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Trophic effect of granulocyte-macrophage colony-stimulating factor on central cholinergic neurons in vitro
Brain Research, 532 (1990) 323-325 Elsevier
323
BRES 24358
Trophic effect of granulocyte-macrophage colony-stimulating factor on central cholinergic neurons in vitro Masahiro Kamegai, Yoshihiro Konishi and Takeshi Tabira Division of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Tokyo 187 (Japan) (Accepted 24 July 1990) Key words." Granulocyte-macrophage colony-stimulating factor; Interleukin-3; Cholinergic; Septal neuron
Granulocyte-macrophage colony-stimulating factor (GM-CSF) elevated choline acetyltransferase (CHAT) activities of mouse septal neurons as well as of cholinergic hybridoma line cells SN6.10.2.2 in vitro. It augmented ChAT activities and neurite extension of interleukin 3-activated cholinergic neurons. Thus, GM-CSF should be added as atrophic factor for central cholinergic neurons.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a T-cell-derived cytokine that has capacity to induce hematopoietic progenitor cells to proliferate and differentiate to granulo,'~'tes and macrophages 11. The effect of G M - C S F mainly, appears at later cell stage, and it induces differentiation and growth of cells generated by interleukin-3 (IL-3) 3. Since we found that IL-3 is a novel trophic factor for central cholinergic neurons in vitro and in vivo 9, we tested the potentiality of G M - C S F and the interaction between G M - C S F and IL-3 on cholinergic n e u r o n s in vitro. The methods for dissociated neuronal culture in the serum-free defined medium was described previously9. Briefly, cells from the septal region of BALB/c mice on the 15th embryonic day were dissociated into dishes coated with 10 ktg/ml poly-L-lysine (PLL, Sigma) and cultured with our serum-free defined medium. To see the effect on neurite formation, cells were seeded at 1 x 105 cells/ml in PLL-coated 12-well plates (Falcon, Becton Dikinson, Oxnard, CA) and 10 U/ml mouse recombinant G M - C S F (Genzyme, Boston, M A ) was added in the m e d i u m at the start of culture. Pictures were taken randomly using a phase-contrast microscope at 0, 20, 40 and 50 h after culture, and neurite-bearing cells were counted on printed pictures at a final magnification of 160x. For choline acetyltransferase (CHAT) assay, cells were seeded at 6 x 105 cells/ml and 50 U/ml h u m a n r e c o m b i n a n t interleukin-3 (IL-3, Genzyme) or 1-100 U/ml G M - C S F was added in the medium. The culture m e d i u m was changed on day 3 and C h A T activities were assayed on day 5 by our slight modification of F o n n u m ' s
method 4. Protein content was m e a s u r e d by Lowry's method. To see the serial effect of IL-3 and GM-CSF, the m e d i u m of 50 U/ml IL-3-treated culture was changed to the one containing 10 U/ml G M - C S F on day 3 and C h A T
50
-
40
.~ 30
'i 20 Z
o-o: control, e - e : none~m GM-CSF, x-x: hlL-3 ~-~: hlL-3~mGM-CSF, D-o: mGM-CSF =-B: hlL-3/mGM-CSF 2~0
4'0
5'0
hr
Fig. 1. Effect of IL-3 and GM-CSF on neurite formation of septal neurons. Culture was started with or without 50 U/ml IL-3, 10 U/ml GM-CSF or a mixture of 50 U/ml IL-3 and 10 U/ml GM-CSF, and the medium was changed with fresh IL-3, GM-CSF or a mixture of IL-3 and GM-CSF at 20 h. Neurite-bearing cells were counte d at 0, 20, 40 and 50 h. Experiments were done twice and mean S.E.M. < 5.7. O---(3, none; ~ - ~ , none --~ GM-CSF; x~x, IL-3 ~ IL-3; ~ , IL-3 ~ GM-CSF; ~ , GM-CSF ~ GM-CSF; ~ , IL-3 + GM-CSF.
