Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice

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BRAIN RESEARCH ELSEVIER

Brain Research ~54 (1994)2(111-206

Research

report

Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice A.E. Williams a,,, A.-M. van D a m b, W.K.H. Man-A-Hing b F. Berkenbosch b,t P. Eikelenboom c, H. Fraser ~' BBSRC and MRC Neuropathogenesis Unit, Institute ¢br Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF, UK, b Research Institute Neurosciences, Free Unil,ersity, Faculty of Medicine, Department of Pharmacology, Van der Boechorststraat 7. 1081 BTAmsterdam, The Netherlands', c Department of Psychiatry, Free UniL,ersity, Faculty of Medicine, Valeriuskliniek, Valeriusplein 9, 107.5 BG Amsterdam, The Netherlands Accepted 10 May 1994

Abstract The presence of cytokines, prostagtandins and lipocortin-1 was investigated in terminally affected mice in two models of scrapie. There was marked induction of glial interleukin-1/3, tumour necrosis factor a, prostaglandin E2, prostagtandin F2,~ and lipocortin-1 immunoreactivity in those areas of the brain showing the characteristic vacuolation of scrapie. A comparison of these staining patterns with those of GFAP and F4/80 showed that their expression occurred predominantly in astrocytes. It is possible that cytokines play a significant role in the pathogenesis of neurodegeneration in scrapie. Key words: Scrapie; Cytokine; Prostaglandin; Lipocortin; Astrocyte; Mouse; Neurodegeneration

I. Introduction

Scrapie, Creutzfeldt-Jakob Disease (CJD) and Alzheimer's disease (AD) are chronic neurodegenerative diseases in which the neuronal degeneration is accompanied by a florid astrocytosis and microgliosis [4,27,34,49,55]. A classical, acute, polymorphonuclear cell inflammatory response is absent. However, expression of /32-integrins by microglia [2,19,49] and the demonstration of various inflammation-related proteins such as activated complement factors [17], al-antichymotrypsin [1], a-2-macroglobulin [4] and ICAM-1 [18] associated with the/3A4 plaques of AD has led to the hypothesis that the microglial response represents a localised, modified inflammatory response. This concept is supported by reports of interleukin-1 (IL-1), IL-6 and tumour necrosis factor a (TNFa) reactivity in both astrocytes and microglia around amyloid deposits in AD brains [5,6,15,30]. Thus it is possible that induction of cytokines, activation of complement, etc., may

* Corresponding author. Fax: (44) (31) 668 3872. t Dr. Berkenbosch died during the preparation of the manuscript. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 6 0 0 - H

initiate a n d / o r augment the cerebral amyloidosis (/3A4 protein deposition) in AD brains and the subsequent pathological cascade that results in neuronal death [4,6]. A number of similar observations have also been made in scrapie and CJD. /32-Integrin expression by microglia associated with the amyloid deposition (here, PrP protein) has been described in both scrapie and CJD [34,55] and TNFa expression by hypertrophic astrocytes associated with myelin dilatation in terminally affected mice infected with the Fujisaki strain of CJD 'virus' has also been reported [40]. Thus /32-integrins and cytokines may also play a role in the progressive neurodegeneration of this disease. This paper reports studies investigating this hypothesis further. Expression of the cytokines IL-1 and TNFa was examined in the brains of terminally affected animals in two models of murine scrapie using immunocytochemical techniques. In addition, the immunocytochemical expression of prostaglandins (PGs) and lipocortin-1 was also studied. The effects of cytokines such as IL-1 are mainly mediated through eicosanoids (PGs and leukotrienes), particularly PGE 2 and PGF2~ [11,16,20]. The cellular source of PGs in the central

A.E. Williams et al. / Brain Research 654 (1994) 200-206

nervous system (CNS) in disease has not been identified although astrocytes have been shown to be a major source in vitro [16,21,46]; cultured microglia can also synthesise PGs [33] and brain endothelial cells express

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P G E 2 following peripheral administration of endotoxin in vivo [54]. Lipocortins are endogenous peptides with anti-inflammatory properties, including an inhibitory action on the effects of cytokines [11,20,48]. This is

Fig. 1. A: vacuolation in hippocampus of 301V/VM model. Haematoxylin and eosin. B: interleukin-1 immunoreactivity in hippocampus of 301V/VM model. Bar = 100/xm.

