CIRCULATING IMMUNE COMPLEXES IN MULTIPLE SCLEROSIS AND OTHER NEUROLOGICAL DISEASES

997 These results indicate that apparently normal people a constant degree of platelet activation that can be decreased by dietary means. The mechanisms whereby these changes are produced is unknown but it may involve prostaglandin synthesis. to It is most important to establish whether these changes are of therapeutic value, especially in patients with highly abnormal test results. Clearly, if ingestion of an unsaturated-fat diet is benefiCIal, these effects on platelets may be, at least in part, rehave

sponsible. fhe

splendid cooperation of

ledged. Thanks are due to

Dr

the volunteers from IBM

is

acknow-

J. Aldndge and the IBM Health Centre;

to .Bhss S. Vrnce, dietician; to Mr E. Haddeman and Mrs Yvonne Giden for technical assistance and to Mr G. Zaalberg for statistical help; to Unilever Research for financial assistance and for the Becel products obtained from van den Bergh & Jurgens N. V. Rotterdam; and to Coulter Electronics Ltd. for the loan of instruments.

REFERENCES

Joint Working Party, Royal College of Physicians of London and British Cardiac Society, J. R Coll. Physns, 1976, 10, 213. 2. O’Brien, J. R., Etherington, M., Jamieson, S Lancet, 1976, i, 878. 3 O’Brien, J. R., Etherington, M , Jamieson, S., Lawford, P., Sussex, J., Lincoln, S. V. J. clin Path. 1975, 28, 975. 4 O’Brien, J. R., Etherington, M. Thromb. Hæmostasis (in the press). 5. O’Brien, J. R., Etherington, M., Jamieson, S., Lawford, P., Lincoln, S V., Alkjaersig, N. Thromb. Diath. hæmorrh. 1975, 34, 483. 6. O’Brien, J R. Jclin. Path. 1961, 14, 140. 7. von Kaulla, E., von Kaulla, K N. Am. J. clin. Path. 1967, 48, 69. 8. Hornstra, G., ten Hoor, F Thromb. Hæmostasis, 1975, 34, 531 9. Hornstra, G., Lewis, B., Chart, A, Turpeinen, O., Karvonen, M. J, Vergroesen, A. J. Lancet, 1973, i, 1155. 10. Willis, A L., Stone, K J., Comai, K., Kuhn, D. C., Hart, M., Harris, V, Marples, P., Botfield, E. in Prostaglandins in Hematology (edited by M. J Silver and J. B. Smith) New York (in the press) 11. Röschlau, P., et al. Z. klin. Chem. klin. Biochem. 1974, 12, 226. 1.

CIRCULATING IMMUNE COMPLEXES IN MULTIPLE SCLEROSIS AND OTHER NEUROLOGICAL DISEASES T. G. TACHOVSKY H. KOPROWSKI

F.

J.

R. P. LISAK A. N. THEOFILOPOULOS DIXON

Multiple Sclerosis Research Center

of the Wistar Institute and Department of Neurology, University of Pennsylvania, Philadelphia, and Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California, U.S.A.

Summary

Circulating

immune

complexes

were

detected in 49% of sera from patients with multiple sclerosis, 45% of monosymptomatic optic neuritis, 45% of Guillain-Barré syndrome, and 15% of normal sera studied. The frequency of immune complexes in multiple-sclerosis sera was not correlated with the clinical status of the patients. Results with serial blood-samples were variable. Introduction A

NUMBER

of diseases have been shown

to

manifest

immunological- tissue injury mediated by immune-complex (i.c.) formation and deposition in various tissues.’ i.c. formation is thought to result from continued antigenic stimulation, sometimes with specificity for host-tissue antigens. A sensitive and simple technique has been described for the detection and measurement of soluble complement (C’)-fixing i.c. and human sera utilising the C’ receptors on the Raji cell-line.3 Circulating i.c.s were identified in the sera of patients with serum hepatitis,

systemic lupus erythematosus, subacute sclerosing panencephalitis, vasculitis, dengue haemorrhagic fever, and malignancies.3 The Raji-cell assay, in addition to quantitating the levels of i.c., facilitates in-vitro identification of the antigenic moiety in the complex. Hypersensitivity has often been proposed as a probable mechanism in the xtiology of multiple sclerosis (M.s.).4 This concept gains support from the finding of elevated levels of gamma-globulins in cerebrospinal fluid,5 the demonstration IgG deposits in periplaque areas,6 the presence of plasma-cells and lymphocytes in the perivascular spaces of the brain,5 the demonstration of myelinotoxic serum factors in some cases,’ and the existence of an experimental immunological model, experimental allergic encephalomyelitis.5 In addition to these humoral mechanisms, lymphocytes of M.s. patients exposed to brain antigens were found by some workers to produce a macrophage-migration-inhibition factor8 9and to undergo blast transformation.10 11

