NASH), the more severe stage of NAFLD, and obese, insulin resistant, Zucker fa/fa rats with steatohepatitis, were also studied with SPIO-MRI and analysed for hepatic uptake of fluorescent microbeads. Immunohistochemical analysis evaluated the numbers of KCs in patients and rat livers. Results Relative signal enhancement (RSE), inversely proportional to KC function, was higher in patients with NAFLD than in controls and with the degree of steatosis on CT. RSE also positively correlated with the degree of steatosis on histology and was similarly higher in rats with induced severe NAFLD (NASH). On immunohistochemistry, defective phagocytic function was the result of reduced phagocytic uptake and not due to reduced KC numbers in rats or patients with NAFLD. Conclusions KC uptake function is significantly impaired in patients with NAFLD and experimental animals with NASH, worsens with the degree of steatosis and is not due to a reduction of KC numbers. Non-alcoholic fatty liver disease (NAFLD), is now the most common cause of chronic liver disease in industrialised nations. 2 It may progress to cirrhosis. The pathogenesis of NAFLD is incompletely understood but may involve hyperendotoxaemia, and indeed obesity and type 2 diabetes, commonly present in NAFLD, are associated with endotoxaemia. It was also recently reported that serum endotoxin levels were elevated, compared with controls, in mice fed a methioninecholine-deficient (MCD) diet to induce non-alcoholic steatohepatitis (NASH)dthe more severe stage of NAFLD. In addition, serum endotoxin levels in patients with NAFLD are greater than in patients without liver disease, and others have shown raised serum levels of lipopolysaccharidebinding protein (LBP) in patients with NAFLD and more so in patients with NASH. Finally, LBP mRNA levels in the livers of rats with high-fat dietinduced NASH were elevated compared with controls. As Gao and colleagues concluded in their study, therefore, the mechanism of injury in NAFLD may be related to increased levels of endotoxin. This observed hyper-endotoxaemia might be secondary to reduced clearance of endotoxin in NAFLD, possibly caused by an impaired Kupffer cell (KC) uptake function, at least in animal models of NAFLD, 9 resulting in overproduction of, and increased sensitivity to, cytokines such as tumour necrosis factor a and interleukin 1b from KCs. 11 KCs are phagocytic macrophages resident in the liver which provide the predominant protection against the egress of endotoxin from the portal to the systemic circulation. The status of KC uptake function in patients with NAFLD is unknown. This has, in part, been owing to an absence of methods to study this parameter reliably in situ. The introduction, however, of super-paramagnetic iron oxide (SPIO) allows such direct studies. SPIO is a liver-specific magnetic resonance imaging (MRI) contrast agent for detecting hepatocellular carcinoma (HCC). 14 The technique relies on the ability of KCs to take up SPIO particles. Since KCs are absent in HCC, differential uptake of SPIO particles allows radiological separation of normal liver from HCC lesions. Following intravenous SPIO, uptake by KCs leads to reduced signal intensity (SI) on T2 MRI sequences, such that, HCC which have no KCs show a high SI. Conversely, areas with abundant KCs show a low T2 SI. In normal liver, therefore, there is a low T2 SI. Since the SI with SPIO depends on uptake by KCs of SPIO contrast, SPIO can serve as a surrogate marker of KC uptake function. In this study, we used SPIO-MRI to test the hypothesis that patients with NAFLD have impaired KC uptake function. To investigate the underlying mechanisms and ascertain that impaired KC uptake function was not due to a reduction in KC numbers, we then used SPIO along with ingestion of fluorescent microbeads in rats fed an MCD diet as a model of NASH, a severe stage of NAFLD 19 and in obese, insulin resistant, steatohepatitic Zucker fa/fa rats, as an additional < Supplementary figures are published online only at http:// gut.bmj. com/content/vol59/ issue2 Department of Radiology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan Department of Veterinary Sciences, University of Miyazaki, Miyazaki, Japan Department of Gastroenterology and Hepatology, Kochi Medical School, Nankoku, Kochi, Japan Department of Radiology, Kochi Medical School, Nankoku, Kochi, Japan Department of Pathology, Kochi Medical School, Nankoku, Kochi, Japan Centre for Hepatology, University College London, UK Guy’s and St. Thomas’ Hospital, London, UK Correspondence to Masafumi Ono, Department of Gastroenterology and Hepatology, Kochi Medical School, Nankoku, Kochi 783-8505, Japan; nom@kochi-u.ac. jp Received 6 January 2009 Accepted 19 October 2009 Published Online First 16 November 2009 258 Gut 2010;59:258e266. doi:10.1136/gut.2009. 176651 Non-alcoholic fatty liver disease group.bmj. com on March 6, 2010 Published by gut.bmj. com Downloaded from