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t h o u g h t to b e m e d i a t e d t h r o u g h inhibition of p h o s p h o lipase A~ a n d the f o r m a t i o n of free a r a c h i d o n i c acid, t h e r e b y r e d u c i n g e i c o s a n o i d synthesis [11,20]. Expression of lipocortin-1 has r e c e n t l y b e e n d e s c r i b e d in

"reactive glial cells' [6] or astrocytes [37] in A D brains with a similar d i s t r i b u t i o n to the cytokincs. 'l'here arc scant d a t a on the e x p r e s s i o n of c y t o k i n e s in s c r a p i e / C J D - i n f e c t e d b r a i n s and thus the c,xpressi(~n

Fig. 2. A: interleukin-1 immunoreactivity in astrocytes (compare with panels E and F). B: tumour necrosis factor ~r immunoreactivity in astrocytes. C: prostaglandin E 2 immunoreactivity in astrocytes. D: lipoeortin-I immunoreaetivity in astrocytes. E: GFAP immunoreactivity showing astrocyte morphology. F: F4/80 immunoreactivity showing mieroglia cell morphology. Bar = 25 #m.

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o f IL-1/3, T N F a a n d o f factors m e d i a t i n g a n d inhibiting the a c t i o n s of c y t o k i n e s w e r e also e x a m i n e d in the p r e s e n t study.

Table 1 Primary antibodies used Dilutions Cytokines:

2. Materials and methods

Sheep anti-mouse IL-1/3 Rabbit anti-mouse TNFa

1:500-1:750 1 : 250

Prostaglandins :

Terminally affected VM/Dk mice infected intracerebrally with a 1% brain homogenate in saline of either 22A or 301V strain of scrapie (i.e., the 22A/VM and 301V/VM 'models') were studied. Both these scrapie models show widespread vacuolation [10,23] (A. Williams, personal observations). VM mice inoculated with a homogenate of normal brain acted as matched controls. Two mice for each scrapie strain and two controls were examined. The mice were deeply anaesthetised and perfused with Tyrode's salt solution (Sigma) followed by Bouin's fixative. The brains were removed and stored in Tris-buffered saline (TBS, pH 7.6), containing 15 mM azide at 4°C. Brains were sectioned coronally at 50 /xm using a vibratome and slices collected in TBS. Sections were thoroughly rinsed in TBS before staining. Immunostaining was performed using a PAP method previously described [53]. Briefly, rinsed, free-floating sections were incubated in TBS-5% bovine serum albumin (BSA) (30 min, room temperature) and TBS containing hydrogen peroxide at a final concentration of 0.3% (20 min, room temperature) before incubating them in the primary antibody (diluted in TBS-0.5% Triton X-100) overnight at 4°C. The primary antibodies used are given in Table 1. Sections stained with preadsorbed antibody, normal serum or without the application of a primary antibody acted as controls. Sections were then rinsed in TBS, incubated in the secondary antibody for 2 h at room temperature, rinsed and incubated in tertiary antibody (peroxidase-anti-peroxidase) for 1 h at room temperature. Immunoreactivity was visualised using DAB as the chromogen. Stained sections were mounted on gelatin-chromalum coated slides, dried at 37°C, dehydrated and embedded in entallan.

3. Results 3.1. M i c e injected with n o r m a l brain

C o n s t i t u t i v e e x p r e s s i o n o f i m m u n o r e a c t i v e IL-1/3 was p r e s e n t in n e u r o n s o f t h e p a r a v e n t r i c u l a r a n d s u p r a - o p t i c nuclei. N o n e u r o n a l reactivity to T N F a , P G E 2, P G F 2 , o r l i p o c o r t i n was o b s e r v e d . S o m e IL-1/3 i m m u n o r e a c t i v e cells w e r e o b s e r v e d in t h e m e n i n g e s a n d a s s o c i a t e d with e p e n d y m a l surfaces. T h e r e was no immunocytochemical expression of cytokines, p r o s t a g l a n d i n s o r l i p o c o r t i n by glia. 3.2. Scrapie-inoculated m i c e

T h e r e was c l e a r i n d u c t i o n o f IL-1/3 a n d T N F a imm u n o r e a c t i v i t y by glial cells in b o t h s c r a p i e m o d e l s s t u d i e d . IL-1 a n d T N F a i m m u n o r e a c t i v e glial cells w e r e p r e s e n t in the h i p p o c a m p u s , c e r e b r a l cortex, p a r a t e r m i n a l body, t h a l a m u s , b a s a l g a n g l i a a n d less p r o m i n e n t l y in m i d b r a i n a n d m e d u l l a , a n d w e r e g e n e r ally a s s o c i a t e d with t h o s e specific a r e a s showing vacuol a t i o n (Figs. 1A,B a n d 2A,B). T h e r e was also m a r k e d

