SUPPRESSION OF RHEUMATOID FACTOR PRODUCTION BY METHOTREXATE IN PATIENTS WITH RHEUMATOID ARTHRITIS

Transcription

1 1156 SUPPRESSION OF RHEUMATOID FACTOR PRODUCTION BY METHOTREXATE IN PATIENTS WITH RHEUMATOID ARTHRITIS Evidence for Differential Influences of Therapy and Clinical Status on IgM and IgA Rheumatoid Factor Expression GRACIELA S. ALARC6N, RALPH E. SCHROHENLOHER, ALFRED A. BARTOLUCCI, JOHN R. WARD, H. JAMES WILLIAMS, and WILLIAM J. KOOPMAN Suppression of rheumatoid factor (RF) production in rheumatoid arthritis (RA) has been variably attributed to the use of remittive agents per se or to clinical improvement associated with their use. There have been conflicting reports with regard to the influence of methotrexate (MTX) on serum RF levels in RA. We determined IgM-RF and IgA-RF levels in paired serum samples (obtained at study entry and completion) from RA patients enrolled in multicenter trials with the Cooperative Systematic Studies of Rheumatic Diseases program. After exclusion of the 14 IgM-RF-negative sera, there were samples from 30 MTX-treated patients and 52 placebo-treated patients. Changes in IgM-RF and IgA-RF levels were weakly associated with each other. Significant decreases in IgM-RF levels were observed in the MTX-treated patients, but not in the From the Division of Clinical Immunology and Rheumatol- OgY, Department of Medicine, and the Department of Biostatistics and Biomathematics, The University of Alabama at Birmingham, and the Veterans Administration Medical Center, Birmingham, Alabama; and the Coordinating Center for the Cooperative Systematic Studies of the Rheumatic Diseases, University of Utah, Salt Lake City. Supported in part by NIH grants AR-20614, AR-03555, and AI and by the Veterans Administration Research Program. Graciela S. Alarcbn, MD, MPH: Department of Medicine, The University of Alabama at Birmingham; Ralph E. Schrohenloher, PhD: Department of Medicine, The University of Alabama at Birmingham and the VA Medical Center; Alfred A. Bartolucci, PhD: Department of Biostatistics and Biomathematics, The University of Alabama at Birmingham; John R. Ward, MD: University of Utah: H. James Williams, MD: University of Utah; William J. Koopman, MD: Department of Medicine, The University of Alabama at Birmingham and the VA Medical Center. Address reprint requests to Graciela S. Alarcbn, MD, MPH, 603 MEB, The University of Alabama at Birmingham, Birmingham, AL Submitted for publication September 7, 1989; accepted in revised form February 14, placebo group. These changes were most significant in the MTX-treated patients who improved clinically. There were significant decreases in IgA-RF levels at study completion among MTX-treated patients who had improved clinically and those who had not improved clinically, but not in the placebo group. The contributions of clinical improvement and MTX treatment to changes in serum IgM-RF and IgA-RF levels were examined using a logistic regression model. Changes in IgM-RF were strongly related to MTX treatment and, to a lesser extent, to clinical improvement; changes in IgA-RF were related only to MTX treatment. These results indicate that MTX treatment per se decreases both IgM-RF and IgA-RF levels, whereas clinical improvement correlates with decreased IgM-RF levels only. The data are consistent with the view that MTX exerts a direct effect on RF production, which may reflect an important therapeutic action of this agent. There is considerable evidence indicating that methotrexate (MTX) is an effective agent for the treatment of rheumatoid arthritis (RA)(14). Although specific effects of MTX on folate metabolism have been extensively delineated (3, the mode of action of this agent in RA is unclear. Increased levels of serum rheumatoid factors (RF), autoantibodies directed against the Fc portion of IgG, are found in -80% of patients with RA, and this is generally correlated with increased disease seventy (for review, see ref. 6). Reductions in serum RF frequently accompany treatment of RA with established disease-modifying antirheumatic drugs (DMARDs), such as gold salts and D-penicillamine (7-12), and may reflect a therapeutically important action of these Arthritis and Rheumatism, Vol. 33, No. 8 (August 1990)

