Measles virus-induced immunosuppression in ... - Semantic Scholar

Journal of General Virology (1999), 80, 2023–2029. Printed in Great Britain ...................................................................................................................................................................................................................................................................................

Measles virus-induced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes Stefan Niewiesk, Hartmut Ohnimus, Jens-Jo$ rg Schnorr, Michaela Go$ tzelmann, Sibylle Schneider-Schaulies, Christian Jassoy and Volker ter Meulen Institute of Virology and Immunobiology, University of Wu$ rzburg, Versbacher Str. 7, 97078 Wu$ rzburg, Germany

Measles virus (MV)-induced immune suppression during acute measles often leads to secondary viral, bacterial and parasitic infections which severely complicate the course of disease. Previously, we have shown that cotton rats are a good animal model to study MV-induced immune suppression, where proliferation inhibition after ex vivo stimulation of cotton rat spleen cells is induced by the viral glycoproteins (fusion and haemagglutinin proteins). We have now tested a variety of putative mechanisms of MV-induced immune suppression in this animal model. Proliferation inhibition is not due to fusion mediated by the MV glycoproteins and subsequent lysis of cells. Other putative mechanisms like classical anergy (unresponsiveness towards IL-2) or apoptosis do not seem to play a role in MV-induced immune suppression. In contrast, it was shown that spleen cells from infected animals preferentially accumulate in the G0/G1 phase and progress more slowly through the cell cycle after mitogen stimulation in comparison to cells from non-infected animals. These data indicate a retardation of the cell cycle which is correlated with proliferation inhibition and might have severe consequences in mounting an effective immune response.

Introduction During and for weeks after acute measles a severe immune suppression is observed in infected individuals. The tuberculin reaction disappears (von Pirquet, 1908) during the acute disease, ex vivo peripheral blood lymphocytes (PBL) show reduced responses towards antigen and mitogen stimulation and individuals are highly susceptible to secondary infections (for review see Griffin, 1995). As only few lymphocytes are infected during natural measles in vivo (Esolen et al., 1993 ; Nakayama et al., 1995) indirect mechanisms rather than virusmediated destruction of lymphocytes seem a likely explanation. For the molecular basis of measles virus (MV)-induced immune suppression experimental evidence for a variety of putative mechanisms has been obtained. Ex vivo studies with PBL from MV-infected patients have shown that supplementation of interleukin-2 (IL-2) led to a reversal of proliferation inhibition indicating classical anergy as the cause of immune suppression (Ward & Griffin, 1991 ; Griffin et al., 1987). In tissue culture experiments, human PBL were inhibited in their response to mitogen after contact with MV-infected cells Author for correspondence : Stefan Niewiesk. Fax j49 931 201 3934. e-mail niewiesk!vim.uni-wuerzburg.de

0001-6223 # 1999 SGM

(Sanchez-Lanier et al., 1988). To induce this unresponsiveness expression of the haemagglutinin (H) and fusion (F) proteins is necessary and sufficient (Schlender et al., 1996). Expression of H and F on human cells leads to cell fusion (Nussbaum et al., 1995) and induces cell cycle arrest (Schnorr et al., 1997) in lymphocytes. This cell cycle arrest has been analysed in great detail in MV-infected B cells and T cells (McChesney et al., 1987, 1988 ; Yanagi et al., 1992). After mitogenic activation infected cells increase in volume, upregulate their mRNA synthesis and express cell surface activation markers such as MHC class II, CD71 (transferrin receptor) and CD25 (IL-2-R) (on T cells). After 48 to 72 h synthesis of total mRNA as well as the level of mRNA for histone 2B (a gene up-regulated during the S phase of the cell cycle) is reduced in comparison to uninfected cells. These data indicate an arrest in the late G " phase of the cell cycle. However, the arrest is incomplete as a low frequency of T cells synthesizes DNA (Yanagi et al., 1992). In addition to induction of cell cycle arrest, MV infection has also been shown to induce apoptosis in tissue culture (Esolen et al., 1995 ; Fugier-Vivier et al., 1997) as well as in a SCID-hu mouse model (Auwaerter et al., 1996). We use the cotton rat (Sigmodon hispidus) model to study MV-induced immune suppression (Niewiesk et al., 1997 a) because these animals are the only rodents in which after CACD

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intranasal infection MV replicates in the respiratory tract. In cotton rats, infection with MV leads to inhibition of mitogenstimulated proliferation of spleen cells ex vivo (Niewiesk et al., 1997 a). 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., 1997 a). 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.

