ARTHRITIS RHEUMATISM

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1 ARTHRITIS & RHEUMATISM OFFICIAL JOUKNXL OF THE AMERICAN RHEUM&%TISM ASSOCIATION SECTION OF?'HE ;IRTHRITIS FOUNDATION 209 INCREASED TOXOPLASMA ANTIBODIES IN IDIOPATHIC INFLAMMATORY MUSCLE DISEASE A Case-Controlled Study PAUL E. PHILLIPS, STUART S. KASSAN, and LAWRENCE J. KAGEN Antibodies to Toxoplasrna gondii were measured in sera from 69 patients with polymyositis, dermatomyositis, and myositis associated with other connective tissue diseases and compared to 69 age-, race-, and sexmatched controls with unrelated diseases. Complement fixation toxoplasma antibodies were significantly more frequent in polymyositis and correlated with high IgM levels. Other distinguishing clinical or laboratory features of these patients were not found. The high toxoplasma antibodies were not associated with generally hyperactive humoral immunity. The serologic data suggested that inflammatory muscle disease was associated with recent active toxoplasma infection in certain patients. The pathogenetic role of the microorganism remains uncertain. From the Department of Medicine, The Hospital for Special Surgery. New York Hospital. and Cornell University Medical College. New York City. Presented in part at the American Rheumatism Association Annual Meeting. New Orleans. June Supported in part by grants from the USPHS (AM AM 14650) and the Muscular Dystrophy Association. Paul E. Phillips, MI>. FAC'P: Associate Professor of Medicine: Stuart S. Kassan. MI>. FACP: Fellow in Rheumatic Diseases, (presently Assistant Clinical Professor of Medicine. University of Colorado Medical Center, Denver); Lawrence J. Kagen, MD, FACP: Associate Professor of Medicine. Cornell University Medical College. The Hospital for Special Surgery. New York City. Address reprint requests to I>r. Paul E. Phillips, The Hospital for Special Surgery. 535 East 70th Street. New York. New York Submitted for publication August : accepted in revised form October The concept that chronic microbial infection may be related to the etiology of connective tissue diseases has been proposed frequently (I). Various animal studies support this concept, e.g., in mice, virus infection is a factor in immunologically mediated disease (2) and Toxoplasma gondii causes inflammatory muscle disease (3). The etiology of human polyrnyositis (PM) is unknown but serologic evidence of recent infection with toxoplasma has been found in some patients (4-10). This might be 1) part of a broadly hyperactive humoral immune state, 2) the result of secondary infection associated with the inflammatory muscle disease or its treatment, or 3) the result of a primary or reactivated toxoplasma infection directly involved in the pathogenesis of PM. This study was an attempt to confirm the original observations in a controlled fashion by comparing toxoplasma antibody levels in patients with PM and other myositis. and in matched controls. We also tried to exclude generally hyperactive humoral immunity as the cause of high toxoplasma antibodies by comparing them with immunoglobulin and virus antibody levels. Finally we investigated other distinguishing features of patients with high toxoplasma antibodies. MATERIALS AND METHODS Pa tien ts Sixty-nine patients with myositis were studied (Table I): 20 with polymyositis (PM), 24 with dermatomyositis (DM), and 25 with rnyositis associated with another disease. The diagnosis of myositis was made clinically by the presence of Arthritis and Rheumatism, Vol. 22. No. 3 (March 1979)