2 MTX SUPPRESSION OF RF 1157 agents. The possibility that MTX might exert an effect on RF production in RA is therefore of interest. Previous studies have generated conflicting evidence with regard to whether changes in RF occur during MTX therapy (2,3). It should be noted that those studies used semiquantitative assays for RF (i.e., latex fixation), which are not highly sensitive. The availability of sensitive quantitative immunoassays for IgM-RF and IgA-RF (13,14) prompted us to reevaluate longitudinally the influence of MTX on RF isotype levels in RA and to determine the relationship between RF changes and the subsequent clinical course. Our results indicate that MTX suppresses both IgM-RF and IgA-RF levels, while clinical improvement is associated with reductions in levels of IgM- RF, but not IgA-RF. These findings are compatible with the notion that suppression of immunologically driven inflammation in RA is an important aspect of the therapeutic action of MTX. PATIENTS AND METHODS Patients. The sera studied were from patients who were participants in double-blind clinical trials for the treatment of RA. All of the patients had definite or classic RA according to the 1958 American Rheumatism Association criteria (15). MTX samples were from 34 patients treated with MTX in the trial of MTX versus placebo (2). Placebo samples were from 62 patients enrolled in either the trial of MTX versus placebo (n = 39, which lasted 18 weeks (2), or the trial of sulfasalazine versus placebo versus parenteral gold (n = 27), which lasted 36 weeks (16). The latter placebo group included an unusually high proportion of patients who exhibited spontaneous clinical improvement. The addition of samples from this placebo group thus allowed us to meaningfully compare changes in RF levels in responders versus nonresponders, for both placebo and MTX groups. All serum samples studied were obtained at random from the total number of eligible patients in that particular trial group, and all participating clinics were represented in a proportion similar to their enrollment of patients in each trial. Sera obtained at each participating clinic at the time of the patient s enrollment in the corresponding trial and at the completion of the study, were stored at -20 C. and were sent periodically to the Coordinating Center for the Cooperative Systematic Studies of the Rheumatic Diseases in Salt Lake City, where they were stored at -70 C. Paired serum samples were shipped to Birmingham for IgM-RF and IgA- RF determinations. Serum samples were assayed for RF without the investigator s knowledge of the treatment modality (placebo or MTX) or clinical response. Patients in either the placebo or the MTX group whose initial serum sample contained <I0 pg of IgM-RF or IgA-RF at baseline were considered to be seronegative and were excluded from subsequent analyses. The final numbers of IgM-RF+ pa- tients in the placebo and MTX groups were 52 and 30, respectively. The numbers of IgA-RF+ patients in these 2 groups were 45 and 24. Clinical data. As described in detail previously (2,161, each participating clinic completed a detailed clinical evaluation of each patient upon enrollment and at the completion of the clinical trial. In addition to demographic data, the following clinical characteristics were recorded: joint counts and scores for pain and swelling; duration of morning stiffness (to the nearest 15 minutes); physician s (blinded observer) overall assessment of disease activity (&5 scale); and patient s overall assessment of disease activity (0-5 scale). For the purpose of this study, a patient s condition was defined as clinically improved if the joint swelling count improved by at least 50% ( responder ) (2); otherwise, the patient was classified as unimproved ( ncnresponder ). When other parameters, such as joint counts for paidtenderness (improved by at least 50%) and patient and physician overall assessments (improved by at least 2 ordinals) (2). were also included in the definition of clinical improvement, patients received the same classification by both definitions in 84% of the cases. However, decrease in joint swelling was chosen as the sole criterion for improvement in this study since it can be regarded as the most objective measure among those obtained. This definition of improvement was used for all analyses performed. RF assays. Serum levels of IgM-RF and IgA-RF were measured by an enzyme-linked immunosorbent assay (ELISA) modification of previously described radioimmunoassays (13,14). Polystyrene microtiter wells (Immulon-2 flat-bottom plates; Dynatech, Alexandria, VA) were coated with 0.2 ml of isolated human IgG (0.05 mg/rnl) in O.15M NaCl buffered with 0.02M phosphate buffered saline (PBS) at ph 7.4, for 1 hour at room temperature. The remaining protein-binding sites on the wells were blocked by incubation for an additional hour with 0.4 ml of 0.1% bovine serum albumin (BSA) in PBS. Duplicate 0.2-ml samples of reference standards, unknowns, and controls, diluted in PBS containing 1% BSA and 0.02% sodium azide (PBS-BSA), were placed in the IgG-coated wells and incubated for 16 hours at room temperature. The wells were then aspirated, washed 3 times with 0.05% Tween 20 in PBS, and reacted for 1 hour with 0.2 ml of alkaline phosphatase-labeled, affinity-purified F(ab ), fragments of rabbit anti-human IgM (p chain-specific; Pel- Freez, Rogers, AR), diluted 1:5,OOO in PBS-BSA, in order