Journal of Neuroscience Research 27:314-323 (1990)

Production of Colony-Stimulating Factor-1 (CSF-1) by Mouse Astroglia In Vitro C. Hao, L.J. Guilbert, and S. Fedoroff Department of Anatomy, University of Saskatchewan, Saskatoon, Saskatchewan, Canada (C.H., S.F.) and Department of Immunology and Cell Biology and Anatomy, University of Alberta and Canadian Red Cross Blood Transfusion Service, Edmonton, Alberta, Canada (L.J.G.)

We investigated whether astroglia produce any of the known macrophage growth factors, CSF-1, GMCSF, and IL-3, and if so, whether any of these cytokines stimulate the growth of CNS macrophages. In this work we used highly enriched cell cultures of C3H/HeJ mouse neopallium: cultures of astroglia and cultures of macrophage-like cells derived from nutritionally deprived astroglia cultures. We found that astroglia in cultures accumulate in the medium, an activity that stimulates the proliferation of macrophage-like cells. The activity has been identified as CSF-1 by using growth assays of cells dependent and nondependent on CSF-1, and by radioreceptor analysis which is highly specific for CSF1. Northern blot analysis demonstrated the presence in astroglia of CSF-1 mRNA and the presence of CSF1 receptor (c-fms) mRNA in macrophage-like cells but not in astroglia. The astroglia did not produce GM-CSF or IL-3. We concluded that a paracrine relationship exists between astroglia production of CSF-1 and the response of macrophage-like cells to the cytokine in culture.

neurotropic virus activate the TNF gene and synthesize TNF (Robbins et al., 1987; Lieberman et al., 1989); thus, these cells, like macrophages (Branch and Guilbert, 1989; Burchett et al., 1989), both respond to and secrete TNF. In similar fashion, astrocytes can be induced to produce interferon ct and p (INF-a$) and, in turn, INF-a@ can enhance the expression of H-2 antigens on subpopulations of astrocytes (Tedeschim et al., 1986). INF-y secreted by activated T lymphocytes can cause the expression of Ia (MHC class 11) antigen on subpopulations of astrocytes (Hirsch et al., 1983; Wong et al., 1984) which then present antigens to T lymphocytes in an MHC-restricted fashion (Fontana et al., 1984). Thus, astrocytes both send and receive humoral signals to and from local and systemic sites. The above observations also place CNS astroglia within the humoral network encompassing the major lymphohematopoietic cytokine secretory cells, T lymphocytes, and macrophages. As is well documented for these two lymphohematopoietic cell types, there is also evidence of humoral cross-talk between astrocytes and CNS macrophages. For example, ameboid microglia can release an IL- l-like activity which stimulates the prolifKey words: astroglia, microglia, CSF-1,c-fms, CSF- eration of astroglia in cultures (Guilian et al., 1986). 1 receptor That the reverse might also be true (astrocytes might control microglia development and function) has not been systematically addressed. IL-3, reported to be reINTRODUCTION leased from cultures of astrocytes (Frei et al., 198.5), is Astrocytes appear to play a central role in the cell- important to macrophage production from hematopoietic cell communications mediated by humoral polypeptide progenitors (Iscove et al., 1982) and is a potent macrofactors (cytokines) in the central nervous system (CNS). phage growth factor in the mouse (Guilbert, 198.5). DeStimulation of astrocytes by bacterial cell wall li- tailed studies of the role of astrocytes in the development popolysaccharide (LPS) activates interleukin- 1 (IL- 1) and function of CNS macrophages have been hindered genes (Lieberman et al., 1989), and stimulation of as- by the heterogeneity of CNS macrophages and the lack trocytes by tumor necrosis factor-cw (TNF) or by infection of suitable in vitro models for studies of this particular with lymphatic choreomeningitis virus (LCMV) leads to interaction. the expression of IL-6 mRNA (Frei et al., 1989), resulting in the production of the respective protein products. By releasing IL-I, astrocytes may be able to modulate Received April 2, 1990; accepted May 29, 1990. the immune response locally by inducing expression of Addrehs reprint requests to S . Fedoroff, Ph.D., D.Sc., Department of the IL-2 receptor and secretion of IL-2 in T-lymphocytes Anatomy, College of Medicine, University of Saskatchewan, Saska(Fontana et al., 1984). Astrocytes stimulated by LPS and toon, Saskatchewan S7N OWO, Canada. 'G 1990 Wiley-Liss, Inc.

