- Email: [email protected]
Interleukin-1β, but not IL-1α, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor
Int. J. Devl Neuroscience 19 (2001) 675– 683 www.elsevier.com/locate/ijdevneu
Interleukin-1b, but not IL-1a, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor Damijana M. Juricˇ, Marija C& arman-Krzˇan * Department of Pharmacology, Faculty of Medicine, Korytko6a 2, SI-1000 Ljubljana, Slo6enia Received 1 June 2001; received in revised form 9 June 2001; accepted 17 July 2001
Abstract In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1b (IL-1b). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1a and IL-1b) and the antagonist (IL-1ra)—members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [125I] IL-1b to cultured astrocytes was saturable and of high affinity. Mean values for the KD and Bmax were calculated to be 60.7 97.4 pM and 2.5 9 0.1 fmol mg-1 protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1a (Ki of 341.1 pM) and IL-1b (Ki 59.9 pM), as well as the antagonist IL-1ra (Ki 257.6 pM), displaced specific [125I] IL-1b binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [125I] IL-1b binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1a and IL-1b revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1b (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1a by itself had no stimulatory action on NGF release. The simultaneous application of IL-1a and IL-1b elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1b. We concluded that IL-1b-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1a and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes. © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Interleukin-1; Interleukin-1 receptor; Astrocytes; Nerve growth factor
1. Introduction In the CNS, nerve growth factor (NGF) (C& armanKrzˇan, 1997) is a neurotrophic factor that promotes the survival and differentiation of the developing basal forebrain cholinergic neurons that are also responsive to and dependent on NGF for phenotypic maintenance and neuronal survival in the adult brain (Thoenen et al., 1987; Whittemore and Seiger, 1987; Cuello, 1993; Lapchak, 1993). During aging, these neurons become atrophic and are susceptible to neurodegenerative disorders such as Alzheimer’s disease. Evidence from both * Corresponding author. Tel.: + 386-1-543-7351; fax: +386-1-5437331. E-mail address: [email protected] (M. C& armanKrzˇan).
‘in vitro’ and ‘in vivo’ studies (Fisher et al., 1987; Gage et al., 1988; Connor and Dragunow, 1998; Hagg and Oudega, 1998) suggests that NGF provides neuroprotection to cholinergic neurons of the basal forebrain by inhibiting intrinsic apoptotic mechanisms, as well as by increasing neuronal metabolism and function. In the lesioned brain, NGF was also found to protect hippocampal and cortical neurons (Pechan et al., 1995; Semkova et al., 1996), suggesting that increasing brain levels of endogenous NGF may be beneficial in certain brain disorders. Under normal physiological conditions NGF is produced predominantly by neurons (Ayer-Lelievre et al., 1988), where the amount of NGF mRNA is regulated by neuronal activity and hormonal influences (Lindholm et al., 1994; Thoenen, 1995). During periods of rapid glial proliferation, i.e. prenatal/early postnatal
0736-5748/01/$20 © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 5 7 4 8 ( 0 1 ) 0 0 0 4 4 - 2
676
D.M. Juricˇ, M. C& arman-Krzˇan / Int. J. De6l Neuroscience 19 (2001) 675–683
period and after injury in the adult brain, astrocytes become a major site of NGF synthesis in the CNS (Furukawa et al., 1987; Lu et al., 1991; OderfeldNowak et al., 1992; Arendt et al., 1995). Previous studies have shown that NGF secretion from astrocytes is susceptible to stimulation by various pharmacological agents including glial cell growth factors, neurotransmitters, and cytokines (C& arman-Krzˇ an et al., 1991; Lipnik-S& tangelj et al., 1998; Lipnik-S& tangelj and C& arman-Krzˇ an 2000; Juricˇ and C& arman-Krzˇ an, 2000; Krzˇ an et al., 2001). IL-1b is one of the most potent stimulators of NGF secretion in cultured neonatal astrocytes (Spranger et al., 1990; C& arman-Krzˇ an et al., 1991; Vige et al., 1991; C& arman-Krzˇ an and Wise, 1993, Juricˇ and C& arman-Krzˇ an, 2000). The action of IL-1b is of interest since it is produced in the CNS and, its stimulation of NGF secretion is both high and specific in comparison to other growth factors in the model system of rat neonatal cortical astrocytes. IL-1b increases the NGF mRNA content by activation of both NGF gene transcription and by posttranscriptional stabilization of NGF mRNA prior to NGF secretion (Lindholm et al., 1988; C& arman-Krzˇ an et al., 1991; Vige et al., 1991; Pshenichkin et al., 1994). Although the precise molecular mechanism regulating NGF elevation in astrocytes by IL-1b is still unresolved, IL-1 receptors are the likely mediators of this response (C& armanKrzˇ an et al., 1991). In the normal healthy brain, the expression of IL-1b and its mRNA is very low (Vitkovic et al., 2000), but it is markedly increased in response to local inflammation, injury, or in disease states including Alzheimer’s disease and stroke (Griffin et al., 1989; Rothwell, 1991; Liu et al., 1993; Rothwell and Hopkins, 1995; Mehlhorn et al., 2000). Based on the finding that an increase in NGF levels in the injured rat brain temporally follows an increase in IL-1b mRNA (Goss et al., 1995) and ‘in vitro’ experiments on the influence of exogenous IL-1b on NGF release, IL-1b produced endogenously could be one of the stimulants of NGF synthesis. In addition to IL-1b, endogenous levels of IL-1a and the IL-1 receptor antagonist (IL-1ra), are also elevated during neurodegenerative conditions (Hopkins and Rothwell, 1995; Rothwell 1999), indicating that endogenous IL-1s play a functional role in disease states of the brain. Therefore, our study was aimed at investigating the involvement of all three members of the IL-1 family (IL-1a, IL-1b and IL-1ra) in NGF secretion from nonneuronal cells (astrocytes) and testing the hypothesis that their action on NGF synthesis and secretion is under the control of IL-1 receptors. Our results showed that signal transducing type I (Sims et al., 1988, 1993), but not a decoy type II (McMahan et al., 1991; Colotta et al., 1993). IL-1 receptors are present on rat neonatal cortical astrocytes in primary culture. We also provide evidence that the stimulation of NGF secretion from
these cells by IL-1b is a type I IL-1 receptor mediated process, whereas IL-1a and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from cultured astrocytes.
2. Experimental procedures
2.1. Materials Timed pregnant Wistar rats were obtained from our own breeding colony. Dulbecco’s phosphate buffered saline (PBS), Dulbecco’s modified Eagle medium and Ham’s nutrient mixture F-12 (DMEM/F12 (1:1)), fetal bovine serum (FBS), and penicillin/streptomycin, were from Gibco BRL, Scotland. Tissue culture plates and immunoplates were from Nunc, Denmark. Recombinant human IL-1a, IL-1b, and IL-1 receptor antagonist (IL-1ra), as well as anti-human type I IL-1 receptor (Ab IL-1RI) and anti-human type II IL-1 receptor (Ab IL-1RII) polyclonal antibodies, were from R&D Systems, UK. Anti-mouse b (2.5S, 7S) nerve growth factor monoclonal antibody, anti-mouse b (2.5S, 7S) nerve growth factor-b-gal monoclonal antibody, nerve growth factor-b (NGF-b) (mouse), and chlorophenol-red-b-dgalactopyranoside (CPRG) were from Boehringer Mannheim, Germany. Iodinated recombinant human interleukin-1b ([125I] IL-1b, specific activity 2142 Ci/ mmol) was from NEN Life Science Products, USA. Bovine serum albumin (BSA) and all other chemicals used were from Sigma.
2.2. Cell culture and treatment of cells Primary cultures of rat neonatal cortical astrocytes were prepared from the brains of newborn Wistar rats according to the method described in C& arman-Krzˇ an et al. (1991). Briefly, after removal of the meninges, the cortices were dissociated by passage through sterile Nitex nylon screens (75 mm mesh size) into 10 ml of the culture medium (DMEM/F12 (1:1), 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin). The cell suspensions were diluted and plated into 35 mm (2× 106 cells/dish) or 100 mm (10×106 cells/dish) tissue culture dishes. Cells were grown at 37 °C in a water saturated air environment containing 10% CO2. Culture media was changed every 3 days of cultivation. After reaching confluency (10–14 days of growing), culture medium in the 35 mm tissue culture dishes was replaced with fresh medium and cells were treated under serum-free conditions with either vehicle (PBS containing 1 mg/ml BSA)-control cells or cytokines IL-1b (0.1–50 U/ml; 1 U/ml equals 5 pg/ml), IL-1a (0.1–50 U/ml) or IL-1ra (0.001–0.6 U/ml; 1 U/ml equals 100 ng/ml) in PBS/BSA. Following a 24 h incubation, the culture medium was collected, frozen,
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
677
and used for NGF determination. Astroglial cells in the 100 mm culture dishes were collected and used for binding studies. Immunocytochemical staining for glial fibrillary acidic protein indicated that 95% of the cells were astrocytes (C& arman-Krzˇ an et al., 1991).
and (IL-1a, IL-1b, or IL-1ra) under the same experimental conditions as described for the saturation binding assay. The radioactivity trapped on the cell homogenates was counted in a g-counter.
3. Binding studies
To differentiate between type I and type II IL-1 receptor binding sites on astrocytes we examined the binding of iodinated IL-1b (30 pM, i.e. 0.5 ng/ml) in the presence of increasing concentrations (1 pg/ml –10 mg/ml) of either specific anti-IL-1R type I (Ab IL-1RI) or anti-IL-1R type II (Ab IL-1RII) polyclonal antibodies. Incubations were carried out under the same experimental conditions as described for the saturation binding assay. The radioactivity trapped on the cell homogenates was counted in a g-counter.
3.1. Saturation binding: Identification and characterization of IL-1 receptors on cell homogenates of neonatal rat cortical astrocytes was performed by receptor-binding experiments according to the modified method of Ban et al. (1993) using iodinated [125I] human recombinant IL-1b as a radiolabel. 100 ml aliquots of cell homogenates containing 100 mg of proteins were incubated at 37 °C in a total volume of 0.2 ml containing PBS (pH 7.4). For the saturation binding assay, increasing concentrations of [125I] IL-1b (10– 200 pM) were added either alone (total binding) or in the presence of 6 nM IL-1b (nonspecific binding) to the incubation mixture. After a 1h incubation, the reaction was terminated by centrifugation for 1 min at 9000 rpm. Cell homogenates were washed with ice-cold PBS and centrifuged. The radioactivity trapped on the cell homogenates was counted in a g-counter. Specific binding was calculated by subtracting nonspecific binding from total binding.
3.2. Kinetics of binding Studying the association of [125I] IL-1b to the IL-1 receptor, cell homogenates were incubated with 30 pM [125I] IL-1b alone or in the presence of unlabeled 6 nM IL-1b. Incubations were carried out for various times between 0 and 60 min. The reaction was terminated and the radioactivity trapped on the cell homogenates was counted in a g-counter. The dissociation of [125I] IL-1b from the IL-1 receptor was examined by incubating cell homogenates for 60 min with 30 pM [125I] IL-1b. At 60 min, unlabelled IL-1b (6 nM) was then added and the reaction was terminated at various times between 1 and 60 min thereafter. The radioactivity trapped on the cell homogenates was counted in a g-counter.
