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Interleukin-1 β and tumor necrosis factor-α synergistically stimulate nerve growth factor (NGF) release from cultured rat astrocytes
Neuroscience Letters, 117 (1990) 335-340
335
Elsevier Scientific Publishers Ireland Ltd. NSL 07156
Interleukin-1 fl and tumor necrosis factor-e synergistically stimulate nerve growth factor (NGF) release from cultured rat astrocytes R.A. Gadient, K.C. C r o n a n d U. O t t e n Department of Physiology, University of Basel, Basel (Switzerland) (Received 1 June 1990; Accepted 6 June 1990)
Key words." Nerve growth factor production; Astrocyte; Interleukin-l; Tumor necrosis factor Nerve growth factor (NGF) may mediate responses to brain injury. To examine regulation of NGF gene expression with respect to neural trauma we examined the effects of interleukin-1 fl (IL-lfl) and tumor necrosis factor-at (TNF-ct) on NGF production in cultures of rat astroglial cells. Purified neocortical astrocytes in serum-free medium were treated with IL-I/¢, TNF-~ or both. Whereas IL-lfl and TNF-~t alone elicited only small effects, simultaneous addition elicited within 48 h a large (3- to 6-fold) increase in NGF content in culture supernatants. Our data are consistent with a role for cytokines in NGF synthesis and release in the injured central nervous system (CNS).
Nerve growth factor (NGF) is the best characterized member [15] of a family of neurotrophic polypeptide factors [14]. It is essential for the differentiation and maintenance of function of sympathetic and sensory neurons in the peripheral nervous system (PNS). In addition, N G F appears to be involved in regenerative events in the PNS [10]. Although it is not clear at present which factors regulate N G F gene expression there is evidence that mediators of inflammation, including interleukin-lfl (ILlfl), are involved [16, 17]. Recent evidence indicates that N G F modulates important functions in the central nervous system (CNS): e.g. it is atrophic agent for cholinergic neurons of the basal forebrain [12, 30] as well as in the caudate-putamen [21]. By in situ hybridization N G F m R N A has been localized to neurons in the CNS [1, 26]. The degeneration of basal forebrain neurons which follows lesion of the septohippocampal pathway can be markedly reduced by N G F [9, 31]. Whether these experiments reflect an effect of N G F in reversing the effects of brain injury is uncertain. However, brain damage does lead to a rapid accumulation of N G F [19, 28, 30] and to increased levels of several cytokines including IL-lfl [7] and tumor necrosis factor(TNF-c0 [11], at sites of neural injury. Recently, it was shown that intrastriatal and Correspondence."U. Otten, Department of Physiology, University of Basel, Vesalgasse I, 4051 Basel, Switzerland. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
336
intraventricular application of IL-lfl stimulated NGF synthesis. These data strongly suggest that IL-lfl regulates NGF synthesis in the CNS [22, 27] and thereby induces events important for reducing neural degeneration, but the locus of cytokine actions and their mode of action is not known. Increasing data indicate that NGF is produced by astrocytes in primary cultures [5, 18, 27]. In the present study the effects of cytokines on N G F production in defined primary astrocyte cultures kept in serumfree medium has been investigated. Astrocyte cultures from the neocortex of newborn Fii albino SPF rats were prepared by the method of McCarthy and deVellis [20]. After 18-20 days incubation, cultures were detached with 10 ml 0.2% ethylenediaminetetraacetate in phosphate buffered saline containing 0.125% trypsin (Gibco) and were seeded in Falcon plastic culture dishes (10 cm diameter). After 3 days, cultures were switched to a serum-free chemically-defined (N2) medium [2] supplemented with 10 mM L-leucinemethylester to eliminate brain macrophages [6]. Primary cultures of astrocytes were grown for 2 days before cytokines were added to the culture medium. The cell density was 4 × 107 cells/plate and purity of cultured cells was determined by immunofluorescence techniques using antibodies against glial fibrillary acidic protein (GFAP), fibronectin, galactocerebroside (GalC) and the specific macrophage markers ED-1, ED-2 [3] as well as Mac-I [23]. Immunohistochemical analysis revealed that cultured cells represented at least 95% astrocytes which were GFAP- and fibronectin-positive. EDl-positive microglia could rarely ( < 1%) be detected in the cultures. In addition, cultured astrocytes were Mac- 1, ED-2 and GalC-negative. In line with previous observations [25] there was no evidence for increased cell death during cytokine incubation. Supernatants of cultured astrocytes were collected and concentrated (10 times) by using Amicon Centriprep concentration units with a cut off of 10 kDa. Recovery of added N G F during this concentration step amounted to more than 80%. NGF determinations were performed by a sensitive two-site immunosorbent assay (ELISA) [29]. 500 -~ 0
E
uo
~d0 300
°~ u_
200
o z
IOO
Control
IL-1fl
TNF-a
IL-li~+ TNF-a
Fig. 1. Cytokine-indueed NGF release by cuRured astrocytes. Ghal cultures were stimulated with either IL-lfl (160 U/ml) or TNF-7 (50 U/ml) or the combination of both TNF-~t (50 U/ml)+ IL-lfl (160 U/ml). Supernatants were collected after a 48 h incubation period. Untreated astrocytes (control) released 3.6_ 0.4 pg/ml NGF. Data are the mean + S.E.M. (bars), values from 6 independent determinations. Statistical analysis was by Student's t-test (*P<0.001).
