Institute of Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany1
Author for correspondence: Stefan Niewiesk.Fax +49 931 201 3934. e-mail niewiesk{at}vim.uni-wuerzburg.de
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Abstract |
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Introduction |
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We use the cotton rat (Sigmodon hispidus) model to study MV-induced immune suppression (Niewiesk et al., 1997a ) because these animals are the only rodents in which after intranasal infection MV replicates in the respiratory tract. In cotton rats, infection with MV leads to inhibition of mitogen-stimulated proliferation of spleen cells ex vivo (Niewiesk et al., 1997a
). Proliferation inhibition correlates with viral titres in lung tissue homogenates and is induced by MV glycoproteins. This has been demonstrated by injection of human fibroblast cells expressing both the MV H and fusion F proteins as well as by infecting cotton rats with a recombinant MV where the viral glycoproteins are replaced by the G protein of vesicular stomatitis virus (Niewiesk et al., 1997a
). In this animal model we have addressed the question whether contact-mediated lysis, IL-2 deficiency, apoptosis or cell cycle arrest are the cause of ex vivo inhibition of mitogen-driven proliferation of cotton rat lymphocytes.
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Methods |
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Cells, viruses and plasmids.
Vero cells (African green monkey) were grown in minimal essential medium (MEM) with 5% foetal calf serum (FCS), human osteosarcoma cells 143B (TK-) and 293 (human transformed primary embryonal kidney) cells in MEM10% FCS. 293 cells stably expressing the F protein of MV Edmonston strain (293-F) were produced as described previously. (Schlender et al., 1996 ).
Vaccinia viruses expressing the H and F protein of MV Edmonston strain [kindly provided by Fabian Wild, Lyon, France (Wild et al., 1992 )] and expressing the reverse transcriptase of human immunodeficiency virus [HIV vCF21, kindly provided by Bernhard Moss, NIH, Bethesda, USA (Walker et al., 1988
)] were grown and titrated according to standard protocols. MV strain Edmonston was passaged and titrated on Vero cells and vaccinia virus on 143B (TK-) cells. All cells and virus stocks were checked for mycoplasmas. PMV87 expressing the haemagglutinin gene from MV strain CAM/RBH (closely related to Edmonston strain) has been described (Niewiesk et al., 1997a
).
Transfection.
293-F cells were transfected with Lipofectin (Gibco BRL) according to the manufacturer's recommendations: 5x106 cells, 5 µg plasmid (pMV87), 10 µl Lipofectin and 1 ml Opti-MEM were mixed by gentle pipetting and left for 4 h. Afterwards, MEM containing 10% FCS was added and cells incubated overnight. Cells were injected into cotton rats when 80% showed cell fusion as estimated by light microscopy examination. Aliquots were stained with monoclonal antibody L77 (H specific) and monoclonal antibody A 504 (F specific) and a secondary FITC-labelled donkey anti-mouse serum and analysed by flow cytometry. Usually, more than 80% of the cells expressed H and F.
Infection of cotton rats.
For intranasal (i.n.) and intraperitoneal (i.p.) infection MV (Edmonston strain) was given in PBS to ether-anaesthetized cotton rats. I.n. inoculations of MV were administered in a volume of not more than 100 µl and for i.p. delivery, MV was injected in a 1 ml volume. For i.p. infection, 106 p.f.u. virus was used and for i.p. injection 107 293 cells were injected in 1 ml PBS. It had previously been shown that immune suppression caused by MV can be induced by i.p. or i.n. infection as well as by injection of cells expressing both the MV H and F proteins (Niewiesk et al., 1997a ). Four days later, animals were asphyxiated using CO2; spleens were removed and spleen cells tested in a proliferation assay. For mock-infection PBS was used. No difference between mock-infected and non-infected animals was observed.
Proliferation assay.
Spleen cells from infected and mock-infected animals were plated in triplicate at 5x105 cells per well in a 96-well-plate in RPMI 1640 with 10% FCS and were left untreated (medium control) or stimulated with mitogen [Concanavalin A (Con A); 2·5 µg/ml]. Where indicated IL-2 was added. After 40 h 0·5 µCi [3H]thymidine per well was added and 1620 h later cells were harvested onto glass-filters and counted with a Betaplate Counter (Wallac). The stimulation index (SI) was calculated as the mean of proliferation of mitogen-stimulated cells in c.p.m./proliferation of cells in medium in c.p.m.. The percentage of proliferation inhibition is expressed by comparing the stimulation indices of an infected to a mock-infected animal. Mock-infected animal are set as 100% and proliferation of cells from infected animals expressed accordingly.
