Granulocyte-macrophage colony stimulating factor inhibits class II major histocompatibility complex expression and antigen presentation by microglia

Journal of Neuroimmunology, 48 (1993) 23-32 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-5728/93/$06.00

23

JNI 02444

Granulocyte-macrophage colony stimulating factor inhibits class II major histocompatibility complex expression and antigen presentation by microglia M a s a h a r u Hayashi, Martin E. D o f f and Sara A b r o m s o n - L e e m a n * Harvard Medical School, Department of Pathology, 200 Longwood Avenue, Boston, MA 02115, USA

(Received 15 March 1993) (Revision received 27 April 1993) (Accepted 27 April 1993)

Key words: Granulocyte-macrophage colony stimulating factor; Regulation of immune responses; Glial cells; Major histocompatibility complex antigens; Experimental autoimmune encephalitis

Summary Granulocyte-macrophage colony stimulating factor (GM-CSF) modulates various functions of monocytes/ macrophages including antigen-presenting capacity. Recently it was found that astrocytes produce GM-CSF in the central nervous system (CNS) and that GM-CSF can induce proliferation and morphological changes of microglia. Here we show that GM-CSF can down regulate the interferon-y-mediated induction of major histocompatibility complex (MHC) class II antigens in microglia, but not in astrocytes. GM-CSF pretreatment completely prevents myelin basic protein-specific T cell proliferation induced by microglia but not astrocytes. GM-CSF did not affect the cell surface expression on microglia of either MHC class I or cell adhesion molecules. The inhibition of microglial MHC class II expression and antigen-presenting function is specific for GM-CSF, as treatment with a different CSF (interleukin-3) did not modulate microglial phenotype or functional capacity. These data suggest that GM-CSF might be involved in the regulation of immune responses within the central nervous system.

Introduction Experimental autoimmune encephalomyelitis (EAE) is an autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). The disease can be induced in a variety of species'by immunization with CNS myelin antigens or by adoptive transfer of myelin antigen-specific, CD4 + T cells (Ben-Nun et al., 1981; Alvord et al., 1984). The potential involvement of cytokines in the pathogenesis as well as the resolution of EAE is supported by results from several in vivo studies which suggest that multiple cytokines can influence EAE. Systemic treatment of S J L / J mice with anti-TNF antibodies prevented transfer of clone-mediated EAE (Ruddle et al., 1990), while neutralizing mAb against interferon (IFN)-y caused an increase in

* Corresponding author.

morbidity rates of EAE in C57BL/6J mice of low susceptibility (Billiau et al., 1988). Administration of TGF-/31 alleviated the clinical signs in chronic relapsing EAE (Racke et al., 1991). The clinical course of EAE is accompanied by several temporally distinct patterns of interleukin (IL)-la, IFN-y and IL-10 mRNA expression within the CNS (Kennedy et al., 1992). In the CNS, microglia and astrocytes can be induced to secrete cytokines such as IL-1, IL-6 and TNF-a (Giulian et al., 1986; Frei et al., 1989; Lieberman et al., 1989; Sawada et al., 1989). Recent studies indicate that both human and murine cultured astrocytes stimulated by LPS, IL-1/3 or TNF-a can secrete GM-CSF (Malipiero et al., 1990; Ohno et al., 1990; Aloisi et al., 1992). Furthermore, GM-CSF has been found to stimulate proliferation of (Frei et al., 1987; Giulian, 1987; Ganter et al., 1992), and to induce morphological changes in purified murine microglia; GM-CSF-stimulated microglia become rod-shaped and elongated (Suzumura et al., 1990, 1991).

24 GM-CSF stimulates a broad range of functional activities of monocytes/macrophages including their tumoricidal activity, accessory cell function, phagocytosis and oxidative metabolism (Ruef et al., 1990). GMCSF augments the expression of H-2A molecules and the antigen-presenting function in murine bone marrow-derived macrophages (Fischer et al., 1988). GMCSF selectively increases HLA-DR and HLA-DP, but not HLA-DQ, expression in human monocytes (Gerrard et al., 1990). By contrast, GM-CSF is reported to down-regulate surface expression of the IFN-y receptor (Fischer et al., 1990) and leukocyte adhesion molecules-1 (LAM-1) on human monocytes (Griffin et al., 1990). Thus GM-CSF can modify the antigen-presenting function of monocytes/macrophages. Astrocytes and microglia in the CNS can be induced by IFN-y to upregulate expression of major histocompatibility complex (MHC) class II molecules and to present antigens to T lymphocytes (Fontana et al., 1984; Fierz et al., 1985; Frei et al., 1987). Similarly, treatment of astrocytes with IFN-y results in increased ICAM-1 expression in humans, mice and rats (Frohman et al., 1989; Satoh et al., 1991; Kraus et al., 1992). Nevertheless, it has not yet been determined whether cytokines other than IFN-y can modulate antigen-presenting capacities of glial cells. The present study attempts to investigate the effects of GM-CSF and IL-3 on the in vitro presentation of antigen by glial cells. Here we demonstrate that GMCSF downregulates the IFN-y-induced expression of MHC class II antigens in microglia, but not in astrocytes, and thus precludes the presentation of antigen to MBP-specific T cell clones by microglia, despite the fact that proliferation and morphological changes are induced in the microglia. This inhibitory effect was not observed with IL-3. GM-CSF has no effect on the surface expression of either MHC class I or cell adhesion molecules. Inasmuch as both astrocytes and MBP-specific T cell clones can produce GM-CSF, GMCSF may play a role in regulating the pathogenesis of EAE.

