Journal of

J. Neurol.,216, 39--46 (1977)

~) by Springer-Verlag1977

Complement Dependent Cytotoxic Antibody Activity against Measles Virus in Multiple Sclerosis* V. Kratzsch and W. R. Kiessling Institut fiJr Virologie und Immunbiologie der Universit~it Wtirzburg, Versbacher Landstr. 7, D-8700 Wtirzburg, Federal Republic of Germany J. Wikstr6m, D. W. Meyer, K. Eickhoff, S. Poser, and H. J. Bauer Neurologische Klinik und Poliklinik der Universit~it G6ttingen, v.-Sieboldt-Str. 5, D-3400 G6ttingen, Federal Republic of Germany

Summary. The presence of measles cytotoxic (CT) and hemagglutination inhibiton (HI) antibodies in 195 multiple sclerosis (MS)patients and 251 controls was tested. The measles virus Lu carrier cells labeled with 5~Cr were exposed to serum specimens in the presence of complement in order to test the presence of CT antibody. The analysis of complement dependent CT antibodies against measles virus revealed significantly (P < 0.01) higher titers in MS patients than in the control group. However, the measles HI test failed to show this difference. Measles CT titers => 1 : 32 among MS patients occured in 54.9% and in 35.5% among the controls. In comparison with this the HI method revealed measles titers => 1 : 128 more often in the control group than in MS cases (27.9 and 17.9%, respectively). The presence of CT antibodies against measles virus in MS proves that these patients have a functional defence mechanism to eliminate virus infected cells. The high measles antibody titer among MS patients could be due to recurrent antigenic stimulation caused by measles virus persistency. Whether this virus persistency plays a role in MS can not be decided on the available data. Key words: Multiple sclerosis - Complement dependent cytotoxic antibodies Measles virus. Zusammenfassung. Die zytotoxischen (CT) und Haemagglutinations-hemmenden (HI) Antik6rper gegen Masernvirus wurden bei 195 Multiple-Sklerose (MS)-Patienten und 251 Kontrollpersonen getestet. Im Testsystem wurden die CT-Antik6rper im Serum mit zugesetztem Komplement, bei Messung des freigesetzten 51Cr aus mit Masernviren infizierten Lu-Zellen, nachgewiesen. Die Analyse der Komplement-abh/ingigen CT-Antik6rper gegen Masernvirus * Supported by Deutsche Forschungsgemeinschaft (Schwerpunkt Atiologie und Pathogenese der multiplen Sklerose und verwandter Erkrankungen)

40

V. Kratzsch et al. zeigte bei MS-Patienten im Vergleich zu Kontrollen eine signifikante (P < 0.01) E r h f h u n g der Titerwerte. Dieser Unterschied war nicht im HI-Test nachzuweisen. Die H~iufigkeit von CT-Titerwerten -> 1 : 32 betrug 54.9% bei MSF~illen und 35.5% bei Kontrollen. Gleichlautend fanden sich HI-Titerwerte - 1:128 nur bei 17.9% der MS-Patienten und 27.9% bei Kontrollen. Der Nachweis von CT-Antik6rper unter MS-Patienten ist ein Beweis daftir, dab sie eine funktionsf~ihige Abwehr gegen virusinfizierte Zellen besitzen. Die erh6hten Titer gegen Masernvirus k6nnen auf eine anhaltende Antigen-Stimulation bei MS-Patienten hinweisen. Die Befunde beweisen jedoch nicht, dab for die Entstehung einer Multiplen Sklerose das Masernvirus verantwortlich ist.

