440
IMMUNE COMPLEXES IN RHEUMATOID ARTHRITIS SERA AND SYNOVIAL FLUIDS A Comparison of Three Methods JAMES T. HALLA, JOHN E. VOLANAKIS, and RALPH E. SCHROHENLOHER Sera and synovial fluids from 88 patients with rheumatoid arthritis were examined for circulating immune complexes by three assays: monoclonal rheumatoid factor radioimmunoassay, C l q binding assay, and Raji cell radioassay. Paired samples were available for 82 patients. Immune complexes were detected with high frequency in the synovial fluid by each assay (75% by the monoclonal rheumatoid factor radioimmunoassay, 95% by the C l q binding assay, and 61% by the Raji cell radioassay). In rheumatoid arthritis sera, immune complexes were detected with high frequency by the Clq binding assay (85%) and the monoclonal rheumatoid factor radioimmunoassay (70%) but infrequently by the Raji cell radioassay (26%). The presence of immune complexes in serum was most frequently accompanied by the presence of complexes in fluid, regardless of the method of detection; moreover, the levels of immune complexes in synovial fluid were generally higher than in paired serum. Further, the levels of immune complexes as measured by the Clq binding assay correlated with certain parameters of clinical activity, while the monoclonal rheumatoid factor radioimmunoassay and Raji cell radioassay correlated with extraarticular features (excluding nodules) of rheumatoid arthritis. From the Department of Medicine, Division of Clinical Immunology and Rheumatology, and the Department of Pathology, University of Alabama in Birmingham. Supported in part by grant number AM-03555 from the U.S. Public Health Service. James T. Halla, MD; John E. Volanakis, MD; Ralph E. Schrohenloher, PhD. Address reprint requests to James T. Halla, MD, Division of Clinical Immunology and Rheumatology, University Station, University of Alabama in Birmingham, Birmingham, Alabama 35294. Submitted for publication November 5, 1978: accepted in revised form January 16, 1979.
Arthritis and Rheumatism, Vol. 22, No. 5 (May 1979)
Available evidence suggests that immunologic processes initiate and/or sustain the inflammation of rheumatoid arthritis. This concept is based in part on the detection of immune complexes and complement abnormalities in both sera and synovial fluids from patients with this disease (1,2). Although numerous methods have been used for the detection of immune complexes, data comparing various procedures in rheumatoid arthritis are limited. The authors have previously presented (3) comparative data on the detection of immune complexes in sera and synovial fluids of rheumatoid arthritis patients by a radioimmunoassay based on reaction with monoclonal rheumatoid factor and gel diffusion procedures that use monoclonal rheumatoid factor and Clq. Immune complexes were detected at similar high frequencies in rheumatoid synovial fluids by precipitin methods and the radioimmunoassay; in contrast, immune complexes were detected with a much higher frequency in rheumatoid sera by the radioimmunoassay than by the precipitin methods. Levels of immune complexes noted by the radioimmunoassay in the sera were generally lower than those found in the synovial fluids. In addition, preliminary data from this and another laboratory have been obtained on the comparison of certain radioassays in various rheumatic diseases including rheumatoid arthritis (4-6). Different patterns of reactivity were observed for various diseases indicating differences in the biologic properties of the immune complexes detected. The frequency and levels of immune complexes by three radioassays (monoclonal rheumatoid factor radioimmunoassay, C 1 q binding assay, and Raji cell radioassay) in rheumatoid arthritis sera and synovial fluids are examined in the present study. Further, the
44 I
IMMUNE COMPLEXES IN RA
relationship between the results o f each assay and various clinical parameters was studied. T h e three radioassays employed in this study recognize different biologic properties of immune complexes. Detection of immune complexes by C 1q is dependent on reaction with classic complement pathway binding sites on either IgG or IgM molecules (7). Paji cell reactivity depends on the presence o f C3 fragments on the surface of immune complexes (8); in addition, the presence of staphylococcal protein-A-reactive immunoglobulin molecules is also required for detection. Monoclonal rheumatoid factors detect immune complexes by reacting with structures on the Fc portion of IgG (9).