Materials and methods

Mice B A L B / c and S J L / J mice were purchased from Harlan Laboratories (Indianapolis, IN) and Jackson Laboratories (Bar Harbor, ME), respectively. Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School, and the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources. National Research Council (Department of Health and Human Services Publication 85-23, revised 1985).

Antigens and cytokines Purified guinea pig and rat myelin basic proteins were prepared at the Center for Neurologic Diseases of the Brigham and Women's Hospital, Boston, MA. Peptides of mouse myelin basic protein were generously provided by Dr. Ariel Miller (Center for Neurologic Diseases of the Brigham & Women's Hospital, Boston, MA) and by Drs. Jeff Alexander and Alessandro Sette (Cytel, San Diego, CA). Subsequent to the initial screening of peptide specificity, a second batch of mouse MBP peptide 137-155 was synthesized by Dr. Charles Dahl, Harvard Medical School, Boston, MA. Recombinant mouse IFN-y was purchased from Collaborative Biomedical Products, Bedford, MA. Recombinant mouse GM-CSF and IL-3 were purchased from Genzyme Corporation, Cambridge, MA. Antibodies Hybridoma cell lines 34-1-2S, MK-D6, 14-4-4S, M17/4.2, YN1/1.7.4 and M1/70.15.11.5.HL, secreting antibodies to H-2D d, I-Ad, I-E ~, LFA-la, ICAM-1 and Mac-l, respectively, were purchased from ATCC. A rabbit mAb to mouse glial fibrillary acidic protein was purchased from Dako, Santa Barbara, CA. FITClabeled goat anti-rat and anti-rabbit immunoglobulins were purchased from Caltag, San Francisco, CA. PElabeled donkey anti-mouse immunoglobulin was purchased from Jackson ImmunoResearch Laboratories, West Grove, PA. MBP-specific T cell clones The establishment of MBP-specific T cell clones used here has been described previously (AbromsonLeeman et al., 1993). Briefly, B A L B / c mice were immunized with an emulsion containing 200 /~g of guinea pig MBP and 50/xg of Mycobacterium tuberculosis H37RA in incomplete Freund's adjuvant (Difco Laboratories, Detroit, MI). Cell suspensions from draining lymph nodes, removed 10 days after immunization, were cultured for 1 week with 25 p.g ml -~ of mouse MBP in complete DME (DME containing 10% FCS). Two days later, viable cells were collected by centrifugation using Ficoll-Hypaque and recultured with syngeneic irradiated spleen cells and 25 ~g ml-1 of mouse MBP in DME. Eight days after restimulation, cells from each line were cloned by limiting dilution. Preparation of microglia and astrocytes The bulk isolation of glial cells was from neonatal mouse brains according to the methods of Suzumura et al. (1984, 1987) with slight modifications. After removal of the meninges, single cell suspensions were prepared by passing brains through nylon mesh of pore size 112 /~m. The primary glial culture was maintained in 75-cm 2 flasks in MEM (minimum essential medium) supple-

25

Growth assay for microglial cells

IL-3 (50 U/ml)

IL-3 (5 U/ml)

Cell p r o l i f e r a t i o n was a s s a y e d using 2 x 10 4 microglial cells c u l t u r e d in 96-well m i c r o t i t e r p l a t e s ( F a l c o n ) . T h e cells w e r e i n c u b a t e d for 5 days in 200 # l o f m e d i u m c o n t a i n i n g r e c o m b i n a n t m o u s e colony stimu l a t i n g factors. F o r t h e final 18 h o f i n c u b a t i o n , t h e cells w e r e p u l s e d with 1 /zCi [3H]thymidine. A f t e r h a r v e s t i n g with an a u t o m a t i c harvester, r a d i o a c t i v i t y was d e t e r m i n e d with a liquid scintillation c o u n t e r .

D

tL-3 (0 5 U/ml) GM-CSF (50 U/ml) GM-CSF (5 U/ml) GM-CSF {0 5 U/ml) none • 0

, 2000

•

, 4000

•

, 8000

•

,

8000

•

,

10000

, 12000

cpm

Fig. 1.3H-thymidine uptake by microglia treated with the indicated doses of mouse rlL-3 or GM-CSF for 5 days. Data are shown as mean + SD of triplicate cultures.

m e n t e d with 10% f e t a l calf s e r u m , 2 m M g l u t a m i n e , 2 m g ml -~ glucose, 5 / z g m1-1 insulin, 50 U m1-1 p e n i cillin a n d 50 /.,g ml -~ s t r e p t o m y c i n in 10% C O 2 at 37°C. A f t e r 14 days, t h e flasks w e r e a g i t a t e d on an o r b i t a l s h a k e r ( L a b - L i n e O r b i t - S h a k e r , L a b L i n e Ins t r u m e n t s Inc., I L ) for 1 h at 250 r p m at 37°C. S u p e r n a t a n t m e d i a c o n t a i n i n g d e t a c h e d cells was p a s s e d t h r o u g h nylon m e s h of p o r e size 3 5 / x m a n d i n c u b a t e d in a n u n c o a t e d p e t r i dish in 10% C O 2 for 20 m i n at 37°C to allow m i c r o g l i a to attach. T h e u n a t t a c h e d cells w e r e r e m o v e d , a n d t h e a d h e r e n t cells w e r e c o l l e c t e d with a cell s c r a p e r a n d w e r e p l a t e d in c u l t u r e vessels. A s t r o c y t e s w e r e p u r i f i e d by 2 - 3 r e p e t i t i o n s o f t r y p s i n i z a t i o n a n d r e p l a t i n g o f t h e p r i m a r y glial cell culture. T h e p u r i t i e s o f t h e m i c r o g l i a a n d a s t r o c y t e s w e r e > 95%, as d e t e r m i n e d by i n d i r e c t i m m u n o f l u o r e s c e n c e with a n t i - M a c - 1 a n d a n t i - G F A P a n t i b o d i e s , respectively.

