Surface markers on lymphocytes from human cerebrospinal fluid

Journal of bleuroimmunology, 5 (1983)325-331

Elsevier

325

JNI 00163 Short Commu:nication

Surface Markers on Lymphocytes from Human Cerebrospinal Fluid Identification by Monoclonal Antibodies Maria Giovanna Marrosu 1, Maria Grazia Ennas 2, Maria Rita Murru 2, Giovanni Marrosu 3, Carlo Cianchetti 3 and Paolo Emilio Manconi 2 t Institute of Neurology, z Chair of GeneralImmunology and 3.Chair of Child Neuropsyc'uatry, Unir~sity of Cagliari, Cagliari(Italy)

(Received20 April, 1983) (Revised,received10 July, 1983) (Accepted8 August. 1983)

Sunaaaff Lymphocyte subpopulations in cerebrospinal fluia (CSF) and peripheral blood (PB) were studied using monoclonal antibodies and the common membrane markers. The results in three groups ot patit'nts were compared: 36 subjects with "non-immunological disorders' (NID), 14 subjects with multiple sclerosis (MS) and 6 with subacute sclerosing panencephalitis (SSPE). It was found that, in patients with NID, (1) 90~ of cells were T lymphoeytes, reactive with OKT3; (2) the helper/suppr--~a.sor (T4/T8) ratios were the same in the CSF and the PB; (3) the OKlal percentage was lower in the CSF than in the PB; and (4) only a few cells were 'immature', reacting with OKT10. Using the membrane'markers (E rosettes, Fc IgG receptors and surface immunoglobulins), on the other hand, it was noted that the majority of cells in the CSF were identified as suppressor T lymphoeytes and surface immunoglobufin-positive B o,'lls were !ess common than the lal marker suggested. There were no significant 'differences between the CSF results in patients with N I D and MS but the OKT3 lymphocytes were reduced in CSF samples from patients with SSPE.

This work was in part fmsnclallysupportedby a CNR (Italy)grant. to: Prof. PltoioEmilioMancmfi,CAtttedradi lmmunologiaGenexlde.Univenfi~di C a g t ~ Via Pocm~ 1~ J-09100OqtUari.lady. 0165.5728/83/S0J.~}© 1983 Else~,r SciencePubEshcrs B.V.

326 Key words: Cerebrospinal fluid - £¥mphocyt¢ ~bpopulations - Monoclonal antibod~ ies - Muh:ple sclerosis - StdTacute sclerosing ponencephalit,:s

Introduction Up to now, lymphocyte populations in human cerebrmpinal fluid (CSF) have been studied using rosette techniques and identification of surfac.e immunoglobulins (slg). The majority of these studies (reviewed in Cook and Brooks 1980) have shown that the CSF contains higher percentages of T cells, and llowerpercentages of B cells, as compared to peripheral blood (PB). There were, lu~,wever, contradictory statements between the various authors about these percetltaf~. Only few data are available about T cell subpopulations in the CSF. Our previous stmlle; (Manconi et aL 197~b, 1980a) demonstrated that, in various 'non.immunol~ical disorders' (NID), about 80~ of CSF lymphocytes form E- and EA-IgG rof~ttes, so that they can be identified as TG (suppressor) cells. The percentage of TG cells was found to be significantly reduced in the CSF from patients with subacute sclerosing panencephalitis (SSPE) (Manconi et ai. 1980b; Marrmu et al, 1983) and with multiple sclerosis (Manconi et ai. 1980a)o Since, ac~.~rding to Mmetta et al. (1977), TG lymphocytes suppress the B cell response to antigens o~ mitogem, we mggested that the decreased numbers of TG ceils in these diseases might a , c ~ t for the abnormally high IgG synthesis found in the central nervous system, The recent availability of hybridoma technology has provid~l a major breakthrough in the precise definition of the lymphocyte subsets (Reinherz et alo 1979, 1980). The use of monoclonal antibodies (MeAbs) results in homologous reagents, codified methods and reproducible data from n~my laboratories in the world. The present study is aimed at the characterization of the CSF lymphocytes using McAbs arid the comparison of these data with that fumiJd~ed by the ¢onunor~ membra~te markers.