3.3. Inhibition binding assay Inhibition binding experiments were carried out in order to assess the ability of IL-1a, IL-1b, and IL-1ra to displace radiolabeled IL-1b from the IL-1 receptor binding sites on astrocytes. Incubations were carried out with 30 pM [125I] IL-1b in the presence of increasing concentrations (0.1 pM– 100 nM) of unlabeled lig-
3.4. Differentiation between type I and type II IL-1 receptor
3.5. Enzyme immunoassay (EIA) of NGF The NGF enzyme immunoassay (NGF-EIA) was performed essentially as described in C& arman-Krzˇ an et al. (1991). Briefly, immunoplates (Nunc) were coated with anti-bNGF monoclonal antibody. Following treatment with 1% BSA, aliquots of astrocyte culture medium or NGF standards (10–320 pg/ml) were added in duplicate to the plates. After incubation overnight, the immunoplate wells were incubated with anti-b NGF monoclonal antibody conjugated with b-galactosidase followed by the enzyme substrate chlorophenol red-b-dgalactopyranoside. The reaction product was measured at 570 nm using a microplate reader. Culture medium samples and NGF standards were prepared as previously described (C& arman-Krzˇ an et al., 1991). Protein concentration in the cell homogenates was determined according to the method of Bradford (1976) using bovine serum albumin as a standard.
4. Calculations The equilibrium dissociation constant (KD) and the maximum binding capacity (Bmax) for [125I] IL-1b were determined according to the method of Scatchard (1949); data was taken from the saturation binding experiments (Fig. 1). Kinetic parameters of [125I] IL-1b binding were calculated as described in Hulme and Birdsall (1992). Ki values (the dissociation constant for a competing ligand) were calculated using the ChengPrusoff equation (Cheng and Prusoff, 1973) from the inhibition binding experiments. IC50 values (concentration of competing ligand that displaces 50% of specific [125I] IL-1b binding) of each competitor were obtained using a logistic equation and a Graph Pad Prism 3.0 program. Data are shown as the means9 SEM. The
678
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
Student’s t-test was used for statistical analysis of the data.
5. Results
5.1. Saturation binding experiments Specific [125I] IL-1b binding to membrane homogenates of neonatal rat cortical astrocytes was saturable, reversible, and of high affinity (Fig. 1). It represented 50% of the total binding. Scatchard’s analysis (Fig. 1) of specific [125I] IL-1b binding at equilibrium revealed a straight line, thus indicating the presence of
Fig. 3. Inhibition of specific [125I] IL-1b binding to rat neonatal cortical astrocytes from primary culture by IL-1a, IL-1b, or IL-1ra. Astroglial cells were incubated with 30 pM [125I] IL-1b alone or in the presence of increasing concentrations (0.1 pM – 100 nM) of unlabelled ligand. Values represent the means 9SEM from five independent experiments, each assayed in triplicate.
a homogenous population of [125I] IL-1b binding sites with a Hill coefficient (nH) near unity (0.949 0.05). The dissociation constant (KD) was 60.79 7.4 pM and the maximal number of binding sites (Bmax) was 2.590.1 fmol/mg protein.
5.2. Kinetics of [ 125I] IL-1i binding
Fig. 1. Saturation analysis of [125I] IL-1b binding to neonatal rat cortical astrocytes at equilibrium. Astroglial cells were incubated with increasing concentrations (10 –200 pM) of [125I] IL-1b alone or in the presence of 6 nM unlabelled IL-1b. Inset: Scatchard’s transformation of the specific binding. Values represent the means 9 SEM of five independent experiments, each assayed in triplicate. See also Materials and Methods.
Kinetic analysis of the binding [125 I] IL-1b to astrocytes showed that the specific binding of 30 pM [125I] IL-1b occurred rapidly with a half-maximal binding within 2.8 min and equilibrium was reached after 20 min (Fig. 2). The observed rate constant (kobs) for the forward reaction was 0.251 min − 1. The dissociation of [125I] IL-1b from the binding sites initiated by IL-1b was rapid (t1/2 of 4.1 min) and yielded a dissociation rate constant (k − 1) of 0.17 min − 1. The association rate constant (k + 1) for [125I] IL-1b of 2.8×109 M − 1 min − 1 was calculated from the k − 1 and kobs. The equilibrium dissociation constant (KD) of 60.8 9 5.4 pM was calculated from the ratio k − 1/k + 1. The KD value is in accordance with the KD calculated from the Scatchard plot of saturation binding data.
5.3. Inhibition binding experiments
Fig. 2. Kinetics of [125I] IL-1b binding to neonatal rat cortical astrocytes. The association reaction was carried out with 30 pM [125I] IL-1b alone or in the presence of unlabeled 6 nM IL-1b and terminated at individual time points (1 –60 s). The dissociation reaction was initiated by the addition of IL-1b (6 nM) to the astrocytes after a 60 min preincubation with 30 pM [125I] IL-1b and terminated at individual time points (0 –60 s). Values at each time point represent the means9SEM of five independent experiments, each assayed in triplicate.
The pharmacological characteristics of the [125I] IL1b labeled IL-1 receptor binding sites were examined by determining the potencies of different IL-1 receptor ligands (IL-1a, IL-1b, and IL-1ra) to displace the radioligand from the receptor binding sites. All tested ligands fully inhibited specific [125I] IL-1b binding to IL-1 receptor binding sites in a monophasic manner (Fig. 3). The rank order of potencies for the inhibition of specific [125I] IL-1b binding was: IL-1b \IL-1ra\IL-1a (Fig. 3). Half-maximally effective doses (IC50) for IL-1b and IL-1a were 89.6 pM. and 518.7 pM, respectively
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
Fig. 4. Inhibition of specific [125I] IL-1b binding by anti-IL-1RI or anti-IL-1RII antibodies to rat neonatal cortical astrocytes from primary culture. Astroglial cells were incubated with 30 pM [125I] IL-1b alone or in the presence of increasing concentrations (1 pg/ml– 10 mg/ml) of unlabelled anti-IL-1RI or anti-IL-1RII antibody. Values represent the means 9 SEM from five independent experiments, each assayed in triplicate.
679
(Fig. 3). The IC50 value of IL-1ra was 385.0 pM. Using the Cheng-Prusoff equation (Cheng and Prusoff, 1973), we calculated the inhibition constants (Ki ) for all the ligands used, which were 59.9 pM for IL-1b, 257.6 pM for IL-1ra and 341.1 pM for IL-1a. To determine which subtype of IL-1 receptors was labeled with the [125I] IL-1b on rat cortical astrocytes, we performed the inhibition binding experiments with iodinated IL-1b (30 pM, i.e. 0.5 ng/ml) in the presence of increasing concentrations (1 pg/ml –10 mg/ml) of the specific polyclonal antibody against either type I (Ab IL-1RI) or type II (Ab IL-1RII) IL-1 receptors. Dosedependent inhibition of the [125I] IL-1b binding by Ab IL-1RI (depicted in Fig. 4) revealed an IC50 value of 6.39 0.1 ng/ml. In contrast, the antibody against the type II IL-1 receptor was not able to block the specific binding of [125I] IL-1b (Fig. 4). These data strongly suggest that the IL-1 receptor we identified is the signal transducing type I IL-1R, whereas the type II IL-1R is notably absent from neonatal rat cortical astrocytes.
5.4. Effects of IL-1h, IL-1i, and IL-1ra on NGF secretion from rat cortical astrocytes in primary culture
Fig. 5. Effects of: (A) IL-1a, IL-1b; and (B) IL-1ra on NGF release from rat neonatal cortical astrocytes in primary culture. Cells were incubated for 24 h in vehicle (PBS/BSA-control cells) or in the culture medium containing IL-1a (0.1–50 U/ml), IL-1b (0.1– 50 U/ml), or IL-1ra (0.001 – 0.6 U/ml). Supernatants were collected and NGF content was determined by ELISA-NGF assay. Values are the means9SEM of five independent determinations. IL-1b (10 U/ml) stimulated NGF secretion represents 703.5 9 28.4 pg NGF/mg protein. Basal secretion was 239.3 9 13.1 pg NGF/mg protein. *P B 0.001; values are significantly different from the control level (Student’s t-test).
As previously demonstrated (C& arman-Krzˇ an et al., 1991), IL-1b stimulates NGF secretion from cultured astrocytes in a dose-dependent manner with the maximal effect between 3 and 10 U/ml. At the maximal concentration of IL-1b (10 U/ml) the level of NGF in the culture medium was increased by 3-fold as compared to the control cells. This effect was specific in comparison to other growth factors and cytokines used, suggesting IL-1 receptor mediated stimulation. The effects of IL-1a and IL-1ra were not addressed in this study (C& arman-Krzˇ an et al., 1991). Since IL-1a and IL-1b are both IL-1 receptor agonists believed, at least in the peripheral nervous system, to exert identical biological effects (Dinarello, 1996) and, as shown in Fig. 3, both have an affinity for the same IL-1 receptor, we tested their potential receptor mediated effect on the regulation of NGF release from astrocytes. NGF release from astrocytes was also determined after treatment with IL-1a or IL-1b in the presence of a natural IL-1 receptor antagonist, IL-1ra. Exposure of cultured cells for 24 h to IL-1b (0.1–50 U/ml) caused a dose-dependent increase in NGF secretion with a half maximal effect at 1.7 U/ml and a maximal effect at 10 U/ml (Fig. 5(A)). IL-1b-stimulated cultures released 3-times more NGF (703.59 28.4 pg NGF/mg protein) than the control cells (239.3913.1 pg NGF/mg protein) (Fig. 5(A)). In contrast, the basal NGF secretion from cultured astrocytes was not altered in response to 24 h stimulation with IL-1a (0.1–50 U/ml) (Fig. 5(A)). No effect was observed even after prolonged treatment (48 h incubation) or when higher concentration of IL-1a (100 U/ml) was used (data not
680
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
concentrations of IL-1a (0.1–50 U/ml) and NGF release was measured 24 h later. As shown in Fig. 7, IL-1a had no significant effect on IL-1b-induced NGF release.
6. Discussion
Fig. 6. Effect of IL-1ra on IL-1b-induced NGF release from rat neonatal cortical astrocytes in primary culture. Cells were incubated for 24 h in culture medium containing IL-1b (10 U/ml) in the presence of IL-1ra (0.001 –0.6 U/ml). Supernatants were collected and tested for NGF content by ELISA-NGF assay. Values are the means9SEM of five independent determinations.
