Clin. exp. Immunol. (1976) 24, 407-414.
Measles antibodies and autoantibodies in autoimmune disorders D. R. TRIGER, T. R. GAMLEN, E. PARASKEVAS, R. S. LLOYD & RALPH WRIGHT Department of Medicine, Southampton University Hospitals, Southampton
(Received 23 October 1975) SUMMARY
Measles CF antibodies have been examined in the sera of patients with a variety of clinical disorders associated with the production of autoantibodies. Previous reports of high-titre reactions in DLE and chronic active hepatitis have been confirmed, the titres in the latter disorder being particularly elevated. Mean antibody titres to measles in patients with rheumatoid arthritis were significantly lower than in matched controls, and an inverse correlation between measles antibody levels and serum globulin levels was found. Measles antibody titres in patients with myasthenia gravis and primary biliary cirrhosis did not differ significantly from those found in controls. However, subdivision of patients with rheumatoid arthritis, myasthenia gravis and primary biliary cirrhosis showed that the presence of anti-nuclear antibody (ANA) was associated with significantly increased measles antibody levels compared with the ANA-negative sera. The presence of gastric parietal cell antibody or thyroid microsomal antibody did not appear to be associated with increased measles antibody levels, whether or not they occurred in association with previous anaemia or thyroid disease. Possible explanations for these findings in terms of immune complex formation and immune hyper-reactivity are discussed.
INTRODUCTION Elevated measles antibody titres have been reported in a number of disease states, notably in DLE (Phillips & Christian, 1970; Hollinger et al., 1971; Hurd et al., 1972; Kalliomaki & Halonen, 1972; Laitinen & Vaheri, 1974) and chronic active hepatitis (Triger et al., 1972b; Closs et al., 1973; Laitinen & Vaheri, 1974). Both conditions are commonly associated with the presence of serum autoantibodies, and a significant correlation between high measles CF titres and strongly positive anti-nuclear and antismooth muscle antibodies has been demonstrated in chronic active hepatitis (Triger, Kurtz & Wright, 1974). Lucas et al. (1972) have shown that there is an increased incidence of high-titre measles haemagglutinating antibodies in the sera of patients with circulating autoantibodies, but they did not attempt to correlate these findings with clinical disorders. The purpose of this paper is to examine complement-fixing antibodies to measles antigen in a number of diseases commonly associated with the presence of autoantibodies in order to see whether the association between elevated measles antibody titres and autoantibodies previously reported in chronic active hepatitis applies to other clinical disorders.
METHODS Patients studied. Rheumatoid arthritis (fifty-five patients). All cases were either 'classical' or 'definite', according to the criteria of the American Rheumatology Association (McEwen, 1972). Most patients were ambulant out-patients, although a few were in-patients. Patients with evidence of complicating disorders (e.g. amyloidosis) were excluded. Nine patients were receiving corticosteroids at the time the serum was taken; of the remainder, all except five were taking some analgesic. These included indomethacin (twenty-three patients), phenylbutazone (seven), salicylates (six), ibufenamic acid (six), Correspondence: Dr D. R. Triger, Professorial Medical Unit, Royal South Hants Hospital, Southampton S09 4PE.
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benoral (five), gold (four), paracetamol (four), and a miscellaneous collection of drugs (four). No patients were receiving immunosuppressive drugs. DLE (thirty-four patients). All patients satisfied the criteria of the American Rheumatology Association (Cohen et al., 1971). Four patients were receiving corticosteroids, but none was on immunosuppressive agents. Chronic active hepatitis (thirty-four patients). These were defined by the criteria previously reported (Triger et al., 1974). Primary biliary cirrhosis (thirty-one patients). The diagnosis was based on the finding of a positive mitochondrial antibody together with compatible clinical and histological features (Klatskin & Kantor, 1972). Alcoholic cirrhosis (twenty-eight patients). All admitted to a history of heavy alcohol intake and had liver biopsy evidence of cirrhosis. Eight patients had biopsy evidence of alcoholic hepatitis in addition to cirrhosis. Anti-nuclear antibodies were present in the serum of four patients and anti-smooth muscle antibodies in five others. Myasthenia gravis (twenty-seven patients). These patients formed part of a previously described survey (Wright & Kerr, 1967). Thyroid disease. Twenty-two patients with clinical and biochemical evidence of thyroid disease (eleven patients with thyrotoxicosis and eleven with myxoedema) together with thyroid microsomal antibodies demonstrable in the serum were included. Pernicious anaemia. Twenty-one patients with pernicious anaemia and gastric parietal cell antibodies, in whom the diagnosis was based on the finding of low serum B12, megaloblastic bone marrow and response to intramuscular B12, were studied. Miscellaneous autoantibody positive sera. A miscellaneous group of patients whose sera contained autoantibodies but who did not have overt autoimmune disease-in particular any evidence of the disorders listed above. These included twenty-four patients with anti-nuclear antibodies, twenty-two with anti-smooth muscle antibodies, twenty-one with thyroid microsomal antibodies, and twenty-eight with gastric parietal cell antibodies. There were insufficient patients with positive anti-mitochondrial antibodies but no evidence of liver disease to permit analysis. Controls. Serum from control subjects was obtained from two sources. (1) Healthy controls-healthy laboratory staff and relatives of hospital patients with no detectable autoantibodies in the serum. (2) Hospital controls-hospital in-patients suffering from a variety of disorders not obviously due to autoimmune mechanisms (e.g. drug overdoses, cerebro- and cardiovascular degenerative diseases). These included patients both with and without serum autoantibodies. For comparison with each group mentioned above, a sample of age- and sex-matched controls were used. All sera were coded and stored at - 20'C until tested. Autoantibodies. Autoantibodies were detected by immunofluorescence (IF), using modifications of the technique of Roitt & Doniach (1969). Sera were diluted 1:10 in phosphate-buffered saline, pH 7-6 (PBS). Fluorescein-conjugated antihuman immunoglobulin, anti-human IgG and IgM were obtained from Wellcome Reagents Ltd. The polyvalent antiserum was used at a dilution of 1: 60, and the anti-IgG and anti-IgM antisera were diluted 1:30 and 1: 15 respectively. The preparations were examined using a Zeiss Standard Universal microscope, an HBO 200 lamp and a IIIRS epi-condenser fitted with standard Zeiss FITC exciting and barrier filters. Anti-nuclear antibody titres were recorded as the reciprocal of the highest dilution of the patient's serum which gave a weakly positive reaction. Rheumatoid factor was quantitated by the sheep cell agglutination test (Ball, 1952) and titres less than 1:16 were considered negative. Serum complement (C3) was measured by radial immunodiffusion, using a commercial kit (Immunoplate C3, Hyland). Measles antibody titres. Complement-fixing antibodies were measured by the method of Bradstreet & Taylor (1962) with a micro-technique in which 0 025 ml dilutions of serum were used. Microtitre plates (U well, Cooke Engineering Company, Alexandria, Virginia) were used throughout. Measles antigen was supplied by the Standards Laboratory for Serological Reagents, Colindale, London. Statistical analysis. The statistical significance of the difference in measles antibody titres between groups of patients was calculated using the Mann-Whitney ranking test (Seigel, 1956). The significance of the number of high-titre reactions recorded in different groups was calculated using the X2 test. Titres of less than 1: 2 were counted as 0.
