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Endocrinology Vol. 140, No. 2 888-896
Copyright © 1999 by The Endocrine Society

ARTICLES

Interleukin-1ß and Catecholamines Synergistically Stimulate Interleukin-6 Release from Rat C6 Glioma Cells in Vitro: a Potential Role for Lysophosphatidylcholine

John W. Zumwalt, Brian J. Thunstrom and Bryan L. Spangelo

Department of Chemistry, University of Nevada Las Vegas, Las Vegas, Nevada 89154-4003

Address all correspondence and requests for reprints to: Bryan L. Spangelo, Department of Chemistry, University of Nevada Las Vegas, 4505 Maryland Parkway, Las Vegas, Nevada 89154-4003. E-mail: spangelb{at}nevada.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) are proinflammatory cytokines that affect the secretion of several neuroendocrine hormones. In addition, glial cells synthesize and release IL-6, suggesting a paracrine role for this cytokine in the brain. We have examined the regulation of IL-6 release from glial cells by cytokines and catecholamines. Forty ng/ml IL-1ß induced a maximal 30-fold stimulation of IL-6 release (P < 0.01); higher and lower concentrations of IL-1ß were less effective. In the presence of (Bu)2cAMP, IL-1ß induced a strongly synergistic response with respect to IL-6 release; thus, the combination of these two agents resulted in a release of IL-6 that was much larger that the release attributed to either agent alone (i.e. 30-fold higher). Similarly, the combination of IL-1ß and the diterpene forskolin (but not the inactive analog 1,9-dideoxyforskolin) or cholera toxin also resulted in a synergistic stimulation of C6 glioma IL-6 release. Thus, increases in intracellular cAMP concentrations act in a synergistic fashion with the IL-1ß signaling pathway for IL-6 release. Because catecholamines increase intracellular cAMP levels, we investigated the effects of dopamine, epinephrine, and norepinephrine on IL-6 release. The combination of 1.0 to 100 µM of each catecholamine with IL-1ß resulted in the synergistic stimulation of IL-6 release. The coincubation of the ß-agonist isoproterenol and IL-1ß resulted in a striking 25-fold synergistic induction of IL-6 release. The synergistic increases in IL-6 release caused by IL-1ß and isoproterenol as well as IL-1ß and norepinephrine were blocked by the pretreatment of C6 cells with the ß-receptor antagonist propranolol. Because lysophosphatidylcholine (LPC) may function as a second messenger for IL-1ß, we also investigated the effects of LPC. Exogenous LPC (5 to 40 µM) stimulated IL-6 release from C6 glioma cells in a concentration-related manner (P < 0.01). The coincubation of LPC with norepinephrine provoked a synergistic release in IL-6 comparable with that obtained with IL-1ß and norepinephrine. Exposure of [3H]choline-labeled C6 cells to IL-1ß resulted in an increase in the [3H]LPC species as well as a decrease in [3H]phosphatidylcholine. Finally, while TNF{alpha} was less efficacious than IL-1ß for the stimulation of IL-6 release from C6 cells, the combination of IL-1ß and TNF{alpha} resulted in a significant synergistic induction of IL-6 release. We have demonstrated that IL-1ß stimulates IL-6 release from rat C6 glioma cells via a noncAMP-mediated mechanism that may involve LPC. The synergistic induction by cytokines and catecholamines of glial cell-derived IL-6 may subsequently affect inflammatory, neurodegenerative or neurotropic processes in the CNS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INTERLEUKINS are multifunctional polypeptides localized in primary and secondary lymphatic tissues as well as neural and glial elements in the CNS. The primary inflammatory cytokines interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-{alpha} (TNF{alpha}) have been implicated in several inflammatory disease states and CNS degenerative disorders including Alzheimer’s disease (reviewed in Ref. 1). IL-1 may contribute to plaque formation and eventual neuronal cell death by increasing ß-amyloid expression. The increased production of IL-6 by IL-1ß or TNF{alpha} may affect the course of neurodegenerative diseases. Amyloid precursor protein messenger RNA (mRNA) is induced in rat cortical neurons by IL-6, indicating a possible role for this cytokine in ß-amyloid deposition in Alzheimer’s disease (2).

The IL-1 family consists of two polypeptides, IL-1{alpha} and IL-1ß, and a receptor antagonist (IL-1ra; reviewed in Ref. 3). IL-1 causes systemic fever and the activation of the hypothalamic-pituitary-adrenal (HPA) axis after peripheral injection (reviewed in Ref. 4). IL-1ß injected sc increases protein transcription in the anterior and posterior lobes of the pituitary as well as in the choroid plexus, subfornical organ, and the dentate gyrus (5). Imposition of stress also activates the biosynthesis and secretion of IL-1ß in the hypothalamic region of the rat brain (6). Additionally, IL-1 is expressed in human microglia following trauma (7).

