Procainamide-lymphocyte reactions. a possible explanation for drug-induced autoimmunityкод для вставкиСкачать
1019 PROCAINAMIDE-LYMPHOCYTE REACTIONS A Possible Explanation for Drug-induced Autoimmunity HARRY G. BLUESTEIN, DOUG REDELMAN, and NATHAN J. ZVAIFLER Procainamide therapy has been associated with a lupus erythematosus (LE) like illness. At least 50% of patients who take the drug for a long time develop antinuclear antibodies and approximately 10%of these develop the complete clinical syndrome (1-3). There has been considerable speculation about the pathogenesis of the drug-induced disease. Most theories implicate drugnucleoprotein interactions, which result in the induction of antinuclear antibodies, but these explanations do not account for other autoantibodies often present in a patient with drug-induced lupus. The autoimmune phenomena of idiopathic lupus have been attributed to impaired immune regulation (4). To determine if similar mechanisms operate in the drug-induced syndrome, we looked for evidence of procainamide-induced lymphocyte abnormalities and found that many individuals taking the drug develop antibodies to cell surface antigens on normal lymphocytes. The frequency with which these antilymphocyte (ALA) antibodies develop and their concentration in the circulation are significantly increased in those patients with the procainamide-induced lupus syndrome. When the drug is discontinued, the antilymphocyte activity diminishes rapidly, in parallel with clinical improvement. We also found that procainamide alters the in vitro proliferative response of normal human lym- From the Rheumatology Division, Department of Medicine, University of California, San Diego. Supported in part by grants from the PHS (AM-07062, AM14916) and an Arthritis Foundation Center Grant. Address reprints to Nathan J. Zvaifler, Rheumatology Division, Department of Medicine, 225 Dickinson Street, San Diego CA 92103 Arthritis and Rheumatism, Vol. 24,No. 8 (August 1981) phocytes to mitogenic and antigenic stimulation. Taken together, these findings raise the possibility that procainamide induces the autoimmune lupus-like syndrome by its direct interaction with lymphocyte membranes. Clinical observations Seventeen patients were identified as having procainamide-induced lupus as evidenced by a history of continued procainamide therapy, the development of new symptoms compatible with lupus, a serum antinuclear antibody titer of 1 :10 or greater, and resolution of symptoms after discontinuation of the drug. The patients ranged in age from 49 to 83 years and 15 were male. The average total procainamide dose per patient in those with the drug-induced lupus syndrome was 695 gm, ranging from 60 to 1320. The clinical features of the procainamide-induced syndrome in our 17 patients included rheumatic complaints (arthralgia, diffuse myalgia, and arthritis) in 16; pleurisy or chest pain in 12; fever and weight loss in 7; and skin rash, mental confusion or headache, and Raynaud's phenomenon in 3 patients each. All subjects had a positive antinuclear antibody test, most with titers of 1:320 or greater. Serum antibodies to native doublestranded DNA (Farr technique) were not detected in any samples. Other immunologic abnormalities included positive direct and indirect Coombs tests, total leukocyte counts of less than 3000/mm3, and mild depressions in C3 or C4. A surprising finding was a circulating anticoagulant in 3 patients and an unexplained prolonged partial thromboplastin time (PTT) in another. BLUESTEIN ET AL 1020 Lymphocytotoxic antibodies Cold reactive (15OC) lymphocytotoxic antibodies were measured by a modification of the Terasaki microdroplet dye exclusion assay described in detail elsewhere (5). Peripheral blood lymphocytes (PBL) obtained from venous blood of normal volunteers by Ficoll-Hypaque density centrifugation were used as target cells. Each serum was tested against lymphocytes from at least 3 donors. The mean percentage of dead cells in triplicate assays was expressed as the percent cytotoxicity. To compare the relative cytotoxic capacity of each serum, a 50% cytotoxicity titer (CC,,) was determined. The CC,, is defined as the log, of the reciprocal of the greatest serum dilution producing 50% cytotoxicity. All sera