Journal of Neuroimmunology, 6 (1984) 151-159 15[ Elsevier

JNI 00174

Prostaglandin Levels in Cerebrospinal Fluid from M dtiple Sclerosis Patients in Remission and

Relapse

Christopher golton 1, Alan M. Turner 2 and John L. Turk t t Department of Pathology, Royal College of Surgeons of Englan~ la'ncoln's Inn Fields, £.tmdms l~:'2.! 3P~,:

and 2 We.:sex Neurological Centre, Southampton S09 4XY (Great Britainb

(Received 26 August, 1983~ (Revised, received I November. 1983)

(Accepted 21 November, 1983)

Summary

Radioimmunoassay (RIA) techniques have been employed to determine pto~. taglandin (PG) levels ul the cerebrospinal fluid (CSF) from multiple sck ro~ (MS) patients in remission ,lnd relapse -'Lnd in subjects with o;her neurological (OND). PGE and PGF2a concentrations in spinal fluid from MS patienl.~ in relapse were significantly lower than values estimated during remission aud in i n d i ~ ~'ith OND of the central nervous system (CNS). These observations are discussed ~a :relation to the clinical state of pat~e~s with demyelinating disease togedun" with a consideration of the concept thai disordered immune mechanisms contribute a central role in the pathogenesis of MS.

Kcy words: Cerebrospinal f l u id - Mul t ip le sclerosis - Pros taglandins

Introduction

Multiple sclerosis (MS) is a chronic inflammatory dise,.se of the central nev,~,us system (CNS) which is frequently characterised by acute ¢finical ~ tp se s foBowed by periods of remission. Pathological changes occurring in CNS tissues an: largc~ restricted to the white matter where lesions consist of fibrous plaques of do- myelinated nerve tracts. One current hypothesis to expla;~n the aetiology o[ MS

T~s study was financially supported by the Multiple Sclerosis Society of Great B~ritain.

0165.5728/84/$03.00 © 1984 Elsevier Science Publishers B.V.

152

suggests that a combination of abnormal immunological responses, possibly precipi- tated by infectious agents in a genetically susceptible individual, may cause onset of the disease (Raine 1977; Iivanainen 1981; Greenstei.n and McFarland 19821). In particular, partial response to immunosuppressive therapy (Eltison and Myers 1980), elevated immunoglobulin levels together with oligoclonal bands ii,l tile cerebrospinal fluid (CSF) (Cuzner and Davison 1979) and the occurrence of lymphocyte popula- tions and activated macrophages in CNS lesions (Prineas and Wright 1978) imply that disordered immune mechanisms may be important in the pathogenesis of MS.

Enzymic factors released as a result of lymphocyte-macrcphage interac,ions are believed to be responsible, in part, for the characteristic d,.'Tnydination of nerve fibres obserw.xl in MS plaques (Arstila et al. 1973). in addition, lymphocyte-~:ctivated macrophages synthesise and release large amounts of prostaglandins (PGs) which are known to act as both mediators and modulators of acute and chronic inflammation (Bonta and Parnham 1978). Furthermore, the prostanoids and their precursor fatty acids have been shown to control cell-mediated immune reactions by ~cting as extra-cellular regulators of T-cell function (Mertin et aL 1973; Pelus and Strausser 1977).

Therefore it is probable that activated inflammatory-type cells within MS plaques and/or during clinical episodes of the disease generate PGs in response to an indirect stimulation by one or more immanogens, The cell-derived prostanoids may then enter the surroun0ing CNS tissues and CSF and attempt to influence the initial cell-mediated immune response by limiting the producticn of lymphocyte-derived inflammatory mediators,

In this study the levels of PGE, PGF2a and 6-oxo-PGFla have been measured in the CSF from MS patients in relapse and remission. Prcnitanoid levels are compared to value.,; obtained from the CSF of individuals with other neurological diseases (OND) of the CNS and the implications of the~e findings are discussed with respect to the pathogenesis of MS.