IL-6 is a glycosylated, monomeric protein that has been implicated in inflammatory disease and is involved in activation of the HPA axis, neural cell differentiation, and B-cell differentiation (reviewed in Refs. 8, 9). As a cytokine, IL-6 is secreted by monocytes, macrophages, fibroblasts, and endothelial cells. IL-1{alpha} and IL-1ß stimulate the release of IL-6 from primary cultures of rat anterior pituitary cells in vitro (10) and from human pituitary adenomas (11). The probable cellular source of IL-6 in nontumoral anterior pituitary tissue is the folliculostellate element, a S-100 protein-containing and glial-like cell thought to represent a form of a resident macrophage. Similarly, the IL-1ß- and endotoxin-induction of neurointermediate pituitary lobe IL-6 is undoubtedly due to the presence of the pituicyte, a resident astroglial cell (12). Thus, IL-1 and IL-6 are produced in central and neuroendocrine structures for the possible paracrine regulation of inflammation and hormone secretion.

Because the increased production of cytokines in the CNS is associated with neurodegenerative disorders, we have investigated the regulation of glial cell-derived IL-6 production by cytokines and neurotransmitters. We used the rat C6 glioma cell line, which has several characteristics in common with the anterior pituitary folliculostellate cell and the posterior lobe pituicyte. C6 cells contain the S-100 protein as well as the glial fibrillary acidic protein (GFAP) (13). IL-1ß stimulated IL-6 release from C6 cells, and in combination with compounds that increase intracellular concentrations of cAMP, evoked a pronounced synergistic induction of IL-6 release. Norepinephrine also generated the synergistic stimulation of IL-6 release in the presence of IL-1ß, an effect mediated by the ß-adrenergic receptor. These effects were mimicked by lysophosphatidylcholine (LPC), a postulated second messenger of IL-1ß in C6 cells. Thus, elevations in CNS cytokines and catecholamines may contribute to the abnormal regulation of IL-6 production characteristic of certain neurodegenerative disorders.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
(Bu)2cAMP, gentamycin sulfate, isoproterenol, lysophosphatidylcholine stearoyl (LPC 18:0), ß-mercaptoethanol, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), penicillin-G, phosphatidylcholine distearoyl (PC), propranolol, and streptomycin sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). DMEM, Ham’s F-10, RPMI-1640, FCS, fungizone, horse serum, and trypsin were purchased from Gibco BRL (Grand Island, NY). Cholera toxin, forskolin, 1,9-dideoxyforskolin, dopamine, epinephrine, norepinephrine, and TNF{alpha} were purchased from Research Biochemicals International (Natick, MA). Recombinant murine IL-6 (rmIL-6) was obtained from R & D Systems (Minneapolis, MN). The 7TD1 cell line was generously provided by Dr. J. Van Snick, Ludwig Institute (Brussels, Belgium). The C6 glioma cell line was obtained from the American Type Culture Collection (Rockville, MD). Recombinant human IL-1ß was obtained from the Biological Response Modifiers Program, National Cancer Institute. IL-1ß was dissolved directly into DMEM at a concentration of 100 µg/ml.

C6 glioma cell culture
For the IL-6 release studies, C6 glioma cells were maintained in continuous culture in a humidified atmosphere of 5% CO2-95% air at 37 C in DMEM supplemented with 10% FCS and antibiotics (7.5 µg/ml steptomycin, 15 µg/ml gentamycin, 19 µg/ml penicillin, 0.6 µg/ml fungizone). After 2–3 days in culture, cells were removed from the tissue culture flasks with 0.25% trypsin in PBS, resuspended in serum-free DMEM, counted by hemacytometer, and either placed in continuous culture (1.0 x 106 cells per flask) or dispersed into 96-well tissue culture plates (Greiner Labortechnik, Intermountain Scientific Corp., Kaysville, UT) at 100 x 103 cells/well with DMEM supplemented with 10% FCS and antibiotics. The cells in 96-well plates were allowed to adhere for 24 h, rinsed twice with serum-free DMEM (vehicle), and then incubated for 24 h with vehicle in the absence or presence of IL-1ß, catecholamines, LPC 18:0, or other agents (250 µl per well). In some experiments, C6 cells were pretreated with propranolol for 0.5 to 1 h in serum-free DMEM medium and subsequently exposed to the compounds of interest in the absence or presence of IL-1ß. Conditioned media were removed and stored at 4 C pending analysis for IL-6.

