CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Lymphocytotoxic ALAN
7, 15-23 (1977)
Antibodies
in Multiple
Sclerosis1
L. SCHOCKET,~ KENNETH
HOWARD L. WEINER,~ JOHN WALKER,~ MCINTOSH, AND PETER F. KOHLER
Division of Clinical Immunology, Pediatrics, University of Colorado
Departments of Medicine, Medical Center, Denver,
Neurology, Colorado
and 80220
Received May 3, 1976 The presence of cold-reactive lymphocytotoxic antibodies has been demonstrated in patients with viral infections and various “autoimmune” diseases thought to be related to viral infection. In the present study we have examined the incidence and possible significance of these antibodies in patients with multiple sclerosis, another disease probably associated with a viral agent. The incidence of lymphocytotoxic antibodies was significantly increased in patients with multiple sclerosis as compared to their siblings, patients with other neurological disease, and normal controls. The level of these antibodies correlated significantly with serum measles antibody titers in the affected patients. No correlation was found between lymphocytotoxic antibody levels and parameters or clinical disease or other viral antibody titers in the serum and cerebrospinal fluid. It was concluded that lymphocytotoxic antibodies in patients with multiple sclerosis probably serve as markers of viral infection.
INTRODUCTION
With the growing interest in the role of the lymphocyte in the pathophysiology of disease, attention has been focused on factors which may modify lymphocyte number and function in vivo and in vitro. Lymphocytotoxic antibody (LCA) is one such factor found in a significant number of sera from patients with a variety of “autoimmune” diseases. The prevalence of these cold-reactive IgM antibodies is increased in patients with systemic lupus erythematosus (SLE) (l-5), rheumatoid arthritis (l), inflammatory bowel disease (6,7), chronic active hepatitis (8), multiple sclerosis (MS) (9), and several other diseases of obscure etiology. One common factor underlying many of these disease entities is their possible induction by latent viral infection. The presence of LCA and its relation to cellular immunity and disease activity has been studied best in SLE (4,10-16) where lymphocytotoxic antibodies have been found in a high percentage of patients. In some of these studies their presence has correlated well with clinical and laboratory parameters of SLE disease activity. Antibodies to viral agents have been shown to
i Supported in part by National Multiple Sclerosis Society Grant No. RG-812-B-2 and National Institutes of Health Grant No. NS-10590. * Supported in part by National Institute of Allergy and Infectious Disease Training Grant No. 5-TOl-AI00013-17. Division of Clinical Immunology, University of Colorado Medical Center, Denver, Colorado 80220. 3 Fellow of the Colorado Multiple Sclerosis Society. Present address: Department of Microbiology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115. 4 Present address: Veterans Administration Hospital, 4500 South Lancaster Road, Dallas, Texas 75216. Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
16
SCHOCliEl
b:T AL
cross react with some lymphocytes from patients with SLE (I?. 18). Further evidence suggests an interrelation between a transmittable agent. the presence of the disease, and the occurrence of LCA. This comes from the demonstration of LC.4 in a high percentage of sera from household contacts of the affected individuals ( 19.20). In addition, the presence of LCA has been demonstrated in patients with acute viral infections (21,22) and in normal individuals following immunizations with viral vaccines (23). Multiple sclerosis IS another disease of obscure etiology. perhaps induced by a latent viral agent (24.35). The incidence of LCA is increased in sera from patients with multiple sclerosis (9). but the correlation of the presence of these antibodies with parameters of clinical disease and viral infection has not been examined. This study was designed to determine the incidence of LCA in sera from patients with multiple sclerosis and their siblings. In addition, the correlations of LCA with age at onset, duration, CSF IgG levels. and extent of disease were examined. To evaluate the relationship of LCA to parameters of specific viral infections, levels of LCA were correlated with serum and CSF measles and vaccinia antibody titers. MATERIALS
Patients. Eighty-seven
AND
METHODS
patients with definite multiple sclerosis as defined by the Schumacher Committee criteria (26) were studied. ,411 clinical data. sera. and cerebrospinal fluid (CSF) were collected in 1972. Sera and CSF were stored at -70°C. The clinical information used in thts study was obtained from the records made at the time of data collection. Age at onset of disease and on admission to the study. sera. and cerebrospinal fluid were available on all 87 patients, but full clinical records were available on only 72 patients. All the subjects were divided into three disease categories as described by Fog and Linneman (27). based on the clinical course of their disease: (i) chronic progressive. (ii) relapsing remitting, and (iii) mixed. Extent of disease as measured by disability was quantitated from data in the available clinical records by the Kurtzke method on a scale of severity from 0 to 7. Sera from 34 siblings of patients with multiple sclerosis were also collected in 1972. The places of residence of the patients and siblings were determined from addresses available in the records. Controls. Serum was obtained from 1X patients on the neurological wards at Colorado General Hospital and