General discussion
5.3 Distinct cell tropism of canine distemper virus strains to adult olfactory ensheathing cells and Schwann cells in vitro
It is well established that OECs play an important role in the guidance of olfactory neuron axons from the nasal mucosa to the olfactory bulb during prenatal and postnatal development (Doucette, 1990). OECs, like Schwann cells, have been shown to display active phagocytosis (Bray et al., 1972; Chuah et al., 1995; Saada et al., 1996; Wewetzer et al., 2005) and to secrete pro- as well as anti-inflammatory cytokines (Cheepudomwit et al., 2008;
Getchell et al., 2002; Li et al., 2005; Meyer zu Hörste et al., 2008; Rutkowski et al., 1999;
Wewetzer et al., 2005). These observations suggested that both cell types may participate in immunoregulatory responses thereby protecting the nervous system from invading pathogens. Canine distemper virus (CDV), a member of the genus Morbillivirus of the family Paramyxoviridae, causes systemic and/or neurological signs (Baumgärtner and Alldinger, 2005; Beineke et al., 2009). Recently, it was shown that CDV enters the brain via the olfactory system in ferrets where transneuronal transmission along the olfactory axons occurs (Rudd et al., 2006). So far, investigations have focussed on olfactory receptor neurons and their role as vehicles for virus infection, while very little is known about the infection of OECs. Whether peripheral glia, such as Schwann cells act as a natural target for CDV has not been reported so far.
In the present study, cultures of adult canine Schwann cells and OECs were exposed to several attenuated (CDV-2544, CDV-R252, CDV-OndeGFP, CDV-Ond) and one mustelid virulent CDV strain (CDV-5804PeGFP). Both cell types were infected by CDV strains albeit to different levels. The cytopathic effect of affected cultures consisted of a few single necrotic cells and prominent monolayer detachment. Formation of multinucleated syncytial cells was absent in infected glial cultures. This is different from cell lines, such as African green monkey kidney cells (Vero cells), marmoset lymphoid cells (B95a) and subcutaneous fibroblasts which frequently show multinucleated syncytial formation following morbillivirus infection (Beineke et al., 2009; Gröne et al., 2002; Nishi et al., 2004). The percentage of CDV-infected cells increased until 10 days post infection as visualized by the co-localization of the cell type-specific marker p75NTR and the CDV nucleoprotein. In addition, all infected cultures produced infectious virus particles.
Following infection in vitro, both Schwann cells and OECs were affected by the different attenuated and virulent CDV strains, albeit to a variable extent. The 2544, CDV-OndeGFP, and CDV-5804PeGFP strains infected significantly more OECs compared to Schwann cells, whereas the CDV-Ond strain mainly targeted Schwann cells. No difference was found between both cell types following CDV-R252 infection and between the virulent
CDV-5804PeGFP and attenuated CDV-OndeGFP strains. The differential susceptibility of the both closely related cell types can be interpreted as evidence for the presence of subtle molecular differences between Schwann cells and OECs. This is in agreement with recent investigations reporting significant differences in the gene expression profile between both cell types at the molecular level in rats (Franssen et al., 2008). However, though CDV can infect the CNS via transneuronal transmission along olfactory neuron axons (Rudd et al., 2006), the localization of CDV in OECs is difficult to demonstrate in situ since the cells have only thin processes encircling bundles of olfactory axons (Field et al., 2003; Raisman, 1985).
Furthermore, no reliable cell type-specific markers for the identification of adult OECs in situ have become available (Wewetzer et al., 2002). Whether OECs become infected during CDV entry into the brain is not clear and has to be addressed by future studies.
The differential susceptibility of Schwann cells and OECs to CDV infection in vitro, as observed in the present study may have two main implications. Firstly, the increased susceptibility of the cells for CDV could be the results of cell isolation and culturing.