Correspondence: T. Tabira, Division of Demyelinating Disease, National Institute of Neuroscience, NCNP, 4-1-10gawahigashi, Kodaira, Tokyo 187, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
324 TABLE I Control
Effect of GM- CSF on ChA T activities of mouse septal neurons
GM-CSF
6 x 105 cells/ml of mouse septal neurons were treated with the indicated dose of GM-CSF and ChAT activities were measured on day 5. Sample
(n)
Specific activity (pmole/ mg/min)
Total protein (~g/ well)
Total Relative ChAT activity activity (%) (pmole/ h/well)
8.5 13.1 20.7 31.2 23.9 25.8
145 188 115 143 119 108
74 148 143 268 171 167
I
IL-Ia IL-I8
I I
IL-2 IL-3
I
IL-6
Control GM-CSF
1U/ml 5U/ml 10U/ml 50U/ml 100U/ml
(2) (2) (2) (2) (2) (2)
100 200 268 362 231 226
l~0
2~0 Relative Activity
(%)
Fig. 2. Effect of cytokines on ChAT activities of cholinergic hybridoma cell line SN6.10.2.2.2 x 105 cells/ml were cultured with serum-free defined medium containing each cytokine, and ChAT activities were measured 48 h later in duplicated dishes each. Values are expressed as relative activities (%) to the total ChAT activities of the control.
activities were assayed similarly on day 5. To confirm the effect, cholinergic hybridoma line cells SN6.10.2.27 kindly gifted from Dr. Wainer (University of Chicago) were cultured in a 35-mm dish (Falcon) at the cell density of 2 x 105/ml. 10 U/ml GM-CSF, 50 U/ml IL-3, 20 U/ml h u m a n recombinant interleukin l a ( I L - l a , G e n z y m e ) , 20 U/ml h u m a n recombinant interleukin lfl (IL-lfl, Genzyme), 20 U/ml h u m a n recombinant interleukin 2 (IL-2, Genzyme), 90 ng/ml h u m a n recombinant interleukin 6 (IL-6, gifted from Dr. Hirano at Osaka
m a x i m u m effect at a dose of 10 U/ml. W h e n G M - C S F was added after IL-3 treatment, it further enhanced ChAT activities (Table II). G M - C S F and IL-3 also elevated C h A T activities of SN6.10.2.2, suggesting direct effect on n e u r o n a l cells (Fig. 2). H u m a n G M - C S F 2'1°'13 is composed of 144 amino acids and the sequence is highly homologous to murine G M - C S F 5'6 (54%). Despite the high degree of homology between h u m a n and murine GM-CSF, the expressed
University), or 100 ng/ml fl nerve growth factor (NGF, gifted from Dr. Furukawa at this institute) was added in the culture medium, and C h A T activities were measured similarly. O n the neurite formation, IL-3 significantly increased the n u m b e r of neurite-bearing cells (55-63%) of the primary cultured septal neurons compared to the controls, while G M - C S F per se did not show such effect (Fig. 1). However, when culture was started with IL-3 and the m e d i u m was changed to the one containing GM-CSF at 20 h, neurite-bearing cells were further increased. As shown in Table I, G M - C S F induced elevation of ChAT activities of primary cultured septal neurons with a
h u m a n gene product has no activity on murine bone marrow cells2. This is why we chose mouse G M - C S F in this experiment. However, as h u m a n IL-3 was active with murine n e u r o n s 9 but not with i m m u n e cells, G M - C S F may also be the same. How G M - C S F acts on cholinergic neurons is as yet to be determined. The producer of G M - C S F in the central nervous system is not k n o w n yet. Since GMoCSF is produced by endothelial cells 1, brain endothelial cells highly probably produce G M - C S E G M - C S F induces macrophage CSF (M-CSF) 8 which induces activation and proliferation of microglia cells 12. Although the biological m e a n i n g of
TABLE II Serial effect of lL-3 and G M-CSF on ChAT activities of mouse septal neurons Culture was started with 50 U/ml IL-3, 10 U/ml GM-CSF, or none, and on day 3, the culture medium was refreshed to the one with the indicated cytokine. ChAT activities were measured on day 5.