337
TABLE I EFFECTS OF IL-lfl AND TNF-a ON NGF PRODUCTION BY CULTURED RAT NEOCORTICAL ASTROCYTES AFTER 48 h Values represent the mean _+ S.E.M. of at least 4 independent experiments. Untreated astrocytes (control) released 3.6+0.4 pg/ml NGF within 48 h. IL, interleukin; TNF, tumor necrosis factor; NGF, nerve growth factor. Treatment
NGF release (% of control)
Control
100.0__+ 10.4
IL-I,8 30 U/ml IL-lfl 160 U/ml IL-lfl 500 U/ml
56.7_+ 10.8" 169.4_+ 13.1"* 76.8 _+ 11. I
TNF-~ 5 U/ml TNF-~ 50 U/mI TNF-~ 400 U/ml
100.3-+ 6.0 80.3-+ 9.0 104.0-+ 6.0
IL- lfl 160 U/ml + TNF-~ 5 U/ml IL-lfl 160 U/ml+TNF-c~ 50 U/ml IL- lfl 160 U/ml + TNF-~ 400 U/ml
355.9_+ 35.4** 453.2_+46.2** 595.6 _+33.6**
*P<0.05, **P< 0.001 as compared to control: Student's t-test,
Biological activity of immunoreactive NGF was monitored by using cultured PC l2 cells [8]. Human recombinant IL-lc~ and IL-lfl (provided by Dr. K. Vosbeck, Ciba Geigy, Basel) and human recombinant TNF-~ and human recombinant IL-6 (provided by Dr. R. Peck, Hoffmann-LaRoche, Basel) were used throughout this study. A variety of different cytokines, including IL-I~, IL-lfl, IL-6 and TNF-~ were tested for the ability to stimulate NGF release by cortical astrocytes. Only IL-lfl at a concentration of 160 U/ml elicited a small but significant (P<0.001) increase in release of immunoreactive NGF (Fig. 1, Table I). All other cytokines including ILl~, TNF-~ and IL-6 failed to significantly affect NGF-release. In several biological systems IL-lfl and TNF-~ interact in a synergistic way [13, 24] and each induces the synthesis of the other [4]. Therefore, it was of interest to investigate whether IL-lfl and TNF-c~ exert synergistic effect on NGF-release by cultivated astrocytes. As shown in Table ! and Fig. 1 combined treatment of astroglial cells with IL-lfl and TNF-~ triggered a marked increase of immunoreactive NGF in the culture medium after 48 h. TNF-~ and IL-lfl at doses as low as 5 U/ml and 160 U/ml respectively induced a 3.5-fold increase in NGF. At the highest dose of TNF-~ tested (400 U/ml) simultaneous addition of 160 U/ml IL-lfl caused a 6-fold increase in NGF. The time course of cytokine induced NGF release is shown in Fig. 2. Within 4 days, the latest point investigated, simultaneous addition of TNF-~ and IL-lfl synergistically stimulated NGF-production 10-fold. In PCI2 cell bioassay system, we found that fiber outgrowth elicited by astrocyte-conditioned medium was completely inhibited by addition of either monoclonal anti-NGF antibodies (mAB23c4) or polyclonal goat-anti NGF-antibodies. These data indicate that cytokine-stimulated astroglial cells release
338
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800-
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1
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2 3 Time (days)
I
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Fig. 2. Time-dependent increase of NGF release by cytokine-stimulated astroglial cultures. Cultures were stimulated by simultaneous addition of IL-lfl (160 U/rnl) and TNF-a (50 U/ml). Culture supernatants were collected after 1, 2 and 4 days incubation periods. Unstimulated astrocytes (control, C) released 5.3 + 0.5 pg/ml NGF. Values given represent the mean + S.E.M. of at least 3 independent determinations, Statistical analysis was by Student's t-test (*P < 0.025, **P < 0.001).