Production of CSF-1 by Astroglia

By most of the criteria of what constitutes a mononuclear phagocyte (Van Furth et al., 1979), there are at least five distinct types in the CNS. Microglia are scattered throughout the CNS and likely represent the resident population. Supracpendymal macrophages are found on the ventricular side of the ciliated ependymal cells; epiplexus cells are associated with choroid plexuses; pericytes are distributed along the cerebral blood vessels; and blood-derived monocyte-macrophages enter the CNS primarily at the site of injury, but may also be present in small numbers in normal nontraumatized CNS. Recent evidence from our laboratory argues for a neuroectodermal , rather than mesodermal (hematopoietic), origin for a population of macrophage-like cells that arises out of rather highly enriched cultures of astrocytes from vascularized and prevascularized neopallium or neuroepithelium (Ha0 et al., 1990). These cells probably correspond to the above mentioned resident population of microglia. Importantly, these experiments also provide the in vitro model for studying humoral interactions between homogeneous populations of astrocytes and a single population of CNS macrophages. Using this model we here ask whether the abovementioned astroglia cultures produce any of the known macrophage growth factors: FSC- 1 , GM-CSF, or IL-3, and if so, whether any of the above cytokines stimulate the growth of the macrophage-like cells derived from the astroglia cultures. We find that while astroglia cultures do not secrete measurable GM-CSF or IL-3, they synthesize large amounts of the macrophage-specific cytokine CSF-1 and that the macrophage-like cells growing out of astroglia cultures express high levels of the CSF- 1 receptor and are growth dependent on CSF-I.

MATERIALS AND METHODS Cell Cultures Astroglia cultures were prepared from C3HIHeJ newborn mice as described previously (Ha0 et al., 1990). The cerebral hemispheres were dissected out and the meninges, hippocampus, basal ganglion, and olfactory bulb were removed. The remaining neopallium was forced through a 75 p m Nitex mesh to form a suspension of the cells in mMEM with 5% horse serum added. The cells were plated in 35 and 60 mm culture dishes with the cell numbers as indicated in the results and the cultures were incubated at 37°C in a humidified atmosphere of 5% CO, in air. The supernatants from the astroglia cultures were collected at the days indicated in the results for the CSF-1 bioassay. Astroglia cultures plated with 2 X lo6 viable cells in each 75 cm flask were incubated for a total of 8 days and used for RNA preparation (see below). Macrophage-like cells were isolated from astroglia

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cultures as described by Hao et al. (1990). To isolate the macrophage-like cells, dense astroglia cultures were incubated for 10 days with medium changed every 2-3 days and for an additional 10 days without medium change. At day 20, the dead and floating astroglia were washed away, leaving the macrophage-like cells attached to the culture surface. Such macrophage-like cell cultures were used for the '251-labeled CSF- l binding assay and the growth assay (see below). Two growth factor-dependent cell lines were used in bioassays for hematopoietic growth factors: 5/10.14, a macrophage cell line which responds to CSF-I , IL-3, and GM-CSF; and DAI .K, a lymphoblastoma cell line responding to IL-3, GM-CSF, G-CSF, erythropoietin, and LIF (Branch and Guilbert, 1987; Branch et al., 1987; Guilbert, unpublished). The 5/10.14 cells were maintained in continuous culture in petri dishes with Iscove's modified Dulbecco's medium (IMDM) containing 20% mouse 929-L fibroblast-conditioned medium and 15% FBS. Adherent 5/10.14 cells were harvested from petri dishes after treatment with 5 ml of cold 2 mM EDTA in PBS for 5 min. Cells of the DA1 .K murine nonadherent lymphoblastoma cell line were maintained in suspension cultures using IMDM supplemented with 20% WEHI3-conditioned medium and 10% heat-inactivated FBS. WEHT-3 is a murine myclomonocytic cell line which releases into the medium IL-3 and CSF-1 (Das et al., 1980). The P388D 1 murine (probably macrophage) cell line was maintained in suspension cultures containing 10% FBS in IMDM (Guilbert and Stanley, 1980) for the CSF- 1 radioreceptor assay (see below). Cells of the EL-4 line that originated from murine carcinogen-induced thymoma (Farrar et al., 1980) were maintained as suspension cultures in DMEM with 10% horse serum. These cells produce IL-2 when stimulated by PMA. They were used as a negative control for CSF-1 mRNA and CSF-1 receptor mRNA.

