Journal of Neuroimmunology, 36 (1992) 179-191

179

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02116

Interleukin-1/3 induction of tumor necrosis factor-alpha gene expression in human astroglioma cells John R. Bethea b, Il Yup Chung b Shaun M. Sparacio a, G. Yancey Gillespie c and Etty N. Benveniste a,b Departments of a Neurology and t, Cell Biology, and ' Division of Neurosurgery, Unicersity of Alabama at Birmingham, Birmingham, AL 35294, USA

(Received 18 July 1991) (Accepted 10 September 1991)

Key words: Cytokine; Astrocyte; Central nervous system

Summary Cells that produce tumor necrosis factor-a (TNF-oD require the presence of signaling molecules since this cytokine is not normally expressed in a constitutive manner. It has been demonstrated that glial cells can produce TNF-~; however, the specific inducing molecules and their mechanism(s) of action have not been clearly defined. In this study, we examined the effect of human recombinant interleukin-1/3 (IL-1/3) on the expression of TNF-a by CH235-MG human malignant glioma cells. CH235-MG cells do not constitutively express TNF-a mRNA or protein; however, upon stimulation with IL-1/3, these cells synthesize and secrete biologically active TNF-a. IL-1/3 induces the expression of a 1.9 kb TNF-a mRNA species. Kinetic analysis demonstrated optimum TNF-a mRNA expression after a 4 h exposure to IL-113, and peak TNF-a protein production at 18 h. Cycloheximide (CHX), an inhibitor of protein synthesis, markedly increased expression of TNF-a mRNA in IL-1/3 stimulated CH235-MG cells, indicating that de novo protein synthesis is not required for astroglioma TNF-a gene expression. Nuclear run-off analysis demonstrates that IL-1/3 causes transcriptional activation of the TNF-a gene, and CHX enhances IL-1/3-induced TNF-a transcription. Studies of TNF-a mRNA stability using actinomycin D show that IL-1/3-induced TNF-a mRNA has a half-life of approximately 30 rain, and CHX increases the half-life of IL-1/3-induced TNF-a mRNA to approximately 210 min. These results indicate that IL-1/3, a cytokine present in the central nervous system during some pathological disease states, is a potent inducer of TNF-a in human malignant glioma cells.

Correspondence to: Dr. Etty N. Benveniste, Department of Cell Biology, University of Alabama at Birmingham, UAB Station, Birmingham, AL 35294, USA. Tel. (205) 934-7667/7668; Fax (205) 934-0950. Abbreviations: ACT-D, Actinomycin-D; CHX, cycloheximide; CNS, central nervous system; EAE, experimental allergic encephalomyelitis; G-CSF, granulocyte-colony stimulating factor; GFAP, glial fibrillary acidic protein; GM-CSF, granulocyte-macrophage colony stimulating factor; IFN-y, interferon-y; IL-1/3, interleukin-1/3; IL-6, interleukin-6; LPS, lipopolysaccharide; MHC, major histocompatibility complex; NU, neutralizing units; TNF-a, tumor necrosis factor-a.

18(]

Introduction

Astrocytes, the most abundant glial cell in the brain, perform a central role in regulating the homeostatic environment in the central nervous system (CNS). One of the more recently ascribed functions of the astrocyte relevant to this investigation has been that of a resident immunocompetent cell of the CNS (for review see Fontana et al., 1987; Benveniste, 1991). Astrocytes express class II major histocompatibility complex (MHC) antigens following exposure to interferon-y (IFNy) or virus (Fierz et al., 1985; Massa et al., 1986), and present antigens in an MHC-restricted fashion to antigen-specific T-cells, resulting in T-cell activation (Fontana et al., 1984; Takiguchi and Frelinger, 1986). Another manner in which the astrocyte can function as an immunocompetent cell is through the ability to synthesize and respond to various immunoregulatory cytokines (Fontana et al., 1982; Robbins et al., 1987; Frei et al., 1989; Lieberman et al., 1989; Benveniste et al., 1990; Chung and Benveniste, 1990; Malipiero et al., 1990). Human glioma cell lines possess some of the same immunoregulatory properties as do astrocytes. Human glioma cells can express H L A - D R antigens on their surface following stimulation with IFN-y (Carrell et al., 1982), and can initiate allogeneic responses in lymphocytes following H L A - D R expression (Takiguchi et al., 1985). Additionally, glioma cells can secrete various cytokines including interleukin-1 (IL-1), tumor necrosis factor- a (TNF- a), interleukin 6 (IL6), and colony stimulating factors (Fontana et al., 1984b; Yasukawa et al., 1987; Bethea et al., 1990; Tweardy et al., 1990). TNF-o~ is a pleiotropic cytokine with a wide variety of biological functions on a broad range of cells (for review see Beutler and Cerami, 1989). T N F - a is recognized to be an important mediator of inflammatory and immunological responses in a number of tissues, including the brain, due to its interaction with astrocytes and oligodendrocytes. T N F - a - m e d i a t e d responses include (1) enhancement of class I MHC antigen expression on astrocytes and oligodendrocytes (Lavi et al., 1988;