Introduction

Serological studies on multiple sclerosis (MS) patients have been carried out by many investigators in order to find a specific antibody response to a possible candidate virus which could etiologically be associated to this disease [2]. Serum and cerebrospinal fluid (CSF) specimens of MS patients have been tested for the frequency of antibodies against all common viruses and compared to that of normal individuals and patients with diseases of the central nervous system (CNS) other than MS. Although results vary they generally indicate that more MS patients tend to have slightly higher antibody titers against measles virus than do the controls [2]. In addition, increased antibody titers against other common viruses such as herpes simplex, vaccinia, varicella zoster, adeno viruses, influenza C, parainfluenza 3, mumps and rubella have also been reported, but not as consistently as measles virus [1]. None of these studies has yet identified a particular causative agent. However, serological studies in the case of measles antibodies have indicated, that measles antigen might be present in the CNS of some MS patients. It has been shown, that in few MS CSF specimens oligoclonal antibodies against measles antigen are detectable, which presumably have been derived from lymphocytes invading the CNS compartment [7]. This finding has led to the hypothesis that measles virus could play a role in the etiology of some MS cases. On the basis of this observation it is necessary to evaluate the immunological response of multiple sclerosis patients to measles virus, since immunological factors are suspected to play a role in the pathogenesis of this disease. So far, in the humoral immune response to measles virus in MS patients, only standard serological tests have been employed to study measles antibody response by measuring the neutralizing, complement-fixing, hemagglutination inhibiting or hemolysin inhibiting activities. Recently, however, another property of measles antibody has been described, which has not yet been studied in MS patients [4]. It has been shown that measles antibodies in the presence of complement are capable of destroying a measles infected cell. This property has been generally regarded as an important host defense mechanism in the elimination of virus infected cells. This study has been designed to detect the presence of measles cytotoxic antibodies in MS patients and control groups. The MS patients selected have

Complement Dependent Cytotoxic Antibody Activity against Measeles Virus Table 1. The age-specific distribution of MS patients and controis

Age group

~30 31----40 41--50 51--60 >60 Total

41

Controls

MS cases % (N)

%

(N)

13 21 31 24 11

(26) (41) (60) (47) (21)

19 22 28 19 12

(47) (55) (69) (49) (31)

100

(195)

100

(251)

Table 2. The disability grade of MS patients according to the scale of Kurtzke [8] Disability grade

MS patients %

(N)

1. 2. 3. 4.

No disability, minimal signs Minimal disability Moderate disability, monoparesis etc. Relatively severe disability not preventing to work

18 15 13 5

(35) (30) (25) (10)

5. 6. 7. 8. 9.

Disability severe enough to preclude working Assistance required for walking Restricted to wheel chair Restricted to bed with effective use of arms Totally helpless bed patients

6 18 14 8 3

(11) (35) (28) (15) (6)

100

(195)

Total

b e e n well d o c u m e n t a t e d c l i n i c a l l y a n d e p i d e m i o l o g i c a l l y , a n d c o m e f r o m a n a r e a o f N o r t h e r n G e r m a n y w h e r e t h e p r e v a l e n c e o f M S has b e e n f o u n d in t h e o r d e r o f 70 p e r 1 0 0 0 0 0 i n h a b i t a n t s .

Materials and Methods Serum Specimens. Sera from 195 patients with MS were tested. The MS cases were selected from a central documentation pool [9] which makes multicentric studies possible if needed. In this the clinical data on MS patients are registered uniformly. The age-specific distribution of MS patients included in this study is given in Table 1. The female to male ratio was 1.2. For both sexes the mean age was 45 years and the mean duration of the disease was 12 years. The clinical course of MS was remitting in 82 cases (42%), remitting progressive in 87 cases (45%), and chronic progressive in 26 cases (I 3%). The disability grade of patients was evaluated according to the scale of Kurtzke [8]. The slight and severe cases were equally represented (Table 2). The control group comprised 161 patients with various neurological disorders other than MS, and 90 patients with psychiatric illnesses. The last group formed a ,,healthy" control group. In the neurological control group there were 40 sciatic syndromes, 26 strokes, 15 tumors,

42

V. Kratzsch et al.

17 convulsive disorders, 9 cases with cerebral contusion, 7 with encephalitis, 5 with cerebral atrophy, 4 with neurolues, and 38 other isolated neurological conditions. The age distribution of the controls is given in Table 1. The female to male ratio was 1.0. All patients came from the same geographic area and were investigated in the same clinic.

