THE DIAGNOSTIC VALUE OF ANTIHISTONE ANTIBODIES IN DRUG-INDUCED LUPUS ERYTHEMATOSUS

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1 158 THE DIAGNOSTIC VALUE OF ANTIHISTONE ANTIBODIES IN DRUG-INDUCED LUPUS ERYTHEMATOSUS ALAN EPSTEIN and PETER BARLAND This study was undertaken to see whether the presence of antihistone antibodies, measured by indirect immunofluorescence assay, could distinguish between patients who were symptomatic from a drug-induced lupus-like illness (DILE) and asymptomatic patients who had drug-induced antinuclear antibodies (DANA). In this study, 82% of the patients with DILE had antihistone antibodies detectable in their sera, compared with 32% of the patients in the DANA group (P < 0.01). When the patients taking procainamide were analyzed separately, it was found that 92% of the patients with DILE had demonstrable antihistone antibodies, contrasted with 33% of the patients in the DANA group (P < 0.01). In those patients with antihistone antibodies (DILE versus DANA patients), there were no differences in the mean antibody titers. We conclude that the presence of antihistone antibodies is strong, statistically significant evidence that the symptoms of a given patient are due to drug-induced lupus erythematosus. It is well known that antinuclear antibodies (ANA) can be detected in the sera of patients with a variety of rheumatic disorders. Moreover, antibodies with different antigenic specificities have been found to characterize specific diseases. Examples of this include the presence of antibodies to native DNA and From the Rheumatology Division, Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York. Supported in part by a grant from the New York Chapter of the Arthritis Foundation. Alan Epstein, MD; Peter Barland, MD. Address reprint requests to Peter Barland, MD, Montefiore Medical Center, 1 I 1 East 210 Street, Bronx, NY Submitted for publication October 4, 1983; accepted in revised form September 13, the Sm antigen in systemic lupus erythematosus (SLE) (l), antibodies to ribonucleoprotein in mixed connective tissue disease (2), and the SS-A and SS-B antibodies in primary Sjogren s syndrome (3). Convincing evidence that drugs can induce a lupus-like syndrome de novo in individuals not otherwise predisposed to develop it began appearing in the literature in 1953, after the introduction of hydralazine for the treatment of hypertension (4). Prospective studies confirming the ability of a drug to induce a lupus-like syndrome have also been reported for procainamide (5) and isoniazid (6). In addition, many other drugs have been suspected of inducing a lupuslike syndrome (based on small series and case reports, reviewed elsewhere [7]). While a significant percentage of patients taking procainamide (8) or hydralazine (9) develop serum antinuclear antibodies, a relatively small percentage of these patients manifest clinical evidence of a lupus-like illness. Recently, there has been an increasing awareness of the role of antihistone antibodies as a marker for clinically significant drug-induced lupus erythematows (10,ll). There have not been many studies, however, on antihistone antibodies in the sera of asymptomatic patients with drug-induced antinuclear antibodies (10). We undertook this study to see whether the presence of antihistone antibodies in the sera of patients with ANA secondary to drug treatment could distinguish those patients symptomatic due to a lupuslike illness (DILE) from asymptomatic patients having drug-induced antinuclear antibodies (DANA). The results of such a study might shed some light on the immunopathogenic process involved in drug-induced lupus erythematosus and clarify the diagnostic implications of a positive test result for antihistone antibod- Arthritis and Rheumatism, Vol. 28, No. 2 (February 1985)

2 HISTONE ANTIBODIES IN DRUG-INDUCED LUPUS 159 ies. We found that although the presence of antihistone antibodies is not absolutely discriminating, their presence remains strong, statistically significant evidence that a patient s symptoms are due to druginduced lupus erythematosus. PATIENTS AND METHODS Patient selection. Clinical information was sought on patients whose sera were sent for antihistone antibody testing to the Immunodiagnostic Laboratory at Montefiore Medical Center during 1981 and All patients were included in the study except under the following conditions: 1) clinical history was unavailable; 2) the patient had a history of DILE which had resolved at the time the specimen was drawn; 3) the patient had SLE; 4) the routine testing of the specimen for ANA gave negative results. The clinical data on each patient were thoroughly reviewed by a physician (AE) who was unaware of the results of the antihistone antibody testing. A diagnosis of DILE was made if