Up-regulation of lipocortin-1 and its mRNA in reactive astrocytes in kainate-lesioned rat cerebellum

Journal of Neuroimmunology ELSEVIER

Journal of Neuroimmunology50 (1994) 25-33

Up-regulation of lipocortin-1 and its mRNA in reactive astrocytes in kainate-lesioned rat cerebellum L y n e t t e M u l l e n s a, D e r e k R . M a r r i o t t b,., K a r e n A . Y o u n g b, L e s l e y T a n n a h i l l c, S t a f f o r d L. L i g h t m a n a,1, G r a h a m P. W i l k i n b a Neuroendocrine Unit, Department of Medicine, Charing Cross & Westminster Medical School, St. Dunstan's Road, London 14:68RF, UK b Department of Biochemistry, Imperial College of Science, Technology & Medicine, South Kensington, London SW7 2AZ, UK c Institute of Neurobiology, ETH, H6nggerberg, 8093, Zurich, Switzerland

(Received 10 August 1993) (Revision received30 September 1993) (Accepted 30 September 1993)

Abstract

We have used a combined molecular and immunocytochemical approach to examine the expression of lipocortin-1 (LC-1) in kainate-lesioned rat cerebellum. Using immunocytochemistry, Western and Northern blotting, we have shown upregulation of LC-1 mRNA and expression of LC-1 localised specifically to reactive astrocytes. These studies suggest that reactive astrocytes are a major synthetic compartment for the expression of LC-1. The well-reported immuno-suppressive effects of lipocortin(s), suggests that reactive astrocytes could serve to negatively modulate inflammatory reactions in the central nervous system. Key words: Lipocortin; Reactive astrocytes; Damage; Central nervous system

1. Introduction

It is well established that physical insult to the central nervous system (CNS) can result in an accumulation of astrocytes within and adjacent to the site of injury. This phenomenon, referred to as reactive gliosis, is a characteristic feature of experimentally induced injury and is implicated in the pathogenesis of several CNS diseases. Reactive astrocytes, which constitute the major cellular component of reactive gliosis, are characterised by hyperplasia, extensive synthesis of the intermediate protein glial fibrillary acidic protein (GFAP) and by hypertrophy of cytoplasmic processes (reviewed Lindsay, 1986; Eng, 1988; Malhotra et al., 1990). The function(s) of reactive astrocytes are unclear. Studies have shown that reactive gliosis may present a

* Corresponding author. Phone (071) 589 5111 ext. 4116, Fax (071) 225 0960 1 Present address: Department of Medicine, University of Bristol, Bristol Royal Infirmary,Upper Mouldlin Street, Bristol BS2 8HW, UK 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI 0165-5728(93)E0152-Y

physical barrier to the regeneration of neurons (Reier and Houle, 1988). However, it has also been suggested that reactive astrocytes may produce factors conducive to neuronal support (see Frautschy et al., 1991). Rapidly accumulating studies suggest that reactive astrocytes express a complex phenotype. These include the upregulation of substance P (Mantyh et al., 1988) and epidermal growth factor (Nieto-Sampedro, 1988) receptors, several growth factors (see Frautschy et al., 1991 and references therein), gangliosides (LeVine et al., 1986) and oxidative enzymes (AI-AIy and Robinson, 1982; Nakamura et al., 1990). Other data suggest that some reactive astrocyte properties are also expressed by cultured astrocytes. For example, in previous studies we have shown that cultured astrocytes express substance P receptors coupled to the accumulation of phosphoinositides and the synthesis of prostaglandins (Marriott et al., 1990). In several systems, these and other eicosanoids, are potent mediators of inflammation (Curtis-Prior, 1988). The observation that, following transection of optic nerve, substance P receptors are up-regulated in reactive astrocytes suggests that expression in vitro could be conserved in reactive astrocytes in vivo (see Mar-