Materials and Metlmds Patients

Thirty-six patients (t8 males and 18 females, mean age 40A years, range 4-70 years) affected with NID, were studied: 8 had epilepsy, 6 Sl~astic paraplegia, 5 poi!yneuropathy, 5 stroke, 4 inyelopathy, 3 neurosis, 3 hydroc~halus, 2 encephalitis, 1 vertigo and 1 disc herniation. Fourteen patients (2 males and 12 females, mean age 28.7 years, range 11-41 years) affected with MS, defined clinically according to tl~ criteria of Rose 4:t ai. (1976), were included in the study. In addition, 6 patients (2 males and 12 female~, mean age 9 years, range 4-13

327 years) who had SSPE, were studied; The diagnosis was based on clinical, electroe~cephalographic a n d laboratory ( C S F I g C conteni, protein electrophoresis, serum and anti,measles antibody titers) findings.

CSF celts The method previoasly described (Manconi et al. 1978b) was followed.

PB lymphocytes PB mononuclear ceils were obtained according to the method previously described (Manconi et al. 1978a).

E, EA-IgG rosettes and sl 3 The methods previously described (Manconi et al. 1978a, b) were used.

McAb OKT3 (pan-T), O K T 4 (T helper-inducer), O K T 8 (T suppressor-cytotoxic;, OKT10 (immature lymphoid cells), O K T l l (E-rosette forming cells) a z d O K l a l ( D R w frameworks: B cells, macrophages and sonle activated T cells). ~ e M c A b s were purchased from O r t h o Diagnostic System (USA). The indirect fluorescence method described b y Reinherz et aL (!979) was followed.

TABLE 1 PERCENTAGES a OF CELLS IDENTIFIED BY McAbs b 1N CSF AND PB FROM PATIENTS WITH NID MeAb

CSF

PB

P¢

OKT3 n

89.0 4-8.6 18

73.7 + 7.1 23

< 0.0005

OKT4 n

60.9 + 20.2 9

53.0 + 8.5 23

NS

OKT8 n

29.8 4.10.3 15

27.9 + 5.4 23

NS

OKTI0 n

4.0 4-3.7 6

7.0 -~1.6 6

OK'FI 1 n

91.7+2.6 6

84.2+ 1.6 6

< 0.005

OKlal n

12.8 4. 4.7 6

14.24. 5.8 23

NS

< 0.1

• MNn+SD. b Moaocloaal an0bodies identifyin8 lymphocyte subsets purchased from ORTHO, U.S.A. © Student t-test. NS -. difference not significant.

328

Results Data on mononuclear cells identified by M:Abs in CSF and PB from patients with Nil) are reported in Table 1. T lymphotytes, identified by both OKT~i aud OKTI1, are more frequent in the CSF than in ~h¢ PB. The percentages of OKTI 1 + cells are"slightly higher than those of the OKT3 + lymFhocytes in both CSF m~d Tj~B.

TABLE 2 CSF CELLS FROM SUBJECTS WITH NID. Comparison of the percentages of EA-RFC with OKT8 + cell~, and of sill+ with OKlai + cells. Sample No.

EA-RFC

OKT8 +

sill +

OKTal ÷

? 2 3 4 5 6 7 M + SD P

66 80 63 90 74 62 80 73.6 :i: 10.3 < 0.001

34 34 17 3 3 5 4 14.3 4.14.2

2 2 4 0 0 1 ND 1.5 + 1.4

12 12 15 21 8 9 ND 12.8 + 4.6 < 0.001

TABLE 3 PERCENTAGES j OF CELLS 'DENTIFIED BY McAbs b IN CSF AND PB FROM PATIENTS WiTH MS, SSPE AND NiD McAb

1

2

3

CSF MS

4

5

6

7

Nir}A ~ NiDCh d

PB MS

S:;PE

A OKT3 !1

86.1+11.6 74.8+9.1 89.0+8.6 9 6 18

SSPE

79.3:/:1.5 3

64,2+11.6 73.75:7.1 71.9-t-7.~ 6 23 17

B OKT4 n

ND ~

55.3:l:!2.7 53.8+9.8 3 6

C OKT 8 n

25.7+14.4 2.'i.6:t:7.1 29.8+10.3 28.7:t:6.6 9 6 15 3

2g.4+12.7 27.94-5.4 28.8+9.4 (, 23 17

D OKlal n

ND

16.3+10.6 14.2+5.8 13.94-6.7 6 23 1:~

ND

ND

NID

ND

ND

10.04.2.0 3

$3.0±8.5 50.4±$.~ 23 17

P t < 0.001:A2-A3 P < 0.05: AS-A7; A2-A5 a b ': d

Mean :t: SD. Monoclonal antibodies identifyinp lymphocyte subsets purchased from ORTHO, U.S.A. Adult subjects with 'non-imm-r:oiogical diseases'. Children with'non-immurtologi.:a~ diw.ases'. ND = not determined. J Unpaired Student t-test.