IL-6 bioassay
The accumulation of IL-6 in the C6 glioma cell conditioned media was quantified using the IL-6-dependent 7TD1 hybridoma bioassay as previously described (14), with minor modifications. This particular IL-6-sensitive hybridoma has not responded to a variety of cytokines or hormones including IL-1, interferon-{alpha} and -ß, TNF-{alpha}, PRL, or GH (15). The 7TD1 cells were maintained in continuous culture in a humidified atmosphere of 5% CO2-95% air at 37 C in 10 ml RPMI-1640 supplemented with 5% FCS (Fetal Clone I; Hyclone Laboratories, Inc., Logan, UT), 50 µM ß-mercaptoethanol, 5 pg/ml rmIL-6, and antibiotics. Approximately 400 x 103 7TD1 cells were distributed in a 25 cm2 flask (Corning, Corning, NY) for continuous culture. Conditioned medium (0.6–10 µl) was cultured in 96-well tissue culture plates in duplicate for 72 h in the presence of 4,000 7TD1 cells/well in 200 µl of RPMI 1640 supplemented with 5% FCS, 50 µM ß-mercaptoethanol, and antibiotics. The extent of 7TD1 proliferation was determined using the tetrazolium salt MTT, which is cleaved in active mitochondria to form a dark blue formazan product. After a 72 h incubation period, 20 µl of 5 mg/ml MTT was added to each well for 4 h. Following removal of 150 µl medium from each well, the dark blue crystals were dissolved by the addition of 150 µl of 0.04 M HCl/isopropanol. After an overnight incubation in the dark, optical densities were obtained in a microelisa instrument using a test wavelength of 570 nm and a reference wavelength of 630 nm (Dynatech Corp. MR5000). A standard curve of rmIL-6 (0.125–16.0 pg/well) was generated in each assay. Maximally effective concentrations of the test agents had no effect on the proliferation of the 7TD1 cells in the absence or presence of rmIL-6.

High performance thin layer chromatography (HPTLC) phospholipid analysis
The C6 cells were removed from the tissue culture flasks and cultured in 12-well plates (800 x 103 cells/2 ml/well) in serum-free Ham’s F-10 for 24 h in the presence of [3H]choline (2–5 µCi/well; New England Nuclear, Boston, MA). The radiolabeling medium was removed and the cells rinsed twice in serum-free Ham’s F-10. Cells were exposed to vehicle (Ham’s F-10) or 50 ng/ml IL-1ß for 30 min (n = 4). The incubations were terminated by withdrawing the medium and adding 400 µl of 5 mM Na3VO4 for 1 min while placing the culture plates on ice. The Na3VO4 was subsequently removed from the wells and the tissue culture plates were stored at -70 C.

In preparation for the measurement of [3H]LPC and the parent [3H]PC compound by HPTLC, the sample plates were allowed to warm to room temperature and the C6 cells were extracted into 300 µl of 5 mM EGTA/15 mM KCl (pH 7.4). The wells were rinsed with 200 µl of this solvent to raise the total extraction volume to 500 µl. Chloroform-methanol (2 ml of a 1:1 solution) was added to each sample in glass tubes, which were vortexed, and subsequently centrifuged (500 x g) for maximal phase separation. The bottom layer of each sample was retained and evaporated under a stream of N2 gas (N-EVAP Analytical Evaporator, Organomation Associates, Inc., Berlin, MA). The sample residue was dissolved in chloroform and subjected to HPTLC.

Precoated HPTLC Silica Gel 60 plates with a concentrating zone for nano-TLC (10 x 20 cm; Merck, Darmstadt, Germany) were scored into multiple lanes and activated at 400 F for 2 h. Samples (supplemented with 20 µg of PC and LPC) were spotted 2–3 mm from the top of the concentration zones and the plates were developed in tanks preequilibrated with HPLC-grade chloroform:methanol:acetic acid:water (25:15:4:1.5 vol/vol). After 20–30 min, the plates were air dried and exposed to iodine vapor for the visualization of individual lipid bands. The bands were removed, dissolved in scintillation cocktail, and the radioactivity in each sample was determined with a liquid scintillation counter (Packard 1600CA, Packard Instruments, Laguna Hills, CA). Using this HPTLC technique in our laboratory the standard Rf values for PC and LPC were 0.266 ± 0.014 and 0.132 ± 0.011, respectively (mean ± SEM, n = 4).

Statistical analysis
ANOVA and the Bonferroni analysis for multiple comparisons were used for statistical evaluation of the data. A P value of <= 0.05 was considered significant. Unless noted otherwise, data are expressed as the mean ± SEM of groups consisting of three to four observations and each experiment was performed independently at least two to three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-1ß stimulates IL-6 release from rat C6 glioma cells in vitro: effect of increasing intracellular cAMP concentrations
IL-1ß increased the release of IL-6 up to 40-fold from 100 x 103 C6 cells/well in a concentration and time-dependent manner (Fig. 1Go). The 40 ng/ml concentration of IL-1ß generated the maximal elevation in IL-6 release which began to plateau after 24 h of incubation. Other concentrations of IL-1ß were less efficacious compared with 40 ng/ml.