Deposits of IgG and C’ 3 have been reported in the kidney of an M.S. patient.12 We report here the demonstration of circulating i.c. in the serum of M.S. and opticneuritis (O.N.) patients with the Raji-cell assay. Also reported are attempts to correlate the levels of complex with the clinical status of M.S. patients. Patients Criteria for clinical classification of patients were as follows. Exacerbation was defined either by the appearance in a definite M.s. patient of a new symptom or by the relatively sudden recurrence of a previous symptom. Such patients fit into the "relapse" group of McAlpine et al. The stable group was defined as those M.s. patients who showed no new symptoms or objective progression of previous symptoms for 6 wk. These patients were included in the "remission" and "latent" phases of McAlpine et a1.5 The progressive Nt.s, category included a patient who displayed repeated acute episodes manifest by an objective progression of previous symptoms. Some patients were also evaluated by the method described by Kurtzke.15 None of the subjects had been on corticosteroids for at least 6 wk before sampling. Diagnosis of the other neurological diseases cited adhered to previously established clinical criteria. 130 serum-samples were obtained from M.s. patients seen at the Multiple Sclerosis Center of the Hospital of the University of Pennsylvania; 19 serum-samples from patients with firstepisode o.N. were provided by Dr D. Santoli of the Wistar Institute. Other serum-samples were supplied by the department of neurology of the Hospital of the University of Pennsylvania and included sera from patients with acute disseminated encephalomyelitis (A.D.E.M.) (5), Guillain-Barre syndrome (G.B.S.) (11), amyotrophic lateral sclerosis (A.L.S.) (10), myasthenia gravis (M.G.) (5), cerebrovascular accident (C.V.A.) (6), Bell’s palsy (1), brain abscess (1), and olivopontine cerebellar atrophy (O.P.C.A.) (1). Normal sera (43) were obtained from healthy medical students, nurses, physicians, and laboratory

personnel. Materials and Methods cultures of the Burkitt-lymphoma-derived Raji maintained in Eagle’s minimal essential medium (M.E.M.) as previously described.3 Cell viability was determined by trypan-blue exclusion. Antiserum to human IgG was prepared in rabbits. IgG frac-

Suspension

cell-line

were

tions of antisera were prepared by chromatography on a D.E.A.E.-52 column equilibrated with phosphate buffer, 0-01 mol/1, pH 7.3. The IgG fractions were conjugated with fluorescem isothiocyanatell or radiolabelled14 with iodine-125.

998

Circulating t.c. was assayed as described.3 50 all M.E.M. containing 2 x 106 Raji cells was reacted with 25 µl of the test sera diluted 1/4 in saline. The serum/cell mixture

was

TABLE I-DETECTION OF I.C. IN SERA OF NORMAL INDIVIDUALS AND PATIENTS WITH NEUROLOGICAL DISEASE

incubated

with gentle shaking for 45 min at 37°C. After three washes with M.E.M., a previously standardised amount of 125I-rabbitanti-human IgG in M.E.M. containing 1’7(, human serum-albumin (M.E.M.-H.S.A.) was added, and incubation was continued with gentle shaking for 30 min at 4°C. The cells were then washed three times with M.E.M.-H.S.A., and the radioactivity in the cell pellet was determined in a gamma counter. The amount of I.C. in the test sera was extrapolated from a standard curve of radioactive-antibody uptake, which was obtained by incubating cells with various amounts of aggregated human globulin (A.H.G.) mixed with an i.c.-negative normal human serum as a C’ source. The amount of i.c. in each test serum was expressed as ;ig A.H.G. equivalent per ml serum; the lower limit of sensitivity of the assay was 12 jig A.H.G. equivalent per ml serum. The mean µg A.H.G. equivalent per ml and standard error of the mean were calculated from triplicate samples. For group data each patient was considered separately; for patients from whom multiple serum-samples had been taken, the mean µg A.H.G. equivalent per ml was used. Probabilities were determined by the 2x22 chi-square contingency test with one degree

*Bell’s

palsy (1), brain abscess (1), olivopontine cerebellar atrophy

1

TABLE II—RELATIONSHIP OF LC. WITH CLINICAL STATU S OF PATIENTS

of freedom. All sera were frozen at -70°C and thawed once before use. The assays for i.c. were done on coded samples; data on diagnosis and disease activity were correlated after completion of the test.