Cell Cultures. The measles virus carrier celline (Lu carrier), kindly supplied by Dr. E. Norrby, Karolinska Institute, Stockholm, was used as a target cell for the cytotoxic assay. The Lu carrier cells were propagated in Eagle's minimun essential medium supplemented with 5% fetal bovine serum (FBS) and 100 units of penicillin and 100 lag of streptomycin/ml. These cells always exhibited on their cell membrane measles antigens, which could be documentated by membrane fluorescence using anti-measles antiserum in the indirect immunofluorescence test. Haemagglutination Inhibition (111). This test was performed according to methods described elsewhere [6].

Cytotoxic Antibody Assay (CT). The technic used in testing sera for the presence of cytotoxic antibody has been described by our laboratory in detail elsewhere [4, 5]. In summary, SlCr labeled Lu carrier cells were exposed to different dilutions of serum specimens in the presence of complement. Percentage of specific release of 5~Cr was used to express the activity of the serum. This was calculated from the average counts per minute (cpm) release into the supernatants of triplicate samples by the formula: % specific release =

cpm (serum + complement) - - cpm (spontaneous release) cpm (total) - - cpm (spontaneous release) x 100

The spontaneous release was calculated as the amount of 5~Cr released from the cells during the 2 h incubation period in the presence of fetal calf serum and complement. The endpoint of cytotoxicity (titer of cytotoxicity) was determined by the dilution of serum causing greater than 5% specific release.

Statistical Method. The antibody titers obtained between MS and control groups were analyzed with the H test of Kruskal and Wallis [11].

Results The differences of geometric m e a n s between MS cases a n d controls according to sex are illustrated in Figure 1. All m e a n values were relatively low a n d sex did n o t have a n y significant influence o n them, at least in the cases of MS. Only in the C T test did measles a n t i b o d y titers reveal higher geometric m e a n values a m o n g the MS patients t h a n a m o n g the controls. The division of the total control material into two separate groups, neurological a n d ,,healthy", did n o t change these relations significantly (Fig. 2). However, the neurological controls showed constantly slightly higher geometric m e a n values t h a n the ,,healthy" group. Measles C T titers of 1 : 32 or m o r e a m o n g MS patients occured in 54.9% a n d in 35.5% a m o n g the controls. I n c o m p a r i s o n with this C T test the H I m e t h o d revealed measles titers of 1 : 128 or higher more often in the c o n t r o l g r o u p t h a n in MS cases (27.9 a n d 17.9%, respectively), as can be seen in Figure 3. The d a t a illustrate that MS patients h a d higher measles a n t i b o d y titers more often t h a n controls only w h e n the C T test was performed. This test revealed a difference between MS patients a n d controls, which was significant (P < 0.01). This was even more m a r k e d w h e n the c o n t r o l g r o u p was c o m p o s e d only of ,,healthy" cases. The two c o n t r o l groups did n o t differ significantly from each other.

Complement Dependent Cytotoxic Antibody Activity against Measeles Virus GM femate mole total

OM/female male total

GM

32-

32-I

32-

16-

16-

16-

iii!iiiiiiii

~:i:i:i:i:i:i~-"xN

8-

D

MS PATIENTS

I

iiiiiiiiiiill ::::.:::::::: !i::::':::::::: izi~!z!i.-~-~-~ !i iii:iiiiii~ii x~_x~_x'~-.'

