Fibronectin Leukemia Virus Type 1-Infected T Cells to Regulates
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SFA-1/PETA-3 (CD151), a Member of the Transmembrane 4 a b Superfamily, Associates Preferentially with 5 1 Integrin and Regulates Adhesion of Human T Cell Leukemia Virus Type 1-Infected T Cells to Fibronectin1 Hitoshi Hasegawa,2 Tetsuhiko Nomura, Kyoko Kishimoto, Kohsuke Yanagisawa, and Shigeru Fujita In this study we have analyzed the adhesion molecules associated with and the biologic function of SFA-1/PETA-3 (CD151) in human T cell leukemia virus type 1 (HTLV-1)-infected T cells and in freshly isolated adult T cell leukemia (ATL) cells using an anti-CD151 a b a mAb. The anti-CD151 mAb coprecipitated 5 1 integrin from HTLV-1-infected T cells. Conversely, an anti- 5 integrin mAb copre- cipitated CD151. The anti-CD151 mAb inhibited the adhesion of HTLV-1-infected T cells to fibronectin but did not have any effect on their adhesion to laminin, collagen type I, or collagen type IV. Moreover, antisense CD151 oligonucleotide-treated HTLV-1-infected T cells showed significant inhibition of adhesion to fibronectin. These findings showed that the CD151 molecule was associated with the a b a b 5 1 integrin molecule and that it enhanced 5 1 integrin-mediated adhesion to fibronectin. In addition, the expression levels of CD151, a b a b 4 1 integrin, and 5 1 integrin on ATL cells from lymph nodes of lymphoma-type ATL patients were significantly higher than those on circulating ATL cells from leukemia-type ATL patients. This suggests that the increased expression of these integrins may contribute to lymphoma formation through the adhesion of ATL cells to the extracellular matrix and dendritic cells, rather than contributing to transmigration. The Journal of Immunology, 1998, 161: 3087–3095. uman T cell leukemia virus type 1 (HTLV-1)3 is an ex- cells as well as probes obtained from normal CD41 T cells and the ogenous human retrovirus closely linked with adult T MOLT-4 cell line (18). SFA-1 and PETA-3 were assigned CD151 at H cell leukemia (ATL). HTLV-1 has also been reported to the Sixth Human Leukocyte Differentiation Antigen Workshop. Hu- be associated with myelopathy, alveolitis, arthropathy, Sjo¨gren man SFA-1 (CD151) was found to be up-regulated upon transforma- syndrome, and uvenitis, which may result from immunologic al- tion by HTLV-1 and is trans-activated by Tax. The mRNA of the terations induced by HTLV-1 infection (1–5). The HTLV-1 ge- human SFA-1 (CD151) gene is comprised of approximately 1.6 kb nome encodes a 40-kDa protein, Tax, that functions as a transcrip- and encodes a protein of 253 amino acids. SFA-1 is a member of the tional trans-activator of viral and cellular gene expression. T cell transmembrane 4 superfamily (TM4SF). The human SFA-1/PETA-3 proliferation and immunologic alterations observed during (CD151) gene is a single gene located on chromosome 11p15.5 (19). HTLV-1 infection appear to be due to the effect of Tax on viral and Moreover, CD151 is conserved between human and mouse (20). cellular gene expression. Tax stimulates the expression of various PETA-3 was originally identified as a human platelet surface glyco- cellular genes, including IL-2, IL-2Ra, granulocyte-macrophage protein, and it has been reported to regulate platelet aggregation and CSF, TNF-b, TGF-b,c-fos,c-jun, Krox-20, and Krox-24, and vi- mediator release (21–23). mentin and suppresses the expression of the genes, b-polymerase, The TM4SF is a family of membrane proteins that are charac- p53, NF-1, and lck (5–17). However, the mechanism of HTLV-1- terized by the presence of four highly conserved transmembrane induced disease still remains to be elucidated. domains (reviewed by Refs. 24–26). This family currently has 19 Previously, to examine the changes in CD41 T cells after HTLV-1 members that are found in species from Schistosoma to human: infection, we have cloned SFA-1 by differential hybridization of a CD9, CD37, CD53, CD63/ME491, CD81/TAPA-1, CD82/C33/ cDNA library, using probes obtained from an HTLV-1-infected T R2/KAI1, CO-029, A15, lbl, CD151/SFA-1/PETA-3, SAS, sm23, sj23, il-TMP, L6, peripherin, Rom-1, uroplakin Ia, and uroplakin First Department of Internal Medicine, Ehime University School of Medicine, Shig- Ib. TM4SF members play roles in signal transduction pathways enobu, Ehime, Japan and regulate cell activation, development, proliferation, motility, Received for publication January 29, 1998. Accepted for publication May 11, 1998. and adhesion of a number of cell types. In addition, TM4SF mem- The costs of publication of this article were defrayed in part by the payment of page bers can form noncovalent associations with each other and with charges. This article must therefore be hereby marked advertisement in accordance other molecules, such as those involved in signal transduction and with 18 U.S.C. Section 1734 solely to indicate this fact. adhesion. In this report, we describe the adhesion molecules asso- 1 This work was supported in part by a grant-in-aid from the Osaka Cancer Research ciated with CD151 and its biologic function in HTLV-1-infected T Foundation and Scientific Research, and the Ministry of Education, Science, and Culture of Japan. cells and freshly isolated ATL cells. 