J~mrnal ol the Neurological Sciences, 1(t3 ( 1991) 209-215

~c 1991 Elsevier Science Publishers B.V. 0022-510X/91/$03.50

209

JNS 03547

Fos R N A accumulation in multiple sclerosis white matter tissue John S. Yu, Tatsuhiko Hayashi, Eric Seboun, Robert M. Sklar, Teresa H. Doolittle and Stephen L. Hauser Neuroimmunologv Unit, Massachusetts General Hospital, Boston, MA 02114 (U.S.A.}

(Received 5 December, 1990) (Revised, received 14 January, 1991) (Accepted 17 January, 1991) Key words: Proto-oncognene; Astrocyte; in vivo RNA expression

Summary In order to better characterize the molecular events that accompany lesion development in multiple sclerosis (MS), we studied the accumulation of RNA specific to the nuclear proto-oncogenes c-fos and c-myb in post mortem white matter brain tissue. RNA was prepared from plaque and periplaque regions of 6 different MS brains, from "normal" white matter regions of 3 MS brains and from 6 normal control samples. Quatitation of specific RNA corresponding to each proto-oncogene was performed by Northern blot hybridization and by scanning densitometry. Results indicate a 2-fold increase in c-fos RNA in MS white matter, compared to control tissue. No c-myb signal was identified in any sample. In situ hybridization studies confirmed the selective upregulation ofcqbs RNA levels in MS tissue, and suggested that glial cells and not inflammatory' cells were responsible for the enhanced cqbs signal. These results suggest that persistent glial cell activation is present within chronic MS lesions irrespective of whether the lesions are active (e.g., inflammatory) or inactive.

Introduction The characteristic cellular pathology of multiple sclerosis (MS) consists of perivascular infiltration by T-cells and macrophages, oligodendroglial depletion, demyelination with relative axonal preservation, and marked astrogliosis (Lumsden 1980, Matthews et al. 1985). Knowledge of the evolution of the MS lesion is incomplete, due in part to the inaccessibility of plaque tissue to serial examination. An earlier view, for example, that oligodendrocytes were the primary "target" of the disease process has been supplanted by recent work indicating that myelin destruction may precede oligodendroglial loss (Raine et al. 1981). The nature of the inflammatory response in situ has also been characterized to some degree, and no consistent evidence of T-lymphocyte activation or of brain-specific reactivity has been identified (Traugott et al. 1983; Hayashi et al., 1988). Finally, the role of the astrocyte in MS lesions has been reconsidered, stimulated by evolving concepts of astrocyte

Corre.v~ondence to: John S. Yu, Neuroimmunology Unit, Warren 352, Masschusetts General Hospital, 32 Fruit Street, Boston, MA 02114, U.S.A. Tel.: (617)726-3787.

participation in immune responses and by evidencc that repair mechanisms may be impaired in MS lesions due to the astroglial response (Reier and Houle 1988). One approach to the study of the cellular pathology of MS is to define the lesion in terms of known molecular responses associated with activation, growth and differentiation. One such class of responses involve proto-oncogenes, or cellular genes homologous to transforming genes of R N A viruses. One category of proto-oncogenes, termed nuclear proto-oncogenes, encode nuclear proteins that influence gene expression. We describe here the quantitation of R N A levels of the nuclear proto-oncogenes c~fi~s and c-mt'b in postmortem MS and control tissue.

Methods Source qf tissue

Postmortem tissue samples of M S plaques from cerebral white matter were studied from 6 MS patients. From three of these patients, regions of "normal" white matter were available. Tissues from all specimens were first examined using routine hematoxylin-eosin-luxol fast blue stains. M S plaques were classified as chronic, active or as chronic,

210 inactive based upon the presence or absence of associated inflammatory cell infiltration (Hayashi et al. 1988). Postmortem specimens of cerebral white matter from six control subjects were also studied. Autolysis times ranged between 1.5 and 12 h and were similar between the MS and control specimens. All tissue was frozen at - 70 °C on the day of autopsy (Clinical histories are summarized in Table 1).

Northern blot analysis R N A was extracted according to the method o f Chirgwin and colleagues (1979). Total R N A was isolated by the CeC1 method (Glisin et al. 1974). Concentrations of R N A were determined by absorbance at 260 nm and purity

TABLE 1 CLINICAL HISTORIES OF MS PATIENTS AND CONTROLS Case

Age

Sex

(A) Normal control N1 69 M

Clinicalhistory

Death secondary to chronic obstructive pulmonary disease.