Denver Veterans Administration Hospital. Diagnoses of these patients included seizure disorder. Huntington’s chorea. head trauma, stroke, and brain tumor. None of these patients had received corticosteroids or immunosuppressive therapy within I month prior to collection of serum. Sera was also obtained from 24 laboratory personnel and volunteers and comprised the normal control group. All sera were stored at --70°C until use. CSF gamma globulin determinution. CSF gamma globulin was measured bv eiectroimmuno-diffusion as described by Schneck and Claman (38). Vucciniu antibodies. Vaccinia antibody titers in the sera and CSF were measured by plaque formation inhibition (29). Measles antibodies. Measles antibody titers m sera and CSF were measured by the hemagglutination inhibition assay (30). Lymphocytotoxic antibodies. LCA were measured by the microdroplet method of Terasaki and McClelland (3 1). The target ceIIs were mononuclear cells obtained
LYMPHOCYTOTOXIC
ANTIBODIES
IN
MULTIPLE
SCLEROSIS
17
by Ficoll-Isopaque gradient separation of 2 cc of heparinized blood from a panel of 20 normal donors. One microliter of the subject’s serum was first incubated with 2 x 103 target cells at 15°C for 30 min in a well of a microtiter plate. Five microliters of normal rabbit serum was then added to each well, and the mixture was incubated for 3 hr at 15°C. The viability of the target cells in each well was then determined by exclusion of the vital dye eosin-Y and the percent killing was recorded. A well with 1 /Al of human serum albumin and 2 x 103 target cells was included as a negative control in each experiment. When the negative control caused 20% or greater killing of target cells, results from the test sera run in parallel were excluded from the study. The serum from a patient with SLE which consistently killed 70 to 100% of target cells on repeated determinations was used as the positive control in all experiments. This serum was stored at -70°C and no diminution of cytotoxicity was observed after repeated freezing and thawing. The final results were expressed as a cytotoxicity index: Cytotoxicity
index =
average % target cell killing average % target cell killing
by test serum
x 100. by positive control serum
A cytotoxicity index of 15 or greater was considered a positive test for the presence of lymphocytotoxic antibodies. The comparison of the cytotoxicity of the test serum to the same positive control in each run rather than the use of the absolute percentage cytotoxicity alone takes into account minor variations of reagents and test conditions which might alter experiments done at different times. In addition, results expressed in this manner provide at least a rough quantitation of LCA in each serum since, in previous studies, percent cytotoxicity of undiluted serum has correlated well with antibody titers determined by serial dilution (31). Statistical analysis. Fisher’s exact method was used to compare the incidence of lymphocytotoxic antibodies in the experimental groups to the control group and the level of significance was derived from standard tables. Standard linear correlations were calculated comparing lymphocytotoxic index of each subject to each variable studied. RESULTS
Incidence of lymphocytotoxic antibodies. Forty of eight-seven (46%) samples of sera from patients with multiple sclerosis had a cytotoxicity index of 15 or greater and were considered positive for lymphocytotoxic antibodies (Fig. 1). This incidence was significant with ap < .OOl when compared to the control groups (Table 1). When the MS patients were separated into three groups according to type of clinical course, no significant difference in the incidence of lymphocytotoxic antibodies was noted among the groups (Table 2). In the sibling group, 6 of 34 (17.6%) sampled had elevated lymphocytotoxic antibody levels (Table 1). This incidence was not significantly greater than that in the control group. Since it has been shown that sera from household contacts of patients with SLE and inflammatory bowel disease have an increased incidence of LCA’s (19,20), the records were examined to determine which siblings shared the same household as the multiple sclerosis patients. Three siblings lived in the same
18
ET AL
SCHOCKET
00 -
70.
60
-
50
-
40
-
30
-
zoI5 10 -
. . . .
:. .. . .:. .. : . 22 %: .f. . .Tt _-_ --*-.? 0:.9%. d .h*
.
l .
A c
FIG. 1. The distribution of cytotoxicity indices within each subject group serum with a cytotoxicity index of 15 or greater is considered positive phocytotoxic antibodies. Fotiy of the eighty-seven patients in the multiple positive for lymphocytotoxic antibodies. This incidence was significantly group with a p value of < ,001. Neither the incidence of positive sera in neurologic group was significantly different from the normal control group.
is plotted on this graph. A for the presence of lymsclerosis group had sera greater than the control the sibling group nor the
house as a patient, and two were positive for LCA. One of these sera had the highest cytotoxicity index (50) of the group. This was an inadequate number of subjects, however, to allow a valid statistical analysis.
Multiple sclerosis Siblings Neurological controls Normal controls
Number of subjects
Number positive for LCA
Positive (5%)
87 34 19 24
40 6 I !
46 17.6 5.3 12.5
LYMPHOCYTOTOXIC
ANTIBODIES
TABLE INCIDENCE
OF
LCA
RELATED
Clinical course
TO TYPE
IN
MULTIPLE
19
SCLEROSIS
2
OF CLINICAL
COURSE
OF MULTIPLE
SCLEROSIS~
Number of pts
Number positive for LCA
Positive (%)
48 13 26 87
22 4 13 40
45.8% 38.5% 50% 46%
Relapsing remitting Chronic progressive Mixed All patients a As described by Fog (26).
