Journal of

J. Neurol, 220, 105--112 (1979)

Neurology © by Springer-Verlag 1979

Measles Antibodies and K-2 Light Chain Distribution in Immunoglobulins of Patients Affected With Multiple Sclerosis F. Bollengier and A. Mahler Laboratorium voor Fysiopathologie van het Zenuwstelsel, Vrije Universiteit Brussel, Brussels, Belgium

Summary. The presence of measles antibodies in serum immunoglobulin G fractions from seven patients affected with multiple sclerosis was investigated with the HI technic. The x-). light chain ratios of all samples under investigation were evaluated. Three multiple sclerosis patients, who displayed either fractionation or a tendency towards fractionation in their serum, had slightly elevated measles antibody titers associated to increased ~c/2 ratios. Key words: Multiple sclerosis - Immunoglobulins - Measles antibodies - Light chain distribution.

Zusammenfassung. Masern-Antik6rper in Immunoglobulin-G-Fraktionen aus Seren von sieben Patienten mit Multipler Sklerose wurden mit der HI-Technik untersucht. Die Relation yon leichter x zu leichter 2 wurde berechnet. Drei Multiple-Sklerose-Patienten, die entweder Immunoglobulin-Unteffraktionen oder eine Tendenz zu Unterfraktionen in ihren Seren aufwiesen, hatten eine leicht erh6hte Masern-Antik6rper-Aktivit~it zugleich mit erh6hter x/),.

Introduction Several studies [5,7,9, 10, 14, 16, 17] have dealt with the quantitation and distribution of measles antibodies in serum and cerebrosplnal fluid (CSF) of patients affected with multiple sclerosis (MS) and the outcome of these investigations is conflicting i.e. it is still not clear whether measles virus is involved in the disease or not. According to Caspary et al. [4] positive or negative results in regard to measles antibodies in MS depend on the cut off level chosen i.e. the significance of the results depends upon the method of analysis. In a previous paper [3] we investigated the presence of measles antibodies in brain extracts and demyelination plaques of several MS patients and we were not able to detect them. However the x-2 light chain distribution of the MS brain specimens were in agreement with the light chain distribution of MS CSF. 0340-5354/79/0220/0105/$01.60

106

F. Bollengier and A. Mahler

In the present p a p e r we l o o k e d for the presence o f measles a n t i b o d i e s in i m m u n o g l o b u l i n G fractions o b t a i n e d b y liquid isoelectric focusing f r o m serum o f several patients affected with MS; in parallel we studied the x - 2 light chain d i s t r i b u t i o n o f the different fractions in o r d e r to l o o k for a possible a s s o c i a t i o n with measles a n t i b o d i e s .

Material and Methods Seven patients affected with MS were studied (ROU, MAR, PAU, LIP, PAE, DEM and REY); the diagnosis was established on the basis of clinical findings, the clinical course of the disease, thorough routine examination of the serum and cerebrospinal fluid, and agar gel electrophoresis. Patients ROU (21 years, •), MAR (39 years, Q) and LIP (28 years, d~) were in an active stage of the disease, but their symptomatology was very mild. Details on the clinical course of PAU (56 years, c~) are not available. In the case of patient PAE (54 years, Q), the disease started in 1972 and worsened progressively without specific bouts; patient DEM (54 years, ~) presented symptoms typical for MS for 10 years and the course of the disease was slowly progressive. In the case of REY (66 years, ~) the disease evolved progressively over a period of 30 years. The IgG were prepared by Na2SO4 precipitation at 18% and 12% [12], followed by liquid column isoelectric focusing. The lyophilized material was submitted to liquid isoelectric focusing in an LKB 8100 Ampholine column (LKB-Produkten, AB, Bromma-Sweden); the pH gradient was established with LKB Ampholines [pH: 9.5--8.0 (60%) - pH: 8.0--5.0 (40%)] and isofocusing proceeded over + 50 h at a constant temperature of 0° C. The proteins were studied by agar gel electrophoresis [23] and thin layer isoelectric focusing [11]. Hemagglutination inhibition (HI) tests were carried out as previously described [21] and light chain ratios were determined as reported elsewhere [1].

