Journal of Neuroimmunology, 39 (1992) 163-174 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

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JNI 02210

Autocrine and paracrine regulation of astrocyte function by transforming growth factor-/3 Maria C. Morganti-Kossmann, Thomas Kossmann, Mary E. Brandes, Stephan E. M e r g e n h a g e n and Sharon M. Wahl Cellular Immunology Section, Laboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD, USA (Received 30 September 1991) (Revised, received 10 March 1992) (Accepted 10 March 1992)

Key words: Astrocyte; Transforming growth factor-/3; Transforming growth factor-/3 receptor; Chemotaxis; Proliferation

Summary Recent evidence indicates that astrocytes have a wide range of functions, usually attributed to ceils of the immune system, which are critical for maintaining a balanced homeostatic environment in the central nervous system (CNS). Moreover, these ceils are known to participate in inflammatory events within the CNS by secreting cytokines such as transforming growth factor-/3 (TGF-/3). In this study we have investigated the ability of TGF-/3 to influence astrocyte functions. TGF-/31 mRNA is constitutively expressed by astrocytes in vitro, and when cultures are stimulated with exogenous TGF-/31 an increase in the expression of this mRNA can be shown, suggesting both autocrine and paracrine regulation. In in vitro assays, TGF-/31 is chemotactic for astrocytes in a dose-dependent fashion and inhibits astrocyte proliferation. These results indicating signal transduction by TGF-/31-prompted studies to explore receptor-ligand interactions on isolated astrocyte populations. In a receptor binding assay, we demonstrate that astrocytes appear to express three distinct TGF-/3 receptor subtypes with nearly 10000 receptors per cell. Thus, TGF-/3 may play an important role in regulating astrocyte functions pivotal to the evolution of intracerebral immune responses including recruitment and activation of glial cells at local inflammatory sites within the CNS.

Introduction The growth strated growth

regulatory functions of transforming factor /3 (TGF-/3) have been demonin numerous biological processes. Cell and differentiation are strongly affected

Correspondence to: S.M. Wahl, Laboratory of Immunology, NIDR, NIH. Building 30, Room 326, Bethesda, MD 20892, USA.

by the presence of TGF-/3 (Roberts et al., 1990). During embryogenesis of the mouse, high levels of three TGF-/3 isoforms, TGF-/31, /32 and /33, have been detected in the nervous system, as well as in other tissues (Flanders et al., 1990; Pelton et al., 1990). However, in the adult human central nervous system (CNS), neither TGF-/31 nor TGF/32 was found expressed in normal brain tissue (Bodmer et al., 1989; Wahl et al., 1991), although TGF-/3 has been shown to be associated with

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acquired immunodeficiency syndrome (AIDS) neuropathology (Wahl et al., 1991). During tumorigenesis, TGF-/3 may also be expressed in the CNS as evidenced by a glioblastoma cell line derived T-cell suppressive factor, which was later identified as TGF-/32 (Wrann et al., 1987; Bodmer et al., 1989). These and other studies suggest that glial cells may be an important source of TGF-/3 within the brain during development, and possibly during injury or inflammation. Much evidence suggests that astrocytes play a central role in the regulation of immune-mediated processes in the CNS (Frei and Fontana, 1989; Hertz et al., 1990). Astrocytes can be induced to express MHC class I and II antigens which mediate cell cooperation by presenting antigen to T cells (Fontana et al., 1987). In vitro studies suggest that astrocytes may also contribute to immune and inflammatory responses by producing cytokines such as IL-1 (Fontana et al., 1982), IL-6 (Benveniste et al., 1990; Hirohata and Miyamoto, 1990) and TNF-a (Robbins et al., 1987; Liebermann et al., 1989; Chung and Benveniste, 1990). Astrocytes in culture have also been found to constitutively express TGF-/31 mRNA and the amount of message increases after stimulation (Wesselingh et al., 1990; Wahl et al., 1991), suggesting that TGF-/3 may act in an autocrine a n d / o r paracrine manner to regulate the activities of these pivotal cells. Earlier studies have demonstrated TGF-/~ production by astrocytes in human immunodeficiency virus (HIV)-infected brains, thus supporting a role for TGF-/3 in AIDS-related dementia (Wahl et al., 1991). These findings were corroborated by in vitro studies showing that TGF-/3 contained in supernatants from HIV-infected macrophages could induce cultured astrocytes to release TGF-/3 (Wahl et al., 1991). Since astrocytes respond to exogenous TGF-/3 stimulation, we initiated studies to characterize which subtypes of TGF-/3 receptors might be present on the astrocyte cell surface (see review, Massagu6, 1990). Two glycoproteins (receptors I and II) of 53 and 70-100 kDa, respectively, and a membrane proteoglycan (type III receptor) have been identified. As functional consequences of receptor-ligand interaction we show that TGF-/31 is chemotactic for astrocytes and inhibits astrocyte growth. These

