Institute for Virology and Immunobiology, University of Würzburg, Versbacher Strasse 7, 97078 Würzburg, Germany1
Author for correspondence: Christian Jassoy. Fax +49 931 201 3934. e-mail jassoy{at}vim.uni-wuerzburg.de
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Previous studies of the mechanism of T-helper cell death were performed primarily with lymphoblast cell lines either infected with virus or virus vectors or transfected with the HIV envelope gene (Cao et al., 1996 ; Koga et al., 1990
; Leonard et al., 1988
; Lifson et al., 1986b
; Sodroski et al., 1986
; Somasundaran & Robinson, 1987
). Alternatively, infected or HIV glycoprotein-expressing lymphoblasts were co-cultured with primary CD4+ T cells (Heinkelein et al., 1995
, 1997
; Ohnimus et al., 1997
). Few studies have been carried out with primary cells (Nardelli et al., 1995
). Since the pathology of HIV infection of primary CD4+ T lymphocytes may differ from that observed in T cell lines, analysis of such cells is crucial to the understanding of T cell death in HIV disease.
We examined whether the cytotoxicity mediated by the HIV envelope glycoprotein occurs in single cells or upon contact with CD4+ lymphocytes. To mimic the in vivo situation better, we used primary CD4+ T lymphocytes.
In a first set of experiments, peripheral blood mononuclear cells were prepared by density-gradient centrifugation from the blood of uninfected individuals and depleted of CD8+ cells by using antibody-covered immunomagnetic beads (Dynal). T cell preparations contained less than 5% CD8+ T cells. Cells were stimulated with PHA (2 µg/ml) and cultured in RPMI-1640 medium supplemented with 10% FCS, HEPES, antibiotics and 100 U/ml interleukin-2 (Proleukin, Eurocetus) for 35 days. Activated CD4+ T cells were infected with HIV-1 strain IIIB/LAI at an m.o.i. of 0·01 TCID50 per cell. More than 70% of the cells were HIV Gag-positive after 34 days of culture. Uninfected CD4+ T cells (3x105) were labelled with 100 µCi Na251CrO4 for 60 min and added to the same number of autologous HIV-infected cells in 96-well round-bottomed microtitre plates. Cells were incubated for 5·515·5 h, after which time supernatants were harvested and counted in a gamma counter. Percentage lysis was determined from the formula 100x(experimental release-spontaneous release)/(maximum release-spontaneous release). Maximum release was determined by lysis of targets in 1·5% Triton X-100.
The results depicted in Fig. 1(a) demonstrate that a significant fraction of uninfected CD4+ T lymphocytes was lysed upon co-culture with HIV-infected cells. The fraction of cells destroyed increased with the duration of culture. However, since co-culture with infected cells may lead to infection of the uninfected cells, single cell lysis due to cytopathicity of virus replication and cell death due to cellcell fusion could not be differentiated in this assay.
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In order to dissect cytolysis upon cell-to-cell contact from cytopathicity due solely to HIV glycoprotein expression, PHA-activated CD4+ T cells were labelled with Na251CrO4 for 60 min, washed twice with medium and split into three fractions. Cells were infected for 1 h either with the envelope vaccinia virus construct or with wild-type vaccinia virus (strain Copenhagen) at an m.o.i. of 15 p.f.u. per cell. The third culture was not infected. Cells (2x105) were placed in duplicate in 96-well plates and the anti-CD4 MAb SIM.2 (60 µg/ml) (McCallus et al., 1992 ), which inhibits gp120CD4 binding, cell-to-cell fusion and cytolysis (Heinkelein et al., 1995
), or the peptide T20 (2 µg/ml), which inhibits post-binding fusion events, were added to some of the cultures. T lymphocytes were incubated for an additional 12 h at 37 °C. Supernatants were harvested and counted in a gamma counter. Percentage lysis was determined from the formula 100x(release from vaccinia virus-infected culture-spontaneous release from uninfected culture)/(maximum release-spontaneous release from uninfected culture). Spontaneous release was less than 20% of maximum release.
In the absence of the anti-CD4 MAb, significant lysis of CD4+ T cells was observed. In contrast, addition of SIM.2 or T20 prevented T cell destruction (Fig. 2). The degree of vaccinia virus infection was unaffected (data not shown). No lysis of the control cells infected with wild-type vaccinia virus was observed during the 13 h incubation in either the absence or the presence of the anti-CD4 MAb or T20 (Fig. 2
). This indicates that the cytolytic effect of vaccinia virus replication did not account for the cytotoxicity described. Analogous experiments with HIV could not be performed, because agents that block CD4gp120 contact or cell-to-cell fusion inhibit cytotoxicity similarly due to prevention of virus infection.