CSF-1 Bioassay The population growth assays of 5/10.14 cells and DA1.K cells were used to specify the CSF-1 activity present in culture supernatants (Branch and Guilbert, 1987). The cells were washed four times with PBS and the number of Y10.14 cells was adjusted to 2 X 104/ml in IMDM containing 15% FBS. The number of DA1.K cells was adjusted to 2 X 105/ml in IMDM containing 5% FBS. Fifty microliters of supernatants from astroglia cultures was added to wells of 96-well culture plates. After 48 hr of incubation, 20 pI of [3H]TdR solution (1 pCi; specific activity of 20 Ciimmol, diluted 1:19 in IMDM) was added to each of the wells and the plates were incubated for an additional 4 hr. After incubation, DA1 .K cells were harvested onto absorbent glass-fiber

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filters using a Titertek cell harvester. The 5I10.14 cells rcquired the addition of 100 pl of 0.1% Triton X-100 in PBS to each well, followed by gentle mixing on a shaker for 2 min before harvesting onto glass-fiber filters with the Titertek cell harvester. The filters were dried in air overnight and the circles containing cells were cut out and placed into scintillation vials with 1 ml of scintillation solution. The radioactivity of each vial was measured using a scintillation counter. Assays were run in quadruplicate and the results expressed as the mean SD.

*

Colony-Stimulating Factors Recombinant human CSF-1, a gift from I h . P. Ralph, Cetus Corp., Emeryville, CA, was used both as a high potency ( 3 X lo6 Uiml) unpurified supernatant from CHO cells plasmid-transfected with the truncated form cDNA of Kawasaki et al. (1985) and as a highly purified preparation. As previously described, CSF- 1 was radioiodinated with chloramine T to approximately 3 X 10'' cpm/mol (Stanley and Guilbert, 1981) and the biologic activity of each iodinated preparation was determined by colorimetric bioassay using 5/10.14 cells (Branch and Guilbert, 1987). IL-3 was prepared by transfection of COS cells with the PCD-MCGF-9 plasmid (Yokota et al., 1984) and used as an unpurified COS cell supernatant of approximdtely 2,500 Uiml. CSF-1 Radioreceptor Assay (RRA) The CSF-1 RRA was carried out as previously described (Das et al., 1980) with modification. Briefly, log phase growing cultures of P388D1 cells were collected and washed twice in ice-cold PBS, resuspended at 2.1 X 10' cellsiml in IB (IMDM lacking bicarbonate and adjusted to 280 mOsm and pH 7.35), and kept on ice. Cell viability was assessed using trypan blue exclusion and was always greater than 95%. All procedures were carried out on ice at 4°C. The assay was performed in 96well microtiter plates. Sample or standards were added in duplicate or triplicate at 50 plIwel1 with control wells having either dilucnt alone (to determine maximum '2sIlabeled CSF- 1 bound) or 2 nM nonradioactive CSF- 1 (to determine nonspecific binding). Freshly harvested and washed P388D1 cells (see above) were added to each well in 50 pl aliquots and incubated for 2 hr prior to the addition of '2s1-labeled CSF- 1 (50 p l containing 5 X lo4 cpmiwell). After an additional 1 hr incubation, a 100 p1 aliquot from each well was layered over 200 pl ice-cold FBS in 500 pl polyethylene microfuge tubes. The tubes were centrifuged (10,000 rpm, 2 min) and rapidly frozen in liquid nitrogen. The tips of the tubes, containing the cell pellet, were cut off and both cell-bound (pellet) and free (supernatant) radioactivity was determined in a gamma counter. The extent of competition of an un-

known sample was expressed as the unitless ratio, boundjfree (BIF). The amount of competing CSF- 1 in the sample was obtained from simultaneously derived standard curves of BIF versus CSF-1 concentration. In standard CSF-1 preparations (obtained from Dr. E.R. Stanley), 1 unit equalled 0.44 fmol. Final values were calculated from the standard curve and expressed as units of CSF-Iiml.

