Glucocorticoid regulation of eicosanoid production by glial cells under basal and stimulated conditions

Journal of Neuroimmunolo~', 40 (1992) 273-280 ,'~ 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

273

JNI 90017

Glucocorticoid regulation of eicosanoid production by glial cells under basal and stimulated conditions T. B r e n n e r ~', A. B o n e h

b E.

S h o h a m i c, O. A b r a m s k y ~ and J. W e i d e n f e l d "

" Department of Neurology and b Department of Pediatrics', Hadassah Unicersity' Hospital and Hebrew Unicersity, Hadassah Medical School, and ' Department of Pharmacolo~', Hebrew Unicersio', Hadassah Medical School, Jerusalem, Israel

Key words: Eicosanoid, Protein kinase C: Phospholipase A 2 glucocorticoid; Glial cell

Summary We measured the production of two eicosanoids, prostaglandin E 2 and thromboxane-B 2, by rat glial cell cultures under basal conditions, following stimulation with phorbol-12-myristate-13-acetate and the bacterial endotoxin lipopolysaccharide, and following treatment with synthetic glucocorticoids. Stimulation of rat glial cells in culture with either phorbol-12-myristate-13-acetate or lipopolysaccharide caused a 1.5-5.0-fold increase in prostaglandin E 2 production, but did not affect thromboxane production. Pretreatment of the cultures with dexamethasone markedly inhibited the stimulated production of prostaglandin E 2 but had only a modest effect on basal production. Dexamethasone did not affect the activity of the enzyme protein kinase C, a putative regulator of eicosanoid synthesis. Our findings show that glucocorticoids have the potential to modulate central nervous system eicosanoid production particularly under conditions of stimulated production, such as inflammatory and demyelinating disorders. This mechanism may explain, at least in part, the therapeutic benefit of glucocorticoids in patients with multiple sclerosis.

Introduction Inflammation occurs in the central nervous system (CNS) in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), as evidenced by the presence of inflammatory infiltrates at the margins of active plaques (Traugott et al., 1983). In addition, elevated levels of eicosanoids and cytokines have been observed in the CNS in MS and EAE (Met-

Correspondence to: T. Brenner, Department of Neurology, Hadassah University Hospital, P.O. Box 12000, Ein Karem, Jerusalem, Israel.

ril et al., t983; Bolton et al., 1984; Hofman et al., 1989; Maimone et al., 1991). Recent works suggest that glial cells, especially astrocytes and microglia, can produce eicosanoids and cytokines, which were once thought to be derived solely from monocytes (Hofman et al., 1989; Barna et al., 1990; Benveniste et al., 1990; Chung and Benveniste, 1990). Thus, it has been suggested that many of the features of the inflammatory process in the CNS in MS and EAE may be attributed to locally produced eicosanoids and cytokines, and are not due only to chemical mediators produced by invading macrophages and lymphocytes.

274 Agents shown to stirnulate the prodttction of eicosanoids and cytokines by glial cells include calcium ionophores (De George, 1986), phorbol esters (Hartung and Toyka. 1987a). neurotropic virus (Lieberman ct al., 1989). and cytokines such as interferon and tumor necrosis factor (Chung and Bcnveniste, 1990). Some of these stimuli as well as cytokines operate at a local level and can also amplify immune responses by inducing the expression of histocompatability antigens in the CNS (Lavi et al., 1988: Vidovic et al., lt)9()). On the other hand, other agents, notably the synthetic glucocorticoid dexamethasone, have been shown by Weidenfeld et al. (1987) to inhibit the synthesis and release of prostaglandins (PG) specifically from brain cortex (in other brain areas this inhibition was variable). Moreover. this inhibitmT effect on PG synthesis was shown to be dexamethasone-specific, since other glucocorticolds failed to affect P G E , release from cortical slices (Weidenfeld et al., 1988). These findings may have clinical relevance in light of the fact that glucocorticoids are widely used in the treatment of CNS inflammatoD.' conditions and in CNS edema. Two enzymes, notably phospholipase A• (PLA~) and protein kinase C (PKC), play a regulatory role in the regulation of eieosanoid production. P L A , . a calcium-activated lipase (Hirata et al., 198(i)), actiw~tes the release of arachidonic acid from cell membrane phospholipids. Arachidonic acid in turn passes through a cascade of enzymatic reactions along the cycloox3'genase pathway to eventually become the eicosanoids PG and thromboxane (TXB•). The role of PKC in the regulation of eicosanoid synthesis in the CNS is less well established but is supported by studies e m p l o y i n g p h o r b o l - 1 2 - m y r i s t a t e - 1 3 acetate (PMA), a tumor-promoting phorbo[ ester; this ester is a known activator of PKC (Hartung and Toyka, 1987b; Jeremy et al., 1987: GebickHaerter et al.. 1988). In the present study, we examined the effect of d e x a m e t h a s o n e on the production of two eicosanoids. P G E , and T X B , , by rat glial cells in vitro. Thus, the production of these eicosanoids by glial cell cultures was measured under basal conditions and following .,,timulation with the phorbol ester PMA or the bacterial endotoxin