Methods

Animals. Cotton rats (inbred strain COTTON\NIco) were obtained from Iffa Credo, France. Animals from 3 weeks up to 7 months of age of both sexes were used. The animals were bought specific pathogen free according to the breeder’s specification. CD46 transgenic rats were produced as described (Niewiesk et al., 1997 a). All animals were maintained in a barrier system. Sentinel mice or rats were examined serologically. Animals were kept under controlled environmental conditions of 22p1 mC, 50p10 % humidity and a 12 h light cycle.

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 MEM–10 % 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., 1997 a).

Transfection. 293-F cells were transfected with Lipofectin (Gibco BRL) according to the manufacturer’s recommendations : 5i10' 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 etheranaesthetized 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, 10' p.f.u. virus was used and for i.p. injection 10( 293 cells were injected in 1 ml PBS. It had previously been CACE

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., 1997 a). Four days later, animals were asphyxiated using CO ; 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 5i10& 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) ; 2n5 µg\ml]. Where indicated IL-2 was added. After 40 h 0n5 µCi [$H]thymidine per well was added and 16–20 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 (10(\ml) incubated in RPMI-10 % FCS with 5i10−& M β-mercaptoethanol and Con A (5 µg\ml for rat and 2n5 µg\ml for cotton rat cells). After 36 h cells were centrifuged, and to the harvested supernatant α-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 (0n1 % Triton-X 100, 0n15 NaCl, 0n1 M HCl), centrifuged, resuspended in 50 µl RNase (RNase A 100 µg\ml, 1 % trisodium citrate) and incubated at 37 mC for 15 min. Cells were washed in 0n1 M Tris–HCl pH 7n4 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 5i10(\ml in RPMI with no protein. A 5 mM stock solution of CFSE in DMSO (stored at k20 mC) was added to a final concentration of 5 µM and incubated at 37 mC for 8 min. At the end of the incubation period, cells were immediately washed three times with RPMI–10 % 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 3n7 MBq Na &"CrO % # (DuPont) for 80 min at 37 mC and washed twice. 10% 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 6n5 h incubation at 37 mC, 100 µl supernatant was harvested and counted. The percentage of lysis was calculated as : 100i(experimentalkspontaneous release)\(totalkspontaneous release).

Apoptosis assay. For the inhibition of apoptosis, 100 µM Z-VADfmk (Enzyme Systems Product, Dublin, CA, USA) was dissolved in PBS0n5 % DMSO and added on day 0 to spleen cells for a proliferation assay.

MV-induced immunosuppression in cotton rats As a control the same volume of PBS–0n5 % DMSO was added to spleen cells.

Results We have shown previously (Niewiesk et al., 1997 a) that injection of cells expressing the MV H and F proteins induces proliferation inhibition in cotton rat spleen cells. This effect is dose dependent. Injection of 10( cells induced a good proliferation inhibition ; injection of 10' cells induced less inhibition and 3i10& cells none at all (data not shown). To define the underlying mechanism we tested whether contactmediated lysis, IL-2 deficiency, apoptosis or cell cycle arrest correlate with MV-induced proliferation inhibition. Contact-mediated lysis does not contribute to MVinduced 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., 1997 b), 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 CD46transgenic 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.

Fig. 1. Coincubation of cotton rat lymphocytes with cells expressing MV glycoproteins does not cause cell lysis. P815 cells were infected with a vaccinia virus recombinant expressing H and F (m.o.i. 10, overnight) and labelled with chromium. Lymphocytes from CD46-transgenic rats, nontransgenic rats and cotton rats were added at various ratios to P815 cells expressing H and F. Supernatant was collected after 6 h and chromium release was measured. Lysis after coincubation of rat and cotton rat lymphocytes with P814 cells infected with a vaccinia virus recombinant expressing the reverse transcriptase of HIV did not exceed 5 % (data not shown).