Materials and Methods

Patients When clinically indicated CSF was obtained by lumbar puncture from MS

patients either in relapse or remission and from individuals suffering from OND. All diagnoses of MS were clinically definite according to the criteria of McDonald and Halliday (1977) and patients were considered to have suffered a relapse if Mthin the last month they had develol',~d increased disability with a new physical sign of neurological impairment. No patient had been treated with corticosteroids or ACTH for at least 6 months before lumbar puncture.

Samples were coded in order to conceal the clinical state o~' each indivk~ual from the investigators. Each CSF '#as immediately frozen *.o -700C after removal of a small aliquot for the determination of cell numbers.

Exlraction of PGs from CSF CSF volumes of 1.25 ml vlere made up to 2 ml with plK~phate-bufi'ered sa}ine

(PBS) and adjusted to pH 3.0 with I N hydrochloric acid. Samples were extrac~e:l 3 times with diethyl ether and, after evaporation of the solvent, the dried extracts were stored under nitrogen ~,t -70°C until assayed. Simultaneous extractions of [3H]PGs in PBS were undertaken to provide an estimate of the percentage of PG recovered from the extracted samples.

Radioimmunoassay (RIA) of extracted CSF Each dried CSF extract was dissolved in 0.7 ml o! 0.1 M tficine-buffered saliine

(TBS) containing 0.1% gelatine and 0.1~ sodium azide. RIA of PGs was undertaken as described by Jose et al. (1981).

Thin-layer chromatography (TLC) of CSF CSF volumes of 1 ml were pooled and extracted as above. The dried extracts were

solubilised in 100 /~1 of methanol and spotted, together with 20 /tl volumes ol" [3H]PG, onto aluminium plates impregnated with 0.2 mm of silica gel (Merck). The organic phase of a solvent system consisting of ethylacetate/disfilled water/iso- octane/glacial acetic acid (11:10:5 :2) was used to separate :he samples aad [3H]standards after double development. The TLC plates were cut into 1.5-~an zones, eluted in 1 ml of methanol for 1 h and evaporated to dryness. Rad/oactivity present in [3H]zones was measured by reconstituting the dried extracts in distilled water and lnstagel and recording the cpm. Extracts from non-radioactive samples were resuspended in TBS and assayed for the presence of PG by RIA.

Reagents The preparation of PG antisera together with the binding and crogs-reactivity

data are as described by Hensby et al. (1981) and Jose et al. (1981).

Radioactive materials [3H]PGE2 (160 Ci/mmol) and [3H]PGF.~a (180 Ci/mmol) were suppfied by the

Radioehenfical Centre, Amersham, U.K. [3H]6-oxo-PGFla (120 Ci/mmol) was obtained from New England Nuclear, Boston, MA, U.S.A.

Statistical analysis The Mann-Whitney U-test was emoloyed to determine sigmficaet differences in

the PG content of CSF between indiv/duals with OND and MS p~tients in re'zpse or remission.

Results

The PG content of CSF from patients w:th MS and OND of the CNS The PGE content of CSF from MS patients in relapse (range: 250-790 l~,/ml)

was significantly lower than values estimated for individuals with OND of the L'~S (range: 500-2200 pg/ml) (P < 0.001) az,d MS patients ill remission (range: 600--1180 pg/nd) (P < 0.02) (Table I). In addition, PGF2a levels in spinal fluid froua MS

154

TABLE 1

THE MEAN CONCENTRATION (pg/ml) (±SEM) OF F¢2E, PGF2a AND 6-OXO-PGFla IN THE CSF FROM M~,; PATIENTS AND INDIVIDUALS WITH OND

Figures in parenthese.t indicate the number of CSF r~alplcs ,qmalyu~d/group.

Clinical state PGE PGFza 6.oxo.POFla

OND 1006 4- 70 (36) 1017 4-149 (36) 55S ~ 70 (39) MS relapse 574 4- 50 (10) 407 4- 79 (10) 731 4- 40 (10) MSremi~sion 8164-74 (8) 6914- 84 (7) 6734-41 (8)

patients suffering an exacerbation of the disease (range: 0-740 pg/ml) were signifi- cantly less than values recorded for patients in remission (range: 470-1050 pg/ml) (P < 0.05) and OND (range: 400-4750 pg/ml) (P < 0,002), In contrast, the levels of 6-oxo-PGFza in CSF from MS patients in relapse (range: 480-900 pg/ml), remis- sion (530-880 pg/ml) and OND (range: 0-1590 pg/ml) were not significantly different.