Flow cytofluorographic analysis G l i a l cells w e r e r e m o v e d f r o m c u l t u r e flasks o r dishes by s t a n d a r d t r y p s i n - E D T A t r e a t m e n t o r by scraping, a n d w a s h e d in c o l d P B S 0.1% B S A 10 m M s o d i u m azide. T h e y w e r e i n c u b a t e d with 1 m g m l - 1 h u m a n g a m m a - g l o b u l i n (Sigma) for 10 m i n at r o o m t e m p e r a t u r e to b l o c k any p o t e n t i a l F c r e c e p t o r b i n d ing. U n l a b e l e d m o u s e o r r a t m A b ( n e g a t i v e c o n t r o l or test) was a d d e d to s a m p l e s o f 5 × 105 cells in t h e p r e s e n c e of 300 /.~g of h u m a n g a m m a - g l o b u l i n for 35 min on ice, t h e n cells w e r e w a s h e d , r e s u s p e n d e d in P E - c o n j u g a t e d d o n k e y a n t i - m o u s e Ig o r g o a t a n t i - r a t Ig, a n d i n c u b a t e d on ice for a f u r t h e r 35 min. A f t e r t h r e e washes, 5 × 103 live cells w e r e a n a l y z e d on a C o u l t e r Profile II flow c y t o m e t e r .

Antigen presentation by glial cells to T cell clones C u l t u r e s w e r e set u p in a 200-/~1 v o l u m e in flat-bott o m e d m i c r o t i t e r wells ( F a l c o n ) in triplicate. F o r the i n d u c t i o n o f M H C class II antigens, 100 U ml - I o f r e c o m b i n a n t I F N - y was a d d e d to glial cell c u l t u r e s for 2 - 3 days p r i o r to c o c u l t u r e with T cells. M i c r o g l i a a n d a s t r o c y t e s i r r a d i a t e d with 4000 r a d w e r e c o - c u l t u r e d at

TABLE 1 The effect of GM-CSF on surface expression of MHC antigens and cell adhesion molecules Glial cells

Pretreatment a

Treatment b

Positive cells c H-2D d

I-A a

ICAM-1

LFA-lt~

Microglia Microglia Microglia Microglia Microglia Microglia

None None None GM-CSF 2 U ml-1 GM-CSF 10 U ml- 1 GM-CSF 50 U ml-1

None IFN-y IFN-y, GM-CSF IFN-y IFN-y IFN-y

58.3% 77.6% 77.3% 78.9% 73.0% 70.3%

2.5% 59.2% 43.7% 17.1% 18.5% 17.8%

0.7% 4.8% 6.1% 0.0% 0.0% 0.0%

80.9% 87.7% 88.9% NT 73.4% NT

48.6% 75.9% 71.8% NT 73.2% NT

Astrocyte Astrocyte Astrocyte Astrocyte

None None None GM-CSF 10 U/ml

None IFN-y IFN-y, GM-CSF IFN-y

58.4% 71.6% 65.8% 71.2%

3.1% 43.5% 51.7% 47.7%

1.9% 37.7% 40.8% 39.2%

30.2% 47.4% 54.0% 54.6%

3.1% 2.7% 4.2% 4.0%

I-E d

BALB/c glial cells were preatreated with mouse rGM-CSF for 3 days. GM-CSF was then washed out before treatment with IFN-y. b Glial cells were treated with 100 U m1-1 of mouse rlFN-y in the presence or absence of GM-CSF for 2-3 days. c Glial cells were prepared for flow cytometric analysis as described in Materials and methods• Percentage of positive cells was calculated by subtracting background staining. Generally, control staining was < 5%. Data from one selected experiment are presented. The mean expression of I-A d on microglia in four separate experiments were 2.3 5: 1.5%, 39.8 + 14.1%, and 17.1 + 4.5% for no treatment, IFN-y alone, and GM-CSF pretreatment followed by IFN-y, respectively (P < 0.05). a

26 0 . 5 - 1 × 104 c e l l s / w e l l with 5 × 104 c l o n e d T cells a n d t h e a p p r o p r i a t e d o s e o f M B P o r its p e p t i d e f r a g m e n t s in c o m p l e t e D M E for 48 h. F o r t h e final 16 h o f

i n c u b a t i o n , cells w e r e p u l s e d with 1 / z C i / w e l l of [3H]thymidine. A f t e r harvesting, r a d i o a c t i v i t y was m e a s u r e d in a liquid b e t a scintillation c o u n t e r . I n s o m e

Fig. 2. Morphological changes in microglia treated with GM-CSF. Isolated microglia (BALB/c-derived) at 2 × 105 cells ml- 1 were incubated in Petri dishes for 3 days without cytokines (top panel) or with 10 U ml-1 of mouse rGM-CSF (bottom panel). Unstimulated microglia were morphologically heterogeneous. Some had short processes. Microglia treated with GM-CSF became enlarged and rounded.

27

experiments, monoclonal anti-Ia, anti-LFA-la or antiICAM-1 hybridoma supernatants were added to microtiter wells at dilutions of 1 / 4 - 1 / 8 .