lipopolysaccharide t l_.PN), either with or without pretreatment with dcxamethasonc. To gain some insight regarding the regulator~ mechanism governing eicosanoid production, we also examined the correlation between the activity Icvels of PKC and PLA~ and the production of eicosanoids.

Materials and methods

Preparatiott of glial cell cultures Dissociated rat brain cells were prepared from fetal (17-21 days gestation) rats as described previously (Eccelston and Silberberg, 1984). Ceils were seeded onto po[y t.-lysine-coated tissue culture dishes or cover slips at a density of 5 × 1()" cells per dish and cultured for 1-3 weeks in MEM supplemented with 10G fetal calf serum, as described previously (Brenner et al., 1986). ,An immunofluorescence assay was performed to identify and quanti~' various cell types in the mixed glial cell cultures (Brenner et al., 1986). This a~say revealed that the mixed glial cell cultures contained 80-95(b astrocytes. 5-I0C~- oligodendrocytes, < lC:,: neurons, < I G fibroblasts, and 1-3c:2 macrophages-microglia. Cell cMture incubation with phorbol esters'. LPS. and hormonal steroMs The medium of the cell cultures was replaced with fresh medium 24 tl prior to all experiments. In some cultures, PMA (1()00 n g / m l : Sigma, USA) or 4a-phorbol-12,13 didecanoate or LPS (20/.zg/ml) was added with the fresh medium. In other experiments, dexamethasonc, progesterone. testosterone, corticosteronc, or pregnenolone (11) # g / ' m l ) (Sigma, USA) were added with the fresh medium. In experiments ira which cells were exposed to hormonal steroids as well as PMA or LPS. the preincubation with the steroids lasted 24 h, after which cells were exposed to either PMA or LPS. After adding the various inducers and/"or hormones, the plates were incubated at 37°C for various times (indicatcd in the figures). Aliquots of media ~verc taken for cicosanoid assays. The cells were scraped on ice using a rubber policeman in 1 nil/dish ice-cold homogenizing buffer and prepared for enzvmatic assays.

275

Determination of eicosanoid production Cell culture supernatants were collected and kept at - 7 0 ° C until assayed by radioimmunoassay using specific antisera and radioligands as described elsewhere (Weidenfeld et al., 1987). Anti-PGE 2 was purchased from Bio-Yeda (Rehovot, Israel) and anti-TXB 2 and anti-6-ketoPGF~a from Dr. Levine (Brandeis University, USA). [3H]-PGE2, [3H]TXB2 and [3H]6-ketoPGF1 to~ (150-200 C i / m m o l ) were obtained from NEN (Boston, USA), and standard prostaglandins from Sigma (USA).

Preparation of cell homogenate and PKC assay Cells were homogenized in ice-cold buffer containing 0.25 M sucrose, 20 mM Tris-Hepes buffer (pH 7.5), 2 mM EGTA, 2 mM EDTA, 10 mM dithiotreitol, and 10 / z g / m l leupeptin. The homogenate was treated with an equal volume of homogenizing buffer containing 0.4% Triton X100 for 60 rain on ice with occasional vortexing, and then further diluted with homogenizing buffer so that the final Triton X-100 in the assay was 0.5%. PKC activity was measured in cell homogehates as the incorporation of 32p from [32p]ATP (Amersham, UK) into calf thymus histone type III-S (Sigma), as described previously (Boneh, 1991). The assay was performed using 1 p,g protein per 100/_tl assay mixture. Following a 1.5-min preincubation, [~2p]ATP (100 /,tmol of approx• 200 c p m / p m o l ) was added and the reaction continued for 10 min. Net PKC activity was determined by subtracting basal activity, i.e. in the absence of calcium and activating phospholipids, from the total activity obtained in the presence of calcium and phospholipids. Phospholipase A 2 assay The assay for PLA~ was described previously (Brenner et al., 1988). Briefly, the assay is based on the cellular uptake of phosphatidylcholine containing a fluorescent labelled hexanoic acid (C6-NBD-PC) at the second carbon of the phospholipid substrate. The labelled product does not incorporate into other lipids and is not metabolized further. Thus, the fluorescent fatty, acid is a direct measure of PLA~ activity. Protein concentration was determined using a

