- Email: [email protected]
Cytotoxic changes in A172 glioblastoma cells exposed to serum Ig from patients with motor neurone disease
JOURNAL OF THE
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 129 (Suppl.)(1995) 136-137
Cytotoxic changes in A172 glioblastoma cells exposed to serum Ig from patients with motor neurone disease A.B. Parkes a,,, C.R. Rickards a, p. Rees b, M.F. Scanlon a ~Autoimmunology Research Unit, Section of Endocrinology, Metabolism and Diabetes, Department of Medicine. University of Wales" College of Medicine. Cardiff CF4 4XN, Wales, UK b traculO2 of Health Care Studies, Cardiff Institute of Higher Education, Cardiff CF5 2YB, Wales, UK Accepted 21 February 1995
Keywords: Glioblastoma A172 cell; Immunoglobulin; Nuclear staining; Amyotrophic lateral sclerosis; Alzheimer's disease; Cytotoxicity; Autoimmunity 1. Introduction
The aetiology of motor neurone disease (MND) is unknown although several hypotheses have been put forward in an attempt to explain the phenomenon. These hypotheses have included intoxication with heavy metal ions (Roelofs-Iverson et al., 1984), mineral deficiencies (Garrato et al., 1985), exposure to neurotoxins (Calne et al., 1986) or the possibility that a previous virus infection (Kennedy, 1990), particularly with the polio virus (Martyn et al., 1988), could be responsible for the later onset of MND. Whatever the primary cause of MND there is evidence to suggest that the immune system could be important in the continuing pathology of MND. We have recently described the presence of antibodies in the serum of patients with MND which interact in an ELISA with epitopes expressed by A172 glioblastoma cells (Rickards et al., 1992). In this study we extend these observations by examining the viability of A172 cells following their exposure to immunoglobulins (Ig) isolated from the serum of patients with MND.
Immunoglobulins were prepared from serum samples using ammonium sulphate precipitation and the protein concentration adjusted 1 m g / m l with PBS before use. A172 cells were seeded onto a Multitest slide (Flow Laboratories) placed in the bottom of a 5-cm petri dish (2.5 × 106 cells in 10 ml of D M E M supplemented with 2 m m o l / l glutamine and pyruvate, 10% fetal calf serum and antibiotics) and incubated at 37°C in a humidified atmosphere containing 5% CO z for 24 h. The slides were washed with Earl's BSS and 10 /~1 of the Ig preparation was added to each well. The slides were further incubated in a humidified chamber for 1 h at 25°C and, after washing with PBS, the cells were stained with ethidium bromide (3.7 /xg/ml 20 /~l/well) for 20 rain at 25°C and examined using a fluorescence microscope. The number of stained nuclei in each of 3 fields examined were enumerated for each culture and the median number of immunocomproraised cells recorded for each serum sample.
3. Results 2. Methods
Serum samples were collected from patients with MND together with samples from patients with Alzheimer's disease and from neurological control subjects.
* Corresponding author. Tel.: (+44-1222) 742 307; Fax: (+441222) 743 819. 0022-510X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0022-51 0 X ( 9 5 ) 0 0 0 8 4 - 4
A172 cells exposed to control Ig preparations showed a background level of nuclear staining (median 1.5 nuclei/field, n = 4). These levels of nuclear staining with ethidium bromide are commensurate with a culture viability of 90-95%. By contrast the exposure of A172 cells to Ig preparations from MND serum showed a much higher degree of nuclear staining (median 16.5 nuclei/field, n = 8) indicating a large decrease in cell viability which was statistically significant ( p < 0.009
A.B. Parkes et al. /Journal of the Neurological Sciences 129 (Suppl.) (1995) 136-137
Mann-Whitney). Exposure of A172 cells to Ig preparations from patients with Alzheimer's disease also showed an increased level of nuclear staining with ethidium bromide (median 4 nuclei/field, n = 5, p < 0.02).