data implicate a role for TGF-/3 in the modulation of astrocytic functions relevant to CNS inflammation and neuropathology.

Materials and methods

Primary glial cell culture Purified astrocytes were obtained from newborn rats as described elsewhere (Morganti et al., 1990). Briefly, brain hemispheres were removed from meningi, trypsinized for 15 rain in a solution of 0.5% trypsin (Gibco, Grand Island, NY) in Hanks' balanced salt solution (HBSS) without Ca 2+ and Mg 2+ (Whittaker, Walkersville, MD) whereafter the tissue was mechanically dissociated in HBSS containing 1 mg/ml DNase I (Sigma Chemical Co., St, Louis, MO) using a fire-polished pasteur pipette. Cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% low endotoxin (< 10 pg/ml) fetal bovine serum (FBS) (Gibco) and finally plated in 75-cm 2 tissue culture flasks (Costar, Cambridge, MA) previously coated with 0.1 mg/ml poly-L-lysine (Sigma). Cultures were maintained for 8-10 days and a mixed glia culture was obtained. Contaminating cells were separated by mechanical agitation to dislodge microglia, oligodendrocytes and precursor cells. Culture purity was determined by immunostaining with a polyclonal antibody to GFAP (Chemicon, E1 Segundo, CA) and > 95% of the cells stained positively for the astrocytic marker. TGF-[3 receptor assay The expression of TGF-/3 receptors was quantified on adherent astrocytes plated in 24-well plates (Costar) (7.5 x 104 cells per well) by measuring the binding of [125I]TGF-/3 as previously described (Brandes et al., 1991a). Purified human TGF-/31 was iodinated (Amersham, Arlington Heights, IL) to a specific activity of 1.7-2.2 /xCi/pmol using a modified chloramine-T method (Frolik et al., 1984). Cultured astrocytes were incubated with constant agitation for 3 h at 4°C with 200 /xl binding buffer (DMEM with 0.1% bovine serum albumin (BSA) (Sigma), 25 mM Hepes (pH 7.4) containing various concentrations

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of [~25I]TGF-/3 up to 250 pM. Initial experiments had shown that the length and the temperature of the binding incubation were sufficient to allow [~25I]TGF-/31 binding to reach equilibrium. Nonspecific binding was determined in the presence of a 400-fold excess of unlabeled TGF-/31. Following the incubation, the supernatant was sampled to determine the amount of unbound [125I]TGF-/31. The cells were washed five times with phosphate-buffered saline (PBS) (pH 7.4) containing 0.1% BSA, and then solubilized with a solution of 1% Triton X-100, 10% glycerol, 0.01% BSA and 20 mM Hepes (Frolik and DeLarco, 1987). The content of each well was counted using a gamma counter to determine the amount of bound [ 125I]TGF-/31.

TGF-Ctl-receptor affinity labeling and molecular mass determination Astrocytes were incubated with 125 pM [125I]TGF-fll in binding buffer as described above. Cross-linking of labeled TGF-/31 to its receptor was performed with a modification of a previously published procedure (Kay et al., 1986). Briefly, cells were incubated for 15 min at 4°C with 0.25 mM disuccinimidyl substrate (Pierce Chemical Co., Rockford, IL) in PBS (pH 7.4). The reaction was stopped by the addition of Tris • HC1 (pH 6.8) and E D T A to final concentrations of 0.3 and 0.06 M, respectively. Cells were washed three times with PBS and then solubilized in a buffer containing 0.1% SDS and 1 mM phenylmethylsulfonyl fluoride and stored at - 2 0 ° C . Human neutrophils were isolated as previously described (Brandes et al., 1991b) and processed in parallel for use as a positive control. SDS-PAGE was performed using an 8% polyacrylamide gel to separate cell proteins. A [14C] methylated protein mix (Amersham) was used as standards. The gel was dried and exposed to X - O M A T A R film (Kodak, Rochester, NY). Chemotaxis Astrocyte chemotaxis was evaluated in 48-well microchamber plates (Neuroprobe, Rockville, MD) by a modification of described methods (Smith et al., 1984; Ohura et al., 1987). Subconfluent astrocyte cultures were trypsinized (0.02% trypsin in HBSS without Ca 2+ and Mg 2+ with

