Lethal Hepatitis After Gene Transfer of IL-4 in the Liver Is Independent of Immune Responses and Dependent on Apoptosis of Hepatocytes: A Rodent Model of IL-4-Induced Hepatitis 1 Ce ´cile Guillot, 2 * He ´le `ne Coathalem, 2 * Je ´ro ˆ me Chetritt,* Anne David,* Pedro Lowenstein, ² Emmanuelle Gilbert,* Laurent Tesson,* Nico van Rooijen, ‡ Maria Cristina Cuturi,* Jean-Paul Soulillou,* and Ignacio Anegon 3 * The putative role of IL-4 in human and animal models of hepatitis has not yet been directly determined. We now report that direct expression of IL-4 in the liver of rats or mice using recombinant adenoviruses coding for rat or mouse IL-4 (AdrIL-4 and AdmIL-4, respectively) results in a lethal, dose-dependent hepatitis. The hepatitis induced by IL-4 was characterized by hepatocyte apoptosis and a massive monocyte/macrophage infiltrate. IL-4-induced hepatitis was independent of T cell-mediated immune responses. Hepatitis occurred even after gene transfer of IL-4 into nude rats, CD8-depleted rats, cyclosporine A-treated rats, or recombinase-activating gene 2 / immunodeficient mice. Peripheral depletion of leukocytes using high doses of cyclophospha- mide, and/or the specific depletion of liver macrophages with liposome-encapsulated dichloromethylene diphosphonate in rats did not block lethal IL-4-induced hepatitis. Direct transduction of hepatocytes with adenoviruses was not essential, since injection of AdrIL-4 into the hind limb induced an identical hepatitis. Finally, primary rat hepatocytes in culture also showed apoptosis when cultured in the presence of rIL-4. IL-4-dependent hepatitis was associated with increases in the intrahepatic levels of IFN-, TNF-, and Fas ligand. Administration of AdmIL-4 to IFN-, TNF- receptor type I, or TNF- receptor type II knockout mice also resulted in lethal hepatitis, whereas a moderate protection was observed in Fas-deficient lpr mice. IL-4-dependent hepatocyte apoptosis could be abolished by treatment with caspase inhibitory peptides. Our results thus demonstrate that IL-4 causes hepatocyte apoptosis, which is only partially dependent on the activation of Apo-1-Fas signaling and is largely independent of any immune cells in the liver. The Journal of Immunology, 2001, 166: 5225–5235. T he attempt to immunomodulate human immune responses using IL-4 has been hampered by the development of clinically relevant hepatic injury in patients and primates (1– 4). Furthermore, various other liver diseases, such as autoim- mune hepatitis (5), chronic hepatitis (5), and primary biliary cir- rhosis (6) have all been associated with increased IL-4 production. Hepatocytes express IL-4R (7), and IL-4 has been shown to mod- ulate the function of hepatocytes in vitro (8, 9) and in vivo (10). Also, IL-4 is essential for the development of hepatic lesions in the widely used model of Con A-induced hepatitis (11). However, the direct role of IL-4 and the mechanisms of IL-4-mediated hepatic injury have not yet been determined. IL-4 is a major cytokine released during T cell responses po- larized toward a Th2 phenotype. IL-4 itself displays a variety of either anti- or proinflammatory actions, mainly on cells of various hemopoietic lineages (8). Its actions include antiapoptotic actions on B cells (12) and proapoptotic effects on macrophages (13), mast cells (14), or eosinophils (15). Although IL-4 has also been de- scribed as modulating functions of nonhemopoietic cells, such as endothelial cells, fibroblasts, and hepatocytes (8), much less is known about its actions on these cells compared with those on hemopoietic cells. We recently showed that adenovirus-mediated IL-4 expression in the liver by intraportal administration of 2.5  10 9 PFU of adenovirus coding for rat IL-4 (AdrIL-4) 4 results in nonlethal, re- versible hepatitis in rats (16). At this dose of AdrIL-4 the hepatitis is partially due to an increase in cellular and humoral anti- adenovirus immune responses (17). In the present study, we show that higher doses of AdrIL-4 (10 10 PFU) induce lethal hepatitis in rats, we reproduced this lethal hepatitis model in mice using an adenovirus coding for mouse IL-4 (AdmIL-4) and investigated the inflammatory and immune mechanisms as well as the role of ap- optosis underlying acute and lethal IL-4-mediated hepatitis. Our present results show that expression of IL-4 in the liver results in extensive hepatocyte apoptosis and mortality, which are *Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 437, Nantes, France; ² Molecular Medicine and Gene Therapy Unit, University of Manchester School of Medicine, Manchester, United Kingdom; and ‡ Department of Cell Biology and Im- munology, School of Medicine, Free University, Amsterdam, The Netherlands Received for publication August 22, 2000. Accepted for publication February 9, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by the Fondation Transvie, the Ligue Nationale Contre le Cancer, the Association pour la Recherche sur le Cancer, and European Union Grant Biomed2 (BMM-CT98-3277). 2 C.G. and H.C. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Ignacio Anegon, Institut Na- tional de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 437, 30 Boulevard Jean Monnet, 44093 Nantes, France. E-mail address: ianegon@nantes.inserm.fr 4 Abbreviations used in this paper: AdrIL-4, adenovirus coding for rat IL-4; AdmIL-4, adenovirus coding for mouse IL-4; FasL, Fas ligand; RAG, recombinase-activating gene; TNF-RI, TNF- receptor type I; ALAT, alanine transaminase; ASAT, aspar- tate transaminase; CL2-MDP, liposome-encapsulated dichloromethylene diphospho- nate; CsA, cyclosporine A; CyP, cyclophosphamide; HES, hematoxylin-eosin-saf- fron; HPRT, hypoxanthine phosphoribosyltransferase; ICE, IL-1-converting enzyme; moi, multiplicity of infection; X-Gal, X-galactosidase. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00