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
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