2 2 10 PHILLIPS ET AL Table 1. Patient groups Diagnosis Number Myositis cases Polymyositis 20 Dermatornyosi tis 24 Other myositis with: Systemic lupus erythematosus 9 Progressive systemic sclerosis 5 Rheumatoid arthritis and overlap connective 6 tissue diseases Other diseases (carcinoma, diabetes mellitus, 5 Sjogren s syndrome) Controls Degenerative joint disease 22 Other orthopedic diseases 18 Endocrine/infection/tumor 16 Other 13 proximal muscle weakness with or without tenderness, and confirmed by elevated serum creatine phosphokinase (CPK) with or without elevated serum lactate dehydrogenase (LDH), glutamic-oxalacetic acid transaminase (GOT) and/or myoglobin, and consistent electromyographic changes and/or characteristic muscle biopsy. Patients with polymyositis had no rash or evidence of other primary connective tissue or muscle disease. DM patients had the characteristic skin rash without evidence of other disease, and other myositis patients had typical features of systemic lupus erythematosus (SLE), systemic sclerosis, rheumatoid arthritis (RA), or the miscellaneous diseases shown (Table 1). The control patients had a variety of disorders not related to inflammatory connective tissue diseases (Table I) and none had signs or symptoms of myositis. They came from the same hospital populations and were individually matched for age, race, and sex with the myositis patients (Table 2): twothirds were white, two-thirds female, and the age was 43 years. Clinical and laboratory data were obtained by review of hospital records. The variables analyzed for their possible relation to toxoplasma antibodies were age, race, sex, birthplace, current residence, total duration of disease, duration and activity of myositis, corticosteroid and immunosuppressive treatment, presence of neoplasm, erythrocyte sedi- Table 2. Demographic characteristics of myositis patients and matched controls Characteristic Myositis patients (n = 69) Controls (n = 69) Mean age, years () 43.3 (4-83) 43.2 (4-80) Race, number white (%) 49 (71%) 49 (71%) Sex, number female (%) 45 (65%) 45 (65%) Birthplace, number born in USA (%) 41 (59%) 44 (64%) Residence, nurn ber living in New York City (%) 45 (65%) 54 (78%) Total disease duration,, years (* SD) 3.9 (& 5.9) 4.4 (26.3) mentation rate (ESR), antinuclear antibody (ANA), CPK, LDH, GOT, and serum myoglobin. Serologic methods Sera. Sera were generally obtained during active disease, stored at -2O C, and coded prior to testing. Sabin-Feldman dye test (SF). A micromodification of the Sabin-Feldman dye test (SF) for toxoplasma antibody was performed as previously described (I 1). Complement fixation test (CF). The complement fixation test (CF) for toxoplasma antibody was performed with antigen prepared from peritoneal exudate of mice infected with Tgondii (12). Virus antibodies. Hemagglutination-inhibition was used to measure antibody to rubella (13), measles (14) (both kindly done by Dr. David Barry), and parainfluenza type 1 viruses (15). Epstein-Barr (EB) virus antibody was measured by indirect immunofluorescence (kindly done by Dr. Yashar Hirshaut) (16). For each of these antibody assays, titers were expressed as the log, of the reciprocal of the highest positive serum dilution (e.g., positive at 1:32 = 5 log,). For calculating antibody titers, sera without detectable antibody at the lowest dilution tested were considered positive at the next lower dilution (e.g., negative at 1:4 = I log,). This accepted convention minimizes differences between titers and thus tends to understate their significance. Serum immunoglobulin (Ig) concentration. IgG and IgM were determined quantitatively by radial immunodiffusion using antisera to purified human IgG and IgM (15). Analysis. The clinical and laboratory data and serologic results were coded and analyzed by computer at the Hunter College Department of Mathematics (kindly performed by Professor Jack Hachigian and his staff). The statistical tests used included Student s t. x2. Wilcoxon signed rank, analysis of variance, and Pearson and Kendall ranked correlations (15). RESULTS Comparison of myositis patients and matched controls. The demographic characteristics of patients and controls were similar (Table 2). Because the frequency of toxoplasma antibody varies geographically, birthplace and current residence were analyzed and found to be similar between patients and controls, as was total duration of disease. Statistical analysis of the I1 other clinical and laboratory variables revealed no unexpected differences between patients and controls, nor between the myositis groups themselves. The frequency of occurrence of SF toxoplasma antibody was similar in the entire myositis group compared to controls (45% versus 43%). However, frequency was somewhat increased in the polymyositis group compared to its controls (Table 3). The SF antibody titer was somewhat higher in the entire myositis group

