Proc. Natl. Acad. Sci. USA Vol. 88, pp. 7016-7020, August 1991 Neurobiology yInterferon promotes proliferation of adult human in vitro and reactive gliosis in the adult mouse in vivo VOON WEE YONG*, ROBERT MOUMDJIAN, FIONA P. YONG, THEODORA C. G. RUIJS, MARK S. FREEDMAN, NEIL CASHMAN, AND JACK P. ANTEL Department of and Neurosurgery, McGill University, Montreal, PQ H3A 2B4, Canada Communicated by Brenda Milner, April 26, 1991

ABSTRACT Reactive gliosis is a characteristic response of TNF has been shown to promote proliferation ofadult human astrocytes to inflammation and trauma of the central nervous astrocytes (21). Thus, it appears possible that certain cyto- system. To investigate whether soluble factors () from kines from inflammatory mononuclear cells can provide the inflammatory mononuclear cells that accumulate at lesion sites cellular signals to initiate the process of reactive gliosis in can provide the cellular signals to initiate gliosis and to identify pathologic brain conditions where these cells are prominently such cytokines, we have tested and found that supernatants featured. derived from subsets ofactivated human T (CD8' A consideration in relating relevance of in vitro prolifera- or CD41) are potent mitogens for cultured human adult tion results to reactive gliosis in vivo is that gliotic scars astrocytes. This effect is blocked by a neutralizing antibody to seldom develop after insults to the embryonic brain (22-24) -interferon (IFN). Recombinant IFN alone can induce prolif- but are common features in adult brain injuries. Thus, it eration of human adult astrocytes in vitro and increase the becomes important to assess proliferation of astrocytes de- extent oftrauma-initiated gliosis in the adult mouse brain. The rived from adult animals, rather than from neonatal ones. We proliferation-inducing activity of supernatants of have adopted such a strategy by using adult human astrocytes glial cultures treated with IUN can be completely blocked with isolated from surgical brain biopsies and have addressed the IFN-neutralizing antibody, suggesting that the proliferative following questions. Do activated human T lymphocytes effect does not require intermediary cytokines or cells. These produce sufficient cytokines to induce proliferation of cul- results implicate IFN as an important mediator of the gliosis tured adult human astrocytes; if so, which (s) are observed in pathologic conditions of the adult central nervous important? Could the cytokine(s) thus identified increase the system associated with infiltrating lymphocytes. extent ofgliosis in vivo in the injured adult mouse brain? Our results implicate y-interferon (IFN) as an important mediator A prominent characteristic following many types of insult to of the reactive gliosis observed in pathologic conditions the is the response of astrocytes associated with infiltrating lymphocytes. (1-5). These cells increase in numbers, become larger, extend more processes, and significantly increase their cytoplasmic MATERIALS AND METHODS content ofglial fibrillary acidic protein (GFAP), an astrocyte- specific (4). The astrocytic response is Human Glial Cell Culture. Dissociated glial cells were referred to as reactive gliosis and the cells are referred to as isolated from the temporal lobe of 12 patients (age, 29 ± 3 reactive astrocytes. In the case of trauma, a situation where years) undergoing surgery to ameliorate intractable , the precise time and site of insult can be documented, by using a trypsin digestion followed by Percoll gradient reactive gliosis is initiated within hours after the injury. In centrifugation (25-27). Cells were plated onto uncoated 25- most cases, the process results over time in a gliotic "scar" cm2 Falcon flasks at 5-10 million cells per flask. After packed with