N2

57

M

Death secondary to pneumonia following surgery.

N3

61

F

Left lower lobe pneumonia and squamous cell carcinoma of right lung. Death from cardiorespiratory arrest.

N4

77

F

Coronary artery disease, hypertension. Death from myocardial infarction.

N5

71

M

Congestive heart failure, adult onset diabetes mellitus, ventricular arrythmia. Death from myocardial infarction.

N6

65

M

(B) Multiple sclerosis MS-I 41 F

History of chronic obstructive pulmonar disease, tuberculosis, and pneumonia. Death from heart failure.

13-year history of chronic progressive MS. History of ulcerative colitis, status post-colectomy and ileostomy.

MS-2

61

F

Multiple sclerosis, inactive at time of death.

MS-3

38

F

Relapsing, progressive MS x 5 years. Acute, active MS at time of death.

MS-4

36

F

Progressive MS × 3 years. Active MS at time of death.

MS-5

60

F

Relapsing-remitting MS. Active MS at time of death.

MS-6

40

F

Chronic, progressive MS x 10 years. Death from cardiorespiratory arrest.

assessed by A26o/280 ratios. Five microgram ,.)f total R N A samples were denatured and resolved through a 1.571, agarose/7.5 m M methylmercury vertical gel (Bailey and Davidson 1976). The gels were stained with ethidium bromide and photographed under UV light. Ti~e separated R N A samples were electrophoretically transfered to nylon membranes (Hybond-N, Amersham). The nylon filters were air-dried and baked at 80 °C in a vacuum for 2 h. The filter was prehybridized for 4 h at 42 °C in a solution containing 100#g/ml salmon sperm D N A (sonicated, sheared and denatured). A 2.0 kb E c o R I fragment from human c-myb, a 1.1 kb SacI/EcoRI fragment from murine c-fos, and a murine /~-actin insert were labeled with deoxycytidine tri[32p]phosphate (3000 Ci/mmol) by the oligolabeling procedure (Feinberg and Vogelstein 1983, 1984). Filters were hybridized for 16h at 4 2 ° C with 3 x 106 cpm probe/ml in a 10 ml solution. Filters were washed twice for 20 rain each at 6 0 ° C with 0.1 x SSC, 0.1 ~o S D S and exposed to X A R - 5 film (Kodak) at - 70 ° C with an intensifying screen. The autoradiograms were scanned in one dimension with an L K B ultroscan XL laser densitometer. A rectangular beam (50 x 800 #m) was positioned at the center of the band and peak integration was optimized using the automatic baseline option with the baseline set at the 16 lowest data points. Two control m R N A s were included on all blots and used as internal controls for determining the level of proto-oncogene expression by all other R N A samples. The hybridization intensity of all samples were arithmetically normalized such that identical control m R N A s run on different blots gave identical signals when analyzed with the same probe.

In situ hybridization Frozen brain tissues were cut into 8-#m sections at -30°C and thaw-mounted onto slides coated with Denhart's solution and pretreated by acetylation. The tissues were then fixed in acetic acid/ethanol ( 1 : 3 ) for 15 rain. Before hybridization, sections were pretreated with 0.2 M H C L for 20 min, and with proteinase K (1 #g/ml in 10 m M Tris, p H 7.4, 2 m M CaC12) for 15 min at 37 °C. They were dehydrated in ethanol and air-dried at room temperature. A 2.0 kb EcoRI fragment from the human c-myb gene and a 1.1 kb SacI/EcoRI fragment from the murine c-fl~s gene were labeled with [35S]dCTP ( N E N ) by the multiprime D N A labeling method (Amersham). The labeled probes were purified on Sephadex G-50 columns. The specific activity ofc-rnyb was 5.34 x 108 cpm/#g, and that of c-los 7.1 × 10Scpm/#g. Probes were dissolved at 0.24 x 104 cpm/#l in hybridization buffer with 0.6 M NaC1, 10 m M Tris p H 7.4, 0.5 m M dithiothreitol, 50~o deionized formamide. Next, 25 #1 of dissolved probe was applied to each section, covered with a siliconized coverslip and sealed