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L. Mullens et al. /Journal of Neuroimmunology 50 (1994) 25-33

riott and Wilkin, 1993). Other studies have shown ATP (Gebicke-Haerter et al., 1988; Pearce et al., 1989) and interleukin-1/3 (Hartung et al., 1989; Katsuura et al., 1989) receptors are also coupled to eicosanoid synthesis in cultured astrocytes. Whilst these observations indicate that astrocytes could initiate a n d / o r potentiate an inflammatory cascade in the CNS, recent studies suggest that astrocytes could also participate in the suppression of inflammatory prostanoids. In this regard, the lipocortins (also called annexins, calpactins, chromobindins, calcimedins and several other terminologies) are a group of highly homologous calcium and phospholipid-binding proteins (reviewed in Martyn Bailey, 1991). The 35-kDa protein lipocortin-1 (LC-1) possesses potent anti-inflammatory activity in several experimental systems (Cirino et al., 1989; Errasfa and Russo-Marie, 1989; Strijbos et al., 1992 and references therein). A recent study has shown marked expression of LC-1 in cultured astrocytes. In contrast, normal brain homogenates from neonatal and adult rats were comparatively devoid of LC-1 (Gebicke-Haerter et al., 1991). These data prompted us to examine the expression of LC-1 in kainate-lesioned rat cerebellum. Kainic acid is a potent neurotoxin which binds specific populations of excitatory amino acid receptors. Administration of kainic acid induces prolonged depolorisations and seizure activity, resulting in lesions of susceptable neuronal populations (reviewed in Coyle et al., 1981). Using a combination of immunocytochemistry, Western and Northern blotting, we show that LC-1 and LC-1 mRNA are present in homogenates of kainate-lesioned tissue and that LC-1 and GFAP are specifically up-regulated in reactive astrocytes in vivo.

allowed to recover and were fed food and water ad libitum. Various coordinates and kainate concentrations were evaluated in preliminary experiments. Penetration and diffusion was evaluated by prior injections of toluidine blue. Preliminary immunocytochemical analysis (see below) established that the temporal expression of reactive gliosis was most marked after 10 days and for up to 3 months thereafter (data not shown). 2.2. Tissue culture Type-1 astrocyte-enriched cultures were prepared essentially as previously described (Marriott and Wilkin, 1993). Cerebral cortices were removed from 2-day-old rat pups. Following mechanical chopping and trituration, the cells were collected and resuspended in DMEM (Imperial Laboratories, Andover, Hants UK) supplemented with: 10% foetal calf serum, 0.35 m g / m l glutamine, 0.1 m g / m l L-valine, 125 IU penicillin/ streptomycin (Sigma) and 2 5 / z g / m l gentamycin (ICN Flow, High Wycombe, Bucks UK). The cells were plated at a density of 2.5 × 105 cells/cm 2 in 150-cm 2 tissue culture flasks (ICN Flow) precoated with 5 / z g / ml poly-L-lysine (Sigma). After 5-6 days in vitro, contaminating fibroblasts were removed by the substitution of o-valine for L-valine in the growth medium (Cholewinski et al., 1989). Contaminating O-2A progenitor cells and microglia were removed from the cultures by shaking the flasks on an orbital shaker (2.5 cm throw; 250 revs/min) for 16-18 h. The remaining type-1 astrocyte monolayers were then washed in PBS, removed by incubation in trypsin and prepared for Northern analysis as described below. Immunocytochemical analysis showed these cultures to comprise > 95% GFAP ÷ astrocytes (data not shown).

2. Materials and methods 2.3. Northern analysis 2.1. Kainate lesions c D N A probes. A 1376-bp human lipocortin 1 cDNA

All procedures were performed on 180-200 g adult male rats (Sprague-Dawley, Imperial College) under aseptic conditions. Animals were anaesthetised with 3% Halothane in medical oxygen:nitrous oxide (2:1 v/v) and positioned in a stereotactic frame. Anaesthesia was maintained with 2% halothane throughout surgery. The head was swabbed with 70% ethanol and a 2-cm anterior-posterior incision was made over the rear of the skull. After clearing the membranes a hole was drilled into the skull 2 mm lateral and 13.7 mm caudal to the bregma, avoiding penetration of the meninges. 2 ~g (in 2 tzl) of filter-sterilised kainic acid (Sigma, Chem Co., Poole, Dorset, UK) in PBS (pH 7.4) was then injected to a depth of 1.8 mm with a Hamilton syringe. The wound was rapidly plugged with bone wax and the skin sutured. The animals were then