2% glucose, 1 mM E D T A ) for 15 min at room temperature. Culture medium containing 10% FBS was added to inactivate the trypsin, and the cells were washed, resuspended in medium with 5% FBS and incubated for 1 h in a humidified incubator at 37°C to recover from the treatment. Cells were then washed in serum-free medium and resuspended (2 × 106/ml) in Gey's buffer (National Institutes of Health Media Unit, Bethesda, MD). The attractants were tested as follows: TGF-/31 0.01-1 p g / m l ( R & D Systems, Minneapolis, MN); rat endotoxin-activated serum containing C5a diluted 1:10 (positive control), and Gey's buffer (negative control). The 48 lower wells were filled with 28/zl of attractants and the upper wells with 50 Izl of cell suspension. The polycarbonate filters (8 txm pore size) which separated the lower and upper wells had previously been coated with poly-L-lysine (0.1 m g / m l ) at room temperature for 4 h. Chemotaxis chambers were incubated for 4 h at 37°C in a humidified incubator. After incubation, filters were fixed and stained with Diff-Quik (American Scientific Products, Stone Mountain, GA). An Optomax Image Analyzer (Optomax, Hollis, NH) was used to quantify the number of cells which had migrated. Chemotactic activity is expressed as the mean number of cells ( + S E M ) which migrated through the pores in three standard fields per filter for triplicate filters. Experiments were repeated four times.

Proliferation assay Astrocytes (15 × 103) were plated in each well of 96-well plates (Costar) and cultured for 24 h in D M E M with 1% FBS. The following day the medium was aspirated and replaced with 1% FBS containing D M E M and TGF-/31 0.001-10 n g / m l in the presence or absence of IL-I¢I 100 U / m l (Genzyme, Cambridge, MA). Reagents contained no detectable endotoxin as determined by Limulus assay (Hochstein et al., 1983). Astrocytes were stimulated for 1-5 days and pulsed with [3H]thymidine (50 ixCi/ml) (New England Nuclear, Boston, MA) for 16 h. Cells were solubilized by adding 1% Triton X-100, 10% glycerol, 0.01% BSA, 20 mM Hepes and harvested with an automated harvester (Tomtec, Orange, CT). The proliferation was quantitated by measuring the

166

amount of [3H]thymidine incorporated in triplicate wells.

Northern blot analysis Astrocyte cultures were trypsinized and 7.5 x 105 cells plated in DMEM containing 10% FBS in 25 cm 2 tissue culture flasks (Costar). Prior to stimulation, the cultures were incubated overnight in DMEM with 1% FBS, and then stimulated with TGF-/31 (1-20 ng/ml) or lipopolysaccharide (LPS, E. coli O55:B5, Difco Lab., Detroit, MI) (10 #g/ml). Following a 4-h exposure to the stimuli, the cells were washed twice with PBS and the total cellular RNA was extracted using a rapid one step acid guanidine isothiocyanatephenol-chloroform procedure (Chomezynski and Sacchi, 1987). RNA samples were electrophoresed (5/zg per lane) in a 1% agarose/10% formaldehyde gel, and blotted onto nitrocellulose. Blots were then prehybridized at 42°C for 4 h in 5 × SSC, 5 × Denhardt's solution (0.05 M NaPO4, 500 p~g/ml salmon sperm DNA, 50% formamide and 0.1% SDS) followed by sequential hybridization with a [32p]-labeled TGF-/31 cDNA probe (kindly provided by Dr. R. Derynck, Genentech Inc., South San Francisco, CA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fort et al., 1985) for 18 h. After washing, autoradiography was performed at

-70°C using Kodak X-OMAT film (Eastman Kodak) for 3-7 days. The relative amounts of hybridizing RNA were determined by scanning the autoradiograms with a laser densitometer (LKB Ultrascan, LKB Instruments, Gaithersburg, MD) and TGF-/31 signals were normalized to the constitutively expressed GAPDH by calculating the ratios of individual samples.