211 with rubber cement. Sections were hybridized at 37 °C for 18-24 h. Slides were washed in 2 x S S C 0.1 m M E D T A for 4 - 8 h at room temperature, followed by 2 x S S C at 5 0 ° C for l h, and d e h y d r a t e d on 0 . 3 M a m m o n i u m acetate/ethanol. After being air-dried, slides were i m m e r s e d into N T B - 2 emulsion ( K o d a k ) and e x p o s e d at 4 °C for 4 - 1 0 days. Slides were d e v e l o p e d in D-19 ( K o d a k ) and fixed ( K o d a k ) . The tissues were stained with toluidine blue and m o u n t e d with Permount. F o r each slide, grain counts from 100 cells were visually examined by 3 observers and fi)rmally quantitated by 1 (TH), under blinded conditions.

"--%%% _%

I

I

I

I

-- 28S

i

c - fos

~

~

Statistical analysis The relative a m o u n t of R N A hybridizing to the c-/os probe was determined by scanning densitometry. The intensity of the b a n d s in given samples were n o r m a l i z e d to /?-actin. Linear regression analysis and S t u d e n t ' s t-test were used to c o m p a r e /?-actin n o r m a l i z e d c-los levels between (1) normal white matter, ( 2 ) " n o r m a l " white matter from M S patients, and (3) M S lesions. F o r c o m p a r i s i o n s of c-fi~s and c-mvb grain counts revealed by in situ hybridization, intergroup differences were determined by the Mantell t a e n s z e l extension test for trends.

Results

Northern blot anal3w& In preliminary studies total R N A was p r e p a r e d from freshly frozen brain tissue derived from 20 different individuals with MS. Autolysis times were in most instances less than 12 h. In m a n y samples R N A was found to be d e g r a d e d and these were excluded. The R N A samples chosen for further study h a d sharp 28s and 18s b a n d s and m a x i m u m molecular weights o f greater than 3 kb. Following hybridization with the c:[bs p r o b e a visible band at 2.2 kb was present in some R N A samples (Fig. 1). This band c o r r e s p o n d s to the k n o w n size of the c:/bs transcript. In addition, R N A specific for M B P and for fi-actin was detected in all white matter samples (Fig. 1). The two R N A p r e p a r a t i o n s derived from normal h u m a n muscle tissue exhibited hybridization only to fl-actin. The relative amount of los R N A , n o r m a l i z e d to fi-actin, is s u m m a r i z e d in Fig. 2 for M S and control white matter samples. A consistent relative increase in los expression was present in M S c o m p a r e d to normal white matter tissue (P < 0.001, t-test). This increase in the densitometry ratio in M S tissue was present in tissue blocks classified as both plaque and " n o r m a l " M S white matter. By contrast, no expression o f myb or myc p r o t o - o n c o genes were detected in either M S or control white matter from any individual.

- 18 s

--28 S

MBP

/3- a c t

~

.......... ~ - 1 8 s

in

Fig. 1. Northern blot analysis of postmortem white maner from normal controls and plaque and non-plaque areas from MS patients. ~2P-labeled eDNA probes corresponding to the jos proto-oneogenc (top), myelin basic protein (middle), and fi-actin (bottom) were hybridized to various preparations of human RNA. Visible RNA bands lbr lbs are detected at 2.2 kb. The first lane represents RNA derived from normal human muscle hence the absence of an MBP signal. The next 3 hines (N I-N3) represent RNA hybridization results from three individuals without neurologic disease. Of the 6 normals studied, only 1 specimen(N3) showed a visible /os hand. By contrast, visible bands were seen in most of the MS white matter samples. The 4 lanes at the right represent MS samples either from non-plaque areas of normal-appearing white matter {NP) or from areas containing MS plaque (P). No no'b or nO'cexpression was observed from any MS or normal white matter sample.

In vivo Jos RNA expression 07 MS To determine the cellular source of los R N A , in situ hybridization of p o s t mortem brain tissue was performed. Tissue was selected from one active plaque, one inactive plaque and one normal control. As shown in Fig. 3, the majority of cells from the normal tissue had 5-10 grains per cell. Cells from the M S lesions (both active and inactive plaque) displayed a greater proportion of cells with higher numbers of grams per cell (P < 0.001). By histomorphological criteria these cells were thought to be of diverse type but at least some cells displayed astroglial morphology, i.e., large multiform cells with multiple processes (Fig. 4A and B). It was noteworthy that the cells comprising perivascular inflammatory cuffs at the edge of the active plaque did not express c-[os above b a c k g r o u n d levels (Fig. 4C). In addition, hybridization of MS and control tissue with a probe c o m p l e m e n t a r y to the mt'b proto-oncogene revealed no difference between normal and MS tissue (Fig. 3).