4. Discussion
Evidence of humoral autoimmune changes, as indicated by the presence in the circulation of autoantibodies directed against nervous tissue antigens, in MND have been reported by several groups in recent years. Digby et al. (1985) were able to show binding of immunoglobulins isolated from the serum of patients with MND to rat spinal cord cells in tissue culture. These same investigators later demonstrated that these autoantibodies were of the IgG class and could bind to synaptic membranes isolated from human motor cortex in an ELISA (Rutter et al., 1987). Others have used antigen separated by electrophoresis and Western blotting to show immune reactivity. A study by Brown et al. (1987) showed autoreactivity in MND serum samples which was directed against human and B A L B / c mouse spinal cord antigens separated by PAGE. The antigens recognised were primarily of molecular weight 70 kDa and 50 kDa but included other higher molecular weight proteins (150-200 kDa). Some control samples also showed autoreactivity in this system but with a much lower frequency than tat seen with MND serum. Inuzuka et al. (1989) also used P A G E and immunoblotting to show reactivity against human spinal cord proteins in the serum of a subgroup of patients with MND. These autoantibodies were all of the IgG class and reacted with both membrane and soluble fraction proteins of molecular weights ranging between 35 and 115 kDa with a strong signal around 55 kDa. Immunohistochemical staining techniques have also been used to demonstrate the presence of autoantibody activity in serum samples obtained from patients with MND using samples of human spinal cord or motor cortex as target antigens (Engelhardt and Appel, 1990; Kawamata et al., 1992). In a study by Greiner et al. (1992) a strong immunohistochemical signal could also be demonstrated in the serum of a proportion of MND patients. However, a positive reaction could only be demonstrated when foetal or neonatal nervous tissue was used as the target antigen. These authors concluded that normal adult CNS tissue does not express the same antigens as foetal or neonatal tissue; antigens expressed on foetal CNS tissue being downregulated early in ontogenesis but might be expressed abnormally in the adult leading to autoreactivity. Using the A172 glioblastoma cell line (Giard et al.,
137
1973) as a source of antigen we recently demonstrated a higher incidence of autoreactivity in NMD serum samples compared to neurological control subjects suggesting that an MND specific antigen was being expressed on these cells in culture (Rickards et al., 1992). In this present study we have extended these observations by showing that the interaction of MND immunoglobulins with A172 cells is cytotoxic. In conclusion, this p a p e r demonstrates that immunoglobulin preparations from MND patients are cytotoxic to A172 glioblastoma cells in tissue culture and further supports an autoimmune basis for the continuing pathology of MND.
References Brown, R.H., Johnson, D., Ogonowski, M. and Weiner, H.L. (1987) Antineural antibodies in the serum of patients with amyotrophic lateral sclerosis. Neurology, 37: 152-155. Calne, D.B., McGeer, E.G., Eisen, A. and Spencer, P. (1986) Alzheimer's disease, Parkinson's disease and motor neurone disease: a biotrophic interaction between aging and environment? Lancet, ii: 1067-1070. Digby, J., Harrison, R., Jehanli, A., Lunt, G.G. and Clifford Rose, F. (1985) Cultures rat spinal cord neurones: Interaction with motor neurone disease immunoglobulins. Muscle Nerve, 8: 595-605. Engelhardt, J.I. and Appel, S.H. (1990) IgG reactivity in the spinal cord and motor cortex in amyotrophic lateral sclerosis. Arch. Neurol., 47: 1210-1216. Garrato, R.M., Swyt, C., Fiori, C.E., Yanagihara, R. and Gajdusek, D.C. (1985) Intraneuronal deposition of calcium and aluminium in amyotrophic lateral sclerosis of Guam. Lancet, ii: 1353. Giard, D.J., Aaronson, S.A., Todaro, G.J., Arnstein, P., Kersey, J.H., Dosik, H. and Parks, W.P. (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst., 51: 1417-1423. Greiner, A., Marx, A., Muller-Hermelink, H.K., Schmausser, B., Petzold, K. and Kruger, H. (1992) Vascular autoantibodies in amyotrophic lateral sclerosis. Lancet, 340: 378-379. Inuzuka, T., Sato, S., Yanagisawa, K. and Miyatate, T. (1989) Antibodies to human spinal cord proteins in sera from patients with motor neurone disease and other neurological diseases. Eur. Neurol., 29: 328-332. Kawamata, T., Akiyama, H., Yamada, T. and McGeer, P.L. (1992) Immunological reactions in amyotrophic lateral sclerosis brain and spinal cord tissue. Am. J. Pathol., 140: 691-707. Kennedy, P.G.E. (1990) On the possible role of viruses in the aetiology of motor neurone disease: a review. J. R. Soc. Med., 83: 784-787. Martyn, C.N., Barker, D.J.P. and Osmond, C. (1988) Motor neuron disease and past poliomyelitis in England and Wales. Lancet, i: 1319-1322. Rickards, C.R., Parkes, A.B., Williams, H., Ham, J. and Scanlon, M.F. (1992) Antibodies against a human glioblastoma cell line in patients with chronic neurological disease. Eur. J. Neurosci., 37. Roelofs-lverson, R.A., Mulder, D.W., EIveback, L.R., Kurland, L.J. and Molygaard, C.A. (1984) ALS and heavy metals: a pilot case-control study. Neurology, 34: 393-395. Rutter, J.V., Jehanli, A., Harrison, R. and Lunt, G.G. (1987) Immunological factors in neuronal degeneration with particular reference to motor neurone disease. Gerontology, 33: 187-192.
