Production of tumor necrosis factor and other cytokines by astrocytes ...

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6348-6352, August 1989 Medical Sciences

Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus (interleukin 1/ype I interferons/interleukin 6)

ANDREW P. LIEBERMAN*, PAULA M. PITHAtt, HYUN S. SHINt, AND MOON L. SHIN* *Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201; and tOncology Center and tDepartment of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

Communicated by Elvin A. Kabat, May 19, 1989

cytotoxin, as well as recombinant human TNF (rHuTNF), kills primary oligodendrocytes, the myelin-forming cells of the CNS (13). In this report, we have characterized the cytotoxin produced by primary astrocytes after stimulation with either LPS or a neurotropic paramyxovirus, Newcastle disease virus (NDV). We demonstrated by functional and Northern blot analyses that stimulated astrocytes produced TNF as well as numerous other cytokines including IL-1, IFN-a, IFN-,B, IL-6, and lymphotoxin. Interestingly, stimulation with endotoxin (LPS) and virus yielded distinct patterns of cytokine induction.

Rat astrocytes, immunologically competent ABSTRACT glial cells of the central nervous system (CNS), released a variety of cytokines after activation. Lipopolysaccharidestimulated astrocytes produced tumor necrosis factor (TNF) as demonstrated by Northern blot analysis using a mouse TNF probe and by functional assay. Biological activity of rat astrocyte-derived TNF was neutralized by rabbit antiserum against recombinant murine TNF. Stimulation of astrocytes by lipopolysaccharide also activated the interleukin 1 and interleukin 6 genes. We have also investigated whether a neurotropic paramyxovirus, Newcastle disease virus, triggers cytokine production by astrocytes. This virus induced astrocytes to produce TNF, lymphotoxin, interleukin 6, and a- and 13-interferons. Thus, stimulation by endotoxin and virus activated distinct, yet overlapping, sets of cytokine genes. We propose that astrocytes and the cytokines they produce may play a significant role in the pathogenesis of immunologically and/or virally mediated CNS disease, in CNS intercellular communication, and in the interactions between the nervous and immune systems.

MATERIALS AND METHODS Cell Cultures. Primary cultures of rat astrocytes were established as described (13). Approximately 95% of the cells obtained by this method stained positively for glial fibrillary acidic protein, an intermediate filament expressed in astrocytes. Staining with MAC-1, a monoclonal antibody that recognizes the iC3b receptor, was negative, indicating that the cultures were free of contaminating macrophages and microglia. Peritoneal macrophages were obtained from adult female Sprague-Dawley rats as described (13). WEHI 164 clone 13, a murine fibrosarcoma line (14), was used for TNF functional assays. RNA Preparation and Analysis. Total RNA was isolated from cells by the guanidinium isothiocyanate method (15). RNA was denatured by formaldehyde treatment, electrophoresed through a 0.8% agarose gel, and transferred to nitrocellulose as described (16, 17). The transferred RNA blots were hybridized with probes of high specific activity. Membranes probed with 32P-labeled DNA fragments were hybridized for 2 days at 370C and washed at 550C, twice in 2X SSC (ix SSC = 0.15 M NaC1/0.015 M sodium citrate)/0.1% SDS and twice in 0.5x SSC/0.1% SDS. Membranes probed with 32P-labeled RNA were hybridized overnight at 650C and washed at 650C as described above. Probes. Mouse cytokine probes were used in all experiments. DNA probes for IL-la, IL-1f3, IL-3, and lymphotoxin were constructed by using an oligolabeling reaction kit (Pharmacia), and RNA probes for TNF and the type I IFN were prepared using an RNA probe vector system (18, 19). Plasmid containing the 1.3-kilobase TNF cDNA was a gift of B. Beutler (University of Texas at Dallas). The plasmid was linearized with HindIII and labeled RNA probes were constructed using T7 polymerase. Plasmids used to construct the type I IFN probes were provided by N. Raj (The Johns Hopkins University) and were linearized with EcoRI. The IFN-a4 probe was constructed from a 776-base-pair (bp) EcoRI/Bgl II fragment of the IFN-a4 genomic clone, and the

