Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Journal of General Virology (2001), 82, 831–835. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION Vascular cell adhesion molecule-1 induced by human T-cell leukaemia virus type 1 Tax protein in T-cells stimulates proliferation of human T-lymphocytes He! le' ne Valentin,1, 2 Samir Hamaia,1 Ste! phane Ko$ nig1 and Louis Gazzolo1 1 Virologie et Pathoge! ne' se Virale (UMR5537), CNRS-UCB Lyon 1, Faculte! de Me! decine Laennec, Rue Guillaume Paradin, 69372 Lyon Cedex 08, France 2 Immunobiologie Fondamentale et Clinique, INSERM U503, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France Human T-cell leukaemia/lymphotropic virus type 1 (HTLV-1), aetiologically linked to lymphoproliferative as well as inflammatory diseases, infects and activates CD4M helper T-cells and thus alters immunoregulatory pathways. The viral regulatory Tax protein has been shown previously to induce the expression of vascular cell adhesion molecule-1 (VCAM-1) by T-cells. To determine the functional role of this adhesion molecule, Jurkat Tcells stably expressing either Tax or both Tax and Rex (another viral regulatory protein) were used in binding and coculture assays performed with either control Jurkat cells or primary human T-lymphocytes. Evidence was provided that VCAM-1 acting in synergy with leucocyte function-associated antigen-3 promotes T-cell–T-cell interactions and increases T-cell proliferation. Interestingly, Rex was found to modulate these events. These data establish that VCAM-1 induced by Tax on T-cells thus contributes to the immunopathological process triggered by HTLV-1 infection. Among individuals infected with human T-cell leukaemia\ lymphotropic virus type 1 (HTLV-1), a small percentage develop a lymphoproliferative disease, adult T-cell leukaemia (ATL) or a chronic inflammatory disease, such as tropical spastic parapesis\HTLV-1-associated myelopathy (TSP\ HAM) (reviewed by Uchiyama, 1997). HTLV-1 is considered to be a retrovirus that alters immunoregulatory pathways. In vivo and in vitro studies have demonstrated that HTLV-1 infects CD4+ helper T-cells, which are then characterized by an increased expression of activation markers and spontaneous proliferation. Both processes are dependent on the activity of Author for correspondence : He! le' ne Valentin at INSERM. Fax j33 4 37 28 23 41. e-mail valentin!cervi-lyon.inserm.fr 0001-7543 # 2001 SGM the viral regulatory protein Tax, which is involved in the transcriptional activation of the provirus and expression of many cellular genes implicated in T-cell activation and proliferation. More specifically, Tax has been shown to intervene in T-cell–T-cell interactions by enhancing the expression of an adhesion molecule, leukocyte functionassociated antigen-3 (LFA-3 or CD58), which is the ligand for CD2 (Tanaka et al., 1995). The interaction of LFA-3 expressed by HTLV-1-infected T-cells with CD2 on resting T-cells defines an important costimulatory pathway in triggering the proliferation of resting T-cells (Wucherpfennig et al., 1992). In addition, other adhesion molecule interactions, such as intercellular adhesion molecule-1 (ICAM-1 or CD54) with LFA-1 (CD11a\CD18), have been suggested to participate, first, in HTLV-1-mediated activation of uninfected T-cells, facilitating lymphoproliferation, and second, in increased adhesion to endothelial cells, resulting in tissue or organ infiltration (Ichinose et al., 1992, 1994). We have reported previously the expression of vascular cell adhesion molecule-1 (VCAM-1 or CD106) by Jurkat cells stably expressing the tax gene and shown that Tax was transactivating the VCAM-1 gene via two NF-κB sites present in the gene promoter (Valentin et al., 1997). Moreover, this VCAM-1 up-regulation has been confirmed in freshly isolated T-cells from TSP\HAM patients (Valentin et al., 1997). VCAM-1 may serve as either an accessory molecule or a potential coreceptor for HTLV-1-induced cell fusion (Daenke et al., 1999 ; Hildreth et al., 1997). VCAM-1 is normally expressed by stimulated endothelial cells, therefore favouring the adhesion of these cells to T-lymphocytes via VLA-4, which is the VCAM-1 ligand that