RESULTS Table 1 compares the antibody titres of healthy controls with those found in hospital in-patients suffering from a variety of non-immunological disorders. There is a tendency for healthy controls to show higher antibody titres, although this does not reach the 5%/0 significance level. The antibody titres themselves have been expressed either as the mean log2 titre or as the incidence of high-titre reactions occurring in a group of patients. Antibody titres equal to or greater than 1:128 were arbitrarily defined as high titres.
Rheumatoid arthritis The measles CF titres in patients with rheumatoid arthritis are shown in Table 2 as compared with
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Measles antibodies and autoantibodies TABLE 1. Measles CF titres in control subjects
Healthy controls Hospital controls
< 1:2
1:2-1:4
1:8-1:16
1:32-1:64
4 18
13 10
29 10
18 24
*
Mean 3 1:128 Total titre log2
4 6
68 68
3-43* 2-96
P=0 1 (n.s.).
age- and sex-matched controls. The mean titre in the arthritis patients was significantly lower than in both the healthy control group and the matched in-patient group. Fifty-two per cent of patients with rheumatoid arthritis show no detectable measles antibody, compared with 4.6% of the healthy controls and 26&1% of the in-patient controls. The group with rheumatoid arthritis comprised patients both with and without anti-nuclear antibodies, in contrast to the control group in which subjects with circulating autoantibodies were specifically excluded. Subdivision of these results according to the presence and class of anti-nuclear antibody (ANA) shows that the mean antibody titre in patients with IgG class positive ANA (log2 4.12) was significantly higher than in the ANA-negative patients (log2 1.03). The TABLE 2. Measles CF titres in rheumatoid arthritis and myasthenia gravis
Rheumatoid arthritis (all patients) Healthy controls Hospital controls Rheumatoid arthritis
Mean 31:128 Total titrelog2
< 1:2
1:2-1:4
1:8-1:16
1:32-1:64
34
8
8
9
6
65
2.05*
3 17 24
13 9 4
28 11 5
17 23 2
4 5 1
65 65 36
3-46 2-96 1-03
4
1
3
4
5
17
4 12t
5
3
1
3
0
12
2-05
0 1 0 0 0
5 6 3 2 3
16 12 12 4 9
4 6 0 4 3
2 2 0 2 1
27 27 15 12 16
3-73 3-51 3-13 4 50+ 3-75
0
2
7
1
1
11
3-91
(ANA - ve) Rheumatoid arthritis (ANA +ve IgG) Rheumatoid arthritis (ANA +ve IgM) Myasthenia gravis Healthy controls Myasthenia ANA- ve Myasthenia ANA +ve Myasthenia skel. muscle antibody - ve Myasthenia skel. muscle antibody +ve
P< 0 001 (v. healthy controls); < 0 03 (v. hospital controls). t P< 0 005 (v. ANA - ye RA patients). t P< 0 01 (v. ANA - ve myasthenia patients). *
mean antibody titre in the IgM ANA-positive sera was log2 2-05. Furthermore, five of the seventeen IgG ANA-positive showed high measles CF titres, compared with only one of the remaining forty-eight sera. The presence of rheumatoid factor has been shown to influence the results of complement-fixation tests (Smiley & Casey, 1969; Haukenes, 1974). Only eight sera in this group were rheumatoid factornegative. The mean measles CF titre in these sera was slightly higher than in the sero-positive group (log2 2-50 v. log2 1.75), but this difference was not statistically significant. A possible correlation between measles antibody titre and serum globulin levels was sought. The results of patients with the lowest
D. R. Triger et al.
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serum globulins (2-3-3.4 g0%) were compared with those found in the patients with the highest globulin levels (3 5-5-0 g). It was found that the former group had significantly higher measles CF titres than the latter (log2 369 v. log2 1-33; P< 0.05), thus suggesting an inverse correlation. The mean serum complement level in the two groups was the same (150 mg%O and 152 mg%0 respectively). Mean serum globulin levels in the sera of patients with anti-nuclear antibodies were lower than those found in patients without ANA (3.27 v. 3-78 g ), but this difference was not statistically significant. The number of patients taking specific analgesics was in most cases too small to permit examination of the correlation between measles titre and drug. The mean measles antibody titre of the twenty-three patients on indomethacin (log2 2 13) did not differ significantly from the mean antibody titre of the other patients with rheumatoid arthritis (log2 2 05). Nine patients with rheumatoid arthritis were receiving corticosteroid therapy. In seven cases no measles antibody was detectable, one showed a titre of 1:2, and one a titre of 1:8. TABLE 3. Measles CF titres in DLE, chronic active hepatitis, alcoholic cirrhosis and primary biliary cirrhosis (PBC)
< 1:2
9 DLE 3 Healthy controls 1 Chronic active hepatitis 2 Healthy controls 2 Alcoholic cirrhosis 2 Healthy controls 9 Primary biliary cirrhosis 3 Healthy controls PBC patients Mitochondrial antibody 8 alone Mitochondrial antibody and smooth muscle anti- I body or antinuclear antiJ body
Mean 31:128 Total titre log2
1:2-1:4
1:8-1:16
1:32-1:64
3 10 0 7 5 8 4 5
10 11 4 11
lot 3 27* 3 3 2 5
13
5 10 1 10 11 7 7 8
4
5
0
1
7 9 6
3 92 3.59 8-73*
2
37 37 33 33 28 28 31 31
3
1
21
2-38
4
4
10
6-10t
3-61 4-18 3 52 3 58 3.46
* P< 0-001 (v. healthy controls). t P< 0 005 (v. healthy controls). t P< 0 005 (v. mitochondrial antibody alone).
DLE Table 3 shows the measles antibody titres in the patients with DLE. The mean titre is higher than in a comparable group of healthy controls, although by the Mann-Whitney test this difference is not significant, due to the large number of patients with very low titres. A much larger number of patients (ten out of thirty-seven) showed high titre measles antibody reactions than was found in the control group, although titres of greater than 1:1000 (log2 10) were found in only two patients. Anti-nuclear antibodies were present in the sera of all patients. These antibodies were of IgG class in all except three cases. Three sera containing IgM class antibody alone had measles titres of 1:4096, 0 and 0. The measles antibody titre in the nineteen sera showing low titres of anti-nuclear antibodies (less than 1:320) was no different from the mean titre in the fifteen sera with high ANA titres (log2 3-82 v. log2 3.83). The measles titres in the patients receiving corticosteroids were not significantly different from those found in patients not on steroids. The mean measles CF titre in those patients with lower serum globulin levels (2.0-3.4 g0%) was higher than that in patients with high globulin levels (3.5-4A6 g0%) (log2 5.5 v. log2 4.0), but this difference was not statistically significant.