TGF-~ bioassay Purified astrocytes were plated in 24-well plates (Costar) at 7.5 × 104 cells/well in medium containing 10% FBS. Confluent cultures were then incubated overnight in 1% FBS and the next day fresh medium containing TGF-/31 (0-20 ng/ml) was added. After a 4-h incubation, cells were vigorously washed two times with DMEM to remove exogenous stimuli and incubated in medium with 1% FBS overnight. Supernatants were collected, aliquoted and frozen at -20°C. Samples were acid activated at pH 2-3 for 30 min by adding 6 M HC1 and then brought to neutral pH with 6 M NaOH and 1 M Hepes prior to assay. Total TGF-/3 activity was determined by measuring the inhibition of IL-l-dependent thymocyte proliferation as described previously (Wahl and Dougherty, 1991). In some experiments, supernatants were pretreated with an antibody which neutralizes TGF-/31 and TGF-/32 (10 /zg IgG

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Fig. 1. Analysis of astrocyte TGF-{31 binding. (A) Astrocyte cultures were incubated with binding buffer, then with indicated concentrations of [125I]TGF-/31 at 4°C for 3 h to obtain equilibrium binding. (B) Scatchard analysis of astrocyte [125I]TGF-~61 binding obtained after reaching equilibrium. The data shown are from representative experiments (n = 3). 1 pmol TGF-/3 corresponds to 25 pg/ml.

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neutralize 5 ng TGF-/3; kindly provided by Dr. J. Dasch, Celtrix Pharmaceuticals, Palo Alto, CA) prior to quantitation of inhibitory activity.

Results

Characterization of TGF-/31 receptors on astrocytes In initial experiments, astrocytes were evaluated for the expression and subtype(s) of TGF-/31 receptors which might be involved in signal transduction. Astrocyte expression of TGF-/3 receptors was examined by incubating the cells with increasing concentrations of [125I]TGF-/31, allowing the binding to reach equilibrium, and then determining the amount of [125I]TGF-/31 bound (Brandes et al., 1991a, b). These experiments showed that [125I]TGF-/31 binding to second passage astrocyte cultures approached equilibrium at 125 pM (Fig. 1A). By Scatchard analysis, astrocytes were found to bind 15.3 _+ 1.2 fmol [125I]TGF-/3/106 cells (n = 3) (Fig. 1B) corresponding to a p p r o x i m a t e l y 9400 +_ 700 receptors/cell. The binding sites appeared to be of a single high affinity, with a dissociation constant ( K d) of 76 _+ 6 pM. To determine which of the three potential subtypes of TGF-/3 receptors were expressed by astrocytes, astrocyte cell surface TGF-/3-binding proteins were affinity labeled. Astrocyte cultures were equilibrated with [125I]TGF-/31, which was then cross-linked to the receptor using the bifunctional cross-linking agent, disuccinimidyl suberate. In parallel, human neutrophils were processed in a like manner to facilitate the identification of the type I TGF-/3 receptor, the subtype primarily expressed by neutrophils (Brandes et al., 1991b). Total cell proteins were separated by S D S - P A G E and the bands representing the l i g a n d / r e c e p t o r protein complexes were visualized by autoradiography. Astrocytes were found to express primarily the type III proteoglycan receptor which appears as a > 200 kDa protein hand (Fig. 2). The specificity of the interaction between [125I]TGF-/31 and the receptor protein is shown by the ability of a 400-fold excess of unlabeled TGF-/31 to virtually eliminate labeled T G F /31 binding. Specific binding was also apparent for