In the control groups, 1 of 19 (5.3%) of the neurological controls and 3 of 24 (12.5%) of the normal controls had sera positive for lymphocytotoxic antibodies (Table 1). This incidence in normal controls is similar to that reported in previous studies (4,9,23). The mean cytotoxicity index value and standard error of the mean are shown in Table 3. Due to the wide range of cytotoxicity indices within each group, as shown in Fig. 2, the mean cytotoxicity indices of the MS, sibling, and control groups did not differ significantly from the mean index of the normal group. Correlation of cytotoxicity index levels with variables. A significant correlation, p < .05, between the cytotoxicity index and serum measles antibody titers was found in the MS patients (Fig. 2). There was no significant correlation between cytotoxic indices and any other of the variables studied (Table 4). The correlation between LCA levels and serum measles antibody titers in the sibling group (Y = .0532) was not significant. An elevated mean titer of antibody to measles in sera from these multiple sclerosis patients and their siblings was reported in an earlier study (32). In the same study, elevated CSF vaccinia antibody titers in the multiple sclerosis patients was also demonstrated. DISCUSSION
The results of this study confirm that cold-reactive antibodies cytotoxic for lymphocytes (LCA) are present in a significantly higher proportion of sera from patients with multiple sclerosis as compared to sera from siblings of MS patients, patients with other neurologic diseases, and normal controls. Of all the clinical and serological variables studied, LCA serum levels bore a significant correlation only to serum measles antibody titers in patients with MS. The finding of an increased incidence of LCA in patients with MS is in agreement with an earlier study by Kuwert and Bet-trams (9). These investigators found significant levels of TABLE DISTRIBUTION
MS patients MS sibs Normal controls Neuro controls
OF CYTOTOXICITY
INDICES
3 WITHIN
EACH
GROUP
STUDIED
N
Mean
SE
87 34 24 19
18.66 10.38 11.00 4.63
1.70 2.07 1.74 1.57
20
SCHOCKET
ET AL
I
80.
.
.
70-
. 60 : D c .-," .u x 2 2
50.
40.
. . .
. . . . .
c 30:
. .
O&3' *1632
5
1 64
. .
128
L 512
256
Reciprocal of Anti-measles Antibody Titer FIG. 2. The cytotoxicity index is plotted against the reciprocal of the antimeasles X intercept = 14.6224, and the slope equals the linear regression coeffkient significant correlation with a p value of < .05.
antibody titer. of .2 160. This
The is a
LCA in 34% of sera from 462 chronic MS patients as compared to 6% of healthy controls and 12% of neurological controls. The antibody was characterized as a cold-reactive IgM maximally cytotoxic at 15°C with little or no activity at 37°C. The relative lack of antibody activity at normal body temperature casts doubt on the potential effect of this LCA upon lymphocyte function and number in viva. Neither our study nor this earlier study has addressed this question in patients TABLE CORRELATION
OF CYTOTOXICITY
INDEX LEVELS WITH MULTIPLE
4 WITH VARIABLES SCLEROSIS
STUDIES
IN 87 PAI-tee-r-s
p Value Serum measles antibody titer Extent of disease Age at onset CSF yG level CSF vaccinia antibody titer Duration of disease Serum vaccinia antibody titer CSF measles antibody titer
.2160 .‘OlO .I435 .I068 - .0732 .0728 .0033
,044 .062 .I85 .325 .500 .SO? .976 ,994
LYMPHOCYTOTOXIC
ANTIBODIES
IN
MULTIPLE
SCLEROSIS
21
with MS. Previous studies have, however, reported no decrease in total circulating lymphocytes, T cells, or B cells in MS patients (33,34). A serum factor has been described in patients with active MS which inhibits protein synthesis by mitogen-stimulated lymphocytes (35-37). On purification, this factor proved not to be an immunoglobulin and consequently is not related to the LCA found in our patients studied. The present evidence suggests therefore that the LCA found in the MS patients has little if any appreciable effect on lymphocyte number or function. Other disease states characterized by the presence of LCA such as pernicious anemia (38) and inflammatory bowel disease (6,7) are also not characterized by significant reductions in total lymphocytes, T or B cells. On the other hand, LCA are present in a high percentage of patients with SLE, a disease characterized by major deficits in lymphocyte number and function. Some studies have shown correlations between LCA and disease activity in these patients (4,l l), suggesting a pathophysiological role for these antibodies. Horowitz and Cousar (15), however, recently reported that in vitro and in vivo deficits in cell-mediated immunity are related to the presence of an IgG antilymphocyte antibody reactive at 37°C and did not correlate with the presence of cold-reactive, IgM lymphocytotoxic antibodies. At present the only diseases in which a correlation has been shown between the presence of this type of LCA and lymphopenia are acute viral infections such as infectious mononucleosis, measles, and rubella (21). It is more likely, however, that this lymphopenia is due to the cytopathic effect of the virus rather than LCA. Since there is little evidence to support the role of LCA as a modulator of lymphocyte