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Chronic active hepatitis The high measles antibody titres described previously have been confirmed by this larger series (Table 3). All sera with low measles titres (less than 1:128) contained low titre anti-nuclear or antismooth muscle antibodies. Most of the measles CF titres were much higher than were found in the sera of patients with DLE-seventeen being greater than 1:1000. Alcoholic cirrhosis The titres of measles antibody in patients with alcoholic cirrhosis is shown in Table 3. The mean titre is somewhat higher than in age- and sex-matched controls, although this difference is not statistically significant. There was no increase in the number of high-titre reactions in this group. The mean titre of the nine patients with anti-nuclear or anti-smooth muscle antibodies did not differ significantly from those without autoantibodies (log2 4-22 v. log2 4.16). Antibody titres in the patients with alcoholic hepatitis were similar to those in the remainder.
Primary biliary cirrhosis Table 3 shows that the mean titre of measles antibodies in patients with positive mitochondrial antibodies was similar to that found in age- and sex-matched healthy controls. Ten of these sera also contained either anti-nuclear (seven), anti-smooth muscle (two) or both (one) autoantibodies-this group had significantly higher measles antibody titres compared with the remainder. Myasthenia gravis Measles antibody titres in myasthenia gravis are compared with healthy controls in Table 2, which shows no significant difference between the two groups. Sera containing antinuclear antibodies show significantly higher antibody titres to measles compared with sera containing no anti-nuclear antibodies. The presence of skeletal muscle antibody or rheumatoid factor makes no significant difference to the measles antibody titre.
Autoantibodies Table 4 shows the measles antibody titres in the sera of patients containing anti-nuclear, anti-smooth TABLE 4. Measles CF titres and autoantibodies (excluding DLE, chronic active hepatitis, rheumatoid arthritis and myasthenia gravis) Mean No. 3 1:128 titre log2
Antinuclear antibody Healthy controls Hospital controls Anti-smooth muscle antibody Healthy controls Hospital controls Gastric parietal cell antibody Pernicious anaemia No pernicious anaemia Healthy controls Hospital controls Thyroid microsomal antibody Thyroid disease No thyroid disease Healthy controls Hospital controls
24 24 24 22 22 22
1 2 1 3 2 1
3 50 3-75 3-05 3-36 3-75 3 06
21 28 25 25
4 2 2 2
3-86 2-86 3-58 3 06
22 21 22 22
3 1 2 1
2-94 3-10 3-76 2-98
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muscle, anti-gastric parietal cell and anti-thyroid microsomal antibodies. Patients with DLE, chronic active hepatitis, rheumatoid arthritis and myasthenia gravis were excluded from these groups. Some of the sera contained more than one auto-antibody, but there was no significant difference in the measles antibody titres in the sera containing ANA, gastric parietal cell antibody or thyroid microsomal antibody alone, compared with sera containing more than one autoantibody. DISCUSSION These results confirm the previous observations of elevated measles antibody titres in chronic active hepatitis, and show that the antibody levels in this condition are very much higher than is found in other putative autoimmune disorders. These titres are higher and persist for longer than in acute measles infections (Triger et al., 1972b; Laitinen & Vaheri, 1974), and only in SSPE are comparable titres observed. The elevation in antibody titres does not appear to be solely related to liver damage, since normal measles titres are found in patients with alcoholic cirrhosis, including patients with florid alcoholic hepatitis. High measles CF titres are also found in some patients with primary biliary cirrhosis associated with anti-nuclear or anti-smooth muscle antibodies, but this may reflect a continuous spectrum between this condition and chronic active hepatitis. We have also confirmed the previous reports of elevated measles antibody levels in DLE. Our results, however, in contrast to earlier reports (Phillips & Christian, 1970; Hollinger et al., 1971) suggest that this is due to the presence of an increased number of high-titre reactions, rather than an increase in geometric mean titre. Our results both in this condition and in rheumatoid arthritis do not show a parametric distribution of antibody titres, and so statistical analysis using the Mann-Whitney test is more appropriate than the Student's t-test. In patients with rheumatoid arthritis, the mean measles antibody titres were significantly lower than in either of the control groups. This differs from the findings of Phillips & Christian (1970) and Hollinger et al. (1971), but their reports are not strictly comparable, due to differences in technique and presentation of results. Kalliomaki & Halonen (1972) reported higher CF titres in patients with rheumatoid arthritis than in controls, but the authors conceded that some of their patients with rheumatoid arthritis may have had DLE. The inverse correlation between measles CF titres and serum globulin levels in patients with rheumatoid arthritis contrasts with our failure to observe any relation between these two indices in patients with DLE. Other workers (Rothfield, Evans & Niederman, 1973; Hurd et al., 1972) have also found no correlation between measles titres and serum globulin or immunoglobulin levels, although Phillips & Christian (1972) observed a direct relationship between measles antibody level and gammaglobulin in sera of patients with DLE and other diseases. The reason for our unexpected observations is uncertain, but it is possible that immune complex formation may play a role. For example, sera in which high gammaglobulin levels are found may contain large numbers of such complexes, which may bind free measles antibody. This is consistent with the finding of Kalliomaki, Halonen & Salmi (1975) that antibody levels to measles in serum and synovial fluid are lower in rheumatoid arthritis patients without total haemolytic complement in the synovial fluid than in the rheumatoid arthritis patients with haemolytic complement. Our inability to find a difference in serum complement levels between the two groups does not invalidate the argument, since serum complement levels may be affected by factors other than immune complex consumption. Nine patients with rheumatoid arthritis were receiving corticosteroids at the time serum was taken, and all showed very low antibody titres to measles. There is no evidence that steroids influence the measles antibody levels in DLE (Phillips & Christian, 1972), and the low titres observed here may reflect the fact that the arthritis was of sufficient clinical severity to warrant the use of steroids. No correlation could be established between measles titres and any other drug, but, with the exception of indomethacin, the number of patients taking other drugs was too small for valid conclusions to be drawn. Subdivisions of the measles titres in our patients with rheumatoid arthritis according to the presence or