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Fig. 2. Molecular mass determination of astrocyte TGF-/3 receptors. Astrocytes were equilibrated with 125 pM [IzSI]TGF-/31in the absence (lane 2) or presence (lane 3) of a 400-fold excess of unlabeled TGF-/31 for non-specific binding determination. Freshly isolated human neutrophils were processed in parallel for use as a positive control (lane 1). After chemically cross-linking the [12SllTGF-/3 to its receptors, cell proteins were separated by SDS-PAGE. The dried gel was exposed to x-ray film for 12 days. The autoradiograph is shown with molecular mass markers indicated to the left and the receptor bands indicated to the right, corresponding to approximately 65, 90 and > 200 kDa. The analysis was repeated three times with similar results.

proteins represented by 65 and 90 kDa bands which may correspond to smaller numbers of the type I and type II TGF-/3 receptors, respectively. These receptor bands are assumed to consist of 53 and 78 kDa proteins cross-linked to a m o n o m e r of TGF-/3 (12 kDa), although the molecular mass of the type II receptor varies with tissue and species. The identification of the 65-kDa band as the type I receptor is aided by the appearance of a band of identical molecular mass in the neutrophil lysate lane. In addition to these three protein bands, numerous minor bands are present on the autoradiograph. Although these bands diminish slightly in intensity upon the addition of a 400-fold excess of unlabeled TGF-/3, they are not eliminated, thus demonstrating that they were the result of non-specific association of

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[125I]TGF-/3 with cell proteins. Thus, astrocyte populations express type I and II signal transducing receptors and also type III TGF-/3 receptors on their cell surface.

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Chemotactic activity of TGF-~ I The identification of type I and II receptors, not previously described on astrocytes, suggested an important pathway for TGF-/3 regulation of astrocyte functions. In this regard, we tested the ability of astrocytes to migrate in response to TGF-/31, an extremely potent chemotactic factor for leukocytes (Wahl et al., 1987; Brandes et al., 1991b) in an in vitro chemotaxis assay. The cells were exposed to increasing concentrations of TGF-/31 as described in Materials and methods. A significant migration towards TGF-/31 was exhibited in a dose-dependent manner within a 4-h period, with maximal response at 0.1 p g / m l (Fig. 3). As is typical of many chemotactic stimuli, including TGF-/3 (Wahl et al., 1987; Brandes et al., 1991b), higher concentrations of the stimuli inhibit, rather than promote, migration. Astrocytes also migrated in response to rat C5a (3-fold

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Fig. 4. Effects of TGF-/31 on astrocyte proliferation. Subconfluent astroglial cells were cultured in D M E M supplemented with 1% FBS in the presence of purified TGF-/31 for 5 days. Cultures were pulsed for 16 h with [3H]thymidine before harvesting. The results show the kinetics of inhibition of astrocyte growth using 10 n g / m l TGF-/31 for 5 days of culture. Control proliferation was measured in the absence of TGF-/3. These data are from a representative experiment

(n = 5).

increased chemotaxis over control) used as the positive control (Armstrong et al., 1990).

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Fig. 3. TGF-/31 induced astrocyte cbemotaxis. Astrocytes (2 × 106/ml) were isolated and assayed for chemotactic activity to TGF-/31 at the indicated concentrations. Data are from a representative experiment (n = 4) and chemotactic activity is defined as the mean n u m b e r ( + SEM) of astrocytes migrated through the filter pores in three fields for each of triplicate wells. Gey's balanced salt solution alone was the negative control.

Since astrocytosis is often associated with CNS injury and disease, we next investigated the effect of TGF-/31 on astrocyte growth. TGF-/31 was added to subconfluent astrocyte monolayers at various concentrations, and the cells were monitored for proliferation by incorporation of [3H]thymidine into DNA over a period of 5 days. Inhibition of cell proliferation was observed for all concentrations of TGF-31 (0.001-10 ng/ml), at all time points tested. Maximal inhibition was evident at day 4 with a 60-78% decrease in [3H]thymidine incorporation compared to control samples (Fig. 4). For comparison, the addition of IL-1/3 (100 U / m l ) to the astrocytic cultures, as a positive control (Giulian and Lachman, 1985), resulted in augmented proliferation with maximal activity at day 5. When TGF-/31 and IL-1 were simultaneously added to the cultures, TGF-/31

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was shown to reverse IL-l-induced proliferation in a dose-dependent fashion (Fig. 5). No toxicity was evident at any of the concentrations of TGF/31 or IL-1 added to the cultures. Thus, TGF-/3 appears to strongly perturbate astrocyte proliferation exhibiting a negative effect.