are defined as being non-responsive to IL-2 (for review see Paul, 1993). Addition of recombinant human IL-2 (100 U\ml) or optimal concentrations of IL-2-containing rat Con A supernatant to mitogen-stimulated cotton rat lymphocytes did not relieve the difference in proliferation of spleen cells from infected versus mock-infected animals (Fig. 2). To exclude the possibility that specifically cotton rat IL-2 is needed we produced IL-2-containing Con A supernatant from cotton rat lymphocytes. Optimal concentrations of cotton rat IL-2 had no effect on MV-induced proliferation inhibition (Fig. 2). It is possible that lymphocytes from infected cells are no longer able to respond to IL-2 (because of, e.g., lack of IL-2 receptor). To test this possibility we used the fact that lymphocytes respond to stimulation with a suboptimal concentration of mitogen only in the presence of IL-2. As shown in Fig. 3 lymphocytes did not respond to suboptimal concentrations of Con A alone, but did so after addition of IL-2 (either human, rat or cotton rat). This indicates that spleen cells from infected animals were able to bind and respond to IL-2. However, the difference in proliferation between spleen cells from infected and non-infected animals remained. These data demonstrate that MV-induced proliferation inhibition is not overcome by addition of IL-2.

Proliferation inhibition in cotton rat spleen cells is not relieved after addition of IL-2

Apoptosis is not involved in MV-induced proliferation inhibition of cotton rat lymphocytes

As expression of H and F is crucial for induction of proliferation inhibition, the contact of H and F with cotton rat lymphocytes might lead to anergy. Classically, anergic T cells

Apoptosis has been observed in tissue culture after infection with MV (Esolen et al., 1995) as well as in hu-SCID mice (Auwaerter et al., 1996). It therefore seemed a possible CACF

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Fig. 2

Fig. 3

Fig. 2. Addition of IL-2 does not relieve proliferation inhibition. Spleen cells from animals injected with 107 293–F cells or 107 293–FjH cells were taken on day 4 and stimulated with Con A (2n5 µg/ml) with or without addition of IL-2 from either humans, rats or cotton rats. Proliferation was measured as thymidine incorporation on day 3. The experiment shown is representative of six experiments with transfected 293 cells or MV infection (2–3i106 p.f.u.). Fig. 3. Proliferation inhibition is not due to unresponsiveness to IL-2. Spleen cells from infected (2–3i106 p.f.u.) and noninfected animals were taken on day 4 and stimulated with an optimal (2n5 µg/ml) or suboptimal (0n5 µg/ml) dose of Con A, IL-2 alone or IL-2 with a suboptimal (0n5 µg/ml) dose of Con A. Proliferation was measured as thymidine incorporation on day 3. The experiment shown is representative of three experiments.

stimulation indices (the ratio of c.p.m. stimulated\unstimulated cells) did not differ and therefore the difference in proliferation was unaffected (Fig. 4). Similarly, with the DNA fragmentation assay and the poly(ADP-ribose) polymerase (PARP) cleavage assay (Ohnimus et al., 1997) we found no difference in apoptosis between spleen cells from infected versus noninfected animals after removal of the spleen or on day 1, 2 and 3 after mitogen stimulation (data not shown). MV infection induces cell cycle retardation in cotton rat spleen cells Fig. 4. Peptide inhibition of apoptosis does not relieve proliferation inhibition. Spleen cells from i.n. and i.p. infected (2i106 p.f.u.) and noninfected animals were taken on day 4 and a proliferation assay was performed with or without addition of 100 µM ZVAD-fmk. The data shown represent the average of four experiments.

mechanism to explain MV-induced proliferation inhibition. However, the numbers of spleen cells isolated from infected versus non-infected animals did not differ. The caspase inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVAD-fmk) has been shown to prevent apoptosis by blocking the activation of caspases in vivo (Rodriguez et al., 1996) and in vitro (Sarin et al., 1996). Addition of ZVAD-fmk to a proliferation assay increased c.p.m. from unstimulated cells or cells stimulated with mitogen by two to three fold. This was true for cells from infected and uninfected animals and indicates that apoptosis occurred in cell cultures of both. However, the CACG