a patient who was taking a drug known to cause this syndrome manifested 1 or both of the following criteria: 1) arthritis that remitted promptly after discontinuation of the offending drug or that remitted after institution of antiinflammatory therapy without recurrence when the treatment was stopped; 2) pleuritis and/or pericarditis that remitted promptly after discontinuation of the offending drug or that remitted after institution of antiinflammatory therapy without recurrence when the treatment was stopped. The presence of fever, anemia, or thrombocytopenia related to drug administration, though frequently observed, was not considered in making a diagnosis of DILE. Those patients not fulfilling the criteria for DILE, but who were taking a drug capable of causing DILE and whose test for ANA was positive, were assigned to the DANA group. A small number of patients were excluded from statistical analysis because there was insufficient retrospective data available to make a valid diagnosis. In no instance was a patient s diagnosis changed after the result of the antihistone antibody test was known. Most of the patients ultimately judged not to have DILE did, in fact, have some symptom(s) compatible with this diagnosis. These patients were not completely asymptomatic. Finally, each patient s clinical diagnosis was correlated with the result of the antihistone antibody testing. Antihistone antibody assay. An indirect immunofluorescent antibody assay technique was used as previously described (1 1). The assay was based on the ability to extract many nuclear proteins, including histones, from acetonefixed mouse kidney sections, using O.1N HCI. This treatment does not remove, but may partially denature, the DNA (12). Acid extraction was performed by gently agitating fixed mouse kidney sections in 0.1N HCI for 30 minutes at 24 C. The sections were then washed with 0.01M phosphate buffered saline (PBS), ph 7.3. Histone reconstitution was performed by incubating the acid-extracted sections in 25 pdml histone followed by washing with PBS. Histones were purchased from Worthington Biochemical Corporation (Freehold, NJ) and were the H-2A fraction. We used this fraction because of its high reactivity with drug-induced lupus sera, as reported by Fritzler and Tan (11). The method of isolation used by Worthington involved sequential extraction and precipitation of calf thymus with ethanolil.25n HCI (4: 1 vo1ume:volume) and, later, with 95% ethanol (13). The reconstituted sections were then used as substrates in an indirect ANA assay. Antihistone antibodies were present if the following conditions were satisfied: 1) a greater than twofold fall in the ANA titer after acid extraction of the kidney substrate, and 2) a greater than twofold return of the activity after histone reconstitution. RESULTS Eighteen patients met our criteria for the diagnosis of drug-induced lupus erythematosus; 4 patients met both criteria, while the remaining 14 met a single criterion. There were 7 females and 11 males, ranging in age from 7-81 years. The drugs believed to be the causative agents were procainamide in 13 patients, hydralazine in 1, penicillamine in 1, diphenylhydantoin in 1, disopyramide in 1, and ethosuximide in I patient. The symptoms, physical findings, and results of routine laboratory investigations of these patients are enumerated in Table 1. Steroids were used in 7 patients. Twenty-eight patients were diagnosed as having DANA. There were 17 females and 11 males, ranging in age from years. The suspected drugs in these patients were procainamide in 12 patients, hydralazine in 10, methyldopa in 1, disopyramide in 1, quinidine in 2, penicillamine in 1, and diphenylhydantoin in 1 patient. These patients either had no symptoms suggestive of lupus erythematosus or had symptoms that were easily explained by another, clearly evident, disease process. Eight patients were excluded from statistical analysis because there was insufficient retrospective data available to make a valid diagnosis. When the results of the antihistone antibody testing were examined, we found that 83% of the patients in the drug-induced lupus erythematosus group showed positive results, compared with 32% of the patients in the DANA group (P < 0.01). Of the 18 patients in the DANA group who did not have demonstrable antihistone antibodies, 16 had antibodies to an extractable nuclear antigen. We then tried to find a measurement that would further discriminate between antihistone antibodypositive patients in each group. We examined 2 measurements: l) the mean of the reciprocal of the log of the antihistone antibody titer in each group, and 2) the amount of reconstitution. There was no statistically significant difference between the 2 groups in either of these measurements (Table 2).