were tested at an initial 1:2 dilution. Lymphocytotoxic antibodies (CC,, L 1) were detected in the serum of l l of the 17 subjects (65%) with procainamide-induced lupus, and one-half of the active sera had 5090 cytotoxicity titers of 1 :8 or greater. In contrast, antilymphocyte activity was found in sera from only 20% of 15 control subjects on long-term procainamide therapy who did not have the lupus syndrome, and none was positive at serum dilutions greater than 1 :2. Another control group consisted of 63 men between the ages of 45 and 65 who had not received procainamide. Only 3 of their sera produced 50% lymphocytotoxicity, none at greater than a 1 :2 serum dilution. There was no correlation between the titer of lymphocytotoxic antibody and the total amount of procainamide taken by the patients with drug-induced disease. Neither was there an association of the antibody titer and the number or severity of symptoms or other laboratory abnormalities. Sera obtained at varying intervals after cessation of drug therapy were available from 6 of the procainamide-induced lupus patients whose serum was Table 1. Sequential serum lymphocytotoxic antibody titers in patients with procainamide-induced lupus _____ ~ Lymphocytotoxicity(CC,,) Patient Sample 1* Sample 2* 2 3 5 0 0 1 5 5 2 3 6 3 Interval (weeks) ~~ RF WK LR GK GE PR 16 7 6 8 3 4 * Sample 1 was obtained at time of diagnosis before procainamide was discontinued. Sample 2 was obtained at the interval indicated after discontinuing procainamide therapy. lymphocytotoxic at the time of diagnosis. The titer of lymphocytotoxic antibody fell abruptly after discontinuation of procainamide (Table 1). Cytotoxic activity in all cases decreased 75% or more on retest after 3 to 16 weeks. The reduction was concomitant with the clearing of symptoms, but was independent of the antinuclear antibody titers which remained elevated and unchanged for many months. Effect of procainamide on mitogen and antigen responses of normal human lymphocytes Peripheral blood lymphocytes from healthy donors were cultured at a density of lo6cells/ml in RPMI1640 medium supplemented with 1oo/o pooled normal human plasma. Purified PHA was added to the cultures to a final concentration of 1, 0.1, or 0.05 pg/ml. Procainamide-HC1 obtained as a sterile aqueous solution was added to the cultures in the concentrations indicated. Cultures were incubated at 37°C in a humidified 5% C0,-in-air atmosphere for 72 hours and 18 to 24 hours before harvesting were pulsed with 1 pCi of 3Hthymidine. The cultured cells were collected onto glass fiber filter papers using an automated multiple sample harvester and the 3H-thymidine incorporated into acid insoluble material enumerated by liquid scintillation spectrometry. The effect of procainamide on PHA-stimulated 3H-thymidineincorporation is expressed as a percentage of the response obtained in the absence of the drug according to the formula: % response = PHA induced ’H CPM with drug PHA induced 3HCPM without drug loo The response of primed lymphocytes to antigenic stimulation in the presence of procainamide was also studied. Peripheral blood lymphocytes from individuals with positive tuberculin skin tests were cultured as described above with purified protein derivative (PPD, Connaught Laboratories) at 2 @culture and the percent response calculated the same as for PHA. Procainamide altered PHA-induced activation of normal human PBL. A biphasic dose response was observed with marked suppression at higher concentrations but enhancement at lower doses (Figure 1). Concentrations of procainamide of 3.75 x lO-’M or greater completely inhibited PHA-induced 3H-thymidineincorporation. However, at a tenfold lower drug concentration the response to PHA was enhanced 60% on the average beyond that obtained with PHA alone (P < 0.01 by Wilcoxon test; 0.02 > P > 0.01 by r-test). Higher PROCAINAMIDE-LYMPHOCYTE REACTIONS mean responses continued as the concentration of procainamide decreased to 10-6M but the differences were not as striking (P< 0.05 by Wilcoxon test and 0.1 > P > 0.05 by paired t-test at 3.75 X lO-$M). Enhancement of the response to PHA was observed best at suboptimal stimulating concentrations of the mitogen (0.05-0.0 1 pg/ml). A similar pattern of complete suppression