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  		Figure 1. Interleukin-1ß stimulates IL-6 release from C6 glioma cells in vitro. Cultured C6 cells (100 x 103 cells/well) were exposed to either vehicle (DMEM containing antibiotics) or different concentrations of IL-1ß for 4–48 h. IL-1ß significantly stimulated IL-6 release at 12 h (vehicle vs. 10 to 100 ng/ml, P < 0.01), 24 h (vehicle vs. 10 to 100 ng/ml, P < 0.01), 36 h (vehicle vs. 1.0 to 100 ng/ml, P < 0.01), and 48 h (vehicle vs. 1.0, P < 0.05; 10 to 100 ng/ml, P < 0.01). The data are expressed as the mean ± SEM of groups consisting of three to four observations.

 
IL-1ß does not increase intracellular concentrations of cAMP in rat anterior pituitary cells, indicating a noncAMP dependent signaling cascade (10). However, the intracellular elevation of cAMP concentrations in rat anterior pituitary and neurointermediate pituitary lobe cells following (Bu)2cAMP treatment results in the synthesis of IL-6 (12, 16). Therefore, we subjected C6 glioma cells to 0.01–10 mM (Bu)2cAMP in the absence and presence of 50 ng/ml IL-1ß for 24 h (Fig. 2Go). IL-1ß stimulated an 8-fold increase in IL-6 release; 1 mM (Bu)2cAMP also significantly increased IL-6 release (P < 0.01). The combination of these two agents resulted in a striking synergistic response that was highly significant (e.g. a 30-fold increase in the presence of 1 mM (Bu)2cAMP and IL-1ß compared with IL-1ß alone). This response was much greater than could be attributed to any additive effect of the two agents acting independently. These data provide evidence that IL-1ß stimulates IL-6 release from rat C6 glioma cells via a cAMP-independent mechanism.



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  		Figure 2. Interleukin-1ß stimulates IL-6 release from C6 glioma cells in vitro: synergistic induction of IL-6 release in the presence of Bu2cAMP. Cultured C6 cells (100 x 103 cells/well) were exposed to either vehicle (DMEM containing antibiotics), IL-1ß, Bu2cAMP, or IL-1ß + Bu2cAMP for 24 h. IL-1ß (50 ng/ml) and Bu2cAMP (1 mM) significantly stimulated basal IL-6 release (P < 0.01). The combination of IL-1ß with 0.1 to 10.0 mM Bu2cAMP resulted in the synergistic induction of IL-6 release (IL-1ß vs. IL-1ß + 0.1 to 10.0 mM Bu2cAMP, P < 0.01). The data are expressed as the mean ± SEM of groups consisting of three to four observations.

 
Forskolin and cholera toxin also act to increase intracellular cAMP concentrations. Forskolin directly activates adenylate cyclase and 5 µM of this agent significantly stimulated IL-6 release from C6 cells (P < 0.01). When forskolin was used in combination with 50 ng/ml IL-1ß, a synergistic induction of IL-6 release was obvious (e.g. a 38-fold increase in the presence of 5 µM forskolin and IL-1ß compared with IL-1ß alone). In contrast, the inactive analog of forskolin, 1,9-dideoxyforskolin, had no significant effect on IL-6 release in the absence or presence of IL-1ß (Fig. 3Go). The G protein ribosylating agent cholera toxin (2.5–250 ng/ml) significantly stimulated IL-6 release (P < 0.01). Once again the combination of cholera toxin and 50 ng/ml IL-1ß resulted in an impressive synergistic induction of IL-6 release from C6 glioma cells (e.g. a 10-fold increase in the presence of 250 ng/ml cholera toxin and IL-1ß compared with IL-1ß alone; Fig. 4Go). Therefore, the elevation of intracellular cAMP in the presence of IL-1ß results in the robust synergistic stimulation of IL-6 release.



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  		Figure 3. Interleukin-1ß stimulates IL-6 release from C6 glioma cells in vitro: synergistic induction of IL-6 release in the presence of forskolin. Cultured C6 cells (100 x 103 cells/well) were exposed to either vehicle (DMEM containing antibiotics), IL-1ß, forskolin, dideoxyforskolin, or IL-1ß + forskolin/dideoxyforskolin for 24 h. IL-1ß (50 ng/ml) and forskolin (5 µM) significantly stimulated basal IL-6 release (P < 0.01). The combination of IL-1ß with 0.5 to 5.0 µM forskolin resulted in the synergistic induction of IL-6 release (IL-1ß vs. IL-1ß + 0.5 to 5.0 µM forskolin, P < 0.01). The inactive analog of forskolin, 1,9-dideoxyforskolin, did not affect either the basal nor the IL-1ß induction of IL-6 release. The data are expressed as the mean ± SEM of groups consisting of three to four observations.