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 129 (Suppl.)(1995) 136-137
Cytotoxic changes in A172 glioblastoma cells exposed to serum Ig from patients with motor neurone disease A.B. Parkes a,,, C.R. Rickards a, p. Rees b, M.F. Scanlon a ~Autoimmunology Research Unit, Section of Endocrinology, Metabolism and Diabetes, Department of Medicine. University of Wales" College of Medicine. Cardiff CF4 4XN, Wales, UK b traculO2 of Health Care Studies, Cardiff Institute of Higher Education, Cardiff CF5 2YB, Wales, UK Accepted 21 February 1995
Keywords: Glioblastoma A172 cell; Immunoglobulin; Nuclear staining; Amyotrophic lateral sclerosis; Alzheimer's disease; Cytotoxicity; Autoimmunity 1. Introduction
The aetiology of motor neurone disease (MND) is unknown although several hypotheses have been put forward in an attempt to explain the phenomenon. These hypotheses have included intoxication with heavy metal ions (Roelofs-Iverson et al., 1984), mineral deficiencies (Garrato et al., 1985), exposure to neurotoxins (Calne et al., 1986) or the possibility that a previous virus infection (Kennedy, 1990), particularly with the polio virus (Martyn et al., 1988), could be responsible for the later onset of MND. Whatever the primary cause of MND there is evidence to suggest that the immune system could be important in the continuing pathology of MND. We have recently described the presence of antibodies in the serum of patients with MND which interact in an ELISA with epitopes expressed by A172 glioblastoma cells (Rickards et al., 1992). In this study we extend these observations by examining the viability of A172 cells following their exposure to immunoglobulins (Ig) isolated from the serum of patients with MND.
Immunoglobulins were prepared from serum samples using ammonium sulphate precipitation and the protein concentration adjusted 1 m g / m l with PBS before use. A172 cells were seeded onto a Multitest slide (Flow Laboratories) placed in the bottom of a 5-cm petri dish (2.5 × 106 cells in 10 ml of D M E M supplemented with 2 m m o l / l glutamine and pyruvate, 10% fetal calf serum and antibiotics) and incubated at 37°C in a humidified atmosphere containing 5% CO z for 24 h. The slides were washed with Earl's BSS and 10 /~1 of the Ig preparation was added to each well. The slides were further incubated in a humidified chamber for 1 h at 25°C and, after washing with PBS, the cells were stained with ethidium bromide (3.7 /xg/ml 20 /~l/well) for 20 rain at 25°C and examined using a fluorescence microscope. The number of stained nuclei in each of 3 fields examined were enumerated for each culture and the median number of immunocomproraised cells recorded for each serum sample.
3. Results 2. Methods
Serum samples were collected from patients with MND together with samples from patients with Alzheimer's disease and from neurological control subjects.
* Corresponding author. Tel.: (+44-1222) 742 307; Fax: (+441222) 743 819. 0022-510X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0022-51 0 X ( 9 5 ) 0 0 0 8 4 - 4
A172 cells exposed to control Ig preparations showed a background level of nuclear staining (median 1.5 nuclei/field, n = 4). These levels of nuclear staining with ethidium bromide are commensurate with a culture viability of 90-95%. By contrast the exposure of A172 cells to Ig preparations from MND serum showed a much higher degree of nuclear staining (median 16.5 nuclei/field, n = 8) indicating a large decrease in cell viability which was statistically significant ( p < 0.009
A.B. Parkes et al. /Journal of the Neurological Sciences 129 (Suppl.) (1995) 136-137
Mann-Whitney). Exposure of A172 cells to Ig preparations from patients with Alzheimer's disease also showed an increased level of nuclear staining with ethidium bromide (median 4 nuclei/field, n = 5, p < 0.02).