(Biogen Inc., Cambridge, MA) was cut from pUC 13 with E c o R I (BCL). The a-tubulin probe was a 1400-bp human cDNA which was cut from the PstI site of plasmid pSP64. The cDNA was separated from plasmid DNA by electrophoresis on a 1% low melting point agarose gel. Eluted cDNA was denatured by heating at 95°C for 5 min and labelled with 50/zCi of [a-32p]dCTP (Amersham Int., Bucks, UK) by random primer elongation (BCL kit) to a specific activity of 1-4 × 10 8 dpm//zg DNA. Northern blotting. Total RNA was extracted from cul-

tured astrocytes, lesioned and control cerebellar hemispheres using the acid guanidinium thiocyanate-phenol chloroform method (Chomczynski and Sacchi, 1987). Homogenates of cultured astrocytes and equal

L. Mullens et al. /Journal of Neuroimmunology 50 (1994) 25-33

amounts of control and lesioned cerebella total RNA were electrophoresed on a 1% agarose gel containing 6% formaldehyde as denaturing agent and 1 x MOPS buffer (0.04 M morpholinopropane sulphonic acid, 10 mM sodium acetate, 1 mM EDTA pH 8.0). RNA was transferred onto Hybond-N nylon membranes

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(Amersham) by capillary action overnight in 20 x SSC (pH 7.0) and bound by exposure to ultra-violet light for 5 min. Blots were pre-hybridized at 65°C for 15 min in hybridization buffer (1 M NaC1 10% dextran sulphate M, 500000 and 1% SDS). Hybridization was performed overnight at 65°C using fresh buffer containing

Fig. 1. Decreased expression of NF (a) and increased expression of GFAP (b) in kainate-lesioned rat cerebellum. 2 p.g of kainic acid was injected into the right cerebellar hemisphere. After 10-14 days animals were killed and processed for double immunocytochemistry.

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L. Mullens et al. /Journal of Neuroimmunology 50 (1994) 25-33

1 0 0 / x g / m l denatured salmon sperm D N A and 1 × 10 6 d p m / m l of radiolabelled denatured c D N A probe. Blots were subsequently washed in 1 × SSC at 65°C for 30 rain prior to autoradiography. The Northern blot hybridized with the LC-1 c D N A was exposed for 3 days. The blot was then stripped of probe by washing in 0.2

M NaOH, re-probed with labelled a-tubulin c D N A and exposed for 5 h for R N A standardisation. 2.4. Cryostat sections

Kainate-lesioned animals were killed 10-14 days following injection. The animals were anaesthetised

Fig. 2. Constitutive expression of LC-1 (a) and GFAP (b) in the contra-lateral, i.e. un-lesioned (left) cerebellar hemisphere. Otherwise, legend as in Fig. 1.

L. Mullens et al. /Journal of Neuroimmunology 50 (1994) 25-33

with sodium pentabarbitone and trans-cardially perfused with PBS at 37°C. Animals were then decapitated and the cerebella removed and frozen in iso-pentane ( - 2 5 ° C ) . 10-tzM sections were cut on a cryostat and thaw-mounted on gelatin-coated slides. Dried sections were stored with desiccant at - 2 0 ° C for up to 3 weeks.

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2.5. Western blotting

A thick coronal slice was removed from a PBS-perfused cerebellum and the tissue prepared for cryostat sectioning as described above. Serial sections were removed from the slice until the lesion was visualised. The tissue was then dissected from the surrounding

Fig. 3. Increased expression of LC-1 (a) and GFAP (b) in kainate-lesioned (right) cerebellar hemisphere. Otherwise, legend as in Fig. 1.

L. Mullens et aL / Journal of Neuroimmunology 50 (1994) 25-33

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area using a cold scalpel. A comparable amount of control tissue was also removed from the same area on the contralateral side of the cerebellum. Frozen tissues from 6-10 cerebella were then pooled and homogenised in buffer (5 mM Tris, 2 mM EDTA, and 2 mM EGTA). Control and lesioned samples were equalised for protein (Bio-rad DC assay kit, Hemel Hempstead, Herts, UK). The tissue was then solubilised by boiling in sample buffer (62.5 mM Tris. HC1, 10% glycerol, 2.5%, SDS 5% mercaptoethanol), and stored at -20°C for up to 1 month. Equal amounts (0.1 mg) of control and lesioned samples were subjected to 10% SDS-PAGE (Laemmli, 1970). Separated proteins were transferred to nylon membranes (ICN Flow) and stained for 2 h in primary antibody. Immunoreactive bands were visualised with the appropriate horseradish peroxidase-conjugated secondary antibody using chloronaphthol as the chromagen. Even loading and transfer of control and lesioned samples was monitored by Coomassie brilliant blue staining of residual proteins following transfer (data not shown).