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Fig. 3 shows that, in the absence of the anti-CD4 MAb, the number of CD4+ T cells decreased rapidly. The fraction of cells expressing the envelope glycoprotein was similar in the absence and presence of the anti-CD4 MAb, reflecting the fact that the antibody did not interfere with vaccinia virus infection and indicating that the T cell loss was not due to destruction of the cells by vaccinia virus infection. The anti-CD4 MAb prevented T-helper cell loss. The degree of T cell loss in the absence of inhibiting antibody was correlated negatively with the level of HIV glycoprotein expression. In this and related experiments with primary cells, T cell destruction and depletion were only marginally affected by an inhibitor of the apoptosis-mediating caspase enzymes (data not shown).
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Since HIV glycoprotein expression on the cell surface is part of the virus particle synthesis process in infected cells, our data suggest that the mechanism of T cell destruction observed in vitro operates in vivo. The T cell death described involves both infected and uninfected cells. The question of whether cell loss in HIV infection is due solely to the death of infected cells or involves both infected and uninfected cells is important. In either model system, T cell loss correlates with the number of infected cells. This compares favourably with the situation observed in infected individuals, where the virus RNA load correlates with the pace of disease progression (Mellors et al., 1996 ). However, in contrast to the situation with single-cell killing, cytotoxicity involving uninfected bystander cells points to a scenario in which T-helper cell death exceeds the number of infected cells significantly at any given time-point. Moreover, if cell death in vivo is mediated by cell-to-cell contact, prevention of virus replication and inhibition of the interaction of infected with uninfected cells may act synergistically in terms of prevention of T-helper cell depletion.
The peptide T20 inhibits HIV infection and syncytium formation by preventing the gp41 molecule from folding adequately after binding of gp120 to its receptor molecules and insertion of the gp41 fusion peptide into the cell membrane (Wild et al., 1994 ). It was observed that treatment of HIV-infected individuals with this agent led to potent suppression of HIV replication in vivo (Kilby et al., 1998
). The results of our study suggest an additional beneficial effect of this and similar agents on T-helper cell numbers in infected individuals, through interference with the lethal consequences of the contact of HIV-infected with uninfected cells.
In conclusion, the results of this study demonstrate rapid cytolysis and cell loss upon contact and fusion of primary HIV-infected and HIV glycoprotein-expressing T cells with uninfected cells. The data support the view that T-helper cell destruction in HIV infection in vitro and in vivo is mediated at least partially by a lethal cell-to-cell contact. Cellcell fusion and cytolysis may represent different aspects of a related intercellular process. Since destruction involves both infected and uninfected cells, cell death is extensive. This may contribute to the large depletion of T-helper cells in HIV-infected individuals.
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References |
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Chesebro, B. & Wehrly, K. (1988). Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity.Journal of Virology 62, 3779-3788.[Medline]
Corbeil, J. & Richman, D. D. (1995). Productive infection and subsequent interaction of CD4gp120 at the cellular membrane is required for HIV-induced apoptosis of CD4+ T cells.Journal of General Virology 76, 681-690.[Abstract]
Frankel, S. S., Wenig, B. M., Burke, A. P., Mannan, P., Thompson, L. D., Abbondanzo, S. L., Nelson, A. M., Pope, M. & Steinman, R. M. (1996). Replication of HIV-1 in dendritic cell-derived syncytia at the mucosal surface of the adenoid.Science 272, 115-117.[Abstract]
Heinkelein, M., Sopper, S. & Jassoy, C. (1995). Contact of human immunodeficiency virus type 1-infected and uninfected CD4+ T lymphocytes is highly cytolytic for both cells. /title>Journal of Virology 69, 6925-6931.[Abstract]
Heinkelein, M., Müller, M., Kutsch, O., Sopper, S. & Jassoy, C. (1997). Rapid and selective depletion of CD4+ T lymphocytes and preferential loss of memory cells on interaction of mononuclear cells with HIV-1 glycoprotein-expressing cells.Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 16, 74-82.[Medline]
Kilby, J. M., Hopkins, S., Venetta, T. M., DiMassimo, B., Cloud, G. A., Lee, J. Y., Alldredge, L., Hunter, E., Lambert, D., Bolognesi, D., Matthews, T., Johnson, M. R., Nowak, M. A., Shaw, G. M. & Saag, M. S. (1998). Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry.Nature Medicine 4, 1302-1307.[Medline]
Koga, Y., Sasaki, M., Yoshida, H., Wigzell, H., Kimura, G. & Nomoto, K. (1990). Cytopathic effect determined by the amount of CD4 molecules in human cell lines expressing envelope glycoprotein of HIV.Journal of Immunology 144, 94-102.