4. Discussion
Evidence of humoral autoimmune changes, as indicated by the presence in the circulation of autoantibodies directed against nervous tissue antigens, in MND have been reported by several groups in recent years. Digby et al. (1985) were able to show binding of immunoglobulins isolated from the serum of patients with MND to rat spinal cord cells in tissue culture. These same investigators later demonstrated that these autoantibodies were of the IgG class and could bind to synaptic membranes isolated from human motor cortex in an ELISA (Rutter et al., 1987). Others have used antigen separated by electrophoresis and Western blotting to show immune reactivity. A study by Brown et al. (1987) showed autoreactivity in MND serum samples which was directed against human and B A L B / c mouse spinal cord antigens separated by PAGE. The antigens recognised were primarily of molecular weight 70 kDa and 50 kDa but included other higher molecular weight proteins (150-200 kDa). Some control samples also showed autoreactivity in this system but with a much lower frequency than tat seen with MND serum. Inuzuka et al. (1989) also used P A G E and immunoblotting to show reactivity against human spinal cord proteins in the serum of a subgroup of patients with MND. These autoantibodies were all of the IgG class and reacted with both membrane and soluble fraction proteins of molecular weights ranging between 35 and 115 kDa with a strong signal around 55 kDa. Immunohistochemical staining techniques have also been used to demonstrate the presence of autoantibody activity in serum samples obtained from patients with MND using samples of human spinal cord or motor cortex as target antigens (Engelhardt and Appel, 1990; Kawamata et al., 1992). In a study by Greiner et al. (1992) a strong immunohistochemical signal could also be demonstrated in the serum of a proportion of MND patients. However, a positive reaction could only be demonstrated when foetal or neonatal nervous tissue was used as the target antigen. These authors concluded that normal adult CNS tissue does not express the same antigens as foetal or neonatal tissue; antigens expressed on foetal CNS tissue being downregulated early in ontogenesis but might be expressed abnormally in the adult leading to autoreactivity. Using the A172 glioblastoma cell line (Giard et al.,
137
1973) as a source of antigen we recently demonstrated a higher incidence of autoreactivity in NMD serum samples compared to neurological control subjects suggesting that an MND specific antigen was being expressed on these cells in culture (Rickards et al., 1992). In this present study we have extended these observations by showing that the interaction of MND immunoglobulins with A172 cells is cytotoxic. In conclusion, this p a p e r demonstrates that immunoglobulin preparations from MND patients are cytotoxic to A172 glioblastoma cells in tissue culture and further supports an autoimmune basis for the continuing pathology of MND.
References Brown, R.H., Johnson, D., Ogonowski, M. and Weiner, H.L. (1987) Antineural antibodies in the serum of patients with amyotrophic lateral sclerosis. Neurology, 37: 152-155. Calne, D.B., McGeer, E.G., Eisen, A. and Spencer, P. (1986) Alzheimer's disease, Parkinson's disease and motor neurone disease: a biotrophic interaction between aging and environment? Lancet, ii: 1067-1070. Digby, J., Harrison, R., Jehanli, A., Lunt, G.G. and Clifford Rose, F. (1985) Cultures rat spinal cord neurones: Interaction with motor neurone disease immunoglobulins. Muscle Nerve, 8: 595-605. Engelhardt, J.I. and Appel, S.H. (1990) IgG reactivity in the spinal cord and motor cortex in amyotrophic lateral sclerosis. Arch. Neurol., 47: 1210-1216. Garrato, R.M., Swyt, C., Fiori, C.E., Yanagihara, R. and Gajdusek, D.C. (1985) Intraneuronal deposition of calcium and aluminium in amyotrophic lateral sclerosis of Guam. Lancet, ii: 1353. Giard, D.J., Aaronson, S.A., Todaro, G.J., Arnstein, P., Kersey, J.H., Dosik, H. and Parks, W.P. (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst., 51: 1417-1423. Greiner, A., Marx, A., Muller-Hermelink, H.K., Schmausser, B., Petzold, K. and Kruger, H. (1992) Vascular autoantibodies in amyotrophic lateral sclerosis. Lancet, 340: 378-379. Inuzuka, T., Sato, S., Yanagisawa, K. and Miyatate, T. (1989) Antibodies to human spinal cord proteins in sera from patients with motor neurone disease and other neurological diseases. Eur. Neurol., 29: 328-332. Kawamata, T., Akiyama, H., Yamada, T. and McGeer, P.L. (1992) Immunological reactions in amyotrophic lateral sclerosis brain and spinal cord tissue. Am. J. Pathol., 140: 691-707. Kennedy, P.G.E. (1990) On the possible role of viruses in the aetiology of motor neurone disease: a review. J. R. Soc. Med., 83: 784-787. Martyn, C.N., Barker, D.J.P. and Osmond, C. (1988) Motor neuron disease and past poliomyelitis in England and Wales. Lancet, i: 1319-1322. Rickards, C.R., Parkes, A.B., Williams, H., Ham, J. and Scanlon, M.F. (1992) Antibodies against a human glioblastoma cell line in patients with chronic neurological disease. Eur. J. Neurosci., 37. Roelofs-lverson, R.A., Mulder, D.W., EIveback, L.R., Kurland, L.J. and Molygaard, C.A. (1984) ALS and heavy metals: a pilot case-control study. Neurology, 34: 393-395. Rutter, J.V., Jehanli, A., Harrison, R. and Lunt, G.G. (1987) Immunological factors in neuronal degeneration with particular reference to motor neurone disease. Gerontology, 33: 187-192.