Rabbit anti-PGE z Rabbit anti-PGF2,~

1:500 1:500

Lipocortins :

Rabbit anti-rat lipocortin-1

1:300

Glia:

Rabbit anti-human GFAP (astrocytes) Rat anti-mouse F4/80 (microglia)

1 : 800 1 : 10

u p r e g u l a t i o n o f i m m u n o r e a c t i v e P G E 2 , PGF2~ a n d l i p o c o r t i n on glia (Fig. 2 C , D ) with a similar d i s t r i b u t i o n to that of t h e cytokines. N o staining was d e t e c t e d in the a b s e n c e o f any p r i m a r y a n t i b o d y a n d staining was m a r k e d l y r e d u c e d o r a b o l i s h e d w h e n the p r i m a r y antib o d y (IL-1/3, T N F a , P G E 2 , P G F 2 , ) was p r e a b s o r b e d with 10 -6 M o f t h e a p p r o p r i a t e ligand. Lipocortin-1 was u n a v a i l a b l e for a d s o r p t i o n of t h e a n t i - l i p o c o r t i n antibody. M a r k e d i n c r e a s e s in G F A P a n d F 4 / 8 0 reactivity were present and confirmed previous observations that astrocytosis a n d microglial activation in s c r a p i e occur in t h o s e b r a i n r e g i o n s showing v a c u o l a t i o n [55]. Since the d i s t r i b u t i o n o f v a c u o l a t i o n t h r o u g h o u t t h e C N S differs b e t w e e n the 2 2 A / V M a n d 3 0 1 V / V M models, t h e exact d i s t r i b u t i o n of t h e i n d u c e d cytokine, P G a n d l i p o c o r t i n i m m u n o r e a c t i v i t y t h r o u g h o u t the b r a i n also d i f f e r e d b e t w e e n t h e two m o d e l s studied. H o w e v e r , in all mice s t u d i e d , the glial cytokine, P G a n d lipocortin i m m u n o r e a c t i v i t y o c c u r r e d in t h o s e b r a i n regions showing v a c u o l a t i o n of n e u r o n s / n e u r o p i l , astrocytosis a n d microglial activation. A c o m p a r i s o n o f the m o r p h o l o g y o f c y t o k i n e / p r o s t a g l a n d i n e x p r e s s i n g cells with G F A P o r F 4 / 8 0 reactivity (Fig. 2 E , F ) i n d i c a t e d that the cytokine, P G a n d l i p o c o r t i n i m m u n o r e a c t i v e gila w e r e astrocytes.

4. Discussion This study clearly d e m o n s t r a t e d t h a t IL-1, T N F a , P G E 2 a n d PGF2~ a r e p r e s e n t i n / o n a s t r o c y t e s in t h e b r a i n s of t e r m i n a l l y affected, s c r a p i e - i n f e c t e d mice. N o staining was d e t e c t e d in n o r m a l b r a i n - i n o c u l a t e d controls a n d t h e o b s e r v a t i o n t h a t cytokine, P G a n d l i p o c o r t i n i m m u n o r e a c t i v i t y s h o w e d a similar distribution to t h e astrocytosis a n d microglial activation in t h e s e m o d e l s , a n d was r e s t r i c t e d to t h o s e a n a t o m i c a l r e g i o n s o f t h e b r a i n showing v a c u o l a t i o n o f n e u r o n s a n d n e u r o p i l , i n d i c a t e d t h a t i m m u n o c y t o c h e m i c a l ex-