Bio-Rad protein assay kit. Statistical evaluation was done using ANOVA.

Results In the first series of experiments, we measured the production of eicosanoids by glial cells under basal conditions and following stimulation with phorbol ester and the bacterial endotoxin LPS, with or without dexamethasone pretreatment. Under basal conditions, PGE 2 and TXB, levels were 215 ___ 13 and 693 + 19 p g / m l , respectively. 6-keto-PGFla, the stable metabolite of prostacyclin, was below the level of detection, Figure 1 shows the production of PGE 2 and TXB~ following a 2-96-h incubation with PMA. A 2-h incubation with PMA elicited a five-fold increase in PGE 2 production. PGE 2 production gradually decreased as the incubation time increased; thus, a 96-h incubation period with PMA resulted in only a 1.3-fold increase compared with control cells. PMA did not affect TXB~ production. Control cultures incubated with 4a-phorbol, a PMA analogue that does not activate PKC, showed no change in either P G E , or TXB, production (data not shown).

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Fig. 1. Effect of phorbol-12-myristate-13-acetate and dexamethasone on prostaglandin Ez (open bars) and thromboxane B2 (hatched bars) production by glia[ cells. Cells were incubated with 1000 ng/m[ phorbol-12-myristate-13-acetate for the time periods indicated or with 10 #g/mi dexamethasone or dexamethasone and phorbol-12-myristate-13-acetate. (Thromboxane B~ was not determined in these experiments.) Each bar represents the mean _+SEM of the percent change in four to eight experiments performed in duplicate. * P < 0.05: • * P < 0.005. compared with control cells.

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Fig. 2. Effect o f phorbol-12-myristatc-13-acetate and dexamethasone on protein kinase C" activity in glial cell homogenates. Cells were incubated vdth 1000 ng..'ml phorbol12-myristate-13-acetate with or without dexamethasone for the time periods indicated. Subsequently, the cells were homogenized and protein kmase C activity was measured. Each bar represents the activity (pmol/#g protein per min) measured in five experiments performed in triplicate. Open bars represent basal activity and hatched bat's represent total activity (i.e. in the absence and presence of calcium and activating pho~,pholipids). Net activity was determined by subtracting basal activity from the total activity. * P < [).[)115. compared with net activity of the control.

Pretreatment with dexamethasone inhibited P G E , production both under basal and PMAactivating conditions (Fig. 1). In non-activated cells, the inhibitory effect was only moderate (26%), yet significant ( P < 0 . 0 5 ) . In PMAactivated cells, the inhibitoi7 effect of dexamethasone was much more pronounced. A 24-h preincubation with dexamethasone markedly rcduced the increase in PGE~, from a five-fold increase to a two-fold increase following a 2-h PMA incubation. Moreover, following 24-h preincubation with dexamethasone alone and a further 24-h incubation with dexamethasone plus PMA, PGE~ production was only 50,q of the control values. Dexamethasone had no effect on TXB~ production, Figure 2 shows the effect of PMA and dexamethasone on PKC activity. In control, nonstimulated cells, basal PKC activity was 232 + 13 p m o l / m g protein per min and the activity in the presence of phospholipid and calcium was 1008 -2-_71: thus, net PKC activity was 736_+ 61. A 2-h incubation with P M A did not elicit a significant change in the overall activity of PKC. Prolonged

incubation of the cells with PMA caused a gradua[ decrease in total PKC activity: after 24-h incubation, net PKC activity was approximately 5(1
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Fig. 3. Effect of lipopolysaccharide and hormonal steroids on prostaglandin E 2 (open bars) and thromboxane B~ (hatched bars) production by glial ceils. The cells were incubated with LPS 20 ~ug/'ml for 20 h. Dexamethasone, corticosterone, and progesterone (l() /xg/ml) were added 24 h prior to lipopolysaccharide. Each bar represents the m e a n + S E M mean of the percent change in three to four experiments performed in duplicate. * P < 0.05:.* :': P < 0.005, compared with controls.