rapidly thawed, fixed for 20 min in 4% paraformaldehyde in PBS, rinsed in PBS and then preincubated for 15 min in PBS containing 0.2% BSA (PBS/BSA). Primary mAbs (GFAP; 1:100, NF; 1:800) and antiLC-1 (1 : 600) were added for 1 h and overnight respectively, followed by the appropriate fiuorochrome-conjugated secondary antibodies for 30 min. The sections were then washed thoroughly and mounted in DABCO (glycerol/PBS 9:1 with 2.5% diazobicyclo-octane v / v / v ) to retard fading. Unless otherwise stated, all washes and antibody dilutions were in PBS/BSA. Preliminary experiments were conducted to define optimal antibody titres and fixation conditions. There were no consistent differences in immunostaining between slide-fixed versus perfused-fixed sections. The absence of fluorescence overlap a n d / o r breakthrough was confirmed by single immunostaining on serial sections. Control experiments were performed by omission of the primary antibodies (data not shown).

3. R e s u l t s

2.6. Immunocytochemistry 3.1. Immunocytochemistry Monoclonal antibody (mAb) RT97 against neurofilament (NF) (Anderton et al., 1982) and polyclonal rabbit anti-LC-1 (842) (Johnson et al., 1989a,b; Bolton et al., 1990) have been previously described. Primary mAbs against GFAP was purchased from a commercial source (Sigma). Indirect double immunofluorescence was performed by a modification of the method described by (Curtis et al., 1988). In brief, sections were

1

2

3

4

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6

7 __

200-

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Injection of kainate into rat cerebellar hemispheres resulted in a marked localised depletion of NF (Fig. la). In contrast, sections double-immunostained for GFAP showed a reciprocal upregulation of GFAP and a pronounced reactive gliosis at the lesion site (Fig. lb). In lesioned cerebellar sections contralateral to the injection site, LC-1 was barely detectable (Fig. 2a), despite pronounced expression of GFAP (Fig. 2b). In contrast, LC-1 was markedly upregulated at the lesion site (Fig. 3a). Co-localisation of upregulated LC-1 with increased expression of GFAP (Fig. 3b), confirmed that these cells were reactive astrocytes. Both Bergmann glia (small arrow heads) in the molecular cell (MC) layer and astrocytes (large arrow heads) in the granule cell (GC) layer were labelled. Though the majority of GFAP-expressing reactive astrocytes were also LC-l-positive, the intensity of LC-1 immunoreactivity was heterogeneous.

I

30--

3.2. Western blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . .

C

L

GFAP

C

L

NF

i.'~ . . . . . . . . . . . . . . . . . .

C

L

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std

LIPOCORTIN-I

Fig. 4. Western blot analysis of contra-lateral and kainate-lesioned cerebellar homogenates. 10-day post-lesion tissue was dissected from frozen contra-lateral and kainate-lesioned cerebellum and identical amounts prepared for SDS-PAGE. Contra-lateral (C) and lesioned (L) tissue were probed as follows: Lanes 1,2, GFAP; lanes 3,4, NF; lanes 5,6, LC-1; lane 7, recombinant LC-1 standard (std).

Western blot analysis of contralateral (control) and kainate-lesioned tissue (Fig. 4), also showed a reciprocal expression of GFAP and NF in tissue homogenates (lanes 1,3 and 2,4). Expression of LC-1 was barely detectable in control tissue, but was markedly upregulated in lesioned tissue (lanes 5,6). The major immunoreactive band precipitated at a molecular mass of 35 kDa. The authenticity of LC-1 was verified with a co-migrating recombinant LC-1 standard (lane 7).

L. MuUens et al. /Journal of Neuroimmunology 50 (1994) 25-33 A. LCI mRNA 1

2

B. ~ - T U B U L I N mRNA

3

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2

3

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---1400 bp---

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Cultured

AstrocyteB

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Cerebellum

- control

3.

