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Cytokines in cerebellar mutant mice
265 responsible for inflammation and edema [3]. The same mediators are released during the immune system's response to viral infection and pass through the circulation to brain receptors where they generate the syndrome, 'sickness behavior' [4]. In animals, this is recognizable by inhibited goal-directed behaviour, and in humans, from feelings of apathy, fatigue and malaise. These symptoms bear a suspicious resemblance to those experienced commonly in SAR. Lymphokines so far identified as promoting sickness behaviour are also pyrogens. However, recent evidence indicates that lymphokine-mediated sickness behaviour can occur in the absence of fever [5]. It is therefore conceivable that the children's learning deficit was another manifestation of sickness behaviour, produced as part of a systemic reaction to the offending allergen. This concept is supported by the discovery that the serum concentration of one lymphokine, -interferon, is chronically elevated in euthermic SAP, patients [6]. References 1. Leutner, D., Geographiedidaktische Forsehungen, 18, 81 (1989). Leutner, D. and Schrettenbrunner, H., Unterrichtswissenschafl, 17, 327 (1989). 2. Lemanske, R.F. and Kaliner, M.A., in Allergy, Principles and Practice, Middelton, E., Reed, C.E., Ellis, E.F., Atkinson, N.F. and Yunginger, J.W., Eds. (Mosby, St. Louis, 1988), p. 236. 3. Askenase, P.W. in Allergy, Principles and Practice, Middelton, E., Reed, C.E., Ellis, E.F., Atkinson, N.F. and Yunginger, J.W., Eds. (Mosby, St. Louis, 1988), p. 253. 4. Dinarell, C.A., Blood, 8, 1627, (1991). 5. Kent, S., Bluthd, R.M., Kelly, K.W. and Danzer, R., Trends in Pharmacological Sciences, 13, 24 (1991). 6. Beatrice, F. et al., Clinical Allergy, 18: pp. 411 (1988.
Cytokines in cerebellar mutant mice
Wollman, E.E., Kopmels, B., Bakalian, A. 1, Delhaye-Bouchaud, N. 1 and Mariani, j.t Laboratoire d'hnmunomodulation, 1NSERM U 283, Hopital Cochin, Paris 75014, France lLaboratoire de Neurobiologie du Ddveloppement, Institut des Neurosciences, URA 1488, Centre National de la Recherche Scientifique et Universitd P. et M. Curie, 9 Quai st. Bernard, 75252 Paris Cedex 05, France Key words: Cytokines; CNS; Mutant mice; Macrophages; Hyperexcitability
Introduction Interleukin-1 (IL-I) and Tumor necrosis factor (TNF), are two major cytokines which are produced by stimulated macrophages and constitute well known immunomodulator factors. They are also supposed to play several roles within the central nervous system. Both are produced by microglial cells and/or astrocytes. Beside its pyrogenic effect and its action in the hypothalamo-hypophysial axis, IL-1 is also a growth factor for astrocytes ill vitro. TNF has also a growth promoting effect on human astrocytoma or glioma cell lines and can enhance class II antigens expression on Interferon (IFN) gamma stimulated astrocytes in vitro. In addition, these two cytokines seem to be involved ill several cases of neuronal degeneration in the brain. Indeed a significant level of IL-1 has been detected at the site of injury in the brain of adult rodents and elevated amounts of IL-1 have been described in diseases involving neuronal degeneration and astroglial proliferation in the brain such as Alzheimer and Down syndrom (Griffin et al., 1989). Robbins et al. (1987) have reported that TNF may participate to the destruction of oligodendroglial cells and the demyelinisation process in the CNS. Genetic mutations in mice appeared to be an efficient tool to obtain models of neuronal degeneration. Especially, a number of these mutants display patterns of neuronal loss in the cerebellum that have been extensively studied in the last decades. Immunological abnormalities, as an atrophy of the thymus and the spleen, a prolonged antibodies response have been
266 described in one of these mutants, the staggerer mouse (Trenkner and Hoffmann, 1986), and thus incited us to inquire about a possible relationship between the dysfunction of the two systems. Materials and Methods
Animals: staggerer (sg/sg) mutant mice were obtained by intercrossing known heterozygous (+/sg) animals and then identified on clinical symptoms. The sg mutation was bred on a C57BL/6J background. Lurcher mutant mice correspond to (Lc/+) animals. They were obtained by intercrossing known mutants (Lc/+) animals that are fertile and then identified on clinical symptoms. The Lc mutation was bred on a B6/CBA background. The other mutants were kindly given by different laboratories. Controls were age and strain matched (+/+) mice. In vitro macrophages stimulation: elicited macrophages were washed from the peritoneal cavity 3 days after intraperitoneal injection of thioglycolate (0.1 ml per gram of body weight). Intraperitoneal macrophages were seeded at a concentration of 106 cells per ml and stimulated, immediately after removal, with 5 ug/ml of LPS. Experiments with MDP or murabutide were performed as follows: the macrophages were prestimulated for 2 hours with one of the muramyl dipeptides using several doses. Then, the cells were pulsed with PLS (5 ug/ml) for 4 hours. RNA extraction: total RNA was extracted from the cells using the guanidinium/cesium chloride (CsCI) method slightly modified as follows: briefly, 50.106 cells in 75 ul of cold physiological saline buffer NaCI (0.9%) were lysed in 