Secondly, the differential infection of both closely related cell types could be interpreted as evidence for subtle differences in the molecular setup between both cell types. It was shown that the p75 neurotrophin receptor (p75NTR) is a functional but not essential receptor for the neurotropic rabies virus (Tuffereau et al., 1998, 2007). This observation is in agreement with a previous report showing the preferential infection of p75NTR-positiveSchwann cell-like glial cells in adult canine brain in vitro by attenuated CDV strains (Orlando et al., 2008). In addition, the up-regulation of p75NTR expression observed in Schwann cells and OECs (Bock et al., 2007; Wewetzer et al., 2005) following cultivation, but its lack of expression in situ, would help to explain the discrepancy between in situ and in vitro CDV infection.
Nevertheless, it still remains to be clarified whether OECs and Schwann cells are targets for CDV infection in situ.
To summarize, the non-virulent and virulent strains of CDV differentially infected Schwann cells and OECs in vitro. Assuming a possible involvement of p75NTR in CDV infection, future studies have to analyze quantitatively p75NTR expression and to study possible correlations between p75NTR expression and susceptibility to CDV infection as well as to determine whether OECs represent a crucial factor during CDV infection in vivo and distemper pathogenesis.
References
Akimov, S. S., Ramezani, A., Hawley, T. S. and Hawley, R. G., 2005. Bypass of senescence, immortalization, and transformation of human hematopoietic progenitor cells. Stem Cells. 23, 1423-1433.
Alexander, C. L., Fitzgerald, U. F. and Barnett, S. C., 2002. Identification of growth factors that promote long-term proliferation of olfactory ensheathing cells and modulate their antigenic phenotype. Glia. 37, 349-364.
Aresu, L., Rastaldi, M. P., Pregel, P., Valenza, F., Radaelli, E., Scanziani, E. and Castagnaro, M., 2008. Dog as model for down-expression of E-cadherin and beta-catenin in tubular epithelial cells in renal fibrosis. Virchows Arch. 453, 617-625.
Argyle, D. J. and Nasir, L., 2003. Telomerase: a potential diagnostic and therapeutic tool in canine oncology. Vet Pathol. 40, 1-7.
Avellana-Adalid, V., Bachelin, C., Lachapelle, F., Escriou, C., Ratzkin, B. and Baron-Van Evercooren, A., 1998. In vitro and in vivo behaviour of NDF-expanded monkey Schwann cells. Eur J Neurosci. 10, 291-300.
Barnett, S. C., Alexander, C. L., Iwashita, Y., Gilson, J. M., Crowther, J., Clark, L., Dunn, L.
T., Papanastassiou, V., Kennedy, P. G. and Franklin, R. J., 2000. Identification of a human olfactory ensheathing cell that can effect transplant-mediated remyelination of demyelinated CNS axons. Brain. 123 ( Pt 8), 1581-1588.
Barnett, S. C. and Riddell, J. S., 2007. Olfactory ensheathing cell transplantation as a strategy for spinal cord repair--what can it achieve? Nat Clin Pract Neurol. 3, 152-161.
Baron-Van Evercooren, A., Avellana-Adalid, V., Lachapelle, F. and Liblau, R., 1997.
Schwann cell transplantation and myelin repair of the CNS. Mult Scler. 3, 157-161.
Baumgärtner, W. and Alldinger, S., 2005. The pathogenesis of canine distemper virus induced demyelination-a biphasic process. In: Lavi, E., Constantinescu, C. S. (Eds), Experimental models of multiple sclerosis, Springer, New York, pp. 871-887.
Beineke, A., Puff, C., Seehusen, F. and Baumgärtner, W., 2009. Pathogenesis and immunopathology of systemic and nervous canine distemper. Vet Immunol Immunopathol. 127, 1-18.
Blackburn, E. H., 1992. Telomerases. Annu Rev Biochem. 61, 113-129.
Bock, P., Beineke, A., Techangamsuwan, S., Baumgärtner, W. and Wewetzer, K., 2007.
