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Raven Press. Ltd.. New York

0 1992 International Society for Neurochemistry

Rapid Communication

Interleukin 1 and Tumor Necrosis Factor-a Stimulate the Production of Colony-Stimulating Factor 1 by Murine Astrocytes Clotilde Thkry, *E. Richard Stanley, and Michel Mallat INSERM U. 114. Chaire de Neurop/iari?iuc~ilo~ie. Coll?ge de Fruticc. Puris, Fruncc; utid *Department of' Developmental Biology utid Cancer. Alhort Einstein College 01Medicine. Bronx. Nciz~York. Cr.S..4.

Abstract: Astrocytes have the ability to secrete colony-stimulating factor I (CSF-I), a growth factor known to stimulate the proliferation of brain macrophages. We have studied the effect of cytokines such as interleukin I (IL-I), tumor necrosis factor-n (TNFn), and interleukin 6 (IL-6) on the production of CSF-I by cultured primary astrocytes and an astrocytic cell line derived from embryonic mouse brain. We observed that both TNFa and IL-l increased CSF-I mRNA and protein levels in the astrocytic cultures. In contrast. IL-6 was ineffective. The CSF-I mRNA levels were strongly reduced by incubating immortalized astrocytic cells with staurosporine, a protein kinase C inhibitor, both in the absence and in the presence of cytokines. Conversely, 12-0-tetradecanoylphorbol 13-acetate, a protein kinase C activator. increased CSF-I mRNA levels. These results suggest a mechanism whereby mononuclear phagocytes could favor their own recruitment in the CNS by producing cytokines. Key Words: Colony-stimulating factor 1 -1nterleukin I-Interleukin 6-Tumor necrosis factor-n-Astrocytes. Thkry C. et al. Interleukin I and tumor necrosis factor-a stimulate the production of colony-stimulating factor I by murine astrocytes. .I. Nei/rochem. 59, I 183- 1 I86 ( I9 92).

Colony-stimulating factor 1 (CSF- 1 : also called macrophage-colony stimulating factor) is a homodimeric glycoprotein that is synthesized in various tissues and that stimulates survival. proliferation. and differentiation of mononuclear phagocytes (for review, see Stanley et al.. 1983). CSF- I transcripts have been detected by northern blot analyses of mouse brain extracts, and astrocytes cultured in serumenriched media secrete CSF-1 (Hao et al.. 1990; Thtry et al., 1990). The known target for CSF- I in the CNS is the brain macrophage, an activated form of microglial cell that is transiently present in the developing brain and can reappear in the adult parenchyma in pathological states (Perry and Gordon. 1988: Sawada et al., 1990). Increasing evidence supResubmitted manuscript received June 8. 1992: accepted June 12. 1992. Address correspondence and reprint requests to Dr. M. Mallat at INSERM U. I 14, Chaire de Neuropharmacologie. Colltge de France. I I place Marcelin Berthelot. 7523 1 Paris Cedex 05. France. .-lbbrei~iationsirsed 8-Br-CAMP, 8-bromo-cyclic AMP: CSF- I .

ports the notion that brain macrophages dramatically influence the survival or the growth of neuronal and macroglial cells (Giulian, 1990: Thtry et al., 1991). Thus, the production of macrophage growth factors in the CNS appears to be important for the nervous tissue remodeling in various contexts. Similar to other mononuclear phagocytes. brain macrophages can release large amounts of tumor necrosis factor-cu (TNFa)and interleukin I (IL-I) (Frei et al., 1987; Giulian et al.. 1988: Hktier et al.. 1988). In contrast to CSF-I, IL-l or TNFcu does not directly promote mitoses of brain macrophages. However. the addition of TNFn and IL-1 to heterogeneous glial cultures increases the proliferation of brain macrophages. suggesting that these cytokines could stimulate the production of macrophage growth factors by neural tube derivatives (Merrill. 1991). TNFu and IL-1 have been shown to share several biological activities on astroglial cells, including the stimulation of mitosis (Selmaj et al., 1990). In the present work we have studied the effect of these cytokines on the production of CSF-I by astrocytes. as well as the influence of interleukin 6 (IL-6). a cytokine produced by both IL- I - and TNFcu-stimulated brain macrophages and astrocytes (Frei et al.. 1989). We report that both IL-I and TNFn strongly up-regulate the production of CSF- 1 by cultured astrocytic cells.