TABLE 2 The effect of IL-3 on mouse glial cell MHC class II antigen expression Glial

Pretreatment a

Treatment b

cells

Results

GM-CSF stimulates microglial proliferation Both GM-CSF and IL-3 are reported to be potent mitogens for microglia (Frei et al., 1987; Giulian, 1987; Ganter et al., 1992) and to cause changes in microglial morphology (Suzumura et al., 1990). The proliferative responses of highly purified (> 95%) microglia to stimulation with GM-CSF and IL-3 were tested. After 5 days of culture, GM-CSF caused a 5-15-fold increase in [3H]thymidine uptake by microglia, but IL-3 stimulated only weak and inconsistent (2-3-fold) proliferative responses (Fig. 1). In addition, microglia treated with GM-CSF demonstrate morphological changes, becoming enlarged and losing dendritic processes (Fig. 2). Pretreatment of microglia but not astrocytes with GMCSF down-regulates IFN-y-induction of MHC class H antigens The expression of surface class II MHC gene products on B A L B / c glial cells was determined by FACS analysis. In the absence of IFN-y, few MHC class II + glial cells were detected ( < 5%). Glial cultures were treated for 3 days with IFN-y (100 U ml-l), then stained with anti-Ia antibodies. Generally, 40-50% of astrocytes expressed I-A and I-E molecules, while 2560% of microglia expressed I-A. Induction of I-E antigens on microglia was extremely low ( < 15%) but consistently above controls (Table 1). Pretreatment of microglia with 2, 10 or 50 U ml-l GM-CSF for 3 days prior to addition of IFN-y reduced the IFN-y-induc-

Microglia Microglia Microglia Microglial Microglia Microglia

Positive cells c H-2D d l-Ad

None None 31.6% None IFN-y 62.1% None IFN-T, GM-CSF 55.3% None IFN-y, IL-3 62.5% GM-CSF 10 U ml I IFN-y 77.0% IL-3 10 U m1-1 IFN-y 56.1%

6.0% 30.1% 23.3% 32.7% 6.4% 25.7%

~,b,c See legends to Table 1.

tion of MHC class II (Table 1 and Fig. 3D). In contrast, GM-CSF pretreatment had no effect on IFN-yinduced class II expression in astrocytes (Table 1). The inhibitory effect of GM-CSF is optimal when GM-CSF is added 2-3 days before treatment with IFN-y (data not shown); only slight effects are noted when IFN-y and GM-CSF are added simultaneously (Table 1). Preincubation with GM-CSF does not significantly inhibit IFN-y augmentation of MHC class I expression on either microglia or astrocytes (Table 1 and Fig. 3C).

GM-CSF does not affect IFN-y induction of cell adhesion molecules in glial cells Cell adhesion molecules such as LFA-1 and ICAM-1 can also be induced on the surface of APC and may be involved in co-stimulation of T cells (Springer, 1990). We therefore investigated the effects of CSF on the surface expression of these molecules on glial cells. 50% of microglia expressed LFA-lo~, which was augmented to 70% following IFN-y treatment. 30-40% of astrocytes constitutively expressed ICAM-1 which could be augmented to 50-60% after treatment with IFN-y.

TABLE 3 Preincubation of microglia with GM-CSF inhibits APC function T cell clone

Antigenpresenting cell (APC)

Pretreatment of APC with GM-CSF a

Addition of anti-Ia b

D1 D1 D1

Microglia Microglia Microglia

+

1E2 1E2 1E2

Microglia Microglia Microglia

+

[3H]thymidine incorporation (cpm) c No antigen

MBP peptide d

Rat MBP d

+ -

1377 + 882 344 + 130 1419 + 688

20928 + 1315 608 + 50 2 626 + 1 472

17160 + 1867 479 + 15 3 248 _+ 1 117

+ -

508 + 35 256 + 182 210 + 59

12167 + 3 141 594 + 342 300_+ 165

15 553 + 2 601 497 + 258 932 + 273

Microglia were pretreated with 10 U m l - 1 of GM-CSF. After 2 days, GM-CSF was removed and cells were cultured with 100 U m l - i of IFN-y for an additional 3 days. b For blocking experiments, conditioned medium from hybridoma cell line 14-4-4S, anti-I-E, was added at a dilution of 1 : 4 from the beginning of T cell clone and microglia co-culture. c 5 × 104 MBP-specific T cell clones from BALB/c were cultured for 48 h with 1 × 104 mmroglia treated with IFN-T and then irradiated with 4000 rad. [3H]thymidine uptake was measured for the final 16 h of incubation. Data are shown as mean + SD of triplicate cultures. a Clone D1 was incubated with microglia in the presence of 1/.~g m l - I of mouse MBP 137-155 peptide or of 10 ~ g m l - 1 of rat MBP. Clone 1E2 was cultured with APC in the presence of 10/.~g m1-1 of either mouse MBP 141-160 peptide or rat MBP. a

28

Importantly, pretreatment of glial cells with GM-CSF failed to change the level of expression of ICAM-1 or LFA-la (Table 2), establishing the specificity of this effect for class II MHC molecules.

Comparison of GM-CSF and 1L-3 on microglial expression of MHC class II antigens To determine whether this inhibitory effect on the induction of class II MHC antigens in microglia is

C

Z

E

F

F l. IJ() It I~ ~ C E N C I ' . Fig. 3. GM-CSF antagonized IFN-~/-induced MHC class II antigen induction in microglia. 2 × 105 microglia were seeded into Petri dishes and cultured in media ( ~ B), 10 U ml - l GM-CSF (C, D) or 10 U m l - I IL-3 (E, F) for 48 h. GM-CSF or IL-3 was removed by extensive washing, and then 100 U m l - 1 IFN-y was added to each group. 3 days later, cells were scraped and analyzed for expression of MHC class I antigen with antibody 34-1-2S ( ~ C, E) or I-Ad with antibody MK-D6 (B, D, F), by flow cytometry. Data from a single representative experiment is shown.