Differential expression of HNK-1 and p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ but not in vitro. J Comp Neurol. 505, 572-585.
Bolin, L. M., Verity, A. N., Silver, J. E., Shooter, E. M. and Abrams, J. S., 1995. Interleukin-6 production by Schwann cells and induction in sciatic nerve injury. J Neurochem. 64, 850-858.
Bray, G. M., Peyronnard, J. M. and Aguayo, A. J., 1972. Reactions of unmyelinated nerve fibers to injury. An ultrastructural study. Brain Res. 42, 297-309.
Cairney, C. J. and Keith, W. N., 2008. Telomerase redefined: integrated regulation of hTR and hTERT for telomere maintenance and telomerase activity. Biochimie. 90, 13-23.
Cannell, E. J., Farrell, P. J. and Sinclair, A. J., 1996. Epstein-Barr virus exploits the normal cell pathway to regulate Rb activity during the immortalisation of primary B-cells.
Oncogene. 13, 1413-1421.
Cheepudomwit, T., Guzelsu, E., Zhou, C., Griffin, J. W. and Hoke, A., 2008. Comparison of cytokine expression profile during Wallerian degeneration of myelinated and unmyelinated peripheral axons. Neurosci Lett. 430, 230-235.
Chen, X. and Thibeault, S. L., 2008. Novel isolation and biochemical characterization of immortalized fibroblasts for tissue engineering vocal fold lamina propria. Tissue Eng Part C Methods.
Chuah, M. I., Tennent, R. and Jacobs, I., 1995. Response of olfactory Schwann cells to intranasal zinc sulfate irrigation. J Neurosci Res. 42, 470-478.
Cotman, C. W. and Head, E., 2008. The canine (dog) model of human aging and disease:
dietary, environmental and immunotherapy approaches. J Alzheimers Dis. 15, 685-707.
Davis, J. B. and Stroobant, P., 1990. Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells. J Cell Biol. 110, 1353-1360.
Di Donna, S., Mamchaoui, K., Cooper, R. N., Seigneurin-Venin, S., Tremblay, J., Butler-Browne, G. S. and Mouly, V., 2003. Telomerase can extend the proliferative capacity of human myoblasts, but does not lead to their immortalization. Mol Cancer Res. 1, 643-653.
Doucette, R., 1990. Glial influences on axonal growth in the primary olfactory system. Glia. 3, 433-449.
Eccleston, P. A., Mirsky, R. and Jessen, K. R., 1991. Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity. Development. 112, 33-42.
Field, P., Li, Y. and Raisman, G., 2003. Ensheathment of the olfactory nerves in the adult rat.
J Neurocytol. 32, 317-324.
Franklin, R. J., Gilson, J. M., Franceschini, I. A. and Barnett, S. C., 1996. Schwann cell-like myelination following transplantation of an olfactory bulb-ensheathing cell line into areas of demyelination in the adult CNS. Glia. 17, 217-224.
Franssen, E. H., De Bree, F. M., Essing, A. H., Ramón-Cueto, A. and Verhaagen, J., 2008.
Comparative gene expression profiling of olfactory ensheathing glia and Schwann cells indicates distinct tissue repair characteristics of olfactory ensheathing glia. Glia.
56, 1285-1298.
Funk, D., Fricke, C. and Schlosshauer, B., 2007. Aging Schwann cells in vitro. Eur J Cell Biol. 86, 207-219.
Getchell, T. V., Subhedar, N. K., Shah, D. S., Hackley, G., Partin, J. V., Sen, G. and Getchell, M. L., 2002. Chemokine regulation of macrophage recruitment into the olfactory epithelium following target ablation: involvement of macrophage inflammatory protein-1alpha and monocyte chemoattractant protein-1. J Neurosci Res. 70, 784-793.
Gorbunova, V. and Seluanov, A., 2003. Telomerase as a growth-promoting factor. Cell Cycle. 2, 534-537.