Results W h e n e x a m i n e d in a g a r gel electrophoresis serum P A E showed definite oligoclonal i m m u n o g l o b u l i n s (3 b a n d s / M c o m p o n e n t s ) , serum R E Y 1 faint b a n d a n d s e r u m D E M a t e n d e n c y t o w a r d s f r a c t i o n a t i o n (Fig. 1). Sera R O U , M A R , P A U a n d L I P s h o w e d no f r a c t i o n a t i o n . In all cases the t o t a l a m o u n t o f I g G isolated f r o m serum b y s o d i u m sulfate p r e c i p i t a t i o n was assayed for x/2 ratios. F o r the isofocusing e x p e r i m e n t s the p H - d e p e n d e n t i m m u n o g l o b u l i n fractions were g r o u p e d in r e g a r d to their measles a n t i b o d y titers ( H I ) a n d their x/2. Table 1 represents the results o b t a i n e d for patients PAE, D E M a n d REY. The first p o i n t t o note is that for all three patients the x/2 o f total I g G is fairly b e l o w the level o f x/2 o f c o n t r o l I g G (Table 3). In a d d i t i o n all three patients s h o w e d slightly increased measles a n t i b o d y titers, which were highest in c o m p a r a b l e p H ranges. One also notices t h a t the measles a n t i b o d y titers are a c c o m p a n i e d with a general increase o f the x / 2 ratios in the c a t h o d i c fractions, when c o m p a r e d to total I g G K/).. In Table 2 the s a m e items are r e p o r t e d for the cases R O U , M A R , P A U a n d LIP. H e r e the p a t t e r n is totally different: measles a n t i b o d y titers were m o s t l y negative, x/2 o f t o t a l I g G was within the range o f c o n t r o l I g G for R O U a n d

Measles Antibodies and x-2 Light Chain Distribution in Immunoglobulins

~

107

~

I~--~

I I

I

< < 0

,~ e~ .~

~i~iJ

-~

I I

I

~

I

~o

,_~ 0

~

~

1

~ ~.~,-~

~

'~

'

~

'

~

~

~

N ,~ ~:m ~-

,~

_ ~ ~ ~ ~

~

~

~

~

~

,2

.~

~

I~1

I I

~ ~

2

a

,.~

N

.~ 0

~

~

~

~

~ ~ ~

.~ .~ .--.

~

~

. . . .

~

d

~

~

~

0

e~ 0 -~ ~

~

~

~

~

~

~

~

~

~

t

.~_

~

,.~

~ ~

"7, N ~ 0

~

~ 0

0

~

~

~ 0

0

~

0

0

0

~

~ O

~

~

O

~

0

0

~

0

~

~

~

108

F. Bollengier and A. Mahler

111

+

4P

O A

O

Q

B

1 C

Fig. 1. A: agar gel electrophoresis of serum PAE. B: agar gel electrophoresis of serum REY. C: agar gel electrophoresis of serum DEM. Oligoclonal IgG is on the side of the cathode ($) M A R , a n d the c a t h o d i c fractions were not characterized b y a p a r t i c u l a r x / 2 increase when c o m p a r e d to t o t a l I g G 1¢/). for all cases u n d e r investigation. Table 3 represents the results o b t a i n e d for controls. The measles a n t i b o d y titers did n o t exceed 1/16 a n d the d i s t r i b u t i o n of the 1¢/2 ratios was r a t h e r regular.