329

OKT4~ and OKT8+ cells arc more frequent in CSF than in PB, but the differenc~ are not significant. The OKT4/OKT8 ratio~ are the same in CSF and PB. Only a low percentage of OKT10 cells, waslfound in either CSF or PB. The data obtained by comparing the percentage of OKT8 + with EA'IgG rosette forming cells (RFC) and the percentage of OKlal + with slg + ceils in the CSF, are shown i n Table 2. The percentage of EA-IgG RFC is sign~ficandy higher than thai ofOKTg+ cells, and the percen~tage of OKla~. + cells is higher than that of slg + ceils. On the other hand, sin~ilar Froportions o~" OKT8 + and EA-IgG RFC, and of OKlal + and slg + ce:lls were nc,ted in the PB of these patients. The percentages of cells reacting with the various McAbs in CSF and PB, from patients with MS and SSPE, compared to the data obtained in patients with NID, are shown in Table 3. No differences were found between MS and NID but i~ patients with SSPE there was a decreased percentage of OKT3 + cells in both CSF and PB.

Discussion

The study of CSF lymphocyte subsets in NID by McAbs shows that. (I) The greater majority (about 90~) of CSF cells can be identified as T lymphocytes. This confirms our previous findings, based on E-rosette formation (Manconi et al. 1976, 1978b, 1980a, b; Marrosu et al. 1983). (2) Based on the reactions ' ~ t h OKT4 and OKT8 McAbs, the helper/suppressor cell ratio in CSF is the same as that found ~n PB. This finding contrasts with our previous results ba.~cl on EA-IgG RFC (Manconi et al. 1978b) but there are data indicating that the OKT8 McAb and IgG Fc receptor identify different cells (reviewed in Moretta et al. 7981). Moreover, it is possible that the CSF itself might modify the expression of dlfferen: Fc receptors. Modulation of other lymphocyte membrane receptors by CSF has b~',en demonstrated recently (Naess 1982). (3) OKIal + cells are present in greater numbers in CSF than are sis + lymphocytes. According to Evans et al. (1978) and Yu et al. (1980), human T cells synthesize ant~ express la antigens when stimulated by mitogens and allogenic cells. Since the sum of the percentages of OKT3 + and of OKlal + cells in some CSF samples is greater than 100, some of the CSF OKlal + cells might be actiwted T cells and not B lymphocytes at all. (4) Based on the reactivity with OKT10 McAb, only a few CSF cells are identified as 'immature' lymphocytes. Con~pariso~t of the percentages of OKT3 + and OKT8 + cells in CSF samples from patient~ with NID and those from MS patients does not show signifr.ant differences, although the low number of patients does not allow d~.f.;nite conclusions. In CSF samples from SSPE patients, a decreased percentage of OKT3 + cells was founds without any alteration is the OKT8 + cell percentage. In previous paper~we have demons=rated ~ decreased percentage of CSF TG cells in patients with MS (Manc(nfi et aL 1980a) and in SSPE (Manconi et al. 19~0b; Marrosu et al. 1983). "D~ hypothesis put fot~vard was that the decrease in TG cells might account for the

330 increased synthesis of oligoclortal l g G in the above ~ . s e s . "lhe p r e s e n t observe.tion, that there are normal proportions of M c A b - d e f i n e d suppressor T cells in the C S F and a normal H e l p e r / s u p p r e s s o r ratio in the PB from M S a n d SSPE patient~;, apparently contradicts the above hyp3thesis. Neverthele~, according to l.~.tl~ Bich-Thuy et al. (1982), F c l g G receptors could have suppressor activity~ irrespective of their cellular origin. T h e decreased p e r c e n t a s e o f T G C S F cells i n M S a n d SSPE rrdght be of some importance in the ~ncreased synthesis of oli~3clona! ISG, even though no relevant alteration in the *3KT8 + cell percentage has been fo~tu].

Acknowled~.qnment The technical assistance of M s G i o v a n n a Milia was o f great aid.

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