3 ~~ ~ TOXOPLASMA ANTIBODIES 21 1 compared to controls (5.25 versus 4.48), but this was almost entirely due to a fourfold (2 log,) higher titer in the PM " eroud (Table 3). The distribution of SF anti- 1, body titers (Figure 1) showed that there was a subgroup of PM patients having very high titers rather than a generalized small increase in titers among all PM patients. The highest titer in the PM controls was 8, while in this subgroup of 6 PM cases titers d from 10 to 16. There were also 3 DM patients and 1 other myositis patient (with Sjogren's syndrome and RA) with titers 210, while the highest titers among their controls were 10 in 2 cases (Figure 1). CF toxoplasma antibody was rarely found except in the PM group, where both frequency and titer were significantly increased (Table 4). The DM and other myositis groups, like their controls, rarely had CF antibody. The highest titer in the controls was 3 log,, whereas 5 myositis patients (4 PM) had titers >3, a level suggesting recent infection (17). Mean IgG and IgM levels were variably increased in each myositis group compared to control> (Table 5), but none of the increases reached statistical significance. However, the entire group of myositis patients did have significantly higher levels of both IgG (17.5 versus 14.0 mg/ml) and IgM (1.90 versus 1.37) compared to controls (P-= 0.05). Virus antibody titers were not significantly increased in any myositis group, although EB antibodies were significantly higher in the entire myositis group compared to controls (Table 6). This was principally due to higher titers in the PM and DM groups. Correlative analysis. An extensive statistical analysis was made of the relationships between the 17 clinical and laboratory variables and the 8 serologic tests, with particular reference to toxoplasma antibody titers. Each myositis and control group was analyzed separately, then as entire myositis and control groups, and then combined. This accumulation of data produced few unexpected findings. Examples of previously Table 3. Frequency and titers of Sabin-Feldman toxoplasma antibody in myositis groups and controls Mean titer Group (no.) No. positive (%) log, (k SD) Polymyositis (20) I2 (60%) 6.70 (+4.33)* controls (20) 9 (45%) 4.65 (k2.14) Dermatomyositis (24) 7 (29%) 4.63 (22.89) controls (24) 8 (33%) 4.25 (22.03) Other myositis (25) 12 (48%) 4.68 (k2.41) controls (25) 13 (52%) 4.56 (k2.13) * PM versus controls, 0.10 > P > negative Y e c.- 4 kl? c m 5 I m - g 12 X + 0 l4 P Controls (69) Polymyositis (20) Dermatomyositis (24) Myositis with other connective tissue diseases (25) I I I I I I I I % of patients Figure 1. Distribution of Sabin-Feldman toxoplasma antibody titers in patients with polymyositis, dermatomyositis, and myositis with other connective tissue diseases and in matched controls. known relationships were age correlated positively with SF, parainfluenza, and EB antibody titers, reflecting the increasing prevalence of prior infection in older populations. Likewise, direct and indirect indicators of disease activity (e.g., ESR, enzymes, steroid dose, and Ig levels) tended to correlate positively with each other. Also as expected, there was a strong direct correlation between SF and CF toxoplasma antibody levels (r = 0.78, P < 0.001). An unexpected finding was a weak di- Table 4. Frequency and titer of complement fixation toxoplasma antibody in myositis groups and controls Mean titer Group (no.) No. positive (%) log, (+ SD) Polymyositis (20) 7 (35%)* 3.55 (k2.87)t controls (20) 0 (0%) I.o Dermatomyositis (24) I(4%) I.20 controls (24) 1(4%) 1.33 Other myositis (25) 2 (8%) 1.86 (k1.70) controls (25) 1(4%) 1.11 * PM versus controls, P < t PM versus controls, P < 0.01.