277

gesterone reduced TXB 2 production but had only a minor effect on P G E 2.

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Fig. 4. Effect of various hormonal steroids on phospholipase A : activity in glial cells. 100/.tg of each steroid was added 24 h prior to the enzymatic assay. Each bar represents the m e a n + S E M of five to ten assays. * P < 0.05, compared with control. Dex, dexamethasone; CS, corticosterone; prog, progesterone; test, testosterone; pregn, pregnenalone.

affect TXB 2 production. Preincubation with dexamethasone reduced this increase to only a 1.5-fold increase above control cells. Corticosterone and progesterone had an effect similar to dexamethasone, and attenuated the large increase in P G E 2 production following stimulation with LPS. The hormonal steroids dexamethasone, corticosterone, and progesterone also had a moderately inhibitory effect on non-stimulated cells: such cells secreted about 70% of the PGE~ as that produced by control cells. LPS did not enhance the production of TXB 2, and steroid hormones neither affected basal TXB 2 production nor TXB 2 synthesis following exposure to LPS. The effect of steroid hormones on PLA 2 activity in cultured glial cells is shown in Fig. 4. Pretreatment with dexamethasone or corticosterone for 48 h caused a slight inhibition of PLA 2 activity (15-20%), and pregnenolone (a biologically inactive precursor) had no effect on enzyme activity. In contrast, incubation with testosterone or progesterone inhibited P L A , activity by 55%. The effect of these hormones on P G E 2 and TXB 2 production under basal conditions paralleled those observed for PLA 2 activity (data not shown). Thus, dexamethasone and corticosterone had a moderate inhibitory effect on P G E 2 and TXB 2 production; testosterone caused a 50% reduction in both P G E 2 and TXB2; pro-

Elevated levels of eicosanoids and cytokines in the CNS have been demonstrated in MS. Studies in vitro and studies using experimental animal models suggest that cytokines and eicosanoids are potent immune mediators which are involved in both the onset and enlargement of demyelinating plaques (Maimone et al., 1991). One way in which eicosanoids and cytokines may enhance the progression of disease is via an effect on the proliferation and maturation of oligodendrocytes and astrocytes. Such an effect could be important since oligodendrocyte hypercellularity, astrocytic proliferation and gliosis have been observed in the plaques and surrounding white matter in the brains of patients with MS. Thus, elucidation of the regulation of eicosanoid production in the CNS by glial cells bears clinical relevance. In the present study, we examined the regulation of eicosanoid production using both stimulators and inhibitors of eicosanoid production. Our results have shown that under basal conditions the level of P G E 2 production by astrocytes was low, whereas TXB~ production was significantly greater. Stimulation of glial cells with either PMA or LPS led to a five-fold increase in P G E 2, which was not paralleled by a similar increase in TXB 2 production. Preincubation of the cells with the synthetic glucocorticoid dexamethasone had a marked inhibitory effect on the stimulated release of P G E 2, and somewhat attenuated basal production. Administration of steroid hormones such as corticosterone and progesterone had an effect similar to that of dexamethasone, causing a significant reduction in the LPS-induced synthesis of P G E 2. The physiological significance of the inhibitory effect of the sex steroids on P L A , and eicosanoid production is not yet understood. It may be speculated that these hormones exert an agonistic effect on the glucocorticoid receptor in glial cells. Further experiments are needed to clarify this notion. These findings indicate that (1) certain stimuli differentially affect P G E , and TXB~ production:

27S (2) g l u c o c o r t i c o i d s have a d i f f e r e n t i a l effect on basal a n d s t i m u l a t e d r e l e a s e of e i c o s a n o i d s : and (31 the sex s t e r o i d p r o g e s t e r o n e and n a t u r a l a n d synthetic g l u c o c o r t i c o i d s have p o t e n t i a l to affect e i c o s a n o i d p r o d u c t i o n by glial cells. A selective r e s p o n s e of P G E , p r o d u c t i o n by C N S tissue following e x p o s u r e to c e r t a i n stimuli or inhibitors w i t h o u t a c o n c o m i t a n t c h a n g e in TXB~ p r o d u c tion was also r e p o r t e d by S h o h a m i and G r o s s (1985) a n d W e i d e n f c l d et al. (1987). O n e e n z y m e directly involved in the l i b e r a t i o n of a r a c h i d o n i c acid from m e m b r a n e p h o s p h o lipids is P L A ~ which in turn is r e g u l a t e d by two i n t r a c e l l u l a r m e c h a n i s m s . T h e first involves PKC, which may p h o s p h o r y l a t e a n d t h e r e b y activate the lipase ( P a r k e r et al., 1987: H a l e n d a et al., 1989), or r e g u l a t e P L A , inhibitory p r o t e i n s such as lipocortin (Toqui et al., 1986) or P L A , stimulatory p r o t e i n s ( C l a r k et al., 1987). T h e s e c o n d m e c h a n i s m by which P L A , activity may be regul a t e d involves a l t e r a t i o n s in calcium flux ( C a r t e r et al., 1989), which may o p e r a t e in t a n d e m with P K C ( Z o r et al., 1990). E x p e r i m e n t s e x a m i n i n g P K C activation s h o w e d that a 2-h i n c u b a t i o n with P M A , a known P K C activator, did not cause an increase in total P K C activity, d e s p i t e the five-fold i n c r e a s e o b s e r v e d in PGE~. T h e increase in e i c o s a n o i d p r o d u c t i o n is p r o b a b l y the result of P K C activation a n d translocation to the p a r t i c u l a t e fraction, as shown by NcaD, et al. (1988). C o n t i n u e d i n c u b a t i o n ( 2 4 - 9 6 h) led to a g r a d u a l d e p l e t i o n and d o w n - r e g u l a t i o n in P K C activity (50c~/- after 24 h and 33C4 after 96 h), as well as a s i m u l t a n e o u s r e d u c t i o n t o w a r d s normal regarding PGE e production. T h e s e findings p o i n t to two c o m p o n e n t s in the r e g u l a t i o n of P G E , p r o d u c t i o n . T h e first is " P M A / d e x a m e t h a s o n e - s e n s i t i v e ' . It is e n h a n c e d by s h o r t - t e r m P M A i n c u b a t i o n a n d inhibited by d e x a m e t h a s o n e , suggesting that this c o m p o n e n t is r e g u l a t e d by P K C - m e d i a t e d activation of P L A . . T h e s e c o n d c o m p o n e n t is a " P M A / d e x a m e t h a sone-insensitivc" activation of the c a s c a d e r e p r e s e n t e d by the r e s i d u a l (approx. 3 3 ~ ) total cellular PKC, which p r o b a b l y acts via a n o t h e r (nonP L A ~) P K C - m e d i a t e d m e c h a n i s m . A l t h o u g h the m e c h a n i s m ( s ) by which dexam e t h a s o n e can affect the C N S a n d a t t e n u a t e MS is not c o m p l e t e l y u n d e r s t o o d , e v i d e n c e from pre-

vious studies and from the p t e s c n t study suggest at least two possible m e c h a n i s m s . T h e first is a r e d u c e d p r o d u c t i o n of P G E , due to the inhibition of P L A , activity ( H i r a t a ct al., 1980). This may be a c c o m p l i s h e d via a redttction in calcium c o n c e n t r a t i o n ( Z o r ct al., 1990) or by the induction of the PLA~ inhibitory p r o t e i n lipocortin ( F l o w e r ct al., 1984: G e b i c k e - H a e r t e r et al., 1988: Smillie ct al., 1989). G l u c o c o r t i c o i d s tnay also d e c r e a s e the affinity of t u m o r necrosis factor to specific cell surface r e c e p t o r s and thus d e c r e a s e the cytotoxic effect of T N F ( F r e d e r i c k and Kull, 1988). A d i m i n i s h e d c e l l u l a r r e s p o n s e in vivo to cytokines a n d o t h e r chemical m e d i a t o r s may contribute to the m e c h a n i s m of g l u c o c o r t i c o i d thcrapy in p a t i e n t s with MS a n d o t h e r d e m y e l i n a t i n g diseases.