function, a second possibility is that LCA may participate in the pathophysiology of the CNS lesion in MS. Evidence from animal experiments as well as human studies using cross absorption techniques has indicated that antigens derived from brain tissue cross-react with antigens on the lymphocyte cell surface (39-42). In the mouse the cross-reacting lymphocyte surface antigen is almost exclusively the theta antigen. Despite earlier studies in man suggesting a similar specific cross-reactivity between brain antigen and the T cell, a recent paper has indicated that in patients with SLE, at least, no such specificity with a particular subset of lymphocytes can be demonstrated (43). In earlier studies antibodies to a specific constituent of brain, myelin, have been found in some patients with multiple sclerosis (44-45). We are not aware of studies examining the reactivity of these antibodies with lymphocyte cell surface. In addition, on a clinical basis there is little evidence in the present study of MS patients linking the presence of LCA with duration of disease or brain dysfunction as measured by extent of disease. Consequently, it is unlikely that these LCA are primarily involved in the pathophysiology of the CNS lesion. A third, and perhaps more plausible, explanation for the presence of LCA in MS patients is the possibility that these antibodies represent markers of latent or persistent viral infection. LCA are present with an increased frequency in patients with acute viral infections such as infectious mononucleosis, measles, and rubella (21,22). In addition, elevated levels of LCA have been demonstrated in normal individuals following immunizations with viral vaccines (23). Further evidence
22
SCHOCKE?‘
E7 AL
relating the occurrence of LCA to a transmittable agent comes from studies of household contacts of patients with SLE and inflammatory bowel disease (19.20). LCA are present in a significantly higher number of sera from both related and nonrelated residents of the households of affected individuals as compared to relatives living in other homes. In reference to multiple sclerosis. support for a viral etiology of this disease has been expounded in the literature for many years (24,25). Elevated antibody levels to various viral agents, especially measles, have been reported in these patients (24), and this finding of increased serum antibody titers has been confirmed in the present group of multiple sclerosis patients. In addition, these antibody levels correlated significantly @ < .05) with serum LCA levels, suggesting an association between LCA and measles infection. Further evidence similar to that found in SLE and inflammatory bowel disease household contacts linking LCA with a presumed infectious agent was sought in the siblings of our patients. Unfortunately, only three of these siblings shared the same household as their relative with the disease. The presence of LCA in two of three of these household contacts only suggests that this is a similar phenomenon to that occurring with contacts of SLE and inflammatory bowel disease patients. This is another clue relating LCA in MS to a presumed transmittable agent. Further study of this relationship is in progress. Finally. further evidence which might elucidate the relationship between LCA and viruses comes from recent studies suggesting that lymphocytes have specific receptors for virus particles and that antibodies raised against a specific virus can react with a small proportion of peripheral blood lymphocytes from SLE patients. First, EB virus receptors have been demonstrated on B lymphocytes (46), and recently the presence of receptors specific for measles virus have been shown on normal human T lymphocytes (47). Secondly, the observation was made by Shwartz that antibodies raised against a C-type virus stained a small percentage of the perhipheral blood lymphocytes from SLE patients (17.18). This finding suggests that there are either viral antigens or a lymphocyte component which cross-reacts with the viral antigen present on the cell surface. One might, therefore, hypothesize that a viral agent bound to a lymphocyte surface receptor serves as a hapten with the cell membrane functioning as the carrier. An antibody induced to this complex might subsequently react with either the viral determinant or normal lymphocyte cell surface. We have provided suggestive evidence that this type of antigen interaction might be responsible for the presence of LCA found in MS and the other disease entities previously associated with LCA mentioned. ACKNOWLEDGMENTS We wish to thank Patterson for typing
Alan M. Suzuki the manuscript.
for his technical
assistance
in preparing
the statistics
and Susan
REFERENCES 1. Terasaki, P. I., Mottironi. V. D.. and Barnett, E. V.. N. Eng1. J. Med. 283, 724. 1970. 2. Mittal, K. K., Rossen, R. D., Sharp, J. T., Lidsky, M. D.. and Butler, W. T., Norure (London) 225, 1255. 1970. 3. Stastny, P.. and Ziff, M., Clin. Exp. Immunol. 8, 543. lY71. 4. Ooi, B. S.. Orlina. A. R., Pesce. A. J., Mendoza. N.. Masaitis. L.. and Pollak. V. E., Clin. Exp. Immunol. 17, 237. 1974.
LYMPHOCYTOTOXIC
ANTIBODIES
IN
MULTIPLE
SCLEROSIS
23