212 1.0

•~---.

0,8 /

"Q

it"

o.6

~

o.z L

I

I

I

CONTROL Muscle

CONTROL White Matter

MS "Normal" White Matter

MS Plaque

--

A

Fig. 2. Fos signal standardized by densitometry to ~-actin signal. On the y-axis, the ratio offos to fl-actin signal is indicated. Each group studied appears on the x-axis. Dotted lines connect samples of MS plaque and non-plaque areas from the same patient, t-test and linear regression analyses indicated that there was a difference between the normal and MS plaque groups (P < 0.001).

Thus, analysis of

c-fos e x p r e s s i o n r e v e a l e d a n a p p r o x i -

o Q

m a t e l y t w o f o l d i n c r e a s e in s i g n a l b e t w e e n M S a n d n o r m a l t i s s u e b y in situ a n a l y s i s , as well a s a 2 - f o l d i n c r e a s e in R N A signal by Northern blot analysis.

(7 60 -

~ Active

c-fos

-

[3

~J Inactive

IL I

K,

I

60

l

I

-

,.I

I

I

I

I

L

I

I

I

I

I

I

c-myb

40-

I

6-10

!

I

I

11-15 16-20

21-25 26-50

GRAINS / CELL Fig. 3. in situ hybridization of 35S-labeled cDNA probes for c-fos and c-myb with active and inactive MS plaque and normal white matter. The number of grains per cell in normal white matter and in MS plaques are shown. On the y-axis is the percentage of cells per field with a given number of grains. On the x-axis, the number of grains is stratified by groups of 5 grains per cell. Using the e-fos probe, both the MS active and MS inactive lesions had a 2-fold increase in grain count compared to that of the control white matter tissue (P < 0.01). By contrast, no intergroup differences were present with respect to c-myb.

Fig. 4. Morphology of c-fos positive cells. (A)Field of cells intensely labeled with c-fos is shown; this region corresponds to the sclerotic center of an MS plaque (x 510). (B)High-power view of labeled cells demonstrates that cells of varying morphology express fos RNA; the cell to the left is identified as an astrocyte ( × 1640). (C) Perivascular inflammatory cuff from the edge of the same lesion is shown; no fos labeling above background levels was detected ( × 780).

Discussion

c-fos ( b u t c-myb) m R N A are p r e s e n t in M S , c o m p a r e d to n o r m a l

T h e s e r e s u l t s i n d i c a t e t h a t i n c r e a s e d levels o f not

213 control, white matter tissue. Results obtained by Northern blot hybridization were confirmed in two lesions by in situ hybridization, in which the c-fi)s RNA accumulation in MS was found to be present in some cells with morphologic characteristics of astrocytes. The c-~s gene is a homologue of the Finkel-BiskisJinkins osteosarcoma virus (Finkel et al. 1966; Curran et al. 1984). Its protein product is a nuclear phosphoprotein that associates with the product ofthejun nuclear oncogene to form a heterodimer that binds to DNA and modulates transcription (Chin etal. 1988; O'Shea etal. 1989). Although its function is not well understood; it has been proposed that c-Jos can prevent a cell from entering into a quiescent state (Greenberg and Ziff 1984). C-fi~s is expressed at a low level in most cell types ; however, a rapid and transient induction occurs during the initial G o to G~ transition in response to growth factors (Kruijer et al. 1984; Muller et al. 1984; Coletta et al. 1986; Greenberg et al.