Fig. 7. Effect of IL-1a on IL-1b-induced NGF release from rat neonatal cortical astrocytes in primary culture. Cells were incubated for 24 h in vehicle (PBS/BSA-control cells) or culture medium containing IL-1b (10 U/ml) in the presence of IL-1a (0.1 –50 U/ml). Supernatants were collected and tested for NGF content by ELISANGF assay. Values are the means 9 SEM of five independent determinations. IL-1b (10 U/ml) stimulated NGF secretion represents 703.5928.4 pg NGF/mg protein. Basal secretion was 239.3 9 13.1 pg NGF/mg protein.
shown). In accordance with its antagonistic function on the IL-1 receptor, IL-1ra (0.001–0.6 U/ml) had no effect on basal NGF release (Fig. 5(B)), but dose-dependently inhibited the stimulatory response induced by IL-1b (10 U/ml) (IC50 of 0.07 U/ml) (Fig. 6). A complete blockade of IL-1b action on NGF secretion occurred at 0.6 U/ml of antagonist. In contrast, IL-1ra showed no influence on IL-1a-treated cells (data not shown). To exclude the possible additive effect of IL-1a on IL-1b-stimulated secretion of NGF, cells were treated with 10 U/ml of IL-1b in the presence of increasing
Neonatal rat cortical astrocytes and neurons in primary culture express NGF mRNA and synthesize NGF. In astrocytes, NGF gene transcription is regulated by different intracellular signaling systems that can be modulated by cytokines. This modulation differs greatly between astrocytes in different stages of development (Wu et al., 1998). The IL-1 family of cytokines consists of IL-1a, IL-1b, and IL-1ra. IL-1b is more predominant form in the brain than IL-1a. IL-1b has been shown to be much more effective than IL-1a in regulating various functions in the normal, healthy brain, as well as in neuroimmune responses to disease and neurodegeneration (for review see Vitkovic et al., 2000; Rothwell, 1999). In the present study, we could demonstrate that the stimulatory action of IL-1b on NGF secretion from primary cultures of astrocytes is mediated through the functional type I IL-1 receptors identified on rat astrocytes. IL-1b-induced NGF secretion was completely inhibited by IL-1ra in a dose dependent manner, thus confirming the IL-1RI receptor mediated process. In addition, we present evidence that IL-1a, which shows a high affinity for the type I IL-1 receptor, is not involved in the regulation of NGF secretion from cultured astrocytes. To identify the IL-1 receptors on rat neonatal cortical astrocytes in primary culture the IL-1 receptor agonist [125I] IL-1b was used. The binding of [125I] IL-1b was saturable, of high affinity, and readily reversible with a KD of 60.797.4 pM and a Bmax of 2.59 0.1 fmol/mg protein (Fig. 1). The KD value of 60.89 5.4 pM from the kinetic data (Fig. 2) is in accordance with the KD value obtained by Scatchard analysis (Fig. 1). The affinity that we observed for [125I] IL-1b binding to IL-1 receptors on cultured rat cortical astrocytes are comparable with the reported affinities for type I IL-1R (KD of 0.02–0.8 nM) in the rodent and human brain (Takao et al., 1990, 1992; Rubio, 1994; Parnet et al., 1994; Hammond et al., 1999). In the inhibition binding experiments (Fig. 3), all unlabeled compounds (IL-1a, IL-1b, and IL-1ra) completely inhibited specific [125I] IL-1b binding in a manner compatible with interaction at a single class of IL-1 binding sites but with differing potencies (IL-1b: Ki of 59.9 pM; IL-1a: Ki of 341.1 pM). This shows that the two IL-1 agonists do not bind equally well to the IL-1 receptor binding sites with IL-1b having a 6-fold higher potency for displacing specifically bound [125I] IL-1b
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
(Fig. 3) than IL-1a. The specificity of IL-1 receptors on astrocytes was confirmed by the competitive binding experiments using increasing concentrations (0.1 pM– 100 nM) of nonspecific IL-1R ligands (IL-2, IL-4 and IL-6). No displacement of specific [125I] IL-1b binding was obtained with IL-2 whereas IL-4 and IL-6 only partially inhibited radioligand binding (data not shown). Vitkovic et al. (2000) suggested that IL-1a binds to the same receptors as IL-1b in the brain but with lower affinity, what we demonstrated in our binding study on astrocytes (Fig. 3). However, studies of IL-1 receptors in the mouse hypocampus (Takao et al., 1990), mouse dendate gyrus (Ban et al., 1991) and mouse astrocyte cultures (Ban et al., 1993) showed that both agonists have the same affinity for the type I IL-1 receptors and therefore differ from our data. The IL-1 receptor antagonist had about a 5-fold lower affinity than the unlabeled IL-1b for IL-1 binding sites (Fig. 3), which is in accordance with observations on rat brain endothelial cells (Van Dam et al., 1996). Identification of functional IL-1 receptors on rat cortical astrocytes raises questions as to the exact type of IL-1 receptor that is expressed on these cells. It is well established that two types of IL-1 receptors play different roles in the regulation of IL-1 activity. Whereas type I is the major signaling receptor for IL-1 (Sims et al., 1993), type II appears to act as a decoy receptor (Colotta et al., 1993). Farrar et al. (1987) first reported IL-1 binding sites in the rodent brain. IL-1 receptors in the CNS were later studied by several groups (for review see Vitkovic et al., 2000) indicating dramatic species differences between the mouse and rat brain in the distribution of IL-1R. In the mouse brain, mRNA of both IL-1 receptor types is expressed, mainly in neuron-rich areas (the granule cell layer of the dentate gyrus and the cerebellum, the pyramidal cell layers of the hippocampus, and within the hypothalamus) (Ban et al., 1991; Parnet et al., 1994). In addition to neuronal cells bodies, IL-1RI expression was also associated with cerebral blood vessels and glial cells, including astrocytes. Ban et al. (1993) first reported IL-1 binding sites on cultured astrocytes from the cerebral hemispheres of newborn mice, whereas Rubio (1994) demonstrated the presence of specific type I IL-1 receptors in newborn murine cortical astrocyte cultures. In contrast to the murine CNS, only the type I IL-1 receptors have been identified in the normal rat brain, whereas the expression of type II IL-1 receptor mRNA has not been detected in any brain region (Yabuuchi et al., 1994; Nishiyori et al., 1997). IL-1RI mRNA is expressed in unidentified neuronal cell groups in several brain regions (the olfactory bulb, the hippocampus, the amygdala, the hypothalamus, and the cerebellum) (Farrar et al., 1987; Yabuuchi et al., 1994) and on the rat brain endothelial and epithelial cells (Yabuuchi et al., 1994; Van Dam et al., 1996; Nishiyori et al., 1997).
681
In order to distinguish different types of IL-1 receptors in the membrane of cultured rat cortical astrocytes, we performed binding studies using antibodies against both IL-1 receptor types (Fig. 4). Polyclonal antibodies allowed us to recognize all allotypic forms of the natural IL-1 receptors. Our results showed that only the anti-IL-1RI antibody dose-dependently inhibited specific [125I] IL-1b binding by selective blocking the binding sites of type I IL-1 receptors while the anti-IL-1RII antibody showed no significant influence on specific radioligand binding (Fig. 4). These results support the conclusion that the signal transducing type I IL-1R is expressed on neonatal rat cortical astrocytes and could therefore be the mediator of the IL-1b stimulated NGF secretion. Type II IL-1R is notably absent from these cells. In the present study, a remarkable difference between the agonists of type I IL-1 receptors (IL-1a and IL-1b) was observed in terms of their ability to induce NGF secretion from cultured rat cortical astrocytes (Fig. 5(A)). Basal NGF secretion from astrocytes was not altered in response to stimulation with IL-1a, while IL-1b potently and specifically stimulated NGF release (3-fold over basal level) (Fig. 5(A)). Simultaneous application of IL-1a and IL-1b elicited no additive response (Fig. 7), confirming that IL-1a had no influence on IL-1b- induced NGF secretion to the astrocyte culture. Therefore, in spite of the high affinity of IL-1a for the type I IL-1 receptors, we conclude that IL-1a is not involved in the regulation of NGF release from rat cortical astrocytes in primary culture. Similar to our results, in rat cultured astrocytes IL-1b was found to be 100-fold more effective than IL-1a on PGE2 release (Katsuura et al., 1989). IL-1b increases also plasma levels of ACTH in rats, while IL-1a has no effect (Uehara et al., 1988). IL-1ra alone had no effect on basal NGF release (Fig. 5(B)), but dose-dependently inhibited the biologic response induced by IL-1b (Fig. 6), thereby supporting our conclusion that the IL-1b-stimulated NGF secretion is a type I IL-1 receptor mediated process. Our observation is in agreement with the ‘in vivo’ study of DeKosky et al. (1996). They demonstrated that an increase in NGF after rat central nervous system trauma is directly mediated through IL-1b and that blocking IL-1b by IL-1ra following brain injury leads to suppression of an NGF-mediated reparative response. In conclusion, the results obtained in this study indicate that cortical astrocytes derived from newborn rats express functional type I IL-1 receptors, but not decoy type II IL-1 receptors. The production and release of NGF in cultured rat neonatal cortical astrocytes is thus regulated by a balance between the pro-inflammatory activities of IL-1b and the ability of IL-1R antagonist to control these activities by occupying the type I IL-1
682
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
receptors. We have also shown that, in contrast to IL-1b, IL-1a has no role in the regulation of NGF secretion from newborn rat astrocytes. The specific stimulatory function of IL-1b on NGF secretion is limited to the neonatal rat astrocytes that in many respects resemble reactive astrocytes. IL-1b was not able to increase NGF mRNA or NGF content in normal adult rat astrocytes (Wu et al., 1998). The group (Wu et al., 1998) did not address the question of the involvement of IL-1R in the regulation of this process. Therefore, the pattern of the expression and the precise function of both types of IL-1 receptor in the regulation of NGF secretion from adult, as well as reactive astrocytes, remains to be determined.
Acknowledgements The authors wish to thank to C. Blazˇ ek and J. Kosˇir for their excellent technical assistance. This study was supported by Ministry of Education, Science and Sport.
References Arendt, T., Bruckner, M.K., Krell, T., Pagliusi, S., Kruska, L., Heumann, R., 1995. Degeneration of rat cholinergic basal forebrain neurons and reactive changes in nerve growth factor expression after chronic neurotoxic injury. II. Reactive expression of NGF gene in astrocytes. Neuroscience 65, 647 – 659. Ayer-Lelievre, C., Olson, I., Ebendal, T., Seiger, A., Persson, H., 1988. Expression of the b-nerve growth factor gene in hippocampal neurons. Science 240, 1339 –1341. Ban, E., Millon, G., Prudhomme, N., Fillion, G., Haour, F., 1991. Receptors for interleukin-1 (a and b) in mouse brain: mapping and neuronal localization in hippocampus. Neuroscience 43, 21 – 30. Ban, E.M., Sarlieve, L.L., Haour, F.G., 1993. Interleukin-1 binding sites on astrocytes. Neuroscience 52, 725 –733. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 –254. Cheng, Y.C., Prusoff, W.H., 1973. Relationship between the inhibition constant (Ki ) and the concentration of inhibitor which cause 50% inhibition (IC50) of the enzymatic reaction. Biochem. Pharmacol. 22, 3099 – 3108. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J.G., Dower, S.K., Siems, J.E., Mantovani, A., 1993. Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science 261, 472 –475. Connor, B., Dragunow, M., 1998. The role of neuronal growth factors in neurodegenerative disorders of the human brain. Brain Res. Rev. 27, 1 – 39. Cuello, C.A., 1993. Trophic responses of forbrain cholinergic neurons: a discussion. Prog. Brain Res. 98, 265 –277. C& arman-Krzˇ an, M., Vige, X., Wise, B.C., 1991. Regulation by interleukin-1 of nerve growth factor secretion and nerve growth factor mRNA expression in rat primary astroglial cultures. J. Neurochem. 56, 636 – 643. C& arman-Krzˇ an, M., Wise, B.C., 1993. Arachidonic acid lipoxygenation may mediate interleukin-1 stimulation of nerve growth factor secretion in astroglial cultures. J. Neurosci. Res. 34, 225 – 232.