MATERIALS AND METHODS Sera and synovial fluids from 88 patients with definite or classic rheumatoid arthritis (RA) were studied for the presence of immune complexes. Paired samples were available in 82 patients. Determination of clinical stages was performed according to Steinbrocker et al(10); joint count was based on the number of tender and swollen joints; radiographic review was performed independently by two rheumatologists on hand films obtained at the time sera were collected; duration of morning stiffness, disease duration, and the presence of extraarticular features were determined by clinical assessment and chart review. Clinical and radiographic review was performed without prior knowledge of results of the immune complex assays. Forty-four (50%) patients were female, 76 (86%) were seropositive, 32 (36%) had antinuclear antibodies, and 39 (44%)had subcutaneous nodules. Fourteen (16%) had extraarticular manifestations of RA (excluding nodules) including pulmonary involvement in 6, vasculitis in 7, and severe constitutional symptoms with Still's type onset in 1. Forty-five non-RA patients were also studied for comparison. Paired sera and synovial fluids were examined in 32. Diagnosis in these patients included osteoarthritis in 27, Reiter's in 6, traumatic synovitis in 5, gout in 3, septic arthritis in 2, osteochondritis in 1, and juvenile rheumatoid arthritis in I . Erythrocyte sedimentation rate was measured by the Westergren method (1 I), rheumatoid factor titers by the latex fixation method (12), and antinuclear antibodies (ANA) by indirect immunofluorescence. After consent, sera and synovial fluids were obtained and aliquots were stored at -70°C. Each specimen was analyzed for the presence of immune complexes by a monoclonal rheumatoid factor radioimmunoassay (MRF-RIA), the C Iq binding assay (Clq-BA), and the Raji cell radioassay (RCRA). The MRF-RIA was based on the inhibition of the binding of '251-labeled heat aggregated human IgG to a monoclonal rheumatoid factor-latex immunoadsorbent (3). The Clq-BA was performed as described by Zubler et a1 (7) by using human C l q purified by affinity chromatography on concanavalin A-Sepharose (13) and labeled with "'1 by the lactoperoxidase method (14). The RC-RA was performed as described by Theofilopoulos et a1 (15) except that 12SI-labeled staphylococcal protein-A (SpA) was used in place of I2'I-la-
beled anti-IgG globulin (16). Analysis of control sera from 28 healthy laboratory and hospital personnel gave 4.4 f 2.3 pg heat aggregated human IgG (HAG) equivalents/ml (mean f SD) for the MRF-RIA, 10.9 f 1.5 percent C l q bound for the Clq-BA, and 1.7 f 3.0 pg HAG equivalents/ml for the RCRA. The upper limit of normal for sera was taken as the mean + 2 SD. The coefficient of variation for the Clq-BA as determined on two pathologic sera assayed nine times each was 11%. The coefficient of variation for the RC-RA determined on 5 pathologic sera assayed 10 times each was IW' at high levels of activity (500 pg HAG equivalents/ml) and 100% at low levels of activity (9 pg HAG equivalents/ml). The average variation for 7 pathologic specimens analyzed on three occasions was 25% for the MRF-RIA. Density gradient ultracentrifugation was performed in a Beckman L2 ultracentrifuge using an SW-50 rotor. Samples (0.2 ml) were applied to linear 12-30% sucrose gradients prepared in pH 7.4 phosphate buffered saline (PBS) and centrifuged at 33,000 rpm and 2°C for 5 hours. Serial fractions were collected by flotation from the top of the tube, and the remaining pellet was dissolved in 0.2 ml of 1% BSA in PBS. The positions of 7 s and 19s proteins were determined by double agar diffusion with anti-IgG and anti-IgM antisera, respectively. Each fraction was tested for immune complexes by the RC-RA as described. The presence of immune complexes by the Clq-BA was evaluated after the addition of an equal volume of normal human serum (NHS). Immune complexes reacting with monoclonal rheumatoid factor were detected by a sandwich-type radioassay (monoclonal rheumatoid factor binding assay on MRF-BA). The latter procedure was based on detecting immune complexes bound to the monoclonal rheumatoid factor-latex immunoabsorbent by reaction with 12'I-MRF. A standard curve was prepared by using heat aggregated human IgG, and results reported as pg HAG equivalents/ml. An unpaired Student's t test was used to compare mean values in the RA and non-RA groups. The difference between the frequency of positive results for each radioassay was analyzed by the chi-square method. Correlation coefficients were calculated by using Spearman's rank correlation test.