1986). Glial cells, neurons, and lymphocytes may express c-/bs under some conditions. Fos RNA has been detected in human astrocytoma cells (Fujimoto et al. 1989) and studies of cultured astrocytoma lines indicate that numerous stimuli, including epidermal growth factor (EGF), the fi-adrenergic agonist carbachol, and phorbol 12-myristate 13-acetate (PMA), are capable of inducing c-flos mRNA expression (Arenander et al. 1989). cq'os has also been localized to the nuclei of neurons (Hunt et al. 1987; Sagar et al. 1988: Sheng and Greenberg 1990), and c-fi)s mRNA has been shown to increase transiently following generalized seizures (Morgan et al. 1987). In the immune system, induction of c-los expression has been shown to occur in both T and B cells in response to mitogenic stimulation (Shipp and Reinherz 1987; Monroe 1988). As c-tbs mRNA levels are rapidly and transiently increased following cellular activation, c-Jbs has been considered to be a marker of an early stage of activation. Current results suggest that persistent astroglial activation is present within established MS lesions, even in lesions that by usual pathologic criteria would be graded as chronic or inactive. These data build upon earlier histologic findings that brisk reactive astrocytosis (Anderson etal. 1980; Matthews et al. 1985; Kostyk etal. 1989) and chronic leakage of serum proteins (Field 1967) may be present in inactive MS lesions. Furthermore, the finding that enhanced c-fi)s levels were present even in mRNA prepared from tissue blocks of normal-appearing MS white matter was unexpected. Small unrecognized plaques may have been present in these tissues; alternatively, c-lbs expression may have been increased in glial cells located outside plaques. As regards the latter possibility, Allen and coworkers (Allen et al.

1981; Allen 1984) have suggested that astrocyte numbers are diffusely increased in normal-appearing white matter beyond the edge of established MS plaques (Prineas and Raine 1976), and that a diffuse astroglial activation characterizes "normal" CNS white matter in this disease (Allen et al. 1981). Other work has indicated a difference in the x-ray diffraction profile of normal-appearing myelin in M S compared to controls, also suggesting the presence of a generalized white matter abnormality (Chia et al. 1984). More convincing evidence will be required, however, before such findings are accepted as core features of the MS process rather than as secondary events. The accumulation of los RNA in glial cells within MS tissue may thus represent a molecular marker for the poliferatiave gliosis that is a cardinal feature of the MS lesion. Some histopathologic studies suggest that the astroglial response occurs early in the evolution of the M S lesion (Dawson 1916; Field 1967) and many investigators have commented upon the unusual degree of gliosis that occurs in MS compared to that present in other neuropathologic conditions (Lumsden 1980). As activated astroeytes in several species have been shown to express class 2 antigens of the major histocompatibility complex (Male et al. 1987: Massa et al. 1987), to present protein antigen to T-cells (Fontana et al. 1984, 1987), and to secrete mterleukin-I (Giulian et al, 1988; Giulian and Lachman 1985) and the myelinotoxic cytokine tumor necrosis factor (Lieberman et al. 1989; Sawada et al. 1989: Selmaj and Raine 1989), it is possible that persistent astroglial activation in MS lesions may amplify or sustain inflammation or tissue destruction. In the single inflammatory MS tissue sample examined by in situ hybridization, no accumulation of cqbs or of the "late" nuclear oncogene c-mvb was noted within cells comprising inflammatory cuffs. While it is possible that the failure to detect these proto-oncogenes was due to their transient expression in mononuclear cells, earlier studies employing immunohistochemical labeling of T-cells within MS lesions have suggested that few activated cells were present (Hayashi et al. 1988). Cultured astrocytes may be stimulated to proliferate by a variety of growth factors and ligands, including phorbol ester tetradecanoyl phorbol acetate (TPA) (Yong et al. 1988), epidermal growth factor (Simpson et al. 1982), and fibroblast growth factor (Yong et al. 1988), in addition to the cytokine interleukin-1 (Giulian et al. 1986). It is possible that los RNA accumulation in MS plaques could result from stimulation by external growth factors, from an autocrine pathway or from other mechanisms. Elucidation of the stimuli responsible for astroglial actiwltion in MS will contribute to an understanding ofgliosis in this disease and may lead to the development of new strategies to control lesion development or extension.

214

Acknowledgements The authors wish to thank E.P. Richardson, Neil Kowall and Robert Ferrante for their help with this project, as well as Leigh Mason for secretarial assistance. This work was supported by the National Multiple Sclerosis Society, the Mathers Foundation, the National Institutes of Health (NS26799) and the Rita Allen Foundation. SLH is a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society.