GFAP filaments. Such scars are thought to be removal of poorly adherent 24 h later, undesirable for central nervous system regeneration (6-10). remaining cells consisting mostly ofastrocytes and , The signal(s) that induce astrocyte reactivity after injury in various proportions for individual donors (see Results), remains incompletely defined. If such signal(s) could be were treated with trysin and seeded on poly(L-lysine)-coated identified, at least for specified injuries, and means were (10 jag/ml) 9-mm Aclar fluorocarbon coverslips at 10,000 available to inhibit their activity, then the detriments of cells per coverslip. These mixed cells were used for the end-stage glial scars might be avoided. In considering the present study; methods for eliminating microglia from rodent identification of such signal(s), it is instructive to note that cultures (leucine methyl ester and silica ingestion) (28- inflammatory mononuclear cells. (lymphocytes, blood- 30) were not effective for human preparations. Culture me- derived , and intrinsic brain microglia cells) are dium was Eagle's minimum essential medium supplemented prominently featured in many pathologic conditions of the with 5% (vol/vol) fetal calf serum, Gentamicin (20 gg/ml), brain. These include inflammatory brain diseases (e.g., mul- and dextrose (1 mg/ml) (all from GIBCO). tiple sclerosis) (11-13) and brain trauma such as those caused Preparation ofT- Culture Supernatant. Human by a penetrating stab injury (14, 15). mononuclear cells were obtained by Ficoll/Hypaque sepa- Secretory products of inflammatory mononuclear cells are ration ofblood donated by healthy individuals. After removal cytokines, and in this regard, supernatants derived from of monocytes by plastic adherence, the CD4' and CD8' activated lymphocytes have been reported to be mitogens for T-lymphocyte subsets were isolated by antibody-dependent neonatal rodent astrocytes (16-18). In addition, specific cytokines that include interleukin (IL)-1 (19), tumor Abbreviations: BrdUrd, bromodeoxyuridine; EGF, epidermal growth factor a (TNF), and IL-6 (20) have been reported to be factor; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic mitogens for neonatal rodent or bovine astrocytes in culture; protein; IFN, -interferon; IL, interleukin; PDGF, platelet-derived growth factor; TNF, a; PI, proliferation index; NU, neutralizing unit(s); IGF-1, insulin-like growth factor 1. The publication costs of this article were defrayed in part by page charge *To whom reprint requests may be addressed at: Montreal Neuro- payment. This article must therefore be hereby marked "advertisement" logical Institute, 3801 University Ave., Montreal, PQ H3A 2B4, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Canada. 7016 Downloaded by guest on October 2, 2021 Neurobiology: Yong et A Proc. Natl. Acad. Sci. USA 88 (1991) 7017 complement lysis (31). One million CD4' or CD8' cells were activated CD8' T lymphocytes from seven blood donors plated in wells precoated with goat anti-mouse IgG (5 ,ug/ml; significantly increased the PI of cultured adult human astro- Cappel Laboratories). OKT3 (5 ng/ml; Ortho Diagnostics) cytes [PI = 22.2 ± 2.3 (mean ± SEM; n = 36 coverslips)] and IL-2 (10 units/ml) were then added and cultures were (Figs. 1 and 2). Supernatant collected from activated CD4' maintained for 5 days at 37TC. The culture medium (lympho- lymphocytes was similarly effective (PI = 16.5 ± 2.5; n = 13 cyte supernatant) was then removed, centrifuged at 2000 rpm coverslips). Whereas the mean percentage BrdUrd incorpo- in a 216 rotor to remove cells, and used. Flow cytometry ration in nontreated control adult human astrocytes was 2%, analyses of cells on the day of supernatant collection indi- incorporation in cultures treated with activated lymphocyte cated that