 


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  		Figure 4. Interleukin-1ß stimulates IL-6 release from C6 glioma cells in vitro: synergistic induction of IL-6 release in the presence of cholera toxin. Cultured C6 cells (100 x 103 cells/well) were exposed to either vehicle (DMEM containing antibiotics), IL-1ß, cholera toxin, or IL-1ß + cholera toxin for 24 h. IL-1ß (50 ng/ml) and cholera toxin (2.5 to 250 ng/ml) significantly stimulated basal IL-6 release (P < 0.01). The combination of IL-1ß with 2.5 to 250 ng/ml cholera toxin resulted in the synergistic induction of IL-6 release (IL-1ß vs. IL-1ß + 2.5 to 250 ng/ml cholera toxin, P < 0.01). The data are expressed as the mean ± SEM of groups consisting of three to four observations.

 
IL-1ß stimulates IL-6 release from rat C6 glioma cells in vitro: effect of catecholamines and isoproteronol
We next investigated the possible synergistic responses of IL-1ß and the catecholamines for IL-6 release from C6 glioma cells. Each catecholamine induced a modest, concentration-responsive basal stimulation in IL-6 release; however, in this and subsequent experiments the level of increase was less than that caused by IL-1ß (Fig. 5Go). In the presence of 50 ng/ml IL-1ß, 100 µM of each catecholamine generated a significant synergistic stimulation of IL-6 release. Thus, the coincubation of 10 and 100 µM norepinephrine with IL-1ß produced 6- and 9-fold increases in IL-6 release compared with IL-1ß alone, respectively. Similarly, 10 and 100 µM epinephrine and 100 µM dopamine in the presence of IL-1ß caused significant synergistic responses in IL-6 release.



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  		Figure 5. Interleukin-1ß stimulates IL-6 release from C6 glioma cells in vitro: synergistic induction of IL-6 release in the presence of catecholamines. Cultured C6 cells (100 x 103 cells/well) were exposed to either vehicle (DMEM containing antibiotics), IL-1ß, dopamine, epinephrine, norepinephrine, or IL-1ß + each of the catecholamines for 24 h. IL-1ß (vehicle vs. 50 ng/ml, P < 0.01), dopamine (vehicle vs. 100 µM, P < 0.05), epinephrine (vehicle vs. 10 µM, P < 0.05; 100 µM, P < 0.01), and norepinephrine (vehicle vs. 100 µM, P < 0.05) each significantly stimulated IL-6 release. The combination of IL-1ß with 1.0 to 100 µM of each of the catecholamines resulted in the synergistic induction of IL-6 release (IL-1ß vs. IL-1ß + 100 µM dopamine, P < 0.01; IL-1ß vs. IL-1ß + 10 and 100 µM epinephrine, P < 0.01; IL-1ß vs. IL-1ß + 10 and 100 µM norepinephrine, P < 0.01). The data are expressed as the mean ± SEM of groups consisting of three to four observations.

 
Norepinephrine enhances intracellular cAMP levels via the ß-adrenergic receptor. To determine the effects of stimulating or inhibiting ß-adrenergic receptors on the norepinephrine-IL-1ß synergistic response, the antagonist propranolol and the agonist isoproterenol were introduced into C6 glioma cell cultures for a 24 h incubation (Fig. 6Go). IL-1ß induced a 15-fold increase in C6 IL-6 release. Compared with IL-1ß alone, isoproterenol (10 µM) and IL-1ß produced a dramatic 25-fold synergistic stimulation of IL-6 release. This synergistic response was completely blocked by the preincubation of 10 µM propranolol, indicating that the ß-adrenergic receptor was essential for the isoproterenol/IL-1ß synergistic induction of IL-6 release (Fig. 6Go). Similarly, norepinephrine (10 µM) and IL-1ß produced a synergistic response that was, however, smaller than the isoproterenol/IL-1ß response. The ß-adrenergic receptor antagonist propranolol also blocked this synergistic induction.



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  		Figure 6. Interleukin-1ß stimulates IL-6 release from C6 glioma cells in vitro: synergistic induction of IL-6 release in the presence of isoproteronol. Cultured C6 cells (100 x 103 cells/well) were exposed to either vehicle (DMEM containing antibiotics), IL-1ß, propranolol (PRO), isoproteronol (IPT), norepinephrine (NE), or IL-1ß + each of these agents for 24 h. IL-1ß (50 ng/ml) significantly stimulated IL-6 release (P < 0.01). The combination of IL-1ß with 10 µM isoproteronol resulted in the synergistic induction of IL-6 release (IL-1ß vs. IL-1ß + 10.0 µM isoproteronol, P < 0.01). Additionally, 10 µM propranolol significantly blocked this synergistic response (isoproteronol + IL-1ß vs. propranolol + isoproteronol + IL-1ß, P < 0.01). The combination of IL-1ß with norepinephrine was similarly inhibited by propranolol. The data are expressed as the mean ± SEM of groups consisting of three to four observations.