Acknowledgements This work was s u p p o r t e d in part by the F a n n i e R u b i n S t e i n p r e s s R e s e a r c h E n d o w m e n t Fund, a n d in p a r t by the Isobel and Marvin Slomowitz R e s e a r c h F u n d in Neurology. T h e a u t h o r s t h a n k Mrs. C. Sicsic for excellent technical assistance.

References Barna, B.P., Estes. M.L, Jacobs, B.S., Hudson, S. and Rat>ahoff, R.M. (1990) Human astrocytes proliferate in response to tumor necro,sis factor alpha. J. Neuroimnmnol. 3(/, 239-243. Benveniste. E.N., Sparacio, S.M., Norris. G.J.. Grenett, H.E. and Fuller. G.M. (1990) Induction and regulation of interleukin-6 gene expression in rat astrocytes. J. Neuroimmunol. 3(L 2(i)1-212. Bolton, C.. Gordon. D. and Turk, J i . (1984) Longitudinal ,,,tudy of ttle prostaghmdin content of the central nervous system tissues from guinea pigs ~',ith acute experimental allergic encephalomyeliti,,, (EAE). Int. J. hnnmnopharmacol. 6, 155-161. Boneh, A. (1991) Inhibition of calcium-activated, phospholipid-dependent protein kinase [protein kinase C] in sphingolipidoses. J. Inherit. Metab. Dis. 14, 393 396. Brenner, T., Lisak, R.P., Rostami, A.. Pleasure, D.E. and Silberberg. D.H. (1986) Astrocytes, oligodendrocytes and Schwann cells share a common antigenic determinant that cross-reacts with myelin basic protein: identification with monoclonal antibod$. J. Neurosci. 6. 1925-1033. Brenner. T.. Yedgar, S.. Weidenfeld, J. and Abramsky, O. (1988) Phospholipase A2 activity in cultured glial cells.

279 Correlation with appearance of myelin markers and effects of steroid hormones. Ann. N.Y. Acad. Sci. 540. 403 404. Carter, T.D., Hallam, T.J. and Pearson, J.D. (19891 Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial cell prostacyclin production to intracellular Ca. Biochem. J. 262. 431-437. Chang, I.Y. and Bemeniste. E.N. ~1990) T u m o r necrosis factor and production of astrocytes. Induction by lipopolysaccharide, interferon gamma and interleukin-beta. J. Immunol. 144. 2999-3007. ('lark, M.A.. Conway, T.M., Short. R.G.L and Crooke, S.T. (1987) Identification and isolation of a mammalian protein which is untigenically and functionally related to the phospholipase A2 stimulatoD' peptide melittin. J. Biol. Chem. 262, 4402 4406. De George, J.J., Morell, P., McCarthy, K.D. and Lapetina. E.G. (198t~) Cholinergic stimulation of arachidonic acid und phosphatidic acid metabolism in C62B glioma cells. J. Biol. Chem. 261, 3428-3433. Eccelston, P.A. and Silberberg, D.H. (l'9841 The differentiation of oligodendrocytes in serum-free hormone-supplemented medium. Dev. Brain Rex. 16, 1-9. Flower, R.J. (19841 Macrocortin and the antiphospholipaxe proteins. In: Advances in Inflammation Research (Weissman, G.. Ed.I, pp. 1-34. Raven Press, New York, NY. Frederick, C and Kull, F.C. Jr. (1988) Reduction in tumor necrosis factor receptor affinity and cytotoxicity by glucocorticoids. Biochem. Biophys. Rex. Commun. 153, 402 400. Gebicke-Haerter, P.J., Seregi, A., Schohert, A. and Hertting, G. (19881 Involvement of protein kinase C m prostaglandin D, synthesis by cultured axtrocytes. Neurochem. Int. 13, 47~ 480. Halenda, S.P.. Banga, H.S., Zavioca, G.B.. t, au, F.-L. and Feinstein, M.S. (19891 Synergistic release of arachidonic acid from platelets by activation of protein kmase C and Ca ionophores. Evidence for a role of protein phosphoD'lation in the activation of phospholipase A , and independence from the Na./H exchanger. Biochemistry 28, 73567363. Hartung, H.-P. and Toyka, K.V. (1987a) Leukotriene production by ct.ltured astroglial cells. Brain Res. 435, 367 37{). Hartung, H.-P. and Toyka, K.V. (1987b) Phorbol ester TPA elicits prostaglandin E release from cultured astrocytes. Brain Res. 417, 347-349. Hirata, F., Schiffman, E., Veukatasubramanian, K., Salomon, D. and Axelrod. J. (19801 A phospholipas¢ A . inhibitoD' protein in rabbit neutrophils induced by glucocorticoids. Proc. Natl. Acad. Sci. USA 77, 2533-2536. Hofman, F.M., Hinton D.R., Johnson, K. and Merrill, J.E. 1089) T u m o r necrosis factor identified m multiple sclerosis brain. J. Exp. Med. 170. 607-612. Jeremy. J., Murphy. S., Pearce, B. and Dandoau, P. (19871 Phorbol ester stimulation of prostanoid synthesis by ctdtured rat CNS astrocytes: role of protein kinase C and calcium. Exp. Res. 419, 364-3e,8. Lavi, E.. Suzumara. A.. Murasko, D.M.. et al. (1988) Tumor necrosis factor induces the expression of MHC class I