Gröne, A., Fonfara, S. and Baumgärtner, W., 2002. Cell type-dependent cytokine expression after canine distemper virus infection. Viral Immunol. 15, 493-505.
Hahn, W. C. and Weinberg, R. A., 2002. Modelling the molecular circuitry of cancer. Nat Rev Cancer. 2, 331-341.
Hoeben, E., Briers, T., Vanderstichele, H., De Smet, W., Heyns, W., Deboel, L., Vanderhoydonck, F. and Verhoeven, G., 1995. Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions. Endocrinology. 136, 2862-2873.
Jeffery, N. D., Lakatos, A. and Franklin, R. J., 2005. Autologous olfactory glial cell transplantation is reliable and safe in naturally occurring canine spinal cord injury. J Neurotrauma. 22, 1282-1293.
Jeffery, N. D., Smith, P. M., Lakatos, A., Ibanez, C., Ito, D. and Franklin, R. J., 2006. Clinical canine spinal cord injury provides an opportunity to examine the issues in translating laboratory techniques into practical therapy. Spinal Cord. 44, 584-593.
Jessen, K. R., Mirsky, R. and Morgan, L., 1991. Role of cyclic AMP and proliferation controls in Schwann cell differentiation. Ann N Y Acad Sci. 633, 78-89.
Jin, X., Lee, J. S., Kwak, S., Lee, S. Y., Jung, J. E., Kim, T. K., Xu, C., Hong, Z., Li, Z., Kim, S. M., Pian, X., Lee, D. H., Yoon, J. T., You, S., Choi, Y. J. and Kim, H., 2006.
Establishment and characterization of three immortal bovine muscular epithelial cell lines. Mol Cells. 21, 29-33.
Kapil, S., Allison, R. W., Johnston, L., 3rd, Murray, B. L., Holland, S., Meinkoth, J. and Johnson, B., 2008. Canine distemper virus strains circulating among North American dogs. Clin Vaccine Immunol. 15, 707-712.
Kotev-Emeth, S., Pitaru, S., Pri-Chen, S. and Savion, N., 2002. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2. Connect Tissue Res. 43, 606-612.
Krakowka, S. and Felsburg, P., 2005. Gnotobiotics and immunopathology: the use of the gnotobiotic environment to study acquired and inherited immunodeficiency diseases.
Vet Immunol Immunopathol. 108, 165-175.
Kreutzer, R., Kreutzer, M., Propsting, M. J., Sewell, A. C., Leeb, T., Naim, H. Y. and Baumgärtner, W., 2008. Insights into post-translational processing of beta-galactosidase in an animal model resembling late infantile human G-gangliosidosis. J Cell Mol Med. 12, 1661-1671.
Krudewig, C., Deschl, U. and Wewetzer, K., 2006. Purification and in vitro characterization of adult canine olfactory ensheathing cells. Cell Tissue Res 326, 687-696.
Kuroki, T. and Huh, N. H., 1993. Why are human cells resistant to malignant cell transformation in vitro? Jpn J Cancer Res. 84, 1091-1100.
Lee, C. J., Suh, E. J., Kang, H. T., Im, J. S., Um, S. J., Park, J. S. and Hwang, E. S., 2002.
Induction of senescence-like state and suppression of telomerase activity through inhibition of HPV E6/E7 gene expression in cells immortalized by HPV16 DNA. Exp Cell Res. 277, 173-182.
Lee, K. M., Choi, K. H. and Ouellette, M. M., 2004. Use of exogenous hTERT to immortalize primary human cells. Cytotechnology. 45, 33-38.
Levi, A. D., Bunge, R. P., Lofgren, J. A., Meima, L., Hefti, F., Nikolics, K. and Sliwkowski, M.
X., 1995. The influence of heregulins on human Schwann cell proliferation. J Neurosci. 15, 1329-1340.
Li, Y., Field, P. M. and Raisman, G., 2005. Olfactory ensheathing cells and olfactory nerve fibroblasts maintain continuous open channels for regrowth of olfactory nerve fibres.