0.5 ml of 6 M guanidinium isothiocyanate. After sonication, the cell extract was layered over 0.5 ml of 5.7 M CSCI. It was ultracentrifuged for 90 minutes in a TL100 at 350,000 g. The pellet was resuspended and precipitated. Northern blots: northern blots were performed with 10 g of total splenocytes RNA. After electrophoretic migration on a 1.2% formaldehyde-agarose gel, the RNA was transferred onto a nylon membrane and hybridized with different cytokines cDNA probes. Prehybridization and hybridization were carried out at 65 ° in 5xDenhart, 6xSSC, 0.1% SDS, 10 ug salmon sperm DNA. In order to ensure that an equal amount of RNA was present in every sample, controls were performed by hybridization with 13 Actin probe. Scanning densitometry procedure: autoradiograms were quantified with an image analyser by measuring the mean optical density of each specific hybridization spot after delimination of their surface area. Normalization with respect to the actin signal was obtained by calculating the ratio between measured optical density of actin and the different cytokines. Biological assays: IL-I activity was determined by measuring the proliferation of a IL-1 dependent D10G4 cell line and IL-6 activity by measuring the proliferation of a IL-6 sensitive B9 cell line. R e s u l t s a n d discussion
To further characterize the alterations in immune function and assess their possible relationship to the neuronal loss affecting cerebellar mutants, we investigated at first the cytokine IL-1 beta expression and production in different cerebellar mutants (staggerer, lurcher, pcd, reeler). At the peripheral immune system level, we performed northern blots with the RNA of LPS-stimulated macrophages, probed with a IL-I beta cDNA. The mutants hyperexpress IL-I beta mRNA throughout the whole life from 3 weeks of age (not before) and hyperproduce IL-1 protein, when compared to wild type ones. Thus, macrophages appear to be a novel category affected directly or indirectly by these mutations. A crucial question then is to know if the IL-1 abnormality is specific of IL-1 expression or if it is due to an abnormal unspecific hyperexcitability of the macrophages. We have tried to distinguish between these possibilities by testing different parameters of the mRNA hyperexpression in the staggerer and lurcher mice: we performed in vitro macrophages stimulation kinetics with a time of stimulation varying form 15' to 24h, dose-response kinetics using different LPS doses between 0.01 to 5 ug/ml, using different stimulating agents (synthetic molecules as Muramyldipeptide), testing the effect of protein synthesis inhibitors like cycloheximide and the level of expression of other cytokines (IL-1 and TNF alpha, IL-6). An hyperexpression w a s detected in every single case, except for the IL-6 mRNA in the lurcher mutant. We checked in other mutants like a muscular one (med) and a myelin one (jimpy), in non genetic models of neuronal degeneration like Wistar rats which cerebellum w a s submitted to postnatal X-ray irradiation of to surgical partial cerebellectomy or rats lesioned with 3-acetyl-pyridine, and no hyperexpression of IL-I beta was found. A striking and puzzling result is that in the heterozygous staggerer +/sg, which exhibits a late onset of cerebellar neuronal loss, hyperexpression was present not only in 12-month old animals but also in 2-month old ones when the number of cerebellar neurons is still normal. Another noticeable feature is that all the mutants displaying IL-1 hyperexpression develop an astrogliosis. In order to check if the same phenomenon was present at the Central Nervous System level, we obtained
267 microglial cells in culture, stimulated them with LPS and performed northern blots with their RNAs. Again, a hyperexpression of IL-1 beta mRNA could be detected in the cells originating from the staggerer mutants versus wild type. The first conclusion is that peripheral immune abnormalities, especially macrophages hyperinducibility for several cytokines can be associated with neuronal degeneration occurring the spinocerebellar region, up to now only in cases of genetic origin. Since in some models, the genes have been shown to act directly in the neurons, the macrophage abnormality could either be due also to a direct expression of the mutated gene in the macrophages or be secondary to the neuronal degeneration. The fact that macrophage abnormality can precede neuronal loss for several weeks in some models (the heterozygous staggerer) make neuronal death per se unlikely to be responsible for immune abnormalities. A possible explanation would be that abnormal neurons are able, long before they die, to modify the responsiveness of immune cells. The pathways and mechanisms involved in this phenomenon remain to be elucidated. References
Griffin WST., Stanley, L.C., Ling, C., White, L., MacLeod, V., Perrot, L.J., White III, C.L. and Araoz, C. (1989). Brain Interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A., 86, 7611-7615. Robbins, D.S., Shirazi, Y., Drysdale, B., Leiberman, A., Shin, H.S. and Shin, M.L. (1987). Production of cytotoxic factor for oligodendrocytes by stimulated astrocytes. J. Immunol., 139, 2593-2597. Trenkner, E. and Hoffmann, M.K. (1986). Defective development of the thymus and immunological abnormalities in the neurological mouse mutation staggerer. J. Neurosci., 6, 1733-1737.