antigens on mouse astrocytes. J. Neuroimmunol. 18, 245253. Liebermun, A.P., Pitha, P.M,, Shin. H.S. and Shin. M.L. (19891 Production of tumor necrosix fuctor und other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus. Proc. Natl. Acad. Sci. LISA 86. 6348 6352, Maimone, D.. Gregory. S., Arnason. B.G.W. and Reder, A.T. (19911 Cytokine levels in the cerebrospinal fluid and serum of patients with mt.ltiple sclerosis. J. Neuroimmunol. 32, 67-74. Merrill, J.E., Gerner, R.H., Myers, L.W. and Ellison, G.W. (19881 Regulation of natural killer cell cytotoxicity by prostaglandin E in peripheral blood and cerebroxpinal fluid of patients with multiple sclerosis and other neurological disease. J. Neuroinlmunol. 4, 223-237. Neary, J.T., Norenberg, L.O.B. and Norenberg. M.D. (1988) Protein kmaxe C in primary astrocyte cultures: cytoplasmic localization and translocation b_~ phorbol ester. J. Neurochem. 50, 1179 1184. Parker. J.. Daniel, L.W. and Waite, M. (1987) Evidence of protein kinuse C involvement in phorbol diester-stimuluted arachidonic acid release and proxtuglandin synthesis. J. Biol. Chem. 262, 5385-5393. Shohami, E. and Gross, J. (19851 An ex vi~o method for evaluating prostuglandin synthetase activity m cortical slices of mouse bruin. J. Neurochem. 45, 132 136. Smillie, F., Bolton. C., Peers, S.H. und Flo~er, R.J. (1989) Distribution of lipocortins 1, 11, and V in tissues of the rat, mouse, and guinea pig. Br. J. Pharmacol. 97, 541. Toqui, L.B.. Rothhut, B., Shaw. A., Fradm, /\., Vargaftig, B.B. and Russo-Marie, F. (1986) Platelet activation - - a role for a 40K anti-phospholipuse A , protein indistinguishable from lipocortin. Nature 321, 177-180. Traugott. U., Reinherz, E.L. and Raine. C.S. (19831 Multiple sclerosis. Distribution of T cell~, T cell subsets and la positive macrophagex m leskms of different ages. J. Neuroimmunol. 4, 2(tl 221. Vidovic, M., Sparacio, S.M.. EMvitz, M. and Benveniste, E.N. (199{)) Induction and regulation of class II major histocompatibility complex m R N A expression in astrocytes b} interferon g a m m a and tumor necrosis factor gamma. J. Neuroimmunol. 311, 18t)-200. Weidenfeld. J., gysy, J. and Shohami, E. (19871 Effect of dexamethasone on proxtaglandin synthesis m various areas of the rat brain. J. Neurochem. 48. 1351 1354. Weidenfeld, J., Abu-Amer, Y. and Shohami, E. { 19881 Inhibition of prostaglandin synthesis m brain of rat by dexamethasone: lack of effect of dexamethasone phosphate ester and various hormonal steroids. Neuropharmacok)gy 27, 1295-1299. Zor. U.. Her, E., Harell, T., Fischer, G., Naor, Z., Braquet, P.. Ferber, E. and Reiss, N. (19901 Arachidonic acid release by basophilic leukemia cells and macrophages stimulated by Ca ionophores, antigen and diacylglyeerol: essential role for protein kinaxe C and prevention by glucocorticoids. Biochim. Biophys. Acta 1091, 385-392.