biologically active NGF. The mechanism whereby TNF-a and IL-1//induce release of biologically active NGF from cortical astrocytes cultured in serum-free medium is not known. A pilot experiment showing that cytokine treatment produced a marked increase in NGF mRNA levels in astrocytes strongly suggests that increased NGF release is a result of increased NGF gene expression. In conclusion, this study reports that IL-lfl and TNF-a interact synergistically to increase NGF release from cultured astrocytes. Whether these inflammatory mediators are involved in the physiological NGF synthesis in vivo remains to be investigated. However, given their presence at sites of neural injury it is possible that they play an important role in increasing NGF levels in lesioned brain tissue. This study was supported by the Swiss National Foundation for Scientific Research (Grant 31-25690.88) and the Deutsche Forschungsgemeinschaft (SFB 325). We would like to thank to Dr. P. Ehrhard and M, Rimle for helpful advice and discussion. 1 Ayer-LeLievre, C.S., Olson, L., Ebendal, T. Seiger, A. and Persson, H., Expression of the ]/-nerve growth factor gene in hippocampal neurons, Science 240 (1988) 1389-1391.
339 2 Bottenstein, J. and Sato, G., Growth of a rat neuroblastoma cell line in serum-free supplemented media, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 514-517. 3 Dijkstra, C.D., Dopp, E.A., Joling, P. and Kraal, G., The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED-1, ED-2 and ED-3, Immunology, 54 (1985) 589-599. 4 Fibbe, W.E., Schaafsma, M.R., Falkenburg, J.H.F. and Wilemze, R., The biological activities ofinterleukin-1, Blut, 59 (1989) 147 156. 5 Furukawa, S., Furukawa, Y., Satoyoshi, E. and Hayashi, K., Synthesis and secretion of nerve growth factor by mouse astroglial cells in culture, Biochem. Biophys. Res. Commun., 136 (1986) 57-63. 6 Giulian, D. and Baker, T.F., Characterization of ameboid microglia isolated from developing mammalian brain, J. Neurosci. 6 (1986) 2163-2178. 7 Giulian, D. and Lachman, L.D., Interleukin-I stimulation of astroglial proliferation after brain injury, Science, 228 (1985)497~499. 8 Green, L.A., A quantitative bioassay for nerve growth factor (NGF) employing a clonal pheochromocytoma cell line, Brain Res., 133 (1977) 351)-353. 9 Heft, F., Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections, J. Neurosci., 6 (1986) 2155 2162. 10 Heumann, R., Lindholm, D., Bandtlow, C., Meyer, M., Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H., Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regeneration: Role of macrophages, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8735-8739. 11 Hofman. F.M., Hinton, D.R., Johnson, K. and Merrill, J.E., Tumor necrosis factor identified in multiple sclerosis brain, J. Exp. Med., 170 (1989) 607~i12. 12 Large, T.H., Bodary, S.C., Clegg, D.O., Weskamp, G., Otten, U. and Reichardt, L.F., Nerve growth factor gene expression in the developing rat brain, Science, 234 (1986) 352 355. 13 Le, J. and Vilcek, J., Biology of disease, tumor necrosis factor and interleukin-1: Cytokines with multiple overlapping biological activities, Lab. Invest., 56 (1987) 234--248. 14 Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer, B., Masiakowski, P., Thoenen, H. and Barde, Y.-A., Molecular cloning and expression of brain-derived neurotrophic factor, Nature, 341 (1989) 149 152. 15 Levi-Montalcini, R., The nerve growth factor 35 years later, Science, 237 (1987) 1154-1162. 16 Lindholm, D., Heumann, R., Meyer, M. and Thoenen, H., Interleukin-I regulates synthesis of nerve growth factor in nonneuronal cells of rat sciatic nerve, Nature, 330 (1987) 658~59. 17 Lindholm, D., Heumann, R., Hengerer, B. and Thoenen, H., Interleukin-I increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts, J. Biol. Chem., 263 (1988) 16348 16351. 18 Lindsay, R.M., Adult rat brain astrocytes support survival of both NGF dependent and NGF insensitive neurones, Nature, 282 (1979) 80-82. 19 Lorez, H.P., von Frankenberg, M., Weskamp, G. and Otten, U., Effect of bilateral decortication on nerve growth factor content in basal nucleus and neostriatum of adult rat brain, Brain Res., 454 (1988) 355-360. 20 McCarthy, K.D. and deVellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. 21 Mobley, W.C., Woo, J.E., Edwards, R.J., Riopelle, R.J. Longo, F.M., Weskamp, G., Otten, U. and Johnston, M.V. Developmental regulation of nerve growth factor and its receptor in the rat caudateputamen, Neuron, 3 (1989) 65~664. 22 Otten, U., Boeckh, C., Ehrhard, P., Gadient, R. and von Frankenberg, M., Molecular mechanisms of nerve growth factor biosynthesis in the rat central nervous system. In H. Kewitz, T. Thomsen and U. Bickel (Eds.), Pharmacological Interventions on Central Cholinergic Mechanisms in Senile Dementia, Proc. Int. Symp., Zuckschwerdt Verlag, Munich, 1989, pp. 20-27. 23 Perry, V.H., Hume, D.A. and Gordon, S., Immunohistochemical localization ofmacrophages and microglia in the adult and developing mouse brain, Neuroscience, 15 (1985) 313-326.