2 Address correspondence and reprint requests to Dr. Hitoshi Hasegawa, First De- partment of Internal Medicine, Ehime University School of Medicine, Shigenobu, Materials and Methods Ehime 791-02, Japan. E-mail address: [email protected] Cells 3 Abbreviations used in this paper: HTLV-1, human T cell leukemia virus type 1; ATL, adult T cell leukemia; TM4SF, transmembrane 4 superfamily; PE, phyco- Human T cell lines, MOLT-4 and Jurkat; a human erythroleukemia cell erythrin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; line, K562; two human myelomonocytoid cell lines, HL60 and U937; and MFI, mean fluorescence intensity. NIH-3T3 cells were obtained from the American Type Culture Collection Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 3088 INTEGRIN ASSOCIATION WITH CD151 MOLECULE (Rockville, MD). A human glioblastoma cell line, A172, and a human renal Preparation of mAbs carcinoma cell line, Caki-1, were obtained from the Japanese Cancer Re- search Resources Bank (Tokyo, Japan). These cell lines were maintained in mAbs were produced by hybridoma technology as described previously RPMI 1640 or DMEM supplemented with 10% heat-inactivated FCS (Life (34). In brief, hybridomas were produced through the fusion of Technologies, Gaithersburg, MD). Three HTLV-1-infected T cell lines, P3X63Ag8.653 cells with spleen cells from BALB/c mice immunized a MT-2, MT-4, and HUT 102, were maintained in RPMI 1640 medium sup- against the NIH-3T3/pL2neoSR IIISFA-1 cells. The hybridoma culture a plemented with 10% FCS. The HTLV-1-infected T cell line, SF-HT (27), supernatants that bound to NIH-3T3/pL2neoSR IIISFA-1 cells but not to a was maintained in RPMI 1640 medium supplemented with 10% FCS and NIH-3T3/pL2neoSR III were screened. After screening, selected colonies 100 U/ml human rIL-2 (Takeda Chemical Industries, Osaka, Japan). Mono- were cloned twice or more by the limiting dilution method. After cloning, cytes were enriched as described previously (28). Normal T and B cells the Ab-producing hybridomas were inoculated into BALB/c mice treated were separated using the sheep erythrocyte-rosetting method (29). previously with pristane (Aldrich, Milwaukee, WI). Ascitic fluid contain- ing Ab was obtained after about 2 wk. The mAbs from the ascitic fluids were purified by affinity chromatography on a DEAE column. ATL patients Immunoprecipitation Mononuclear cells were isolated from peripheral blood samples from 14 patients with leukemia-type and from the lymph nodes of 10 patients with Cell surface proteins were labeled with 125I using carrier-free Na 125I and lymphoma-type by a Ficoll-Conray density gradient centrifugation. Con- lactoperoxidase (ICN, Costa Mesa, CA) following the instructions of the trol PBMCs were obtained from 10 normal healthy volunteers. The control manufacturer. Labeled cells were lysed in either Nonidet P-40 lysis buffer lymph nodes samples were prepared from the reactive lymph nodes of (10 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 3 mM MgCl2, 2 mM PMSF, eight HTLV-1-seronegative individuals who had undergone abdominal 0.5% Nonidet P-40), or CHAPS lysis buffer (10 mM Tris-HCl (pH 8.0), surgery. ATL was diagnosed according to the following clinical criteria: 0.15 M NaCl, 2 mM PMSF, 1 mg/ml antipain, 1 mg/ml pepstatin, 1 mg/ml serum Abs against HTLV-1-associated Ags; morphologic characteristics leupeptin, 1 mg/ml chymostatin, 10 mM iodoacetamine, and 1% CHAPS; showing highly convoluted nuclei; phenotypic analysis of ATL cells with all reagents were purchased from Sigma, St. Louis, MO). The lysates were anti-CD2, anti-CD4, and anti-CD25 mAbs; and monoclonal integration of centrifuged, and the supernatants were precleared overnight at 4°C with the HTLV-1 proviral genome in the cells. Using Shimoyama’s criteria (30), protein G-Sepharose (Pharmacia, Piscataway, NJ) precoated with normal 11 of 14 leukemia-type ATL patients were diagnosed with acute ATL, and rabbit serum. The lysates were then incubated with protein G-Sepharose 3 had chronic or smoldering ATL. Six of the leukemia-type ATL patients precoated with each mAb. After washing extensively with lysis buffer, had involvement of the skin, gut, or lymphoid organs such as the lymph samples were boiled for 2 min in Laemmli’s electrophoresis sample buffer nodes, liver, or spleen. The infiltration of ATL cells into the skin, gut, or and fractionated by SDS-PAGE.