References Allen, I.M. (1984) Demyelinating diseases. In: S.H. Adams, J.A.N. Cornelius and L.W. Duchen (Eds.), Greenfield's Neuropathology, 4th edn., Edward Arnold, London, pp. 338-384. Allen, I.V., G. Glover and R. Anderson (1981) Abnormalities in the macroscopically normal white matter in cases of mild or spinal multiple sclerosis. Acta Neuropathol. (Berl.), Suppl., 7: 176-178. Anderson, M., B. Hughes, M. Jefferson, W.T. Smith and A.H. Waterhouse (1980) Gliomatous transformation and demyelinating diseases. Brain, 103: 603-622. Arenander, A.T., R.W. Lim, B.C. Varnum, R. Cole, J. de Vellis and H.R. Herschman (1989) TIS gene expression in cultured rat astrocytes: multiple pathways of induction by mitogens. J. Neurosci. Res., 23: 257-265. Bailey, J.M. and N. Davidson (1976) Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal, Biochem., 70: 75-85. Chia, L.S., J.E., Thompson and M.A. Moscarello (1984) Alteration of lipid-phase behavior in multiple sclerosis myelin revealed by wideangle x-ray diffraction. Proc. Natl. Acad. Sci. USA, 81 : 1871-18714. Chirgwin, J.M., A.E. Przybyla, R.L.J. MacDonald and W.J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18: 5294-5299. Chiu, R., W.J. Boyle, J. Meek, T. Smeal, T. Hunter and M. Karin (1988) The c-fos protein interacts with c-jun/AP- 1 responsive genes to stimulate transcription of AP-1 responsive genes. Cell, 54: 541-522. Coletta, G., A.M. Cirafici and B. Vecchio (1986) Induction of the c-fos oncogene by thyrotropic hormone in rat thyroid cells in culture. Science, 233: 436-440. Curran, T., A.D. Miller, L. Zokas and I.M. Verma (1984) Viral and cellular fos proteins: a comparative analysis. Cell, 36: 259-268. Dawson, J.W. (1916) The histology of disseminated sclerosis. Trans. R. Soc. Edinb., 50: 517-740. Feinberg, A.P. and B. Vogelstein (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132: 6-13. Feinberg, A.P. and B. Vogelstein (1984) A technique for radiolabeling DNA restrict endonluclease fragments to high specific activity. Addendum. Anal. Biochem., 137: 266-267. Field, E.J. (1967) The significance of astroglial hypertrophy in scrapie, multiple sclerosis and old age together with a note on the possible nature of the scrapie agent. D. Z. Nervenheilk., 192: 265-74. Finkel, M.P., B.O. Biskis and P.B. Jinkins (1966) Virus induction of osteosarcomas in mice. Science, 151: 698-701. Fontana, A., W. Fierz and H. Wekerle (1984) Astrocytes present myelin basic protein to encephalitogenic T-cell lines. Nature, 307: 273-276. Fontana, A., K. Frei, S. Bodmer and E. Hofer (1987) Immune mediated encephalitis: on the role of antigen-presenting cells in brain tissue. lmmunol. Rev., 100: 185-201. Fujimoto M., P.J. Sheridan, Z.D. Sharp, F.J. Weaker, K.S. Kagan-Hallet and J.L. Story (1989) Proto-oncogene analysis in brain tumors. J. Neurosurg., 70: 910-915.