4 212 PHILLIPS ET AL Table 5. Serum IgG and IgM levels in myositis groups and controls Mean IgG level Mean IgM level Group (no.) mg/ml (k SD) mg/ml (ksd) Polymyositis (20) 17.9 (k13.4) 2.53 (k2.51) controls (20) 13.4 (k 4.2) 1.31 (k1.05) Dermatomyositis (24) 14.9 (k12.3) 1.83 (20.89) controls (24) 13.5 (k 4.0) 1.47 (k0.75) Other myositis (25) 19.9 (k14.0) 1.49 (k0.89) controls (25) 15.0 (& 4.6) 1.33 (k0.58) rect correlation of IgM levels with SF antibody (r = 0.22, P < 0.01), and a quite strong correlation with CF antibody (r = 0.50, P < 0.001). A weak correlation of CF antibodies with IgG was probably related to the stronger correlation with IgM, since IgG and IgM levels also correlated directly (r = 0.34, P < 0.001). Except for a weak negative correlation between parainfluenza and CF antibody (r = -0.29, P < 0.05), virus antibody titers correlated with neither toxoplasma antibody nor IgM. Taken together, this analysis suggested that the high toxoplasma antibodies in myositis were not part of a nonspecifically hyperactive immune response. Their association with high IgM levels suggested that the CF antibodies particularly were manifestations of a primary immune response to recent toxoplasma infection. Further analysis was then done to discover if the myositis patients with high SF titers had other distinctive characteristics. Clinical and laboratory correlates of high toxoplasma antibody titers in adult myositis. There were 59 adult myositis patients who were divided by their SF toxoplasma antibody levels into high, low, and negative groups (Table 7). All 10 children under age 16 lacked SF antibody and thus toxoplasma could not be implicated in their disease (1 PM, 7 DM, 2 other). Therefore they were excluded from further analysis in order to make the groups of adult myositis more comparable. Although the characteristics of these three groups were still heterogeneous, the high SF group did have some Table 6. Virus antibody titers in all myositis cases and controls Mean antibody titer, log2 (k SD); no. tested Antibody Myositis Controls Rubella 7.46 (k2.19) (k2.51) 23 Measles 7.31 (k2.04) (k2.36) 26 Parainfluenza type (k1.29) (21.04) 67 Epstein-Barr 8.93* (k2.70) (k2.63) 39 * Myositis versus controls, P < distinguishing features. More of them had polymyositis and they were somewhat younger, although age d widely in all three groups (Table 7). Race, sex, birthplace, and current residence were similar in all three groups. Total disease duration was much shorter in the high SF group, whereas duration of myositis itself was much shorter in both the high and low groups compared to the negative antibody group (Table 7). Thus SF antibody was more often found early in the course of myositis. Although the myositis was active in 75 to 100% patients of each group, those in the high SF group were treated more frequently with corticosteroids and with higher doses (Table 7). None received cytotoxic agents. Table 7. Characteristics of adult myositis patients by toxoplasma SF antibody level Toxoplasma antibody level* High Low Negative Characteristic (n = 10) (n = 21) (n = 28) Diagnosis polymyositis, % dermatomyositis, % other myositis, % Age, years Disease duration, years Total Myositis Corticosteroid % treated prednisone dose, mg/day Cytotoxic treatment, % Malignancy, % IgG, mg/ml IgM, mg/ml Antibodies Toxoplasma CF, % positive titer Mean titer of rubella measles parainfluenza Epstein-Barr I t * High, 210 log2 SF titer; low, 4-8; negative, c4. t P < 0.05 versus low or negative groups I.o I 8. I

5 TOXOPLASMA ANTIBODIES 213 These therapeutic differences in the high SF group could in part be the result of the acuteness or severity of their illness. However, the low group also has a short disease duration. The severity of myositis, as judged from the laboratory characteristics (ESR, ANA, CPK, LDH, GOT, myoglobin), was basically similar in all three groups, although both the high and low groups tended to have somewhat higher enzyme and myoglobin levels. Thus the possibility remains that corticosteroid treatment may have predisposed certain patients to primary or reactivated toxoplasma infection. Interestingly, a disproportionate number of the patients with associated neoplasia (6/10) had low SF titers (Table 7). IgG levels were elevated in both the