Mauerhoff et al., 1988), (2) induction of ICAM-I on human fetal astrocytes (Frohman et al., 1989), (3) enhancement of class II MHC expression on astrocytes through its synergistic interaction with IFN-y or virus (Massa et al., 1987; Benveniste et al., 1989), (4) proliferation of adult astrocytes (Selmaj et al., 1990), (5) induction of IL-6 production by astrocytes (Frei et al., 1989; Benveniste et al., 1990), (6) lysis of oligodendrocytes, the myelin producing cell in the CNS (Robbins et al., 1987), and (7) myelin damage (Selmaj and Raine, 1988). Due to the wide ranging effects of TNF-c~ on cells of the CNS, this cytokine may be an important mediator of intracerebral inflammation and immune responses. Additionally, T N F - a may act as an autocrine regulator of astrocyte function as these cells both produce TNF-o~ (Robbins et al., 1987; Lieberman et al., 1989; Bethea et al., 1990; Chung and Benveniste, 1990) and express high affinity TNF-c~ receptors (Benveniste et al., 1989; Bethea et al., 1990). A large number of stimuli have been reported to induce TNF-o~ expression in various cell types, of which the m o n o c y t e / m a c r o p h a g e has been most intensively studied (for review see Vilcek and Lee, 1991). Lipopolysaccharide (LPS) appears to be the most active stimulus, and the one used by most investigators. Cytokines such as IL-I, T N F - a and IFN-y have been shown to induce T N F - a in human monocytes (Philip and Epstein, 1986), although little is known regarding the cellular and molecular mechanisms by which cytokines induce and regulate T N F - a gene expression, especially in cell types other than the monocyte. The aim of this study was to examine TNF-c~ gene expression in human astroglioma cells in response to biological stimuli, particularly cytokines known to be present in the CNS during disease states. We report that the cytokine IL-1/3 induces T N F - a m R N A and protein expression in CH235-MG glioma cells in a time- and dose-dependent manner. Nuclear run-off assays demonstrate that IL-1/3 acts by inducing transcription of the T N F - a gene, and transcriptional activation is not dependent on de novo protein synthesis. IL-

181

1/3-induced T N F - a expression is also regulated at the posttranscriptional level, with effects on T N F - a m R N A stability.

Materials and methods

Human malignant glioma cell lines The CH235-MG tumor cell line was derived from a glioblastoma multiforme obtained from a white female in 1973. CH235-MG glioma ceils are grown in a 50 : 50 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 ( D M E M / F - 1 2 ) supplemented with 10 mM Hepes (pH 7.2), 2 mM L-glutamine and 10% fetal bovine serum (FBS, Hyclone). All media were determined to be free of endotoxin contamination ( < 30 p g / m l ) and the cell line was routinely screened by the Hoechst dye method (Chen, 1977) to ensure that it was free of mycoplasma contamination. Ceils in confluent monolayers were harvested using 1 x trypsin (1 : 2 5 0 ) / E D T A (0.02%) in phosphate buffered saline (PBS, pH 7.2); the trypsin was neutralized in 10% FBS and viable cells estimated on a h e m o c y t o m e t e r by trypan blue exclusion. Other human glioma cells used in this study include the U373-MG astrocytoma cell line (obtained from ATCC, Rockvillle, MD, USA), and the U251-MG cell line.