Giulian, D. and L.B. Lachman (1985) lnterleukin 1 stimulation of as troglial proliferation after brain injury. Science, 220: 497-499. Giulian, D., T.J. Baker, L.N. Shih and L.B. Lachman (1986) lnterleukin- l of the central nervous system is produced by ameboid microglia. J. Exp. Med., 164: 594-604. Giulian, D., D.G. Young, J. Woodward, D.C. Brown and L.B. Lachman (1988) lnterleukin-1 is an astroglial growth factor in the developing brain. J. Neurosci., 8: 709-714. Glisin, V., R. Crkvejakov and C. Byns (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry, 13: 26533-2637. Greenberg, M.E. and E.B. Ziff (1984) Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature, 311: 433-438. Greenberg, M.E., E.B. Ziff and L.A. Greene (1986) Stimulation of neuronal acetylcholine receptors induces rapid gene transcription. Science, 234: 80-83. Hayashi, T., C. Morimoto, J.S. Burks, C. Kerr and S.L. Hauser (1988) Dual-label immunocytochemistry of the active multiple sclerosis lesion: major histocompatibility complex and activation antigens. Ann. Neurol., 24: 523-531. Hunt, S.P., A. Pini and G. Evan (1987) Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature, 328: 632-634. Kostyk, S.K.N.W. Kowall and S.L. Hauser (1989) Substance P immunoreactive astrocytes are present in multiple sclerosis plaques. Brain Res., 504: 284-288. Kruijer, W., J.A. Cooper, T. Hunter and I.M. Verma (1984) Plateletderived growth factor induces rapid but transient expressions of the c-fos gene and protein. Nature, 312:711-716. Lieberman, A.P., P.M. Pitha, H.S. Shin and M.L. Shin (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. Lumsden, C.E. (1980) The neuropathology of multiple sclerosis. In: P,J, Vinken and G.W. Bruyn (Eds.), Handbook of Clinical Neurology, vol. 9, Multiple Sclerosis and Allied Demyelinating Diseases. Elsevier, Amsterdam, pp. 217-309. Male, D.K., G. Pryce and C.C.W. Hughes (1987) Antigen presentation in brain: MHC induction on brain endothelium and astrocytes compared. Immunology, 60: 453. Massa, P.T., V. ter Meulen and A. Fontana (1987) Hyperinducibility of la antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis, Proc. Natl. Acad. Sci. USA, 84: 4219-4223. Matthews, W.B., E.D. Acheson, J.R. Batcheler and R.O. Weller (1985) McAlpine's Multiple Sclerosis, Churchill Livingstone, Edinburgh, pp. 321-322, 358. Monroe, J.G. (1988) Up-regulation of c-fos expression ios a component of the mlg signal transduction mechanism but is not indicative of competence for proliferation. J. Immunol., 140: 1454-1460. Morgan, J.I.. D.R. Cohen, J.L Hempstead and T. Curran (1987) Mapping of patterns of c-fos expression in the central nervous system after seizure. Science, 237: 192-197. Muller, R.T., R. Bravo, J. Buckhardt and T. Curran (1984) Induction of c-fos gene and protein by growth factors precedes activation ofc-myc. Nature, 312: 716-720. Prineas, J.W. and C.S. Raine (1976) Electron microscopy and immunoperoxidase studies of early multiple sclerosis lesions. Neurol., 26 (Suppl.): 29-32. O'Shea, E.K., R. Rlutkowski, W.F. Stafford and P. Kim (1989) LSI: preferential heterodimer formation by isolated zippers from los and jun. Science, 245: 646-648. Raine, C.S., L. Scheinberg and J.M. Waltz (1981) Multiple sclerosis-

215 oligodendrocyte survival and proliferation in an active established lesion. Lab. lnvest., 45: 534-546. Reier, P.J. and J.D. Houle (1988) The glial scar: its bearing on axonal elongation and transplantation approaches to CNS repair. In: S.G. Waxman (Ed.), Functional Recovery in Neurological Diseases, Raven Press, New York, pp. 87-138. Sagar, S.M., F.R. Sharp and T. Curran (1988) Expressions of c-los protcins in bruin: metabolic mapping at the cellular level. Science. 240: 1328-1330. Sawada, M., N. Kondo. A. Suzumura and T. Marunouchi (1989) Production of tumor necrosis factor-alpha by microglia and astrocytes in culture. Brain Res., 491:394-397. Selmaj, K. and C. Raine (1989) Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann. Neurol., 23: 339-346. Sheng, M. and M.E. Greenberg (1990) The regulation and function of

coqbs and other immediate early genes in the nervous system. Neuron, 4: 477-485. Shipp, M.A. and E.L. Reinherz (1987) Differential expression of nuclear proto-oncogenes in T-cells triggered with mitogenic and nonmitogenic T3 and T l l activation signals. J. lmmunol., 139: 2143-2148. Simpson, DE., R. Morrison, J.L. deVellis and H.R Herschman (1982) Epidermal growth factor binding and mitogenic activity of purified populations of cells from the central nerwms system. J. Neurosci. Rcs., 8: 453-462. Traugott, V., E.L. Reinherz and C.S. Rainc (1983) Multiple sclerosis: distribution ofT cell subsets with active chronic Icsions. Scicncc, 219: 308-310. Yong, V.W., S.U. Kim, M.W. Kim and O.S. Shir (19881 Growth filctors for human glial cells in culture. Gila, 1: 112-123.