Cerebellum

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lesioned

Fig. 5. Northern blot analysis of cultured astrocytes, contra-lateral and 10-day post kainate-lesioned cerebellar homogenates. Lane 1, 20 /zg total RNA from cultured astrocytes; lane 2, 20 /zg total RNA from contra-lateral hemispheres; lane 3, 20 /xg total RNA from kainate-lesioned hemispheres. (A) Hybridized with 32P-LC-1 cDNA. Autoradiogram exposed for 3 days. (B) The same blot stripped and re-hybridized with [a-32p]tubulin cDNA. Autoradiogram exposed for 6h.

3.3. Northern blot analysis Northern blot analysis of cultured astrocytes, control and lesioned cerebellar homogenates showed marked hybridization with 32p-LC-1 cDNA to a single band of 1.7 kb, the correct size for LC-1 mRNA, in lesioned tissue and cultured astrocytes only (Fig. 5a). In contrast, the same blot stripped and re-hybridized with [a-32p]tubulin cDNA showed similar expression of atubulin mRNA in cUltured cells and both the control and lesioned tissue (Fig. 5b).

4. Discussion

Using several independent approaches, we have described the presence of LC-1 mRNA and the expression of LC-1 protein in reactive astrocytes. In Western blots, LC-1 antibody bound only the protein derived from lesioned tissue homogenates. Northern blot analysis of total RNA probed with a 1376-bp human LC-1 cDNA confirmed this restricted distribution. In contrast, blots stripped and re-probed with a-tubulin cDNA showed a similar signal in both control and lesioned tissue indicating specific upregulation of LC-1 mRNA. Double immunocytochemistry on sections of kainate-lesioned cerebellum showed co-localisation of GFAP and LC-1 in reactive astrocytes. We have also confirmed increased expression of LC-1 mRNA restricted to the lesioned site by in situ hybridization of kainate-lesioned cerebellar sections using a LC-1 antisense riboprobe (data not shown).

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Our observations that both LC-1 mRNA and LC-1 were absent or minimal in control tissue homogenates confirms several previous observations (Johnson et al., 1988a,b; Pepinsky et al., 1988; Gebicke-Haerter et al., 1991). However, using antibodies against a recombinant N-terminal fragment, Rothwell and colleagues have reported a high density of LC-l-like immunoreactivity in glial and neuronal cells, particularly in the hippocampus (see Relton et al., 1991). It is likely that this discrepancy relates to differences in epitope specificity between antibodies. Alternatively, it is possible that these antibodies recognise isoforms of LC-1 which are differentially expressed in normal brain. In contrast, there has been more consensus on the expression of lipocortins in the injured CNS. Previous studies have suggested inflammatory cells of the immune system as being prominent sources of these proteins (Bolton et al., 1990). Studies using diseased human CNS tissue have showed increased LC-1 associated with invading and resident macrophages. The same authors also suggested increased LC-1 associated with glial tumours (Johnson et al., 1989a) and astrocytosis in diseased human CNS (Johnson et al., 1989b). However, previous immunocytochemical studies were not combined with cell specific markers. Consequently, the identity of LC-l-expressing ceils has remained undefined. In this regard, this is the first study to unequivocally co-localise LC-1 to GFAP-expressing reactive astrocytes in vivo. With regard to other cellular sources of LC-1, our study does not preclude a possible contribution from invading monocytes or intrinsic tissue macrophages (microglia) to the LC-1 content of tissue homogenates. However, in agreement with others (see Malhotra et al., 1990), we have noted that complement receptorpositive ceils (peripheral macrophages and microglia), whilst initially numerous, are absent or minimal at later time points (personal observations). Rather, the colocalisation of the majority of LC-1 to GFAP-expressing reactive astrocytes 10-14 days post lesion suggests that the temporal expression of this protein could be maintained by reactive astrocytes. In confirmation of previous immunocytochemical studies (GebickeHaerter et al., 1991), we have also shown a marked expression of LC-1 mRNA in cultured astrocytes. These data support the view that cultured astrocytes, at least in part, express a reactive astrocyte phenotype and implicate these cells as a major compartment for the synthesis of LC-1 both in vivo following damage and in vitro. Various methods have been reported for inducing experimental reactive gliosis (see Malhotra et al., 1990). It is apparent that reactive astrocytes express a diverse array of marker molecules (see Introduction). However, it should be noted that the aforementioned reactive astrocyte properties are a composite account de-