In tissue culture MV infection leads to cell cycle arrest in the G \G phase (McChesney et al., 1987, 1988 ; Yanagi et al., ! " 1992). We attempted to correlate the proliferative capacity of spleen cells from either MV- or mock-infected cotton rats with the number of cells in the G \G phase. After propidium iodide ! " staining we analysed the DNA content of mitogen-stimulated lymphocytes by flow cytometry. The percentage of cells in the G \G phase was higher in cells from infected animals (Fig. 5 ! " a). Cells from mock-infected animals differed significantly from i.n. infected animals on day 3 and 4 and from i.p. infected animals on day 2 and 3 (Fig. 5 b). Proliferation inhibition of 40–60 % was measured on day 3 and correlated with changes in the cell cycle. To investigate whether all cells replicate slowly or only some cells are arrested in the G \G phase we ! " used the fluorescent dye CSFE, which binds to cytosolic proteins and allows cell division over time to be followed (Lyons & Parish, 1994). Flow cytometry analysis showed that all cells from infected animals go through the cell cycle.

MV-induced immunosuppression in cotton rats

(a)

(b)

Fig. 5. Cell cycle retardation in lymphocytes from infected cotton rats. Spleen cells from i.n. and i.p. infected and noninfected animals were taken on day 4 and stimulated with Con A. (a) An example from cells stained with propidium iodide on day 3 is shown with spleen cells from a noninfected (above) and an infected animal (below). The brackets indicate the percentage of cells in the G0/G1 phase. (b) The percentage of cells in the G0/G1 phase is shown from day 2 to day 4 (four to six animals per time-point). The asterisks indicate a significant difference (two-tailed paired t-test) with respect to cells from non-infected animals.

However, they progress more slowly in comparison with cells from non-infected animals (Fig. 6). This was also true if instead of Con A the superantigen SEC 3, which stimulates only a subpopulation of T cells, was used (data not shown).

Discussion Immune suppression during acute measles is a welldocumented phenomenon in man. Ex vivo, proliferation of PBL in response to mitogen and to antigen from infected individuals is inhibited (for review see Griffin, 1995). Addition of IL-2 has in some instances relieved this proliferation inhibition (Ward & Griffin, 1991 ; Griffin et al., 1987). So far, no other data have been obtained from patients in respect to the mechanism of MV-induced immune suppression. In a SCID mouse model secretion of immunoglobulins by human B cells is reduced in infected animals and cell cycle arrest has been suggested to be responsible (Tishon et al., 1996). An alternative mechanism has been found in SCID mice with implants of human foetal tissue (Auwaerter et al., 1996). Infection of thymic epithelium with a strongly replicating wild-type strain of MV induced apoptosis

in the implanted human thymocytes. Most putative mechanisms of MV-induced immune suppression are derived from tissue culture experiments. In contrast to the in vivo situation where virus is relatively scarce (Esolen et al., 1993 ; Nakayama et al., 1995), in vitro cultures are used where most or all of the cultured PBLs are infected (McChesney et al., 1987, 1988 ; Yanagi et al., 1992 ; Esolen et al., 1995 ; Fugier-Vivier et al., 1997). In an alternative approach UV-inactivated MV or inactivated MV-infected cells have been used to investigate immune suppression (Sanchez-Lanier et al., 1988 ; Schlender et al., 1996). With all these tissue culture systems various mechanisms such as contact-mediated lysis, IL-2 deficiency, apoptosis and cell cycle arrest have been observed and suggested to play a role in vivo. 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 MVinduced 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 CACH

Day 3

Day 2

Day 0

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Fig. 6. Progression of cell cycle in cells from infected and non-infected animals. Spleen cells from i.n. and i.p. infected and non-infected animals were taken on day 4 after infection, stained with CSFE and stimulated with Con A. An experiment representative of four is shown with cells from a non-infected animal (left) and an infected animal (right). Unstimulated cells move from the right (day 0, top) after stimulation towards the left (day 3, bottom).

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 RT–PCR that 1 in 10$ to 10' spleen cells are positive for viral RNA (Niewiesk et al., 1997 a). That means that 0n5 to 50 viral RNA molecules are present per 5i10& spleen cells (l 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., CACI

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 G \G 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 G \G ! " 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. S. N. was supported by Stipendienprogramm Infektionsforschung (Bundesministerium fu$ r Bildung, Wissenschaft, Forschung und Technologie). This work was in part supported by Deutsche Forschungsgemeinschaft, Bundesministerium fu$ r Bildung, Wissenschaft, Forschung und Technologie, Pfleger-Stiftung and World Health Organization.

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