to detect IgM bound to the wells. For determination of IgA-RF, the wells were reacted with alkaline phosphatase-labeled, affinity-purified F(ab ), fragments of rabbit anti-human IgA (a chain-specific; Pel-Freez), diluted 15,000, to detect bound IgA. Unbound enzyme-labeled antibody was removed by washing 3 times with 0.05% Tween 20, and the wells were reacted with 0.2 ml of disodium paranitrophenyl phosphate, 1 mdml in 0.05M sodium carbonate buffer (ph 9.8) containing 1 mm magnesium chloride, for 30 minutes at room temperature. Color development was stopped by adding 0.05 ml of IN sodium hydroxide, and absorbance was measured at 405 nm using a Dynatech MR580 or Bio-Rad (Richmond, CA) model 2550 microplate reader. Quantities of IgM-RF were estimated by comparison with curves constructed using a monoclonal IgM-RF refer-

3 ~ 1158 ALARC6N ET AL ence standard (protein LEW), over a dose range of ng/ml (13). Quantities of IgA-RF were estimated using a monoclonal polymeric IgA paraprotein with RF activity (protein SCH) as the reference standard, over the same dose range (14). All results were corrected for nonspecific binding of serum IgM or IgA by parallel analysis using microtiter plates coated with BSA only (13,14). Results for both IgM-RF and IgA-RF are expressed in pg/ml. Interassay variation was precluded by simultaneous analysis of the paired sera, and RF values were calculated from results obtained with 2 replicate dilutions (usually 1:1,OOO) of each serum, in order to further increase the precision of the results. The intraassay coefficient of variation, as indicated by the analysis of 8 replicate dilutions for each of 3 RA sera, was 4.2% for IgM-RF and 4.9% for IgA-RF. For the purpose of this study, RF levels were considered to have changed significantly if the values at study entry and study completion differed by at least 10% or 10 pg/ml, whichever was greater. The 10% level was chosen because it represents roughly twice the coefficient of variation for both IgM-RF and IgA-RF assays; 10 pg was chosen to minimize errors when changes in lower values were considered. In order to corroborate the analysis and eliminate the possible effect of using smaller percentage changes to represent significant differences, data were also analyzed using changes of 15% and 20% as arbitrary definitions of significance. Statistical analysis. Differences between means were examined using Student's z-test. Differences between proportions were examined using the chi-square distribution. A 1-tailed 1-test (decrease in serum levels) was used to compare the difference between mean values of RF isotypes at study entry and study completion. P values less than 0.05 were considered significant. The kappa statistic was calculated to determine comparability of changes between serum levels of IgM-RF and IgA-RF; this statistic is an indicator of agreement between 2 sets of measurements, where a value of I indicates perfect agreement and 0 indicates none. Since placebo samples were obtained from patients enrolled in 2 different studies with different durations of followup (18 weeks and 36 weeks), a regression analysis involving only the placebo group was performed, in which changes in serum IgM-RF and IgA-RF levels were the dependent variables, and clinical response and duration of followup were the independent variables. Duration of followup did not significantly influence changes in IgA-RF or IgM-RF levels, Therefore, the samples from the placebotreated patients were treated as a single group in subsequent analyses. The relationships between changes in serum IgM- RF and IgA-RF levels (dependent variable) and clinical improvement and therapeutic modality (independent variables) were subsequently analyzed. RESULTS Of the 96 patients originally selected for study, 14 were found to be seronegative for IgM-RF and were excluded from further analyses. Thus, the results reported are for 82 pairs of sera (52 from the placebo Table 1. Selected clinical and demographic features of the rheumatoid arthritis patients studied* Age, mean 2 SD Feature Meth- Placebo otrexate group group (n = 52) (n = 30) % female % Caucasian * Years of disease, mean 2 SD % with extraarticular manifestationst % with clinical improvement * All patients had serum IgM rheumatoid factor levels of 210 &ml. There was no significant difference between the 2 treatment groups for any of the features shown. t Defined as vasculitis, sicca syndrome, andor orgadsystem involvement. group and 30 from the MTX group). Forty-five of the placebo-treated patients and 24 of the MTX-treated patients, respectively, were positive for IgA-RF. The 2 treatment groups had similar demographic characteristics (Table I). The majority of the patients in both groups were Caucasian women. Age and disease duration were comparable in the 2 groups, as was the frequency