C& arman-Krzˇ an, M., 1997. Nerve growth factor and related neurotrophins —their role in the CNS. Zdrav. Vestn. 66, 1 – 4. DeKosky, S.T., Styren, S.D., O’Malley, M.E., Goss, J.R., Kochanek, P., Marion, D., Evans, C.H., Robbins, P.D., 1996. Interleukin-1 receptor antagonist suppresses neurotrophin response in injured rat brain. Ann. Neurol. 39, 123 – 127. Dinarello, C.A., 1996. Biologic basis for Interleukin-1 in disease. Blood 87, 2095 – 2147. Farrar, W.L., Killian, P.L., Ruff, M.R., Hill, J.M., Pert, C.B., 1987. Visualization and characterization of interleukin 1 receptors in brain. J. Immunol. 139, 459 – 463. Fisher, W., Victorin, A., Bjo¨ rklund, A., Williams, L.R., Varon, S., Gage, F.H., 1987. Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor. Nature 329, 65 – 68. Furukawa, S., Furukawa, Y., Satoyashi, E., Hayaski, K., 1987. Synthesis/secretion of nerve growth factor is associated with cell growth in cultured mouse astroglial cells. Biochem. Biophys. Res. Commun. 142, 395 – 402. Gage, F.H., Armstrong, D.M., Williams, L.R., Varon, S., 1988. Morphological response of axotomized septal neurons to nerve growth factor. J. Corp. Neurol. 269, 147 – 155. Griffin, W.S., Stanley, L.C., Ling, C., White, L., MacLeod, V., Perrot, L.J., White, C.L., Araoz, C., 1989. Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc. Natl. Acad. Sci. USA 86, 7611 –7615. Goss, J.R., Styren, S.D., Miller, P.D., Kochanek, P.M., Palmer, A.M., Marion, D.W., DeKosky, S.T., 1995. Hypothermia attenuates the normal increase in interleukin 1 beta RNA and nerve growth factor following traumatic brain injury in rat. J. Neurotrauma 12, 159 – 167. Hagg, T. and Oudega, M., Neurotrophic factors and CNS regeneration. In: Stalberg E., Sharma H.S., Olssen Y. (Eds.), Spinal Cord Monitoring, Springer, New York, 1998, pp. 129 – 155. Hammond, E.A., Smart, D., Toulmond, S., Suman-Chauhan, N., Hughes, J., Hall, M.D., 1999. The interleukin-1 type I receptor is expressed in human hypothalamus. Brain 122, 1697 – 1707. Hopkins, S.J., Rothwell, N.J., 1995. Cytokines and the nervous system I: expression and recognition. Trends Neurosci. 18, 83 –88. Hulme, E. C. and Birdsall, N. J. M., Strategy and tactics in receptor binding studies. In: Hulme, E. C. (Ed.), Receptor – ligand interactions, A practical approach, IRL Press at Oxford University Press Oxford, Tokyo, 1992, pp. 63 – 176. Juricˇ , D.M., C& arman-Krzˇ an, M., 2000. Cytokine-regulated secretion of nerve growth factor from cultured rat neonatal astrocytes. Pfluegers Arch. – Eur. J. Physiol. 440, R96 – R98. Katsuura, G., Gottshal, P.E., Dahl, R.R., Arimura, A., 1989. Interleukin-1 beta increase prostaglandin E2 in rat astrocytes cultures: modulatory effect of neuropeptides. Endocrinology 124, 3124 – 3127. Krzˇ an, M., Wu, V.W., Schwartz, J.P., 2001. Serotonin regulation of nerve growth factor synthesis in neonatal and adult astrocytes: comparison to the b-adrenergic agonist isoproterenol. J. Neurosci. Res. 64, 261 – 267. Lapchak, P.A., 1993. Nerve growth factor pharmacology: application to the treatment of cholinergic neurodegeneration in Alzheimer’s disease. Exp. Neurol. 124, 16 – 20. Lipnik-S& tangelj, M., Juric, D.M., C& arman-Krzˇ an, M., 1998. Histamine-induced synthesis and secretion of nerve growth factor from astrocytes. Inflamm. Res. 47, S34 – S35. Lipnik-S& tangelj, M., C& arman-Krzˇ an, M., 2000. The effects of histamine and interleukin-6 on NGF release from cortical astrocytes in primary culture. Pfluegers Arch. – Eur. J. Physiol. 440, R99 – R100. Lindholm, D., Heumann, R., Hengerer, B., Thoenen, H., 1988. Interleukin 1 increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts. J. Biol. Chem. 263, 16348 – 16351.
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683 Lindholm, D., Castren, E., Berzaghi, M., Blo¨ chl, A., Thoenen, H., 1994. Activity-dependent and hormonal regulation of neurotrophin mRNA levels in the brain —implications for neuronal plasticity. J. Neurobiol. 25, 1362. Liu, T., McDonnell, P.C., Young, P.R., White, R.F., Siren, A.L., Hallenbeck, J.M., Barone, F.C., Feurestein, G.Z., 1993. Interleukin-1 beta mRNA expression in ishemic rat cortex. Stroke 24, 1746 – 1750. Lu, B., Yokoyama, M., Dreyfus, C.F., Black, I.B., 1991. NGF gene expression in actively growing brain glia. J. Neurosci. 11, 318 – 326. McMahan, C.J., Slack, J.L., Mosley, B., Cosman, D., Lupton, S.D., Brunton, L.L., Grubin, C.E., Wignall, J.M., Jenkins, N.A., Brannan, C.I., et al., 1991. A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types. EMBO J. 10, 2821 – 2832. Mehlhorn, G., Hollborn, M., Schliebs, R., 2000. Induction of cytokines in glial cells surrounding cortical b-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology. Int. J. Dev. Neurosci. 18, 423 – 431. Nishiyori, A., Minami, M., Takami, S., Satoh, M., 1997. Type 2 interleukin-1 receptor mRNA is induced by kainic acid in the rat brain. Brain Res. Molec. Brain Res. 50, 237 –245. Oderfeld-Nowak, B., Bacia, A., Gradkowska, M., Fusco, M., Vantani, G., Leon, A., Aloe, L., 1992. In vivo activated brain astrocytes may produce and secrete nerve growth factor-like molecules. Neurochem. Int. 21, 455 –461. Parnet, P., Amindari, S., Wu, C., Brunke-Reese, D., Goujon, E., Weyhenmeyer, J.A., Dantzer, R., Kelley, K.W., 1994. Expression of type I and type II interleukin-1 receptors in mouse brain. Molec. Brain. Res. 27, 63 –70. Pechan, P.A., Yoshida, T., Panahian, N., Moskowitz, M.A., Breakefield, X.O., 1995. Genetically modified fibroblasts producing NGF protect hippocampal neurons after ishemia in the rat. NeuroReport 6, 669 – 672. Pshenichkin, S.P., Szekely, A.M., Wise, B.C., 1994. Transcriptional and posttranscriptional mechanisms involved in the interleukin-1, steroid, and protein kinase C regulation of nerve growth factor in cortical astrocytes. J. Neurochem. 63, 419 –428. Rothwell, N.J., 1991. Functions and mechanisms of interleukin-1 in the brain. Trends Pharmacol. Sci. 12, 430 –436. Rothwell, N.J., Hopkins, S.J., 1995. Cytokines and the nervous system II: actions and mechanisms of action. Trends Neurosci. 18, 130 – 136. Rothwell, N.J., 1999. Cytokines – killers in the brain? J. Physiol. 514, 3–17. Rubio, N., 1994. Demonstration of the presence of an interleukin-1 receptor on the surface of murine astrocytes and its regulation by cytokines and Theiler’s virus. Immunology 82, 178 –183. Scatchard, G., 1949. The attraction of proteins for small molecules and ions. Ann. NY Acad. Sci. 51, 600 –672. Semkova, I., Wolz, P., Schilling, M., Kriegelstein, J., 1996. Selegiline enhances NGF synthesis and protects central nervous system neurons from excitotoxic and ischemic damage. Eur. J. Pharma-
683
col. 315, 19 – 30. Sims, J.E., March, C.J., Cosman, D., Widmer, M.B., MacDonald, H.R., McMahan, C.J., Grubin, C.E., Wignall, J.M., Jackson, J.L., Call, S.M., et al., 1988. CDNA expression cloning for the IL-1 receptor, a member of the immunoglobulin superfamily. Science 241, 585 – 589. Sims, J.E., Gayle, M.A., Slack, J.L., Alderson, M.R., Bird, T.A., Giri, J.G., Colotta, F., Re, F., Mantovani, A., Shanebeck, K., Grabstein, K.H., Dower, S.K., 1993. Interleukin-1 signaling occurs exclusively via the type I receptor. Proc. Natl. Acad. Sci. USA 90, 6155 – 6159. Spranger, M., Lindholm, D., Bandtlow, C., Heumann, R., Gnahn, H., Naher-Noe, M., Thoenen, H., 1990. Regulation of nerve growth factor (NGF) synthesis in the rat central nervous system: comparison between the effects of interleukin-1 and various growth factors in astrocyte cultures and in vivo. Eur. J. Neurosci. 2, 69 – 76. Takao, T., Tracey, D.E., Mitchell, W.M., De Souza, E.B., 1990. Interleukin-1 receptors in mouse brain: characterization and neuronal localization. Endocrinology 127, 3070 – 3078. Takao, T., Culp, S.G., Newton, R.C., De Souza, E.B., 1992. Type I interleukin-1 receptors in the mouse brain-endocrine-immune axis labelled with [125I] recombinant human interleukin-1 receptor antagonist. J. Neuroimmunol. 41, 51 – 60. Thoenen, H., 1995. Neurotrophins and neuronal plasticity. Science 27, 593 – 598. Thoenen, H., Bandtlow, C., Heumann, R., 1987. The physiological function of nerve growth factor in the central nervous system: comparison with the pheriphery. Rev. Physiol. Biochem. Pharmacol. 109, 145 – 178. Uehara, A., Gottschall, P.E., Dahl, R.K., Arimura, A., 1988. Stimulation of ACTH release by human interleukin-1beta, but not by interleukin-1alpha, in conscious, freely moving rats. Biochem. Byophys. Res. Commun. 146, 1286 – 1290. Van Dam, A.-M., de Vries, H.E., Kupier, J., Ziljstra, F.J., de Boer, A.G., Tilders, F.J.H., Berkenbosch, F., 1996. Interleukin-1 receptors on rat brain endothelial cells: a role in neuroimmune interaction? FASEB J. 10, 351 – 356. Vige, X., Costa, E., Wise, B.C., 1991. Mechanism of nerve growth factor mRNA regulation by interleukin-1 and basic fibroblast growth factor in primary cultures of rat astrocytes. Mol. Pharmacol. 40, 186 – 192. Vitkovic, L., Bockaert, J., Jacque, C., 2000. Inflammatory cytokines: neuromodulators in normal brain? J. Neurochem. 74, 457 – 471. Whittemore, S.R., Seiger, A., 1987. The expression, localization and functional significance of b-nerve growth factor in the central nervous system. Brain Res. Rev. 12, 439 – 446. Wu, V.W., Nishiyama, N., Schwartz, J.S., 1998. A culture model of reactive astrocytes: increased nerve growth factor synthesis and reexpression of cytokine responsivness. J. Neurochem. 71, 749 – 756. Yabuuchi, K., Minami, M., Katsumata, S., Satoh, M., 1994. Localization of type I interleukin-1 receptor mRNA in the rat brain. Mol. Brain Res. 27, 27 – 36.