the purity oflymphocytes was between 95 and 98% supernatants was as high as 60%. The lymphocyte culture of CD3+CD4' or CD3+CD8' cells (CD3 is a marker for all T medium containing IL-2 and OKT3 did not increase the lymphocytes). proliferation of astrocytes (control, 1.8%; IL-2 and OKT3, Assessment of Proliferation of Astrocytes. To assess astro- 2.0%). The PI induced by activated lymphocyte supernatants cytic proliferation, a double immunofluorescence GFAP/ did not correlate with the degree of microglial contamination bromodeoxyuridine (BrdUrd) technique was used (32, 33). in the astrocyte cultures (Fig. 2). Noncytokine growth factors Cultures were incubated with test agents for 4 days, with 10 (PDGF, FGF, EGF, and IGF-1) could at most evoke a 3-fold gM BrdUrd (a thymidine analog) added during the last 48 h increase in PI (Fig. 3). so that proliferating cells may incorporate this label; prelim- Proliferative Response to Activated Lymphocyte Superna- inary experiments had indicated that this time frame was tants Can Be Blocked by the Neutralization of IFN: IFN optimal to observe the low basal rate ofproliferation ofadult ofboth human astrocytes. Cultures were then immunolabeled, Promotes Astrocyte Proliferation. Supernatants CD4' coded, and analyzed using a Leitz dual immunofluorescence and CD8' cell cultures, as expected (35, 36), contained IFN; microscope. Negative controls for staining included omission human IFN at 100-200 units/ml was detected using a radio- of both primary antibodies and cultures where the 10 gM immunoassay kit (Centocor, Malvern, PA). To examine BrdUrd was not added. To quantify proliferation, the per- whether a neutralizing antibody to human IFN could inhibit centage ofGFAP+ cells (astrocytes) that had BrdUrd labeling the proliferative response of astrocytes to the lymphocyte in their nuclei was determined. For some experiments, these supernatants, the polyclonal antibody at 10, 100, or 500 results were expressed as the proliferation index (PI), which neutralizing units (NU)/ml was preincubated for 30 min at was the percentage of GFAP+BrdUrd' cells in test cultures 370C with supernatants before their addition to astrocyte divided by similar values from sister control cultures (i.e., cultures. Fig. 2 shows that this antibody at 100 and 500 cultures maintained in the same feeding medium but without NU/ml blocked the stimulating effects of the supernatants. addition of test factors). Use of the PI allowed results to be pooled from several different human specimens. All results were subjected to statistical analyses using a one-way anal- ysis of variance with Duncan's multiple comparisons; signif- icance was set at the P <0.05 level. The following cytokines were tested: supernatants from CD4+ or CD8+ lymphocytes, recombinant human IFN [Rorer Biotechnology (King of Prussia, PA) or Boehringer Mannheim], and recombinant human IL-1 and IL-2 (Gen- zyme). Neutralizing human cytokine antibodies against IFN (rabbit polyclonal, Genzyme), IL-1 (Genzyme), and TNF (Genzyme) were used. The following noncytokine growth factors were also tested for comparison: platelet-derived growth factor (PDGF) (Collaborative Research), fibroblast growth factor (FGF) (Boehringer Mannheim), epidermal growth factor (EGF) (Boehringer Mannheim), and insulin- like growth factor 1 (IGF-1) (Collaborative Research). To correlate the astrocyte proliferation results and the amount of microglia cells in the cultures, we identified microglia by surface immunofluorescence for Leu-M5, a human monocyte marker (Becton Dickinson) at a 1:10 dilu- tion (26). Modulation of Reactive Gliosis in Vivo. Adult 2-month-old mice were anesthetized with pentobarbital (50 mg/kg, intra- peritoneally) and the head was restrained in a rodent stero- taxic device. The skull was exposed and a 