of extraarticular manifestations. Although more patients in the MTX group than in the placebo group had clinical improvement, the difference was not statistically significant. Relationship between serum changes in IgA-RF and serum changes in IgM-RF. IgA-RF and IgM-RF values tended to change in the same direction in the majority of the patients. However, the level of agreement, though significant, was modest (K = 0.607,95% confidence interval ). Treatment modality, clinical response, and changes in RF. The relationship of treatment and clinical response to changes in the IgM-RF level and the relationship of treatment and clinical response to changes in the IgA-RF level are depicted in Tables 2 and 3, respectively. There was no significant decrease in serum IgM-RF levels in the placebo-treated patients (mean f SD 247 f 277 pg/ml at entry versus &ml at completion), but a significant decrease in IgM-RF levels was observed in the MTX-treated patients (311 f 426 pg/ml at entry versus 210 * 453 pg/ml at completion; P = 0.02) (Table 2). Similarly, a decrease in the level of IgA-RF ( LLglml at entry versus pg/ml at completion) was observed in the MTX-treated patients (P = 0.001) (Table 3). When patients in the MTX treatment group were categorized according to clinical response, there was a significant -

4 MTX SUPPRESSION OF RF 1159 Table 2. Serum levels of IgM rheumatoid factor (IgM-RF) in the rheumatoid arthritis patients studied Study group, clinical status IgM-RF (mean f SD &nl) at Study final visit n entry Final visit Change Methotrexate Improved * Unimproved f f k 283 All f ' -101? 252 Placebo Improved ? f Unimproved * All f f * P = 0.02 versus study entry. decrement in the serum levels of IgM-RF only in those who improved clinically (Table 2). Decreases in the serum IgA-RF levels were significant both in clinically improved and clinically unimproved patients in the MTX group (Table 3). In the placebo group, no significant decrease in IgM-RF or IgA-RF was observed in either the clinically improved or the clinically unimproved patient subgroups. Results from the multiple logistic regression analyses are shown in Table 4. Changes in IgM-RF were strongly related to treatment modality (MTX) (P = ) and, to a lesser extent, to clinical improvement (P = ). In contrast, changes in IgA-RF were related only to treatment modality (MTX) (P = ) and not to clinical improvement (P = ). Similar results were obtained using changes of 15% and 2W0, instead of lo%, as arbitrary definitions of significant changes in RF levels (data not shown). Results of the multiple logistic regression were consis- Table 3. Serum levels of IgA rheumatoid factor (IgA-RF) in the rheumatoid arthritis patients studied Study group. clinical status IgA-RF (mean f SD pg/ml) at Study Final final visit n entry visit Change Methotrexate Improved C f 38' Unimproved t -12 & I1 All f41t Placebo Improved Unimproved f f All f 65 3 f 29 * P = versus study entry. t f = versus study entry. Table 4. Logistic regression analyses of changes in IgM rheumatoid factor (IgM-RF) and IgA-RF levels, according to treatment modality and clinical status, in the rheumatoid arthritis patients studied AlgM-RF AlgA-RF Independent variable X2 P X2 P Treatment modality O.OOO Clinical improvement tent with the descriptive data presented in Tables 2 and 3, in that the major modifier for serum levels of IgM-RF and IgA-RF was treatment modality. DISCUSSION We studied serial serum samples obtained from RA patients treated with either methotrexate or placebo, in order to determine the effect of MTX on the production of RF. The definition of improvement used in our study is regarded as standard in clinical rheurnatologic trials and was chosen from among several parameters as being the most objective measure of clinical improvement. The inclusion of a large number of placebo-treated patients, with an unusually high proportion of individuals who improved clinically (161, permitted analysis of the relative influence of both variables (MTX treatment and clinical improvement) on RF levels. Approximately one-third of the patients in the MTX group in the present study responded to this treatment; this proportion is essentially identical to that observed in the overall group of patients receiving MTX in the clinical trial from which our patients were drawn (2). Our data indicate that IgA-RF and IgM-RF levels are differentially influenced by therapy per se and clinical status. Clearly the most significant variable affecting serum levels of IgM-RF and IgA-RF was treatment with MTX, whereas clinical improvement showed a modest relationship with IgM-RF levels and was essentially not related to IgA-RF levels. Previous studies of MTX-treated patients (2,3) have sequentially analyzed the serum levels of IgM-RF using the latex fixation test. In the Cooperative Clinics study, there was a significant decrease in RF titer in the MTX-treated patients (2), whereas no significant changes in RF titer were observed in the study by Weinblatt et a1 (3). It is conceivable that changes in IgM-RF occurred in the latter study as well, but were