Glia. 52, 245-251.
Masamune, A., Satoh, M., Kikuta, K., Suzuki, N. and Shimosegawa, T., 2003. Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization. World J Gastroenterol. 9, 2751-2758.
Mathon, N. F. and Lloyd, A. C., 2001. Cell senescence and cancer. Nat Rev Cancer. 1, 203-213.
Mathon, N. F., Malcolm, D. S., Harrisingh, M. C., Cheng, L. and Lloyd, A. C., 2001. Lack of replicative senescence in normal rodent glia. Science. 291, 872-875.
McEachern, M. J., Krauskopf, A. and Blackburn, E. H., 2000. Telomeres and their control.
Annu Rev Genet. 34, 331-358.
Meyer zu Hörste, G., Hu, W., Hartung, H. P., Lehmann, H. C. and Kieseier, B. C., 2008. The immunocompetence of Schwann cells. Muscle Nerve. 37, 3-13.
Monje, P. V., Bartlett Bunge, M. and Wood, P. M., 2006. Cyclic AMP synergistically enhances neuregulin-dependent ERK and Akt activation and cell cycle progression in Schwann cells. Glia. 53, 649-659.
Nasir, L., 2008. Telomeres and telomerase: Biological and clinical importance in dogs. Vet J.
175, 155-163.
Nishi, T., Tsukiyama-Kohara, K., Togashi, K., Kohriyama, N. and Kai, C., 2004. Involvement of apoptosis in syncytial cell death induced by canine distemper virus. Comp Immunol Microbiol Infect Dis. 27, 445-455.
Olson, J. K., Zamvil, S. S. and Miller, S. D., 2003. Efficient technique for immortalization of murine microglial cells relevant for studies in murine models of multiple sclerosis. J Neurosci Methods. 128, 33-43.
Opii, W. O., Joshi, G., Head, E., Milgram, N. W., Muggenburg, B. A., Klein, J. B., Pierce, W.
M., Cotman, C. W. and Butterfield, D. A., 2008. Proteomic identification of brain proteins in the canine model of human aging following a long-term treatment with antioxidants and a program of behavioral enrichment: relevance to Alzheimer's disease. Neurobiol Aging. 29, 51-70.
Orlando, E. A., Imbschweiler, I., Gerhauser, I., Baumgärtner, W. and Wewetzer, K., 2008. In vitro characterization and preferential infection by canine distemper virus of glial precursors with Schwann cell characteristics from adult canine brain. Neuropathol Appl Neurobiol. 34, 621-637.
Ouyang, H., Mou, L., Luk, C., Liu, N., Karaskova, J., Squire, J. and Tsao, M. S., 2000.
Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype. Am J Pathol. 157, 1623-1631.
Pringproa, K., Kumnok, J., Ulrich, R., Baumgärtner, W. and Wewetzer, K., 2008. In vitro characterization of a murine oligodendrocyte precursor cell line (BO-1) following spontaneous immortalization. Int J Dev Neurosci. 26, 283-291.
Prowse, K. R. and Greider, C. W., 1995. Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc Natl Acad Sci U S A. 92, 4818-4822.
Raisman, G., 1985. Specialized neuroglial arrangement may explain the capacity of vomeronasal axons to reinnervate central neurons. Neuroscience. 14, 237-254.
Rubio, M. P., Muñoz-Quiles, C. and Ramón-Cueto, A., 2008. Adult olfactory bulbs from primates provide reliable ensheathing glia for cell therapy. Glia. 56, 539-551.
Rudd, P. A., Cattaneo, R. and von Messling, V., 2006. Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion. J Virol. 80, 9361-9370.
Rutkowski, J. L., Tuite, G. F., Lincoln, P. M., Boyer, P. J., Tennekoon, G. I. and Kunkel, S. L., 1999. Signals for proinflammatory cytokine secretion by human Schwann cells. J Neuroimmunol. 101, 47-60.