29

common to colony stimulating factors, we tested the effect of IL-3 on the induction of class II antigens in glial cells. Pretreatment with 1L-3 failed to inhibit the induction of class II antigens on microglia (Table 3 and Fig. 3F) or astrocytes (data not shown) by IFN-~,. Neither GM-CSF alone nor IL-3 alone induced MHC class II antigens or affected MHC class I or ICAM-1 expression on glial cells (data not shown).

GM-CSF inhibits antigen-presenting cell function in microglia BALB/c-derived MBP-specific MHC class II-restricted T cell clones D1, 1E2 and 2C2 were incubated with antigen and cytokine-treated syngeneic B A L B / c glial cells. Antigen-dependent T cell proliferation is obtained with IFN-3,-treated microglia, but virtually no stimulation of T cell clones is observed with untreated glial cells (data not shown). Cultures were stimulated with varying amounts of either rat MBP or mouse MBP peptide fragments. Results of a representative experiment are shown in Table 3. The proliferative responses of D1 and 1E2 clones to IFN-y-stimulated microglia were completely blocked with anti-Ia antibody, demonstrating the requirement for class II recognition. Next, the effect of GM-CSF pretreatment on antigen-presenting capacity of microglia was examined. Pretreatment of microglia with GM-CSF for 2 days prior to incubation with IFN-y abolished the presentation of MBP peptide 137-155 and MBP to clone D1, and that of MBP peptide 141-160 and MBP to clones 1E2 and 2C2 (Tables 3 and 4). In contrast, pretreatment with IL-3 did not affect antigen presentation to clones D1

and 2C2 by IFN-3,-treated microglia (Table 4). Finally, no inhibition of T cell proliferative responses to antigens presented by IFN-,/-treated astrocytes was observed after GM-CSF pretreatment (Table 4). Discussion

GM-CSF has been reported to induce the de novo synthesis of H-2A molecules and enhance the antigenpresenting capacity of murine bone marrow macrophages (Fischer et al., 1988) as well as HLA-DR and HLA-DP expression in human monocytes (Gerrard et al., 1990). Recently, Lee et al. (1993) reported that CSF-1 (M-CSF) reduced the basal expression of HLADR on human fetal microglia and that GM-CSF also had a slight down regulatory effect on microglial HLADR. CSF-1 had a less pronounced effect on IFN-~/-induced HLA-DR expression. In the present study, pretreatment of murine microglia with GM-CSF antagonized IFN-y-induction of MHC class II antigen and completely abolished antigen presentation by microglia treated with IFN-y (Tables 1 and 3). Inasmuch as astrocytes can produce GM-CSF and M-CSF in the CNS (Malipiero et al., 1990; Aloisi et al., 1992; Lee et al., 1993), CSFs may play an important role in modulating development and function of microglia in pathological processes within the CNS. MHC class II antigen induction by IFN-~ was shown to be selectively down-regulated by neurotransmitters such as norepinephrine and glutamate (Frohman et al., 1988; Lee et al., 1992) as well as reagents known to activate intracellular cAMP and protein kinase C

TABLE 4 Effects of G M - C S F and IL-3 on A P C function of glial cells T cell clone

Antigenpresenting cell (APC)

Pretreatment of A P C with CSF a

[3H]thymidine incorporation (cpm) b No antigen

MBP peptide c

rat MBP c

D1 D1 D1

Microglia Microglia Microglia

None GM-CSF IL-3

508 + 202 1013 -+ 974 190 + 54

7 8 6 4 + 2176 1388 5: 513 1 3 2 1 6 + 1621

2894+ 469 + 2347__+

586 274 482

D1 D1 D1

Astrocytes Astrocytes Astrocytes

None GM-CSF IL-3

2 728 -+ 362 3 219 -+ 572 2 201 _+ 404

8109 + 484 15 553 __+ 2 999 15628__+ 1159

4 702 + 4 263 __+ 4404_+

327 64 61

2C2 2C2 2C2

Microglia Microglia Microglia

None GM-CSF IL-3

579 + 288 816 _+ 326 1666 __+382

23591 _+ 18804 1352 __+ 197 32506 + 3214

2C2 2C2 2C2

Astrocytes Astrocytes Astrocytes

None GM-CSF IL-3

3 325 -+ 954 2 631 + 664 2 374 + 212

10 738 _+ 2 276 13816__+ 2336 2 2 7 4 8 + 1518

60904 + 7739 2 021 -+ 523 55807 -+ 11022 6 946 + 6913-+ 8320-+

770 409 375

a Microglia were pretreated with 10 U m l - 1 of G M - C S F or IL-3. After 2 days, G M - C S F or IL-3 was removed and cells were cultured with 100 U m l - i of IFN-y for an additional 3 days. b See legend 'c' to Table 3. c Clone D1 was incubated with microglia in the presence of 10/~g m1-1 of either m o u s e M B P 137-155 peptide or rat MBP. Clone 2C2 was cultured with A P C in the presence of 2 0 / z g m1-1 of m o u s e MBP 141-160 peptide or 10/~g m1-1 of rat MBP.