MATERIALS AND METHODS Cell cultures Astrocyte primary cultures (2 weeks in vitro) were derived from the C3H/HeJ (lipopolysaccharide-resistant) strain of mice according to a previously reported procedure (Thiry et al.. 1990). In brief. dissociated cells derived from the cerebral cortex of 16-day-old embryos were seeded at low dencolony-stimulating factor I: DMEM. Dulbecco's modified Eagle's medium: FCS. fetal calf serum: IL-I. interleukin 1: 1L-6. interleukin 6: PKC. protein kinase C: RIA. radioimmunoassay: TNFLY. tumor necrosis factor-a: TPA. 12-0-tetradecanoylphorbol 13-acetate.

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sity (3.5 X 104/cm2) in 100- or 35-mm-diameter culture dishes and cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% fetal calf serum (FCS; Imperial Laboratory). The culture medium was changed once a week. Based on immunofluorescence studies, using rabbit polyclonal anti-glial fibrillary acidic protein antibodies (Dakopatts) to stain astrocyte-specific filaments and anti-F4/80 antiserum (provided by Dr. P. Crocker, Institut Pasteur, Paris, France), which binds selectively to macrophages, >95% of the cells in the 2-week-old cultures were identified as astrocytes, and these cultures were virtually devoid of contaminating microglial cells. The immortalized astrocytic cell line C.LT.T. 1.1 (provided by Dr. P. Rouget, College de France, Paris) was derived from the cerebral cortex of a transgenic mouse embryo expressing the polyoma virus T oncogene. This clonal cell line displays typical features of astrocytes. including expression of glial fibrillary acidic protein (Galiana et al., 1990: ThCry et al., 1990). It was grown in the medium used for astrocyte primary cultures and kept for 10 days at confluency before treatment and biochemical analyses.

Astrocyte treatments The C.LT.T. 1.1 clone or 2-week-old primary cultures were washed twice with phosphate-buffered saline and kept overnight in DMEM containing 0.2% FCS. The next day, the cells were washed again, and the medium was replaced with the same medium containing various reagents: recombinant human cytokines including IL-I@,TNFa, and IL-6 (all from Genzyme): 8-bromo-cyclic AMP (8-Br-CAMP0.1 mM; Sigma): 12-0-tetradecanoylphorbol 13-acetate (TPA: 200 nM, Sigma): staurosporine (Boehringer); or FCS (8%).

RNA extraction and northern blot analyses Total RNA was prepared from cells grown in 100-mmdiameter culture dishes, following 4-6 h of cell treatment (see above). RNAs were then separated by electrophoresis on a I % agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized with a mouse cDNA CSF-I probe as reported previously (ThCry et al., 1990). The 28s and 18s RNAs were visualized by ethidium bromide staining of the filters. Quantification of CSF- I mRNA and 18s RNA was performed using an image analyzer (IMSTAR, Paris). Autoradiograms and ethidium bromide staining were transformed into digitalized images ( 5 I2 X 5 I2 pixels. 256 gray levels per pixel), and the mean optical density of the bands above film background was determined with a computer.

Assay for astrocyte release of CSF-1 Culture media conditioned by primary astrocytes or cells of the C.LT.T. 1.1 clone ( 1 ml in 35-mm-diameter culture dishes) were collected after 18-24 h of cell incubation with different reagents, cleared of any cell debris by centrifugation, and lyophilized. Radioimmunoassay (RIA) of CSF- l in reconstituted culture media was performed as previously described (Stanley, 1979. 1985). One unit of CSF-I was equal to 0.44 fmol or 12 pg of CSF-I protein.

RESULTS AND DISCUSSION Influence of cytokines and serum on astrocytic synthesis of CSF-1 Figure IA shows the detection of CSF-1 transcripts by northern blot hybridization in 2-week-old astrocyte priJ . Nutirochem. Vol. 59. No. 3. 1992

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FIG. 1. Effect of cytokines and FCS on CSF-1 mRNA. Upper panel:Astrocytes in primary cultures derived from C3H/HeJ mice (A) or cells from the astrocytic clone (B) were incubated in DMEM plus 0.2% FCS (lane 1) supplemented with 70 U/ml of IL-1 (lane 2), 100 U/ml of TNFa (lane 3). 200 U/ml of IL-6 (lane 4), or 8% FCS (lane 5). After 4 h of incubation, total cellular RNA was harvested and hybridizedwith a CSF-1 probe. 18 S indicates the 18s RNA present in each lane and visualized by ethidium bromide staining. The effect of any treatment is representative of results from at least three independentexperiments. Lower panel: CSF1 mRNA expression in the astrocytic cell line incubated with increasing concentrations of IL-1 or TNFcu. Hybridized CSF-1 mRNA was quantified by densitometry, and the values were normalized to optical densities of stained 18s RNA. Control levels correspond to cells untreated with cytokines. Data are mean k SEM (bars) values from five independent experiments. ' p < 0.05 by Student's t test for values significantly above control levels.