3-mm2 piece overlying the left occipital cortex was excised using a dental drill. A piece (2 mm3) of left occipital cortex (corticectomy) was then resected with a scalpel. Absorbable Gelfoam (Up- john; 2 mm3, when soaked) soaked with recombinant IFN (100 units/ml) was then packed into the brain cavity. Con- trols were corticectomized mice whose wounds were packed with Gelfoam containing saline. Seven days later' formalin- fixed paraffin-embedded 10-,um sections were obtained for GFAP FIG. 1. Double immunofluorescence detects proliferation of as- immunoperoxidase staining (34). trocytes. (A and C) GFAP immunofluorescence. (B and D) BrdUrd immunolabeling of cells in A and C, respectively. A and B are RESULTS controls; C and D are a sister culture exposed to an activated CD8' T-cell supernatant. Double-positive cells are indicated by arrows. Activated T-Lymphocyte Supernatants Are Potent Mitogens Note that more astrocytes in the treated group (C and D) have for Adult Human Astrocytes. Supernatant conditioned by incorporated BrdUrd. Downloaded by guest on October 2, 2021 7018 Neurobiology: Yong et al. Proc. Natl. Acad. Sci. USA 88 (1991)

A B 50 CD8 Control - (12)* -; 45 (6) Activated CD8 S/N (11)

a 40 Activated CD8 S/N (4) * anti-IFN(500NU/ml) (4) * f Activated CD8 S/N o 35 * anti-IFN(100NU/mi) (7)* t 10 IT (4) Activated CD8 S/N X 30 (3) + antl-IFN (IONU/m1) (4)

- 25 CD4 x '4 20 - 3 (5) Control- =D (5) T (5) .2o 15 Activated CD4 S/N - 1(4) Activated CD4 S/N EC (4) 1.1 + anti-IFN(500NU/ml) - 10; Activated CD4 S/N ()_ (4) t 0- + - . anti-IFN(100NU/mi) Activated CD4 S/N -1() dII * anti-IFN (10NU/mi) (4) 0 1 1 2a 2b 5617 0 5 10 15 20 25 CD8 -CD4-- % (GFAP+ BrdU+)/GFAP+

FIG. 2. Potent mitogenic effect of activated lymphocyte supernatants can be blocked by a neutralizing antibody to IFN. (A) Activated CD8' T-cell supernatants from seven lymphocyte donors (bars 1-7) were tested on an astrocyte culture series [one lymphocyte donor (bars 2a and 2b) was assayed on two cultures], and activated CD4' T-cell supernatant from one lymphocyte donor was tested on three astrocyte cultures. These supernatants were used at a 1:1 dilution with astrocyte feeding medium. All results in A were significantly different from controls. The degree of microglial contamination in each culture is indicated above the histograms as follows: +, <20% of total cell population; + +, 20-50%o; + + +, 50-80%; + + + +, >80%. There was no apparent correlation between the PI of astrocytes and the degree of microglial contamination. (B) Neutralizing antibody (100 and 500 NU/ml) to IFN obliterated the proliferative response to CD8+ or CD4+ supernatants (S/N). None of the concentrations of anti-IFN neutralizing antibody by themselves resulted in values significantly different from controls. Values significantly different from activated CD8+ (*) or CD4+ (t) supernatants or not significant from normal controls (t) are indicated. Values are mean ± SEM, with the number of coverslips analyzed shown in parentheses.

The antibody did not decrease astrocytic cell numbers, due to IFN, we assessed the capability of purified recombi- mitigating against a nonspecific cytotoxic effect. nant human IFN (Rorer Biotechnology or Boehringer Mann- A neutralizing ant ibody to human IL-1 (62.5 gg/ml) did not heim) to evoke a proliferative response on human astrocytes. inhibit the astrocytie response to CD8' supernatants (acti- Despite reports of various interferons being negative regu- vated CD8' supern atant, 17.9 2.4% of astrocytes incor- lators of cell growth (37-40), both preparations of IFN