5 1160 ALARC6N ET AL missed due to the relative insensitivity of the agglutination assay employed. We considered the possibility that changes in serum levels of IgM-RF and IgA-RF over time could be nonspecific, related to changes in the overall production of immunoglobulin. To address this issue, total serum levels of both IgM and IgA were measured by ELISA in all sample pairs that were available for testing (16 from the MTX group and 52 from the placebo group). Briefly, the changes observed in IgM- RF and IgA-RF did not correlate with changes in total IgM and IgA levels in the MTX group, and were correlated only moderately in the placebo group. Since relatively fewer samples were available from the MTX-treated patients than from the placebo-treated patients, no definite conclusions can be reached regarding the specificity of the changes in serum levels of IgM-RF and IgA-RF observed in this study. In vitro studies have demonstrated a decrease in spontaneous and pokeweed mitogen-induced production of IgM-RF by peripheral blood mononuclear cells obtained from RA patients being treated with either MTX or other DMARDs (17,18). The influence of clinical improvement per se on RF production was not investigated in those patients. In another group of prospectively studied RA patients undergoing MTX treatment, a rapid decrease in serum IgM-RF levels and in vitro production of RF was observed, but the relationship of these changes to clinical improvement was not determined (18). More recently, Olsen and colleagues observed an independent association of both DMARD therapy and in vitro IgM-RF production with disease activity in a cross-sectional study of 133 patients with RA, 48 of whom were taking a DMARD (including 22 taking MTX) (12). Although that study clearly demonstrated a correlation between disease activity and in vitro RF production, the possibility of an association between DMARD therapy per se and suppression of RF production was not excluded by the data. Results of these previous studies, together with the data reported here, suggest that MTX exerts a direct effect on RF production in RA. Although the mechanism for this suppressive influence is unclear, a recent report suggesting that MTX inhibits the activity of interleukin-1 (19) offers an attractive possibility. Although MTX treatment per se strongly influenced both IgM-RF and IgA-RF levels in this study, it is evident that ciinical improvement also contributed to decreases in IgM-RF. These results are consistent with those of previous studies indicating a relationship between IgM-RF levels and clinical status (2b22). In contrast, the lack of correlation between IgA-RF levels and clinical status conflicts with previous reports that IgA-RF is associated with disease activity (23-25). It should also be noted that, in some of the latter studies, the patients had early disease (23), and in others, the study populations included patients with rheumatic diseases other than RA (24,25). In contrast, our patients had well-established RA, and the treatment modality may have influenced the results. Nevertheless, our data are consistent with other reports suggesting that IgM-RF and IgA-RF levels are independently regulated (26). In summary, methotrexate exerts a direct effect on the production of both IgM-RF and IgA-RF, and this may be an important aspect of the therapeutic efficacy of this drug in RA. Further studies of the mechanisms underlying this effect will, hopefully, foster the development of more effective, less toxic therapies for this disease. ACKNOWLEDGMENTS We are indebted to Drs. D. 0. Clegg (University of Utah), R. F. Willkens (University of Washington), J. Klippel (NIH), L. M. Billingsley (Johns Hopkins University), J, F. Singer (State University of New York Health Sciences Center at Brooklyn), V. D. Steen (University of Pittsburgh), M. E. Luggen (University of Cincinnati), R. P. Polisson (Duke University), and H. E. Paulus (University of California, Los Angeles), who were the directors of the participating clinics at the time this study was performed. We are also indebted to Miki Kaarg (Coordinating Center, Cooperative Systematic Studies of the Rheumatic Diseases, University of Utah) and to Ella Henderson (The University of Alabama at Birmingham) for their expert assistance in conducting the study and preparing the manuscript. REFERENCES 1. Thompson RN, Watts C, Edelman J, Esdaile 3, Russell AS: A controlled two-centre trial of parenteral methotrexate therapy for refractory rheumatoid arthritis. 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