5. Nies. K. M., Brown, J. C., Dubois, E. L., Quismorio, F. P., Frion, G. J.. and Terasaki, P. I., Arthritis Rheum. 17, 397, 1974. 6. Korsmeyer, S., Strickland, R. G., Wilson, I. D., and Williams, R. C., Jr., Gastroenterology 67, 578, 1974. 7. Strickland, R. G., Friedler, E. M., Henderson, C. A., Wilson, I. D., and Williams, R. C., Jr., Clin. Exp. Immunol. 21, 384, 1975. 8. Husby, G., Pierce, P. E., and Williams, R. C., Jr., Ann. Int. Med. 83, 801, 1975. 9. Kuwert, E.. and Bertrams, J., Europ. Neurol. 7, 65. 1972. 10. Stastny, P., and Ziff, M., Arthritis Rheum. 14, 733, 1971. Il. Butler, W. T., Sharp, J. T., Rossen, R. D.. Lidsky, M. D., Mittal, K. K., and Gard, D. A., Arthritis Rheum. 15, 231, 1972. 12. Williams, R. C.. Lies, R. B., and Messner, R. P., Arthritis Rheum. 16, 597, 1973. 13. Winfield, J. B., Winchester. R. J., Wernet, P., Fu, S. M., and Kunkel, H. G., Arthritis Rheum. 18, 1, 1975. 14. Messner, R. P., Kennedy, M. S., and Jelinek, J. G., Arthritis Rheum. 18, 201, 1975. 15. Horwitz, D. A., and Cousar, J. B., Amer. J. Med. 58, 829, 1975. 16. Glinski, W. M., Gershwin, M. E., and Steinberg, A. D., J. Clin. Invest. 57, 604, 1976. 17. Lewis, R. M., Tannenberg, W., Smith, C., and Schwartz, R. S.. Nature (London) 252, 78, 1974. 18. Schwartz, R. S., N. Engl. J. Med. 293, 132, 1975. 19. De Horatius, R. J., and Messner, R. P., J. Clin. Invest. 55, 1254, 1975. 20. Korsmeyer, S. J.. Williams, R. C., Jr., Wilson, I. D., and Strickland, R. G., N. Engl. J. Med. 293, 1117, 1975. 21. Huang, S. W., Lattos, D. B., Nelson, D. B., Reeb, K., and Hang, R., J. Clin. Invest. 52, 1033, 1973. 22. Mottironi, V. D., and Terasaki, P. I., “Histocompatibility Testing 1970” (P. I. Terasaki, Ed.), p. 301, Munksgaard, Copenhagen, 1970. 23. Kreisler, M. J., Hirata, A. A., and Terasaki, P. I., Transpluntation 10, 411, 1970. 24. Cathala, F.. and Brown, P., J. Clin. Pathol. 25, Suppl. 6. 141, 1972. 25. Koldovsky, V., Koldovsky, P., Henle. G., Henle, W., Ackerman, R., and Haase, G., Infect. Immunity 12, 1355, 1975. 26. Schumacher, G. A., Beebe, G., Kebler, R. F., et al., Ann. N. Y. Acad. Sci. 122, 552, 1965. 27. Fog, T., and Linneman, F., Acta Neurol. Stand. 46, Suppl. 47, 1, 1970. 28. Schneck, S. A., and Claman, H. N., Arch. Neural. 20, 132, 1969. 29. Takabayashi, K., and McIntosh, K., Infect. Immunity 8, 582, 1973. 30. Norrby, E., and Gollmar, Y., Infect. Immunity 6, 240, 1972. 31. Terasaki, P. I., and McClelland, J. D., Nature (London) 204, 998, 1964. 32. Mujamoto, H., Walker, J. E., Ginsberg, A. H., Burks, J. S., McIntosh, K., and Kempe, C. H., Arch. Neural., in press. 33. Oger, J. F., Amason, B. G. W., Wray, S. H., and Kistler, J. P., Neurology 25, 444, 1975. 34. Lisak, R. P., Levinson, A. I., Zweiman, B., Abdou, N. I., Clin. Exp. Zmmunol. 22, 30, 1975. 35. Van den Noort, S., and Stjemholm. R. L., Neurology 21, 783, 1971. 36. Bums, C. P., Armentrout, S. A., Gross, S., Van den Noort, S., and Stjernholm, R. L., J. Lab. Clin. Med. 78, 508, 1971. 37. White, G., Armentrout, S., and Van den Noort, S., Neurology 25, 37, 1975. 38. Goldberg, L. S., Cunningham, J. E., and Terasaki, P. I., Blood 39, 862, 1972. 39. Golub, E. S., Cellular Zmmunol. 2, 353, 1971. 40. Golub, E. S., J. Zmmunol. 109, 168, 1972. 41. Kongshavin, P. A. L., Gold, P., Shuster, J., Colquhoun, B., Freedman, S. 0.. Clin. Zmmunol. Zmmunopathol. 3, 1, 1974. 42. Takada, A., Takada, Y., Ito, U., and Minowada, J., Clin. Exp. Zmmunol. 18, 491, 1974. 43. Bluestein, H. G., and Zvaifler, N. J., J. Clin. Invest. 57, 509, 1976. 44. Bornstein, M. B., and Appel, S. H., Ann. N. Y. Acad. Sci. 122, 280, 1965. Sclerosis, a Reappraisal,” 45. McAlpine, D., Lumsden, C. E., and Acheson, E. D., “Multiple pp. 556-568, Williams and Wilkins, Baltimore, 1972. 46. Jondal, M., and Klein, G., J. Exp. Med. 138, 1365, 1973. 47. Valdimarsson, H., Gudrun, A., and Lachmann, P. J., Nature (London) 225, 554, 1975.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Lymphocytotoxic ALAN
7, 15-23 (1977)
Antibodies
in Multiple
Sclerosis1
L. SCHOCKET,~ KENNETH
HOWARD L. WEINER,~ JOHN WALKER,~ MCINTOSH, AND PETER F. KOHLER
Division of Clinical Immunology, Pediatrics, University of Colorado
Departments of Medicine, Medical Center, Denver,
Neurology, Colorado
and 80220
Received May 3, 1976 The presence of cold-reactive lymphocytotoxic antibodies has been demonstrated in patients with viral infections and various “autoimmune” diseases thought to be related to viral infection. In the present study we have examined the incidence and possible significance of these antibodies in patients with multiple sclerosis, another disease probably associated with a viral agent. The incidence of lymphocytotoxic antibodies was significantly increased in patients with multiple sclerosis as compared to their siblings, patients with other neurological disease, and normal controls. The level of these antibodies correlated significantly with serum measles antibody titers in the affected patients. No correlation was found between lymphocytotoxic antibody levels and parameters or clinical disease or other viral antibody titers in the serum and cerebrospinal fluid. It was concluded that lymphocytotoxic antibodies in patients with multiple sclerosis probably serve as markers of viral infection.