mary cultures derived from lipopolysaccharide-resistant mice. Each lane was loaded with the same amount of RNA, as shown by visualization of the ethidium bromide-stained 18s RNA. A 4.5-kb CSF- I transcript was detected in astrocyte cultures kept for 24 h in a medium containing low FCS (0.2%) (lane I). Both IL-I and TNFa increased the abundance of the 4.5-kb transcript within 4 h of their addition to the cultures (lanes 2 and 3). Increased expression was also observed when the cells were cultured in high FCS (8%) (lane 5 ) . In contrast. IL-6 had no effect on the expression of CSF-I mRNA (lane 4). This pattern of astrocytic responses to FCS and cytokines was confirmed using the cloned astrocytic C.LT.T. I . 1 cell line (Fig. 1 B). Irrespective of the cell preparation used (primary cultures or immortalized cells), the involvement oftrace amounts ofheat-resistant lipopolysaccharide-contaminating recombinant products was ruled out by the fact that heat-denatured IL- 1 or TNFa failed to increase CSF-I mRNA expression by cultured cells (data not shown). Both IL-l and TNFa stimulated the CSF-1 mRNA accumulation in a dose-dependent manner. At a threshold concentration of 10 U/ml. both cytokines significantly increased the CSF-I mRNA levels in immortalized astrocytic cells (Fig. 1 ). As shown in Fig. 2. CSF- 1 was detectable by RIA in culture media conditioned by serum-deprived (0.2%) astro-

CSF-1 IN ASTROCYTE CULTURES cytes. This result is of particular interest given the isolation of the CNS by the blood-brain barrier. In agreement with the results from northern blot analyses, addition of IL- 1, TNFa, or FCS to the cultures led to marked increases of CSF-1 levels measured in the media harvested 18 h after addition. The effect of cytokines on CSF-I levels did not reflect an increase in cell number because there was no increase in astrocyte mitoses after 24 h of treatment with IL- 1 or TNFa (data not shown). In fact, increases of cell number in highly pure astrocyte cultures were observed only following longer periods of incubations with cytokines (Selmaj et al., 1990). IL-1 and TNFa appear to trigger astrocytic synthesis of IL-6 as a short-term response (Frei et al., 1989). IL-6 by itself was shown to stimulate astrocyte mitosis and production of nerve growth factors (Frei et al., 1989; Selmaj et al., 1990). However. our results suggest that the effects of IL-I or TNFa are not mediated by IL-6 because IL-6 added to astrocyte cultures did not significantly alter the CSF-1 levels (Fig. 2 ) .

Influence of protein kinase modulators on CSF-1 mRNA levels Studies performed with nonneural cell types have shown that TNFa and IL- 1 can induce an increase of cyclic AMP levels or the activation of protein kinase C (PKC) as early postreceptor events (Pravinkumar et al., 1987; Shirakawa et al., 1988; Vilcek and Lee, 1991). Therefore, we looked for the possible influences of intracellular protein kinases on the levels of CSF- 1 mRNA accumulation using the astrocytic cell line. As shown in Fig. 3, CSF- 1 mRNA expression wasdramatically reduced if staurosporine. a PKC inhibitor, was added to the cultures at a concentration of I p M , with or without cytokines (lanes 1-6). The decrease of CSF-1 mRNA levels was still obvious when the staurosporine concentration was reduced to 0.1 pM (lanes 8 and 12). The effect of PKC on CSF- 1 mRNA accumulation was further investigated by treating the cultures with TPA, an activator of PKC. TPA clearly increased the concentration of CSF-I mRNA in the astrocytic cell line following 4 h of treatment. The TPA stimulation was blocked in the presence of staurosporine (lanes 8-1 I). In contrast, we observed that 8-Br-cAMP, a lipophilic protein kinase A activator, reduced CSF- 1

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FIG. 2. RIA of CSF-1 in the media of astrocyte cultures after an 18-h incubation. The culture media consisted of DMEM plus 0.2% FCS (column 1) supplemented with 70 U/ml of IL-1 (column 2). 100 U/ml of TNFn (column 3), 200 U/ml of IL-6 (column 4), or 8% FCS (column 5). A: Astrocyte primary cultures. Data are mean f SD (bars) values from three independentexperiments with two determinations per condition in each experiment. 6:Astrocytic cell line. Data are mean f SD (bars)values of triplicates from one representativeexperiment. *p < 0.05 by Student's t test.