Control IL-3 --~ IL-3 none--~ GM-CSF IL-3 --~ GM-CSF GM-CSF--~ GM-CSF
(n)
Specific activity (pmole/mg/min )
Total protein (l~g/well)
Total ChA T activity (pmole/h/well)
Relative activity (%)
(5) (3) (3) (3) (3)
12.2 + 2.5 29.5 + 2.0 16.0 + 3.2 53.6 + 13.5 43.5 + 7.5
97.8 + 18.5 110 + 16.7 104 + 21.4 122 + 3.9 106 + 6.5
71.1 + 17.6 193 + 23.7 97.4 + 10.7 389 + 85.3 279 + 63.7
100 272 137 548 392
325 G M - C S F in vivo is not k n o w n yet, it must be considered as a t r o p h i c factor for central cholinergic neurons, and cytokine network in the brain should be studied.
1 Burgess, A.W. and Metcalf, D., The nature and action of granulocyte-macrophage colony stimulating factors, Blood, 56 (1980) 947-958. 2 Cantrell, M.A., Anderson, D., Cerretti, D.P., Price, V., McKereghan, K., Tushinski, R.J., Mochizuki, D.Y., Larsen, A., Grabstein, K., Gillis, S. and Cosman, D., Cloning, sequence, and expression of a human granulocyte/macrophage colonystimulating factor, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 6250-6254. 3 Clark, S.C. and Kamen, R., The human hematopoietic colonystimulating factors, Science, 236 (1987) 1229-1237. 4 Fonnum, E, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 5 Gough, N.M., Gough, J., Metcalf, D., Kelso, A., Grail, D., Nicola, N.A., Burgess, A.W. and Dunn, A.R., Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor, Nature, 309 (1984) 763-767. 6 Gough, N.M., Metcalf, D., Gough, J., Grail, D. and Dunn, A.R., Structure and expression of the mRNA for murine granulocyte-macrophage colony stimulating factor, EMBO J., 4 (1985) 645-653. 7 Hammond, D.N., Wainer, B.H., Tonsgard, J.H. and Heller, A., Neuronal properties of clonal hybrid cell lines derived from central cholinergic neurons, Science, 234 (1986) 1237-1240.
This work was partially supported by a grant from Health Science Foundation and a grant (Dementia) from the Department of Health and Welfare, Japan. The authors thank Miss Kakeba for typing this manuscript.
8 Horiguchi, J., Warren, M.K. and Kufe, D., Expression of the macrophage-specific colony-stimulating factor in human monocytes treated with granuiocyte-macrophage colony-stimulating factor, Blood, 69 (1987) 1259-1261. 9 Kamegai, M., Niijima, K., Kunishita, T., Nishizawa, M., Ogawa, M., Araki, M., Ueki, A., Konishi, Y. and Tabira, T., Interleukin 3 as atrophic factor for central cholinergic neurons in vitro and in vivo, Neuron, 4 (1990) 429-436. 10 Lee, F., Yokota, T., Otsuka, T., Gemmell, L., Larson, N., Luh, J., Arai, K. and Rennick, D., Isolation of cDNA for a human granulocyte-macrophage colony-stimulating factor by functional expression in mammalian cells, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 4360-4364. 11 Metcalf, D., The granulocyte-macrophage colony-stimulating factors, Science, 229 (1985) 16-22. 12 Sawada, M., Suzumura, A., Yamamoto, H. and Marunouchi, T., Activation and proliferation of the isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase C, Brain Research, 509 (1990) 119-124. 13 Wong, G.G., Witek, J.S., Temple, P.A., Wilkens, K.M., Leary, A.C., Luxenberg, D.P., Jones, S.S., Brown, E.L., Kay, R.M., Orr, E.C., Shoemaker, C., Golde, D.W., Kaufman, R.J., Hewick, R.M., Wang, E.A. and Clark, S.C., Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins, Science, 228 (1985) 810-815.
