Unité de Neurovirologie et Régénération du Système Nerveux, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France1
Authors for correspondence: Thérèse Couderc (e-mail tcouderc{at}pasteur.fr) and Bruno Blondel (e-mail bblondel@pasteur.fr). Fax +33 1 40 61 34 21.
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Apoptosis is an active process of cell death, which occurs in response to a variety of stimuli, including viral infections. It is characterized by a number of distinct morphological features and biochemical processes, such as cell shrinkage, plasma membrane blebbing, chromatin condensation and intranucleosomal cleavage (Roulston et al., 1999 ). A family of proteases, called caspases (cysteine proteases with aspartate specificity), are universal effectors of apoptotic cell death (Cryns & Yuan, 1998
; Earnshaw et al., 1999
). In vitro, PV can either induce or inhibit apoptosis in HeLa cells, an epithelial cell line, according to the conditions of the viral infection (Tolskaya et al., 1995
; Agol et al., 1998
, 2000
). Moreover, PV-induced apoptosis has been observed in the CaCo-2 enterocyte-like cell line and in the U937 promonocyte cell line (Ammendolia et al., 1999
; Lopez-Guerrero et al., 2000
). However, no in vitro model has been available to investigate PV-induced apoptosis in nerve cells. The primary cultures of human foetal brain cells that have been described (Pavio et al., 1996
) are not a convenient model for investigating PV-induced apoptosis in nerve cells because of the difficulty of obtaining human foetal tissues. Here we describe the development of a model of mixed nerve cell cultures prepared from the cerebral cortex of neonatal TgCD155 mice.
Mixed mouse primary nerve cell cultures were prepared from the cerebral cortex of neonatal TgCD155 mice, as described by Kiss et al. (1994) . Briefly, the cerebral hemispheres were gently dissociated by passage through a Pasteur pipette to obtain a single-cell suspension in Hanks balanced salts solution (Gibco). Cells were resuspended in DMEM containing 4·5 g/l glucose (Sigma) and 10% foetal calf serum, and then filtered through 80 µm pore size nylon mesh to remove tissue clumps. Viable cells were plated at approximately 1·5x105 cells/cm2 and incubated at 37 °C in a humidified atmosphere of 5% CO2. Culture medium was changed every 3 days until cell confluence, and then replaced with serum-free medium [DMEM containing 4·5 g/l glucose, 5 µg/ml insulin, 20 µg/ml transferrin, 20 nM progesterone, 100 µM putrescine, 30 mM sodium selenite, 1% penicillinstreptomycin (Gibco)]. Cell cultures were used 811 days after plating.
These cultures consisted of a bilayer made up of mixed neuronal and glial cells. To identify further the neural cell types in the primary cultures, we performed triple immunofluorescence labelling using specific cell markers for neuronal and glial cell lineages, as previously described (McKinnon et al., 1990 ; Ben-Hur et al., 1998
). Neuronal cells were detected with TUJ1, a mouse monoclonal antibody (IgG2a) against neuron-specific class III
-tubulin (1/500 dilution; BAbCO). Oligodendrocytes and astrocytes were identified with a mouse monoclonal antibody (IgM, 1/5 dilution; Boehringer Mannheim) recognizing the cell surface sulfatide O4 and a rabbit antibody to glial fibrillary acidic protein (GFAP) (1/200 dilution; DAKO), respectively. Primary antibodies were stained with the appropriate fluorescent secondary antibody. Nuclei were stained with DAPI. Control experiments included omitting the primary antibodies from the staining procedure. No non-specific labelling was observed in controls.
The confluent underlying layer comprised flat type 1 astrocytes with an epitheloid shape; these cells strongly expressed GFAP (Fig. 1A). They constituted about 50% of the total cell population. The surface of this monolayer was predominantly populated by neuronal cells (about 30%) characterized by expression of
-tubulin antigen. The majority of the neurons were grouped in clusters of small, round cells with short processes, but some more mature neurons with long neurites were dispersed among these clusters. Other glial cells (about 20%), including oligodendrocyte progenitors, were also observed dispersed within the upper monolayer. These cells expressed O4 antigen on their surface. Rare non-neural cells such as macrophages, fibroblasts and endothelial cells were also occasionally found in the upper monolayer (Kiss et al., 1994
).
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In infected cell cultures, viral antigen staining was observed in the cell cytoplasm, the site of virus replication, in all three cell types (Fig. 1B). While most astrocytes and oligodendrocytes (more than 80%) were infected at 8 h and 16 h post-infection, only 1520% of neurons were positive for viral antigens. Viral antigens were also detected in the processes of infected oligodendrocytes and neurons, as illustrated in Fig. 1(B)
for the 16 h time point. At later time points (22 h and 28 h post-infection), the percentages of infected cells were similar to those at earlier time points. However, most astrocytes exhibited an altered morphology with a decrease in both the intensity and the fibrillary aspect of the GFAP staining. Moreover, neurons and the rare surviving oligodendrocytes showed degeneration of cell processes.