Measles antibodies and autoantibodies
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absence of anti-nuclear antibodies (ANA) showed that the mean titre in the sera possessing ANA was significantly higher than in the ANA-negative group, and that five of the six high-titre sera were ANApositive. Since high measles CF titres are known to occur in the sera of patients with DLE (which is almost invariably associated with ANA), it is important to be certain that the patients with rheumatoid arthritis and ANA do not have DLE. This seems unlikely, because: (a) none of the patients with rheumatoid arthritis satisfied the recognized criteria for DLE; and (b) all six rheumatoid arthritis sera containing high measles antibody titres contained rheumatoid factor (in contrast to only two out of ten of the high measles titre DLE sera). Retrospective analysis of the patients' clinical history assessed by severity or duration of symptoms failed to reveal any significant difference between patients with and without anti-nuclear antibodies. The overall frequency of high-titre reactions, and the geometric mean titre in patients with myasthenia gravis, were similar to those found in controls, but once again division of the patients according to the presence of anti-nuclear antibodies showed two subpopulations. No correlation, however, was found between the measles antibody levels and the presence or absence of skeletal muscle antibody. Both the geometric mean antibody titre and the incidence of high-titre reactions to measles in sera containing anti-nuclear or anti-smooth muscle antibodies are similar to those found in healthy controls if patients with the disorders already described are specifically excluded. However, other conditions may exist in which the presence of autoantibodies may be associated with elevated measles antibody levels, but this study does not include sufficient data from other clinical disorders for appropriate analysis. Similarly, the presence of gastric parietal cell or thyroid microsomal antibodies was not associated with an increased incidence of high-titre measles CF reactions, in contrast to the findings of Lucas et al. (1972) in their study of haemagglutinating antibodies. This may reflect the difference in method, as Salmi (1973) has shown elevated measles HI antibody levels in patients with multiple sclerosis, whereas the CF titres are normal. We did not find any significant difference in geometric mean titre or in the frequency of high-titre reactions in patients with pernicious anaemia or thyroid disease in comparison with either healthy or in-patient control subjects. The finding of high bacterial antibody titres in different forms of liver disease (Triger, Alp & Wright, 1972a) prompted the suggestion that the intact liver was able to sequester exogenous antigens, and damage to this organ resulted in increased amounts of antigen reaching other parts of the reticuloendothelial system with consequent enhanced antibody production. The marked disparity between the measles antibody levels found in chronic active hepatitis, compared with other liver diseases, suggests a different explanation. Liver disease is commonly associated with depressed cell-mediated immunity (Dudley, Fox & Sherlock, 1972), thus raising the possibility that the enhanced B cell response to measles antigen might be compensating for a reduced T-cell response. Our attempts to examine this by measuring leucocyte migration inhibition to measles antigen in patients with DLE and chronic active hepatitis have been unsuccessful and recent reports (Fuccillo et al., 1975) have cast doubt on the validity of this test as a measure of cellular immunity to measles. In conclusion, we have demonstrated the association of anti-nuclear antibodies with increased measles antibody titres in several different clinical disorders. The possibility of cross-reaction has been ruled out by studies in which antibodies to mouse nuclei (Hollinger et al., 1971) and measles antigen (Triger et al., 1974) have been adsorbed out without affecting the titre of the remaining antibodies. The results are consistent with the hypothesis that anti-nuclear antibody may be a marker representing either excessive antigenic stimulation (Christian et al., 1965; Fournie, Lambert & Miescher, 1974), or a hyperreactive response to a normal antigenic stimulus such as measles (Triger & Wright, 1973). The amount of measles antibody formed may also be influenced by the formation of immune complexes, which may contain varying ratios of antigen-antibody. In non-hepatic disorders, such as myasthenia gravis and rheumatoid arthritis, these complexes will be taken up by the liver, although alterations in the ratio of antigen to antigen-antibody may affect the ability of the liver to sequester these complexes (Thomas & Vaez-Zadeh, 1974). The extraordinarily high titres to measles found in chronic active hepatitis may reflect loss of the ability of the liver to sequester these complexes in addition to the other immunological disturbances.
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We thank Mrs Teresa Gregory, Miss Celia Lang, Mrs Lindsay Scott-Morgan and Mr Frank Paul for technical assistance. We are grateful to Dr C. M. P. Bradstreet (Central Public Health Laboratory, Colindale) for kindly providing measles CF antigen. T.R.G. was in receipt of an M.R.C. Research Fellowship. This work was supported by grants from the Medical Research Council and the Nuffield Foundation. REFERENCES BALL, J. (1952) Sheep cell agglutination test for rheumatoid arthritis: clinicopathological study. Ann. rheum. Dis. 11, 97. BRADSTREET, C.M.P. & TAYLOR, C.E.D. (1962) Technique of complement fixation test applicable to the diagnosis of virus diseases. Month. Bull. Min. Health and P.H.L.S.
21,96. CHRISTIAN, C.L., ABRUZZO, J.L., DE SIMONE, A.R. & HowES, E.L. (1965) Studies of hyperimmunization. Ann. N.Y. Acad. Sci. 124, 143. CLOSS, O., HAUKENES, G., BLOMHOFF, J.P. & GJoNE, E. (1973) High titres of antibodies against rubella and morbilli virus in patients with chronic hepatitis. Scand. J. Gastroenterol. 8, 523. COHEN, A.S., REYNOLDS, W.E., FRANKLIN, E.C., KULKA, J.P., ROPES, M.W., SHULMAN, I.E. & WALLACE, S.L. (1971) Preliminary criteria for the diagnosis of systemic Jupus erythematosus. Bull. rheum. Dis. 21, 643. DUDLEY, F.J., Fox, R.A. & SHERLOCK, S. (1972) Cellular immunity and hepatitis associated Australia antigen liver disease. Lancet, i, 723. FOURNIt, G.J., LAMBERT, P.H. & MIESCHER, P.A. (1974) Release of D.N.A. in circulating blood and induction of anti-D.N.A. antibodies after injection of bacterial lipopolysaccharides. J. exp. Med. 140, 1189. FuccILLO, D.A., ABELA, J.E., TRAUB, R.G., GILLESPIE, M.M., BEADLE, E.L. & SEVER, J.L. (1975) Cellular immunity in multiple sclerosis. Lancet, i, 980. HAUKENES, G. (1974) Negative complement fixation tests with rheumatoid factor positive sera. Ann. rheum. Dis. 33,