'%Labeled CSF-1 Binding Assay The assay was carried out as described previously (Guilbert and Stanley, 1986). In brief, macrophage-like cells in 75 cm2 flasks were treated with 0.25070 trypsin for 10 min, replated in 35 mm culture dishes with 5 x lo5 cells per dish in medium containing 50% astroglia-conditioncd medium and incubated for 24 hr. Then these cultures and astroglia cultures in 35 mm culture dishes were washed with ice-cold PBS, transferred to damp tissue paper on ice, and 1 ml of ice-cold IB containing 15% FBS (IB-FBS) was added. After 20?1 min of incubation, 0.1 ml of ice-cold '251-labeled CSF-1 (final concentration approximately 300 pM in FBS-TB) was added and the dishes were incubated for an additional 2 hr. The nonspecific '2sI-labeled CSF- 1 binding was determined after a 2 hr preincubation with 1 nM unlabeled CSF-1 in IB-FBS. The binding reaction was stopped by washing five times with a total of 10 ml of ice-cold PBS. The cells were removed from the dishes by the addition of 0.1 M NaOH and the Iz5Icontent of the pooled extract was determined on a gamma counter. Northern Blot Analysis The total RNA from astroglia or macrophage-like cells was prepared by acid guanidinium thiocyanate-phenol-chloroform extraction by the procedure modified from Chomczynski and Sacchi (1987). TE buffer (pH 8), DEPC-treated water, prehybridization solution, and SSC solution were prepared as described by Maniatis et al. (1 982). The cells were washed with cold PBS three times and lysed directly in the culture flask (75 cm) by the addition of 1.8 ml of the denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). The solution of lysed cells was transferred into a polypropylene tube and 0.18 ml of 2 M sodium acetate, pH 4, 1.8 ml of phenol (equilibrated with 1 M Tris-HC1, pH 8 , then stored under TE buffer), and 0.36 ml of chloroforn-isoamyl alcohol mixture (49: 1) were added sequentially. After centrifugation at 10,OOOg for 20 min at 4"C, the aqueous phase was transferred to a fresh tube, mixed with 1.8 ml of isopropanol, and kept at - 20°C overnight. Sedimentation at 10,OOOg for 20 min was again performed and the RNA pellet was dissolved in 0.3 ml of denaturing solution, transferred into a Eppendorf tube, and precipitated with 0.3 ml of isopropanol at -20°C for 1 hr. After

Production of CSF-1 by Astroglia

centrifugation in an Eppendorf centrifuge for 10 min at 4"C, the RNA pellet was washed with 75% ethanol, vacuum dried, and dissolved in 0.5% SDS at 65°C for 10 min. The RNA was loaded (1.5 pg), and electrophoresed through a I % agarose gel containing 2.2 M formaldehyde, then transferred to nylon membrane. The transferred RNA blots were incubated with prehybridization solution for 4 hr and hybridized overnight at 40°C with probe labeled with 32Pby random priming (Feinberg and Vogelstein, 1982). The membranes were washed twice in 2 x SSC containing 0.1% SDS, twice in 1 X SSC with 0.1% SDS, and once in 0.2 X SSC with 0.1 % SDS at 50°C. X-Ray film was exposed to the washed nylon membrane blot overnight. Total RNA from mouse L-929 fibroblasts was used for the CSF-1 mRNA positive control and the total RNA from the S- 1 cell line, a subclone of 5110.14, for the c-fms positive control. The total RNA from mouse EL-4 cell line was used as negative control for both murine cTfms and CSF-1.

cDNA Probes cDNA probes were obtained as fragments excised from the following plasmids: mouse CSF-1: a gift from CETUS Corporation, used as a 4.4 kb EcoRI fragment from PGE142MCSF10 (Ladner et al., 1988); mouse c,fms: a 4.2 kb SalI-BamHI fragment from PGT37 (Rothwell and Rohrschneider, 1987); and chicken p-actin: a 2 kb PstI fragment from the PA-1 plasmid (Cleveland et al., 1987).