Cytokines in cerebellar mutant mice
Wollman, E.E., Kopmels, B., Bakalian, A. 1, Delhaye-Bouchaud, N. 1 and Mariani, j.t Laboratoire d'hnmunomodulation, 1NSERM U 283, Hopital Cochin, Paris 75014, France lLaboratoire de Neurobiologie du Ddveloppement, Institut des Neurosciences, URA 1488, Centre National de la Recherche Scientifique et Universitd P. et M. Curie, 9 Quai st. Bernard, 75252 Paris Cedex 05, France Key words: Cytokines; CNS; Mutant mice; Macrophages; Hyperexcitability
Introduction Interleukin-1 (IL-I) and Tumor necrosis factor (TNF), are two major cytokines which are produced by stimulated macrophages and constitute well known immunomodulator factors. They are also supposed to play several roles within the central nervous system. Both are produced by microglial cells and/or astrocytes. Beside its pyrogenic effect and its action in the hypothalamo-hypophysial axis, IL-1 is also a growth factor for astrocytes ill vitro. TNF has also a growth promoting effect on human astrocytoma or glioma cell lines and can enhance class II antigens expression on Interferon (IFN) gamma stimulated astrocytes in vitro. In addition, these two cytokines seem to be involved ill several cases of neuronal degeneration in the brain. Indeed a significant level of IL-1 has been detected at the site of injury in the brain of adult rodents and elevated amounts of IL-1 have been described in diseases involving neuronal degeneration and astroglial proliferation in the brain such as Alzheimer and Down syndrom (Griffin et al., 1989). Robbins et al. (1987) have reported that TNF may participate to the destruction of oligodendroglial cells and the demyelinisation process in the CNS. Genetic mutations in mice appeared to be an efficient tool to obtain models of neuronal degeneration. Especially, a number of these mutants display patterns of neuronal loss in the cerebellum that have been extensively studied in the last decades. Immunological abnormalities, as an atrophy of the thymus and the spleen, a prolonged antibodies response have been
266 described in one of these mutants, the staggerer mouse (Trenkner and Hoffmann, 1986), and thus incited us to inquire about a possible relationship between the dysfunction of the two systems. Materials and Methods
Animals: staggerer (sg/sg) mutant mice were obtained by intercrossing known heterozygous (+/sg) animals and then identified on clinical symptoms. The sg mutation was bred on a C57BL/6J background. Lurcher mutant mice correspond to (Lc/+) animals. They were obtained by intercrossing known mutants (Lc/+) animals that are fertile and then identified on clinical symptoms. The Lc mutation was bred on a B6/CBA background. The other mutants were kindly given by different laboratories. Controls were age and strain matched (+/+) mice. In vitro macrophages stimulation: elicited macrophages were washed from the peritoneal cavity 3 days after intraperitoneal injection of thioglycolate (0.1 ml per gram of body weight). Intraperitoneal macrophages were seeded at a concentration of 106 cells per ml and stimulated, immediately after removal, with 5 ug/ml of LPS. Experiments with MDP or murabutide were performed as follows: the macrophages were prestimulated for 2 hours with one of the muramyl dipeptides using several doses. Then, the cells were pulsed with PLS (5 ug/ml) for 4 hours. RNA extraction: total RNA was extracted from the cells using the guanidinium/cesium chloride (CsCI) method slightly modified as follows: briefly, 50.106 cells in 75 ul of cold physiological saline buffer NaCI (0.9%) were lysed in 0.5 ml of 6 M guanidinium isothiocyanate. After sonication, the cell extract was layered over 0.5 ml of 5.7 M CSCI. It was ultracentrifuged for 90 minutes in a TL100 at 350,000 g. The pellet was resuspended and precipitated. Northern blots: northern blots were performed with 10 g of total splenocytes RNA. After electrophoretic migration on a 1.2% formaldehyde-agarose gel, the RNA was transferred onto a nylon membrane and hybridized with different cytokines cDNA probes. Prehybridization and hybridization were carried out at 65 ° in 5xDenhart, 6xSSC, 0.1% SDS, 10 ug salmon sperm DNA. In order to ensure that an equal amount of RNA was present in every sample, controls were performed by hybridization with 13 Actin probe. Scanning densitometry procedure: autoradiograms were quantified with an image analyser by measuring the mean optical density of each specific hybridization spot after delimination of their surface area. Normalization with respect to the actin signal was obtained by calculating the ratio between measured optical density of actin and the different cytokines. Biological assays: IL-I activity was determined by measuring the proliferation of a IL-1 dependent D10G4 cell line and IL-6 activity by measuring the proliferation of a IL-6 sensitive B9 cell line. R e s u l t s a n d discussion