30 (Sasaki et al., 1990) on astrocytes but not on microglia. Inhibition of expression of HLA-DR by CSF-1 was observed for microglia, but not astrocytes (Lee et al., 1993). These observations indicate that regulation of MHC class II antigen expression on astrocytes may differ from that of microglia. Here, GM-CSF is shown to selectively inhibit the IFN-y induction of MHC class II antigens and antigen-presenting capacity of microglia, but not astrocytes. The mechanism of this inhibitory effect is not clear. In human monocytic cell lines or freshly isolated monocytes, 24 h to 48 h of GM-CSF treatment down-regulated IFN-y receptor expression (Fischer et al., 1990). Fischer et al. (1993) recently reported that microglia isolated from neonatal mouse brain cultures of mixed glial cells grown in GM-CSF for 12-14 days are very efficient inducers of antigen-specific T cell proliferation independent of IFN-y. The inclusion of GM-CSF early in the culture of mixed glial cells presumably affects that differentiation of microglia and possibly other cells that may interact with them, resulting in a population with different characteristics. Data presented here show that pretreatment of microglia with GM-CSF did not affect the IFN-3,-induced augmentation of MHC class I, ICAM-1 and L F A - l a expression (Table 1). Even after IFN-y induction, H-2E expression on microglia was very low (< 15%), whereas that of H-2A antigens was 30-60% (Table 1). However, the H-2E molecules are functional since the MBP-specific BALB/c T cell clones used in this study recognize antigen in association with H-2E d (Abromson-Leeman et al., 1993), and their proliferative responses were completely blocked with anti-I-E antibody (Table 3). B A L B / c IFN-y-induced microglia express lower levels of H-2E d than H-2Ad, however, astrocytes can express comparable levels of these MHC class II molecules. Pretreatment with GM-CSF can abolish phenotypic and functional expression of these class II molecules on microglia but not on astrocytes (Table 4). The interaction between the TCR complex and antigen presented in the context of class II molecules on APC may be insufficient for T cell activation; participation of additional cell-surface molecules that mediate adhesion and signal transduction may be required for optimal activation (Schwartz, 1990; Springer, 1990). ICAM-1 is readily up-regulated on astrocytes with proinflammatory stimuli (Frohman et al., 1989; Satoh et al., 1991; Kraus et al., 1992). Until recently, little information on the surface expression of adhesion molecules on microglia was available. We found that microglia constitutively express ICAM-1 and LFA-la, both of which are augmented by IFN-y (Table 1). These may function as accessory molecules for antigen presentation. We failed to detect ICAM-2 on B A L B / c glial cells (data not shown). GM-CSF is reported to induce rapid and complete loss of leukocyte adhesion

molecule-1 (LAM-I) in human monocytes, and the loss of LAM-1 was temporally correlated with up-regulation of another adhesion molecule, C D l l b / C D 1 8 (Griffin et al., 1990). In this study, GM-CSF did not affect either basal expression or IFN-~/-induction of ICAM-1 or L F A - l a by glial cells (Table 1). Rio-Hortega proposed that microglia were found in two predominant forms, the ameboid, or macrophagelike, cell and the ramified, or process-bearing, type (Rio-Hortega, 1932). Ramified microglia are generally considered to be resting cells, which revert to an ameboid form when they are activated in the case of injury or inflammation in the CNS (Giulian, 1987). Although it is known that IL-3 also stimulates rat microglia to proliferate in the same way as GM-CSF (Giulian, 1987; Ganter et al., 1992), IL-3 does not affect the morphology or enzymatic activity and has little or no effect on the proliferation of murine microglia (Suzumura et al., 1991). We confirmed that GM-CSF was mitogenic for microglia (Fig. 1), and that microglia treated with GMCSF acquired an ameboid appearance (Fig. 2). It is intriguing that while GM-CSF appears to activate microglia, as judged by their proliferation and morphological changes, it also inhibits IFN-y-induced immunological function as judged by class II expression and presentation of antigen to T cells. Fontana postulated that astrocytes might play a major role in autoimmune reactions within the CNS (Fontana et al., 1987). The amount of MHC class II induced by IFN-y on astrocytes in vitro correlated with susceptibility to EAE in vivo (Massa et al., 1987). However, in situ class II MHC expression during EAE is restricted to microglia (Konno et al., 1989) and astrocytes appear to limit microglia proliferation (Matsumoto et al., 1992). Furthermore, it was demonstrated by bromodeoxyuridine staining in situ that the proliferation of microglia was continuous throughout the course of EAE, whereas that of T cells decreased rapidly at a later stage of the disease (Ohmori et al., 1992). These findings suggest that microglia, but not astrocytes, might initiate or augment T cell proliferation in an antigen-specific manner in EAE. Therefore, the mechanisms regulating immunological functioning of microglia may be of central importance to understand the susceptibility to EAE. GM-CSF is predominantly produced by astrocytes when stimulated with LPS or TNF-a (Malipiero et al., 1990; Aloisi et al., 1992). In addition, most MBPspecific T cell clones can produce GM-CSF (Martin, Kuchroo, Abromson-Leeman, unpublished data). These findings suggest that GM-CSF may play an important role in modulating EAE. We can speculate that the inhibitory effect of GM-CSF on APC function of microglia may be one of the mechanisms for down-regulation of the onset of immunological disease in the CNS.

31

Acknowledgements We wish to thank Drs. Howard Weiner, Ariel Miller, Ahmad AI-Sabbagh, Alessandro Sette and Jeff Alexander for generously providing MBP and MBP peptides for this study. This study was supported by NIH Grant CA56057 and a gift from the Multiple Sclerosis Foundation.