Reagents Human recombinant IL-1/3 (specific activity: 5.0 x 108 U / r a g ) was purchased from Amgen Biologicals (Thousands Oaks, CA, USA), and human recombinant T N F - a (specific activity: 5.6 x 10 7 U / m g ) was the generous gift of Genentech (South San Francisco, CA, USA). Polyclonal antibody to human r T N F - a ( > 1.5 x 104 N U / m g ) and human rIL-1/3 ( > 1.0 x 104 N U / m g ) was purchased from Endogen (Boston, MA, USA). Actinomycin-D mannitol (ACT-D), cycloheximide (CHX) and MTT (3-(4,5-dimethylthiazol-2-YL)2,5-diphenyl-tetrazolium bromide) were purchased from Sigma Chemical Company (St. Louis, MO, USA).

TNF-a protein production by human glioma cells Human glioma cells were resuspended in D M E M / F - 1 2 containing 10% FBS, and plated at

1 × 10 6 cells/well into 6-well (35 mm) plates (Costar, Cambridge, MA, USA). When the cells reached confluency, the original medium was aspirated off, and 2 ml of D M E M containing 10% FBS was added to the wells. Glioma cells were treated with IL-1/3 (0-2 n g / m l ) for various time periods (0-24 h). Supernates were collected, centrifuged to remove contaminating cells, and stored at - 70 ° C until use.

Measurement of TNF-a activity T N F - a activity in glioma-derived culture supernates was determined in a biologic assay using W E H I 164 clone 13 (WEHI 164.13) mouse fibrosarcoma cells as previously described (Bethea et al., 1990; Chung and Benveniste, 1990). T N F - a activity was expressed as pg T N F - a / m l culture supernate. The absolute concentration of T N F - a (pg/ml) was determined by extrapolation from the standard curve which was generated by using known amounts of recombinant human T N F - a . The lower level of sensitivity of our assay system for T N F - a ranged from 4 to 20 p g / m l . All samples were tested in triplicate and are presented as the mean + SD.

Neutralization of TNF-a activity 1-100 neutralizing units ( N U ) / m l of anti-human T N F - a polyclonal antibody or varying concentrations of normal rabbit serum were incubated with dilutions of glioma-derived culture supernates for 1 h at 37°C prior to adding to the W E H I 164 clone 13 cells. The extent of neutralization was expressed as: Neutralization(%) = 1_ [

[

T N F - a activity with antibody

wit-- -ou

1x

]

100

RNA isolation and analysis Total cellular R N A was isolated from confluent monolayers of CH235-MG cells that had been incubated for various intervals (0-8 h) with or without IL-1/3 (0-2 ng/ml). RNA isolation followed the procedure of Chomczynski and Sacchi

182 (1987) as previously described (Bethea et al., 1990). Briefly, R N A was extracted with guanidinium isothiocyanate and phenol, and precipitated with ethanol. Samples (20/xg) of total cellular R N A were denatured with formaldehyde for 15 min at 55°C, and R N A was size fractionated by electrophoresis through a 1.0% agarose gel containing ethidium bromide for visualization of 28S and 18S ribosomal RNA. The R N A was then transferred to nitrocellulose in 20 × standard saline citrate solution (SSC) (3 M NaCl and 0.3 M sodium citrate). Prehybridization was performed at 42°C in a solution containing 50% ( v / v ) formamide, 5 × SSC, 1 × Denhardt's solution, 50 /xg/ml of denatured salmon sperm DNA, and 0.1% sodium dodecyl sulfate (SDS) for 24 h. Hybridization was carried out at 42°C for 48 h in prehybridization solution containing 10% dextran sulfate and denatured 32P-labeled human T N F c D N A probe (2 × 10 ~' c p m / m l ) . The blots were washed at 42-55°C in 1 × SSC containing 0.1% SDS for 2 - 3 h, then 0.1 × SSC for 30 min, and exposed at - 7 0 ° C for variable times to Kodak X-Omat A R film plus intensifying screens. The filters were then stripped to remove bound T N F c~ probe, and rehybridized with a second control probe, cyclophilin, to ascertain thc amount of R N A in each lane that transferred to the filters. Quantification of autoradiographs was determined by scanning densitometry with the Bio-Rad model 620 video densitometer, using different exposures to insure linearity. Density values are normalized to the value for cyclophilin hybridization within each experiment.