Astrocytes are macroglial cells of the central nervous system (CNS) that express a variety of immunological characteristics. Astrocytes stimulated with y-interferon (IFN-y) express class I and class II major histocompatibility complex (MHC) antigens in rodents (1-3). Astrocytes expressing class II antigens can present foreign antigen to T cells in a MHCrestricted fashion (3, 4). Lipopolysaccharide (LPS) stimulates astrocytes to produce prostaglandins (5), complement components C3 and factor B (6), and cytokines with biological activities similar to interleukin 1 (IL-1) (5) and IL-3 (7). These observations indicate that astrocytes are immunologically competent cells that share many important functional characteristics with macrophages. Accumulating evidence has revealed that astrocytes, like macrophages (8), are activated by viral infection. Murine coronaviruses that cause primary demyelination stimulate expression of class I and class II MHC antigens on astrocytes (9, 10). The expression of class II MHC molecules and subsequent antigen presentation by astrocytes may play a central role in the development of an immune response within the CNS. Interestingly, the inducibility of class II expression by coronavirus JHM (10), like that of IFN-y (11), is strain dependent and correlates with susceptibility to experimental autoimmune encephalomyelitis in rats and mice. It has been proposed that the induction of class I antigen by murine coronavirus occurs through the release of soluble factor(s) by infected astrocytes (12). We have previously demonstrated that astrocytes stimulated with LPS and/or Ca2' ionophore A23187 produce a cytotoxic factor that is functionally and antigenically related to tumor necrosis factor (TNF) (13). This astrocyte-produced

Abbreviations: CNS, central nervous system; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; moi, multiplicity of infection; NDV, Newcastle disease virus; TNF, tumor necrosis factor; rTNF, recombinant TNF; rMuTNF, recombinant murine TNF.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6348

Medical Sciences: Lieberman et al. IFN-pS probe was constructed from a 500-bp Pvu II fragment of IFN-(3 cDNA. Labeled RNA probes for the type I IFN were generated by using SP-6 promoter vectors. The IL-la probe was constructed from a 440-bp Pst I/Pvu II fragment of IL-la cDNA provided by P. Lomedico (Roche Research Center). The IL-1p8 probe was constructed from a 775-bp EcoRI fragment of IL-13 cDNA obtained from J. Huang (E. I. du Pont de Nemours). The IL-3 probe was constructed from a 400-bp Xba I/HindIII cDNA fragment of IL-3 cDNA that was a gift from K. Leslie (University of British Columbia). The IL-6 probe was constructed from a 650-bp EcoRI/ Bgl II fragment of IL-6 cDNA that was obtained from J. van Snick (Ludwig Institute for Cancer Research, Brussels). The lymphotoxin probe was constructed from a 1.4-kb BamHI/ EcoRI cDNA fragment of lymphotoxin cDNA that was provided by N. Ruddle (20). Cell Induction. Cells (5 x 106) were induced with NDV New Jersey LaSota strain, at a multiplicity of 30 unless otherwise indicated. LPS (Sigma) was used at a final concentration of 10 ,ug/ml. Cell death was assessed by measuring the release of lactate dehydrogenase as described (21). Cytokine Functional Assays. TNF concentration was determined by the WEHI 164 clone 13 cytotoxicity assay (14). In brief, 4 x 105 cells per ml of medium containing 1 jig of actinomycin D per ml (Sigma) were placed in each well of 96-well flat bottom plates and 50 A.l of appropriately diluted recombinant murine TNF (rMuTNF) (ICN) or test sample was added. Replicate samples were pretreated with an equal volume of a 1:100 dilution of rabbit antiserum to rMuTNF (Genzyme) for 2 hr at 37TC. The medium from NDVstimulated cells was inactivated by UV light prior to assay. After a 20-hr incubation at 370C, 10 j.l of MTT (thiazolyl blue) (5 mg/ml) (Sigma) in phosphate-buffered saline was added to each well and incubated for 4 hr. The plates were then spun

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Table 1. Production of TNF by stimulated astrocytes Stimulant TNF, ng/ml None 0.007 ± 0.004 LPS (10 Ag/ml) 1.74 ± 0.164 NDV (moi 30)* 7.00 ± 1.20 Astrocytes were stimulated for the length of time required to achieve maximum TNF concentration: 8 hr for NDV (Fig. 4) and 20 hr for LPS (13). TNF concentration was determined by the WEHI cytotoxicity assay and is expressed as mean ± SD. *Supernatant was exposed to UV light prior to TNF assay. moi, Multiplicity of infection.