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FIG. 3. Effect of protein kinase modulators on CSF-1 mRNA levels in the astrocytic cell line. RNA was extracted following a 4-h incubation with DMEM plus 0.2% FCS alone (lanes 1 and 8)or with DMEM plus 0.2% FCS supplemented with 1 pM staurospor0.1 pM staurosporine (lane 12). 100 U/ml of ine (lanes 2 and 1l), IL-1 (lane 3). 100 U/ml of IL-1 and 1 pM staurosporine (lane 4), 100 U/ml of TNFa (lane 5), 100 U/ml of TNFa and 1 pM staurosporine (lane 6), 0.1 mM 8-Br-CAMP(lane 7), 200 nM TPA (lane 9), or 200 nM TPA and 1 pM staurosporine (lane 10). The data are representativeof three independentexperiments. Lanes 1-6 and 7-12 are from separate experiments.

mRNA levels in the culture (lanes 7 and 8). Therefore, our results strongly suggest that PKC activity up-regulates astrocyte CSF-I mRNA expression under both basal conditions and cytokine stimulations. Moreover, an intracellular accumulation ofcyclic AMP is unlikely to be responsible for the cytokine stimulations. Altogether, our results indicate striking similarities between astrocytes and fibroblasts in terms of cytokine regulation of CSF-I production. Thus, it has also been reported that fibroblasts in culture produce CSF- 1 and that FCS, IL1, and TNFa all strongly enhance their synthesis of CSF- 1, whereas IL-6 appears inefficient (Akashi et al., 1989; Schaafsma et al., 1989: Falkenburg et al., 1990). PKC activators were also shown to increase the level of CSF-I mRNA in a human fibroblastic cell line (Akashi et al.. 1989). The regulation of CSF-I production by astrocytes provides a molecular mechanism whereby macrophages secreting cytokines could amplify their own recruitment in the CNS. This mechanism could operate during development or in pathological circumstances. In this respect. it should be noted that expression of the CSF-I gene occurs in the immature brain and that detection of IL- 1 is correlated with the transient outgrowth of macrophage populations in different CNS regions (Giulian et al., 1988; Thtry et al., 1990). With regard to human neuropathologies, it is interesting to note that bacterial meningitis and AIDS-dementia complex, which involve brain macrophage reactions, are also associated with the appearance of CSF- I and TNFa or IL- 1 in the cerebrospinal fluid (Gallo et al., 1990). Acknowledgment: We thank Reza Zadeh for excellent technical assistance. We also thank Dr. Pierre Rouget for providing the C.LT.T. I . 1 clone and Drs. Brigitte Chamak. Michele Gelman, and Matthieu Levi-Strauss and Prof. Jacques Glowinski for their constant help. This work was supported by INSERM. MRT grant 89.C.0715. and an ANRS grant (to M. Mallat) and National Institutes of Health grant CA 3255 1, Albert Einstein Core Cancer Grant P30-CA 1330. and the Lucille P. Markey Charitable Trust (to E. R. Stanley).

REFERENCES Akashi M.. Sail0 M.. and Koeffler H. P. (1989) Lymphotoxin: stimulation and regulation of colony-stimulating factors in fibroblasts. Blood 74, 2383-2390.