461. HOLLINGER, F.B., SHARP, J.T., LIDSKY, M.D. & RAWLS, W.E. (1971) Antibodies to viral antigens in systemic lupus erythematosus. Arthr. Rheum. 14, 1. HURD, E.R., DowDLE, W., CASEY, H. & ZIFF, M. (1972) Virus antibody levels in S.L.E. Arthr. Rheum. 15, 267. KALLIOMAKI, J.L. & HALONEN, P. (1972) Antibody levels to parainfluenza, herpes simplex, varicella-zoster, cytomegalo virus and measles virus in patient with connective tissue diseases. Ann. rheum. Dis. 31, 192. KALLIOMAKI, J.L., HALONEN, P. & SALMI, M. (1975) Virus antibodies in serum and synovial fluid of patients with rheumatoid arthritis and other connective tissue disorders. Ann. rheum. Dis. 34, 43. KLATSKIN, G. & KANTOR, F.S. (1972) Mitochondrial antibody in primary biliary cirrhosis and other disorders. Ann. intern. Med. 77, 533. LAITINEN, 0. & VAHERI, A. (1974) Very high measles and
rubella virus antibody titres associated with hepatitis, systemic lupus erythematosus, and infectious mononucleosis. Lancet, i, 194. LucAs, C.J., BROUWER, R., FELTKAMP, T.E.W., TEN VEEN, J.H. & VAN LOGHEM, J.J. (1972) Measles antibodies in serum from patients with autoimmune disease. Lancet, i, 115. McEwEN, C. (1972) The diagnosis and differential diagnosis of rheumatoid arthritis. Arthritis and Allied Conditions (ed. by J. C. Hollander and D. J. McCarthy), 8th edn, p. 403. Lea & Febiger, Philadelphia. PHILLIPS, P.E. & CHRISTIAN, C.L. (1970) Myxovirus antibody increases in human connective tissue disease. Science, 168, 982. PHILLIPS, P.E. & CHRISTIAN, C.L. (1972) The influence of serum immunoglobulin concentration on measles antibody level. Proc. Soc. exp. Biol. (N.Y.), 140, 1340. RoITT, I.M., & DONIACH, D. (1969) Immunofluorescent tests for the detection of antibodies. Immunology Techniques, p. 1. W.H.O., Geneva. ROTHFIELD, N.F., EvANs, A.S. & NIEDERMAN, J.C. (1972) Clinical and laboratory aspects of raised virus antibody titres in systemic lupus erythematosus. Ann. rheum. Dis. 32, 238. SALMI, M. (1973) Virus antibodies in patients with multiple sclerosis. Ann. clin. Res. 5, 319. SIEGEL, S (1956) Non-parametric Statistics, p. 116. McGrawHill, New York. SMILEY, J.D. & CASEY, H.L. (1969) Decreased C.F. antibodies in sera and decreased lymphocyte transformation to herpes simplex in patients with rheumatoid arthritis. Arthr. Rheum. 12, 698. THOMAS, H.C. & VAEZ-ZADEH, F. (1974) A homeostatic mechanism for the removal of antigen from the portal circulation. Immunology, 26, 375. TRIGER, D.R., ALP, M.H. & WRIGHT, R. (1972a) Bacterial and dietary antibodies in liver disease. Lancet, i, 60. TRIGER, D.R., KURTZ, J.B., MACCALLUM, F.O. & WRIGHT, R. (1972b) Raised antibody titres to measles and rubella viruses in chronic active hepatitis. Lancet, i, 665. TRIGER, D.R., KURTZ, J.B. & WRIGHT, D. (1974) Viral antibodies and autoantibodies in chronic liver disease. Gut, 15, 94. TRIGER, D.R. & WRIGHT, R. (1973) Hyperglobulinaemia in liver disease. Lancet, i, 1494. WRIGHT, R. & KERR, J.H. (1967) Autoimmunity in myasthenia gravis: a family study. Clin. exp. Immunol. 2, 93.
Measles antibodies and autoantibodies in autoimmune disorders D. R. TRIGER, T. R. GAMLEN, E. PARASKEVAS, R. S. LLOYD & RALPH WRIGHT Department of Medicine, Southampton University Hospitals, Southampton
(Received 23 October 1975) SUMMARY
Measles CF antibodies have been examined in the sera of patients with a variety of clinical disorders associated with the production of autoantibodies. Previous reports of high-titre reactions in DLE and chronic active hepatitis have been confirmed, the titres in the latter disorder being particularly elevated. Mean antibody titres to measles in patients with rheumatoid arthritis were significantly lower than in matched controls, and an inverse correlation between measles antibody levels and serum globulin levels was found. Measles antibody titres in patients with myasthenia gravis and primary biliary cirrhosis did not differ significantly from those found in controls. However, subdivision of patients with rheumatoid arthritis, myasthenia gravis and primary biliary cirrhosis showed that the presence of anti-nuclear antibody (ANA) was associated with significantly increased measles antibody levels compared with the ANA-negative sera. The presence of gastric parietal cell antibody or thyroid microsomal antibody did not appear to be associated with increased measles antibody levels, whether or not they occurred in association with previous anaemia or thyroid disease. Possible explanations for these findings in terms of immune complex formation and immune hyper-reactivity are discussed.
INTRODUCTION Elevated measles antibody titres have been reported in a number of disease states, notably in DLE (Phillips & Christian, 1970; Hollinger et al., 1971; Hurd et al., 1972; Kalliomaki & Halonen, 1972; Laitinen & Vaheri, 1974) and chronic active hepatitis (Triger et al., 1972b; Closs et al., 1973; Laitinen & Vaheri, 1974). Both conditions are commonly associated with the presence of serum autoantibodies, and a significant correlation between high measles CF titres and strongly positive anti-nuclear and antismooth muscle antibodies has been demonstrated in chronic active hepatitis (Triger, Kurtz & Wright, 1974). Lucas et al. (1972) have shown that there is an increased incidence of high-titre measles haemagglutinating antibodies in the sera of patients with circulating autoantibodies, but they did not attempt to correlate these findings with clinical disorders. The purpose of this paper is to examine complement-fixing antibodies to measles antigen in a number of diseases commonly associated with the presence of autoantibodies in order to see whether the association between elevated measles antibody titres and autoantibodies previously reported in chronic active hepatitis applies to other clinical disorders.
METHODS Patients studied. Rheumatoid arthritis (fifty-five patients). All cases were either 'classical' or 'definite', according to the criteria of the American Rheumatology Association (McEwen, 1972). Most patients were ambulant out-patients, although a few were in-patients. Patients with evidence of complicating disorders (e.g. amyloidosis) were excluded. Nine patients were receiving corticosteroids at the time the serum was taken; of the remainder, all except five were taking some analgesic. These included indomethacin (twenty-three patients), phenylbutazone (seven), salicylates (six), ibufenamic acid (six), Correspondence: Dr D. R. Triger, Professorial Medical Unit, Royal South Hants Hospital, Southampton S09 4PE.