References Abromson-Leeman, S.R., Hayashi, M., Sobel, R., AI-Sabbagh, A., Weiner, A. and Dorf, M.E. (1993) T cell responses to myelin basic protein in experimental autoimmune encephalomyelitis-resistant BALB/c mice. J. Neuroimmunol. 45, 89-102. Aloisi, F., Care, A., Borsellino, G., Gallo, P., Rosa, S., Bassani, A., Cabibbo, A., Testa, U., Levi, G. and Peschle, C. (1992) Production of hemolymphopoietic cytokines (IL-6, IL-8, colony stimulating factors) by normal human astrocytes in response to IL-l-beta and tumor necrosis factor-alpha. J. Immunol. 149, 2358-2366. Alvord, E.C., Kies, M.W. and Suckling, A.J. (1984) Experimental allergic encephalomyelitis - a useful model for multiple sclerosis. Proceedings of clinical biology research, vol 146. New York, NY: Alan R. Liss Inc. Ben-Nun, A., Wekerle, H. and Cohen, I.R. (1981) The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur. J. Immunol. 11, 195-199. Bernard, C.C.A. (1976) Experimental autoimmune encephalomyelitis in mice: Genetic control of susceptibility. J. Immunogen. 3, 263-274. Billiau, A., Heremans, H., Vandeckerckhove, F., Dijkmans, R., Sobis, H., Meulepas, E. and Carton, H. (1988) Enhancement of experimental allergic encephalomyelitis in mice by antibodies against IFN-gamma. J. Immunol. 140, 1506-1510. del Rio-Hortega, P. (1932) Microglia. In Cytology and Cellular Pathology of the Nervous System, W. Penfield, ed. New York, NY: Hocker, pp. 481-584. Fierz, W., Endler, B., Reske, K., Wekerle, H. and Fontana, A. (1985) Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation. J. Immunol. 134, 3785-3793. Fischer, H.G., Frosch, S., Reske, K. and Reske-Kunz, A.B. (1988) Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function. J. Immunol. 141, 3882-3888. Fischer, H.G., Nitzgen, B., Germann, T., Degitz, K., Daubener, W. and Hadding, U. (1993) Differentiation driven by granulocytemacrophage colony-stimulating factor endows microglia with interferon-3,-independent antigen presentation function. J. Neuroimmunol. 42, 87-96. Fischer, T., Wiegmann, K., Bottinger, H., Morens, K., Burmester, G. and Pfizenmaier, K. (1990) Regulation of IFN-gamma receptor expression in human monocytes by granulocyte-macrophage colony-stimulating factor. J. Immunol. 145, 2914-2919. Fontana, A., Fierz, W. and Wekerle, H. (1984) Astrocytes present myelin basic protein to encephalitogenic T-cell lines. Nature 307, 273-276. Fontana, A., Frei, K., Bodmer, S. and Hofer, E. (1987) Immunemediated encephalitis: On the role of antigen-presenting cells in brain tissue. Immunol. Rev. 100, 185-201. Frei, K., Malipiero, U.V., Leist, T.P., Zinkernagel, R.M., Schwab,

M.E. and Fontana, A. (1989) On the cellular source and function of interleukin 6 produced in the central nervous sytem in viral diseases. Eur. J. Immunol. 19, 689-694. Frei, K., Siepl, C., Groscurth, P., Bodmer, S., Schwerdel, C. and Fontana, A. (1987) Antigen presentation and tumor cytotoxicity by interferon-gamma-treated microglial cells. Eur. J. Immunol. 17, 1271-1278. Fritz, R.B., Chou, C-H.J. and McFarlin, D.E. (1983) Induction of experimental allergic encephalomyelitis in P L / J and ( S J L / J × PL/J)F1 mice by myelin basic protein and its peptides: Localization of a second encephalitogenic determinant. J. Immunol. 130, 191-194. Frohman, E.M., Frohman, T.C., Dustin, M.L., Vayuvegula, B., Chjoi, B., Gupta, A., van den Noort, S. and Gupta, S. (1989) The induction of intercellular adhesion molecule-l(ICAM-1) expression on human fetal astrocytes by interferon-gamma, tumor necrosis factor-alpha, lymphotoxin andinterleukin-l: Relevance to intracerebral antigen presentation. J. Neuroimmunol. 23, 117124. Frohman, E.M., Vayuvegula, B., Gupta, S. and van den Noort, S. (1988) Norepinephrine inhibits gamma interferon-induced major histocompatibility class II (Ia) antigen expression on cultured astrocytes via beta-two-adrenergic signal transduction mechanisms. Proc. Natl. Acad. Sci (USA) 85, 1292-1296. Ganter, S., Northoff, H., Mannel, D. and Gebicke-Harter, P.J. (1992) Growth control of cultured microglia. J. Neurosci. Res. 33, 218230. Gerrard, T.L., Dyer, D.R. and Mostowski, H.S. (1990) IL-4 and granulocyte-macrophage colony-stimulating factor selectively increase HLA-DR and HLA-DP antigens but not HLA-DQ antigens on human monocytes. J. Immunol. 144, 4670-4674. Giulian, D. (1987) Ameboid microglia as effectors of inflammation in the central nervous system. J. Neurosci. Res. 18, 155-171. Giulian, D., Baker, T.J., Shih, L.N. and Lachman, L.B. (1986) Interleukin-1 of the central nervous system is produced by ameboid microglia. J. Exp. Med. 164, 594-604. Griffin, J.D., Spertini, O., Ernst, T.J., Belvin, M.P., Levine, H.B., Kanakura, Y. and Tedder, T.F. (1990) Granulocyte-macrophage colony-stimulating factor and other cytokines regulate surface expression of the leukocyte adhesion molecule-1 on human neutrophils, monocytes, and their precursors. J. Immunol. 145, 576584. Kennedy, M.K., Torrance, D.S., Picha, K.S. and Mohler, K.M. (1992) Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery. J. Immunol. 149, 2496-2505. Konno, H., Yamamoto, T., Iwasaki, Y., Suzuki, H. and Terunuma, H. (1989) Ia-expressing microglial cells in experimental allergic encephalomyelitis in rats. Acta Neuropathol. 77, 472-479. Kraus, E., Schneider-Schaulies, S., Miyasaka, M., Tamatani, T. and Sedgwick, J. (1992) Augmentation of major histocompatibility complex class I and ICAM-1 expression on glial cells following measles virus infection: Evidence for the role of type-1 interferon. Eur. J. Immunol. 22, 175-182. Lee, S.C., Collins, M., Vanguri, P. and Shin, M.L. (1992) Glutamate differentially inhibits the expression of class II MHC antigens on astrocytes and microglia. J. Immunol. 148, 3391-3397. Lee, S.C., Liu, W., Roth, P., Dickson, D.W., Berman, J.W. and Brosnan, C.F. (1993) Macrophage colony-stimulating factor in human fetal astrocytes and microglia. Differential regulation by cytokines and lipopolysaccharide, and modulation of class II MHC on microglia. J. Immunol. 150, 594-604. Lieberman, A.P., Pitha, P.M., Shin, H.S. and Shin, M.L. (1989) Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus. Proc. Natl. Acad. Sci. USA 86, 6348-6352.