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SN 8 SN 10 SN 12 SN 14

Fig. I. Effect of supernatants (SNs) from astroglia cultures on 5110.14 cell growth (measured by [3H]TdR uptake). Macrophage growth factor activity was asscsscd by incubating 51 10.14cells for 2 days with the indicated biological preparations at 25% viv then quantifying [ 'H]TdR uptake after a 4 hr pulse as described in methods. Supernatants were obtained from astroglia cells cultured as described in methods and the text. SN6, SN8, SNlO, SN12, and SN14 denote supernatants harvested from astroglia cultures at day 6, 8, 10, 12, and 14, respectively. Fresh astroglia culture medium [5% horse serum in mMEM (Med)] was used as the negative control. Medium conditioned by mouse L929 cells (LCM) served as the positive control.

glia formed a dense monolayer, which took approximately 10 days of culturing. The supernatants were also collected from astroglia cultures initiated with a variable number of cells. It was observed that supernatant-stimGrowth Assay ulatcd 5/10.14 cell growth increased with increase in the Macrophage-like cells were harvested, washed number of astroglia in cultures (Fig. 2). The Y10.14 cell with PBS, and planted in a 96-well plate with 6.5 X lo4 line, originally isolated from immature (day 3) bone marcellsiwell . Astroglia cultures were incubated in 96-well row-derived macrophages, was selected because of its culture plates for 6 days and washed with PBS. The absolute growth requirement for CSF- 1, 1L-3, or GMcultures were incubated for 24 hr after CSF-1 was added CSF (Branch and Guilbert, 1987). Therefore, the stimto the cultures and then [3H]TdR incorporation was eval- ulating effect of the supernatants from astroglia cultures uated by the procedure described above for bioassay. on the 5110.14 cell growth indicates that astrocytes secreted either CSF- I , IL-3, odand GM-CSF. To further define the factor(s) secreted by astroglia, RESULTS the effect of the supernatants was evaluated on line Production of CSF-1 by Astroglia DA 1.K cell growth. DA 1 .K is a nonadherent murine cell To determine the production of CSF- 1 by astroglia, line growth sensitive to both IL-3 and GM-CSF (Branch the supernatants from astroglia cultures were evaluated and Guilbert, 1987). The results (Fig. 3) showing that for their ability to stimulate growth of cells of the 5110.14 astrocyte supernatants do not stimulate DAI .K cell mdcrophage cell line. In the assay, astroglia cultures growth suggest that there was no IL-3 or GM-CSF were planted with 8 x lo5 viable cells per 60 mm culture present. To eliminate the possibility that an inhibitor of dish containing 3 ml of medium and the supernatants DAl .K cell growth might be present in the supernatants were collected at day 6, 8, 10, 12, and 14 of culturing. and thereby mask any growth activity present, known The data in Figure 1 indicate that supernatants from as- amounts of IL-3 were added to the supernatants and the troglia cultures stimulated the growth of 310.14 cells. mixtures were tested for their effect on the DA1.K cell The stimulatory effect present in the supernatants of as- growth. The data in Figure 3 clearly show that there is troglia cultures was observed in astroglia cultures as very little inhibition of DA1 .K cell growth by supernaearly as 6 days, and the effect increased until the astro- tants in the presence of TL-3, indicating that neither IL-3

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Fig. 2. Effect of supernatants (SNs) from cultures initiated with various numbers of cells on 5110.14 cell growth (measured by [3H]TdR uptake). Astroglia cultures were initiated with 2 X los (SN 2 x ) , 4 x lo5 (SN 4 x ) , and 8 x 10' (SN 8 x ) cells, and SNs were collected after 8 days of incubation. Macrophage growth factor activity was assessed as discussed in the methods and the legend to Figure 1.

SN 8

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IL-3

Fig. 3. Effect of supernatants (SNs) from astroglia cultures and IL-3 on DA1.K cell growth (measured by [3H]TdR uptake). 2 : DA1 .K cells incubated with SNs, or medium (Med) as control; m: DA1.K cells incubated with SNs and IL-3, or IL-3 alone as positive control. Supernatantswere added at 25% viv and cultures were incubated for 48 hr, and for 4 hr with ['HITdR as detailed in methods. The supernatant designations are explained in Figure 1. nor GM-CSF was present in the supernatants. Thesc observations, i.e., the response of 5110.14 cells but not DAl .K cells to the supernatants from astroglia cultures, demonstrate the presence of a rnacrophage growth factor that is not GM-CSF or IL-3, and therefore most likely is CSF-I. The lack of DAl .K cell growth activity in astroglia supernatants also indicates the absence of erythropoietin, G-CSF, and leukemia inhibitory factor (LIF), which are also DA1.K cell growth factors (Branch and Guilbert, 1980, Janowski-Wieczovek et al., 1988; A . B . Croy, L.J. Guilbert, M.A. Brown, N.M. Goush, O.T.