 
LPC stimulates IL-6 release from rat C6 glioma cells in vitro: effect of norepinephrine
IL-1ß stimulates phospholipase A2 activity and arachidonic acid mobilization in rat anterior pituitary cells in vitro (17). The putative by-product of arachidonic acid formation, LPC 18:0, increased IL-6 release from rat anterior pituitary cells in a concentration-related manner (18). In the present study, 20 and 40 µM LPC 18:0 caused a significant increase in C6 glioma cell IL-6 release (Fig. 7Go). The coincubation of 10 µM norepinephrine with either 20 or 40 µM LPC 18:0 produced a synergistic response in IL-6 release compared with LPC 18:0 alone (P < 0.01). In addition, the stimulation of IL-6 release due to the norepinephrine/LPC 18:0 coincubation was comparable in magnitude to that obtained by the norepinephrine/IL-1ß treatment group (Fig. 7Go). Therefore, similar to IL-1ß, LPC 18:0 in the presence of catecholamine induced a synergistic stimulation of IL-6 release from C6 glioma cells.



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  		Figure 7. Interleukin-1ß stimulates IL-6 release from C6 glioma cells in vitro: synergistic induction of IL-6 release in the presence of LPC. Cultured C6 cells (100 x 103 cells/well) were exposed to either vehicle (DMEM containing antibiotics), IL-1ß, norepinephrine, LPC, IL-1ß + norepinephrine, or LPC + norepinephrine for 24 h. IL-1ß (50 ng/ml), and LPC (20 and 40 µM) significantly stimulated basal IL-6 release (P < 0.01). The combination of IL-1ß with 10 µM norepinephrine resulted in the synergistic induction of IL-6 release (IL-1ß vs. IL-1ß + 10.0 µM norepinephrine, P < 0.01). The combination of LPC with 10 µM norepinephrine also resulted in the synergistic induction of IL-6 release (20 µM LPC vs. 20 µM LPC + 10.0 µM norepinephrine, P < 0.01; 40 µM LPC vs. 40 µM LPC + 10.0 µM norepinephrine, P < 0.01). The data are expressed as the mean ± SE of groups consisting of three to four observations.

 
IL-1ß-induction of LPC synthesis
Because IL-1ß and LPC 18:0 were both synergistic with respect to IL-6 release in the presence of catecholamines, we hypothesized that IL-1ß would increase the accumulation of cell associated LPC 18:0. Following a 30 min incubation, IL-1ß (50 ng/ml) induced a modest increase in LPC 18:0 production in C6 glioma cells prelabeled for 24 h with [3H]choline (P < 0.05; control: 1268 ± 157 vs. IL-1ß: 1835 ± 175). In addition, IL-1ß exposures resulted in a significant decrease in PC content (P < 0.05; control: 20340 ± 1660 vs. IL-1ß: 13922 ± 1427). Thus, IL-1ß stimulates the accumulation of LPC 18:0 as well as the decrease in the parent species of this lysophospholipid.

IL-1ß stimulates IL-6 release from rat C6 glioma cells in vitro: effect of TNF{alpha}
To illustrate the influence of another inflammatory cytokine on IL-6 release, C6 glioma cells were incubated with 1–100 ng/ml TNF{alpha} for 24 h (Fig. 8Go). Treatment with TNF{alpha} resulted in a concentration-dependent increase in IL-6 release that was smaller in magnitude compared with IL-1ß and significant only at 100 ng/ml TNF{alpha} (P < 0.05). However, when TNF{alpha} was coincubated with 50 ng/ml of IL-1ß, a significant synergistic response was observed at all three concentrations of TNF{alpha} (P < 0.01).



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  		Figure 8. Interleukin-1ß stimulates IL-6 release from C6 glioma cells in vitro: synergistic induction of IL-6 release in the presence of TNF{alpha}. Cultured C6 cells (100 x 103 cells/well) were exposed to either vehicle (DMEM containing antibiotics), IL-1ß, TNF{alpha}, or IL-1ß + TNF{alpha} for 24 h. IL-1ß (vehicle vs. 50 ng/ml, P < 0.01) and TNF{alpha} (vehicle vs. 100 ng/ml, P < 0.05) significantly stimulated IL-6 release (P < 0.01). The combination of IL-1ß with 1.0 to 100 ng/ml TNF{alpha} resulted in the synergistic induction of IL-6 release (IL-1ß vs. IL-1ß + 1.0 to 100 ng/ml TNF{alpha}, P < 0.01). The data are expressed as the mean ± SEM of groups consisting of three to four observations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that IL-6 release from C6 glioma cells is synergistically induced by IL-1ß in the presence of agents that increase intracellular levels of cAMP. Increasing IL-1ß concentrations to approximately 50 ng/ml maximally stimulated IL-6 release, whereas higher concentrations of IL-1ß (e.g. 100 ng/ml) were markedly less effective. Because this effect was noticeable after a 24 h incubation, this may represent a desensitization of the IL-1ß receptor by the cognate ligand. We stimulated various components of the cAMP generating pathway to confirm whether C6 glioma IL-6 production is also induced by this second messenger. The coadministration of IL-1ß and (Bu)2cAMP generated a remarkably synergistic stimulation of glioma cytokine release (i.e. the combination of these two agents resulted in a release of IL-6 that was much larger than the release attributed to either agent alone). Similarly, activation of adenylate cyclase either indirectly by cholera toxin or directly with forskolin increased basal IL-6 concentrations and resulted in the synergistic induction of IL-6 release when each was combined with IL-1ß. Therefore, increasing intracellular cAMP concentrations by a variety of mechanisms provides for the synergistic induction of IL-6 release in the presence of IL-1ß. These results suggest that IL-1ß stimulates IL-6 release in a noncAMP dependent manner.