high and negative SF groups (Table 7) but when the single high SF and the two negative cases with very high IgG (>50 mg/ml) were excluded, IgG was similar in all three groups. IgM levels were significantly higher in the high SF group compared to the other two (Table 7). Even when the single high and negative SF cases with very high IgM (>5 mg/ml) were excluded, this difference persisted ( IgM 2.2 and 1.5 mg/ml, respectively). As expected from the correlation analysis, CF antibodies were present principally in the high SF group, rarely in the low, and not in the negative group (Table 7). Four of the five cases with CF antibodies >3 logz had PM. The high SF group tended to have somewhat lower virus antibody titers, although the three groups were basically similar in this regard (Table 7). Thus the high SF group tended to have high IgM levels, which were even more strongly associated with high CF antibodies. Otherwise there was no evidence of a generally hyperactive humoral immune state in the high SF group. DISCUSSION Previous reports of definite or presumed Toxoplasma gondii infection with myopathic symptoms (4-9) support the hypothesis that this infectious agent can produce muscle disease in human beings. In some cases the agent was found in skeletal muscle associated with focal inflammatory reactions and necrosis (5,9). Attempts at isolation of toxoplasma from muscle tissue have rarely been successful (4-9). Chemotherapy directed against the agent was of marginal benefit in 2 of our earlier patients (4), but was associated with a fall in antibody titer and clinical improvement in 3 patients reported by others (4-6, 10). Lymphocytes from a patient with PM and high toxoplasma antibody showed a de- pressed in vitro response to toxoplasma antigen and other B- and T-cell mitogens compared to asymptomatic subjects with SF antibodies (5). The ing of these abnormalities is not as yet clear. The present study confirmed the observation that serologic evidence of recent or reactivated toxoplasma infection is found more frequently in patients with myositis, especially polymyositis (4). The PM group had both higher frequency and titers of both SF and CF toxoplasma antibodies compared to matched controls. The dermatomyositis and other myositis groups were not different from their matched controls. The possible explanations of these findings are that they may be due to 1) a nonspecific reaction related either to the toxoplasma antibody tests themselves or to a generally hyperactive humoral immune response in myositis, like that found in SLE (15), 2) recent primary or reactivated toxoplasma infection, occurring secondary to either the inflammatory muscle disease itself or its treatment, or 3) direct involvement of Toxoplasma gondii in the pathogenesis of myositis in certain patients. Regarding the first possibility, the antigenic specificity of the SF and CF methods for toxoplasma antibodies has been shown previously (18, 19). No evidence of generally hyperactive humoral immunity was found: both SF and CF antibody levels correlated best with IgM, and less well or not at all with IgG. Virus antibodies were not elevated in the myositis cases, except for EB, and these did correlate with IgG levels as had been found in SLE (16). Overall none of the virus antibodies correlated with either toxoplasma antibody and, in fact, virus antibodies were lower in the high toxoplasma antibody group. Thus the elevated toxoplasma antibodies in myositis appeared to be a specific immune response to toxoplasma infection. Regarding the possibility of recent primary or reactivated infection, the most specific test is the IgM-fluorescent antibody method (20), which unfortunately was not available for this study. However, high CF antibody titers (>3 log,) have been correlated with recent infection (17, 19). In this study, both high SF titers and CF antibody were found in 10 myositis cases but only 2 controls, with high CF antibody in 5 cases and no controls. Furthermore, an association of high SF and, more strongly, high CF antibody with high IgM levels was found, which suggests a primary immune response. Together with previous reports (4-10) these data suggest that recent toxoplasma infection, whether primary or reactivated, had occurred in a subgroup of myositis patients.