Induction of TGF-/31 mRNA and TGF-/3 peptide secretion To determine if binding of TGF-/31 to astrocyte receptors could influence TGF-/3 expression by autocrine a n d / o r paracrine mechanisms, we monitored the induction of TGF-/31 mRNA and peptide secretion following exposure to increasing concentrations of exogenously added TGF-/31. As shown by Northern blot analysis, astrocytes which have been isolated and cultured as adherent monolayers constitutively express TGF-/31 mRNA (Fig. 6A). However, an upregulation of mRNA expression was observed following a 4-h incubation with purified TGF-/31 at concentrations from 1 to 20 ng/ml (Fig. 6A, B). Densitometric analysis showed a 4-fold increase of TGF/31 mRNA for samples stimulated with TGF-/31 (10 ng/ml) expressed as the ratio of the cytokine signal to the GAPDH housekeeping signal. For comparison, LPS (10 ~ g / m l ) which is known to stimulate astrocyte production of certain cy-

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Fig. 6. TGF-/31 mRNA expression by stimulated astrocytes. Expression of TGF-/3 transcripts obtained after confluent astrocytic cultures were exposed for 4 h to purified TGF-/3 at 0-20 ng/ml. Northern analysis of mRNA hybridized with a TGF-/31 cDNA probe and ribosomal RNA of the corresponding lanes to indicate equivalent loading of RNA for two separate experiments (A and B).

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TGF-~ (ng/ml) Fig. 5. Effect of TGF-/31 on IL-I-induced astrocyte proliferation. TGF-/31 at indicated concentrations was simultaneously added with IL-1 (100 U / m l ) and suppressed IL-1 augmented proliferation in a dose-dependent fashion with maximal activity on day 5 of culture. The data shown are from a representative experiment (n = 3) and are expressed as percent + SEM of IL-l-induced proliferation (11 192 cpm).

tokines (Fontana et al., 1982; Wesselingh et al., 1990) was used as a positive control and induced a 4-fold increase in TGF-/3 mRNA (data not shown). The ability of stimulated astrocytes to secrete TGF-/3 was monitored by quantitation of TGF-/3

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Fig. 7. Production of TGF-/3 by purified astrocytes. Cultures were stimulated with TGF-/31 for 4 h, washed and fresh medium was added for an additional 16 h. Supernatants were acid activated and then assayed untreated or treated with anti-TGF-/3 (10 p.g/ml) for 30 min at 22°C prior to assay. TGF-/3 activity in the supernatants was determined as described in Materials and methods in the thymocyte proliferation assay.

levels in culture supernatants. As shown in Fig. 7, levels of TGF-/3 in acid-activated astrocyte supernatants correlated with the changes observed in the expression of TGF-/31 m R N A under the same conditions. Whereas little or no TGF-/3 was detectable in unstimulated astrocyte supernatants, TGF-/3 activity was measured in the supernatants derived from cultures treated with purified T G F /31. Since negligible levels of bioactivity were observed in supernatants which were not acidactivated, the majority of the inducible TGF-/3 activity was secreted in a latent form. Following treatment of the supernatants with a neutralizing antibody to TGF-/3, the anti-proliferative activity in the stimulated astrocyte supernatants was completely eliminated, documenting the production of TGF-/3 by the astrocytes. The presence of TGF-/3 in the TGF-/31-stimulated astrocyte cultures could not be attributed to residual exogenous TGF-/31 since the cultures were vigorously washed after a 4-h pulse with TGF-/31 and no activity was found in the fresh culture medium immediately after being added to the astrocyte monolayers. Consistent with our Northern analysis and with previous studies (Wesselingh et al., 1990), LPS was also found to induce astrocyte TGF-/3 secretion (data not shown).