porating BrdUrd; as:tivated CD8+ supemnatant plus antibody promoted the proliferation of human adult astrocytes in a ± a to to IL-1, 15.9 1.9'7). Similarly, neutralizing antibody dose-dependent manner (Fig. 4). However, the magnitude of TNF (2000 and 10,0)00 NU/ml), another microglia cell prod- uct that can producSe proliferation of bovine calf astrocytes astrocyte proliferative response to IFN (7-fold at best) was (20), did not attenudate the effects of CD8' supernatants. much less than the average 22-fold response caused by To further suppc rt the hypothesis that the proliferative activated CD8' lymphocyte supernatants. Of other cyto- action ofthe activateed CD4t andiCD8s cell supernatantswas kines tested, recombinant human IL-1 was found to be mitogenic for adult human astrocytes; again, the magnitude of the astrocyte response to IL-1 was less than that of PDGF, 20 mU/ml (2) lymphocyte supernatants. IFN Can Act Directly on Astrocytes to Exert Its Proliferative Response. We further addressed whether the mitogenic action PDGF, 100 mU/ml __(5) of IFN on human adult astrocytes was by a direct effect or indirect mechanism. The latter would be that IFN was stimulating microglia cells, also present in culture, to release EGF, 100 ng/ml (5) IL-1 or other cytokines mitogenic for human adult astrocytes. We exposed a human adult culture containing 60% microglia cells to recombinant human IFN at 100 units/ml (to ensure a FGF, 100 ng/ml 1(4) proliferative response) for 6, 24, 48, 72 or 96 h (n = 4 coverslips per time point). The IFN-treated cell-conditioned medium was collected; half(0.75 ml) was promptly incubated IGF-1, 20 ng/ml _(10) with the neutralizing antibody to IFN (100 NU/ml) and the l other half was left untreated. These media were reconstituted

0 1 2 3 4 5 with fresh feeding medium [1:1 (vol/vol)] and added to another series of human cultures containing <10%o microglia Proliferation Index cells. Four days later, cells were immunolabeled for GFAP (fold increase over controls) and BrdUrd. Fig. 5 illustrates that whereas the nonneutral-

FIG. 3. Noncytokmine growth factors (PDGF, EGF, FGF, and ized medium provoked proliferation of astrocytes, the cell- IGF-1) are poor mito~gens for human adult astrocytes. None of the conditioned medium preincubated with the neutralizing an- values is significantly different from controls. mU, milliunits. Num- tibody to IFN did not result in increases of GFAP/BrdUrd bers in parentheses ai re number of coverslips used. double-labeled cells. The results thus favor IFN acting di- Downloaded by guest on October 2, 2021 Neurobiology: Yong et al. Proc. Natl. Acad. Sci. USA 88 (1991) 7019 rh X-Interferon 1 U/ml -H (6) 10 U/ml (3) * I 100 U/ml (13) * J l-~ - 1000 U/ml I (3) *

e rh Interleukin-1 50 mU/ml (10) 500 mU/ml _ _ (14[) * 5 U/ml (4 100 U/ml - E. q (3) * 500 U/ml -] (4) rh Interleukin-2 500 mU/mI --_ (4) b ~~~~~~~~~V-;% ,41 10 U/ml- (6) FIG. 6. GFAP immunoperoxidase staining to assess the extent of 50 U/mI _1 (5) reactive gliosis in vivo in corticectomized adult mice treated with Control Level saline (A and B) or IFN at 100 units/ml (C and D). A and C represent I T- areas abutting the corticectomy (indicated by *); B and D are the 0 1 2 3 4 5 6 7 8 9 10 lateral part of ipsilateral hemisphere of the same coronal section. Note that IFN enhanced the degree ofgliosis close to the cavity (C) Proliferation Index as well as increased the spread of the reactive process (D). (fold increase over controls) (6, 7, 10), the early phase of the reactive gliosis process may FIG. 4. Recombinant human (rh) IFN is a mitogen for human adult astrocytes. Results were pooled from four astrocyte culture actually be an attempt by astrocytes to promote recovery, series. U, unit(s); mU, milliunits. Values significantly different from given the many neurotrophic properties of this cell type that controls are indicated (*). are increasingly being appreciated (41-44). A means to resolve the role of astrocyte reactivity may come from the rectly on human adult astrocytes