340 24 Pfeilschifter, J., Pignat, W., Vosbeck, K. and M/irki, F., Interleukin-1 and tumor necrosis factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release from rat renal mesangial cells, Biochem. Biophys. Res. Commun., 159 (1989)385-394. 25 Selmaj, K.W., Farooq, M., Norton, W.T., Raine, C.S. and Brosnan, C.R., Proliferation of astrocytes in vitro in response to cytokines, J. lmmunol., 144 (1990) 129-135. 26 Spillantini, M.G., Aloe, L., Alleva, E., DeSimone, R., Goedert, M. and Levi-Montalcini, R., Nerve growth factor mRNA and protein increase in hypothalamus in a mouse model of aggression, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 8555-8559. 27 Spranger, M., Lindholm, D., Bandtlow, C., Heumann, R., Gnahn, H., N~iher-No+, M. and Thoenen, H., Regulation of nerve growth factor (NGF) synthesis in the rat central nervous system: comparison between the effects of interleukin-I and various growth factors in astrocyte cultures and in vivo, Eur. J. Neurosci., 2 (1990) 69-76. 28 Weskamp, G., Gasser, U.E., Dravid, A.R. and Otten, U., Fimbria-fornix lesion increases nerve growth factor content in the adult rat septum and hippocampus, Neurosci. Lett., 70 (1986) 121 -126. 29 Weskamp, G. and Otten. U., An enzyme-linked immunoassay for nerve growth factor (NGF): A tool for studying regulatory mechanism involved in NGF production in brain and in peripheral tissues, J. Neurochem., 48 (1987) 1779 1786. 30 Whittemore, S.R. and Seiger, A., The expression, localization and functional significance of beta nerve growth factor in the central nervous system, Brain Res. Rev., 12 (1987) 439-464. 31 Williams, L.R., Varon, S., Peterson, G.M., Wictorin, K., Fischer, W., Bj6rklund, A. and Gage, F.H., Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection, Proc. Natl. Acad. Sci. U.S.A., 82 (1986) 9231-9235.
335
Elsevier Scientific Publishers Ireland Ltd. NSL 07156
Interleukin-1 fl and tumor necrosis factor-e synergistically stimulate nerve growth factor (NGF) release from cultured rat astrocytes R.A. Gadient, K.C. C r o n a n d U. O t t e n Department of Physiology, University of Basel, Basel (Switzerland) (Received 1 June 1990; Accepted 6 June 1990)
Key words." Nerve growth factor production; Astrocyte; Interleukin-l; Tumor necrosis factor Nerve growth factor (NGF) may mediate responses to brain injury. To examine regulation of NGF gene expression with respect to neural trauma we examined the effects of interleukin-1 fl (IL-lfl) and tumor necrosis factor-at (TNF-ct) on NGF production in cultures of rat astroglial cells. Purified neocortical astrocytes in serum-free medium were treated with IL-I/¢, TNF-~ or both. Whereas IL-lfl and TNF-~t alone elicited only small effects, simultaneous addition elicited within 48 h a large (3- to 6-fold) increase in NGF content in culture supernatants. Our data are consistent with a role for cytokines in NGF synthesis and release in the injured central nervous system (CNS).