INTRODUCTION
With the growing interest in the role of the lymphocyte in the pathophysiology of disease, attention has been focused on factors which may modify lymphocyte number and function in vivo and in vitro. Lymphocytotoxic antibody (LCA) is one such factor found in a significant number of sera from patients with a variety of “autoimmune” diseases. The prevalence of these cold-reactive IgM antibodies is increased in patients with systemic lupus erythematosus (SLE) (l-5), rheumatoid arthritis (l), inflammatory bowel disease (6,7), chronic active hepatitis (8), multiple sclerosis (MS) (9), and several other diseases of obscure etiology. One common factor underlying many of these disease entities is their possible induction by latent viral infection. The presence of LCA and its relation to cellular immunity and disease activity has been studied best in SLE (4,10-16) where lymphocytotoxic antibodies have been found in a high percentage of patients. In some of these studies their presence has correlated well with clinical and laboratory parameters of SLE disease activity. Antibodies to viral agents have been shown to
i Supported in part by National Multiple Sclerosis Society Grant No. RG-812-B-2 and National Institutes of Health Grant No. NS-10590. * Supported in part by National Institute of Allergy and Infectious Disease Training Grant No. 5-TOl-AI00013-17. Division of Clinical Immunology, University of Colorado Medical Center, Denver, Colorado 80220. 3 Fellow of the Colorado Multiple Sclerosis Society. Present address: Department of Microbiology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115. 4 Present address: Veterans Administration Hospital, 4500 South Lancaster Road, Dallas, Texas 75216. Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
16
SCHOCliEl
b:T AL
cross react with some lymphocytes from patients with SLE (I?. 18). Further evidence suggests an interrelation between a transmittable agent. the presence of the disease, and the occurrence of LCA. This comes from the demonstration of LC.4 in a high percentage of sera from household contacts of the affected individuals ( 19.20). In addition, the presence of LCA has been demonstrated in patients with acute viral infections (21,22) and in normal individuals following immunizations with viral vaccines (23). Multiple sclerosis IS another disease of obscure etiology. perhaps induced by a latent viral agent (24.35). The incidence of LCA is increased in sera from patients with multiple sclerosis (9). but the correlation of the presence of these antibodies with parameters of clinical disease and viral infection has not been examined. This study was designed to determine the incidence of LCA in sera from patients with multiple sclerosis and their siblings. In addition, the correlations of LCA with age at onset, duration, CSF IgG levels. and extent of disease were examined. To evaluate the relationship of LCA to parameters of specific viral infections, levels of LCA were correlated with serum and CSF measles and vaccinia antibody titers. MATERIALS
Patients. Eighty-seven
AND
METHODS
patients with definite multiple sclerosis as defined by the Schumacher Committee criteria (26) were studied. ,411 clinical data. sera. and cerebrospinal fluid (CSF) were collected in 1972. Sera and CSF were stored at -70°C. The clinical information used in thts study was obtained from the records made at the time of data collection. Age at onset of disease and on admission to the study. sera. and cerebrospinal fluid were available on all 87 patients, but full clinical records were available on only 72 patients. All the subjects were divided into three disease categories as described by Fog and Linneman (27). based on the clinical course of their disease: (i) chronic progressive. (ii) relapsing remitting, and (iii) mixed. Extent of disease as measured by disability was quantitated from data in the available clinical records by the Kurtzke method on a scale of severity from 0 to 7. Sera from 34 siblings of patients with multiple sclerosis were also collected in 1972. The places of residence of the patients and siblings were determined from addresses available in the records. Controls. Serum was obtained from 1X patients on the neurological wards at Colorado General Hospital and Denver Veterans Administration Hospital. Diagnoses of these patients included seizure disorder. Huntington’s chorea. head trauma, stroke, and brain tumor. None of these patients had received corticosteroids or immunosuppressive therapy within I month prior to collection of serum. Sera was also obtained from 24 laboratory personnel and volunteers and comprised the normal control group. All sera were stored at --70°C until use. CSF gamma globulin determinution. CSF gamma globulin was measured bv eiectroimmuno-diffusion as described by Schneck and Claman (38). Vucciniu antibodies. Vaccinia antibody titers in the sera and CSF were measured by plaque formation inhibition (29). Measles antibodies. Measles antibody titers m sera and CSF were measured by the hemagglutination inhibition assay (30). Lymphocytotoxic antibodies. LCA were measured by the microdroplet method of Terasaki and McClelland (3 1). The target ceIIs were mononuclear cells obtained
LYMPHOCYTOTOXIC
ANTIBODIES
IN
MULTIPLE
SCLEROSIS
17
by Ficoll-Isopaque gradient separation of 2 cc of heparinized blood from a panel of 20 normal donors. One microliter of the subject’s serum was first incubated with 2 x 103 target cells at 15°C for 30 min in a well of a microtiter plate. Five microliters of normal rabbit serum was then added to each well, and the mixture was incubated for 3 hr at 15°C. The viability of the target cells in each well was then determined by exclusion of the vital dye eosin-Y and the percent killing was recorded. A well with 1 /Al of human serum albumin and 2 x 103 target cells was included as a negative control in each experiment. When the negative control caused 20% or greater killing of target cells, results from the test sera run in parallel were excluded from the study. The serum from a patient with SLE which consistently killed 70 to 100% of target cells on repeated determinations was used as the positive control in all experiments. This serum was stored at -70°C and no diminution of cytotoxicity was observed after repeated freezing and thawing. The final results were expressed as a cytotoxicity index: Cytotoxicity
index =
average % target cell killing average % target cell killing
by test serum
x 100. by positive control serum
A cytotoxicity index of 15 or greater was considered a positive test for the presence of lymphocytotoxic antibodies. The comparison of the cytotoxicity of the test serum to the same positive control in each run rather than the use of the absolute percentage cytotoxicity alone takes into account minor variations of reagents and test conditions which might alter experiments done at different times. In addition, results expressed in this manner provide at least a rough quantitation of LCA in each serum since, in previous studies, percent cytotoxicity of undiluted serum has correlated well with antibody titers determined by serial dilution (31). Statistical analysis. Fisher’s exact method was used to compare the incidence of lymphocytotoxic antibodies in the experimental groups to the control group and the level of significance was derived from standard tables. Standard linear correlations were calculated comparing lymphocytotoxic index of each subject to each variable studied. RESULTS
Incidence of lymphocytotoxic antibodies. Forty of eight-seven (46%) samples of sera from patients with multiple sclerosis had a cytotoxicity index of 15 or greater and were considered positive for lymphocytotoxic antibodies (Fig. 1). This incidence was significant with ap < .OOl when compared to the control groups (Table 1). When the MS patients were separated into three groups according to type of clinical course, no significant difference in the incidence of lymphocytotoxic antibodies was noted among the groups (Table 2). In the sibling group, 6 of 34 (17.6%) sampled had elevated lymphocytotoxic antibody levels (Table 1). This incidence was not significantly greater than that in the control group. Since it has been shown that sera from household contacts of patients with SLE and inflammatory bowel disease have an increased incidence of LCA’s (19,20), the records were examined to determine which siblings shared the same household as the multiple sclerosis patients. Three siblings lived in the same
18
ET AL
SCHOCKET
00 -
70.