for 10 min at 2500 rpm. Medium (100 AL) was removed from each well and 100 jp. of 2-propanol/0.04 M HCl was added to dissolve formazan crystals prior to reading the optical density at 540 nm. TNF concentration was determined by comparison with a standard curve. IFN activity in cell supernatant was quantitated as described (22) following acid inactivation (pH 2 at 40C for. 5 days) of the NDV. One international unit of acid-stable IFN, as assessed by comparison with a type I IFN standard (Lee Biomolecular Laboratories, San Diego, CA), yielded 50% protection from cytotoxicity following challenge with encephalomyocarditis virus. RESULTS Production ofTNF by Rat Astrocytes After LPS Stimulation. To characterize the cytotoxic factor produced by LPSstimulated astrocytes (13), RNA from uninduced and LPSinduced primary astrocytes was analyzed by Northern blot. As shown in Fig. 1 and Table 1, stimulated astrocytes weakly expressed 18S TNF mRNA and released significant amounts of functional protein. Induction with LPS produced almost a 250-fold increase in the cytotoxic activity present in the supernatant of stimulated as compared with unstimulated cells. The specificity of this cytotoxic activity was confirmed by functional neutralization with rabbit antiserum against mouse rTNF (Fig. 1B). These data indicated that TNF production by LPS-stimulated astrocytes occurred through transient expression of low levels of 18S TNF mRNA, which were avidly translated into functional protein. In view of previous reports that LPS induces the production of IL-1- and IL-3-like factors by astrocytes (5, 7), we examined RNA from stimulated and unstimulated cells for expression of these genes (Fig. 2). In contrast with TNF, a

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FIG. 1. Rat astrocytes produce TNF after stimulation with LPS. (A) Northern blot analysis of total cellular RNA (10 ug per lane) isolated from unstimulated (lane 0) or LPS-stimulated cells at the indicated times. Lane C, total cellular RNA (10 j.g) isolated from RAW 264.7, a mouse macrophage cell line, stimulated with LPS for 2 hr. Film was exposed for 4 days. (B) Supernatants were collected 20 hr after LPS stimulation and assayed for TNF activity (o). The supernatants were also preincubated with an antiserum against rMuTNF and tested for cytotoxic activity (o). Means ± SD of WEHI cell death are presented. Background antiserum cytotoxicity of 20% has been subtracted from the neutralization data.

0 1 3 5 7 C Time (Hr) FIG. 2. Detection of IL-la and IL-1,8 mRNAs in astrocytes after stimulation with LPS. Northern blot analysis of total cellular RNA (25 jig per lane) isolated from unstimulated (lane 0) or LPSstimulated cells at the indicated times. Lane C, total cellular RNA (10 ,ug) from rat peritoneal macrophages stimulated with LPS for 2 hr. Blots were probed for either IL-la (A) or IL-1,3 (B) mRNA and film was exposed overnight.

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substantial amount of mRNA for IL-la and IL-1f8 appeared after LPS induction and remained prominent for up to 7 hr (note that Figs. 1 and 2 were exposed for different lengths of time; described in legends). IL-3 mRNA was not detected after LPS stimulation for up to 44 hr (data not shown). Production of TNF by Astrocytes Induced with NDV. While the initiation of primary demyelination is poorly understood, circumstantial evidence indicates that infectious agents, notably viruses, may play a pathogenic role in humans (23). Since TNF can induce oligodendrocyte death (13) and myelin A