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L. Mullens et al. /Journal of Neuroimmunology 50 (1994) 25-33

rived from studies which have used a variety of experimental paradigms. Consequently, it is possible that many of these studies are sufficiently different to preclude a consistent interpretation. For example, although upregulation of GFAP is a widely reported property of reactive gliosis (reviewed in Eng, 1988), its temporal expression can be permanent (Vijayan et al., 1990) or transient (Hozumi et al., 1990; O'Callaghan et al., 1990) depending on the site a n d / o r method of injury. In this present study, the expression of LC-1 was co-localised with GFAP elevation within a single time point (10-14 days post-lesion). We cannot exclude the possibility that the temporal expression of LC-1 and GFAP are asynchronous at other time points. Experiments are currently underway to determine if LC-1 induction precedes, follows or is in frame with GFAP elevation. The presence of LC-1 m R N A and LC-1 in reactive astrocytes is particularly significant in the light of accumulating evidence implicating astrocytes as a major cellular compartment for the synthesis of prostanoids (reviewed in Murphy et al., 1988). These compounds, generated by phospholipase A2-mediated release of arachidonic acid, constitute an important group of inflammatory mediators released at local sites of tissue injury in response to a variety of agonists (Curtis-Prior, 1988). In this regard, several neuro and immuno-active compounds have been shown to stimulate the release of prostanoids from cultured astrocytes (see Marriott et al., 1991 and references therein). The ability of exogenous lipocortins to block prostanoid synthesis is also well-documented. For example, LC-1 or its active N-terminal fragments have variously been shown to be anti-pyretic (Carey et al., 1990; Davidson et al., 1991; Strijbos et al., 1992), anti-inflammatory (Cirino et al., 1989; Browning et al., 1990) and protective against neuronal damage (Relton et al., 1991; Black et al., 1992). The precise mechanism(s) whereby LC-1 exerts these effects are unclear, though several have been proposed including phospholipid and calcium binding (see Davidson et al., 1990; Martyn Bailey, 1991). The original proposal that lipocortins mediate the suppressive activity of glucocorticoids via a direct inhibition of PLA 2 has not been sustained in the majority of studies (see Martyn Bailey et al., 1991). Nevertheless, whatever their mechanism of action, the efficacy of lipocortins as potent anti-inflammatory mediators has not been disputed. In contrast, a role for lipocortins as a major physiologic target for the E G F receptor has gained considerably more consensus. It is known that E G F receptor activation initiates an intrinsic protein kinase activity, which in turn, leads to phosphorylation of numerous intracellular proteins. Of these, LC-1 is one of the best characterised substrates for the E G F receptor (Haigler et al., 1987; Varticovski et al., 1988; Johnson et al.,

1990). Phosphorylation of LC-1 by EGF receptor kinase (Pepinsky and Sinclair, 1986) or by protein kinase C (Touqui et al., 1986), has been shown to block the inhibitory activity of LC-1. Significantly, reactive astrocytes also express elevated levels of E G F receptors (Nieto Sampedro, 1988). Additionally, it has also been suggested that expression of LC-1 in cultured astrocytes could account for the inability of some agonists to stimulate the synthesis of prostanoids (Gebicke-Haerter et al., 1991). In summary, we have used a combined molecular and immunocytochemical approach to demonstrate upregulation of LC-1 m R N A and LC-1 immunoreactivity in reactive astrocytes from kainate-lesioned rat cerebellum. These studies suggest that reactive astrocytes are a major synthetic compartment for the expression of LC-1 and that these cells could serve to negatively modulate inflammatory reactions in the CNS.

Acknowledgements We wish to thank Dr. J. Browning (Biogen, Cambridge, MA) for the gift of LC-1 antibodies and human recombinant LC-1, and Mr. David Green for excellent technical assistance. This work was supported by a Wellcome Trust post-doctoral award (D.M.), a Wellcome Trust Clinical Research Training Fellowship (L.M.) and a Nuffield research bursary (K.A.Y).

