Acta Neurol Scand., 1983:67:218-221

Key words: Brain antigens; multiple sclerosis; lymphocytes; polymorphonuclear leucocytes.

Thymidine uptake by lymphocytes in the presence of Supernatants of polymorphonuclear leucocytes incubated with multiple sclerosis brain antigens

S. C. Rastogi and J. Clausen

The Neurochemical Institute, Copenhagen, Denmark

ABSTRACT - The effects of factors secreted by polymorphonuclear leuco- cytes (PMNL) incubated with multiple sclerosis (MS) and control brain antigens (MSG2 and KG2 respectively) were evaluated by assay of thymid- ine uptake by lymphocytes from normal individuals and MS patients. It was found that the cellular uptake of thymidine of both MS and normals was significantly lower in the presence of MSG2-PMNL supernatants compared to that in the presence of KG2-PMNL or without antigen-PMNL super- natant. The findings indicate that an inhibitor of lymphocyte proliferation either may be released from PMNL by the action of MSG2 or may be synthesized in response to challenge by MSG2.

Accepted for publication December 2, I982

Multiple sclerosis (MS) may be considered an acquired inflammatory disease of the human central nervous system (CNS) as- sociated with demyelination (1, 2). In initial phases of MS, perivascular infiltration with hematogenous cells occurs in CNS. We have recently traced 2 antigens specific for MS in CNS, which have been partially puri- fied (3, 4). Our finding have been con- firmed by a blinded study (5). The partially purified MS specific brain antigens signif- icantly stimulated active-E-rosette forming lymphocytes of MS patients (6) , and the monokines released stimulated the helper

cell function of MS lymphocytes (7). Fur- thermore, polymorphonuclear leucocytes (PMNL) incubated with MSG2 lost neutral proteinases (8). The present communication describes the effect of soluble factors re- leased from PMNL incubated with MS brain antigens on the thymidine uptake by lymphocytes. Serumless medium supple- mented with lactoalbumin hydralysate (9) was employed both for the PMNL and lymphocyte cultures. Hereby the possibility of the binding of proteinases released from PMNL (8) with serum antiproteinases was excluded.

THYMIDINE UPTAKE 219

Material and methods

MS patients and control subjects The study comprised 12 clinically definite MS patients (median age 34 years) diag- nosed according to the criteria of Schu- macher et a1 (10). All patients were in a stable phase of MS and none of them were receiving any immunosuppressive drugs. 10 laboratory personnel were used as control subjects (median age, 27 years). Heparin- ized fresh blood, approximately 70 mI on day 1 and 30 ml on day 2, was used for all subjects.

Brain antigens Partially purified MS specific brain antigen, MSG2, isolated from 6 MS brains (4) were used in the present study. The correspond- ing fraction from control brains, i.e. KG2 used was isolated from 5 brains (2 Hunting- ton’s disease, 1 each of amyotrophic lateral sclerosis, progressive multifocal leucoence- phalopathy, and normal). The antigens (1 mg/ml) in distilled water were frozen at -20°C and diluted to 100 yg/ml in serumless medium (Gibco-Bio-Cult, Scotland) before use.

Isolation of lymphocytes and PMN leucocytes Monocyte-deficient lymphocytes from hep- arinized blood were isolated and suspended at a concentration of 2 x 106 cells/ml serum- less medium containing 100 IU penicillin/ml and 100 yg streptomycin/ml (7). The via- bility of the cells as judged by the trypan blue exclusion test was found to be >96% in all preparations.

PMN leucocytes (granulocytes >90, mononuclear lymphocytes <2 %) from heparinized blood were isolated as previous-

ly described (11). They were washed with serumless medium and suspended at a con- centration of 3 x 106 cells/mi in serumless medium containing streptomycin and peni- cillin.

Cell cultures The doses of MSG2 and KG2 used per culture was as indicated in previous studies (7, 8). The standard method of culturing the cells was as follows:

Day I . 12 tissue culture bottles (NUNC A/S, Denmark) containing 6 x 106 lymphocytes/3 ml serumless medium were incubated with 1 yg MSG2 (2 bottles), 1 yg KG2 (2 bottles), and without any antigen (8 bottles).

Day 2. 15 x 106 PMN leucocytes/5 ml se- rumless medium were incubated in the pre- sence of 2 kg MSG2 or 2 pg KG2 or in the absence of any antigen. After 4 h incuba- tion, 1 ml cell-free supernatant from day-2 cultures was added to autologous lympho- cytes (from previous day) as described below:

2 x lymphocytes without antigen + 1 ml serumless medium; 2 x lymphocytes without antigen + PMNL supernatant without antigen; 2 x lymphocytes without antigen + MSGZPMNL supernatant; 2 x lymphocytes without antigen + KG2-PMNL supernatant; 2 x lymphocytes with MSG2 + MSGZPMNL supernatant; 2 x lymphocytes with KG2 + KGZPMNL supernatant.

Thereafter, the lymphocytes were incubated for further 48 h. After this period, 5 pCi (6-3H) thymidine (0.2 mM thymidine, spe- cific activity 100 pCi/ml) were added to each culture, and the thymidine uptake by the cells in the next 20 h was determined. The harvesting of the cells and the counting

220 S. RASTOGI AND J. CLAUSEN

of 3H-thymidine incorporated in lympho- cytes has been described previously (7). The viability of cells was estimated before harvesting to be >95% in all cases.