The clir~icai conditions constituting OND of the CNS and the two phases of MS are described in Fig. 1 (A-C), Patients were assigned to their appropriate clinical group and the PG levels in each CSF sample were individually recorded. Mean values for PGE and PGF2a in CSF from MS patients in relapse were consistently lower than levels noted for individuals affected by other pathological states (Fi 8, 1A, B). Conversely, spinal fluid from relapsing MS patients contained mean levels of 6-oxo-PGFlc¢ comparable to those occurring in the CSF from patients in the remaining chnical groups (Fig, 1C),

TLC studies on pooled CSF Figure 2 (A-C) demonstrates that material quantitated by RIA as PGE. PGF~a

and 6-oxo-PGFla co-chromatographed with their authentic [3HIPGs. For example. the total amount of immunoreactive PGFza present in pouted CSF was recover~ within the area in which maximum radioactivity for [ 3H|PGFza was detected (Fig, 2A),

Leucocyte numbers and PG levels in CSF samples The clinical condition of the patient at the time of sampling and during occa..

TABLE 2

THE LEVELS OF PGs (pg/ml:t:SEM) AND THE NUMBER OF LEUCOCYTES (/ram 3) IN CSF SAMPLt~

Figures in parentheses indicate the numbe~ of sampled/group.

Leucocyte number 13(3 levels

0 (13) 852+ 124 612+ 79 792+ llO 1-10(3"7) 855+ 69 805+141 6814- 4g >10 (5) 918+':'.16 6404. 33 7264- 63

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Fig. 2. TLC of immunoreactive PGF2a(A), FGF~(B), and 6-oxo-PGF, a(C) present in extract of p o o ~ CSF. Solid lines trace the separation of au;hcntic [~H]PG standm'ds and dashed liras indicate :oncentration of immunoreactive PG(pg/ml ).

sional traumatic lumbar puncture resulted in the presence of variable nun,,ba~ of white cells in the majority of CSFs. It was cor~sidered that certain populations of leucocytes in the spinal fluids may directly or indirectly lead to the production and subsequent increase in the PC content of the samples. Table 2 shows there wa~ no correlation between the levels of the 3 PGs estimated and the number of white ¢ ~ present in the CSFs at the rime of sampling. Therefore, an)" changes in the prostanoid content of CSF samples could not be attributq~ to contamination of spinal fluid by leucocytes.

Discussion

The current observations show that CSF taken from MS patients during, a relapse contains significantly lower levels of the primary PGs, E aJld F2a, compared to values obtained for patients in remission and individuals with OND of the CNS. Several workers have shown that PGE is undetectable in the CSF from normal subjects and PGF2a levels are below 100 pg/ml (Wolfe and Mamer 1975; Wolfe 1978; Egg et al. 1980). Therefore it would appear that the PGE and PGF2a content of spinal fluid from patients during an exacerbation of MS is significantly elevated compared to normal values but not to the high levels d~tected in CSF from MS patients in remission and others with unrelated neurological disorders. This mgges- tion gains support from the findings of Rosnowska et al. 1,1981) who showed that the PGE 2 and PGF2a content of CSF from MS patients in relapse was significantly increased compared to control values. In addition, Egg et al. (1980) found that mean levels of PGF2a in the CSF of patients with MS exceeded normal limits. However, observations were not made on patients in definite remission. The latter study and the investigations of Wolfe and Mamer (1975) and Aizawa and Yamada (1976) agree with the data presentc.d in Fig. 1 (A-C) that spinal fluid from subjects ,~-ith abnormal CNS pathology, excluding MS, contain increased conce~ltrations of E and F type PGs. In addition, the study by Egg etal. (1980) is in accordance with present observations that levels of PGF2a in CSF from patient, s with OND are higher than in individuals with relapsing MS.