32 Malipiero, U.V., Frei, K. and Fontana, A. (1990) Production of hemopoietic colony-stimulating factors by astrocytes. J. Immunol. 144, 3816-3821. Massa, P.T., Meulen, V. and Fontana, A. (1987) Hyperinducibility of Ia antigen on astrocytes correlate with strain-specific susceptibility to experimental allergic encephalomyelitis. Proc. Natl. Acad. Sci. USA 84, 4219-4223. Matsumoto, Y., Ohmori, K. and Fujiwara, M. (1992) Microglial and astroglial reactions to inflammatory lesions of experimental autoimmune encephalomyelitis in the rat central nervous system. J. Neuroimmunoi. 37, 23-33. Ohmori, K., Hong, Y., Fujiwara, M. and Matsumoto, Y. (1992) In situ demonstration of proliferating cells in the rat central nervous system during experimental autoimmune encephalomyelitis. Evidence suggesting that mostinfiltrating T cells do not proliferate in the target organ. Lab. Invest. 66, 54-62. Ohno, K., Suzumura, A., Sawada, M. and Marunouchi, T. (1990) Production of granulocyte/macrophage colony-stimulating factor by cultured astrocytes. Biochem. Biophys. Res. Commu. 169, 719-724. Racke, M.K., Dhib-Jalbut, S., Cannela, B., Albert, P.S., Raine, C.S. and McFarlin, D.E. (1991) Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta-1. J. Immunol. 146, 3012-3017. Raine, C.S., Barnett, L.B., Brown, A., Behar, T. and McFarlin, D.E.(1980) Neuropathology of experimental allergic encephalomyelitis in inbredstrains of mice. Lab. Invest. 43, 150-157. Ruddle, N.H., Bergman, C.M., McGrath, K.M., Lingenheld, E.G., Grunet, M.L., Padula, S.J. and Clark, R.B. (1990) An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis. J. Exp. Med. 172, 1193-1200. RueI, C. and Coleman, D.L. (1990) Granulocyte-macrphage colony-

stimulating factor: Pleiotropic cytokine with potential clinical usefulness. Rev. Infect. Dis. 12, 41-62. Sasaki, A., Levison, S.W. and Ting, J. P-Y. (1990) Differential suppression of interferon-gamma-induced la antigen expression on cultured rat astrogliaand microglia by second messengers. J. Neuroimmunol. 29, 213-222. Satoh, J., Kim, S.U., Kastrukoff, L.F. and Takei, F. (1991) Expression and induction of intercellular adhesion molecules (ICAMs) and major histocompatibility complex (MHC) antigens on cultured murine oligodendrocyte sand astrocytes. J. Neurosci. Res. 29, 1-12. Sawada, M., Kondo, N., Suzumura, A. and Marunouchi, T. (1989) Production of tumor necrosis factor-alpha by microglia and astrocytes in culture. Brain Res. 491,394-397. Schwartz, R.H. (1990) A cell culture model for T lymphocyte clonal anergy. Science 248, 1349-1356. Springer, T.A. (1990) Adhesion receptors of the immune system. Nature 346, 425-434. Suzumura, A., Marunouchi, T. and Yamamoto, H. (1991) Morphological transformation of microglia in vitro. Brain Res. 545, 301-306. Suzumura, A., Metitus, S.G.E., Gonatas, N.K. and Silberberg, D.H. (1987) MHC antigen expression on bulk isolated macrophage-microglia from newbornmouse brain: Induction of Ia antigen expression by gamma-interferon. J. Neuroimmunol. 15, 263-278. Suzumura, A., Sawada, M., Yamamoto, H. and Marunouchi, T. (1990) Effects of colony stimulating factors on isolated microglia in vitro. J. Neuroimmunol. 30, 111-120. Suzumura, Bhat, S., Eccleston, P.A., Lisak, R.P. and Silberberg, D.H. (1984) The isolation and long-term culture of oligodendrocytes from newborn mouse brain. Brain Res. 324, 379-383.