Results

Previous work has established the Raji-cell radioimassay as a sensitive method for the detection of circulating immune complexes in human sera.3 Working from a baseline value of radioactive-antibody uptake by cells incubated with normal human serum or a pool of normal human serum, we found that quantitative estimates of i.c. are possible. The lower limit of the assay was determined to be 12 µg A.H.G. equivalent per ml-a level that exceeded i.c. values in most normal sera.3 mune

The

population distributions of i.c. in sera of normal people, M.S. patients, and patients with monosymptomatic O.N. are shown in the figure. 4 of 27 normal sera were found to possess circulating i.c. above the lower limit of the test-i.e., above 12 µg A.H.G. equivalent per m!; the range for the normal sera was < 12 to 40 µg R.rt.c. equivalent per ml (table I). 33 of 67 M.S. sera and5 of 11 found

contain measurable levels of I.C The range of M. s. sera was < 12-17 5 [g A.H.G. equ’ivalent per ml; the range of o.N. sera was <12-80 µg A.H.G. equivalent per ml (table I). The difference between the normal and M.S. groups was highly signifiO.N. sera were

cant

distributions of circulating immune complexes detected by the Raji cell assay in sera of normal individuals and patients with multiple sclerosis (M.S.) and optic neuritis

Population (O.N.).

to

(P>0001).

Sera from patients with neurological disorders other than M.S. and o.N. are also shown in table 1.5 of 11 G.B.s. sera had i.c. with a range of <12-135 µg A.H.G. ml. 1 of of 1 10 sera from 1 and of 5, 6, equivalent per with A.L.S. and A.D.E.M., C.V.A., patients respectivelv were positive for i.c. by the Raji-cell assay. No complexes were detected in the sera of patients with Ai.G. (5, Bell’s palsy (1), brain abscess (1), and o.p.c.A. (11.’). The findings that a greater percentage of M.s. sera than normal sera contained i.C., and that the range ui values of i.c. was quite different, prompted an attempt to correlate the presence of i.c. with the clinical presentation of the M.s. patients. The relationship of !.c te clinical status and the total Kurtzke value are sholln in table 11. A slightly higher percentage of sera from panenv judged to be clinically stable or progressive had detectable i.c. than did sera from patients in exacerbation When compared on the Kurtzke scale, sera of paner.’.with total Kurtzke values (i.e., combined disabilu and functional scales) of 11-20 and 21-35 showed a grea:,’

999 TABLE III-IMMUNE COMPLEXES IN SERIAL BLOOD-SAMPLES OF M.S. PATIENTS

In this report we have demonstrated the presence of detectable amounts of circulating i.c. in the serum of patients with M.S. and some other neurological diseases. The incidence of i.c. in M.S. sera is statistically significant when compared with sera from normal individuals (P>0001). Our finding of detectable amounts of i.c. in 15% of normal serum samples is higher than the previously reported value of 3%.3 The reason for this disparity is unknown but may be attributed to the small size of our sample (27 vs 120) and/or the possibility of continued high exposure to disease of the individuals in the sample. The incidence of i.c. in A.L.S. sera in this study (table I) is in contrast to the findings of Oldstone et a1.22 Using the Clq precipitation test, they found i.c. in 10 of 25 A.L.S. sera, whereas we, using the Raji-cell assay, found detectable complexes in only 1 of 10 sera. This discrepancy is unexplained but is presumably a function of the specificities and/or sensitivities of these two tests for i.c. No strict correlation was found between the incidence of i.c. and the clinical status of M.S.; patients judged to be clinically stable and/or with higher Kurtzke values were found to possess I.C. up to 175 µg A.H.G. equivalent per ml.

*11=progressive, S=stable, E=acute exacerbation.

percentage containing detectable i.c.; the difference was, however, not significant. In addition, the range of values in these latter groups was greater than that observed in the group with Kurtzke values of 1-10. Table m lists the results obtained by the Raji-cell assay of serial blood-samples. Also shown is the clinical status of the patient on the date the blood-sample was obtained. These results support the previously noted variability in the presentation and course of the disease. Sera of 3 patients (nos. 1-3) displayed detectable levels of circulating I.C. over a period of 2-11 mo. Patients 4 and 5 were observed for a shorter time but were negative (<12 .gA.H.G. equivalent/ml), except on one occasion in patient 4. 4 serial samples from 2 normal individuals taken over a period of a year were negative.