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pression of cytokines, prostaglandins and lipocortin was induced following infection of neurons by the scrapie agent. The present results suggest that in the terminal stages of scrapie infection, it is predominantly astrocytes that express these cytokines and lipocortin-l. These findings confirm and extend the observations made of TNFa expression in astrocytes in mice terminally affected with the Fujisaki strain of CJD [40] and lipocortin expression in astrocytes in AD [6,37]. Studies of cytokine expression in AD have variously described immunoreactivity in /3A4 plaques [4], microglia around these plaques [15], glial cells both associated and unassociated with /3A4 plaques [6] or in astrocytes and microglia around /3A4 plaques and in more widely scattered microglia [30]. Either astrocytes or microglia or both have been shown to express ILs and TNFa in various pathological conditions, including multiple sclerosis, ischaemia, endotoxaemia, HIV-related encephalopathies, meningitis and AD [4,6,15,24,30,51,52, 53]. Thus the significance of the present results where cytokine expression was mainly, if not only, in astrocytes, is unclear. One factor to consider is that both scrapie models studied here show PrP immunoreactivity in both neurons and astrocytes (P.A. McBride, personal communication; A. Williams, personal observations). However, a previous report of TNFa expression in a murine model of CJD also reported that cytokine staining occurred in astrocytes [40]. It is also possible that different glial cell types express cytokines in acute vs. chronic insults. Thus the predominant celt type that express cytokines following acute insults/inflammation would be microglia a n d / o r invading monocytes/macrophages whereas astrocytes would be the main cytokine-expressing cell type in more chronic lesions. The contribution of induced cytokine expression following post mortem delay a n d / o r any acute cause of death to the overall picture of cytokine immunoreactivity observed in AD cases [15] clearly requires further investigation in this context. The cytokine immunoreactivity observed here may explain the marked astrocytosis and microglial activation present in scrapie-infected brains. IL-1 is a potent regulator of astrocyte proliferation and differentiation [7,28] and microglia also become activated and upregulate MHC Class II in response to various cytokines [15]. The roles of cytokines/prostaglandins and lipocortin in the genesis of the neuronal lesions in scrapie - - neuronal loss, vacuolation of neurons and neuropil, accumulations of a disease-specific isoform of the PrP protein (PrP ~c or prions) - - remain unclear. The induction of cytokines may occur late-on in the development of lesions and be the consequence of widespread neurodegeneration. However, it is also possible that cytokines play a significant role in earlier events of neuronal dysfunction in a way analogous to

that proposed in AD [6], a disease that shows many similar features to scrapie/CJD [91. Major features of AD include ~A4 amyloid deposition, abnormal tau-phosphorylation, neurofibrilla1.-y tangle formation and neuronal death. Tile/3A4 deposition may be due to overproduction of amyloid precursor protein (APP) a n d / o r abnormal post-translational processing [31]. The expression of activated complement factors, acute phase proteins, cell stress proteins and cytokines observed in AD brains [5,6,12,15,30,32, 44,47] may cause increased APP synthesis, since the promoter region of the APP gene contains AP-I sites, acute phase elements and heat shock elements [29,50]. IL-1 has been shown to up-regulate the synthesis of APP mRNA in vitro [29], possibly via PGs and a nerve growth factor (NGF)-mediated mechanism [25,26] and intracerebral administration of NGF shown to increase levels of APP mRNA in the CNS in vivo [45]. In turn, NGF itself enhances the toxic effects of APP accumulation in vitro [56]. In scrapie/CJD, 'pathological' post-translational modifications of the PrP protein result in PrP ~c depositions in areas of the brain that subsequently show vacuolation [8,9,13,36,43]. Marked astrocytosis and microglial activation, induction of/32-integrins, increased cell stress protein expression and ubiquitin conjugation have all been reported in scrapie/CJD [14,27,34,35, 39-42,55]. Examination of the published sequence of the promoter region of the PrP gene in the hamster [3] reveals the presence of an AP-1 site. Intracerebral injection of hamsters with NGF has also been shown to increase levels of PrP mRNA [45]. Furthermore it has been reported that a fragment of PrP, like some APP fragments, can be cytotoxic to CNS cultures [14,22,38,57]. It is tempting to speculate, therefore, that cytokines play an important role in the pathogenesis of the neurodegeneration in scrapie/CJD also. Thus neuronal infection by the scrapie agent may provide the initial stimulation for glial cytokine production that would be sustained by the amplification of infection in neurons that become irreversibly damaged. It is also possible that infection of glia by the scrapie agent could result in sustained cytokine production. Further studies are clearly required to investigate the hypothesis that glial cytokine production affects PrP processing, the development of lesions and neuronal survival/death in scrapie.

Acknowledgements We thank S. Poole (National Institute for Biological Standards and Controls, South Mimms, Hertfordshire, UK), F. Carey and R. Forder (Zeneca Pharmaceuticals, Macclesfield, UK), N. Rothwetl (Department of Physiological Sciences, University of Manchester, UK)

A.E. Williams et al. / Brain Research 654 (1994) 200-206

and W. Buurman and F. Ramakers (State University of Limburg, Maastricht, The Netherlands) for their gifts of antibodies. We also thank L. Doughty for photographic assistance. F4/80 antibody was obtained from Serotec, UK.

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