Iso Iso Iso Iso Iso Iso Iso

1 2 3 4 5 6 7

Total IgG

Control pH range

K/2

HI

10.09--8.93 8.83--8.63 8.52--8.31 8.20--7.99 7.89--7.68 7.54--7.34 7.12--5.32

2.1 1.6 1.6 1.8 1.4 1.3 1.5

1/16--1/8 1/16--1/8 1/16--1/8 1/8 --1/4 1/8 --1/4 1/4 --1/2 1/4 --1/2

1.9

1/16

Table 3. pH-dependent immunoglobulin fractions obtained by column isofocusing in control serum

In o r d e r to visualize the different fractions o b t a i n e d b y c o l u m n isofocusing, aliquots were s u b m i t t e d to thin layer isofocusing in parallel with their t o t a l I g G . I m m u n o g l o b u l i n s R O U , M A R , P A U a n d L I P gave the t r a d i t i o n a l diffuse isofocusing p a t t e r n o f h e t e r o g e n e o u s i m m u n o g l o b u l i n s (Fig. 2), b u t the i m m u n o globulins PAE, D E M a n d R E Y showed the restricted h e t e r o g e n o u s type, such as is o b s e r v e d with subacute sclerosing p a n e n c e p h a l i t i s (SSPE): each fraction is r e d u c e d to clearcut m y e l o m a - l i k e areas, c o r r e s p o n d i n g to well defined parts o f the total I g G picture (Fig. 2).

109

Measles Antibodies and x-2 Light Chain Distribution in Immunoglobulins

~D I

A

1

2

3

4

5

6

Fig. 2. Thin layer isoelectric focusing: total IgG ROU B: total IgG iso 1 ROU 1': iso 1 iso 2 ROU 2': iso 2 iso 3 ROU 3': iso 3 iso 4 ROU 4': iso 4 iso 5 ROU 5': iso 5 iso 6 ROU 6': iso 6 iso 7 ROU 7': iso 7

A: 1: 2: 3: 4: 5: 6: 7:

7

7'6'5'4

3'

2'

I'B

PAE PAE PAE PAE PAE PAE PAE PAE

Discussion

The investigation for the presence of measles antibodies in isolated serum immunoglobulins of seven MS patients does not add convincing arguments to the measles virus etiology of the disease, since more than half the patients investigated did not display particular HI measles antibody titers, or were even frankly negative. On the one hand, these results do not agree with those reported by Salmi et al. [18--20] who identified different measles virus-specific antibodies in the serum and CSF from patients with SSPE and MS. However those authors carried out neutralization, HLI, HI tests with tween 80-ether treated antigen, and CF tests were carried out with crude virus material, purified nucleocapsids, and small particle hemagglutinin as antigen. They reported a certain diversity in the relative content of antibodies against different virus products. On the other hand Vandvik and Degr6 [22] in their study on measles antibodies in MS failed to show a rise of measles HI antibody levels in MS patients compared to non MS patients.