60
-
50
-
40
-
30
-
zoI5 10 -
. . . .
:. .. . .:. .. : . 22 %: .f. . .Tt _-_ --*-.? 0:.9%. d .h*
.
l .
A c
FIG. 1. The distribution of cytotoxicity indices within each subject group serum with a cytotoxicity index of 15 or greater is considered positive phocytotoxic antibodies. Fotiy of the eighty-seven patients in the multiple positive for lymphocytotoxic antibodies. This incidence was significantly group with a p value of < ,001. Neither the incidence of positive sera in neurologic group was significantly different from the normal control group.
is plotted on this graph. A for the presence of lymsclerosis group had sera greater than the control the sibling group nor the
house as a patient, and two were positive for LCA. One of these sera had the highest cytotoxicity index (50) of the group. This was an inadequate number of subjects, however, to allow a valid statistical analysis.
Multiple sclerosis Siblings Neurological controls Normal controls
Number of subjects
Number positive for LCA
Positive (5%)
87 34 19 24
40 6 I !
46 17.6 5.3 12.5
LYMPHOCYTOTOXIC
ANTIBODIES
TABLE INCIDENCE
OF
LCA
RELATED
Clinical course
TO TYPE
IN
MULTIPLE
19
SCLEROSIS
2
OF CLINICAL
COURSE
OF MULTIPLE
SCLEROSIS~
Number of pts
Number positive for LCA
Positive (%)
48 13 26 87
22 4 13 40
45.8% 38.5% 50% 46%
Relapsing remitting Chronic progressive Mixed All patients a As described by Fog (26).
In the control groups, 1 of 19 (5.3%) of the neurological controls and 3 of 24 (12.5%) of the normal controls had sera positive for lymphocytotoxic antibodies (Table 1). This incidence in normal controls is similar to that reported in previous studies (4,9,23). The mean cytotoxicity index value and standard error of the mean are shown in Table 3. Due to the wide range of cytotoxicity indices within each group, as shown in Fig. 2, the mean cytotoxicity indices of the MS, sibling, and control groups did not differ significantly from the mean index of the normal group. Correlation of cytotoxicity index levels with variables. A significant correlation, p < .05, between the cytotoxicity index and serum measles antibody titers was found in the MS patients (Fig. 2). There was no significant correlation between cytotoxic indices and any other of the variables studied (Table 4). The correlation between LCA levels and serum measles antibody titers in the sibling group (Y = .0532) was not significant. An elevated mean titer of antibody to measles in sera from these multiple sclerosis patients and their siblings was reported in an earlier study (32). In the same study, elevated CSF vaccinia antibody titers in the multiple sclerosis patients was also demonstrated. DISCUSSION
The results of this study confirm that cold-reactive antibodies cytotoxic for lymphocytes (LCA) are present in a significantly higher proportion of sera from patients with multiple sclerosis as compared to sera from siblings of MS patients, patients with other neurologic diseases, and normal controls. Of all the clinical and serological variables studied, LCA serum levels bore a significant correlation only to serum measles antibody titers in patients with MS. The finding of an increased incidence of LCA in patients with MS is in agreement with an earlier study by Kuwert and Bet-trams (9). These investigators found significant levels of TABLE DISTRIBUTION
MS patients MS sibs Normal controls Neuro controls
OF CYTOTOXICITY
INDICES
3 WITHIN
EACH
GROUP
STUDIED
N
Mean
SE
87 34 24 19
18.66 10.38 11.00 4.63
1.70 2.07 1.74 1.57
20
SCHOCKET
ET AL
I
80.
.
.
70-
. 60 : D c .-," .u x 2 2
50.
40.
. . .
. . . . .
c 30:
. .
O&3' *1632
5
1 64
. .
128
L 512
256
Reciprocal of Anti-measles Antibody Titer FIG. 2. The cytotoxicity index is plotted against the reciprocal of the antimeasles X intercept = 14.6224, and the slope equals the linear regression coeffkient significant correlation with a p value of < .05.