at higher doses (3.75 X lO-’M and greater) and enhancement at lower concentrations was observed in studies of antigen stimulated lymphocyte activation. The in vitro response of PBL from tuberculin skin test positive individuals to PPD was 180% of normal at 3.75 x 10-4M procainamide (P < 0.01 by Wilcoxon and paired ttests). Effect of procainamide on in vitro antigenspecific antibody producing cell induction in rabbit spleen cells Procainamide has two effects on in vitro plaqueforming cell (PFC) responses of sheep erythrocyte (SRBC) primed rabbit lymphocytes. The normal kinetics of antiSRBC responses are a sharp peak on day 4, with a rapid decline by day 5 to less than 15% of the peak (Table 2). At t 2 mM procainamide, no effect on the peak response was observed at day 4 but there was a significant enhancement of the number of PFC on days 5 and 6. As the procainamide concentration increased, the total PFC response dropped dramatically. At 2.7 mM the day 4 response fell 8896, while the day 5 and day 6 responses rose 2- to 3-fold higher than the cultures that did not receive the drug. Discussion Procainamide administration to humans induces antinuclear antibodies and a disease resembling systemic lupus erythematosus. Antibodies to histones and denatured, but not native, DNA are a regular finding (6-8). These autoantibodies appear to result from shared drug-nucleoprotein antigenicity or drug-nucleoprotein interactions which enhance the immunogenicity of nucleic acid antigens (9-13). The relationship between the antinuclear antibodies and the drug-induced disease is ill defined. Those antibodies appear before clinical findings and persist in high titers for months to years after the drug is withdrawn and all symptoms have gone. Moreover, patients with procainamide-lupus show few stigmata of immune complex disease (3,14,15). Attributing the pathogenesis of procainamidelupus to antinuclear antibody-mediated immune com- 1021 w m z 0 n m w CT I- z W 80 0 a w n 1 I 10-6 10-5 10-4 10-3 PROCAINAMIDE CONC. (3.75 X M) Figure 1. The effect of procainamide on the in vitro response of normal human peripheral blood lymphocytes to phytohemagglutinin (PHA). For each procainamide concentration, the results are expressed as a percentage of the response to PHA (0.5 pg/ml) in the absence of added procainamide. Each point represents the mean of experiments, and the vertical bars represent the standard error (*). plexes disregards the findings of other autoantibodies in the drug-induced syndrome. As Blomgren and Vaughan noted, some patients have antibodies to IgG (rheumatoid factor) and red blood cells (13). Others have reported false positive serologic tests for syphilis, leukopenia, and thrombocytopenia (15). Three of our 17 Table 2. Effect of procainamide on sheep erythrocyte plaqueforming cell responses of primed splenic lymphocytes from the rabbit Procainamide concentration (M) 0 0 1.2 x 1.7 x 2.2 x 2.7 x 3.2 x PFC/106 cultured cells SRBC 0 10-3 10-3 10-3 10-3 10-3 + + + + + + Day3 Day4 Day5 Day6 158 226 3309 3800 2744 1629 713 99 20 523 1110 1346 985 1054 332 194 430 579 743 716 310 867 440 321 202 83 24 11 1022 patients had a circulating lupus anticoagulant. These observations suggest impaired immune regulation in patients with procainamide lupus analogous to that seen in the idiopathic form of the disease. Antilymphocyte antibodies are autoantibodies that can alter lymphocyte function (16). Individuals treated with procainamide have a 5 fold increased frequency of lymphocytotoxicantibodies compared to ageand sex-matched control subjects. Most patients with the lupus-like syndrome have circulating antilymphocyte activity at titers considerably higher than the clinically unaffected individuals. Furthermore, procainamide withdrawal is followed by a rapid disappearance of the lymphocytotoxic antibody concomitant with clinical improvement. Thus the presence, amount, and persistence of lymphocytotoxic antibodies parallel the exposure to procainamide and the development and resolution of disease. Procainamide-lymphocyte interactions have not been documented by others, but chlorpromazine and lidocaine were shown to inhibit cytotoxic murine T cells and the activation of murine spleen cells by various mitogens and bacterial lipopolysaccharides (17). The effects are dose-related, reversible, and not due to interference with mitogen binding to the lymphocyte membrane or impaired cell viability. Suppression of mitogen-induced activation of human peripheral blood lymphocytes and antigen-specific antibody responses of rabbit splenocytes by procainamide occurs at similar molar concentrations. Hyperresponsiveness of human lymphocytes to PHA was seen at procainamide concentrations that approximate therapeutic blood levels. A similar exaggerated effect on the activation of human lymphocytes has been observed with chlorpromazine (18)The biphasic effects of procainamide raise the possibility that at lower concentrations the drug interacts preferentially with a subpopulation of immunoregulatory cells. The effects of procainamide on the in vitro antibody-producing cell assay are under investigation to determine if selective alteration of helper or suppressor cell function can be documented. The preliminary results using sheep erythrocyte primed rabbit splenic lymphocytes document a profound suppression at drug concentrations above 3mM. Under 2mM, however, helper function is preserved. The increase in the PFC response later in the time course raises the possibility of selective loss of suppressor cell activity, since the normally rapid fall-off of the PFC response from its peak has been attributed to T cell-mediated suppression. However, a more detailed analysis of the effects of BLUESTEIN ET AL procainamide on isolated lymphocyte subpopulations will be needed to document selective immunoregulatory activity. The procainamide effects on lymphocyte activation are predicted by its “anesthetic” characteristics. Anesthetics as a class are lipid soluble drugs that are incorporated into, and exert their effect upon, cell membranes. Many tranquilizers, anticonvulsants, and antiarrhythmics are included in this class of compounds (19,20). Presumably procainamide intercalates into the lipid membrane bilayer, thereby altering its architecture. As a result, the immune system may be continuously exposed to new lymphocyte membrane determinants that stimulate the production of autoreactive lymphocyte antibodies. The antigenic stimulus should cease when the drug is removed. Based on this model, we would propose the following sequence of events in procainamide-induced lupus. Antinucleic acid antibodies appear either because of drug-nucleoprotein interactions, as previously suggested (6), or as an early indication of drug-induced alteration of immune regulation. The antinuclear antibodies serve as a marker of the illness but are not sufficient to induce the clinical symptoms. Further deterioration in immune regulation, caused by the direct effect of procainamide on lymphocyte membranes leads to development of disease. Antilymphocyte antibodies are a consequence of the drug-induced membrane alteration. While in the circulation they further impair normal lymphocyte function, as has been shown for the antilymphocyte antibodies in the idiopathic form of the disease (21,22). Thus a positive feedback loop, amplifying abnormalities of immune function, is established and persists only so long as the drug is present. The actual immunologic events responsible for tissue injury still require explanation, but we suggest looking beyond antinucleoprotein antibodies for the answer. REFERENCES 1. Blomgren SE, Condemi JJ, Bignall MC, Vaughan JH: Antinuclear antibody induced by procainamide: a prospective study. N Engl J Med 281:64-66, 1969 2. Molina J, Dubois EL, Bilitch M: Procainamide-induced serologic changes in asymptomatic patients. Arthritis Rheum 12:608-614, 1969 3. Lee SL, Chase PH: Drug-induced systemic lupus erythematosus: a critical review. Semin Arthritis Rheum 5:83-103, 1975 4. Tala1 N: Disordered immunologic regulation and autoimmunity. Transplant Rev 3 1:240-263, 1976 PROCAINAMIDE-LYMPHOCYTE REACTIONS 5 . Zvaifler NJ, Bluestein HG: Lymphocytotoxic antibody activity in cryoprecipitates in serum of patients with SLE. Arthritis Rheum 19:844-850, 1976 6. Tan EM: Drug-induced autoimmune disease. Fed Proc 33:1894-1897, 1974 7. Winfield JB, Davis JS: Anti-DNA antibody in procainamide-induced lupus erythematosus. Arthritis Rheum 17:97-110, 1974 8. Fitzler MJ, Tan EM: Antibodies to