323
BRES 24358
Trophic effect of granulocyte-macrophage colony-stimulating factor on central cholinergic neurons in vitro Masahiro Kamegai, Yoshihiro Konishi and Takeshi Tabira Division of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Tokyo 187 (Japan) (Accepted 24 July 1990) Key words." Granulocyte-macrophage colony-stimulating factor; Interleukin-3; Cholinergic; Septal neuron
Granulocyte-macrophage colony-stimulating factor (GM-CSF) elevated choline acetyltransferase (CHAT) activities of mouse septal neurons as well as of cholinergic hybridoma line cells SN6.10.2.2 in vitro. It augmented ChAT activities and neurite extension of interleukin 3-activated cholinergic neurons. Thus, GM-CSF should be added as atrophic factor for central cholinergic neurons.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a T-cell-derived cytokine that has capacity to induce hematopoietic progenitor cells to proliferate and differentiate to granulo,'~'tes and macrophages 11. The effect of G M - C S F mainly, appears at later cell stage, and it induces differentiation and growth of cells generated by interleukin-3 (IL-3) 3. Since we found that IL-3 is a novel trophic factor for central cholinergic neurons in vitro and in vivo 9, we tested the potentiality of G M - C S F and the interaction between G M - C S F and IL-3 on cholinergic n e u r o n s in vitro. The methods for dissociated neuronal culture in the serum-free defined medium was described previously9. Briefly, cells from the septal region of BALB/c mice on the 15th embryonic day were dissociated into dishes coated with 10 ktg/ml poly-L-lysine (PLL, Sigma) and cultured with our serum-free defined medium. To see the effect on neurite formation, cells were seeded at 1 x 105 cells/ml in PLL-coated 12-well plates (Falcon, Becton Dikinson, Oxnard, CA) and 10 U/ml mouse recombinant G M - C S F (Genzyme, Boston, M A ) was added in the m e d i u m at the start of culture. Pictures were taken randomly using a phase-contrast microscope at 0, 20, 40 and 50 h after culture, and neurite-bearing cells were counted on printed pictures at a final magnification of 160x. For choline acetyltransferase (CHAT) assay, cells were seeded at 6 x 105 cells/ml and 50 U/ml h u m a n r e c o m b i n a n t interleukin-3 (IL-3, Genzyme) or 1-100 U/ml G M - C S F was added in the medium. The culture m e d i u m was changed on day 3 and C h A T activities were assayed on day 5 by our slight modification of F o n n u m ' s
method 4. Protein content was m e a s u r e d by Lowry's method. To see the serial effect of IL-3 and GM-CSF, the m e d i u m of 50 U/ml IL-3-treated culture was changed to the one containing 10 U/ml G M - C S F on day 3 and C h A T
50
-
40
.~ 30
'i 20 Z
o-o: control, e - e : none~m GM-CSF, x-x: hlL-3 ~-~: hlL-3~mGM-CSF, D-o: mGM-CSF =-B: hlL-3/mGM-CSF 2~0
4'0
5'0
hr
Fig. 1. Effect of IL-3 and GM-CSF on neurite formation of septal neurons. Culture was started with or without 50 U/ml IL-3, 10 U/ml GM-CSF or a mixture of 50 U/ml IL-3 and 10 U/ml GM-CSF, and the medium was changed with fresh IL-3, GM-CSF or a mixture of IL-3 and GM-CSF at 20 h. Neurite-bearing cells were counte d at 0, 20, 40 and 50 h. Experiments were done twice and mean S.E.M. < 5.7. O---(3, none; ~ - ~ , none --~ GM-CSF; x~x, IL-3 ~ IL-3; ~ , IL-3 ~ GM-CSF; ~ , GM-CSF ~ GM-CSF; ~ , IL-3 + GM-CSF.