SN 6

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SN 10 SN 2x SN 4 x S N 8x

Fig. 4. Determination of CSF-1 in supernatants from astroglia cultures by radioreceptor assay (RRA). (a) SN 6, SN 8, and SN 10 were harvested from astroglia cultures at day 6, 8, or 10, respectively. (Compare with Fig. 1.) (b) SN 2 X , S N 4 X , and SN 8 X were the supernatants from astroglia cultures initiated with 2 X los, 4 X lo5, or 8 x lo5 cells. (Compare with Fig. 2.) Stinchcomb, N. Reed, and T.G. Wegmann, unpublished). That CSF-I is the macrophage growth factor produced by astroglia was confirmed, and the concentrations of CSF-1 in the supernatants were determined, by a specific RRA based on the ability of CSF-1 to compete for the binding of '251-labeled CSF-1 to CSF-1 receptors on P388D1 cells (Stanley and Guilbert, 1981). Between 100 and 300 Uiml CSF-1 was detected (Fig. 4) in astroglia supernatants with the relative levels detected by RRA paralleling the amount of 5110.14 cell growth activity detected above (Figs. 1 and 2). Thc production of CSF-1 by astroglia was again confirmed at the mRNA level. Total RNA was prepared from astroglia cultures, and size separated on agarose gel by electrophoresis. RNA blots were hybridized with mouse CSF-1 cDNA probes (Ladner et al., 1988). Figure 5 shows that astrocytic RNA (lane As), similar to mouse L-929 fibroblasts (lane L), contained 4 kb CSF-1 mRNA. The blots were hybridized again with p-actin cDNA probes as a control for the loading of RNA samples (Fig. 5 ) .

The Response of Macrophage-Like Cells and Astroglia to CSF-1 To assess the potential of astroglia and macrophage-like cells derived from astrocyte cultures (see methods) to respond to thc CSF-1 produced by the astrocytes, it was first determined whether either population expresses mRNA to the CSF-1 receptor. CSF-1 receptor is a single subunit glycoprotein of M , 165,000, identical or closely related to the c-fms protooncogene

Production of CSF-1 by Astroglia

p-Actin

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Fig. 5 . Northern blot analysis of CSF-1 expression in mouse astrocytes. Astroglia cultures were incubated for a total of 8 days and used for total R N A preparation (Lane As). Lane E, total RNA from human EL-4 cells which do not produce CSF1. Lane L, total RNA from mouse L-929 cells which do produce CSF-1, The RNA blots were hybridized first with CSF-1 probes (a). This probe removed and then rehybridized with a P-actin probe (b). The film was exposed overnight.

Mi

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Fig. 6. Northern blot analysis of CSF-1 receptor expression. Total RNA was prepared from 8 day old astroglia cultures (Lane AS) or macrophage-like cells (Lane Mi) isolated from astroglia cultures subjected to nutrition deprivation. Lane E. total RNA from EL-4 cells. Lane S , total RNA from 5110.14S1 (a CSP-I-dependentmacrophage cell line, Branch et al., 1989) cells. The blots were probed with cTfmscDNA probes and film was exposed overnight.

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(Morgan and Stanley, 1984; Sherr et al., 1985). Northern blot analysis of RNA prepared from both types of cells was carried out using a murine c-jms cDNA probe (Rothwell and Rohrschneider, 1987). Figure 6 shows that macrophage-like cells (lane Mi), but not astroglia (lane As), contain detectable c-fms mRNA. To determine whether the c-fms message is translated in macrophage-like cells, the cells were tested for the presence of CSF-1 receptors by a '251-labeled CSF-1 binding assay. As expected, no specific '2sI-labeled CSF-1 binding was found on the astroglia, i.e., there was no significant difference between the '251-labeled CSF- 1 binding on astrocytes preincubated with high levels of unlabeled CSF- 1 and astroglia incubated directly with '251-labeled CSF-1 (Fig. 7). In contrast, specific binding of 12'I-labeled CSF- 1, was detected on macrophage-like cells (Fig. 7), indicating that these cells have translated cTfmsto functional cell surface receptors. To assess function of the CSF-1 receptors on the macrophage-like cells, CSF- 1 stimulation of their proliferation was determined. The effects of various concentrations of CSF-1 on [3H]TdR uptake by purified preparations of macrophage-like cells and astroglia after a 2 day incubation were evaluated. The results indicate that macrophage-like cells (Fig. 8a), but not astroglia (Fig. 8b), proliferate in response to CSF-1. The maximum growth response of macrophage-like cells to CSF-1 was reached at about 1000 U/ml of CSF-1 (440 pM). The growth response was accompanied by a morphological change of the macrophage-like cells. In the presence of CSF- 1, the macrophage-like cells became long, crescentshaped spindles (Fig. 9).