The ß-adrenergic receptor class contains three subtypes, each of which are G protein-coupled. The ß1-, ß2-, and ß3-adrenergic receptors activate adenylate cyclase through Gs. Because C6 glioma cells express the ß1- and ß2-adrenergic receptors (19), we investigated the catecholamine induction of C6 cell IL-6 release. Both norepinephrine and epinephrine induced modest stimulations of IL-6 release. However, in the presence of IL-1ß and either norepinephrine or epinephrine, IL-6 concentrations were synergistically elevated. Although epinephrine has an approximately 10-fold higher affinity than norepinephrine for the ß2 receptor (20), both of these catecholamines had similar potencies for the synergistic stimulation of IL-6 release. Because the C6 glioma cells express the ß1 adrenergic receptor predominately (ß1 to ß2 ratio is 7:3, 19), we suggest that the ß1 receptor is central to the catecholamine/IL-1ß synergistic induction of IL-6 release. Dopamine receptors are either D1-like (D1 and D5 receptors) or D2-like (D2, D3, and D4). The predominant effecter pathway activated by the D1-like receptors is the stimulation of adenylate cyclase. Thus, similar to norepinephrine and epinephrine, dopamine in the presence of IL-1ß induced a synergistic stimulation of IL-6 release. Because D2-like receptors inhibit adenylate cyclase, dopamine probably increases C6 intracellular cAMP concentrations via the D1 receptor class for the synergistic production of IL-6.

Isoproteronol is an agonist for ß-adrenergic receptors while propranolol acts as an antagonist. Although isoproteronol had little effect on basal IL-6 release, in the presence of IL-1ß this ß-agonist induced a dramatic synergistic release of IL-6. Importantly, this synergistic induction was completely blocked (i.e. to IL-1ß stimulated levels) by the general ß-adrenergic receptor antagonist propranolol. Similarly, propranolol blocked the norepinephrine/IL-1ß synergistic stimulation of IL-6 release. These data confirm the importance of the ß-adrenergic receptor class to the isoproteronol and norepinephrine synergistic inductions of IL-6 release. Other investigators have described the IL-1ß and norepinephrine synergistic activation of IL-6 release in rat astrocytes (21, 22).

We previously reported that IL-1ß enhanced IL-6 release and phospholipase A2 (PLA2)-mediated hydrolysis of PC in rat anterior pituitary cells in vitro (17). In addition, we found no effect of IL-1ß on PLC or PLD activities specific for PC. We subsequently demonstrated that LPC 18:0 stimulated IL-6 release from rat anterior pituitary cells (18). Thus, we hypothesize that LPC may function as a second messenger for IL-1ß. In the present study, this lysophospholipid species also stimulated IL-6 release from C6 glioma cells in a concentration-dependent manner. Importantly, LPC 18:0 in the presence of norepinephrine increased IL-6 release to concentrations similar to those obtained by IL-1ß and norepinephrine. Because IL-1ß and LPC 18:0 possessed similar capacities to stimulate the synergistic release of IL-6 in the presence of norepinephrine, we investigated the possible production of LPC 18:0 in C6 glioma cells. LPC 18:0 levels significantly increased by 45% while the amount of the PC parent species was decreased. Because the decrease in the absolute number of cpm in the PC lipid class was not matched by a similar increase in cpm in the LPC 18:0 species, we suggest that LPC is rapidly degraded resulting in the relatively modest measured increase in this lysophospholipid. For example, in rat anterior pituitary cells, IL-1ß induced the complete deacylation of PC to glycerophosphorylcholine (17).