References AI-AIi, S.Y.A. and Robinson, N. (1982) Ultrastructural study of enzymes in reactive astrocytes: clarification of astrocytic activity. Histochem. J. 14, 311-321. Anderton, B.H., Breinburg, D., Downes, M.J., Green, P.J., Tomlinsop., B.E., Ulrich, J., Wood, J.N. and Kahn, J. (1982) Monoclonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants. Nature 298, 84-86. Black, M.D., Carey, F., Crossman, A.R., Relton, J.K. and Rothwell, N.J. (1992) Lipocortin-1 inhibits NMDA receptor-mediated neuronal damage in the striatum of the rat. Brain Res. 585, 135-140. Bolton, C., Elderfield, A.-J. and Flower, R.J. (1990) The detection of lipocortins 1, 2 and 5 in central nervous system tissues from Lewis rats with acute experimental allergic encephalomyelitis. J. Neuroimmunol. 29, 173-181. Browning, J.L., Ward, M.P., Wallner, B.P. and Pepinsky, R.B. (1990) Studies on the structural properties of lipocortin-1 and its synthesis by steroids. In: Parente, L. and Merelli, L. (Eds.), Lipocortins, Cytokines and Inflammation. Alan R. Liss, New York, NY. Carey, F., Forder, R., Edge, M.D., Greene, A.R., Horan, M.A., Strijbos, P.J.L.M. and Rothwell, N.J. (1990) Lipocortin-1 modifies the pyrogenic actions of cytokines in the rat. Am. J. Physiol. 259, R266. Cholewinski, A.J., Reid, J.C., McDermott, A.M. and Wilkin, G.P. (1989) Purification of astroglial-cell cultures from rat spinal cord: the use of D-valine to inhibit fibroblast growth. Neurochem. Int. 15, 365-369.

L. Mullens et al. /Journal of Neuroimmunology 50 (1994) 25-33 Chomczynski, P. and Sacci, N. (1987) Single-step method of RNA isolation by acid and guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159. Cirino, G., Peers, S.H., Flower, R.J., Browning, R.J. and Pepinski, R.B. (1989) Human recombininant Lipocortin-1 has acute local anti-inflammatory properties in the rat paw oedema test. Proc. Natl. Acad. Sci. USA 86, 3428. Coyle, J.T., Bird, S.J., Evans, R.H., Gulley, R.L., Nadler, J.V., Nicklas, W.J. and Olney, J.W. (1981) Excitatory amino acid neurotoxins: selectivity, specificity and mechanism of action. Neurosci. Res. Prog. Bull. 19, 4. Curtis-Prior, P.B. (1988) Prostaglandins. Biology and Chemistry of Prostaglandins and Related Eicosanoids. Churchill Livingstone. Davidson, J., Flower, R.J., Milton, A.S., Peers, S.H. and Rotondo, D. (1991) Antipyretic actions of human recombinant lipocortin 1. Br. J. Pharmacol. 102, 7-9. Eng, L. (1988) Astrocytic response to injury. In: Current Issues in Neural Regeneration Research, Alan R. Liss, New York, NY, pp. 247-255. Errasfa, M. and Russo-Marie, F. (1989) A purified lipocortin shares the anti-inflammatory effect of glucocorticosteroids in vivo in mice. Br. J. Pharmacol. 97, 1051-1058. Frautschy, S.A., Walicke, P.A. and Baird, A. (1991) Localization of basic fibroblast growth factor and its mRNA after CNS injury. Brain. Res. 553, 291-299. Gebicke-Haerter, P.J., Wurster, S., Schobert, A. and Hertting, G. (1988) P2-purinoceptor induced prostaglandin synthesis in primary rat astrocyte cultures. Arch. Pharmacol. 338, 704-707. Gebicke-Haerter, P.J., Schobert, A., Dieter, P., Honegger, P. and Hertting, G. (1991) Regulation and glucocorticoid-independent induction of lipocortin 1 in cultured astrocytes. J. Neurochem. 57, 175-183. Haigler, H.T., Schlaepfer, D.D. and Burgess, W.H. (1987) Characterisation of lipocortin 1 and an immunologicaily unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A 2 inhibitors. J. Biol. Chem. 262, 6921-6930. Hartung, H.-P., Sch~ifer, B., Heininger, K. and Toyka, K.V. (1989) Recombinant interleukin-1/3 stimulates eicosanoid production in rat primary culture astrocytes. Brain Res. 489, 113-119. Hoffmann, M.-C., Nitsch, C., Scotti, A.L., Reinhard, E. and Monard, D. (1992) The prolonged presence of glia-derived nexin, an endogenous protease inhibitor, in the hippocampus after ischaemia-induced delayed neuronal death. Neuroscience 48, 397408. Hozumi, I., Aquino, D.A. and Norton, W.T. (1990) GFAP mRNA levels following stab wounds in rat brain. Brain Res. 534, 291-294. Johnson, M.D., Kamso-Pratt, J., Pepinsky, R.B. and Whetsell, W.O. (1989a) Lipocortin-1 immunoreactivity in central and peripheral nervous system glial tumours. Human Pathol. 20, 772-776. Johnson, M.D., Kamso-Pratt, J.M., Whetsell, W.O. and Pepinsky, R.B. (1989b) Lipocortin-1 immunoreactivity in the normal human central nervous system and lesions with astrocytosis. Am. J. Clin. Pathol. 92, 424-429. Johnson, M.D., Gray, M.E., Carpenter, G., Pepinsky, R.B. and Stahlman, M.T. (1990) Ontogeny of epidermal growth factor receptor and lipocortin-1 in fetal and neonatal human lungs. Human Pathol. 21, 182-191.