110

F. Bollengier and A. Mahler

Paty et al. [14] showed that there is a relationship between HLA type and high measles antibody titers in MS and their siblings, suggesting that a constitutional factor is not only important in determining susceptibility to MS, but that the same factor may determine the level of measles antibodies in the patients of the siblings. Their data did not support the hypothesis that measles virus is latent in the central nervous system. In a previous paper [3] we reported the investigation of the presence of measles antibodies in brain specimens from several MS patients and we were not able to detect them. Recently Eldridge et al. [6] reported that no relationship was noted between H L A type or DW2 phenotype and antimeasles antibody titer within their families and they could not demonstrate significant differences in measles titers between affected and unaffected individuals. When mentally reviewing (since reporting all of them is not the object of this paper) the most recent investigations as to whether measles virus is a general agent in the production of MS, it seems that the answer is most probably negative. Moreover the occurrence of false positive measlesspecific immunoglobulins is not to be excluded, since Fraser [8] demonstrated them in MSspecific serum. He concluded that MSIgM in absorbed serum represented a residual rheumatoid factor like protein and he regarded the properties formerly ascribed to it as those of measlesvirus antihemolysin. However his studies dealt with IgM, and this phenomenon has yet to be demonstrated for IgG. In the present report three patients examined, who showed definite oligoclonal immunoglobulins or a tendency towards fractionation, yielded immunoglobulin fractions which were characterized by slightly elevated measles antibody titers, coincident with increased x/2 ratios. In SSPE, a neurological disease which is characterized not only by the appearance of oligoclonal imunoglobulins in CSF and serum, but also by high measles antibody titers and disturbed x/2, hyperimmunization against measles or measles-like virus is generally accepted. In SSPE serum we considered x enrichment in immunoglobulin fractions corresponding to high measles antibody titers, as the result of the formation of sharply restricted predominant antimeasles clones [2]. Now in the MS cases mentioned above, an analogue situation is observed, although the picture is not as outspoken as in SSPE. Measles antibodies, when they do occur in MS, are also characterized by similar pH ranges as in SSPE, and by a x increase; consequently they could thus also be considered to be immunoglobulins resulting from the formation of restricted xpredominant antimeasles clones, although to a less degree than in SSPE. The thin layer isoelectric focusing (Fig. 2) reinforces this clonal hypothesis. It is difficult to know whether a measles virus etiology can be generally accepted for MS, with certain patients reacting more strongly tO the causative agent, or whether in certain MS patients, there is, next to and independent of the disease, a particular immunological response to measles virus. According to McDermott et al. [13] elevated measles titers in MS could be due to the fact that breakdown products, especially the encephalitogenic factor (EF)

Measles Antibodies and x-2 Light Chain Distribution in Immunoglobulins

111

m a y share certain antigenic d e t e r m i n a n t s with measles virus; cross reactivity o f s e r u m a n t i b o d i e s to such b r e a k d o w n p r o d u c t s c o u l d be i n t e r p r e t e d as elevated measles titers. A l t h o u g h they gave evidence o f antigenic d e t e r m i n a n t sharing between measles virus a n d E F , this h y p o t h e s i s nonetheless does n o t entirely cope with the MS cases which are measles-negative. Raine et al. [15] in i m m u n o c h e m i c a l studies on the localization o f measles antigen in MS p l a q u e s , "failed to o b t a i n evidence o f expressed measles antigens in b o t h c h r o n i c lesions f r o m 2 p a t i e n t s with typical relapsing M S a n d in actively d e m y e l i n a t i n g lesions f r o m a p a t i e n t with acute M S . " This r e p o r t once m o r e emphasizes the fact t h a t it is very h y p o t h e t i c a l to c o n s i d e r measles antigen to be a general agent responsible for MS.

Acknowledgement. We are greatly indebted to Prof. Dr. A.Lowenthal (Laboratorium voor Fysiopathologie van het Zenuwstelsel, Vrije Universiteit Brussel) and Drs. R. Medaer (Neurologisch Centrum MS Kliniek, Overpelt, Belgium) and C. Moerman (Brugman Hospitaal, Vrije Universiteit Brussel) for the precious gift of the MS sera.