histones in drug-induced and idiopathic lupus erythematosus. J Clin Invest i2:560-567, 1978 9. Yamauchi Y, Litwin A, Adams K, Zimmer H, Hess EV: Induction of antibodies to nuclear antigen in rabbits by immunization with hydralazine-human serum albumin conjugates. J Clin Invest 56:958-969, 1975 10. Gold EF, Ben-Efraim S, Faivisewitz A, Steiner Z, Klajman A: Experimental studies on the mechanism of induction of antinuclear antibodies by procainamide. Clin Immunol Immunopathol7: 176-186, 1977 11. Waring MJ: Drugs which affect the structure and function of DNA. Nature 219:132&1325, 1968 12. Eldredge NT, Robertson WB, Miller JJ 111: The interaction of lupus inducing drugs with deoxyribonucleic acid. Clin Immunol Immunopathol 3:263-271, 1964 13. Blomgren SE, Condemi JJ, Vaughan JH: Procainamideinduced lupus erythematosus. Am J Med 52:338-348, 1972 14. Utsinger PD, Zvaifler NJ, Bluestein HG: Hypocomple- DISCUSSION Dr. Weigle: The sequence system of cellular events with pokeweed mitogen is complex. An accessory T cell requirement involves interactions among T cells, macrophages, and other accessory cells. Dr.ZvaMer: Our studies so far suggest that each of the factors is affected to some degree. Unanue has examined the effect of both lidocaine and chlorpromazine on macrophage function and on capping and patching in lymphocytes (Nature 25056, 1976). A group in Minneapolis (Ferguson RM,Schmidtke JR, Simmons RL: J Immunol 116:627-634, 1976) has looked extensively at pokeweed mitogen, phytohemagglutinin, and other responses. Lidocaine particularly and chlorpromazine are also good drugs for these studies. Dr. Reidenberg: Is the effect of NAPA identical to that of procainamide? Dr. Zvaifler: Yes. We originally thought it would be interesting to dissect the two, but the effect was essentially the same with each. Dr. Stollar: If there is an effect on lymphocytes, would one expect a wide spectrum of increased antibody or 1023 mentemia in procainamide-associated SLE. Ann Intern Med 84293-294, 1976 IS. Alarcon-Segovia D: Drug-induced lupus syndromes. Mayo Clin Proc 44:664-681, 1969 16. Bluestein HG: Autoantibodies to lymphocyte membrane antigens: pathogenetic implications. Clin Rheum Dis 4643459, 1978 17. Ferguson RM, Schmidtke JR, Simmons RL: Inhibition of mitogen produced lymphocyte transformation by local anesthetics. J Immunol 116:627-634, 1976 18. Ferguson RM, Schmidtke JR, Simmons RL: Effects of psychoactive drugs on in vitro lymphocyte activation. Original Article Series, XIV,pp 379406, 1978 19. Seeman P: The membrane actions of anesthetics and tranquilizers. Pharmacol Rev 24584-655, 1972 20. Ryan CB, Unanue ER, Karnovsky MJ: Inhibition of surface capping of macromolecules by local anesthetics and tranquilizers. Nature 2505657, 1974 21. Twomey JJ, Laughter AH, Steinberg AD: A serum inhibitor of immune regulation in patients with systemic lupus erythematosus. J Clin Invest 62:713-715, 1978 22. Sakane T, Steinberg AD, Reeves JP, Green I: Studies of immune functions of patients with systemic lupus erythematosus: complement-dependent immunoglobulin-'M anti-thymus-derived cell antibodies preferentially inactivate suppressor cells. J Clin Invest 63:954-965, 1979 immunoglobulin production or could the drug select a small population of factors? Dr. Zvaifler. One problem with antilymphocyte antibodies as significant pathogens is understanding their selectivity. It is quite clear that antilymphocyte antibodies show no specificity for lymphocyte subsets, and yet in a functional sense they clearly do. Whether this is because the turnovers are more rapid or the antigen density on some lymphocyte subsets is greater is not clear. But if you simply add antilymphocyte antibodies in vitro to lymphocytes, they react equally with any subset, such as suppressor or helper, as measured by cytotoxic assay. A number of antilymphocyte antibodies, particularly IgG, may well be more important than the ones we are measuring. Dr. Talal: Do these patients have hypergammaglobulinemia? Dr. Zvaifler: Yes. Some. Dr. Tala]: It would be interesting to make an anti-idiotype to an anti-lymphocyte antibody and study those with hypergammaglobulinemia but without other autoantibodies such as antinuclear antibodies. Perhaps other gamma globulins express this idiotype.