The capacity for normal cerebral tissues to generate prost,'moids, in vivo, is ne-ofigible beca'Jse of the low activity of PG synthesising e1~yme.s. With the occur- rence of CNS disorders, endogenous PG synthesis increases leadi~lg to the/r accumu- lation in the CSF (Wolfe 1978). A variety of explanations have been offered to account for these changes including cellular damage of brain tiss~Je due to hypor2a and electrolytic imbalance associated with oedema. However, in MS, it is probable that the significant changes in PGE and PGF2a levels betwo.~n relapse and remission are ro.lated to direct or indirect variations in the production of these cor~mnds by lymphocytes and macrophages present in the perivascular and parenchymal CN$ infiltrates (Traug0tt et al. 1982) and/or neuronal components residual in the target tissue. Recently Dore-Duffy (1982) has shown that peripheral blood monoc3aes of MS patients before relapse synthesise increased amounts of E-type PGs, the levels of which dramatically fall with the appearance of clinical symptoma. F ~ as

standardize RF preparations. Compar~ with these mctluxh. Kg is heat-stable a~ad easy to handle.

Kg is found in normal bovine sera and is known to have a high affini% for C31bi bound to immune complexes in the presence of Ca ions (l.achmann 196"0 Using Kg has allowed us to develop a simple and sensitive method to meagre CIC

The methc~l which uses a microtiter plate ELIgA ( ~ l i n k : ¢ , ImmLnosmber~t Assay) systera for Ciq binding as.~ay or Raji call assay has been dt~.ribed (Singh and Tingle 1982; Rote and Caudle 1983). However, the gg binding assay using microtitcr plate EL1SA has not been described, in this project for establishing a simpler and more sensitive method, we investigated the use of a Kg-micrctiter plme ELISA for measurement of CIC and applied this system to surw.-yin$ sever~.l neurological patients" sera.

Materials and Methods

Conglutinin purification The purified Kg from whole bovine .~'ra was obtained ~.¢.ording to Lachmann

(1967). Approximately 20 g of zymo~an {Sigma, St. Louis, U.S.A.) w,.'re used for treating 1600 ml of heat-inactivated bovine f, era. gedtw.cd and alkylaeed zymosan suspension ,~as mixed ~vith bovine sera and stirred at room temperature for I h. After mixing with 1 00~ ml of PBS (Dulbecco), containing 0.2 mM CaCI~. ¢luated protein was obtained from the zymosan r, edlmcm by adding 240 ml of FBS IpH 7.2L containing 0.01 M EDI'A. The ¢luated protein was ~¢¢il~t~tcd by 4g.h dialysis against 0.01 M phosphate buffer solution (pH 5,4), "lhe precipitated protein was dissolved in 10 ml of 0,03 M phosphate buff~ s,~lution (pH 8.0). containing 0.01 M EDTA and 0.03 M NaCi. Finally. this p~otein .,~olutio.I was aql~plied to a 1:$0 ml of DE-23 (Whatman, London, U.K.) ¢oluma and ¢luated with 0.0~-9.d M NaCI gradient. The first single peak was pooled.

Heat.aggregated human lgG CA HG) AHG was obtained b~," heat aggregation of human ling at 63°C for 25 rain. The

monometric lgO component of AHG was eliminated by a gel filtrai/a:m em a column of Sephacryl S-300 (Pharmacia, Uppsala, Sweden). Pgtified Kg and AHG stored at -70°C until use.

Duffer Stock vernal buffer solution (VBS): 0.7 M NaCI. 0.02~i M h~b~tal ~ i e m .

adjusted to pH 7.3 with 0.1 N HCI~ -15 VBS++'. 5-fold dilution of VBS wi;h dislilleA water, conlh'thai~ 0,15 mM C,~CI~

and 0.5 mM MgCI 2- Kg coating solution: 1 s VBS + +. crmtaining 0.,02% (w/v) NaN 3. adjttstcd 1o pit 9~$

with 1 N NaOH. -~VBS ÷+. Tween: ~VBS + +, containing 0.05% {w,/v) Tween 20, IVBS++.Tween.OA " ~ [VBS *~ .Tween contailfing 2~ (w/v) 0~*al alb~n~n

(Sigma, St. Lot, is, U.S.A.L

159

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