Discussion

The significance of these results remains to be shown. The detection of IgG-C’ deposits in the brain tissue of M.S. patients6 and in the kidney of 1 M.S. patient’2 suggests continuous antigenic stimulation. Alternatively, the presence of circulating i.c. may be an epiphenomenon not directly involved in the pathogenesis of M.S. but reflecting injury to the central nervous system or a secondary bacterial infection. The latter is suggested by the variability of i.c. detection observed among serial blood-samples (table in), although no coincident bacterial infection was documented in these cases. The results of sucrose density-gradient centrifugation of sera possessing i.c. (unpublished results) suggest that, whatever the antigen, it is apparently of small size. Nevertheless, the demonstration of i.c. in M.S. sera affords an opportunity for experimental analysis of the complexassociated antigen. This work is underway. Requests for reprints should be at Spruce, Philadelphi,

36th Street

T. G. T., Wistar Instutute, 19104, U.S.A.

sent to

PA

REFERENCES

Immune-complex disease is mediated by the formation of soluble antigen-antibody aggregates within the circulation. Once formed, these aggregates become localised in tissues, especially in glomeruli, arterial walls, and choroid plexus. 12 Refinements in the methods of detecting immune complexes have led to a greater appreciation of the role that i.c. plays in disease.’ 16 The bases for the understanding of immune complexes in disease are derived from the study of experimental serum sickness. Soluble antigen-antibody complexes formed when a single large dose (or multiple small doses) of antigen circulates throughout the body, eventually becoming deposited at certain sites, where tissue injury ensues. 17-19 Similarly, virus-antibody complexes play a prominent part in the pathogenesis of var20

viral diseases of both man and animals.16 Imcomplexes in viral diseases of man have been detected by the Raji-cell radioimmune assay in dengue hæmorrhagic fever, Burkitt’s lymphoma, subacute sclerosmg panencephalitis, and hepatitis B.21 Circulating i.c. have also been demonstrated with this technique in systemic lupus erythematosus and rheumatoid arthritis.3 ious

mune

1. Dixon, F. J. Invest Derm 1973, 59, 413 2. Cochrane, C. G., Koffler, D. Adv Immun 1973, 16, 186 3 Theofilopoulos, A N, Wilson, C B., Dixon, F. J. J. clin Invest 1976, 57, 169 4. Panelius, M Med Biol. 1975, 53, 187. 5 McAlpine, D., Lumsden, C. E., Acheson, E D. Multiple Sclerosis. A Reappraisal. Baltimore, 1972. 6. Tavolato, B. F J neurol. Sci 1975, 24, 1 7 Lisak, R. P, Heinze, R. G., Falk, G A., Kies, M W Neurology, Minneap. 1968, 18, 122. 8 Bartfeld, H., Atoynatan, T. Int Archs Allergy appl. Immun 1970, 39, 361. 9 Rocklin, R. E, Sheremata, W. A , Feldman, R. G New Engl. J. Med 1971,

284, 803 10 Dow, P C., Peterson, R. D A. Archs Neurol. 1970, 23, 32. 11. Barteld H., Atoynatan, I. Br med. 1970, J ii, 91. 12. Whitaker, J N, Dowling, P. C., Cook, S D J Neuropath exp Neurol. 129 1971, 30, 13 Clark, H F , Shepard, C. C Virology, 1963, 20, 642 14. McConahey, P. J, Dixon, F J Int Archs Allergy appl Immun. 1966, 29, 185

Kurtzke, J. F Neurology, Minneap 1961, 11, 686. Oldstone, M B A Prog med Virol. 1975, 19, 84 Dixon, F J, Feldman, J D , Vazquez, J. Jexp. Med 1961, 113, 899 Wilson, C B, Dixon, F J J Immun 1970, 105, 279 Wilson, C B., Dixon, F J J exp Med 1971, 134, 7s. Oldstone, M. B A, Dixon, F J in Viral Immunology and Immunobiology edited by A N Notkins, p 34 New York, 1975 21 Oldstone, M. B A, Theofilopoulos, A. N., Gunver, P, Klein, G Intervirol. 15 16 17 18. 19. 20.

1974, 4, 202 22. Oldstone, M B A. W ilson, C B., Perrin, L. H . Norris, F H., 1976, ii, 169

Jr, Lancet,