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benoral (five), gold (four), paracetamol (four), and a miscellaneous collection of drugs (four). No patients were receiving immunosuppressive drugs. DLE (thirty-four patients). All patients satisfied the criteria of the American Rheumatology Association (Cohen et al., 1971). Four patients were receiving corticosteroids, but none was on immunosuppressive agents. Chronic active hepatitis (thirty-four patients). These were defined by the criteria previously reported (Triger et al., 1974). Primary biliary cirrhosis (thirty-one patients). The diagnosis was based on the finding of a positive mitochondrial antibody together with compatible clinical and histological features (Klatskin & Kantor, 1972). Alcoholic cirrhosis (twenty-eight patients). All admitted to a history of heavy alcohol intake and had liver biopsy evidence of cirrhosis. Eight patients had biopsy evidence of alcoholic hepatitis in addition to cirrhosis. Anti-nuclear antibodies were present in the serum of four patients and anti-smooth muscle antibodies in five others. Myasthenia gravis (twenty-seven patients). These patients formed part of a previously described survey (Wright & Kerr, 1967). Thyroid disease. Twenty-two patients with clinical and biochemical evidence of thyroid disease (eleven patients with thyrotoxicosis and eleven with myxoedema) together with thyroid microsomal antibodies demonstrable in the serum were included. Pernicious anaemia. Twenty-one patients with pernicious anaemia and gastric parietal cell antibodies, in whom the diagnosis was based on the finding of low serum B12, megaloblastic bone marrow and response to intramuscular B12, were studied. Miscellaneous autoantibody positive sera. A miscellaneous group of patients whose sera contained autoantibodies but who did not have overt autoimmune disease-in particular any evidence of the disorders listed above. These included twenty-four patients with anti-nuclear antibodies, twenty-two with anti-smooth muscle antibodies, twenty-one with thyroid microsomal antibodies, and twenty-eight with gastric parietal cell antibodies. There were insufficient patients with positive anti-mitochondrial antibodies but no evidence of liver disease to permit analysis. Controls. Serum from control subjects was obtained from two sources. (1) Healthy controls-healthy laboratory staff and relatives of hospital patients with no detectable autoantibodies in the serum. (2) Hospital controls-hospital in-patients suffering from a variety of disorders not obviously due to autoimmune mechanisms (e.g. drug overdoses, cerebro- and cardiovascular degenerative diseases). These included patients both with and without serum autoantibodies. For comparison with each group mentioned above, a sample of age- and sex-matched controls were used. All sera were coded and stored at - 20'C until tested. Autoantibodies. Autoantibodies were detected by immunofluorescence (IF), using modifications of the technique of Roitt & Doniach (1969). Sera were diluted 1:10 in phosphate-buffered saline, pH 7-6 (PBS). Fluorescein-conjugated antihuman immunoglobulin, anti-human IgG and IgM were obtained from Wellcome Reagents Ltd. The polyvalent antiserum was used at a dilution of 1: 60, and the anti-IgG and anti-IgM antisera were diluted 1:30 and 1: 15 respectively. The preparations were examined using a Zeiss Standard Universal microscope, an HBO 200 lamp and a IIIRS epi-condenser fitted with standard Zeiss FITC exciting and barrier filters. Anti-nuclear antibody titres were recorded as the reciprocal of the highest dilution of the patient's serum which gave a weakly positive reaction. Rheumatoid factor was quantitated by the sheep cell agglutination test (Ball, 1952) and titres less than 1:16 were considered negative. Serum complement (C3) was measured by radial immunodiffusion, using a commercial kit (Immunoplate C3, Hyland). Measles antibody titres. Complement-fixing antibodies were measured by the method of Bradstreet & Taylor (1962) with a micro-technique in which 0 025 ml dilutions of serum were used. Microtitre plates (U well, Cooke Engineering Company, Alexandria, Virginia) were used throughout. Measles antigen was supplied by the Standards Laboratory for Serological Reagents, Colindale, London. Statistical analysis. The statistical significance of the difference in measles antibody titres between groups of patients was calculated using the Mann-Whitney ranking test (Seigel, 1956). The significance of the number of high-titre reactions recorded in different groups was calculated using the X2 test. Titres of less than 1: 2 were counted as 0.
RESULTS Table 1 compares the antibody titres of healthy controls with those found in hospital in-patients suffering from a variety of non-immunological disorders. There is a tendency for healthy controls to show higher antibody titres, although this does not reach the 5%/0 significance level. The antibody titres themselves have been expressed either as the mean log2 titre or as the incidence of high-titre reactions occurring in a group of patients. Antibody titres equal to or greater than 1:128 were arbitrarily defined as high titres.
Rheumatoid arthritis The measles CF titres in patients with rheumatoid arthritis are shown in Table 2 as compared with
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Measles antibodies and autoantibodies TABLE 1. Measles CF titres in control subjects
Healthy controls Hospital controls
< 1:2
1:2-1:4
1:8-1:16
1:32-1:64
4 18
13 10
29 10
18 24
*
Mean 3 1:128 Total titre log2
4 6
68 68
3-43* 2-96
P=0 1 (n.s.).
age- and sex-matched controls. The mean titre in the arthritis patients was significantly lower than in both the healthy control group and the matched in-patient group. Fifty-two per cent of patients with rheumatoid arthritis show no detectable measles antibody, compared with 4.6% of the healthy controls and 26&1% of the in-patient controls. The group with rheumatoid arthritis comprised patients both with and without anti-nuclear antibodies, in contrast to the control group in which subjects with circulating autoantibodies were specifically excluded. Subdivision of these results according to the presence and class of anti-nuclear antibody (ANA) shows that the mean antibody titre in patients with IgG class positive ANA (log2 4.12) was significantly higher than in the ANA-negative patients (log2 1.03). The TABLE 2. Measles CF titres in rheumatoid arthritis and myasthenia gravis
Rheumatoid arthritis (all patients) Healthy controls Hospital controls Rheumatoid arthritis
Mean 31:128 Total titrelog2
< 1:2
1:2-1:4
1:8-1:16
1:32-1:64
34
8
8
9
6
65
2.05*
3 17 24
13 9 4
28 11 5
17 23 2
4 5 1
65 65 36
3-46 2-96 1-03
4
1
3
4
5
17
4 12t
5
3
1
3
0
12
2-05
0 1 0 0 0
5 6 3 2 3
16 12 12 4 9
4 6 0 4 3
2 2 0 2 1
27 27 15 12 16
3-73 3-51 3-13 4 50+ 3-75
0
2
7
1
1
11
3-91
(ANA - ve) Rheumatoid arthritis (ANA +ve IgG) Rheumatoid arthritis (ANA +ve IgM) Myasthenia gravis Healthy controls Myasthenia ANA- ve Myasthenia ANA +ve Myasthenia skel. muscle antibody - ve Myasthenia skel. muscle antibody +ve
P< 0 001 (v. healthy controls); < 0 03 (v. hospital controls). t P< 0 005 (v. ANA - ye RA patients). t P< 0 01 (v. ANA - ve myasthenia patients). *
mean antibody titre in the IgM ANA-positive sera was log2 2-05. Furthermore, five of the seventeen IgG ANA-positive showed high measles CF titres, compared with only one of the remaining forty-eight sera. The presence of rheumatoid factor has been shown to influence the results of complement-fixation tests (Smiley & Casey, 1969; Haukenes, 1974). Only eight sera in this group were rheumatoid factornegative. The mean measles CF titre in these sera was slightly higher than in the sero-positive group (log2 2-50 v. log2 1.75), but this difference was not statistically significant. A possible correlation between measles antibody titre and serum globulin levels was sought. The results of patients with the lowest