antibody titer. of .2 160. This
The is a
LCA in 34% of sera from 462 chronic MS patients as compared to 6% of healthy controls and 12% of neurological controls. The antibody was characterized as a cold-reactive IgM maximally cytotoxic at 15°C with little or no activity at 37°C. The relative lack of antibody activity at normal body temperature casts doubt on the potential effect of this LCA upon lymphocyte function and number in viva. Neither our study nor this earlier study has addressed this question in patients TABLE CORRELATION
OF CYTOTOXICITY
INDEX LEVELS WITH MULTIPLE
4 WITH VARIABLES SCLEROSIS
STUDIES
IN 87 PAI-tee-r-s
p Value Serum measles antibody titer Extent of disease Age at onset CSF yG level CSF vaccinia antibody titer Duration of disease Serum vaccinia antibody titer CSF measles antibody titer
.2160 .‘OlO .I435 .I068 - .0732 .0728 .0033
,044 .062 .I85 .325 .500 .SO? .976 ,994
LYMPHOCYTOTOXIC
ANTIBODIES
IN
MULTIPLE
SCLEROSIS
21
with MS. Previous studies have, however, reported no decrease in total circulating lymphocytes, T cells, or B cells in MS patients (33,34). A serum factor has been described in patients with active MS which inhibits protein synthesis by mitogen-stimulated lymphocytes (35-37). On purification, this factor proved not to be an immunoglobulin and consequently is not related to the LCA found in our patients studied. The present evidence suggests therefore that the LCA found in the MS patients has little if any appreciable effect on lymphocyte number or function. Other disease states characterized by the presence of LCA such as pernicious anemia (38) and inflammatory bowel disease (6,7) are also not characterized by significant reductions in total lymphocytes, T or B cells. On the other hand, LCA are present in a high percentage of patients with SLE, a disease characterized by major deficits in lymphocyte number and function. Some studies have shown correlations between LCA and disease activity in these patients (4,l l), suggesting a pathophysiological role for these antibodies. Horowitz and Cousar (15), however, recently reported that in vitro and in vivo deficits in cell-mediated immunity are related to the presence of an IgG antilymphocyte antibody reactive at 37°C and did not correlate with the presence of cold-reactive, IgM lymphocytotoxic antibodies. At present the only diseases in which a correlation has been shown between the presence of this type of LCA and lymphopenia are acute viral infections such as infectious mononucleosis, measles, and rubella (21). It is more likely, however, that this lymphopenia is due to the cytopathic effect of the virus rather than LCA. Since there is little evidence to support the role of LCA as a modulator of lymphocyte function, a second possibility is that LCA may participate in the pathophysiology of the CNS lesion in MS. Evidence from animal experiments as well as human studies using cross absorption techniques has indicated that antigens derived from brain tissue cross-react with antigens on the lymphocyte cell surface (39-42). In the mouse the cross-reacting lymphocyte surface antigen is almost exclusively the theta antigen. Despite earlier studies in man suggesting a similar specific cross-reactivity between brain antigen and the T cell, a recent paper has indicated that in patients with SLE, at least, no such specificity with a particular subset of lymphocytes can be demonstrated (43). In earlier studies antibodies to a specific constituent of brain, myelin, have been found in some patients with multiple sclerosis (44-45). We are not aware of studies examining the reactivity of these antibodies with lymphocyte cell surface. In addition, on a clinical basis there is little evidence in the present study of MS patients linking the presence of LCA with duration of disease or brain dysfunction as measured by extent of disease. Consequently, it is unlikely that these LCA are primarily involved in the pathophysiology of the CNS lesion. A third, and perhaps more plausible, explanation for the presence of LCA in MS patients is the possibility that these antibodies represent markers of latent or persistent viral infection. LCA are present with an increased frequency in patients with acute viral infections such as infectious mononucleosis, measles, and rubella (21,22). In addition, elevated levels of LCA have been demonstrated in normal individuals following immunizations with viral vaccines (23). Further evidence
22
SCHOCKE?‘
E7 AL
relating the occurrence of LCA to a transmittable agent comes from studies of household contacts of patients with SLE and inflammatory bowel disease (19.20). LCA are present in a significantly higher number of sera from both related and nonrelated residents of the households of affected individuals as compared to relatives living in other homes. In reference to multiple sclerosis. support for a viral etiology of this disease has been expounded in the literature for many years (24,25). Elevated antibody levels to various viral agents, especially measles, have been reported in these patients (24), and this finding of increased serum antibody titers has been confirmed in the present group of multiple sclerosis patients. In addition, these antibody levels correlated significantly @ < .05) with serum LCA levels, suggesting an association between LCA and measles infection. Further evidence similar to that found in SLE and inflammatory bowel disease household contacts linking LCA with a presumed infectious agent was sought in the siblings of our patients. Unfortunately, only three of these siblings shared the same household as their relative with the disease. The presence of LCA in two of three of these household contacts only suggests that this is a similar phenomenon to that occurring with contacts of SLE and inflammatory bowel disease patients. This is another clue relating LCA in MS to a presumed transmittable agent. Further study of this relationship is in progress. Finally. further evidence which might elucidate the relationship between LCA and viruses comes from recent studies suggesting that lymphocytes have specific receptors for virus particles and that antibodies raised against a specific virus can react with a small proportion of peripheral blood lymphocytes from SLE patients. First, EB virus receptors have been demonstrated on B lymphocytes (46), and recently the presence of receptors specific for measles virus have been shown on normal human T lymphocytes (47). Secondly, the observation was made by Shwartz that antibodies raised against a C-type virus stained a small percentage of the perhipheral blood lymphocytes from SLE patients (17.18). This finding suggests that there are either viral antigens or a lymphocyte component which cross-reacts with the viral antigen present on the cell surface. One might, therefore, hypothesize that a viral agent bound to a lymphocyte surface receptor serves as a hapten with the cell membrane functioning as the carrier. An antibody induced to this complex might subsequently react with either the viral determinant or normal lymphocyte cell surface. We have provided suggestive evidence that this type of antigen interaction might be responsible for the presence of LCA found in MS and the other disease entities previously associated with LCA mentioned. ACKNOWLEDGMENTS We wish to thank Patterson for typing
Alan M. Suzuki the manuscript.
for his technical
assistance
in preparing
the statistics
and Susan
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