3 160 EPSTEIN AND BARLAND Table 1. Clinical and laboratory features (routine testing) of 18 patients with drug-induced lupus erythematosus* Clinical feature n Laboratory feature n Fever 13 Elevated ESR 10 Constitutional symptoms 7 Anemia I Arthralgias 9 Leukopenia 0 Arthritis 9 Thrombocytopenia 1 Myalgias 1 H ypergammaglobulinemia 2 Skin rash 2 Positive Coombs test 3 Malar rash 0 False-positive VDRL 0 Oral ulcers 0 Circulating anticoagulant 1 Alopecia 0 Pleuropericarditis 13 Pulmonary infiltrate 1 Renal involvement 1 Hepatomegaly 1 Splenomegal y 1 Lymphadenopathy 1 Mononeuritis multiplex 0 * Not all laboratory tests were available on every patient. ESR = erythrocyte sedimentation rate. During the course of our study, we became aware of some new observations reported by Portanova et a1 (14), indicating that the immunofluorescence histone reconstitution assay detects only antihistone antibodies directed against the histone 2A-2B (H-2A- H-2B) fraction. These investigators had also compared the antihistone antibodies found in patients with DILE induced by procainamide with those found in patients with DILE induced by hydralazine. They found that most of their patients with DILE secondary to procainamide treatment developed antibodies to the H- 2A-H-2B fraction, detectable by a radioimmunoassay. These same sera were also positive on immunofluorescence assay using total histones for reconstitutiofi. The patients with DILE due to hydralazine treatment did not develop antibodies to the H-2A-H-2B fraction and were negative for antihistone antibodies by immunofluorescence assay. There is no literature on the specificity of the antihistone antibodies induced by other drugs. For these reasons we analyzed separately our patients who were taking procainamide. It was found that of the 13 patients with DILE caused by procainamide, 12 (92%) had demonstrable antihistone antibodies. By contrast, of the 12 procainamide-treated patients in the DANA group, only 4 (33%) had antihistone antibodies (P < 0.01 by Fisher s exact test). DISCUSSION In a previous study from this laboratory (lo), antihistone antibodies were measured in a group of patients with drug-induced lupus erythematosus and in asymptomatic patients with drug-induced antinuclear antibodies, using an indirect immunofluorescence assay and acid-extracted, histone-reconstituted mouse kidney sections as substrate. It was found that 67% of the patients with DILE had antihistone antibodies as the major, or the only, ANA in their sera, but these Table 2. Results of antihistone antibody (AHA) testing* Diagnosis DILE DANA P ~ No. AHA-positive/no. with diagnosis 15/18 (83%) 9/28 (32%) <O.Ol 1 Mean ~ log AHA NS Amount of reconstitution NS * DILE = drug-induced lupus-like illness; DANA = drug-induced antinuclear antibody. P value calculated using Fisher s exact test. NS = not significant. Amount of reconstitution calculated as: 1 1 log AHA after reconstitution log AHA after extraction

4 HISTONE ANTIBODIES IN DRUG-INDUCED LUPUS 161 antibodies were not seen in the sera from patients with DANA. The present study was undertaken for 2 reasons. First, the original study involved only a small number of patients. Subsequently, we have observed that an occasional patient with DANA has detectable serum antihistone antibodies. Second, the original group of DANA patients was completely asymptomatic, whereas in the present study, most of the DANA patients showed some symptoms that caused us to consider drug-induced lupus erythematosus as a possible explanation. Such patients represent a more important diagnostic group. The present study does not include any of the patients from the original series. The data from this study show that the majority of patients with drug-induced lupus erythematosus have antihistone antibodies, which is in general agreement with our original study (10) and the study by Fritzler and Tan (1 1). In this study, 83% of the patients had antihistone antibodies, compared with 67% in the original study and 96% in the series reported by Fritzler and Tan. The present study differs from the original series in that antihistone antibodies were found in the sera of 32% of the patients in the DANA group. This further contrasts with the original series data showing a complete absence of antihistone antibodies in the 12 asymptomatic patients who were tested. Therefore, one can conclude that, although the presence of antihistone antibodies is not absolutely discriminating, as suggested by our original series, it nevertheless remains strong, statistically significant evidence that a given patient s symptoms are due to drug-induced lupus erythematosus. The studies of Portanova et a1 (14) indicate that patients with drug-induced lupus erythematosus caused by procainamide develop antibodies to the histone 2A-2B fraction. This histone complex also appears to be the antigenic component reacting in the immunofluorescence assay. We therefore analyzed separately our patients who were taking procainamide. Again, there was a statistically significant, but not absolute, difference in the frequency of antihistone antibodies between the DILE and DANA groups. The clinical features of these patients were similar to those previously reported (15,16); 2 features deserve special comment. First, the high incidence of pleuropericardial involvement in the DILE patients probably reflects the fact that 13 of the 18 