D. R. Triger et al.
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serum globulins (2-3-3.4 g0%) were compared with those found in the patients with the highest globulin levels (3 5-5-0 g). It was found that the former group had significantly higher measles CF titres than the latter (log2 369 v. log2 1-33; P< 0.05), thus suggesting an inverse correlation. The mean serum complement level in the two groups was the same (150 mg%O and 152 mg%0 respectively). Mean serum globulin levels in the sera of patients with anti-nuclear antibodies were lower than those found in patients without ANA (3.27 v. 3-78 g ), but this difference was not statistically significant. The number of patients taking specific analgesics was in most cases too small to permit examination of the correlation between measles titre and drug. The mean measles antibody titre of the twenty-three patients on indomethacin (log2 2 13) did not differ significantly from the mean antibody titre of the other patients with rheumatoid arthritis (log2 2 05). Nine patients with rheumatoid arthritis were receiving corticosteroid therapy. In seven cases no measles antibody was detectable, one showed a titre of 1:2, and one a titre of 1:8. TABLE 3. Measles CF titres in DLE, chronic active hepatitis, alcoholic cirrhosis and primary biliary cirrhosis (PBC)
< 1:2
9 DLE 3 Healthy controls 1 Chronic active hepatitis 2 Healthy controls 2 Alcoholic cirrhosis 2 Healthy controls 9 Primary biliary cirrhosis 3 Healthy controls PBC patients Mitochondrial antibody 8 alone Mitochondrial antibody and smooth muscle anti- I body or antinuclear antiJ body
Mean 31:128 Total titre log2
1:2-1:4
1:8-1:16
1:32-1:64
3 10 0 7 5 8 4 5
10 11 4 11
lot 3 27* 3 3 2 5
13
5 10 1 10 11 7 7 8
4
5
0
1
7 9 6
3 92 3.59 8-73*
2
37 37 33 33 28 28 31 31
3
1
21
2-38
4
4
10
6-10t
3-61 4-18 3 52 3 58 3.46
* P< 0-001 (v. healthy controls). t P< 0 005 (v. healthy controls). t P< 0 005 (v. mitochondrial antibody alone).
DLE Table 3 shows the measles antibody titres in the patients with DLE. The mean titre is higher than in a comparable group of healthy controls, although by the Mann-Whitney test this difference is not significant, due to the large number of patients with very low titres. A much larger number of patients (ten out of thirty-seven) showed high titre measles antibody reactions than was found in the control group, although titres of greater than 1:1000 (log2 10) were found in only two patients. Anti-nuclear antibodies were present in the sera of all patients. These antibodies were of IgG class in all except three cases. Three sera containing IgM class antibody alone had measles titres of 1:4096, 0 and 0. The measles antibody titre in the nineteen sera showing low titres of anti-nuclear antibodies (less than 1:320) was no different from the mean titre in the fifteen sera with high ANA titres (log2 3-82 v. log2 3.83). The measles titres in the patients receiving corticosteroids were not significantly different from those found in patients not on steroids. The mean measles CF titre in those patients with lower serum globulin levels (2.0-3.4 g0%) was higher than that in patients with high globulin levels (3.5-4A6 g0%) (log2 5.5 v. log2 4.0), but this difference was not statistically significant.
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Chronic active hepatitis The high measles antibody titres described previously have been confirmed by this larger series (Table 3). All sera with low measles titres (less than 1:128) contained low titre anti-nuclear or antismooth muscle antibodies. Most of the measles CF titres were much higher than were found in the sera of patients with DLE-seventeen being greater than 1:1000. Alcoholic cirrhosis The titres of measles antibody in patients with alcoholic cirrhosis is shown in Table 3. The mean titre is somewhat higher than in age- and sex-matched controls, although this difference is not statistically significant. There was no increase in the number of high-titre reactions in this group. The mean titre of the nine patients with anti-nuclear or anti-smooth muscle antibodies did not differ significantly from those without autoantibodies (log2 4-22 v. log2 4.16). Antibody titres in the patients with alcoholic hepatitis were similar to those in the remainder.
Primary biliary cirrhosis Table 3 shows that the mean titre of measles antibodies in patients with positive mitochondrial antibodies was similar to that found in age- and sex-matched healthy controls. Ten of these sera also contained either anti-nuclear (seven), anti-smooth muscle (two) or both (one) autoantibodies-this group had significantly higher measles antibody titres compared with the remainder. Myasthenia gravis Measles antibody titres in myasthenia gravis are compared with healthy controls in Table 2, which shows no significant difference between the two groups. Sera containing antinuclear antibodies show significantly higher antibody titres to measles compared with sera containing no anti-nuclear antibodies. The presence of skeletal muscle antibody or rheumatoid factor makes no significant difference to the measles antibody titre.
Autoantibodies Table 4 shows the measles antibody titres in the sera of patients containing anti-nuclear, anti-smooth TABLE 4. Measles CF titres and autoantibodies (excluding DLE, chronic active hepatitis, rheumatoid arthritis and myasthenia gravis) Mean No. 3 1:128 titre log2
Antinuclear antibody Healthy controls Hospital controls Anti-smooth muscle antibody Healthy controls Hospital controls Gastric parietal cell antibody Pernicious anaemia No pernicious anaemia Healthy controls Hospital controls Thyroid microsomal antibody Thyroid disease No thyroid disease Healthy controls Hospital controls
24 24 24 22 22 22
1 2 1 3 2 1
3 50 3-75 3-05 3-36 3-75 3 06
21 28 25 25
4 2 2 2
3-86 2-86 3-58 3 06
22 21 22 22
3 1 2 1
2-94 3-10 3-76 2-98
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muscle, anti-gastric parietal cell and anti-thyroid microsomal antibodies. Patients with DLE, chronic active hepatitis, rheumatoid arthritis and myasthenia gravis were excluded from these groups. Some of the sera contained more than one auto-antibody, but there was no significant difference in the measles antibody titres in the sera containing ANA, gastric parietal cell antibody or thyroid microsomal antibody alone, compared with sera containing more than one autoantibody. DISCUSSION These results confirm the previous observations of elevated measles antibody titres in chronic active hepatitis, and show that the antibody levels in this condition are very much higher than is found in other putative autoimmune disorders. These titres are higher and persist for longer than in acute measles infections (Triger et al., 1972b; Laitinen & Vaheri, 1974), and only in SSPE are comparable titres observed. The elevation in antibody titres does not appear to be solely related to liver damage, since normal measles titres are found in patients with alcoholic cirrhosis, including patients with florid alcoholic hepatitis. High measles CF titres are also found in some patients with primary biliary cirrhosis associated with anti-nuclear or anti-smooth muscle antibodies, but this may reflect a continuous spectrum between this condition and chronic active hepatitis. We have also confirmed the previous reports of elevated measles antibody levels in DLE. Our results, however, in contrast to earlier reports (Phillips & Christian, 1970; Hollinger et al., 1971) suggest that this is due to the presence of an increased number of high-titre reactions, rather than an increase in geometric mean titre. Our results both in this condition and in rheumatoid arthritis do not show a parametric distribution of antibody titres, and so statistical analysis using the Mann-Whitney test is more appropriate than the Student's t-test. In patients with rheumatoid arthritis, the mean measles antibody titres were significantly lower than in either of the control groups. This differs from the findings of Phillips & Christian (1970) and Hollinger et al. (1971), but their reports are not strictly comparable, due to differences in