cDNA probes" A c D N A probe specific for human T N F - a (Pennica et al., 1984) was the generous gift of Genentech (South San Francisco, CA, USA). The 820 base pair EcoRI insert was excised from the plasmid, purified, and labeled with [a-~2P]deoxy CTP using a Stratagene random priming kit according to the manufacturer's instructions. A specific activity of 1.0-2.0 × l0 s c p m / / x g D N A was routinely attained. A c D N A probe for rat cyclophilin (plB15) (Danielson et al., 1988) was the generous gift of Dr. Jim Douglass, The Oregon Health Sciences University.

Nuclear run-off analysis Nuclear run-off assays were performed as described by Woodward et al. (1989). Nuclei were isolated from confluent monolayers of CH235MG cells that were incubated for 4 h with medium alone, IL-113 (0.2 ng/ml), C H X (5 # g / m l ) , or IL-1/3 plus CHX. The cells (8 × 107) were collected, washed twice with cold PBS, and pelletcd. Nuclei were isolated by lysing the ceils in 0.01 M Tris (pH 8.4), 1.5 mM MgCI 2, 0.14 M NaCI and 0.3% Nonidet P-40, followed by centrifugation at 1000 × g . The nuclei were either used immediately or stored at 7 0 ° C in buffer containing 0.02 M Tris, pH 8.0, 20% glycerol, 0.14 M KCI, 0.01 M MgC12, I mM MnC12, and 14 mM fimercaptoethanol. To perform run-off transcriptional analysis, the nuclei were incubated (30 min, 30 ° C) in reaction buffer containing 0.033 M each of ATP, GTP, and CTP, 2.5 × storage buffer, and 0.5 mCi of [32p]UTP (3000 C i / m M , A m e r s h a m Corp., Arlington Heights, 1L, USA). After the reaction, nuclei were lysed with guanidine thiocyanate, and the D N A sheared using a 22-gauge needle. Samples were loaded onto a cushion of 5.7 M CsC1, 0.01 M EDTA, pH 7.5, and spun at 36,000 rpm for at least 16 h at 20°C. R N A pellets were recovered, hydrolyzcd with 1 M N a O H , neutralized with 1 M Hepes, and labeled transcripts were purified by ethanol precipitation. Denatured linear insert D N A and linear vector D N A were immobilized onto nitroccllulose paper using a Milliblot S system following the manufacturer's instructions (Millipore, Bedford, MA, USA). After UV crosslinking the D N A to nitrocellulose, prehybridization was performed at 65°C for 3 h in a solution of 10 mM Tris, pH 8.0, 10 mM EDTA, 300 mM NaCI, 1 m g / m l Ficoll, 1 m g / m l polyvinylpyrrolidone, 1 m g / m l BSA, and 100 U / m l RNasin (Promega Biotech, Madison, WI, USA). For hybridization, 2.0-3.0 × l 0 7 cpm of labeled RNA was used in 2 ml of hybridization solution and incubated at 65°C for 48-72 h. The filters were washed in 2 x SSC for 10 min at room temperature, 5(/ rain at 65°C, 30 min at 37°C in 2 × SSC plus 10 m g / m l ribonuclease A, and finally for 30 min at 37°C in 2 × SSC. The filters were exposed to Kodak X-Omat A R film plus intensifying screens at 70°C for variable times. Quantification of the autoradiographs was

183

by scanning densitometry, and values normalized to the value of cyclophilin hybridization within each experiment.

lmmunofluorescence staining CH235-MG cells (5 × 104) were plated on tissue culture chamber slides, and after 2 days of incubation in medium alone, the ceils were examined for phenotypic characterization as previously described (Benveniste et al., 1988). Cytoplasmic staining was performed on fixed cells (10 s in cold acetone) with rabbit antibody to glial fibrillary acidic protein (GFAP) (1:200), monoclonal antibody to S-100 (1 : 10), and monoclonal antibody to vimentin (1 : 10) for 40 min. Surface staining was performed on live cells with monoclonal antibodies to A2B5 (1:100), class II MHC (1:10), /32 microglobulin (1 : 100), and CD4 (1:10). Secondary antibody was either goat anti-mouse IgFITC (1:20) or goat anti-rabbit Ig-FITC (1:20) (Southern Biotechnology, Birmingham, AL, USA) for 30 min. The cells were then mounted in 30% glycerol, and visualized by fluorescence microscopy.

