that infiltrating T cells are activated and produce inflamma-CD4 / T lymphocytes have been identified as being retory lymphokines. 3,4 In experimental autoimmune hepatitis, sponsible for organ damage in the murine model of ex-T-cell-mediated liver damage could be demonstrated. 5 Howperimental liver injury induced by intravenous injection ever, the early mechanisms leading to liver injury are poorly of concanavalin A (Con A). Liver sinusoidal endothelial characterized. Recruitment of antigen-specific and bystander cells (SEC) and Kupffer's cells (KC) are among the first T cells into the liver and the initiation of the inflammatory cells that come into contact with lymphocytes in the process are poorly understood. liver sinusoid. We aimed to investigate the respective
The recently described model of concanavalin A (Con A)role of these cell populations in the initial steps of Tcell-mediated liver injury in Con A-induced hepatitis. induced experimental liver injury in mice has been shown to By electron microscopy, we could show that intravebe mediated by activated T cells. 6 Con A is a T-lymphocyte nously applied Con A bound predominantly to SEC but mitogen in vitro that leads to production of cytokines (innot to KC. KC depletion by gadolinium chloride treatterleukin-2, tumor necrosis factor a [TNF-a], interferon ment of mice did not result in protection from liver ingamma) and lymphocyte proliferation. 7 Eight hours after injury, indicating that KCs are not primarily involved in travenous injection of Con A, a fulminant hepatitis develops the generation of liver injury. We could show that a CD4 / in mice with no other organs being affected. The organ speci-T-cell line (LNC.2) displayed selective cytotoxicity toficity is attributed to the preferential binding of Con A in the ward SEC (ú50%) but not KC (12%) or fibroblasts (5%) in liver. 8 Both the presence and the functional integrity of T the presence of Con A in vitro. Microscopic observation cells are required for Con A-induced hepatitis because imrevealed that the SEC monolayer was rapidly destroyed munodeficient mice (SCID or athymic mice) or mice treated by LnC 2 in the presence of Con A. Specificity of the Con with immunosuppressive drugs such as cyclosporin, FK506, A-induced cytotoxicity was shown by the ability of a or corticosteroids do not develop hepatitis after Con A chalcompetitive ligand, methyl-a-D-mannopyranoside, to relenge. 6 By cell-depletion experiments, CD4 / T cells were idenduce T-cell-mediated cytotoxicity to SEC by more than tified as effector cells. 6 TNF-a is a cytokine produced early 50%. Tumor necrosis factor a (TNF-a) was produced by in the course of Con A-induced hepatitis, and blockade of LnC 2 in high amounts after Con A stimulation (ú6 ng/ TNF-a action by antiserum was shown to protect against mL), but antiserum to TNF-a did not reduce LnC 2 -medidevelopment of liver injury. 8,9 ated cytotoxicity toward SEC. In conclusion, we could
The aim of the present study was to characterize the differshow for the first time that liver SECs have accessory ential role of sinusoidal endothelial cells and Kupffer's cells function and are selectively destroyed by CD4 / T lym-(KCs) in the initial steps of T-cell-mediated liver injury in phocytes in the presence of Con A. We speculate that the model of Con A hepatitis.
SEC damage is an early event in T-cell-mediated liver injury recruiting T lymphocytes from the sinusoidal cir-MATERIALS AND METHODS
culation. Loss of the SEC barrier function then exposes underlying hepatocytes to further attack by activated T
Reagents. All lectins (Con A, Wheat-Germ-Agglutinin I [WGA I], lymphocytes. These results offer a model of initiating Ulex-Europa ¨us I [UEA I]), whether native/fluorescein isothiocya- events in T-cell-mediated liver diseases, such as viral or nate-labeled or Au -labeled, were purchased from Sigma (Mu ¨nchen, autoimmune hepatitis, and suggest an important role Germany). Gadolinium chloride (GdCl 3 ) came from Aldrich (Steinheim, Germany). Lipopolysaccharide (B55:50) S. minnesota for sinusoidal endothelial cells. (HEPATOLOGY 1996;24: was from Sigma. The monoclonal antibodies for TNF-a were from
824-829.)
Pharmingen (San Diego, CA), and a specific sandwich enzyme-linked Activated T lymphocytes appear to be responsible for organ immunosorbent assay was performed according to the manufactur- er's instructions. Antiserum to TNF-a was kindly provided by Prof. damage in chronic viral hepatitis and autoimmune liver dis-Dr. Wendel, Konstanz. Recombinant TNF-a was supplied by Dianova ease. They are found as infiltrating cells in the periportal (Hamburg, Germany). 51 Chromium was purchased from Amersham area in all forms of chronic active hepatitis. 1,2 It was shown (Braunschweig, Germany). Methyl-a-D-mannopyranoside was purchased from ICN Flow (Meckenheim, Germany).
Sheep red blood cells were obtained from BAG-Hessen (Lich, Ger-Abbreviations: Con A, concanavalin A; TNF-a, tumor necrosis factor a; KC, Kupffer's many), and antiserum to sheep red blood cells was obtained from cell; WGA I, Wheat-Germ-Agglutinin I; UEA I, Ulex Europa ¨us I; FITC, fluorescein isothiocyanate; SEC, sinusoidal endothelial cell; PBS, phosphate-buffered saline.
Camon (Wiesbaden, Germany).