Production and testing of IL-2.
Rat and cotton rat IL-2 were produced from spleen cells (107/ml) incubated in RPMI-10% FCS with 5x10-5 M ß-mercaptoethanol and Con A (5 µg/ml for rat and 2·5 µg/ml for cotton rat cells). After 36 h cells were centrifuged, and to the harvested supernatant -methylmannoside (10 mg/ml) was added. IL-2 content was measured using the IL-2-dependent CTLL clone 3 cell line and the optimal concentration (just enough to reach the plateau of the growth curve) was used. Human IL-2 was purchased from Eurocetus, Frankfurt, Germany.
Cell cycle analysis.
Mitogen-stimulated cotton rat spleen cells were mixed with detergent solution (0·1% Triton-X 100, 0·15 NaCl, 0·1 M HCl), centrifuged, resuspended in 50 µl RNase (RNase A 100 µg/ml, 1% trisodium citrate) and incubated at 37 °C for 15 min. Cells were washed in 0·1 M TrisHCl pH 7·4 and stained for 10 min (50 µg/ml propidium iodide in 1% trisodium citrate) (Taylor & Milthorpe, 1980 ). For CFSE [5(6)-carboxyfluorescein diacetate succinimidyl ester] staining, mitogen-stimulated cotton rat spleen cells were resuspended at 5x107/ml in RPMI with no protein. A 5 mM stock solution of CFSE in DMSO (stored at -20 °C) was added to a final concentration of 5 µM and incubated at 37 °C for 8 min. At the end of the incubation period, cells were immediately washed three times with RPMI10% FCS (Lyons & Parish, 1994
). After propidium iodide and CFSE staining lymphocytes were analysed by flow cytometry.
Fusion and lysis assay.
For the fusion assay P815 cells (mouse mastocytoma cell line) were infected with vaccinia virus recombinants expressing MV H and F or the HIV reverse transcriptase (m.o.i. 10). For 24 h after infection no fusion between infected P815 cells was observed. After overnight infection P815 cells were incubated at the indicated ratios with lymphocytes from CD46-transgenic rats, non-transgenic rats or cotton rats. After 6 h fusion was observed by light microscopy. For the lysis assay infected P815 cells were labelled with 3·7 MBq Na251CrO4 (DuPont) for 80 min at 37 °C and washed twice. 104 labelled target cells in a volume of 100 µl were added to varying numbers of spleen cells in 100 µl volumes in U-bottomed microtitre plates. After 6·5 h incubation at 37 °C, 100 µl supernatant was harvested and counted. The percentage of lysis was calculated as: 100x(experimental-spontaneous release)/(total-spontaneous release).
Apoptosis assay.
For the inhibition of apoptosis, 100 µM Z-VAD-fmk (Enzyme Systems Product, Dublin, CA, USA) was dissolved in PBS-0·5% DMSO and added on day 0 to spleen cells for a proliferation assay. As a control the same volume of PBS0·5% DMSO was added to spleen cells.
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Results |
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Contact-mediated lysis does not contribute to MV-induced proliferation inhibition
MV-infected cells expressing the H and F proteins of a vaccine strain are able to fuse with non-infected cells expressing the receptor for MV vaccine strains, the human CD46 molecule (Nussbaum et al., 1995 ). Flow cytometry analysis with monoclonal antibodies as well as polyclonal antisera specific for human CD46 did not reveal a homologous structure on cotton rat lymphocytes (data not shown). However, it is not possible to exclude a functional homologue of human CD46 by antibody staining. In order to test for a functional homologue we incubated P815 cells (mouse mastocytoma cell line) expressing MV H and F proteins from a vaccinia virus recombinant with lymphocytes from a CD46-transgenic rat (Niewiesk et al., 1997b
), lymphocytes from non-transgenic rats or cotton rat lymphocytes. After 6 h fusion between P815 cells expressing H and F and CD46-expressing lymphocytes occurred (data not shown). No fusion was seen between P815 cells expressing H and F and lymphocytes from non-transgenic rats or cotton rats. Neither did control P815 cells expressing the reverse transcriptase of HIV fuse with CD46-expressing rat lymphocytes (data not shown). HIV infection mediates fusion between infected and non-infected cells leading to unstable cell aggregates which rapidly undergo cell lysis (Ohnimus et al., 1997
). We therefore incubated lymphocytes from CD46-transgenic or non-transgenic rats or cotton rats with chromium-labelled H- and F-expressing target cells. Only coculture of CD46-expressing lymphocytes resulted in lysis of H- and F-expressing P815 cells in a dose-dependent manner (Fig. 1
). These data indicate that proliferation inhibition is not due to a direct lytic effect of infected cells or virus on cotton rat lymphocytes.
