Mice Lpr Disease in MRL- Lymphoproliferation but Not Autoimmune Transgenic Expression of Fas in T Cells Blocks

Transgenic Expression of Fas in T Cells Blocks Lymphoproliferation But Not Autoimmune Disease in MRL-lpr Mice This information is current as Hidehiro Fukuyama, Masashi Adachi, Sachiko Suematsu, Keiko of September 25, 2021. Miwa, Takashi Suda, Nobuaki Yoshida and Shigekazu Nagata J Immunol 1998; 160:3805-3811; ; http://www.jimmunol.org/content/160/8/3805 Downloaded from References This article cites 52 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/160/8/3805.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Transgenic Expression of Fas in T Cells Blocks Lymphoproliferation But Not Autoimmune Disease in MRL-lpr Mice1 Hidehiro Fukuyama,*† Masashi Adachi,* Sachiko Suematsu,2‡ Keiko Miwa,* Takashi Suda,* Nobuaki Yoshida,‡ and Shigekazu Nagata3*† Fas is a member of the TNF receptor family. Binding of Fas ligand to Fas induces apoptosis in Fas-bearing cells. Fas is expressed in various cells, including thymocytes, peripheral T cells, and activated B cells. The mouse lpr mutation is a loss of function mutation of Fas. MRL-lpr/lpr mice develop lymphadenopathy and splenomegaly, and produce multiple autoantibodies, which results in autoimmune disease. In this report, we describe the establishment of a line of Fas transgenic MRL-lpr mice in which mouse Fas cDNA was expressed using the T cell-specific murine lck promoter. The transgenic mice expressed functional Fas in Downloaded from thymocytes and peripheral T cells, but not in B cells. The transgenic mice did not accumulate abnormal T cells (Thy-11 B2201), but still accumulated B cells (Thy-12 B2201); they produced a large quantity of Igs (IgG1 and IgG2a), including anti-DNA Abs, and developed glomerulonephritis. These results suggest that autoreactive or activated B cells must be killed through Fas expressed in the B cells by the Fas ligand expressed in activated T cells. The Journal of Immunology, 1998, 160: 3805–3811. omeostasis in the immune system is maintained not only They develop lymphadenopathy and splenomegaly and suffer from http://www.jimmunol.org/ by growth of lymphocytes but also by cell death. Fas autoimmune disease caused by the production of autoantibodies H ligand (FasL)4 is a type II membrane protein belonging (autoimmune lymphoproliferative syndrome) remarkably similar to the TNF family (1, 2). It is predominantly expressed in activated to those found in mouse lpr and gld mutants. T cells and NK cells (3–5). Fas, the receptor for FasL, is a type I Animals carrying the mutation in Fas or FasL accumulate cells that membrane protein, and a member of the TNF receptor family (2, have the surface phenotypes Thy-11 B2201 CD42 CD82 and are of 6). It is expressed in various cells, including thymocytes, mature T T cell origin (11). The cells also express various activation Ags of T cells, and activated B cells (3, 7–9). Binding of FasL to Fas in- cells, such as CD69 and FasL (20, 21). The lymphoproliferation in the duces apoptosis in Fas-bearing cells and kills them within hours Fas-deficient animals is explained as follows. The activation of T cells by guest on September 25, 2021 (2). This is one of the effector systems of cytotoxic T cells and NK by foreign Ags activates CD41-type T cells to produce various lym- cells (10). phokines and cell surface molecules that stimulate the proliferation Mice carrying the mutation of lymphoproliferation (lpr) or gen- and differentiation of lymphocytes. CD81 T cells are also activated by eralized lymphoproliferative disease ( gld) develop lymphadenop- foreign Ags and kill the target cells. These activated T cells must be athy and splenomegaly (11). They also produce large amounts of removed after they have accomplished their tasks. This deletion pro- Igs, including anti-dsDNA or anti-ssDNA Abs, and suffer from cess, called activation-induced suicide of T cells, is mainly mediated autoimmune diseases such as nephritis and vasculitis, particularly by Fas and FasL (22–24). The activated T cells express FasL, which in the mouse strain MRL. The lpr and gld mutations are loss of kills the activated cells by an autocrine or paracrine mechanism. The function mutations of Fas and FasL, respectively (12). Fas-null activated T cells in animals that have defects in Fas or FasL cannot mice, established by gene targeting, show phenotypes that are undergo activation-induced suicide, and they accumulate