Interleukin-1b, but not IL-1a, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor Damijana M. Juricˇ, Marija C& arman-Krzˇan * Department of Pharmacology, Faculty of Medicine, Korytko6a 2, SI-1000 Ljubljana, Slo6enia Received 1 June 2001; received in revised form 9 June 2001; accepted 17 July 2001
Abstract In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1b (IL-1b). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1a and IL-1b) and the antagonist (IL-1ra)—members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [125I] IL-1b to cultured astrocytes was saturable and of high affinity. Mean values for the KD and Bmax were calculated to be 60.7 97.4 pM and 2.5 9 0.1 fmol mg-1 protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1a (Ki of 341.1 pM) and IL-1b (Ki 59.9 pM), as well as the antagonist IL-1ra (Ki 257.6 pM), displaced specific [125I] IL-1b binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [125I] IL-1b binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1a and IL-1b revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1b (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1a by itself had no stimulatory action on NGF release. The simultaneous application of IL-1a and IL-1b elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1b. We concluded that IL-1b-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1a and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes. © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Interleukin-1; Interleukin-1 receptor; Astrocytes; Nerve growth factor
1. Introduction In the CNS, nerve growth factor (NGF) (C& armanKrzˇan, 1997) is a neurotrophic factor that promotes the survival and differentiation of the developing basal forebrain cholinergic neurons that are also responsive to and dependent on NGF for phenotypic maintenance and neuronal survival in the adult brain (Thoenen et al., 1987; Whittemore and Seiger, 1987; Cuello, 1993; Lapchak, 1993). During aging, these neurons become atrophic and are susceptible to neurodegenerative disorders such as Alzheimer’s disease. Evidence from both * Corresponding author. Tel.: + 386-1-543-7351; fax: +386-1-5437331. E-mail address: [email protected] (M. C& armanKrzˇan).
‘in vitro’ and ‘in vivo’ studies (Fisher et al., 1987; Gage et al., 1988; Connor and Dragunow, 1998; Hagg and Oudega, 1998) suggests that NGF provides neuroprotection to cholinergic neurons of the basal forebrain by inhibiting intrinsic apoptotic mechanisms, as well as by increasing neuronal metabolism and function. In the lesioned brain, NGF was also found to protect hippocampal and cortical neurons (Pechan et al., 1995; Semkova et al., 1996), suggesting that increasing brain levels of endogenous NGF may be beneficial in certain brain disorders. Under normal physiological conditions NGF is produced predominantly by neurons (Ayer-Lelievre et al., 1988), where the amount of NGF mRNA is regulated by neuronal activity and hormonal influences (Lindholm et al., 1994; Thoenen, 1995). During periods of rapid glial proliferation, i.e. prenatal/early postnatal
0736-5748/01/$20 © 2001 ISDN. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 5 7 4 8 ( 0 1 ) 0 0 0 4 4 - 2
676
D.M. Juricˇ, M. C& arman-Krzˇan / Int. J. De6l Neuroscience 19 (2001) 675–683
period and after injury in the adult brain, astrocytes become a major site of NGF synthesis in the CNS (Furukawa et al., 1987; Lu et al., 1991; OderfeldNowak et al., 1992; Arendt et al., 1995). Previous studies have shown that NGF secretion from astrocytes is susceptible to stimulation by various pharmacological agents including glial cell growth factors, neurotransmitters, and cytokines (C& arman-Krzˇ an et al., 1991; Lipnik-S& tangelj et al., 1998; Lipnik-S& tangelj and C& arman-Krzˇ an 2000; Juricˇ and C& arman-Krzˇ an, 2000; Krzˇ an et al., 2001). IL-1b is one of the most potent stimulators of NGF secretion in cultured neonatal astrocytes (Spranger et al., 1990; C& arman-Krzˇ an et al., 1991; Vige et al., 1991; C& arman-Krzˇ an and Wise, 1993, Juricˇ and C& arman-Krzˇ an, 2000). The action of IL-1b is of interest since it is produced in the CNS and, its stimulation of NGF secretion is both high and specific in comparison to other growth factors in the model system of rat neonatal cortical astrocytes. IL-1b increases the NGF mRNA content by activation of both NGF gene transcription and by posttranscriptional stabilization of NGF mRNA prior to NGF secretion (Lindholm et al., 1988; C& arman-Krzˇ an et al., 1991; Vige et al., 1991; Pshenichkin et al., 1994). Although the precise molecular mechanism regulating NGF elevation in astrocytes by IL-1b is still unresolved, IL-1 receptors are the likely mediators of this response (C& armanKrzˇ an et al., 1991). In the normal healthy brain, the expression of IL-1b and its mRNA is very low (Vitkovic et al., 2000), but it is markedly increased in response to local inflammation, injury, or in disease states including Alzheimer’s disease and stroke (Griffin et al., 1989; Rothwell, 1991; Liu et al., 1993; Rothwell and Hopkins, 1995; Mehlhorn et al., 2000). Based on the finding that an increase in NGF levels in the injured rat brain temporally follows an increase in IL-1b mRNA (Goss et al., 1995) and ‘in vitro’ experiments on the influence of exogenous IL-1b on NGF release, IL-1b produced endogenously could be one of the stimulants of NGF synthesis. In addition to IL-1b, endogenous levels of IL-1a and the IL-1 receptor antagonist (IL-1ra), are also elevated during neurodegenerative conditions (Hopkins and Rothwell, 1995; Rothwell 1999), indicating that endogenous IL-1s play a functional role in disease states of the brain. Therefore, our study was aimed at investigating the involvement of all three members of the IL-1 family (IL-1a, IL-1b and IL-1ra) in NGF secretion from nonneuronal cells (astrocytes) and testing the hypothesis that their action on NGF synthesis and secretion is under the control of IL-1 receptors. Our results showed that signal transducing type I (Sims et al., 1988, 1993), but not a decoy type II (McMahan et al., 1991; Colotta et al., 1993). IL-1 receptors are present on rat neonatal cortical astrocytes in primary culture. We also provide evidence that the stimulation of NGF secretion from
these cells by IL-1b is a type I IL-1 receptor mediated process, whereas IL-1a and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from cultured astrocytes.
2. Experimental procedures
2.1. Materials Timed pregnant Wistar rats were obtained from our own breeding colony. Dulbecco’s phosphate buffered saline (PBS), Dulbecco’s modified Eagle medium and Ham’s nutrient mixture F-12 (DMEM/F12 (1:1)), fetal bovine serum (FBS), and penicillin/streptomycin, were from Gibco BRL, Scotland. Tissue culture plates and immunoplates were from Nunc, Denmark. Recombinant human IL-1a, IL-1b, and IL-1 receptor antagonist (IL-1ra), as well as anti-human type I IL-1 receptor (Ab IL-1RI) and anti-human type II IL-1 receptor (Ab IL-1RII) polyclonal antibodies, were from R&D Systems, UK. Anti-mouse b (2.5S, 7S) nerve growth factor monoclonal antibody, anti-mouse b (2.5S, 7S) nerve growth factor-b-gal monoclonal antibody, nerve growth factor-b (NGF-b) (mouse), and chlorophenol-red-b-dgalactopyranoside (CPRG) were from Boehringer Mannheim, Germany. Iodinated recombinant human interleukin-1b ([125I] IL-1b, specific activity 2142 Ci/ mmol) was from NEN Life Science Products, USA. Bovine serum albumin (BSA) and all other chemicals used were from Sigma.
2.2. Cell culture and treatment of cells Primary cultures of rat neonatal cortical astrocytes were prepared from the brains of newborn Wistar rats according to the method described in C& arman-Krzˇ an et al. (1991). Briefly, after removal of the meninges, the cortices were dissociated by passage through sterile Nitex nylon screens (75 mm mesh size) into 10 ml of the culture medium (DMEM/F12 (1:1), 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin). The cell suspensions were diluted and plated into 35 mm (2× 106 cells/dish) or 100 mm (10×106 cells/dish) tissue culture dishes. Cells were grown at 37 °C in a water saturated air environment containing 10% CO2. Culture media was changed every 3 days of cultivation. After reaching confluency (10–14 days of growing), culture medium in the 35 mm tissue culture dishes was replaced with fresh medium and cells were treated under serum-free conditions with either vehicle (PBS containing 1 mg/ml BSA)-control cells or cytokines IL-1b (0.1–50 U/ml; 1 U/ml equals 5 pg/ml), IL-1a (0.1–50 U/ml) or IL-1ra (0.001–0.6 U/ml; 1 U/ml equals 100 ng/ml) in PBS/BSA. Following a 24 h incubation, the culture medium was collected, frozen,
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
677
and used for NGF determination. Astroglial cells in the 100 mm culture dishes were collected and used for binding studies. Immunocytochemical staining for glial fibrillary acidic protein indicated that 95% of the cells were astrocytes (C& arman-Krzˇ an et al., 1991).
and (IL-1a, IL-1b, or IL-1ra) under the same experimental conditions as described for the saturation binding assay. The radioactivity trapped on the cell homogenates was counted in a g-counter.
3. Binding studies
To differentiate between type I and type II IL-1 receptor binding sites on astrocytes we examined the binding of iodinated IL-1b (30 pM, i.e. 0.5 ng/ml) in the presence of increasing concentrations (1 pg/ml –10 mg/ml) of either specific anti-IL-1R type I (Ab IL-1RI) or anti-IL-1R type II (Ab IL-1RII) polyclonal antibodies. Incubations were carried out under the same experimental conditions as described for the saturation binding assay. The radioactivity trapped on the cell homogenates was counted in a g-counter.
3.1. Saturation binding: Identification and characterization of IL-1 receptors on cell homogenates of neonatal rat cortical astrocytes was performed by receptor-binding experiments according to the modified method of Ban et al. (1993) using iodinated [125I] human recombinant IL-1b as a radiolabel. 100 ml aliquots of cell homogenates containing 100 mg of proteins were incubated at 37 °C in a total volume of 0.2 ml containing PBS (pH 7.4). For the saturation binding assay, increasing concentrations of [125I] IL-1b (10– 200 pM) were added either alone (total binding) or in the presence of 6 nM IL-1b (nonspecific binding) to the incubation mixture. After a 1h incubation, the reaction was terminated by centrifugation for 1 min at 9000 rpm. Cell homogenates were washed with ice-cold PBS and centrifuged. The radioactivity trapped on the cell homogenates was counted in a g-counter. Specific binding was calculated by subtracting nonspecific binding from total binding.
3.2. Kinetics of binding Studying the association of [125I] IL-1b to the IL-1 receptor, cell homogenates were incubated with 30 pM [125I] IL-1b alone or in the presence of unlabeled 6 nM IL-1b. Incubations were carried out for various times between 0 and 60 min. The reaction was terminated and the radioactivity trapped on the cell homogenates was counted in a g-counter. The dissociation of [125I] IL-1b from the IL-1 receptor was examined by incubating cell homogenates for 60 min with 30 pM [125I] IL-1b. At 60 min, unlabelled IL-1b (6 nM) was then added and the reaction was terminated at various times between 1 and 60 min thereafter. The radioactivity trapped on the cell homogenates was counted in a g-counter.