Results and discussion In the initial experiments (using peripheral blood from 2 patients), the addition of 0.25, 0.5, 1.0 and 1.5 ml supernatant of PMNL incubated with or without brain antigens to syngenic lymphocyte cultures resulted in a dose-dependent increase in thymidine in- corporation in these cells up to a 1 ml dose. Further stimulation of thymidine uptake of lymphocytes by higher doses of PMNL supernatant was not significant. Therefore, 1 ml of PMNL supernatant was added in lymphocyte cultures in further experiments. The effect of supernatants of PMNL in- cubated over 4 h was not investigated be- cause more than 10% of the cells were found to be non-viable following 6 h in- cubation of the cells.

The stimulatory effect of PMNL super- natants on lymphocyte proliferation, ex- pressed as a stimulatory index (SI), is shown in Fig. 1.

thymidine uptake by lymphocytes with PMNL supernatant

thymidine uptake by lymphocytes without PMNL supernatant

SI =

The thymidine uptake by lymphocytes of all controls and MS patients was found to be increased in the presence of all cell-free supernatants of PMNL incubated with or without any antigen compared to that in the absence of any cell-free supernatant of PMNL. However, the thymidine uptake of both MS and normal lymphocytes cultured in the presence of MSGZPMNL was signif- icantly lower than those cultured with the supernatants of PMNL incubated without

I . MS

! . .

. . . a . I, 0

a . . . . a 1 ' a . a

" O A ' B ' C ' L I E Fig. 1. Stimulatory index (SI) of lymphocytes in the presence of supernatants of PMNL incubated without antigen (A), with MSG2 (B), with KG2 (C). D: SI of lymphocytes incubated with MSG2 prior to addition of MSG2-PMNL supernatant; E: SI of lymphocytes incubated with KG2 prior to addition of KG2-PMNL supernatant.

antigen (Wilcoxon's test, PMs < 0.02, PnOrmal < 0.05). Futhermore, the thymidine uptake of MS lymphocytes in the presence of MSGZPMNL supernatant was significantly lower than that with KG2-PMNL super- natant (Wilcoxon's text, P < 0.05). The KG2-PMNL supernatant-induced thymidine uptake of lymphocytes was not significantly different from that induced without antigen- PMNL supernatants. The p H of the cell-free supernatants of brain antigen-PMNL was not found to be changed in any experiments. Moreover, the vitality test revealed that > 95 % cells were viable in all cases. Thus, the above-mentioned phenomenon of impaired

THYMIDINE UPTAKE 221

cellular thymidine uptake may be associated with the presence of an inhibitor (regulator) of lymphocyte proliferation in MS brain antigen-PMNL supernatants. Such an in- hibitor (regulator) may be present in PMNL or it may be synthesized in response to the action of MS brain antigens with PMNL. The inhibitor may be released from PMNL during inflammation, then modulating the lymphocyte response. Further studies will be necessary to determine the nature of the inhibitor.

By preincubating lymphocytes for 24 h with or without antigen, suppressor and helper functions may be augmented (12). The thymidine uptake of all MS lympho- cytes incubated (24 h) with MSG2 prior to culturing with MSGZPMNL supernatant was found to be decreased, 1 0 4 3 % (25.2 k 9.1) compared to that of the correspond- ing lymphocytes not preincubated with MSG2. This indicated that lymphocytes of MS patients, all in a stable phase, were sensitized to MSG2.

Acknowledgements We thank Miss L. J~rgensen for skilful technical assist- ance.

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Rastogi S C, Clausen J , Offner H, Konat G, Fog T. Partial purification of MS specific brain antigens. Acta Neurol Scand 1979:59:281-296, Rastogi S C, Clausen J. A simple method for the isolation of MS specific brain antigens. Clin Chim Acta 1980:101:8S92. Rastogi S C, Clausen J, Tourtellotte W W, Potvin A R. Multiple sclerosis-specific central nervous system antigens (MSG2): a blind study. Eur Neurol 1983: in press. Offner H, Rastogi S C, Konat G, Clausen J. Stimula- tion of active E-rosette forming lymphocytes by myelin basic protein and specific antigens from MS brains. J Neurol Sci 1979:42:349-355. Rastogi S C, Clausen J. In vitro stimulation of multi- ple sclerosis (MS) T and B lymphocytes by auto- logous monokines generated in the presence of MS- specific brain antigens. CIin Immunol Immunopathol 1982:22:68-74. Rastogi S C, Clausen J. Loss of lysosomal neutral proteinase from leucocytes induced by the action of multiple sclerosis specific brain antigens. Clin Exp Immunol 1980:42:50-56. Neumann R E, Tytell A A. Serumless medium for cultivation of cells of normal and malignant origin. Proc Exp Biol Med 1960:/04:252-259.

10. Schumacher G A, Beebe G, Kibler R F, et al. Problems of experimental therapy in multiple scle- rosis. Ann NY Acad Sci 1965:122:552-568.

1 1 . Rastogi S C, Clausen J, Melchior J C, Dyggve H V. Jensen G E. Lysosomal (leucocyte) proteinase and sulfatase levels in Dyggve-Melchior-Clausen (DMC) syndrome. Acta Neurol Scand 1977:56:389-396.

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Address S. C. Rastogi, Ph.D. The Neurochemical Institute Raadmandsgade 58 2200 Copenhagen N Denmark