rather than by a microglia ability to diminish or enhance the gliotic process and to assess intermediary. subsequent functional aspects. This may be achieved by the Modulation of Reactive Gliosis in Vivo by IFN. Having identification of cellular mediator(s) of the reactivity. identified IFN as an important mitogen for adult brain- In this report, we show that supernatants from activated T derived astrocytes in vitro, we examined whether this cyto- lymphocytes can provide the cellular signal to promote the kine might increase the extent of reactive gliosis in vivo. The proliferation of adult human astrocytes in vitro (Fig. 2). This results in Fig. 6 show that IFN significantly enhanced the result is interesting, given that accumulation ofinflammatory extent of reactive gliosis in vivo at the lesion site when mononuclear cells is observed around lesions not only in compared to saline controls. In addition, the degree ofspread inflammatory brain diseases (11-13) but also in traumatic ofreactive gliosis in the ipsilateral hemisphere was extensive; brain injuries, such as those caused by a stab wound (14, 15). an effect not observed for the controls. Although the basal rate of proliferation of adult human astrocytes is low, a finding that correlates well with the low rate of proliferation of astrocytes in adult brain in vivo (45, DISCUSSION 46), activated CD8+ lymphocyte supernatants increased the The common occurrence of reactive gliosis after injuries to extent of proliferation by 22-fold on average. This high the central nervous system poses the question as to the role potency is even more impressive given that noncytokine of the astrocyte changes. Although the end-stage astrocyte growth factors for neonatal rodent astrocytes (EGF, FGF, scars are thought to be impediments to functional recovery PDGF, and IGF-1) (for review, see ref. 47) could at best 25 No IFN antibody Plus anti-IFN 20 a_ U- * FIG. 5. Medium containing IFN (100 units/ 11|* * ml) and previously exposed to cells for 6-96 h | | 0|-- increased BrdUrd (BrdU) incorporation in as- O0 | | trocytes over a 4-day treatment period when m | E compared to normal feeding medium control 15 | | (time 0, no IFN). However, ifthese IFN-treated cell-conditioned media were preincubated with I a neutralizing antibody (100 NU/ml) to IFN, 5 t then their proliferative effects were substan- | 7 t tially reduced. Values significantly different 1 | S t from normal feeding medium without any added X IFN (*) or significantly different from IFN- 0 ._. -.= ._ | treated medium at its respective time period (t) are indicated. All values are mean ± SEM of 24h 48h 72h 96h three or four coverslips. Downloaded by guest on October 2, 2021 7020 Neurobiology: Yong et al. Proc. Natl. Acad. Sci. USA 88 (1991)

evoke a 3-fold increase in proliferation of adult human 3. O'Callaghan, J. P., Miller, D. B. & Reinhard, J. F., Jr. (1990) Brain Res. 521, 73-80. astrocytes (Fig. 3).' Of note, not all astrocytes could be 4. Bignami, A., Eng, L., Dahl, D. & Uyeda, C. (1972) Brain Res. 43, induced to incorporate BrdUrd. This may represent subpop- 429-435. ulations of astrocytes; currently, we are unable to classify 5. Nathaniel, E. J. H. & Nathaniel, D. R. (1981) Adv. Cell. Neurobiol. 2, different types astrocytes 249-301. of adult human other than by 6. Reier, P. J., Stensaas, L. J. & Guth, L. (1983) in Recon- morphology (27). struction, eds. Kao, C. C., Bunge, R. P. & Reier, P. J. (Raven, New The cytokine contained within the activated lymphocyte York), pp. 163-196. supernatants appears 7. Liuzzi, F. J. (1990) Brain Res. 512, 277-283. that important for proliferation of adult 8. Pollen, D. A. & Trachtenberg, M. C. (1970) Science 167, 1252-1253. human astrocytes is IFN. Thus, a neutralizing antibody to 9. Grisar, T. & Delgado-Escueta, A. Y. (1986) Brain Res. 364, 1-11. IFN could completely abrogate the proliferative effect of the 10. Raine, C. S. & Bornstein, M. B. (1970 J. Neuropathol. Exp. Neurol. 