3.3. Inhibition binding assay Inhibition binding experiments were carried out in order to assess the ability of IL-1a, IL-1b, and IL-1ra to displace radiolabeled IL-1b from the IL-1 receptor binding sites on astrocytes. Incubations were carried out with 30 pM [125I] IL-1b in the presence of increasing concentrations (0.1 pM– 100 nM) of unlabeled lig-
3.4. Differentiation between type I and type II IL-1 receptor
3.5. Enzyme immunoassay (EIA) of NGF The NGF enzyme immunoassay (NGF-EIA) was performed essentially as described in C& arman-Krzˇ an et al. (1991). Briefly, immunoplates (Nunc) were coated with anti-bNGF monoclonal antibody. Following treatment with 1% BSA, aliquots of astrocyte culture medium or NGF standards (10–320 pg/ml) were added in duplicate to the plates. After incubation overnight, the immunoplate wells were incubated with anti-b NGF monoclonal antibody conjugated with b-galactosidase followed by the enzyme substrate chlorophenol red-b-dgalactopyranoside. The reaction product was measured at 570 nm using a microplate reader. Culture medium samples and NGF standards were prepared as previously described (C& arman-Krzˇ an et al., 1991). Protein concentration in the cell homogenates was determined according to the method of Bradford (1976) using bovine serum albumin as a standard.
4. Calculations The equilibrium dissociation constant (KD) and the maximum binding capacity (Bmax) for [125I] IL-1b were determined according to the method of Scatchard (1949); data was taken from the saturation binding experiments (Fig. 1). Kinetic parameters of [125I] IL-1b binding were calculated as described in Hulme and Birdsall (1992). Ki values (the dissociation constant for a competing ligand) were calculated using the ChengPrusoff equation (Cheng and Prusoff, 1973) from the inhibition binding experiments. IC50 values (concentration of competing ligand that displaces 50% of specific [125I] IL-1b binding) of each competitor were obtained using a logistic equation and a Graph Pad Prism 3.0 program. Data are shown as the means9 SEM. The
678
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
Student’s t-test was used for statistical analysis of the data.
5. Results
5.1. Saturation binding experiments Specific [125I] IL-1b binding to membrane homogenates of neonatal rat cortical astrocytes was saturable, reversible, and of high affinity (Fig. 1). It represented 50% of the total binding. Scatchard’s analysis (Fig. 1) of specific [125I] IL-1b binding at equilibrium revealed a straight line, thus indicating the presence of
Fig. 3. Inhibition of specific [125I] IL-1b binding to rat neonatal cortical astrocytes from primary culture by IL-1a, IL-1b, or IL-1ra. Astroglial cells were incubated with 30 pM [125I] IL-1b alone or in the presence of increasing concentrations (0.1 pM – 100 nM) of unlabelled ligand. Values represent the means 9SEM from five independent experiments, each assayed in triplicate.
a homogenous population of [125I] IL-1b binding sites with a Hill coefficient (nH) near unity (0.949 0.05). The dissociation constant (KD) was 60.79 7.4 pM and the maximal number of binding sites (Bmax) was 2.590.1 fmol/mg protein.
5.2. Kinetics of [ 125I] IL-1i binding
Fig. 1. Saturation analysis of [125I] IL-1b binding to neonatal rat cortical astrocytes at equilibrium. Astroglial cells were incubated with increasing concentrations (10 –200 pM) of [125I] IL-1b alone or in the presence of 6 nM unlabelled IL-1b. Inset: Scatchard’s transformation of the specific binding. Values represent the means 9 SEM of five independent experiments, each assayed in triplicate. See also Materials and Methods.
Kinetic analysis of the binding [125 I] IL-1b to astrocytes showed that the specific binding of 30 pM [125I] IL-1b occurred rapidly with a half-maximal binding within 2.8 min and equilibrium was reached after 20 min (Fig. 2). The observed rate constant (kobs) for the forward reaction was 0.251 min − 1. The dissociation of [125I] IL-1b from the binding sites initiated by IL-1b was rapid (t1/2 of 4.1 min) and yielded a dissociation rate constant (k − 1) of 0.17 min − 1. The association rate constant (k + 1) for [125I] IL-1b of 2.8×109 M − 1 min − 1 was calculated from the k − 1 and kobs. The equilibrium dissociation constant (KD) of 60.8 9 5.4 pM was calculated from the ratio k − 1/k + 1. The KD value is in accordance with the KD calculated from the Scatchard plot of saturation binding data.
5.3. Inhibition binding experiments
Fig. 2. Kinetics of [125I] IL-1b binding to neonatal rat cortical astrocytes. The association reaction was carried out with 30 pM [125I] IL-1b alone or in the presence of unlabeled 6 nM IL-1b and terminated at individual time points (1 –60 s). The dissociation reaction was initiated by the addition of IL-1b (6 nM) to the astrocytes after a 60 min preincubation with 30 pM [125I] IL-1b and terminated at individual time points (0 –60 s). Values at each time point represent the means9SEM of five independent experiments, each assayed in triplicate.
The pharmacological characteristics of the [125I] IL1b labeled IL-1 receptor binding sites were examined by determining the potencies of different IL-1 receptor ligands (IL-1a, IL-1b, and IL-1ra) to displace the radioligand from the receptor binding sites. All tested ligands fully inhibited specific [125I] IL-1b binding to IL-1 receptor binding sites in a monophasic manner (Fig. 3). The rank order of potencies for the inhibition of specific [125I] IL-1b binding was: IL-1b \IL-1ra\IL-1a (Fig. 3). Half-maximally effective doses (IC50) for IL-1b and IL-1a were 89.6 pM. and 518.7 pM, respectively
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
Fig. 4. Inhibition of specific [125I] IL-1b binding by anti-IL-1RI or anti-IL-1RII antibodies to rat neonatal cortical astrocytes from primary culture. Astroglial cells were incubated with 30 pM [125I] IL-1b alone or in the presence of increasing concentrations (1 pg/ml– 10 mg/ml) of unlabelled anti-IL-1RI or anti-IL-1RII antibody. Values represent the means 9 SEM from five independent experiments, each assayed in triplicate.
679
(Fig. 3). The IC50 value of IL-1ra was 385.0 pM. Using the Cheng-Prusoff equation (Cheng and Prusoff, 1973), we calculated the inhibition constants (Ki ) for all the ligands used, which were 59.9 pM for IL-1b, 257.6 pM for IL-1ra and 341.1 pM for IL-1a. To determine which subtype of IL-1 receptors was labeled with the [125I] IL-1b on rat cortical astrocytes, we performed the inhibition binding experiments with iodinated IL-1b (30 pM, i.e. 0.5 ng/ml) in the presence of increasing concentrations (1 pg/ml –10 mg/ml) of the specific polyclonal antibody against either type I (Ab IL-1RI) or type II (Ab IL-1RII) IL-1 receptors. Dosedependent inhibition of the [125I] IL-1b binding by Ab IL-1RI (depicted in Fig. 4) revealed an IC50 value of 6.39 0.1 ng/ml. In contrast, the antibody against the type II IL-1 receptor was not able to block the specific binding of [125I] IL-1b (Fig. 4). These data strongly suggest that the IL-1 receptor we identified is the signal transducing type I IL-1R, whereas the type II IL-1R is notably absent from neonatal rat cortical astrocytes.
5.4. Effects of IL-1h, IL-1i, and IL-1ra on NGF secretion from rat cortical astrocytes in primary culture
Fig. 5. Effects of: (A) IL-1a, IL-1b; and (B) IL-1ra on NGF release from rat neonatal cortical astrocytes in primary culture. Cells were incubated for 24 h in vehicle (PBS/BSA-control cells) or in the culture medium containing IL-1a (0.1–50 U/ml), IL-1b (0.1– 50 U/ml), or IL-1ra (0.001 – 0.6 U/ml). Supernatants were collected and NGF content was determined by ELISA-NGF assay. Values are the means9SEM of five independent determinations. IL-1b (10 U/ml) stimulated NGF secretion represents 703.5 9 28.4 pg NGF/mg protein. Basal secretion was 239.3 9 13.1 pg NGF/mg protein. *P B 0.001; values are significantly different from the control level (Student’s t-test).
As previously demonstrated (C& arman-Krzˇ an et al., 1991), IL-1b stimulates NGF secretion from cultured astrocytes in a dose-dependent manner with the maximal effect between 3 and 10 U/ml. At the maximal concentration of IL-1b (10 U/ml) the level of NGF in the culture medium was increased by 3-fold as compared to the control cells. This effect was specific in comparison to other growth factors and cytokines used, suggesting IL-1 receptor mediated stimulation. The effects of IL-1a and IL-1ra were not addressed in this study (C& arman-Krzˇ an et al., 1991). Since IL-1a and IL-1b are both IL-1 receptor agonists believed, at least in the peripheral nervous system, to exert identical biological effects (Dinarello, 1996) and, as shown in Fig. 3, both have an affinity for the same IL-1 receptor, we tested their potential receptor mediated effect on the regulation of NGF release from astrocytes. NGF release from astrocytes was also determined after treatment with IL-1a or IL-1b in the presence of a natural IL-1 receptor antagonist, IL-1ra. Exposure of cultured cells for 24 h to IL-1b (0.1–50 U/ml) caused a dose-dependent increase in NGF secretion with a half maximal effect at 1.7 U/ml and a maximal effect at 10 U/ml (Fig. 5(A)). IL-1b-stimulated cultures released 3-times more NGF (703.59 28.4 pg NGF/mg protein) than the control cells (239.3913.1 pg NGF/mg protein) (Fig. 5(A)). In contrast, the basal NGF secretion from cultured astrocytes was not altered in response to 24 h stimulation with IL-1a (0.1–50 U/ml) (Fig. 5(A)). No effect was observed even after prolonged treatment (48 h incubation) or when higher concentration of IL-1a (100 U/ml) was used (data not
680
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
concentrations of IL-1a (0.1–50 U/ml) and NGF release was measured 24 h later. As shown in Fig. 7, IL-1a had no significant effect on IL-1b-induced NGF release.
6. Discussion
Fig. 6. Effect of IL-1ra on IL-1b-induced NGF release from rat neonatal cortical astrocytes in primary culture. Cells were incubated for 24 h in culture medium containing IL-1b (10 U/ml) in the presence of IL-1ra (0.001 –0.6 U/ml). Supernatants were collected and tested for NGF content by ELISA-NGF assay. Values are the means9SEM of five independent determinations.