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Falkenburg J. H. F.. Harrington M. A,. Walsh W. K.. Daub R.. and Broxmeyer H. E. ( 1990) Gene expression and release of macrophage-colony stimulating factor in quiescent and proliferating fibroblasts. J. Imtnimol. 144, 4657-4662. Frei K.. Siepl C.. Groscurth P.. Bodmer S.. Swerdel C.. and Fontana A. (1987) Antigen presentation and tumor cytotoxicity by interferon-y treated microglial cells. Eirr. J. ltntnirnol. 17, I27 I 1278. Frei K., Malipiero U. V.. Leist T. P.. Zinkernagel R. M., Schwab M. E., and Fontana A. (1989) On the cellular sourcc and function of interleukin 6 produced in the central nervous system in viral diseases. Eirr. J. Imtnirnol. 19, 689-694. Galiana E., Borde I.. Mann P.. Rassoulzadegan M.. Cuzin F.. Gros F., Rouget P.. and Evrard C. (1990) Establishment of permanent astroglial cell lines. able to differentiate in vitro, from transgenic mice carrying the polyoma virus large T gene: an alternative approach to brain cell immortalization. J. Ntwrosei. Rex 26, 269-277. Gallo P., Pagni S., Giometto B., Piccinno M. G.. Bozza F.. Argentiero V., and Tavolato B. (1990) Macrophage-colony stimulat29, ing factor in the cerebrospinal fluid. J . N~~irr~~itntnirnol. 105-1 12. Giulian D. (1990) Microglia and tissue damage in the central nervous system. in Nmr010gj~and Neiirohiolo,q.~:Vol. S5: Difjivenliaiion and Firnc/ions oj'GliaI Cclls (Levi G., ed), pp. 379389. Wiley-Liss. New York. Giulian D., Young D. G.. Woodward J.. Brown D. C.. and Lachman L. B. ( 1 988) Interleukin-1 is an astroglial growth factor in the developing brain. J . Ncirrosci. 8, 709-7 14. Hao C..Guilbert L. J., and Fedoroff S. (1990) Production of colony-stimulating factor-I (CSF-I ) by mouse astroglia in vitro. J . Neirrosci. Res. 27, 3 14-323. Hetier E.,Ayala J.. Denefle P., Bousseau A,. Rouget P.. Mallat M.. and Prochiantz A. (1988) Brain macrophages synthesize interleukin-l and interleukin-1 mRNAs in vitro. J . Ncwrosci. RPS. 21, 391-397. Merrill J. E. (199 1) Effects of interleukin- I and tumor necrosis factor-tu on astrocytes. microglia, oligodendrocytes. and glial precursors in vitro. Dev. Nriirosci. 13, 130- 137. Perry V. H. and Gordon S. (1988) Macrophages and microglia in the nervous system. Trcwds Neirrosci. 11, 273-277.

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Pravinkumar B. S.. WaltherZ..andTammI.(1987)Rapidenhancenient of @?-interferon/B-cell differentiation factor BSF-2 gene expression in human fibroblasts by diacylglycerols and the calcium ionophore A23 187. I'roc. Null. Acad Sci. LiSA 84, 36633667. Sawada M.. Suzumura A.. Yamamoto H., and Marunouchi T. ( 1990) Activation and proliferation ofthe isolated microglia by colony stimulating factor- I and possible involvement of protein kinase C. Brain RPS.509, 1 19-1 24. Schaafsma M. R.. Fibbe W. F.. Van Damme J., Duinkerken N., Ralph P.. Kaushansky K., Altrock B. W., Willemze R., and Falkenburg J. H. F. (1989) Interleukin-6 is not involved in the interleukin- I induced production of colony-stimulating factors by human bone marrow stromal cells and fibroblasts. B l d 74, 26 19-2623. Selmaj K. W., Farooq M.. Norton W. T.. Raine C. S., and Brosnan C. F. (1990) Proliferation of astrocytes in vitro in response to cytokines. A primary role for tumor necrosis factor. J . I m m i u d 144, 129-135. Shirakawa F.. Yamashita U., Chedid M.. and Mizel S. B. (1988) Cyclic AMP-an intracellular second messenger for interleukin- I . Proc. Null. .-lccrcl.Sci. L'S.4 85, 820 1-8205. Stanley E. R. (1979) Colony-stimulating factor (CSF) radioimmunoassay: detection of a CSF subclass stimulating macrophage production. Proc,. Natl. ..lead. Sci. L'SA 16, 2969-2973. Stanley E. R. ( 1985) The macrophage colony-stimulating factor. CSF- I . in Mclliods in Enzjonology Vol. 116: Immimocliernicul Twlrriiqire~(Colowick S. 0. and Kaplan N. 0..eds), pp. 564587. Academic Press. San Diego. Stanley E. R.. Guilbert R.. Tushinski R.. and Bartelmez S. (1983) CSF- 1 : a mononuclear phagocyte lineage-specific hematopoietic growth factor. J . C'c.N. Biochcrn. 21, 15 1-1 59. Th6ry C..Hetier E.. Evrard C.. and Mallat M. (1990) Expression of macrophage colony-stimulating factor gene in the mouse brain during devclopment. J . Nmrosci. Rev. 26, 129-1 33. ThCry C.. Chamak B.. and Mallat M. (1991) Cytotoxic effect of brain macrophages on developing neurons. Eirr. J . Neiirosci. 3, 1155-1 164. Viicek J . and Lee T. H. ( I99 I ) Tumor necrosis factor-new insights into the molecular mechanisms of its multiple actions. J. Biol. C / I P I266, ~ Z . 73 13-73 16.