Saada, A., Reichert, F. and Rotshenker, S., 1996. Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells. J Cell Biol.
133, 159-167.
Shay, J. W. and Wright, W. E., 2007. Hallmarks of telomeres in ageing research. J Pathol.
211, 114-123.
Smith, L. L., Coller, H. A. and Roberts, J. M., 2003. Telomerase modulates expression of growth-controlling genes and enhances cell proliferation. Nat Cell Biol. 5, 474-479.
Smith, P. M., Sim, F. J., Barnett, S. C. and Franklin, R. J., 2001. SCIP/Oct-6, Krox-20, and desert hedgehog mRNA expression during CNS remyelination by transplanted olfactory ensheathing cells. Glia. 36, 342-353.
Sobue, G., Shuman, S. and Pleasure, D., 1986. Schwann cell responses to cyclic AMP:
proliferation, change in shape, and appearance of surface galactocerebroside. Brain Res. 362, 23-32.
Sonigra, R. J., Kandiah, S. S. and Wigley, C. B., 1996. Spontaneous immortalisation of ensheathing cells from adult rat olfactory nerve. Glia. 16, 247-256.
Tang, D. G., Tokumoto, Y. M., Apperly, J. A., Lloyd, A. C. and Raff, M. C., 2001. Lack of replicative senescence in cultured rat oligodendrocyte precursor cells. Science. 291, 868-871.
Techangamsuwan, S., Imbschweiler, I., Kreutzer, R., Kreutzer, M., Baumgärtner, W. and Wewetzer, K., 2008. Similar behaviour and primate-like properties of adult canine Schwann cells and olfactory ensheathing cells in long-term culture. Brain Res. 1240, 31-38.
Techangamsuwan, S., Kreutzer, R., Kreutzer, M., Imbschweiler, I., Rohn, K., Wewetzer, K.
and Baumgärtner, W., 2009. Transfection of adult canine Schwann cells and olfactory ensheathing cells at early and late passage with human TERT differentially affects growth factor responsiveness and in vitro growth. J Neurosci Methods. 176, 112-120.
Tuffereau, C., Benejean, J., Blondel, D., Kieffer, B. and Flamand, A., 1998. Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus.
Embo J. 17, 7250-7259.
Tuffereau, C., Schmidt, K., Langevin, C., Lafay, F., Dechant, G. and Koltzenburg, M., 2007.
The rabies virus glycoprotein receptor p75NTR is not essential for rabies virus infection. J Virol. 81, 13622-13630.
Uebing-Czipura, A. U., Dawson, H. D. and Scherba, G., 2008. Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb. J Neurosci Methods. 170, 262-276.
Wewetzer, K. and Brandes, G., 2006. Axonal signaling and the making of olfactory ensheathing cells: a hypothesis. Neuron Glia Biol. 2, 217-224.
Wewetzer, K., Grothe, C. and Claus, P., 2001. In vitro expression and regulation of ciliary neurotrophic factor and its alpha receptor subunit in neonatal rat olfactory ensheathing cells. Neurosci Lett. 306, 165-168.
Wewetzer, K., Kern, N., Ebel, C., Radtke, C. and Brandes, G., 2005. Phagocytosis of O4+
axonal fragments in vitro by p75- neonatal rat olfactory ensheathing cells. Glia. 49, 577-587.
Wewetzer, K., Radtke, C., 2009. Translating basic research into clinical practice or what else do we have to learn about olfactory ensheathing cells? Neurosci Lett.
doi:10.1016/j.neulet.2008.07.097.
Wewetzer, K., Verdú, E., Angelov, D. N. and Navarro, X., 2002. Olfactory ensheathing glia and Schwann cells: two of a kind? Cell Tissue Res. 309, 337-345.
Xiang, H., Wang, J., Mao, Y., Liu, M., Reddy, V. N. and Li, D. W., 2002. Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway. Oncogene. 21, 3784-3791.