As the majority of neurons did not stain positive for viral antigen, we verified that PV receptor was expressed on their surface. CD155 was detected in cultures by immunofluorescence with the mouse monoclonal antibody IgG1 404.19 (1/100 dilution, kindly supplied by M. Lopez) (Lopez et al., 1997 ), followed by biotinylated antibody (1/100 dilution, Southern Biotechnology Associates, Inc.) and with streptavidinCY2 conjugate (1/200 dilution, Jackson ImmunoResearch). Neurons were identified with TUJ1 and the appropriate fluorescent secondary antibody in the same culture. CD155 was detected at the surface of all cells expressing neuron-specific
-tubulin (data not shown), indicating that the low level of neuron infection was not due to the lack of PV receptors but rather to a restriction in a post-binding event of the virus cycle. The role of the stage of maturation of the neuronal population in the culture on the susceptibility to PV needs to be studied further.
In the mixed mouse primary nerve cell cultures, the three main cell types of the CNS, neurons, astrocytes and oligodendrocytes, are thus all susceptible to PV infection. In contrast, in the CNS, the main cell target of PV is the motor neuron. However, no clear experiments using glial cell markers have yet been described and it would be interesting to perform double labelling for glial cell markers and viral antigens in the CNS of infected TgCD155 mice, even if the CD155 mRNA could not be detected by in situ hybridization in the glial cells of the TgCD155 mouse CNS (Koike et al., 1994 ).
To investigate whether PV infection of neural cells was associated with apoptosis, we analysed DNA fragmentation in infected cultures by testing for oligonucleosomal laddering, an indicator of apoptosis. At 16 h and 28 h post-infection, cells (6x106) were washed and suspended in a buffer containing 50 mM TrisHCl, pH 7·5, and 20 mM EDTA and lysed by addition of NP40 to a final concentration of 1%. After pelleting intact chromatin in an Eppendorf centrifuge (1200 g, 5 min, 4 °C), SDS was added to the supernatant to a final concentration of 0·5%. Supernatants were treated with 0·1 mg/ml proteinase K for 2 h at 50 °C and DNA was precipitated and end-labelled with terminal transferase (25 U) and digoxigenin-11-dUTP (50 µM final concentration) (Boehringer Mannheim) according to the manufacturers instructions. The DNA was treated with RNase A (0·1 mg/ml, 37 °C, 30 min) and the samples were electrophoresed on a 1·8% agarose gel, transferred to a Hybond-N nylon membrane (Amersham Life Science) and visualized by the digoxigenin luminescent detection kit (Boehringer Mannheim) with alkaline phosphatase-conjugated antibody and Lumin-phos Plus as the chemiluminescent substrate for the alkaline phosphatase.
No DNA laddering was visualized in mock-infected mixed primary nerve cell cultures (Fig. 3, lane 2). In cultures infected for 16 h, only a slight DNA laddering was detected (data not shown), whereas distinct DNA laddering, manifest as mono- (180200 bp), di-, and trinucleosome fragments, was clearly observed in cultures infected for 28 h (Fig. 3
, lane 3). Thus, PV infection caused apoptosis in mixed nerve cell cultures and DNA fragmentation appeared to be delayed, lagging behind the virus growth curve for PV-induced death in HEp-2 cells. In fact, late PV-induced apoptosis has also been previously observed in the CaCo-2 enterocyte-like cell line and in the U937 promonocyte cell line (Ammendolia et al., 1999
; Lopez-Guerrero et al., 2000
). The delay in PV-induced death in these cells might be based on the fact that CaCo-2 and U937 cells, as well as mixed nerve cells in our cultures, are more differentiated than HEp-2 cells.
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To determine whether PV-induced apoptosis was caspase-dependent, we studied the effect of the irreversible and cell-permeable pan-caspase inhibitor Z-VAD.FMK (benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethylketone) on apoptosis in PV-infected cell cultures. The inhibitor was used at a concentration of 100 µM, which has been shown to inhibit caspases completely in cultured mammalian cells (Slee et al., 1996 ) but which did not affect PV growth (data not shown). Apoptosis was determined by evaluating DNA fragmentation in samples 18 h post-infection by quantitative ELISA. Inhibition of caspases was not analysed after 18 h post-infection because Z-VAD.FMK had a toxic effect on these cell cultures after this time point. Infected cell cultures exhibited substantial oligonucleosome DNA fragmentation in the absence of caspase inhibitor, but in the presence of Z-VAD.FMK there was no detectable DNA fragmentation (Fig. 4
). Thus, the PV-induced apoptosis was caspase-dependent.
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References |
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Agol, V. I., Belov, G. A., Bienz, K., Egger, D., Kolesnikova, M. S., Romanova, L. I., Sladkova, L. V. & Tolskaya, E. A. (2000). Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle. Journal of Virology 74, 5534-5541.
Ammendolia, M. G., Tinari, A., Calcabrini, A. & Superti, F. (1999). Poliovirus infection induces apoptosis in CaCo-2 cells. Journal of Medical Virology 59, 122-129.[Medline]
Barco, A., Feduchi, E. & Carrasco, L. (2000). Poliovirus protease 3Cpro kills cells by apoptosis. Virology 266, 352-360.[Medline]
Ben-Hur, T., Rogister, B., Murray, K., Rougon, G. & Dubois-Dalcq, M. (1998). Growth and fate of PSA-NCAM+ precursors of the postnatal brain. Journal of Neuroscience 18, 5777-5788.