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Katsuura, G., Gottschal, P.E., Dahl, R.R. and Ariura, A. (1989) Interleukin-1/3 increases prostaglandin E 2 in rat astrocyte cultures: modulatory effects of neuropeptides. Endocrinology 124, 2125-3127. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. LeVine, S.M., Seyfried, T.N., Yu, R.K. and Goldman, J.E. (1986) Immunocytochemical Iocalisation of GD 3 ganglioside to astrocytes in cerebellar mutants. Brain Res. 374, 260-269. Lindsay, R.M. (1986) Reactive gliosis. In: S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. 3, pp. 231-261. Academic Press, New York, NY. Malhotra, S.K., Shnitka, T.K. and Elbrink, J. (1990) Reactive Astrocytes - a review. Cytobios 61, 133-160. Marriott, D.R. and Wilkin, G.P. (1993) Substance P-stimulated PI accumulation in O-2A progenitor cells and type-2 astrocytes in vitro. J. Neurochem. 61,826-834. Marriott, D.R. Wilkin, G.P. and Wood, J.N. (1991) Substance P-induced release of prostaglandins from astrocytes: regional specialisation and correlation with phosphoinositol metabolism. J. Neurochem. 56, 259-265. Martyn Bailey, J. (1991) New mechanisms for effects of anti-inflammatory glucocorticoids. Biofactors 3, 97-102. Nakamura, S., Kawamata, T., Akiguchi, I., Kameyama, M., Nakamura, N. and Kimura, H. (1990) Expression of monoamine oxidase B activity in astrocytes of senile plaques. Acta Neuropathol. 80, 419-425. Nieto-Sampedro, M (1988) Astrocyte mitogen inhibitor related to epidermal growth factor receptor. Science 240, 1784-1786. Pearce, B., Murphy, S., Jeremy, J., Morrow, C. and Dandona, P. (1989) ATP-evoked Ca 2÷ mobilisation and prostanoid release from astrocytes: P2-purinergic receptors linked to phosphoinositide hydrolysis. J. Neurochem. 52, 971-977. Pepinsky, R.B. and Sinclair, L.K. (1986) Epidermal growth factor-dependent phosphorylation of lipocortin. Nature 321, 81-84. Reier, P.J. and Houle, J.D. (1988) The glial scar: Its bearing on axonal elongation and transplantation approachs to CNS repair. Adv. Neurol. 47, 87-138. Relton, J.K., Strijbos, P.J.L.M., O'Shaughnessy, C.T., Carey, F., Forder, F.A., Tilders, F.J.H. and Rothwell, N.J. (1991) Lipocortin-1 is an endogenous inhibitor of ischaemic damage in rat brain. J. Exp. Med. 174, 305-310. Strijbos, P.J., Hardwick, A.J., Relton, J.K., Carey, F. and Rothwell, N.J. (1992) Inhibition of central actions of cytokines on fever and thermogenesis by lipocortin-1 involves CRF. Am J. Physiol. 263, E632-E636. Varticovski, L., Chahwala, S.B., Whitman, M., Cantley, L., Schindler, D., Pingchang Chow, E., Sinclair, L.K. and Pepinsky, R.B. (1988) Location of sites in human lipocortin 1 that are phosphorylated by protein kinases and protein kinases A and C. Biochemistry 27, 3682-3690. Vijayan, V.K., Lee, Y.-L. and Eng, L.F. (1990) Increase in glial fibrillary acidic protein following neural trauma. Mol. Chem. Neuropathol. 13, 107-117.