References 1. Bollengier, F., Lowenthal, A., Henrotin, W.: Bound and free light chains in subacute sclerosing panencephalitis and multiple sclerosis serum and cerebrospinal fluid. Z. klin. Chem. klin. Biochem. 13,305--310 (1975) 2. Bollengier, F., Karcher, D., Rabinovitch, N.: Imbalance of x/2 ratios associated with high measles antibody titers in fractionated serum immunoglobulins of patients with subacute panencephalitis. Ann. Microbiol. (Institut Pasteur) 128A, 89--96 (1977) 3. Bollengier, F., Mahler, A., Clinet, G., Lowenthal, A.: Multiple sclerosis: oligoclonal IgG, x-2 light chain distribution and measles antibodies in brain extracts. Brain Research 152, 133--144 (1978) 4. Caspary, E. A., Chambers, E. M., Field, E. J.: Antibodies to measles antigen, control antigen, and monkey kidney antigen. Studies in patients with multiple sclerosis and other neurological diseases and normal healthy individuals. Neurology 19, 1038--1042 (1969) 5. Cendrowski, W., Polna, I., Nowicka, K.: Measles virus infection and MS-serologicalstudies. J. Neurol. 213, 369--376 (1976) 6. Eldridge, R., McFarland, H., Sever, J., Sadowski, D., Krebs, H.: Familial multiple sclerosis: clinical, histocompatibility and viral serological studies. Ann. Neurology 3, 72--80 (1978) 7. Fraser, K. B.: Multiple sclerosis: a virus disease? Br. Med. Bull. 33, 34---39 (1977) 8. Fraser, K. B.: False-positive measles-specific IgM in multiple sclerosis. Lancet 1978 I, 91--92 9. Haire, M.: Significance of virus antibodies in multiple sclerosis. Br. Med. Bull. 33, 40--44 (1977) 10. Hutchinson, W. M., Haire, M.: Measles-virus specific IgG in optic neuritis and in multiple sclerosis after optic neuritis. Brit. Med. J. 1976 I, 64---66 11. Karlsson, C., Davis, H., Ohman, J., Andersson, U. B.: LKB 2117 Multiphor I. Analytical thin layer gel electrofocusing in polyacrylamide gel. LKB Application Note - - March 29, 1973 12. Keckwick, R. A.: The serum proteins in multiple myelomatosis. Biochem. J. 34, 1248--1257 (1940) 13. McDermott, J. R., Field, E. J., Caspary, E. A.: Relation of measles virus to encephalitogenic factor with reference to the aetiopathogenesis of multiple sclerosis. J. Neurol. Neurosurg. Psychiat. 37, 282--287 (1974) 14. Paty, D. W., Furisz, J., Boucher, D. W., Rand, G. R., Stiller, C. R.: Measles antibodies as related to HLA types in multiple sclerosis. Neurology 26, 651--656 (1976)

112

F. Bollengier and A. Mahler

15. Raine, C. S., Prineas, J. W., Sheppard, R. D., Bornstein, M. B., Dubois-Dalcq, M.: Immunochemical studies for the localization of measles antigen in MS plaques and measles virusinfected CNS-tissue. J. Neurol. Sci. 33, 13--20 (1977) 16. Reunanen, M., Arstila, P., Hakkarainen, H., Niskokelainen, J., Salmi, A., Panelius, M.: A longitudinal study on antibodies to measles and rubella viruses in patients with multiple sclerosis. A preliminary report. Acta neurol. Scand. 54, 1--12 (1976) 17. Roberts-Thomson, P. J., Esiri, M. M., Young, A. C., Mac Lennan, I. C. M.: Cerebrospinal fluid immunoglobulin quotients, kappa/lambda ratios, and viral antibody titers in neurological disease. J. Clin. Path. 29, 1105--1115 (1976) 18. Salmi, A. A., Panelius, M., Halonen, P., Rinne, U. K., Penttinen, K.: Measles virus antibody in cerebrospinal fluids from patients with multiple sclerosis. Brit. Med. J. 19721, 477---479 19. Salmi, A. A., Norrby, E., Panelius, M.: Identification of different measles virus-specific antibodies in the serum and cerebrospinal fluid from patients with subacute sclerosing panencephalitis and multiple sclerosis. Infect. Immunity 6, 248--254 (1972) 20. Salmi, A. A.: Virus antibodies in patients with multiple sclerosis. Ann. Clin. Research 5, 319--329 (1973) 21. Thiry, L., Dachy, A., Lowenthal, A.: Measles antibodies in patients with various types of measles infection. Arch. ges. Virusforsch. 28,278--284 (1969) 22. Vandvik, B., Degr6, M.: Measles virus antibodies in serum and cerebrospinal fluid in patients with multiple sclerosis and other neurological disorders, with special reference to measles antibody synthesis within the central nervous system. J. Neurol. Sci. 24, 201--219 (1975) 23. Wieme, R. J.: Agar gel electrophoresis. Amsterdam: Elsevier 1964

Received June 30, 1978