The cytokine TNF{alpha} stimulated C6 IL-6 release in a concentration-dependent manner, and in combination with IL-1ß, produced a maximal 60-fold synergistic increase in IL-6 release. The TNF{alpha} signaling pathway may involve the stimulation of a sphingomyelinase activity, leading to the production of ceramide that activates a ceramide-activated protein kinase (23). Although we have not measured sphingomyelinase activity in IL-1ß and TNF{alpha}-treated cells, the synergistic stimulation of IL-6 release by these two cytokines indicates that IL-1ß may not activate the sphingomyelinase pathway in C6 glioma cells. Several other neuropeptides were evaluated for their effects on IL-6 release in C6 glioma cells (data not shown). Oxytocin, vasopressin, GHRF, PRL, CRF, and vasoactive intestinal polypeptide (VIP) had no significant effect on IL-6 release. Other neurotransmitters and growth factors reportedly affect the expression of glial-derived IL-6. Serotonin increased IL-6 mRNA accumulation in rat astrocytes (24). Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulated IL-6 release from microglial cells but not astrocytes (25). Substance P increased IL-6 release from the human astrocytoma cell line U373MG (26), and this neuropeptide potentiated IL-1ß-stimulated IL-6 release from human astrocytes (27). Calcitonin increased IL-6 release from and cAMP accumulation in human A172 glioblastoma cells (28). Adenosine but not glutamate increased IL-6 release from murine astrocytes (29). In contrast, somatostatin inhibited basal IL-6 release from rat cortical type I astrocytes (30). Thus, glial-derived IL-6 expression is potentially subject to complex regulatory pathways mediated by multiple neurotransmitters and cytokines.

The exact pathway of IL-1ß signal transduction in glial cells is unknown. The participation of inositol 1,4,5-trisphosphate (31), cAMP (32), non-cAMP (33), protein kinase C (PKC; 34) and tyrosine kinase (35, 36) pathways in IL-1ß signal transduction have been reported in astrocytes and glial cells. In general, IL-1ß activates PKC isoforms, the MAP kinases or tyrosine kinases for IL-6 production (reviewed in Ref. 37). In C6 glioma cells, IL-1{alpha} and IL-1ß activated the NF-{kappa}B transcription factor (38). Sparacio et al. (39) reported that the IL-1ß and TNF{alpha} stimulations of IL-6 promoter activity in astrocytes required an NF-{kappa}B binding site. We hypothesize that the synergistic stimulation of C6 glioma IL-6 release may result from the interaction of cAMP and LPC (Fig. 9Go); the exact nature of this interaction is unknown. Occupancy of the IL-1ß type I receptor leads to the activation of PLA2 and consequently to the rapid formation of arachidonic acid and LPC (17). The transient formation of LPC may stimulate membrane-associated PKC isoforms, the MAP kinases or tyrosine kinases to phosphorylate trans-acting enhancers (e.g., NF-{kappa}B, NFIL-6). The LPC stimulation of IL-6 release from rat anterior pituitary cells was blocked by the PKC inhibitors H7 and chelerythrine chloride (18).



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  		Figure 9. Possible synergistic signaling pathways resulting in IL-6 release from C6 glioma cells. AA, Arachidonic acid; ß-AdR, ß-adrenergic receptor; IL-1ß, interleukin-1ß; IPT, isoproteronol; LPC, lysophosphatidylcholine; MAPK, mitogen-activated protein kinases; NE, norepinephrine; NF-IL-6; nuclear factor IL-6; NF-{kappa}B, nuclear factor {kappa}B; PC, phosphatidylcholine; PKC, protein kinase C; PLA2, phospholipase A2; PRO, propranolol.

 
The potential synergistic elevation of cytokine levels may affect disease processes in the CNS. Alzheimer’s disease progression is correlated with the appearance of neuritic plaques and neurofibrillary tangles that are diagnostic for this neurodegenerative disorder. The neuritic plaques contain dystrophic neurites as well as extracellular deposits of ß-amyloid protein. Also intrinsic to the neuritic plaques are activated glial cells that overexpress cytokines. Microglial cells have been localized to Alzheimer’s disease plaques, whereas astrocytes encircle and extend processes into the cores of these structures (1). Production of IL-1 and IL-6 in senile plaques may contribute to the pathogenesis of this disease (reviewed in 1, 40). The enhanced accumulation of IL-1 and IL-6 may increase levels of ß-amyloid precursor protein in neuronal cells leading to eventual ß-amyloid deposition and neuronal cell death (2). An elevation in central IL-1ß levels may increase catecholamime concentrations (41), which could allow for the synergistic induction of IL-6 release. IL-6 expression has been localized largely to the diffuse plaque population of Alzheimer’s disease patients, indicating that this cytokine is present in plaques before the onset of neuritic degeneration (42). The synergistic induction of IL-6 release by IL-1ß and the catecholamines begins to elucidate a potential mechanism for IL-6 overproduction in diffuse plaques before neuritic plaque formation.


   Acknowledgments
 
We thank Dr. J. Van Snick (Ludwig Institute, Brussels, Belgium) for supplying the 7TD1 cell line. This work was supported by a grant (to B.L.S.) from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-42059).

Received June 16, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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