The absence of detectable TGF-/31 in normal adult brain tissue and its recent identification in virally infected CNS suggests a role for this cytokine in brain pathology (Wahl et al., 1991). In this regard, astrocytes were identified as a source of TGF-/3, yet little is known of the mechanisms of synthesis or regulation of TGF-/3 by these cells. In the present study astrocytes were shown to express multiple subtypes of TGF-/3 receptors. The type III receptor, which is expressed in the greatest number, is thought to be involved in cell-matrix interactions and in TGF-/3 storage, delivery or clearance (Andres et al., 1989), but not involved in signal transduction (Laiho et al., 1990, 1991). This proposal is supported by the recent determination that the cytoplasmic portion of the type III receptor is very short and contains no known signaling domains (Wang et al., 1991; Lopez-Casillas et al., 1991). Astrocytes also express the type I TGF-/3 receptor and probably the type II receptor as well. The expression of one or both of these receptor subtypes correlates with cell responsiveness to TGF-/3 (Laiho et al., 1990, 1991). While the differential function of each receptor type has not been fully elucidated, it is known that type I receptors alone are sufficient on leukocytes to induce a migratory response (Wahl et al., 1987; Brandes et al., 1991a, b). Astrocytes migrate toward TGF-/3 in a dose-dependent manner, with the greatest response occurring at concentrations of TGF-/3 similar to those reported for monocytes (Wahl et al., 1987). Tissue inflammation and wound healing are characterized by an ordered accumulation of inflammatory cells in which TGF-/3 plays a key role (Wahl et al., 1989; Wahl, 1992). Cell migration is also an intrinsic part of CNS development; glial precursor cells, as well as astrocytes, migrate through different regions of the brain to reach their final destination. The factors involved in the guidance of glial migration have not yet been identified. The high expression of TGF-/3 during morphogenesis, and the ability of TGF-/3 to control the synthesis of extracellular matrix components (Massagu6, 1990; Roberts et al. , 1990), suggest that TGF-/3 might be important in regulating cell migration for cells of the nervous system.

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In addition to recruitment in development and inflammation, TGF-/3 may also influence the mitotic activity of astrocytes as it does for many other cell types (Roberts et al., 1985; Moses et al., 1985). Consistent with recent studies (ToruDelbauffe et al., 1990), we found that TGF-/31 can negatively modulate the growth of astrocytes in culture. Since astrocytes secrete TGF-/3, although in a latent complex, they may be able to regulate their own growth potential following release of the active homodimer. Although not detected in adult brain (Bodmer et al., 1989; Wahl et al., 1991), TGF-/31 mRNA was found to be constitutively expressed in unstimulated cultured astrocytes, implicating a post-transcriptional regulation of TGF-/3 synthesis and secretion as recently reported for monocytes (McCartney-Francis et al., 1990). Thus, the potent ability of TGF-/3 to recruit astrocytes to a site of injury or inflammation, thereby contributing to astrogliosis, may be tempered by the ability of this cytokine to impair their proliferative response. Furthermore, TGF-/3 may regulate astrocyte growth by functioning as an antagonist to certain other inflammatory cytokines including IL-1/3. In pathological disorders of the CNS, recruitment of infiltrating monocytes is often described despite the presence of the blood-brain barrier (Moench and Griffin, 1984; Wahl et al., 1991). Locally produced TGF-/3 may not only contribute to mononuclear cell recruitment, but could also represent a possible mechanism for activating astrocytes to participate in these events. In this regard, our findings suggest that the expression of TGF-/3 receptors allows astrocytes to respond to TGF-/3 by migrating toward injury or inflammatory loci and then locally contributing to the evolving events including the synthesis of extracellular matrix components. The delicate balance between beneficial and potentially pathological regulation of these pathways is likely influenced by the autocrine and paracrine interactions of TGF-/3 with the astrocytes. Whether the presence of TGF-/3 in the inflammatory foci within the brain actually contributes to tissue injury or alternatively, prevents severe nervous tissue damage by inactivating T cells such as those specific for the myelin basic protein (Racke et al., 1991), by limiting excessive

astrocytosis, or by inducing NGF and other neurotrophic factors (Lindholm et al., 1990) continues to be explored. Clearly, TGF-/3 is emerging as a fundamental cytokine in events associated initially with development (Roberts et al., 1990) and subsequently, in pathological disorders of the CNS (Wahl et al., 1991).

Acknowledgements We thank Sue Dougherty and Claire Schuster for expert technical assistance. Dr. T. Kossmann is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft, Bonn, Germany (KO 1078/1-1).

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