29, lymphocyte supernatants (Fig. 2); recombinant IFN alone 552-574. 11. Traugott, U., Reinherz, E. L. & Raine, C. S. (1983) Science 219, showed mitogenic effect on adult human astrocytes (Fig. 4) 308-310. and enhanced the extent of astrocyte reactivity in vivo in the 12. Traugott, U., Scheinberg, L. C. & Raine, C. S. (1985)J. Neuroimmunol. injured adult mouse brain (Fig. 6). The finding that IFN is a 8, 1-14. 13. Cuzner, M. L., Hayes, G. M., Newcombe, J. & Woodroofe, M. N. mitogen for any cell type, to our knowledge, has not been (1988) J. Neuroimmunol. 20, 203-209. reported previously. 14. Giulian, D., Chen, J., Ingeman, J. E., George, J. K. & Noponen, M. The observation that purified IFN could evoke only a (1989) J. Neurosci. 9, 4416-4429. 15. Tsuchihashi, Y., Kitamura, T. & Fujita, S. (1981) Acta Neuropathol. 53, 7-fold increase in the PI of human adult astrocytes whereas 213-219. the neutralizing antibody to IFN could completely inhibit the 16. Fontana, A., Grieder, A., Arrenbrecht, S. & Grob, P. (1980) J. Neurol. 22-fold average effect of CD8 supernatants may relate to the Sci. 46, 55-62. of IFN cytokine contained 17. Merrill, J. E., Kutsunai, S., Mohlstrom, C., Hofman, F., Groopman, J. cooperative'effects and another & Golde, D. W. (1984) Science 224, 1428-1430. within the activated lymphocyte supernatants. Cooperation 18. Benveniste, E. N., Whitaker, J. N., Gibbs, D. A., Sparacio, S. M. & of cytokines, especially with regard to IFN, has been docu- Butler, J. L. (1989) Int. Immunol. 1, 219-228. mented. Thus, whereas IFN or TNF was unable to induce 19. Giulian, D. & Lachman, L. B. (1985) Science 228, 497-499. 20. Selmaj, K. W., Farooq, M., Norton, W. T., Raine, C. S. & Brosnan, major histocompatibility complex class II on human C. F. (1990) J. Immunol. 144, 129-135. islet cells, their combination could (48). Similarly, IFN and 21. Barna, B. P., Estes, M. L., Jacobs, B. S., Hudson, S. & Ransohoff, IL-2 act synergistically to induce selective monokine secre- R. M. (1990) J. Neuroimmunol. 30, 239-243. tion in murine 22. Berry, M., Maxwell, W. L., Logan, A., Mathewson, A., McConnell, P., peritoneal macrophages (49). Ashhurst, D. E. & Thomas, G. H. (1983) Acta Neurochir. Suppl. 32, Is the proliferative effect of IFN a direct mechanism or an 31-53. indirect mechanism mediated by the release of microglia 23. Barrett, C. P., Donati, E. J. & Guth, L. (1984) Exp. Neurol. 84, 374-385. intermediaries? Our data suggest that a direct mechanism 24. Smith, G. M. & Silver, J. (1988) Prog. Brain Res. 78, 353-361. 25. Yong, V. W., Sekiguchi, S., Kim, M. W. & Kim, S. U. (1988) J. does exist, given the lack of correlation between the propor- Neurosci. Res. 19, 187-194. tion of microglia cells in culture and the magnitude of the 26. Grenier, Y., Ruijs, T. C. G., Robitaille, Y., Olivier, A. & Antel, J. P. CD8' response (Fig. 2). In addition, the 'astrocyte- (1989) J. Neuroimmunol. 21, 103-115. proliferation-inducing activity of supernatants of glial cul- 27. Yong, V. W., Yong, F. P., Olivier, A., Robitaille, Y. & Antel, J. P. (1990) J. Neurosci. Res. 27, 678-688. tures treated with IFN can be completely blocked with 28. Giulian, D. & Baker, T. J. (1986) J. Neurosci. 6, 2163-2178. IFN-neutralizing antibody, suggesting that microglia cells or 29. Tansey, F. A. & Brosnan, C. F. (1982) J. Neuroimmunol. 3, 169. their cytokine products were not required for an astrocyte 30. Thiele, D. L., Kurosaka, M. & Lipsky, P. E. (1983) J. Immunol. 131, proliferative response to occur. 2282-2290. 31. Freedman, M. S., Ruijs, T. C. G., Blain, M. & Antel, J. P. (1991) Cell. 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