Correspondence: T. Tabira, Division of Demyelinating Disease, National Institute of Neuroscience, NCNP, 4-1-10gawahigashi, Kodaira, Tokyo 187, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
324 TABLE I Control
Effect of GM- CSF on ChA T activities of mouse septal neurons
GM-CSF
6 x 105 cells/ml of mouse septal neurons were treated with the indicated dose of GM-CSF and ChAT activities were measured on day 5. Sample
(n)
Specific activity (pmole/ mg/min)
Total protein (~g/ well)
Total Relative ChAT activity activity (%) (pmole/ h/well)
8.5 13.1 20.7 31.2 23.9 25.8
145 188 115 143 119 108
74 148 143 268 171 167
I
IL-Ia IL-I8
I I
IL-2 IL-3
I
IL-6
Control GM-CSF
1U/ml 5U/ml 10U/ml 50U/ml 100U/ml
(2) (2) (2) (2) (2) (2)
100 200 268 362 231 226
l~0
2~0 Relative Activity
(%)
Fig. 2. Effect of cytokines on ChAT activities of cholinergic hybridoma cell line SN6.10.2.2.2 x 105 cells/ml were cultured with serum-free defined medium containing each cytokine, and ChAT activities were measured 48 h later in duplicated dishes each. Values are expressed as relative activities (%) to the total ChAT activities of the control.
activities were assayed similarly on day 5. To confirm the effect, cholinergic hybridoma line cells SN6.10.2.27 kindly gifted from Dr. Wainer (University of Chicago) were cultured in a 35-mm dish (Falcon) at the cell density of 2 x 105/ml. 10 U/ml GM-CSF, 50 U/ml IL-3, 20 U/ml h u m a n recombinant interleukin l a ( I L - l a , G e n z y m e ) , 20 U/ml h u m a n recombinant interleukin lfl (IL-lfl, Genzyme), 20 U/ml h u m a n recombinant interleukin 2 (IL-2, Genzyme), 90 ng/ml h u m a n recombinant interleukin 6 (IL-6, gifted from Dr. Hirano at Osaka
m a x i m u m effect at a dose of 10 U/ml. W h e n G M - C S F was added after IL-3 treatment, it further enhanced ChAT activities (Table II). G M - C S F and IL-3 also elevated C h A T activities of SN6.10.2.2, suggesting direct effect on n e u r o n a l cells (Fig. 2). H u m a n G M - C S F 2'1°'13 is composed of 144 amino acids and the sequence is highly homologous to murine G M - C S F 5'6 (54%). Despite the high degree of homology between h u m a n and murine GM-CSF, the expressed
University), or 100 ng/ml fl nerve growth factor (NGF, gifted from Dr. Furukawa at this institute) was added in the culture medium, and C h A T activities were measured similarly. O n the neurite formation, IL-3 significantly increased the n u m b e r of neurite-bearing cells (55-63%) of the primary cultured septal neurons compared to the controls, while G M - C S F per se did not show such effect (Fig. 1). However, when culture was started with IL-3 and the m e d i u m was changed to the one containing GM-CSF at 20 h, neurite-bearing cells were further increased. As shown in Table I, G M - C S F induced elevation of ChAT activities of primary cultured septal neurons with a
h u m a n gene product has no activity on murine bone marrow cells2. This is why we chose mouse G M - C S F in this experiment. However, as h u m a n IL-3 was active with murine n e u r o n s 9 but not with i m m u n e cells, G M - C S F may also be the same. How G M - C S F acts on cholinergic neurons is as yet to be determined. The producer of G M - C S F in the central nervous system is not k n o w n yet. Since GMoCSF is produced by endothelial cells 1, brain endothelial cells highly probably produce G M - C S E G M - C S F induces macrophage CSF (M-CSF) 8 which induces activation and proliferation of microglia cells 12. Although the biological m e a n i n g of
TABLE II Serial effect of lL-3 and G M-CSF on ChAT activities of mouse septal neurons Culture was started with 50 U/ml IL-3, 10 U/ml GM-CSF, or none, and on day 3, the culture medium was refreshed to the one with the indicated cytokine. ChAT activities were measured on day 5.