Results

Fig. 1. CH235-MG cells stained for vimentin. 5 × 104 cells were plated on tissue culture chamber slides, fixed (10 s in cold acetone) and stained with a monoclonal antibody to vimentin (1:10) for 40 min. Secondary antibody was goat anti-mouse Ig-FITC (1:20) for 30 min. The cells were visualized by fluorescence microscopy.

Effect of IL-1/3 on TNF-a protein production by CH235-MG cells We have recently demonstrated that primary rat astrocytes (Chung and Benveniste, 1990) and human glioma cells (Bethea et al., 1990) are capable of producing biologically active TNF-a protein in response to various stimuli. Since IL-1/3 is produced in the CNS by activated astrocytes (Fontana et al., 1982) and microglia (Giulian et al., 1986), we wished to examine the effect of this cytokine on TNF-a production by CH235-MG cells. CH235-MG cells were incubated with IL-1/3 (0-2 ng/ml) for varying times (0-24 h), at which

Phenotypic characterization of CH235-MG cells Immunofluorescence analysis was performed to determine the phenotype of the CH235-MG ceils. The cells were analyzed for a variety of glial-specific cell markers and activation antigens. Staining demonstrates the presence of vimentin in 100% of the cells (Fig. 1, Table 1). Vimentin is a marker of both embryonic and adult astrocytes (Schnitzer et al., 1981). The majority of the ceils were weakly positive for GFAP and $100, both astrocyte antigens (Moore, 1965; Bignami et al., 1972; Ludwin et al., 1976), and negative for A2B5, an antigen which identifies bipotential glial progenitor cells (Raft et al., 1983) (Table 1). CH235MG cells were positive for/32 microglobulin and negative for class II MHC and CD4. These resuits indicate that the CH235-MG cells are of astrocytic origin.

TABLE 1 PHENOTYPIC CELLS

CHARACTERIZATION

OF

Marker

CH235-MG cells "

Vimentin GFAP S-100 A2B5 fl2-microglobulin Class I! M H C CD4

+ + + b + +

CH235-MG

+ + +

a 5 X 104 cells were plated onto tissue culture chamber slides, incubated for 2 days in control medium, then processed for staining as described in Materials and methods. u Symbols: ( + + + ) 100%, bright staining; ( + + ) medium to bright staining; ( + ) weak to medium staining; (_+) weak staining; ( - ) no staining.

184

point the supernates were harvested and assayed for TNF-c~ protein. As shown in Table 2, CH235MG cells do not constitutively produce T N F - a , but can be induced to produce biologically active TNF-c~ in a dose-dependent manner. Optimal TNF-c~ production is seen using IL-1/3 concentrations between 0.2 and 2 n g / m l . T N F - a protein was detected when CH235-MG cells were stimulated with IL-1/3 (0.2 n g / m l ) either in the presence (10%) or absence of serum, indicating that a component(s) of serum is not responsible for lL-13-induced TNF-c~ production (data not shown). This recombinant source of IL-1/3 has previously been determined to be free of endotoxin c o n t a m i n a t i o n in experiments using polymyxin B sulfate (Chung and Benveniste, 1990). TNF-c~ protein was detected by 12 h, and was maximal after an 18 h stimulation with IL-1/3 (data not shown). The specificity of this IL-1/3-induced response was confirmed using a polyclonal antibody directed against human recombinant ILl/3. The inclusion of this antibody effectively inhibits the ability of IL-1/3 to induce TNF-c~ production (96.7% inhibition). The addition of antihuman TNF-c~ antisera to CH235-MG cell-derived supernates neutralized the cytotoxicity of CH235-MG-derived TNF-c~ for WEHI 164 cells (Table 3). Normal rabbit serum had no neutralizing effect in this system (data not shown). These

TABLE 2 EFFECT OF IL-I/3 ON T N F - a P R O T E I N P R O D U C T I O N BY CH235-MG CELLS Cell treatment Control b 1L-13 ( n g / m l ) c

T N F - a activity ;' (pgflml per 1 × 10 ~' cells)