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Discussion |
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In vitro, the presence of CD46, F and H are necessary to mediate fusion (Nussbaum et al., 1995 ) and lysis (this paper). However, in a tissue culture system with human cells it was shown that CD46-negative cells are also susceptible to MV-induced immune suppression (Schlender et al., 1996
). Similarly, MV induces proliferation inhibition in cotton rats, which do not express molecules structurally similar to human CD46 on the cell surface (as shown by antibody staining) and express no functional homologue (as shown by fusion and lysis assay).
In cotton rats virus replication takes place in the lung whereas in other organs only viral RNA can be found. We have shown previously by RTPCR that 1 in 103 to 106 spleen cells are positive for viral RNA (Niewiesk et al., 1997a ). That means that 0·5 to 50 viral RNA molecules are present per 5x105 spleen cells (=one well) in a proliferation assay. The putative expression of H and F on these cells would be below the detection limit of flow cytometry and we were never able to recover infectious virus by cocultivation. These data seem to point towards an indirect mechanism like, e. g., induction of unresponsiveness. Thus, replenishing ex vivo human PBL from MV-infected individuals partly restores their proliferative capacity (Ward & Griffin, 1991
; Griffin et al., 1987
). In vitro, it has been shown that T cells produce less IL-2 after contact with MV-infected cells (Schnorr et al., 1997
). However, addition of IL-2 did not overcome proliferation inhibition of human cells in vitro (Schnorr et al., 1997
) or in cotton rat lymphocytes ex vivo.
Apoptosis is thought to be an important regulatory mechanism in cell growth and regulation. MV-infected dendritic cells (DC) undergo apoptosis in vitro (Fugier-Vivier et al., 1997 ). Apoptosis is enhanced by contact with T cells and T cells become apoptotic themselves without being infected. Cultured Vero cells infected with MV become apoptotic, too (Esolen et al., 1995
). In SCID mice with implants of human foetal thymic tissue infection of thymic epithelium with MV leads to thymocytes undergoing apoptosis (Auwaerter et al., 1996
). So far, it is not clear whether apoptosis is an effect induced by MV in particular in contrast to other viruses or whether apoptosis of thymocytes, activated T cells and DCs is a general regulatory mechanism of the immune response. However, in cotton rats apoptosis does not seem to be responsible for MV-induced proliferation inhibition.
Lymphocytes have been demonstrated to arrest in the G0/G1 phase of the cell cycle after infection with MV (McChesney et al., 1987 , 1988
; Yanagi et al., 1992
) or contact with MV-infected cells (Schnorr et al., 1997
). In cotton rats, all spleen cells from infected animals divide more slowly than those from non-infected animals. This indicates that the observed `arrest' is, rather, a retardation of cells in the G0/G1 phase. So far it is not known how MV affects the cell cycle. In vitro experiments with a two-chamber system have shown that MV-induced proliferation inhibition is induced by direct contact between lymphocytes and infected cells or cells expressing the viral glycoprotein (Schlender et al., 1996
) and that soluble factors smaller than 70 kDa do not play a role. However, inhibition of antigen-specific T cell lines in vitro seems to be mediated by an unidentified 100 kDa protein (Sum et al., 1998
). Therefore, the mechanism underlying the cell cycle retardation remains to be solved. Most likely the cell cycle retardation of T cells described here is only one of the factors contributing to MV-induced immune suppression in vivo. At least in vitro, the nucleocapsid protein inhibits B cell responses (Ravanel et al., 1997
) and MV infection has been shown to induce aberrant cytokine expression in macrophages (Karp et al., 1996
) and to reduce their ability to present antigen (Leopardi et al., 1993
).
In summary, we have shown that the proliferation inhibition of lymphocytes from MV-infected cotton rats is due a retardation of the cell cycle and not to virus-mediated lysis, apoptosis or IL-2 deficiency.
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Acknowledgments |
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References |
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Received 2 February 1999;
accepted 28 April 1999.