Fig. 7. Effect of IL-1a on IL-1b-induced NGF release from rat neonatal cortical astrocytes in primary culture. Cells were incubated for 24 h in vehicle (PBS/BSA-control cells) or culture medium containing IL-1b (10 U/ml) in the presence of IL-1a (0.1 –50 U/ml). Supernatants were collected and tested for NGF content by ELISANGF assay. Values are the means 9 SEM of five independent determinations. IL-1b (10 U/ml) stimulated NGF secretion represents 703.5928.4 pg NGF/mg protein. Basal secretion was 239.3 9 13.1 pg NGF/mg protein.
shown). In accordance with its antagonistic function on the IL-1 receptor, IL-1ra (0.001–0.6 U/ml) had no effect on basal NGF release (Fig. 5(B)), but dose-dependently inhibited the stimulatory response induced by IL-1b (10 U/ml) (IC50 of 0.07 U/ml) (Fig. 6). A complete blockade of IL-1b action on NGF secretion occurred at 0.6 U/ml of antagonist. In contrast, IL-1ra showed no influence on IL-1a-treated cells (data not shown). To exclude the possible additive effect of IL-1a on IL-1b-stimulated secretion of NGF, cells were treated with 10 U/ml of IL-1b in the presence of increasing
Neonatal rat cortical astrocytes and neurons in primary culture express NGF mRNA and synthesize NGF. In astrocytes, NGF gene transcription is regulated by different intracellular signaling systems that can be modulated by cytokines. This modulation differs greatly between astrocytes in different stages of development (Wu et al., 1998). The IL-1 family of cytokines consists of IL-1a, IL-1b, and IL-1ra. IL-1b is more predominant form in the brain than IL-1a. IL-1b has been shown to be much more effective than IL-1a in regulating various functions in the normal, healthy brain, as well as in neuroimmune responses to disease and neurodegeneration (for review see Vitkovic et al., 2000; Rothwell, 1999). In the present study, we could demonstrate that the stimulatory action of IL-1b on NGF secretion from primary cultures of astrocytes is mediated through the functional type I IL-1 receptors identified on rat astrocytes. IL-1b-induced NGF secretion was completely inhibited by IL-1ra in a dose dependent manner, thus confirming the IL-1RI receptor mediated process. In addition, we present evidence that IL-1a, which shows a high affinity for the type I IL-1 receptor, is not involved in the regulation of NGF secretion from cultured astrocytes. To identify the IL-1 receptors on rat neonatal cortical astrocytes in primary culture the IL-1 receptor agonist [125I] IL-1b was used. The binding of [125I] IL-1b was saturable, of high affinity, and readily reversible with a KD of 60.797.4 pM and a Bmax of 2.59 0.1 fmol/mg protein (Fig. 1). The KD value of 60.89 5.4 pM from the kinetic data (Fig. 2) is in accordance with the KD value obtained by Scatchard analysis (Fig. 1). The affinity that we observed for [125I] IL-1b binding to IL-1 receptors on cultured rat cortical astrocytes are comparable with the reported affinities for type I IL-1R (KD of 0.02–0.8 nM) in the rodent and human brain (Takao et al., 1990, 1992; Rubio, 1994; Parnet et al., 1994; Hammond et al., 1999). In the inhibition binding experiments (Fig. 3), all unlabeled compounds (IL-1a, IL-1b, and IL-1ra) completely inhibited specific [125I] IL-1b binding in a manner compatible with interaction at a single class of IL-1 binding sites but with differing potencies (IL-1b: Ki of 59.9 pM; IL-1a: Ki of 341.1 pM). This shows that the two IL-1 agonists do not bind equally well to the IL-1 receptor binding sites with IL-1b having a 6-fold higher potency for displacing specifically bound [125I] IL-1b
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
(Fig. 3) than IL-1a. The specificity of IL-1 receptors on astrocytes was confirmed by the competitive binding experiments using increasing concentrations (0.1 pM– 100 nM) of nonspecific IL-1R ligands (IL-2, IL-4 and IL-6). No displacement of specific [125I] IL-1b binding was obtained with IL-2 whereas IL-4 and IL-6 only partially inhibited radioligand binding (data not shown). Vitkovic et al. (2000) suggested that IL-1a binds to the same receptors as IL-1b in the brain but with lower affinity, what we demonstrated in our binding study on astrocytes (Fig. 3). However, studies of IL-1 receptors in the mouse hypocampus (Takao et al., 1990), mouse dendate gyrus (Ban et al., 1991) and mouse astrocyte cultures (Ban et al., 1993) showed that both agonists have the same affinity for the type I IL-1 receptors and therefore differ from our data. The IL-1 receptor antagonist had about a 5-fold lower affinity than the unlabeled IL-1b for IL-1 binding sites (Fig. 3), which is in accordance with observations on rat brain endothelial cells (Van Dam et al., 1996). Identification of functional IL-1 receptors on rat cortical astrocytes raises questions as to the exact type of IL-1 receptor that is expressed on these cells. It is well established that two types of IL-1 receptors play different roles in the regulation of IL-1 activity. Whereas type I is the major signaling receptor for IL-1 (Sims et al., 1993), type II appears to act as a decoy receptor (Colotta et al., 1993). Farrar et al. (1987) first reported IL-1 binding sites in the rodent brain. IL-1 receptors in the CNS were later studied by several groups (for review see Vitkovic et al., 2000) indicating dramatic species differences between the mouse and rat brain in the distribution of IL-1R. In the mouse brain, mRNA of both IL-1 receptor types is expressed, mainly in neuron-rich areas (the granule cell layer of the dentate gyrus and the cerebellum, the pyramidal cell layers of the hippocampus, and within the hypothalamus) (Ban et al., 1991; Parnet et al., 1994). In addition to neuronal cells bodies, IL-1RI expression was also associated with cerebral blood vessels and glial cells, including astrocytes. Ban et al. (1993) first reported IL-1 binding sites on cultured astrocytes from the cerebral hemispheres of newborn mice, whereas Rubio (1994) demonstrated the presence of specific type I IL-1 receptors in newborn murine cortical astrocyte cultures. In contrast to the murine CNS, only the type I IL-1 receptors have been identified in the normal rat brain, whereas the expression of type II IL-1 receptor mRNA has not been detected in any brain region (Yabuuchi et al., 1994; Nishiyori et al., 1997). IL-1RI mRNA is expressed in unidentified neuronal cell groups in several brain regions (the olfactory bulb, the hippocampus, the amygdala, the hypothalamus, and the cerebellum) (Farrar et al., 1987; Yabuuchi et al., 1994) and on the rat brain endothelial and epithelial cells (Yabuuchi et al., 1994; Van Dam et al., 1996; Nishiyori et al., 1997).
681
In order to distinguish different types of IL-1 receptors in the membrane of cultured rat cortical astrocytes, we performed binding studies using antibodies against both IL-1 receptor types (Fig. 4). Polyclonal antibodies allowed us to recognize all allotypic forms of the natural IL-1 receptors. Our results showed that only the anti-IL-1RI antibody dose-dependently inhibited specific [125I] IL-1b binding by selective blocking the binding sites of type I IL-1 receptors while the anti-IL-1RII antibody showed no significant influence on specific radioligand binding (Fig. 4). These results support the conclusion that the signal transducing type I IL-1R is expressed on neonatal rat cortical astrocytes and could therefore be the mediator of the IL-1b stimulated NGF secretion. Type II IL-1R is notably absent from these cells. In the present study, a remarkable difference between the agonists of type I IL-1 receptors (IL-1a and IL-1b) was observed in terms of their ability to induce NGF secretion from cultured rat cortical astrocytes (Fig. 5(A)). Basal NGF secretion from astrocytes was not altered in response to stimulation with IL-1a, while IL-1b potently and specifically stimulated NGF release (3-fold over basal level) (Fig. 5(A)). Simultaneous application of IL-1a and IL-1b elicited no additive response (Fig. 7), confirming that IL-1a had no influence on IL-1b- induced NGF secretion to the astrocyte culture. Therefore, in spite of the high affinity of IL-1a for the type I IL-1 receptors, we conclude that IL-1a is not involved in the regulation of NGF release from rat cortical astrocytes in primary culture. Similar to our results, in rat cultured astrocytes IL-1b was found to be 100-fold more effective than IL-1a on PGE2 release (Katsuura et al., 1989). IL-1b increases also plasma levels of ACTH in rats, while IL-1a has no effect (Uehara et al., 1988). IL-1ra alone had no effect on basal NGF release (Fig. 5(B)), but dose-dependently inhibited the biologic response induced by IL-1b (Fig. 6), thereby supporting our conclusion that the IL-1b-stimulated NGF secretion is a type I IL-1 receptor mediated process. Our observation is in agreement with the ‘in vivo’ study of DeKosky et al. (1996). They demonstrated that an increase in NGF after rat central nervous system trauma is directly mediated through IL-1b and that blocking IL-1b by IL-1ra following brain injury leads to suppression of an NGF-mediated reparative response. In conclusion, the results obtained in this study indicate that cortical astrocytes derived from newborn rats express functional type I IL-1 receptors, but not decoy type II IL-1 receptors. The production and release of NGF in cultured rat neonatal cortical astrocytes is thus regulated by a balance between the pro-inflammatory activities of IL-1b and the ability of IL-1R antagonist to control these activities by occupying the type I IL-1
682
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683
receptors. We have also shown that, in contrast to IL-1b, IL-1a has no role in the regulation of NGF secretion from newborn rat astrocytes. The specific stimulatory function of IL-1b on NGF secretion is limited to the neonatal rat astrocytes that in many respects resemble reactive astrocytes. IL-1b was not able to increase NGF mRNA or NGF content in normal adult rat astrocytes (Wu et al., 1998). The group (Wu et al., 1998) did not address the question of the involvement of IL-1R in the regulation of this process. Therefore, the pattern of the expression and the precise function of both types of IL-1 receptor in the regulation of NGF secretion from adult, as well as reactive astrocytes, remains to be determined.
Acknowledgements The authors wish to thank to C. Blazˇ ek and J. Kosˇir for their excellent technical assistance. This study was supported by Ministry of Education, Science and Sport.
References Arendt, T., Bruckner, M.K., Krell, T., Pagliusi, S., Kruska, L., Heumann, R., 1995. Degeneration of rat cholinergic basal forebrain neurons and reactive changes in nerve growth factor expression after chronic neurotoxic injury. II. Reactive expression of NGF gene in astrocytes. Neuroscience 65, 647 – 659. Ayer-Lelievre, C., Olson, I., Ebendal, T., Seiger, A., Persson, H., 1988. Expression of the b-nerve growth factor gene in hippocampal neurons. Science 240, 1339 –1341. Ban, E., Millon, G., Prudhomme, N., Fillion, G., Haour, F., 1991. Receptors for interleukin-1 (a and b) in mouse brain: mapping and neuronal localization in hippocampus. Neuroscience 43, 21 – 30. Ban, E.M., Sarlieve, L.L., Haour, F.G., 1993. Interleukin-1 binding sites on astrocytes. Neuroscience 52, 725 –733. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 –254. Cheng, Y.C., Prusoff, W.H., 1973. Relationship between the inhibition constant (Ki ) and the concentration of inhibitor which cause 50% inhibition (IC50) of the enzymatic reaction. Biochem. Pharmacol. 22, 3099 – 3108. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J.G., Dower, S.K., Siems, J.E., Mantovani, A., 1993. Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science 261, 472 –475. Connor, B., Dragunow, M., 1998. The role of neuronal growth factors in neurodegenerative disorders of the human brain. Brain Res. Rev. 27, 1 – 39. Cuello, C.A., 1993. Trophic responses of forbrain cholinergic neurons: a discussion. Prog. Brain Res. 98, 265 –277. C& arman-Krzˇ an, M., Vige, X., Wise, B.C., 1991. Regulation by interleukin-1 of nerve growth factor secretion and nerve growth factor mRNA expression in rat primary astroglial cultures. J. Neurochem. 56, 636 – 643. C& arman-Krzˇ an, M., Wise, B.C., 1993. Arachidonic acid lipoxygenation may mediate interleukin-1 stimulation of nerve growth factor secretion in astroglial cultures. J. Neurosci. Res. 34, 225 – 232.