MEASLES (CT)

43

MEASLES (HI)

WCONTROLS

...........

iiiiiiiiiiliN MEASLES (CT) MEASLES(HI) D

MS PATIENTS I--7CONTROLS(healthy)

[]CONTROLS (with neurological diseases) Fig. 1. The sex-specific distribution of geometric means (GM) of measles CTandHItiters among MS patients and controls Fig. 2. The geometric means (GM) of measles CT and H I titers among MS patients and "healthy" and neurological controls

The age-specific distribution of geometric mean titers is illustrated in Figures 4 and 5. The CT titers were consistently higher by decades in the MS group than in the control group. This was not the case only in the age group above 60 years. The slope of the CT curve was decreasing for controls when compared to measles HI curve, which did not reveal this tendency. The MS patients showed a slight peak of CT titers before the age of 60 years in comparison with controls. With regard to CT titers of 1 : 32 or over, the MS patients in age groups from 41 to 60 years made up 57.9% of all MS cases at that titer level and the corresponding controls accounted for 36.0% (Fig. 5). On the basis of the clinical course, 63.4% of MS patients with remissions showed CT titers of 1:32 or above, whereas in patients with the remitting progressive or chronic progressive form the figure was 49.4 and 46.2% respectively (Fig. 6). The disability grade did not have any influence on the corresponding geometric mean values.

Discussion The analysis of complement dependent cytotoxic antibodies against measles virus in multiple sclerosis patients revealed significantly higher titers in MS patients than in the control groups. However, the measles hemagglutination inhibition test failed to show these differences. A similar discrepancy has been observed by Vandvik and Degr6 [12] measuring measles HI and CF antibodies in MS patients. These authors found a higher measles antibody titer in their MS groups in comparison to control persons only in the CF test, but not in the HI assay, whereas in many other investigations only a higher HI titer was observed [2].

44 o/°

302010-

V. Kratzsch et al.

Im ~8 16 32 MEASLES (CT)

6~

7MS

PATIENTS

T CONTROLS

m

128 >128 TITERS

°/o

30-

& ,,, 8 ~16 32 MEASLES (HI)

64

128 256

~ ~ __ 512 1024 2048 TITERS

Fig. 3. The frequency in per cent of various titers in measles CT and HI test among MS patients and controls

GM

64-

• • MS PATIENTS o - - - - o CONTROLS

GM 6/-.-

"-----.L~,_____.__~."

32-

NO'~ ~ __O f

16-

8-

8-

~:30 31:40 41:50 51:60 >60 AGE GROUPS MEASLES (CT)

~-½0 31:40 41'-50 51:60 >60 AGE GROUPS MEASLES (HI)

Fig. 4. The age-specific distribution of geometric means (GM) of measles CT and HI titers among MS patients and controls

From these studies one can assume that different measles antigens are responsible in MS patients for the recurrent stimulation of antibodies. Measles virus antigens are located in the virus envelope and nucleocapsid, and in the event of a regular measles virus infection, antibodies are produced against all viral antigen determinants. However, it is conceivable that in the case of persistent or abortive infection not each virus antigen is synthesized or released at the same rate from an infected cell into the extracellular space. This would lead to a different immune response to the individual antigen. Such events could account for the differences observed by the serological surveillances. The presence of cytotoxic antibodies against measles virus in multiple sclerosis proves that these patients have at least one functional immunological

Complement Dependent Cytotoxic Antibody Activity against Measeles Virus

r-i

°/o

I

70-

45

IMS PATIENTS CONTROLS

5030-

r-h

1oi ,~30

31-40 41-50 51-60 >60 AGE GROUPS

Fig. 5. The age-specific distribution of CT titers >- 1 : 32 a m o n g MS patients and controls. The frequency of these titers is expressed in per cent of each age group separately