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Fig. 7. '251-labeled CSF-1 binding on astroglia or macrophage-like cells. Binding was carried out at 4°C with 5 X lo5 cells as detailed in methods. 0:Nonspecific 1251-labeledCSF-1 binding. Cultures preincubated with 1 nM unlabeled CSF-I. C : Total '251-labeledCSF-1 binding. (For details see text.) The evidence above indicates that the c-jms protooncogene is expressed in the macrophage-like cells at the mRNA and functional cell surface levels, but is absent at all levels in astroglia.

DISCUSSION In this paper we report that cultures of astroglia from cerebra of C3WHeJ mice accumulate an activity that stimulates the proliferation of macrophage-like cells derived from astroglia cultures. We have identified the activity as CSF- I by several criteria: (1) Although medium from astroglia cultures stimulated the proliferation of a macrophage cell line growth dependent on CSF-1, GM-CSF, or IL-3, it did not stimulate proliferation of a lymphoblastoma cell line dependent on the latter two cytokines. (2) Medium from astroglia cultures contained an activity detected by a radioreceptor assay (RRA) highly specific to CSF-1. ( 3 ) The astroglia cultures pro-

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prior activation by agents such as bacterial cell wall lipopolysaccharide (LPS) since the cells used in this study 4 were derived from LPS-insensitive C3HIHeJ mice. On the assumption that the situation in vitro can be 3 transposed to the situation in situ, the question arises, what is the function of CSF-1 in the CNS'? Which cells of the CNS are targets for CSF-l? It is well established that within hematopoiesis CSF- I is a macrophage-specific growth factor (Byrne et al., 1981; Shadduck et al., 1983) b and that in our cultures it also affects macrophage-like cells (present study; Hao et al., 1990). Thus it would seem reasonable that CSF-1 should target these cells in 4 the CNS. Arguing against this extrapolation are the observations that the macrophage-like cells in the astroglia cultures appear to be of neuroectodermal rather than mesodermal (hematopoietic) origin and to be counterparts 4 I of microglia in situ (Hao et al . , 1990). However, there is 1 2 4 8 1 6 3 2 6 4 128 2 5 6 Med precedent for the expression of CSF-1 receptors on cells Dilution o f CSF-1 (1=5000 u/ml) of ectodermal origin. Placental trophoblasts, of trophecFig. 8. Effect of CSF-1 on growth of macrophage-like cells todermal origin, express cTfmsmRNA, bind '2'I-labeled and astroglia (measured by [3H]TdRuptake). (a) Macrophage- CSF-1 at high affinity, and stimulate production of plalike cells were plated in 96-well plates (5 X 10' cellsiwell). (b) cental hormones (human placental lactogen) (Regenstrief The astroglia cultures were initiated with I x lo3 cellsiwell. et al., 1989; Guilbert et al., 1990). Therefore we hypothBoth a and b were incubated with senal dilutions of recombi- esize that astrocytes may regulate the production, surnant CSF-I for 48 hr and with ['HITdR for 4 hr. vival, and function of ectodermally derived microglia by secreting CSF-1 . In support of this hypothesis we find that macroducing the above two activities contained mRNA that phage-like cells derived from astroglia cultures, but not strongly hybridizes to a full length cDNA probe for mu- astrocytes, express functional receptor to CSF-1. The rine CSF-I, indicating the presence of CSF-1 mRNA and CSF-1 receptor is a single subunit glycoprotein of M , thus the de novo synthesis of CSF-I . These results dem- 165,000, identical to the c-fms protooncogene (Morgan onstrate the potential of astroglia cultures to produce and Stanley, 1984; Sher et al., 1985). We have here CSF-1 and indicates their inability to produce the other demonstrated the expression of mRNA encoding the macrophage growth factors, GM-CSF and IL-3. The lat- CSF-1 receptor in macrophage-like cells derived for aster observations are in variance with the claim of Frei et troglia cultures by Northern blot analysis using a murine cTfmscDNA probe (Rothwell and Rohrschneider, 1987). al. (1985) that nonstimulated astroglia release IL-3. Since 95% of the cells in the astroglia cultures ex- Translation of c-fms into functional cell surface product press the astrocyte-specific protein GFAP, it appears has been demonstrated by specific binding of 1251-labeled highly probable that the cell type producing CSF-1 is the CSF- 1 at 4°C to sites on the macrophage-like cells and by astrocyte. However, since monocytesimacrophages are demonstrating mitogenic response to pure CSF- 1 . Therealso able to secrete CSF-1 (Horiguchi et a]., 1987) and fore, the secretion of CSF-1 by astrocytes is part of an in macrophage-like cells can be derived from the astroglia vitro paracrine process with the above-mentioned maccultures, we looked specifically for macrophages in the rophage-like (microglia) cells being the principal target. cultures. Fewer than 1% of the cells stained positive for These findings are in line with those of Guilian and MAC-1, a common marker for murine macrophages Tngeman (1988) and Frei et al. (1988), who also observe (Springer et al., 1979). In addition, we found that cells in that CSF-1 promotes the growth of macrophages isolated the astroglia cultures expressed no significant high affin- by shaking astroglia cultures at specific developmental ity CSF- 1 binding, a general marker for mononuclear stages. In conclusion, this study demonstrates the paraphagocytes (Stanley and Guilbert, 198 I ) . We therefore conclude that the activity from astro- crine relationship between astrocyte production of CSF- 1 glia cultures that stimulates the growth of astroglia-de- and the response of microglia to this cytokine in culture. rivcd macrophage-like cells is CSF-1 and that it is syn- Based on these observations, and those of others (Liethesized and released by astrocytes. Further, the berman et al., 1989; Tedeschim et al., 1986), it appears astrocytes appear to release CSF-1 in cultures without that astrocytes have the genetic information to synthesize 5 ,