is constitutively expressed by T-cells. Thus, this adhesion pathway may be important in mediating emigration of leukaemic T-cells into the organs of ATL patients and entry of activated T-cells into the central nervous system of individuals with TSP\HAM (Ishikawa et al., 1993 ; Uchiyama, 1997). Furthermore, a recent report has underlined the role of VCAM-1 in the induction of matrix metalloproteinase-2, which is an important mediator for the extravagation of T-cells and the degradation of the subendo- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 13:06:36 IDB H. Valentin and others Fig. 1. Comparative analysis by flow cytometry of cell surface (A) LFA-3 (CD58), (B) VCAM-1 (CD106) and (C) VLA-4 (CD29/CD49d) expression by parental Jurkat cells, by control neomycin-resistant Jurkat cells (C9), and by Jurkat cells stably expressing either Tax (50) or both Tax and Rex (C11 and E12). Bars represent the mean of fluorescence intensity. Data represent the meanpSD from 3 to 10 experiments. The asterisks (C) indicate a statistically significant difference compared with control, as calculated by Student’s t-test (P 0n05). thelial basement membrane (Kambara et al., 1999). Alternatively, we have proposed that VCAM-1 expressed by HTLV1-infected T-cells might favour the interaction of these cells with each other and with uninfected T-cells expressing VLA-4. IDC Indeed such interactions may lead to cell fusion and\or transmission of HTLV-1. In the present study, we have investigated the functional consequences of Tax-induced VCAM-1 expression by T-cells in the activation and proliferation of normal resting T-cells. To that aim, we have used, in addition to parental Jurkat and Taxnegative neomycin-resistant Jurkat cells, three clones of Jurkat cells stably expressing either Tax (clone 50) or Tax and Rex, another viral regulatory protein, but acting at a posttranscriptional level (clones C11 and E12) (Lemasson et al., 1997). We first performed a flow cytometry analysis to compare the expression of VCAM-1 to that of LFA-3 by the different Jurkat cell lines. We confirmed that VCAM-1 was significantly expressed by Tax-expressing Jurkat cells (C11, E12 and 50) compared with the Tax-negative C9 and parental T-cells (Fig. 1 B) (Valentin et al., 1997). Furthermore, the expression of LFA-3 was found to be up-regulated in the three Tax-positive clones when compared to the Tax-negative cells (Fig. 1 A). We then analysed the profile of VLA-4 (CD29\ CD49d) expression. In contrast to CD29 (β1 integrin) expression, CD49d (α4 integrin) down-regulation was statistically correlated, by the Student t-test, to Tax expression in T-cells (Fig. 1 C). These data propose that Tax expression might induce a switch of VLA-4 expression to its VCAM-1 ligand on T-cells. As the level of CD49d expression has been reported to vary among ATL and HTLV-1-infected cases (AlFahim et al., 1999 ; Dhawan et al., 1993 ; Ishikawa et al., 1993 ; Uchiyama et al., 1996 ; Wake et al., 1995), it cannot be excluded that the expression of this integrin may be dependent on the level of Tax or of any viral or cellular protein. Next, we investigated the functional consequences of VCAM-1 and\or LFA-3 expressed by Jurkat cells stably expressing Tax. We first evaluated the ability of Jurkat Taxpositive cells to adhere to Tax-negative T-cells by rosette assay analysis. Briefly, 1i10& Tax-negative T-cells were labelled with Hoechst H33342 fluorochrome (Sigma), as described previously (Weston & Parish, 1990), and then mixed with either Tax-negative or Tax-expressing T-cells for 15 min at 37 mC. No rosette formation was observed between parental Jurkat cells, between parental Jurkat and Tax-negative neomycin-resistant Jurkat (C9) cells or between C9 cells. On the contrary, from 15 to 48 % of Tax-negative T-cells were found to bind to C11, E12 and 50 Tax-expressing T-cells (Table 1 and data not shown). These results underline the fact that Tax is mediating T-cell–T-cell interactions. To determine the involvement of adhesion molecules, Tax-expressing cells were first incubated with specific MAbs (10 µg\ml for 40 min at 4 mC) and then with labelled Tax-negative T-cells. Contrary to an incubation with an irrelevant MAb (to CD46) which was unable to interfere with rosette formation, inhibitions of 38 and 48 % were observed when Tax-expressing T-cells were preincubated with blocking MAbs to LFA-3 or VCAM-1, respectively (Table 1). This inhibition increased up to 75 % when Tax-expressing cells were treated with both MAbs. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 13:06:36 Tax-induced VCAM-1 and T-cell proliferation Table 1. Effect of blocking VCAM-1 and LFA-3 MAbs on binding to parental Jurkat cells Control parental Jurkat cells were labelled with Hoechst H33342 fluorochrome and incubated with Jurkat E12 cells stably expressing Tax and Rex, at a ratio of 10 : 1, respectively, for 30 min at 4 mC. E12 cells were pre-incubated with medium (none), an irrelevant MAb to CD46 (MCI20.6) or with blocking MAbs to LFA-3 (AICD58) or VCAM-1 (1G11) at 10 µg\ml. Cells were then observed with a fluorescence microscope (Nikon-Diaphot ELW D 0.3). A rosette was scored when at least three labelled cells were binding one unlabelled cell. Data represent the mean of three separate experiments (SD 10 %). Results are expressed as percentage rosette formation (labelled parental Jurkat cells bound to E12 cells) or percentage inhibition of rosette formation observed with MAb-treated E12 cells to that observed with untreated E12 cells. Pre-treatment of E12 cells None Anti-CD46 Anti-LFA-3 Anti-VCAM-1 Anti-LFA-3 j anti-VCAM-1 Rosette formation (%) Inhibition of rosette formation (%) 36n5 34n6 22n5 19n0 9n0 – 5n2 38n3 47n9 75n3 These data clearly provide evidence that VCAM-1 induced in Tax-positive T-cells is involved in T-cell–T-cell interactions and that both VCAM-1 and LFA-3 are acting synergistically in this adhesion process. These data suggest that these adhesion molecules may increase T-cell proliferation by enhancing binding of Taxexpressing T-cells to normal T-cells. To verify this hypothesis, human T-lymphocytes were purified and cultured, as described elsewhere (Huet et al., 1986). Briefly, mononuclear cells, obtained by Ficoll–hypaque centrifugation of peripheral blood from normal healthy donors (E! tablissement de Transfusion Sanguine de Lyon), were incubated for 1 h at 37 mC in plastic culture dishes. Nonadherent cells were then depleted of phagocytic cells by carbonyl iron ingestion and passage over a magnet. Accessory cells were lysed after incubation with leucine methyl ester. After eliminating residual HLA-DR cells, this preparation was analysed by flow cytometry and found to contain more than 95 % CD2+CD3+ cells (data not shown). Under these conditions, these T-lymphocytes were unable to proliferate when stimulated via the CD3\TCR pathway unless they were incubated with accessory cells. We therefore investigated whether the Tax-expressing Jurkat cells were endowed with such a property. To investigate this, Taxnegative or -positive cells were fixed with 1 % paraformaldehyde. No significant [$H]thymidine incorporation by these cells could be detected (less than 300 c.p.m.). Then, 2i10% fixed cells were incubated with 5i10% T-lymphocytes in the Fig. 2. Comparative analysis of the ability of parental Jurkat cells, control neomycin-resistant Jurkat cells (C9) and Jurkat cells stably expressing either Tax (50) or both Tax and Rex (C11 and E12) to induce proliferation of normal human T-lymphocytes. (A) Purified T-lymphocytes incubated in 96-well plates coated with anti-CD3 MAb for 1 day and then cocultivated for 3 days at 37 mC with paraformaldehyde-fixed Jurkat cells. Cells were then incubated with 1 µCi [3H]thymidine for 16 h. Data are expressed as [3H]thymidine (c.p.m.) incorporated by T-lymphocytes. (B) Inhibition of T-lymphocyte proliferation as stimulated in (A) and cocultivated with Jurkat cells (stably expressing Tax or Tax and Rex) pretreated for 1 h with anti-CD46, anti-VCAM-1 (1G11) or anti-LFA-3 (AICD58) MAb. Data are shown as percentage proliferation of Tlymphocytes cocultivated with untreated Jurkat cells. Data represent the meanpSD of three separate experiments. presence of 5 µg\ml CD3 MAb pre-adsorbed onto a 96-well plate. The proliferation of these T-lymphocytes was assessed following the addition of [$H]thymidine. As shown in Fig. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 13:06:36 IDD H. Valentin and others 2 (A), Tax-negative Jurkat cells were unable to induce DNA synthesis whereas Tax-positive cells induced a significant level of [$H]thymidine incorporation. These observations indicate that a costimulatory signal is delivered by Tax-positive Jurkat cells. To determine whether VCAM-1 induced by Tax is involved in the proliferation of CD3-stimulated Tlymphocytes, we performed blocking experiments in which Tax-expressing Jurkat cells were pre-treated with 10 µg\ml VCAM-1-blocking MAb for 30 min at 4 mC, fixed and then cocultivated with CD3-stimulated T-lymphocytes. A 15 to 20 % inhibition of the proliferation of CD3-stimulated Tlymphocytes cocultivated with C11 and E12 cells (which express both Tax and Rex) was observed, whereas a 90 % inhibition was reached in the presence of cells from clone 50 (which expresses only Tax) (Fig. 2 B). Likewise, [H$]thymidine incorporation by CD3-stimulated T-lymphocytes was reduced by about 70 % only with fixed cells of clone 50 which had been pre-treated with the LFA-3-blocking MAb (Fig. 2 B). The discrepancy in the inhibition of T-cell proliferation between Jurkat cells expressing Tax and Rex and those expressing only Tax proposes that the stimulatory pathway induced by VCAM-1 and LFA-3 is the only one operating in cells expressing Tax alone. Consequently, other molecules in cells expressing both Tax and Rex might be required to fully activate resting T-cells (reviewed by Ho$ llsberg, 1999). Collectively, these data indicate that Rex may be involved in the expression of these molecules and\or in the down-regulation of the stimulatory potential linked to VCAM-1 and LFA-3. This former hypothesis is supported by previous observations indicating that Rex has the capability to increase cellular gene expression. Indeed, it has been reported that Rex may contribute to human interleukin-2 gene expression regulation and in stabilizing the interleukin-2 receptor α-chain mRNA in T-cells (Kanamori et al., 1990 ; McGuire et al., 1993). More interestingly, a recent study has underlined that the concomitant expression of Tax and Rex in Jurkat cells resulted in the overexpression of a protein tyrosine kinase, FynB, probably through a splicing mechanism controlled by Rex (Weil et al., 1999). In conclusion, VCAM-1 induced by HTLV-1 Tax protein in infected T-cells is able to promote T-cell–T-cell adhesion, thus contributing to the proliferation of uninfected T-lymphocytes. Such events may have particular relevance to an understanding of the immunopathogenesis of HTLV-1 and of the spontaneous proliferation of T-cells of HTLV-1-infected individuals, which contributes to an amplification of immune system activation in an antigen-independent manner, thus favouring HTLV-1 dissemination and maintenance of the disease. References Al-Fahim, A., Cabre, P., Kastrukoff, L., Dorovini-Zis, K. & Oger, J. (1999). Blood mononuclear cells in patients with HTLV-I-associated myelopathy : lymphocytes are highly activated and adhesion to endothelial cells is increased. Cellular Immunology 198, 1–10. Daenke, S., McCracken, S. A. & Booth, S. (1999). Human T-cell leukaemia\lymphoma virus type 1 syncytium formation is regulated in a cell-specific manner by ICAM-1, ICAM-3 and VCAM-1 and can be inhibited by antibodies to integrin β or β . Journal of General Virology 80, # ( 1429–1436. Dhawan, S., Weeks, B. S., Abbasi, F., Gralnick, H. R., Notkins, A. L., Klotman, M. E., Yamada, K. M. & Klotman, P. E. (1993). Increase expression of alpha4-beta1 and alpha5-beta1 integrins on HTLV-Iinfected lymphocytes. Virology 197, 778–781. Hildreth, J. E., Subramanium, A. & Hampton, R. A. (1997). Human Tcell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation mediated by vascular cell adhesion molecule-1 : evidence for involvement of cell adhesion molecules in HTLV-1 biology. Journal of Virology 71, 1173–1180. Ho$ llsberg, P. (1999). Mechanisms of T-cell activation by human T-cell lymphotropic virus type I. Microbiology and Molecular Biology Reviews 63, 308–333. Huet, S., Wakasugi, H., Sterkers, G., Gilmour, J., Tursz, T., Boumsell, L. & Bernard, A. (1986). T cell activation via CD2 (T, gp50) : the role of accessory cells in activating resting T cells via CD2. Journal of Immunology 137, 1420–1428. Ichinose, K., Nakamura, T., Kawakami, A., Eguchi, K., Nagasato, K., Shibayama, K., Tsujihata, M. & Nagataki, S. (1992). Increased adherence of T cells to human endothelial cells in patients with human Tcell lymphotropic virus type I-associated myelopathy. Archives of Neurology 49, 74–76. Ichinose, K., Nakamura, T., Nishiura, Y., Nagasato, K., Ohishi, K., Watanabe, H., Fujita, A., Kurouji, K.