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destruction (24), we examined whether NDV, a neurotropic paramyxovirus (25), triggers TNF production by astrocytes. As shown in Fig. 3, abundant 18S TNF mRNA appeared within 3 hr of astrocyte exposure to NDV, reached a maximum level around 8 hr, and then declined by 20 hr. This induction was dependent on viral multiplicity and was accompanied by the release of TNF into the supernatant. As shown in Fig. 4, functional protein, produced in the absence of cell death, peaked 8 hr after exposure to the virus, and by 20 hr decreased to 4 ng/ml. The amount of TNF released by NDV-induced astrocytes was -4-fold more than that released by LPS-stimulated cells (Table 1). The apparent discrepancy between TNF mRNA and protein levels detected after induction with NDV may have resulted from an inhibition of cellular protein synthesis in virally infected astrocytes (25). Activation of Numerous Cytokine Genes in Astrocytes by Virus. Genes for a variety of cytokines, including type I IFNs, TNF, lymphotoxin, and IL-6, contain within their regulatory regions a repeated hexanucleotide with a consensus sequence of AAG/AT/GGA (26). This hexamer is believed to play an important role in virally mediated cytokine induction (26-28). To evaluate whether NDV also activated genes for these other cytokines, RNA from stimulated astrocytes was analyzed by hybridization with specific cytokine probes. As shown in Fig. 5, astrocytes exposed to virus expressed mRNA for IFN-a, IFN-/3, and IL-6 along with TNF. Although NDV exposure provided the initial inducing signal, the level of induction and the longevity of mRNA expression differed for the cytokines examined. In addition, astrocytes expressed very low levels of lymphotoxin mRNA, which were detected only at the 20-hr time point (data not shown). Astrocytes stimulated by NDV for 20 hr also released 1667 international units of acid-stable IFN per ml. In contrast to the induction with live NDV, UV-inactivated virus did not induce mRNAs for the cytokines examined (data not shown). Interestingly, activation of cytokine genes in astrocytes by NDV differed significantly from that induced by LPS. While both LPS and NDV induced TNF and IL-6 mRNAs (Figs. 1, 3, and 5; IL-6 mRNA induction by LPS is not shown) and failed to activate the IL-3 and IFN-y genes (data not shown), neither IL-la nor IL-lP mRNA was detected in virally stimulated astrocytes. In addition, the induction of type I

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FIG. 3. Astrocytes produce TNF after induction with NDV. (A) Total cellular RNA from unstimulated (lane 0) and NDV-stimulated cells was isolated at the indicated times and analyzed by Northern blot (10 ,ug per lane). Lane C, total cellular RNA (10 ,g) from RAW 264.7 cells stimulated with LPS for 2 hr. (B) Total cellular RNA from unstimulated (lane 0) and NDV-stimulated cells was collected 6 hr after induction at the indicated multiplicities (moi, multiplicity of infection) and analyzed by Northern blot (10 ,ug per lane). Lane C, total cellular RNA (10 ,ug) from RAW 264.7 cells stimulated with LPS for 2 hr. (C) Supernatants were collected 20 hr after NDV stimulation and assayed for TNF activity (o). The supernatants were also preincubated with an antiserum to rMuTNF and tested for cytotoxic SD of WEHI cell death are presented. activity (o). Means Background antiserum cytotoxicity of 20%o has been subtracted from the neutralization data. ±

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Time (Hr) FIG. 4. Kinetics of TNF protein synthesis and astrocyte viability after induction with NDV. TNF concentration (o) in the supernatant is expressed as mean ± SD. Cell death (o) was determined by the release of lactate dehydrogenase and is expressed as the average of duplicate determinations.

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k1*o 0 1 2 3 4 8 20 Time (Hr FIG. 5. Astrocytes induced with NDV express mRNAs for IFN-a, IFN-pB, and IL-6. Total cellular RNA from unstimulated (lane 0) or NDV-stimulated cells was isolated at the indicated times and analyzed by Northern blot (10 ug per lane) for the expression of IFN-a (A), IFN-f3 (B), or IL-6 (C) mRNA.

IFN and lymphotoxin mRNA was not observed in LPSinduced cells (data not shown).

DISCUSSION We have characterized cytokine production by primary rat astrocytes in vitro. These glial cells of the CNS responded to LPS and NDV with distinct, yet overlapping, patterns of cytokine induction. Several lines of evidence indicated that these cytokines were produced by astrocytes and not by minor contaminating cells. The population of MAC-i- and GFAP-negative cells (-'5% of the total) may consist of glial progenitors. Even if this were not the case, the intensities of the cytokine signals induced by virus and detected in 10 ;kg of total RNA were not consistent with their generation by a minor cell population. In addition, TNF mRNA was potently induced in astrocytes by NDV and weakly induced by LPS, a pattern which is different from that seen in macrophages and which suggested that the TNF gene is differentially regulated in these two cell types. Interestingly, the weak induction of TNF mRNA by LPS was accompanied by the production of significant amounts of functional protein. This differential effect may be due to the combination of TNF mRNA instability and enhanced posttranscriptional processing in LPS-stimulated cells (29, 30). Stimulation of astrocytes with LPS resulted in a striking difference in the relative levels of TNF and IL-i mRNAs. While the expression of TNF mRNA was weak, LPS induced the production of high levels of IL-la and IL-13 mRNAs. These quantitative differences may reflect the existence of separate induction pathways for these genes in astrocytes, as demonstrated in macrophages (31). The existence of independently regulated induction pathways is further supported by our finding that virally induced astrocytes expressed abundant TNF mRNA (Fig. 3) but undetectable levels of IL-i mRNA. These data also indicate that stimulation with LPS and NDV may be coupled to the activation of distinct messenger signal pathways, which stimulate gene activation by separate mechanisms. For example, the failure of NDV to activate the IL-i genes may be due to the absence of virus-responsive elements in their regulatory regions. One