Nerve growth factor (NGF) is the best characterized member [15] of a family of neurotrophic polypeptide factors [14]. It is essential for the differentiation and maintenance of function of sympathetic and sensory neurons in the peripheral nervous system (PNS). In addition, N G F appears to be involved in regenerative events in the PNS [10]. Although it is not clear at present which factors regulate N G F gene expression there is evidence that mediators of inflammation, including interleukin-lfl (ILlfl), are involved [16, 17]. Recent evidence indicates that N G F modulates important functions in the central nervous system (CNS): e.g. it is atrophic agent for cholinergic neurons of the basal forebrain [12, 30] as well as in the caudate-putamen [21]. By in situ hybridization N G F m R N A has been localized to neurons in the CNS [1, 26]. The degeneration of basal forebrain neurons which follows lesion of the septohippocampal pathway can be markedly reduced by N G F [9, 31]. Whether these experiments reflect an effect of N G F in reversing the effects of brain injury is uncertain. However, brain damage does lead to a rapid accumulation of N G F [19, 28, 30] and to increased levels of several cytokines including IL-lfl [7] and tumor necrosis factor(TNF-c0 [11], at sites of neural injury. Recently, it was shown that intrastriatal and Correspondence."U. Otten, Department of Physiology, University of Basel, Vesalgasse I, 4051 Basel, Switzerland. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
336
intraventricular application of IL-lfl stimulated NGF synthesis. These data strongly suggest that IL-lfl regulates NGF synthesis in the CNS [22, 27] and thereby induces events important for reducing neural degeneration, but the locus of cytokine actions and their mode of action is not known. Increasing data indicate that NGF is produced by astrocytes in primary cultures [5, 18, 27]. In the present study the effects of cytokines on N G F production in defined primary astrocyte cultures kept in serumfree medium has been investigated. Astrocyte cultures from the neocortex of newborn Fii albino SPF rats were prepared by the method of McCarthy and deVellis [20]. After 18-20 days incubation, cultures were detached with 10 ml 0.2% ethylenediaminetetraacetate in phosphate buffered saline containing 0.125% trypsin (Gibco) and were seeded in Falcon plastic culture dishes (10 cm diameter). After 3 days, cultures were switched to a serum-free chemically-defined (N2) medium [2] supplemented with 10 mM L-leucinemethylester to eliminate brain macrophages [6]. Primary cultures of astrocytes were grown for 2 days before cytokines were added to the culture medium. The cell density was 4 × 107 cells/plate and purity of cultured cells was determined by immunofluorescence techniques using antibodies against glial fibrillary acidic protein (GFAP), fibronectin, galactocerebroside (GalC) and the specific macrophage markers ED-1, ED-2 [3] as well as Mac-I [23]. Immunohistochemical analysis revealed that cultured cells represented at least 95% astrocytes which were GFAP- and fibronectin-positive. EDl-positive microglia could rarely ( < 1%) be detected in the cultures. In addition, cultured astrocytes were Mac- 1, ED-2 and GalC-negative. In line with previous observations [25] there was no evidence for increased cell death during cytokine incubation. Supernatants of cultured astrocytes were collected and concentrated (10 times) by using Amicon Centriprep concentration units with a cut off of 10 kDa. Recovery of added N G F during this concentration step amounted to more than 80%. NGF determinations were performed by a sensitive two-site immunosorbent assay (ELISA) [29]. 500 -~ 0
E
uo
~d0 300
°~ u_
200
o z
IOO
Control
IL-1fl
TNF-a
IL-li~+ TNF-a
Fig. 1. Cytokine-indueed NGF release by cuRured astrocytes. Ghal cultures were stimulated with either IL-lfl (160 U/ml) or TNF-7 (50 U/ml) or the combination of both TNF-~t (50 U/ml)+ IL-lfl (160 U/ml). Supernatants were collected after a 48 h incubation period. Untreated astrocytes (control) released 3.6_ 0.4 pg/ml NGF. Data are the mean + S.E.M. (bars), values from 6 independent determinations. Statistical analysis was by Student's t-test (*P<0.001).