To further characterize the alterations in immune function and assess their possible relationship to the neuronal loss affecting cerebellar mutants, we investigated at first the cytokine IL-1 beta expression and production in different cerebellar mutants (staggerer, lurcher, pcd, reeler). At the peripheral immune system level, we performed northern blots with the RNA of LPS-stimulated macrophages, probed with a IL-I beta cDNA. The mutants hyperexpress IL-I beta mRNA throughout the whole life from 3 weeks of age (not before) and hyperproduce IL-1 protein, when compared to wild type ones. Thus, macrophages appear to be a novel category affected directly or indirectly by these mutations. A crucial question then is to know if the IL-1 abnormality is specific of IL-1 expression or if it is due to an abnormal unspecific hyperexcitability of the macrophages. We have tried to distinguish between these possibilities by testing different parameters of the mRNA hyperexpression in the staggerer and lurcher mice: we performed in vitro macrophages stimulation kinetics with a time of stimulation varying form 15' to 24h, dose-response kinetics using different LPS doses between 0.01 to 5 ug/ml, using different stimulating agents (synthetic molecules as Muramyldipeptide), testing the effect of protein synthesis inhibitors like cycloheximide and the level of expression of other cytokines (IL-1 and TNF alpha, IL-6). An hyperexpression w a s detected in every single case, except for the IL-6 mRNA in the lurcher mutant. We checked in other mutants like a muscular one (med) and a myelin one (jimpy), in non genetic models of neuronal degeneration like Wistar rats which cerebellum w a s submitted to postnatal X-ray irradiation of to surgical partial cerebellectomy or rats lesioned with 3-acetyl-pyridine, and no hyperexpression of IL-I beta was found. A striking and puzzling result is that in the heterozygous staggerer +/sg, which exhibits a late onset of cerebellar neuronal loss, hyperexpression was present not only in 12-month old animals but also in 2-month old ones when the number of cerebellar neurons is still normal. Another noticeable feature is that all the mutants displaying IL-1 hyperexpression develop an astrogliosis. In order to check if the same phenomenon was present at the Central Nervous System level, we obtained
267 microglial cells in culture, stimulated them with LPS and performed northern blots with their RNAs. Again, a hyperexpression of IL-1 beta mRNA could be detected in the cells originating from the staggerer mutants versus wild type. The first conclusion is that peripheral immune abnormalities, especially macrophages hyperinducibility for several cytokines can be associated with neuronal degeneration occurring the spinocerebellar region, up to now only in cases of genetic origin. Since in some models, the genes have been shown to act directly in the neurons, the macrophage abnormality could either be due also to a direct expression of the mutated gene in the macrophages or be secondary to the neuronal degeneration. The fact that macrophage abnormality can precede neuronal loss for several weeks in some models (the heterozygous staggerer) make neuronal death per se unlikely to be responsible for immune abnormalities. A possible explanation would be that abnormal neurons are able, long before they die, to modify the responsiveness of immune cells. The pathways and mechanisms involved in this phenomenon remain to be elucidated. References
Griffin WST., Stanley, L.C., Ling, C., White, L., MacLeod, V., Perrot, L.J., White III, C.L. and Araoz, C. (1989). Brain Interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A., 86, 7611-7615. Robbins, D.S., Shirazi, Y., Drysdale, B., Leiberman, A., Shin, H.S. and Shin, M.L. (1987). Production of cytotoxic factor for oligodendrocytes by stimulated astrocytes. J. Immunol., 139, 2593-2597. Trenkner, E. and Hoffmann, M.K. (1986). Defective development of the thymus and immunological abnormalities in the neurological mouse mutation staggerer. J. Neurosci., 6, 1733-1737.