technique and presentation of results. Kalliomaki & Halonen (1972) reported higher CF titres in patients with rheumatoid arthritis than in controls, but the authors conceded that some of their patients with rheumatoid arthritis may have had DLE. The inverse correlation between measles CF titres and serum globulin levels in patients with rheumatoid arthritis contrasts with our failure to observe any relation between these two indices in patients with DLE. Other workers (Rothfield, Evans & Niederman, 1973; Hurd et al., 1972) have also found no correlation between measles titres and serum globulin or immunoglobulin levels, although Phillips & Christian (1972) observed a direct relationship between measles antibody level and gammaglobulin in sera of patients with DLE and other diseases. The reason for our unexpected observations is uncertain, but it is possible that immune complex formation may play a role. For example, sera in which high gammaglobulin levels are found may contain large numbers of such complexes, which may bind free measles antibody. This is consistent with the finding of Kalliomaki, Halonen & Salmi (1975) that antibody levels to measles in serum and synovial fluid are lower in rheumatoid arthritis patients without total haemolytic complement in the synovial fluid than in the rheumatoid arthritis patients with haemolytic complement. Our inability to find a difference in serum complement levels between the two groups does not invalidate the argument, since serum complement levels may be affected by factors other than immune complex consumption. Nine patients with rheumatoid arthritis were receiving corticosteroids at the time serum was taken, and all showed very low antibody titres to measles. There is no evidence that steroids influence the measles antibody levels in DLE (Phillips & Christian, 1972), and the low titres observed here may reflect the fact that the arthritis was of sufficient clinical severity to warrant the use of steroids. No correlation could be established between measles titres and any other drug, but, with the exception of indomethacin, the number of patients taking other drugs was too small for valid conclusions to be drawn. Subdivisions of the measles titres in our patients with rheumatoid arthritis according to the presence or
Measles antibodies and autoantibodies
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absence of anti-nuclear antibodies (ANA) showed that the mean titre in the sera possessing ANA was significantly higher than in the ANA-negative group, and that five of the six high-titre sera were ANApositive. Since high measles CF titres are known to occur in the sera of patients with DLE (which is almost invariably associated with ANA), it is important to be certain that the patients with rheumatoid arthritis and ANA do not have DLE. This seems unlikely, because: (a) none of the patients with rheumatoid arthritis satisfied the recognized criteria for DLE; and (b) all six rheumatoid arthritis sera containing high measles antibody titres contained rheumatoid factor (in contrast to only two out of ten of the high measles titre DLE sera). Retrospective analysis of the patients' clinical history assessed by severity or duration of symptoms failed to reveal any significant difference between patients with and without anti-nuclear antibodies. The overall frequency of high-titre reactions, and the geometric mean titre in patients with myasthenia gravis, were similar to those found in controls, but once again division of the patients according to the presence of anti-nuclear antibodies showed two subpopulations. No correlation, however, was found between the measles antibody levels and the presence or absence of skeletal muscle antibody. Both the geometric mean antibody titre and the incidence of high-titre reactions to measles in sera containing anti-nuclear or anti-smooth muscle antibodies are similar to those found in healthy controls if patients with the disorders already described are specifically excluded. However, other conditions may exist in which the presence of autoantibodies may be associated with elevated measles antibody levels, but this study does not include sufficient data from other clinical disorders for appropriate analysis. Similarly, the presence of gastric parietal cell or thyroid microsomal antibodies was not associated with an increased incidence of high-titre measles CF reactions, in contrast to the findings of Lucas et al. (1972) in their study of haemagglutinating antibodies. This may reflect the difference in method, as Salmi (1973) has shown elevated measles HI antibody levels in patients with multiple sclerosis, whereas the CF titres are normal. We did not find any significant difference in geometric mean titre or in the frequency of high-titre reactions in patients with pernicious anaemia or thyroid disease in comparison with either healthy or in-patient control subjects. The finding of high bacterial antibody titres in different forms of liver disease (Triger, Alp & Wright, 1972a) prompted the suggestion that the intact liver was able to sequester exogenous antigens, and damage to this organ resulted in increased amounts of antigen reaching other parts of the reticuloendothelial system with consequent enhanced antibody production. The marked disparity between the measles antibody levels found in chronic active hepatitis, compared with other liver diseases, suggests a different explanation. Liver disease is commonly associated with depressed cell-mediated immunity (Dudley, Fox & Sherlock, 1972), thus raising the possibility that the enhanced B cell response to measles antigen might be compensating for a reduced T-cell response. Our attempts to examine this by measuring leucocyte migration inhibition to measles antigen in patients with DLE and chronic active hepatitis have been unsuccessful and recent reports (Fuccillo et al., 1975) have cast doubt on the validity of this test as a measure of cellular immunity to measles. In conclusion, we have demonstrated the association of anti-nuclear antibodies with increased measles antibody titres in several different clinical disorders. The possibility of cross-reaction has been ruled out by studies in which antibodies to mouse nuclei (Hollinger et al., 1971) and measles antigen (Triger et al., 1974) have been adsorbed out without affecting the titre of the remaining antibodies. The results are consistent with the hypothesis that anti-nuclear antibody may be a marker representing either excessive antigenic stimulation (Christian et al., 1965; Fournie, Lambert & Miescher, 1974), or a hyperreactive response to a normal antigenic stimulus such as measles (Triger & Wright, 1973). The amount of measles antibody formed may also be influenced by the formation of immune complexes, which may contain varying ratios of antigen-antibody. In non-hepatic disorders, such as myasthenia gravis and rheumatoid arthritis, these complexes will be taken up by the liver, although alterations in the ratio of antigen to antigen-antibody may affect the ability of the liver to sequester these complexes (Thomas & Vaez-Zadeh, 1974). The extraordinarily high titres to measles found in chronic active hepatitis may reflect loss of the ability of the liver to sequester these complexes in addition to the other immunological disturbances.
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We thank Mrs Teresa Gregory, Miss Celia Lang, Mrs Lindsay Scott-Morgan and Mr Frank Paul for technical assistance. We are grateful to Dr C. M. P. Bradstreet (Central Public Health Laboratory, Colindale) for kindly providing measles CF antigen. T.R.G. was in receipt of an M.R.C. Research Fellowship. This work was supported by grants from the Medical Research Council and the Nuffield Foundation. REFERENCES BALL, J. (1952) Sheep cell agglutination test for rheumatoid arthritis: clinicopathological study. Ann. rheum. Dis. 11, 97. BRADSTREET, C.M.P. & TAYLOR, C.E.D. (1962) Technique of complement fixation test applicable to the diagnosis of virus diseases. Month. Bull. Min. Health and P.H.L.S.
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