Control IL-3 --~ IL-3 none--~ GM-CSF IL-3 --~ GM-CSF GM-CSF--~ GM-CSF
(n)
Specific activity (pmole/mg/min )
Total protein (l~g/well)
Total ChA T activity (pmole/h/well)
Relative activity (%)
(5) (3) (3) (3) (3)
12.2 + 2.5 29.5 + 2.0 16.0 + 3.2 53.6 + 13.5 43.5 + 7.5
97.8 + 18.5 110 + 16.7 104 + 21.4 122 + 3.9 106 + 6.5
71.1 + 17.6 193 + 23.7 97.4 + 10.7 389 + 85.3 279 + 63.7
100 272 137 548 392
325 G M - C S F in vivo is not k n o w n yet, it must be considered as a t r o p h i c factor for central cholinergic neurons, and cytokine network in the brain should be studied.
1 Burgess, A.W. and Metcalf, D., The nature and action of granulocyte-macrophage colony stimulating factors, Blood, 56 (1980) 947-958. 2 Cantrell, M.A., Anderson, D., Cerretti, D.P., Price, V., McKereghan, K., Tushinski, R.J., Mochizuki, D.Y., Larsen, A., Grabstein, K., Gillis, S. and Cosman, D., Cloning, sequence, and expression of a human granulocyte/macrophage colonystimulating factor, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 6250-6254. 3 Clark, S.C. and Kamen, R., The human hematopoietic colonystimulating factors, Science, 236 (1987) 1229-1237. 4 Fonnum, E, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 5 Gough, N.M., Gough, J., Metcalf, D., Kelso, A., Grail, D., Nicola, N.A., Burgess, A.W. and Dunn, A.R., Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor, Nature, 309 (1984) 763-767. 6 Gough, N.M., Metcalf, D., Gough, J., Grail, D. and Dunn, A.R., Structure and expression of the mRNA for murine granulocyte-macrophage colony stimulating factor, EMBO J., 4 (1985) 645-653. 7 Hammond, D.N., Wainer, B.H., Tonsgard, J.H. and Heller, A., Neuronal properties of clonal hybrid cell lines derived from central cholinergic neurons, Science, 234 (1986) 1237-1240.
This work was partially supported by a grant from Health Science Foundation and a grant (Dementia) from the Department of Health and Welfare, Japan. The authors thank Miss Kakeba for typing this manuscript.
8 Horiguchi, J., Warren, M.K. and Kufe, D., Expression of the macrophage-specific colony-stimulating factor in human monocytes treated with granuiocyte-macrophage colony-stimulating factor, Blood, 69 (1987) 1259-1261. 9 Kamegai, M., Niijima, K., Kunishita, T., Nishizawa, M., Ogawa, M., Araki, M., Ueki, A., Konishi, Y. and Tabira, T., Interleukin 3 as atrophic factor for central cholinergic neurons in vitro and in vivo, Neuron, 4 (1990) 429-436. 10 Lee, F., Yokota, T., Otsuka, T., Gemmell, L., Larson, N., Luh, J., Arai, K. and Rennick, D., Isolation of cDNA for a human granulocyte-macrophage colony-stimulating factor by functional expression in mammalian cells, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 4360-4364. 11 Metcalf, D., The granulocyte-macrophage colony-stimulating factors, Science, 229 (1985) 16-22. 12 Sawada, M., Suzumura, A., Yamamoto, H. and Marunouchi, T., Activation and proliferation of the isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase C, Brain Research, 509 (1990) 119-124. 13 Wong, G.G., Witek, J.S., Temple, P.A., Wilkens, K.M., Leary, A.C., Luxenberg, D.P., Jones, S.S., Brown, E.L., Kay, R.M., Orr, E.C., Shoemaker, C., Golde, D.W., Kaufman, R.J., Hewick, R.M., Wang, E.A. and Clark, S.C., Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins, Science, 228 (1985) 810-815.