6 2 14 PHILLIPS ET AL The third possibility contains the crucial question: are toxoplasma directly involved in the pathogenesis of myositis in this subgroup of patients? This analysis failed to identify any other uniform characteristics of these patients. Most had polymyositis of relatively short duration, which may have been the reason they received more corticosteroids more often. However, such treatment might also have predisposed them either to acquiring primary toxoplasma infection or to reactivating a latent one (2 I). The inflammatory muscle disease itself might also cause a similar predisposition. As of now it seems clear that active toxoplasma infection occurs in certain, possibly genetically predisposed, myositis patients. The hypothesis that this treatable infection is related to the pathogenesis of inflammatory muscle disease merits further investigation. ACKNOWLEDGMENTS We gratefully acknowledge the help of Dr. Anne C. Kimball and Mrs. Pat Daniels (Tropical Medicine Unit, New York Hospital) for the toxoplasma antibody tests, Dr. Charles R. Steinman and Harry Spiera (Mt. Sinai Hospital Medical Center, New York, New York) in obtaining sera, Drs. David W. Barry (Bureau of Biologics, U.S. Food and Drug Administration, Bethesda, Maryland) and Yashar Hirshaut (Memorial-Sloan-Kettering Cancer Center, New York, New York) for virus antibody testing, Drs. Jack Hachigian and Janet Wittes and Mr. Paul Nani (Department of Mathematics, Hunter College of the City University, New York. New York) for computer programming and statistical analysis. and Dr. Charles L. Christian for advice and support. REFERENCES Phillips PE, Christian CL: Infectious agents in chronic rheumatic diseases, Hollander s Arthritis and Allied Conditions. Ninth edition. Edited by DJ McCarty. Philadelphia, Lea & Febiger Buchmeier MJ. Oldstone M BA: Virus-induced immune complex disease: identification of specific viral antigens and antibodies deposited in complexes during chronic lymphocytic choriomeningitis virus infection. J Immunol 120: , I978 Henry L, Beverley JKA: Experimental toxoplasmic myocarditis and myositis in mice. Br J Exp Pathol 50: , Kagen LJ. Kimball AC, Christian CL: Serologic evidence of toxoplasmosis among patients with polymyositis. Am J Med 56: , Greenlee JE, Johnson WD, Campa JF, Adelman LS, Sande MA: Adult toxoplasmosis presenting as polymyositis and cerebellar ataxia. Ann Intern Med 82: , Chandar K, Mair HJ, Mair NS: Case of toxoplasma polymyositis. Br Med J 1: , Rowland LP, Greer M: Toxoplasmic polymyositis. Neurology I 1 : , 196 I 8. McNicholl B. Underhill D: Toxoplasmic polymyositis. Isr J Med Sci , Fonseca RC, Mullner F. Segura JJ. Ruiz PJ. Ingianna M, Acuni RA: Miositis Toxoplasmica aguda en un Adulto. Acta Medica Cost 16:75-78, Samuels BS, Rietschel RL: Polymyositis and toxoplasmosis. JAMA 235: II. Feldman HA, Lamb GA: A micromodification of the Toxoplusmu dye test. J Parasitol 52:415, Cooney MK, Kimball AC, Bauer H: Studies on toxoplasmosis. I. Complement fixation tests with peritoneal exudate antigen. J Immunol 81: Stewart GL. Parkman PD. Hopps HE, et al: Rubella virus hemagglutination inhibition test. N Engl J Med 276: Rosen L: Hemagglutination and hemagglutination-inhibition with measles virus. Virol 13: Phillips PE, Christian CL: Virus antibodies in systemic lupus erythematosus and other connective tissue diseases. Ann Rheum Dis 32: , Phillips PE. Hirshaut Y: Epstein-Barr virus antibody levels in systemic lupus erythematosus. Arthritis Rheum 16:97-101, Kean BH, Kimball AC: The complement fixation test in the diagnosis of congenital toxoplasmosis. Am J Dis Child , Jacobs L. Cook MH. Wilder HC: Serologic data on adults with histologically diagnosed toxoplasmic chorioretinitis. Trans Am Acad Opthalmol Otolaryngol 58: Lunde MN: Laboratory methods in the diagnosis of toxoplasmosis. Health Lab Sci 10: , Remington JS. Miller MJ, Brownlee I: IgM antibodies in acute toxoplasmosis. 11. Prevalence and significance in acquired cases. J Lab Clin Med 71: Gleason TH, Hamlin WB: Disseminated toxoplasrnosis in the compromised host. Arch Intern Med 134: , 1974