Blondel, B., Akacem, O., Crainic, R., Couillin, P. & Horodniceanu, F. (1983). Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat inactivated virions. Virology 126, 707-710.[Medline]
Bodian, D. & Howe, H. A. (1955). Emerging concept of poliomyelitis infection. Science 122, 105-108.[Medline]
Castelli, J. C., Hassel, B. A., Wood, K. A., Li, X. L., Amemiya, K., Dalakas, M. C., Torrence, P. F. & Youle, R. J. (1997). A study of the interferon antiviral mechanism apoptosis activation by the 25a system. Journal of Experimental Medicine 186, 967-972.
Castelli, J., Wood, K. A. & Youle, R. J. (1998). The 25A system in viral infection and apoptosis. Biomedicine and Pharmacotherapy 52, 386-390.
Couderc, T., Christodoulou, C., Kopecka, H., Marsden, S., Taffs, L. F., Crainic, R. & Horaud, F. (1989). Molecular pathogenesis of neural lesions induced by poliovirus type 1. Journal of General Virology 70, 2907-2918.[Abstract]
Couderc, T., Delpeyroux, J., Le Blay, H. & Blondel, B. (1996). Mouse adaptation determinants of poliovirus type 1 enhance viral uncoating. Journal of Virology 70, 305-312.[Abstract]
Cryns, V. & Yuan, J. (1998). Proteases to die for. Genes and Development 12, 1551-1570.
Earnshaw, W., Martins, L. & Kaufmann, S. (1999). Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annual Review of Biochemistry 68, 383-424.[Medline]
Girard, S., Couderc, T., Destombes, J., Thiesson, D., Delpeyroux, F. & Blondel, B. (1999). Poliovirus induces apoptosis in the mouse central nervous system. Journal of Virology 73, 6066-6072.
Goldstaub, D., Gradi, A., Bercovitch, Z., Grosmann, Z., Nophar, Y., Luria, S., Sonenberg, N. & Kahana, C. (2000). Poliovirus 2A protease induces apoptotic cell death. Molecular and Cell Biology 20, 1271-1277.
Kiss, J. Z., Wang, C., Olive, S., Rougon, G., Lang, J., Baetens, D., Harry, D. & Pralong, W. F. (1994). Activity-dependent mobilization of the adhesion molecule polysialic N-CAM to the cell surface of neurons and endocrine cells. EMBO Journal 13, 5284-5292.[Abstract]
Koike, S., Ise, I. & Nomoto, A. (1991). Functional domains of the poliovirus receptor. Proceedings of the National Academy of Sciences, USA 88, 4104-4108.[Abstract]
Koike, S., Aoki, J. & Nomoto, A. (1994). Transgenic mouse for the study of poliovirus pathogenicity. In Cellular Receptors for Animal Viruses , pp. 463-479. Edited by E. Wimmer. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Lopez, M., Jordier, F., Bardin, F., Coulombel, L. & Chabannon, C. (1997). CD155 workshop: identification of a new class of IgG superfamily antigens expressed in hemopoiesis. In Leukocyte Typing VI, White Cells Differentiation Antigen, pp. 10811083. Garland Publishing.
Lopez-Guerrero, J. A., Alonso, M., Martin-Belmonte, F. & Carrasco, L. (2000). Poliovirus induces apoptosis in the human U937 promonocytic cell line. Virology 272, 250-256.[Medline]
McKinnon, R. D., Matsui, T., Dubois-Dalcq, M. & Aaronson, S. A. (1990). FGF modulates the PDGF-driven pathway of oligodendrocyte development. Neuron 5, 603-614.[Medline]
Pavio, N., Buc-Caron, M.-H. & Colbère-Garapin, F. (1996). Persistent poliovirus infection of human fetal brain cells. Journal of Virology 70, 6395-6401.[Abstract]
Ren, R. B., Costantini, F., Gorgacz, E. J., Lee, J. J. & Racaniello, V. R. (1990). Transgenic mice expressing a human poliovirus receptor: a new model for poliomyelitis. Cell 63, 353-362.[Medline]
Roulston, A., Marcellus, R. & Branton, P. E. (1999). Virus and apoptosis. Annual Review of Microbiology 53, 577-628.[Medline]
Slee, E. A., Zhu, H., Chow, S. C., MacFarlane, M., Nicholson, N. D. & Cohen, G. M. (1996). Benzyloxycarbonyl-val-ala-asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochemical Journal 315, 21-24.[Medline]
Tolskaya, E. A., Romanova, L., Kolesnikova, M. S., Ivannikova, T. A., Smirnova, E. A., Raikhlin, N. T. & Agol, V. I. (1995). Apoptosis-inducing and apoptosis-preventing functions of poliovirus. Journal of Virology 69, 1181-1189.[Abstract]
Received 17 January 2002;
accepted 22 March 2002.