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Production of CSF-1 by Astroglia

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Fig. 9. Morphological changes in macrophage-like cells cxposed to CSF-I . (a) Purified macrophage-like cells grown in &EM with 5% horse serum and 50% astroglia culture-con-

ditioned medium; (b) macrophage-like cells exposed to CSF-I (1 nM) for 2 days; (c) high magnification of macrophage-like cells from b.

potent cytokines such as CSF-1, INF, TL-1, and TNF. Whether astrocytes in situ release cytokines constitutively or only when activated by injury or disease processes is still not known. It also remains to be determined whether microglia bear the CSF-I receptor in situ. The accumulating evidence of cytokine production by astrocytes argues for cytokine production as an important role for astrocytes in the CNS. The findings presented here not only support this emerging view, but directly point to a probable target for one of the secreted cytokines. The above-mentioned cytokines secreted by astrocytes are all potent signal mediators and, when secreted, could participate in the modulation of both steady-state physiological processes (such as microglia production and survival) and in extraordinary events involving inflammatory and immune responses in the CNS.

of resting ramified microglia following the first postnatal days; however, they did not provide supporting data. In our paper we report that astroglia in cultures not only express CSF-1 mRNA but also express functional CSF- 1 polypeptide, which is secreted into the culture medium. We also report that microglia in cultures express CSF-1 receptor mRNA and functional CSF-1 receptor and that CSF- 1 stimulates microglia proliferation in cultures.

ADDENDUM

REFERENCES

While this paper was in press, a paper by ThCry et al. (1990) was published in which they reported that mouse brain expresses mRNA for CSF-1 from day 14 gestation until 2 weeks after birth. Using primary cultures from mouse cerebrum and two established astrocyte cells lines, Mes 3-1 and C.LT.T. I . 1, they demonstrated that the CSF-1 mRNA is expressed in astrocytes. They also demonstrated that brain macrophages in culture normally do not express CSF-1 mRNA unless they are stimulated by lipopolysaccharides. They suggested that the production of CSF- 1 by astrocytes may support survival

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ACKNOWLEDGMENTS This work was supported by Grant MT4235 from the Medical Research Council of Canada (S.F.) and by a Grant from the National Cancer Institute (Canada) (L.J.G.). We would like to thank Shirley West for typing the manuscript and Bonnie Winkler-Lowen and Anne Smith for technical assistance.

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