in the more severe than the leaky lpr mutation (13). Human patients car- periphery (20, 25). rying mutations in Fas or FasL have also been identified (14–19). In contrast to the lymphoproliferation of T cells, it is not clear how animals that are deficient in Fas and FasL produce autoantibodies. Activated B cells express Fas, and they are sensitive to *Department of Genetics, Osaka University Medical School, Yamadaoka, Suita; †Osaka Bioscience Institute, Furuedai, Suita; and ‡Osaka Medical Center for Maternal Fas-induced apoptosis. The number of B cells is increased in lpr and Child Health, Izumi, Osaka, Japan and Fas-null mice (13, 26), and a large number of autoantibody- Received for publication September 8, 1997. Accepted for publication December producing cells was found within the T cell zone of the spleen in 22, 1997. lpr mice (27). From these results, it was suggested that the auto- The costs of publication of this article were defrayed in part by the payment of page reactive B cells are killed through the interaction of FasL-express- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ing T cells with Fas-expressing B cells (28, 29). In this report we have established Fas transgenic MRL-lpr mice 1 This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan, and by Special Coordination Funds from the Science in which Fas was specifically expressed in T cells but not in B and Technology Agency of the Japanese Government. cells. The mice did not accumulate Thy-11 B2201 T cells, yet 2 Present address: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA produced large amounts of Igs, causing glomerulonephritis. These 92037. results indicate that lymphoproliferation of T cell origin and the 3 Address correspondence and reprint requests to Dr. Shigekazu Nagata, Department production of autoantibodies are independent processes caused by of Genetics, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. E-mail address: [email protected] defective Fas in T cells and B cells, respectively, and support the 4 Abbreviations used in this paper: FasL, Fas ligand; gld, generalized lymphoprolif- proposal that the autoreactive B cells are removed, at least in part, erative disease; lpr, lymphoproliferation; PE, phycoerythrin; mFas, mouse Fas. by Fas-mediated apoptosis. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 3806 TRANSGENIC EXPRESSION OF FAS IN T CELLS OF lpr MICE Materials and Methods 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in 10 mM 1-methylimi- 3 3 Transgenic mice dazole, pH 7.0. The plates were extensively washed with 5 SSC (1 SSC is composed of 150 mM NaCl and 15 mM sodium citrate) containing The expression vector, p1017 (30), carrying the proximal murine lck pro- 0.25% SDS at 55°C. Nonspecific binding sites on the DNA-coated plates moter and the human growth hormone gene sequence, was provided by Dr. were blocked by incubation for 60 min at room temperature with blocking R. M. Perlmutter (University of Washington, Seattle, WA). The coding solution (Tris-buffered saline (10 mM Tris-HCl, pH 7.4, and 140 mM sequence of mouse Fas cDNA (EcoRI-AccI fragment of pMF1) (31) was NaCl) containing 1% BSA and 3 mM EDTA). Mouse sera were diluted 100 inserted into the BamHI site of p1017 using a BamHI linker, and the re- times with the blocking solution, and 50-ml aliquots were applied to the sulting construct was designated p1017Fas. The p1017Fas plasmid DNA DNA-coated plates and incubated for 60 min at room temperature. After was digested with NotI, and a 6.7-kb DNA fragment (5 ng/ml) carrying washing three times with the washing buffer (Tris-buffered saline contain- mouse Fas cDNA between the lck promoter and human growth hormone ing 0.1% Nonidet P-40, 3 mM EDTA, and 1% gelatin), the plates were sequence was isolated. This fragment was microinjected into the pronuclei incubated for 60 min with peroxidase-conjugated goat anti-mouse Igs of fertilized eggs from MRL-lpr mice. Transgenic mice were screened by (Cappel) at a dilution of 1/4000. The peroxidase activity was detected using Southern blot analysis with mouse Fas cDNA as the probe, using tail DNA o-phenylenediamine as a substrate with a peroxidase-detecting kit (Sumi- prepared as previously described (32). Two transgene-positive lines were tomo, Tokyo, Japan), and the absorbance was measured at 492 nm using a established by backcrossing founder animals with MRL-lpr mice.

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