-i., Tsujihata, M. & Nagataki, S. (1994). Characterization of adherent T cells to human endothelial cells in patients with HTLV-I-associated myelopathy. Journal of the Neurological Sciences 122, 204–209. Ishikawa, T., Imura, A., Tanaka, K., Shirane, H., Okuma, M. & Uchiyama, T. (1993). E-selectin and vascular cell adhesion molecule-1 mediate adult T-cell leukemia cell adhesion to endothelial cells. Blood 82, 1590–1598. Kambara, C., Nakamura, T., Furuya, T., Migita, K., Ida, H., Kawakami, A., Shirabe, S., Nakane, S., Kinoshita, I. & Eguchi, K. (1999). Vascular cell adhesion molecule-1-mediated matrix metalloproteinase-2 induction in peripheral blood T cells is up-regulated in patients with HTLV-Iassociated myelopathy. Journal of Neuroimmunology 99, 242–247. Kanamori, H., Suzuki, N., Siomi, H., Nosaka, T., Sato, A., Sabe, H., Hatanaka, M. & Honjo, T. (1990). HTLV-1 p27rex stabilizes human interleukin-2 receptor alpha chain mRNA. EMBO Journal 9, 4161–4166. Lemasson, I., Robert-Hebmann, V., Hamaia, S., Duc Dodon, M., Gazzolo, L. & Devaux, C. (1997). Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax. Journal of Virology 71, 1975–1983. McGuire, K. L., Curtiss, V. E., Larson, E. L. & Haseltine, W. A. (1993). Influence of human T-cell leukemia virus type 1 tax and rex on interleukin-2 gene expression. Journal of Virology 67, 1590–1599. Tanaka, Y., Fukudome, K., Hayashi, M., Takagi, S. & Yoshie, O. (1995). We gratefully acknowledge D. Gerlier and B. Horvat for helpful discussions and critical reading of the manuscript. This work was supported in part by grants from the Association pour la Recherche sur le Cancer (ARC), Association Nationale de Recherches sur le SIDA (ANRS) and Fondation de France. IDE Induction of ICAM-1 and LFA-3 by Tax1 of human T-cell leukemia virus type 1 and mechanism of down-regulation of ICAM-1 or LFA-1 in adultT-cell-leukemia cell lines. International Journal of Cancer 60, 554–561. Uchiyama, T. (1997). Human T cell leukemia virus type I (HTLV-I) and human diseases. Annual Review of Immunology 15, 15–37. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 13:06:36 Tax-induced VCAM-1 and T-cell proliferation Uchiyama, T., Ishikawa, T. & Imura, A. (1996). Cell adhesion molecules in HTLV-I infection. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 13 (Suppl. 1), S114–S118. Valentin, H., Lemasson, I., Hamaia, S., Casse, H., Ko$ nig, S., Devaux, C. & Gazzolo, L. (1997). Transcriptional activation of the vascular cell adhesion molecule-1 gene in T lymphocytes expressing human T-cell leukemia virus type 1 Tax protein. Journal of Virology 71, 8522–8530. Wake, A., Tanaka, Y., Nakatsuka, K., Misago, M., Oda, S., Morimoto, I. & Eto, S. (1995). Calcium-dependent homotypic adhesion through leukocyte function-associated antigen-1\intracellular adhesion molecule1 induces interleukin-1 and parathyroid hormone-related protein production on adult T-cell leukemia cells in vitro. Blood 86, 2257–2267. Weil, R., Levraud, J. P., Dodon, M. D., Bessia, C., Hazan, U., Kourilsky, P. & Israel, A. (1999). Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines. Journal of Virology 73, 3709–3717. Weston, S. A. & Parish, C. R. (1990). New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. Journal of Immunological Methods 133, 87–97. Wucherpfennig, K. W., Ho$ llsberg, P., Richardson, J. H., Benjamin, D. & Hafler, D. A. (1992). T-cell activation by autologous human T-cell leukemia virus type-I-infected T-cell clones. Proceedings of the National Academy of Sciences, USA 89, 2110–2114. Received 13 November 2000 ; Accepted 26 December 2000 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 13:06:36 IDF