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such element contains a repeated hexamer with a consensus sequence of AAG/AT/GGA (26-28, 32). These regions may confer viral inducibility by binding specific transactivators such as IRF-1 (33). In addition, gene activation following stimulation with virus or LPS likely involves many undefined factors, which may be expressed in a tissue-specific manner (26). Cytokine production by astrocytes after stimulation with NDV identifies a new and important response by cells of the CNS to viral infection. We have been particularly interested in the production of TNF within the CNS because of the potential role of TNF as a mediator of demyelination. Primary oligodendrocytes are highly susceptible to the cytotoxic activity ofTNF, a finding that is unusual among primary cells (13). TNF also induces oligodendrocyte degeneration and myelin vesiculation in murine CNS explants (24). Viral infection is often cited as a probable cause of demyelination in humans, as exemplified by measles virus, another paramyxovirus, which induces subacute sclerosing panencephalitis, and by human immunodeficiency virus, which causes a degenerative myelopathy (34, 35). Demyelination can also be induced with distemper virus in dogs and with coronavirus or Theiler's virus in rodents (34, 35). We speculate that the multiple cytokines released by astrocytes during viral infection may function as effectors or modulators in virally-mediated CNS diseases. These cytokines may increase capillary permeability, attract inflammatory cells, and directly damage oligodendrocytes. In addition, different insults to the CNS may directly affect the nature of immune responses by selectively activating certain cytokine genes. The physiological importance of these cytokines within the disease-free CNS, however, may be their broad spectrum of biomodulatory effects. These include pyrogenic activity (36, 37), stimulation of neurite outgrowth (38), and regulation of the expression of MHC molecules on glial cells (39-41). These astrocytederived cytokines may also function as communicators between the nervous and immune systems. We thank Dr. N. B. K. Raj and Dr. Daniel Bednarik for advice and discussion during the course ofthis work. The following investigators are thanked for generously providing plasmids used to construct cytokine probes: Dr. B. Beutler, TNF; Dr. J. Huang, IL-1,8; Dr. P. Lomedico, IL-la; Dr. K. Leslie, IL-3; Dr. N. B. K. Raj, IFN-a and -B; Dr. N. Ruddle, lymphotoxin; and Dr. J. van Snick, IL-6. We also thank Ms. Theresa Hess for typing this manuscript. This work was supported by NS 15662 and NS 200205 to M.L.S. and by U.S. Public Health Service-National Institute of Allergy and Infectious Diseases Al 19737 to P.M.P. 1. Hirsch, M. R., Wietzerbin, J., Pierres, M. & Gordis, C. (1983) Neurosci. Lett. 41, 199-204. 2. Wong, G., Bartlett, P., Clark-Lewis, I., Battye, F. & Schrader, J. (1984) Nature (London) 310, 688-691. 3. Fierz, W., Endler, B., Reske, K., Wekerle, H. & Fontana, A. (1985) J. Immunol. 134, 3785-3793. 4. Fontana, A., Fierz, W. & Wekerle, H. (1984) Nature (London) 307, 273-276. 5. Fontana, A., Kristensen, F., Dubs, R., Gemsa, D. & Weber, E. (1982) J. Immunol. 129, 2413-2419. 6. Levi-Strauss, M. & Mallat, M. (1987) J. Immunol. 139, 23612366. 7. Frei, K., Bodmer, S., Schwerdel, C. & Fontana, A. (1985) J. Immunol. 135, 4044-4047. 8. Aderka, D., Holtmann, H., Toker, L., Hahn, T. & Wallach, D. (1986) J. Immunol. 136, 2938-2942. 9. Suzumura, A., Lavi, E., Weiss, S. & Silberberg, D. (1986) Science 232, 991-993. 10. Massa, P., Brinkman, R. & ter Meulen, V. (1987) J. Exp. Med.

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