Yan, H., Bunge, M. B., Wood, P. M. and Plant, G. W., 2001. Mitogenic response of adult rat olfactory ensheathing glia to four growth factors. Glia. 33, 334-342.
You, S., Moon, J. H., Kim, T. K., Kim, S. C., Kim, J. W., Yoon, D. H., Kwak, S., Hong, K. C., Choi, Y. J. and Kim, H., 2004. Cellular characteristics of primary and immortal canine embryonic fibroblast cells. Exp Mol Med. 36, 325-335.
Young, J. I., Sedivy, J. M. and Smith, J. R., 2003. Telomerase expression in normal human fibroblasts stabilizes DNA 5-methylcytosine transferase I. J Biol Chem. 278, 19904-19908.
SUMMARY
Immortalization and proliferation of adult canine Schwann cells and olfactory ensheathing cells and their infection with canine distemper virus
Somporn Techangamsuwan
A variety of studies have shown that Schwann cells and olfactory ensheathing cells (OECs) are closely related glial cell types that promote axonal growth and remyelination in vivo following transplantation into the lesioned nervous system. However, the vast majority of these studies have been done using the rodent model and it is not clear in how far the data can be extrapolated to large animals and humans. Canine distemper virus (CDV) infection of adult dogs is considered an alternative animal model to study demyelinating diseases, including multiple sclerosis. Recently, it was shown that CDV can enter the brain via infection of olfactory neurons. Whether associated OECs or Schwann cells of the peripheral nerve are natural targets for CDV infection has not been reported so far. Based on these considerations, Schwann cells and OECs were transfected with ectopic human telomerase reverse transcriptase (hTERT) to induce immortalization thereby establishing a stable source of regenerative adult canine glial cells. The proliferation and differentiation of transfected (1) and non-transfected adult canine glia (2) was analyzed to reveal putative alterations induced by the immortalization procedure and to characterize species-specific properties of non-transfected glia. Finally, Schwann cells and OECs were exposed to different CDV strains in order to study the susceptibility of both cell types to CDV infection in vitro (3).
(1) Both Schwann cells and OECs were successfully immortalized by hTERT transfection.
However, both transfected cell types only displayed infinite growth in the presence of fibroblast growth factor-2 (FGF-2). Interestingly, adverse effects on cellular growth following hTERT transfection were observed. Though transfection of late passage Schwann cells and OECs induced FGF-2-dependent immortalization, transfection of early passage cells reduced proliferation independent of FGF-2. Immortalization was associated with subtle differences in the expression profile. Whereas the expression of p75 neurotrophin receptor (p75NTR), GFAP, p53, and p16 was unaltered, there were significant changes in the expression of O4 and A2B5. It is concluded that hTERT transfection is an efficient mean for establishing a stable source of adult canine glial cells and that hTERT plays distinct functional roles depending on the replicative age of the cultured cells.
(2) Adult canine non-transfected Schwann cells and OECs were analyzed regarding long-term proliferation, growth factor responses and antigenic expression. Both glial cell types increased proliferation in response to the same growth factors, including FGF-2 and heregulin-1ß, and displayed a similar expression of cell surface markers, such as p75NTR, O4 and A2B5. This underscored the assumption that both cell types are closely related to each other. Surprisingly, canine Schwann cells and OECs maintained long-term proliferation both under serum-containing and serum-free medium in the absence of any exogenous mitogens
(2) Adult canine non-transfected Schwann cells and OECs were analyzed regarding long-term proliferation, growth factor responses and antigenic expression. Both glial cell types increased proliferation in response to the same growth factors, including FGF-2 and heregulin-1ß, and displayed a similar expression of cell surface markers, such as p75NTR, O4 and A2B5. This underscored the assumption that both cell types are closely related to each other. Surprisingly, canine Schwann cells and OECs maintained long-term proliferation both under serum-containing and serum-free medium in the absence of any exogenous mitogens