GM

femate

male

total

°/oJ

32-

ox'x o

16-

. ' ~

20 8-

10 II [II

I IIIII

I IIIIl

~8

18

32

84

128 >128 TITERS

Fig. 6. The geometric mean (GM) and frequency in per cent of measles CT titers in the three different clinical courses of MS. I = . • (remitting), H = o .. o (remitting progressive), III = x - - - x (chronic progressive)

defence mechanism to eliminate virus infected cells. It is k n o w n from in vitro studies that measles infection can lead, under certain conditions, to viral persistency in tissue culture cells [10]. This persistency can be established in the presence of antiviral antibodies or as a result of an unknown virus host relationship. In order to prevent virus persistency it is important for the organism to destroy an infected cell. This can be established by complement dependent cytotoxic antiviral antibodies and by T cell mediated cytotoxicity [3]. But it is essential that viral antigens are present on the outer surface of the infected cells. This is the case with measles virus since, in the course of viral replication, viral antigens are incorporated into the cell membrane before the budding process takes place. H o w this immunomechanism functions if an infection takes place in the central nervous system, is unknown. In addition to its anatomical particularities the brain is relatively isolated from the immune system. In the case of a positive immune reaction to a virus infection in the central nervous system the blood brain barrier has to be crossed by antibodies and immune cells to reach the infected areas. This takes place in acute CNS infections and obviously also in slow virus infections such as SSPE, but whether the response is sufficient to eliminate virus infected cells in the CNS is questionable.

46

v. Kratzsch et al.

T h e p r e s e n t s t u d y has shown t h a t the h u m o r a l i m m u n e response to measles virus, as far as the c o m p l e m e n t d e p e n d e n t c y t o t o x i c a n t i b o d i e s are concerned, is o b v i o u s l y not impaired. F r o m this p o i n t o f view MS patients d o n o t differ f r o m c o n t r o l groups. T h e significantly higher a n t i b o d y titers however c o u l d be due to r e c u r r e n t antigenic s t i m u l a t i o n caused b y measles virus persistence. W h e t h e r measles virus persistence p l a y s a role in eliciting the clinical m a n i f e s t a t i o n o f MS can n o t be d e c i d e d at p r e s e n t on the l a b o r a t o r y d a t a available.

References 1. Adams, C. W. M., Leibowitz, S.: In Research on Multiple Sclerosis, pp. 135--148. Springfield (Ill.): Thomas 1972 2. Brody, I. A.: Epidemiologic and serologic data on MS and their possible significance. Multiple Sclerosis: Immunology, Virology and Ultrastructure, pp. 127--141 (F. Wolfgram, G. W. Ellison, J. G. Stevens, J. M. Andrews, eds.). UCLA Forum in Medical Science No. 16. New York-London: Academic Press 1972 3. Brunner, K. T., Cerottini, J. C.: Cytotoxic lymphocytes as effector cells of cell mediated immunity. In Progress in Immunology, p. 985 (B. Amos, ed.). New York: Academic Press 1971 4. Kibler, R., ter Meulen, V.: Antibody mediated cytotoxicity after measles virus infection. J. Immunol. 114, 93--98 (1975) 5. Kibler, R., Deller, A., ter Meulen, V.: Cytotoxic antibody activity in measles and subacute sclerosing panencephalitis (SSPE) infection. Med. Microbiol. !mmunol. 160, 179--190 (1974) 6. Norrby, E., Gollmar, Y.: Appearance and persistence of antibodies against different virus components after regular measles infection. Infec. Immun. 6, 240--247 (1972) 7. Norrby, E., Link, H., Olsson, J. E.: Measles virus antibodies in multiple sclerosis. Arch. Neurol. 30, 285--292 (1974) 8. Kurtzke, J. F.: On the evaluation of disability in multiple sclerosis. Neurology (Minneap.) 11, 686--694 (1961) 9. Poser, S., Hauptvogel, H.: Clinical data from 418 MS patients in relation to the diagnosis. First experiences with an optical mark reader documentation system. Acta neurol, scand. 49, 473---479 (1973) 10. Rustigian, R. J.: Persistent infections of cells in culture by measles virus. J. Bacteriol. 92, 1792 (1966) 11. Sachs, L.: Angewandte Statistik. Planung und Auswertung, Methoden und Modelle, pp. 238--240. Berlin-Heidelberg-New York: Springer 1974 Received January 15, 1977