patients in this group were taking procainamide, which has been reported to show a propensity for causing pleuroperi- carditis (15,17). Second, an elevated erythrocyte sedimentation rate was frequently found in those patients who were ultimately judged as having DILE. These results, as well as the results of previously reported studies (10,l l), indicate that antihistone antibodies account for a large part of the ANA activity in patients with DILE. The antigenic specificity of the ANA activity in patients with DANA remains undefined. Previous studies, using sera from patients with DANA, failed to reveal precipitating antibody activity toward a crude rabbit thymus nuclear extract containing a number of nonhistone nuclear antigens, including Sm, RNP, and La (10). Several observations lead us to believe that the ANA activity in these patients is probably also directed toward a histone component, but that these antibodies differ qualitatively from those found in patients with DILE. Among the patients who were antihistone antibody-positive, there was no significant difference in the mean titers between the 9 patients with DANA and the 15 patients with DJLE. These results would argue against a simple quantitative difference between the 2 groups. A recent study by Portanova et a1 described a group of patients receiving hydralazine whose ANA activity, by immunofluorescence assay, was lost after acid extraction of the mouse kidney section, but could not be restored by histone reconstitution. When these sera were tested for antihistone antibodies, using a radioimmunoassay, all were positive. The explanation for this discrepancy appeared to be that the immunofluorescence assay, which uses histone-reconstituted nuclei, is selective for antihistone antibodies that react with a mixture of histones 2A and 2B. The low levels of antk(h-za-h-2b) activity in the sera of patients taking hydralazine appeared to account for the failure of most, but not all, of the sera to react in the immunofluorescence assay. It should be noted that the same pattern of reactivity on immunofluorescence assay that Portanova reported for hydralazine-treated patients was also observed in 16 of our 18 DANA patients who were antihistone antibody-negative on immunofluorescence assay. These data could be taken to suggest that many of the patients in the DANA group had antibodies to histone fractions other than the H-2A-H-2B mixture. Preliminary investigations in our laboratory, using an enzyme-linked immunosorbent assay technique to detect antihistone antibodies, have shown that most of our DANA patients do indeed have antihistone antibody titers comparable with those found in the DILE

5 162 EPSTEIN AND BARLAND patients. Currently, we are further defining the antigenic specificity of these antibodies. ACKNOWLEDGMENTS We gratefully acknowledge the critical advice of Dr. Mark Burns and Dr. Arthur Grayzel, the excellent technical assistance of June Arias, and the expert assistance of Lynne Enoch in the preparation of the manuscript. REFERENCES 1. Notman DD, Kurata MD, Tan EM: Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann Intern Med 83: , Sharp GC, Irvin WD, Tan EM, Gould RG, Holman HR: Mixed connective tissue disease: an apparently distinct rheumatic disease syndrome associated with specific antibody to an extractable nuclear antigen (ENA). Am J Med 52: , Alspaugh MA, Tala1 N, Tan EM: Differentiation and characterization of autoantibodies and their antigens in Sjogren s syndrome. Arthritis Rheum 19: , Morrow JD, Schroeder HA, Perry HM Jr: Studies on the control of hypertension by Hyphex. 11. Toxic reactions and side effects. Circulation 8: , Fakhro AM, Ritchie RF, Lown B: Lupus-like syndrome induced by procainamide. Am J Cardiol 20: , Rothfield NF, Bierer WF, Garfield JW: Isoniazid induction of antinuclear antibodies: a prospective study. Ann Intern Med 88: , Lee SC, Chase PH: Drug-induced systemic lupus erythematosus: a critical review. Semin Arthritis Rheum 5:83-103, Blomgren SE, Condemi JJ, Bignall MC, Vaughan JH: Antinuclear antibody induced by procainamide: a prospective study. N Engl J Med 281:64-66, Perry HM Jr, Tan EM, Carmody S, Sakamoto A: Relationship of acetyl transferase activity to antinuclear antibodies and toxic symptoms in hypertensive patients treated with hydralazine. J Lab Clin Med 76: , Grossman L, Barland P: Histone reactivity of druginduced antinuclear antibodies: a comparison of symptomatic and asymptomatic patients. Arthritis Rheum 24: , Fritzler MJ, Tan EM: Antibodies to histones in druginduced and idiopathic lupus erythematosus. J Clin Invest , Tan EM, Robinson J, Robitaille P: Studies on antibodies to histones by immunofluorescence. Scand J Immunol 5: , Jones EW: Preparative methods for histone fractions from calf thymus. Biochem J 92:55-59, Portanova JP, Rubin RL, Josh FG, Agnello VD, Tan EM: Reactivity of anti-histone antibodies induced by procainamide and hydralazine. Clin Immunol Immunopathol 25:67-79, Blomgren SE, Condemi JJ, Vaughan JH: Procainamideinduced lupus erythematosus: clinical and laboratory observations. Am J Med 52: , Alarcon-Segovia D, Wakim KG, Worthington JW, Ward LE: Clinical and experimental studies on the hydralazine syndrome and its relationship to systemic lupus erythematosus. Medicine (Baltimore) 46: 1-33, Byrd RB, Schanzer B: Pulmonary sequelae in procaine amide induced lupus-like syndrome. Dis Chest 55: , 1969