C& arman-Krzˇ an, M., 1997. Nerve growth factor and related neurotrophins —their role in the CNS. Zdrav. Vestn. 66, 1 – 4. DeKosky, S.T., Styren, S.D., O’Malley, M.E., Goss, J.R., Kochanek, P., Marion, D., Evans, C.H., Robbins, P.D., 1996. Interleukin-1 receptor antagonist suppresses neurotrophin response in injured rat brain. Ann. Neurol. 39, 123 – 127. Dinarello, C.A., 1996. Biologic basis for Interleukin-1 in disease. Blood 87, 2095 – 2147. Farrar, W.L., Killian, P.L., Ruff, M.R., Hill, J.M., Pert, C.B., 1987. Visualization and characterization of interleukin 1 receptors in brain. J. Immunol. 139, 459 – 463. Fisher, W., Victorin, A., Bjo¨ rklund, A., Williams, L.R., Varon, S., Gage, F.H., 1987. Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor. Nature 329, 65 – 68. Furukawa, S., Furukawa, Y., Satoyashi, E., Hayaski, K., 1987. Synthesis/secretion of nerve growth factor is associated with cell growth in cultured mouse astroglial cells. Biochem. Biophys. Res. Commun. 142, 395 – 402. Gage, F.H., Armstrong, D.M., Williams, L.R., Varon, S., 1988. Morphological response of axotomized septal neurons to nerve growth factor. J. Corp. Neurol. 269, 147 – 155. Griffin, W.S., Stanley, L.C., Ling, C., White, L., MacLeod, V., Perrot, L.J., White, C.L., Araoz, C., 1989. Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc. Natl. Acad. Sci. USA 86, 7611 –7615. Goss, J.R., Styren, S.D., Miller, P.D., Kochanek, P.M., Palmer, A.M., Marion, D.W., DeKosky, S.T., 1995. Hypothermia attenuates the normal increase in interleukin 1 beta RNA and nerve growth factor following traumatic brain injury in rat. J. Neurotrauma 12, 159 – 167. Hagg, T. and Oudega, M., Neurotrophic factors and CNS regeneration. In: Stalberg E., Sharma H.S., Olssen Y. (Eds.), Spinal Cord Monitoring, Springer, New York, 1998, pp. 129 – 155. Hammond, E.A., Smart, D., Toulmond, S., Suman-Chauhan, N., Hughes, J., Hall, M.D., 1999. The interleukin-1 type I receptor is expressed in human hypothalamus. Brain 122, 1697 – 1707. Hopkins, S.J., Rothwell, N.J., 1995. Cytokines and the nervous system I: expression and recognition. Trends Neurosci. 18, 83 –88. Hulme, E. C. and Birdsall, N. J. M., Strategy and tactics in receptor binding studies. In: Hulme, E. C. (Ed.), Receptor – ligand interactions, A practical approach, IRL Press at Oxford University Press Oxford, Tokyo, 1992, pp. 63 – 176. Juricˇ , D.M., C& arman-Krzˇ an, M., 2000. Cytokine-regulated secretion of nerve growth factor from cultured rat neonatal astrocytes. Pfluegers Arch. – Eur. J. Physiol. 440, R96 – R98. Katsuura, G., Gottshal, P.E., Dahl, R.R., Arimura, A., 1989. Interleukin-1 beta increase prostaglandin E2 in rat astrocytes cultures: modulatory effect of neuropeptides. Endocrinology 124, 3124 – 3127. Krzˇ an, M., Wu, V.W., Schwartz, J.P., 2001. Serotonin regulation of nerve growth factor synthesis in neonatal and adult astrocytes: comparison to the b-adrenergic agonist isoproterenol. J. Neurosci. Res. 64, 261 – 267. Lapchak, P.A., 1993. Nerve growth factor pharmacology: application to the treatment of cholinergic neurodegeneration in Alzheimer’s disease. Exp. Neurol. 124, 16 – 20. Lipnik-S& tangelj, M., Juric, D.M., C& arman-Krzˇ an, M., 1998. Histamine-induced synthesis and secretion of nerve growth factor from astrocytes. Inflamm. Res. 47, S34 – S35. Lipnik-S& tangelj, M., C& arman-Krzˇ an, M., 2000. The effects of histamine and interleukin-6 on NGF release from cortical astrocytes in primary culture. Pfluegers Arch. – Eur. J. Physiol. 440, R99 – R100. Lindholm, D., Heumann, R., Hengerer, B., Thoenen, H., 1988. Interleukin 1 increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts. J. Biol. Chem. 263, 16348 – 16351.
D.M. Juricˇ , M. C& arman-Krzˇ an / Int. J. De6l Neuroscience 19 (2001) 675–683 Lindholm, D., Castren, E., Berzaghi, M., Blo¨ chl, A., Thoenen, H., 1994. Activity-dependent and hormonal regulation of neurotrophin mRNA levels in the brain —implications for neuronal plasticity. J. Neurobiol. 25, 1362. Liu, T., McDonnell, P.C., Young, P.R., White, R.F., Siren, A.L., Hallenbeck, J.M., Barone, F.C., Feurestein, G.Z., 1993. Interleukin-1 beta mRNA expression in ishemic rat cortex. Stroke 24, 1746 – 1750. Lu, B., Yokoyama, M., Dreyfus, C.F., Black, I.B., 1991. NGF gene expression in actively growing brain glia. J. Neurosci. 11, 318 – 326. McMahan, C.J., Slack, J.L., Mosley, B., Cosman, D., Lupton, S.D., Brunton, L.L., Grubin, C.E., Wignall, J.M., Jenkins, N.A., Brannan, C.I., et al., 1991. A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types. EMBO J. 10, 2821 – 2832. Mehlhorn, G., Hollborn, M., Schliebs, R., 2000. Induction of cytokines in glial cells surrounding cortical b-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology. Int. J. Dev. Neurosci. 18, 423 – 431. Nishiyori, A., Minami, M., Takami, S., Satoh, M., 1997. Type 2 interleukin-1 receptor mRNA is induced by kainic acid in the rat brain. Brain Res. Molec. Brain Res. 50, 237 –245. Oderfeld-Nowak, B., Bacia, A., Gradkowska, M., Fusco, M., Vantani, G., Leon, A., Aloe, L., 1992. In vivo activated brain astrocytes may produce and secrete nerve growth factor-like molecules. Neurochem. Int. 21, 455 –461. Parnet, P., Amindari, S., Wu, C., Brunke-Reese, D., Goujon, E., Weyhenmeyer, J.A., Dantzer, R., Kelley, K.W., 1994. Expression of type I and type II interleukin-1 receptors in mouse brain. Molec. Brain. Res. 27, 63 –70. Pechan, P.A., Yoshida, T., Panahian, N., Moskowitz, M.A., Breakefield, X.O., 1995. Genetically modified fibroblasts producing NGF protect hippocampal neurons after ishemia in the rat. NeuroReport 6, 669 – 672. Pshenichkin, S.P., Szekely, A.M., Wise, B.C., 1994. Transcriptional and posttranscriptional mechanisms involved in the interleukin-1, steroid, and protein kinase C regulation of nerve growth factor in cortical astrocytes. J. Neurochem. 63, 419 –428. Rothwell, N.J., 1991. Functions and mechanisms of interleukin-1 in the brain. Trends Pharmacol. Sci. 12, 430 –436. Rothwell, N.J., Hopkins, S.J., 1995. Cytokines and the nervous system II: actions and mechanisms of action. Trends Neurosci. 18, 130 – 136. Rothwell, N.J., 1999. Cytokines – killers in the brain? J. Physiol. 514, 3–17. Rubio, N., 1994. Demonstration of the presence of an interleukin-1 receptor on the surface of murine astrocytes and its regulation by cytokines and Theiler’s virus. Immunology 82, 178 –183. Scatchard, G., 1949. The attraction of proteins for small molecules and ions. Ann. NY Acad. Sci. 51, 600 –672. Semkova, I., Wolz, P., Schilling, M., Kriegelstein, J., 1996. Selegiline enhances NGF synthesis and protects central nervous system neurons from excitotoxic and ischemic damage. Eur. J. Pharma-
683
col. 315, 19 – 30. Sims, J.E., March, C.J., Cosman, D., Widmer, M.B., MacDonald, H.R., McMahan, C.J., Grubin, C.E., Wignall, J.M., Jackson, J.L., Call, S.M., et al., 1988. CDNA expression cloning for the IL-1 receptor, a member of the immunoglobulin superfamily. Science 241, 585 – 589. Sims, J.E., Gayle, M.A., Slack, J.L., Alderson, M.R., Bird, T.A., Giri, J.G., Colotta, F., Re, F., Mantovani, A., Shanebeck, K., Grabstein, K.H., Dower, S.K., 1993. Interleukin-1 signaling occurs exclusively via the type I receptor. Proc. Natl. Acad. Sci. USA 90, 6155 – 6159. Spranger, M., Lindholm, D., Bandtlow, C., Heumann, R., Gnahn, H., Naher-Noe, M., Thoenen, H., 1990. Regulation of nerve growth factor (NGF) synthesis in the rat central nervous system: comparison between the effects of interleukin-1 and various growth factors in astrocyte cultures and in vivo. Eur. J. Neurosci. 2, 69 – 76. Takao, T., Tracey, D.E., Mitchell, W.M., De Souza, E.B., 1990. Interleukin-1 receptors in mouse brain: characterization and neuronal localization. Endocrinology 127, 3070 – 3078. Takao, T., Culp, S.G., Newton, R.C., De Souza, E.B., 1992. Type I interleukin-1 receptors in the mouse brain-endocrine-immune axis labelled with [125I] recombinant human interleukin-1 receptor antagonist. J. Neuroimmunol. 41, 51 – 60. Thoenen, H., 1995. Neurotrophins and neuronal plasticity. Science 27, 593 – 598. Thoenen, H., Bandtlow, C., Heumann, R., 1987. The physiological function of nerve growth factor in the central nervous system: comparison with the pheriphery. Rev. Physiol. Biochem. Pharmacol. 109, 145 – 178. Uehara, A., Gottschall, P.E., Dahl, R.K., Arimura, A., 1988. Stimulation of ACTH release by human interleukin-1beta, but not by interleukin-1alpha, in conscious, freely moving rats. Biochem. Byophys. Res. Commun. 146, 1286 – 1290. Van Dam, A.-M., de Vries, H.E., Kupier, J., Ziljstra, F.J., de Boer, A.G., Tilders, F.J.H., Berkenbosch, F., 1996. Interleukin-1 receptors on rat brain endothelial cells: a role in neuroimmune interaction? FASEB J. 10, 351 – 356. Vige, X., Costa, E., Wise, B.C., 1991. Mechanism of nerve growth factor mRNA regulation by interleukin-1 and basic fibroblast growth factor in primary cultures of rat astrocytes. Mol. Pharmacol. 40, 186 – 192. Vitkovic, L., Bockaert, J., Jacque, C., 2000. Inflammatory cytokines: neuromodulators in normal brain? J. Neurochem. 74, 457 – 471. Whittemore, S.R., Seiger, A., 1987. The expression, localization and functional significance of b-nerve growth factor in the central nervous system. Brain Res. Rev. 12, 439 – 446. Wu, V.W., Nishiyama, N., Schwartz, J.S., 1998. A culture model of reactive astrocytes: increased nerve growth factor synthesis and reexpression of cytokine responsivness. J. Neurochem. 71, 749 – 756. Yabuuchi, K., Minami, M., Katsumata, S., Satoh, M., 1994. Localization of type I interleukin-1 receptor mRNA in the rat brain. Mol. Brain Res. 27, 27 – 36.