Laurent-Crawford, A. G., Krust, B., Muller, S., Rivière, Y., Rey-Cuillé, M.-A., Béchet, J.-M., Montagnier, L. & Hovanessian, A. G. (1991). The cytopathic effect of HIV is associated with apoptosis.Virology 185, 829-839.[Medline]
Laurent-Crawford, A. G., Krust, B., Rivière, Y., Desgranges, C., Muller, S., Kieny, M. P., Dauguet, C. & Hovanessian, A. G. (1993). Membrane expression of HIV envelope glycoproteins triggers apoptosis in CD4 cells.AIDS Research and Human Retroviruses 9, 761-773.[Medline]
Laurent-Crawford, A. G., Coccia, E., Krust, B. & Hovanessian, A. G. (1995). Membrane-expressed HIV envelope glycoprotein heterodimer is a powerful inducer of cell death in uninfected CD4+ target cells.Research in Virology 146, 5-17.[Medline]
Leonard, R., Zagury, D., Desportes, I., Bernard, J., Zagury, J.-F. & Gallo, R. C. (1988). Cytopathic effect of human immunodeficiency virus in T4 cells is linked to the last stages of virus infection.Proceedings of the National Academy of Sciences, USA 85, 3570-3574.[Abstract]
Lifson, J. D., Feinberg, M. B., Reyes, G. R., Rabin, L., Banapour, B., Chakrabarti, S., Moss, B., Wong-Staal, F., Steimer, K. S. & Engleman, E. G. (1986a). Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein.Nature 323, 725-728.[Medline]
Lifson, J. D., Reyes, G. R., McGrath, M. S., Stein, B. S. & Engleman, E. G. (1986b). AIDS retrovirus induced cytopathology: giant cell formation and involvement of CD4 antigen.Science 232, 1123-1127.[Medline]
McCallus, D. E., Ugen, K. E., Sato, A. I., Williams, W. V. & Weiner, D. B. (1992). Construction of a recombinant bacterial human CD4 expression system producing a bioactive CD4 molecule.Viral Immunology 5, 163-172.[Medline]
Mellors, J. W., Rinaldo, C. R.Jr, Gupta, P., White, R. M., Todd, J. A. & Kingsley, L. A. (1996). Prognosis in HIV-1 infection predicted by the quantity of virus in plasma.Science 272, 1167-1170.[Abstract]
Nardelli, B., Gonzales, C. J., Schechter, M. & Valentine, F. T. (1995). CD4+ blood lymphocytes are rapidly killed in vitro by contact with autologous human immunodeficiency virus-infected cells.Proceedings of the National Academy of Sciences, USA 92, 7312-7316.[Abstract]
Navia, B. A., Cho, E.-S., Petito, C. K. & Price, R. W. (1986). The AIDS dementia complex: II. Neuropathology.Annals of Neurology 19, 525-535.[Medline]
Ohnimus, H., Heinkelein, M. & Jassoy, C. (1997). Apoptotic cell death upon contact of CD4+ T lymphocytes with HIV glycoprotein-expressing cells is mediated by caspases but bypasses CD95 (Fas/Apo-1) and TNF receptor 1.Journal of Immunology 159, 5246-5252.[Abstract]
Pantaleo, G. (1999). Unraveling the strands of HIVs web.Nature Medicine 5, 27-28.[Medline]
Sodroski, J., Goh, W. C., Rosen, C., Campbell, K. & Haseltine, W. A. (1986). Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity.Nature 322, 470-474.[Medline]
Somasundaran, M. & Robinson, H. L. (1987). A major mechanism of human immunodeficiency virus-induced cell killing does not involve cell fusion.Journal of Virology 61, 3114-3119.[Medline]
Wild, C. T., Shugars, D. C., Greenwell, T. K., McDanal, C. B. & Matthews, T. J. (1994). Peptides corresponding to a predictive -helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection.Proceedings of the National Academy of Sciences, USA 91, 9770-9774.
Yang, O. O., Kalams, S. A., Rosenzweig, M., Trocha, A., Jones, N., Koziel, M., Walker, B. D. & Johnson, R. P.(1996). Efficient lysis of human immunodeficiency virus type 1-infected cells by cytotoxic T lymphocytes.Journal of Virology70, 5799-5806.[Abstract]
Yoffe, B., Lewis, D. E., Petrie, B. L., Noonan, C. A., Melnick, J. L. & Hollinger, F. B. (1987). Fusion as a mediator of cytolysis in mixtures of uninfected CD4+ lymphocytes and cells infected by human immunodeficiency virus.Proceedings of the National Academy of Sciences, USA 84, 1429-1433.[Abstract]
Received 28 December 1999;
accepted 12 April 2000.