337
TABLE I EFFECTS OF IL-lfl AND TNF-a ON NGF PRODUCTION BY CULTURED RAT NEOCORTICAL ASTROCYTES AFTER 48 h Values represent the mean _+ S.E.M. of at least 4 independent experiments. Untreated astrocytes (control) released 3.6+0.4 pg/ml NGF within 48 h. IL, interleukin; TNF, tumor necrosis factor; NGF, nerve growth factor. Treatment
NGF release (% of control)
Control
100.0__+ 10.4
IL-I,8 30 U/ml IL-lfl 160 U/ml IL-lfl 500 U/ml
56.7_+ 10.8" 169.4_+ 13.1"* 76.8 _+ 11. I
TNF-~ 5 U/ml TNF-~ 50 U/mI TNF-~ 400 U/ml
100.3-+ 6.0 80.3-+ 9.0 104.0-+ 6.0
IL- lfl 160 U/ml + TNF-~ 5 U/ml IL-lfl 160 U/ml+TNF-c~ 50 U/ml IL- lfl 160 U/ml + TNF-~ 400 U/ml
355.9_+ 35.4** 453.2_+46.2** 595.6 _+33.6**
*P<0.05, **P< 0.001 as compared to control: Student's t-test,
Biological activity of immunoreactive NGF was monitored by using cultured PC l2 cells [8]. Human recombinant IL-lc~ and IL-lfl (provided by Dr. K. Vosbeck, Ciba Geigy, Basel) and human recombinant TNF-~ and human recombinant IL-6 (provided by Dr. R. Peck, Hoffmann-LaRoche, Basel) were used throughout this study. A variety of different cytokines, including IL-I~, IL-lfl, IL-6 and TNF-~ were tested for the ability to stimulate NGF release by cortical astrocytes. Only IL-lfl at a concentration of 160 U/ml elicited a small but significant (P<0.001) increase in release of immunoreactive NGF (Fig. 1, Table I). All other cytokines including ILl~, TNF-~ and IL-6 failed to significantly affect NGF-release. In several biological systems IL-lfl and TNF-~ interact in a synergistic way [13, 24] and each induces the synthesis of the other [4]. Therefore, it was of interest to investigate whether IL-lfl and TNF-c~ exert synergistic effect on NGF-release by cultivated astrocytes. As shown in Table ! and Fig. 1 combined treatment of astroglial cells with IL-lfl and TNF-~ triggered a marked increase of immunoreactive NGF in the culture medium after 48 h. TNF-~ and IL-lfl at doses as low as 5 U/ml and 160 U/ml respectively induced a 3.5-fold increase in NGF. At the highest dose of TNF-~ tested (400 U/ml) simultaneous addition of 160 U/ml IL-lfl caused a 6-fold increase in NGF. The time course of cytokine induced NGF release is shown in Fig. 2. Within 4 days, the latest point investigated, simultaneous addition of TNF-~ and IL-lfl synergistically stimulated NGF-production 10-fold. In PCI2 cell bioassay system, we found that fiber outgrowth elicited by astrocyte-conditioned medium was completely inhibited by addition of either monoclonal anti-NGF antibodies (mAB23c4) or polyclonal goat-anti NGF-antibodies. These data indicate that cytokine-stimulated astroglial cells release
338
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o k_ cO 0
800-
600-
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U_ L9 Z
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aO0-
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../fl |/'/
C
I
1
I
I
2 3 Time (days)
I
4
Fig. 2. Time-dependent increase of NGF release by cytokine-stimulated astroglial cultures. Cultures were stimulated by simultaneous addition of IL-lfl (160 U/rnl) and TNF-a (50 U/ml). Culture supernatants were collected after 1, 2 and 4 days incubation periods. Unstimulated astrocytes (control, C) released 5.3 + 0.5 pg/ml NGF. Values given represent the mean + S.E.M. of at least 3 independent determinations, Statistical analysis was by Student's t-test (*P < 0.025, **P < 0.001).
biologically active NGF. The mechanism whereby TNF-a and IL-1//induce release of biologically active NGF from cortical astrocytes cultured in serum-free medium is not known. A pilot experiment showing that cytokine treatment produced a marked increase in NGF mRNA levels in astrocytes strongly suggests that increased NGF release is a result of increased NGF gene expression. In conclusion, this study reports that IL-lfl and TNF-a interact synergistically to increase NGF release from cultured astrocytes. Whether these inflammatory mediators are involved in the physiological NGF synthesis in vivo remains to be investigated. However, given their presence at sites of neural injury it is possible that they play an important role in increasing NGF levels in lesioned brain tissue. This study was supported by the Swiss National Foundation for Scientific Research (Grant 31-25690.88) and the Deutsche Forschungsgemeinschaft (SFB 325). We would like to thank to Dr. P. Ehrhard and M, Rimle for helpful advice and discussion. 1 Ayer-LeLievre, C.S., Olson, L., Ebendal, T. Seiger, A. and Persson, H., Expression of the ]/-nerve growth factor gene in hippocampal neurons, Science 240 (1988) 1389-1391.
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