RESULTS Individual values given by each assay for sera from R A and non-RA patients are indicated in Figure 1. Elevated values by the M R F - R I A were found in 62/ 88 (70%) R A sera and in 7/36 (20%) non-RA sera. Similarly, 74/88 (85%) R A sera and 11/35 (31%) non-RA sera gave elevated values by the Clq-BA. W h e n analyzed by the RC-RA, 26/88 (30%) R A sera and only l / 36 (3%) non-RA sera gave elevated values. T h e difference in the frequency of positive results in R A and nonR A sera was significant for each assay (P < 0.001 for M R F - R I A and Clq-BA and P = 0.01 for RC-RA). The levels by the M R F - R I A and C l q - B A were generally higher in RA patients, and the mean levels by both as-
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IMMUNE COMPLEXES IN RHEUMATOID ARTHRITIS SERA AND SYNOVIAL FLUIDS A Comparison of Three Methods JAMES T. HALLA, JOHN E. VOLANAKIS, and RALPH E. SCHROHENLOHER Sera and synovial fluids from 88 patients with rheumatoid arthritis were examined for circulating immune complexes by three assays: monoclonal rheumatoid factor radioimmunoassay, C l q binding assay, and Raji cell radioassay. Paired samples were available for 82 patients. Immune complexes were detected with high frequency in the synovial fluid by each assay (75% by the monoclonal rheumatoid factor radioimmunoassay, 95% by the C l q binding assay, and 61% by the Raji cell radioassay). In rheumatoid arthritis sera, immune complexes were detected with high frequency by the Clq binding assay (85%) and the monoclonal rheumatoid factor radioimmunoassay (70%) but infrequently by the Raji cell radioassay (26%). The presence of immune complexes in serum was most frequently accompanied by the presence of complexes in fluid, regardless of the method of detection; moreover, the levels of immune complexes in synovial fluid were generally higher than in paired serum. Further, the levels of immune complexes as measured by the Clq binding assay correlated with certain parameters of clinical activity, while the monoclonal rheumatoid factor radioimmunoassay and Raji cell radioassay correlated with extraarticular features (excluding nodules) of rheumatoid arthritis. From the Department of Medicine, Division of Clinical Immunology and Rheumatology, and the Department of Pathology, University of Alabama in Birmingham. Supported in part by grant number AM-03555 from the U.S. Public Health Service. James T. Halla, MD; John E. Volanakis, MD; Ralph E. Schrohenloher, PhD. Address reprint requests to James T. Halla, MD, Division of Clinical Immunology and Rheumatology, University Station, University of Alabama in Birmingham, Birmingham, Alabama 35294. Submitted for publication November 5, 1978: accepted in revised form January 16, 1979.
Arthritis and Rheumatism, Vol. 22, No. 5 (May 1979)
Available evidence suggests that immunologic processes initiate and/or sustain the inflammation of rheumatoid arthritis. This concept is based in part on the detection of immune complexes and complement abnormalities in both sera and synovial fluids from patients with this disease (1,2). Although numerous methods have been used for the detection of immune complexes, data comparing various procedures in rheumatoid arthritis are limited. The authors have previously presented (3) comparative data on the detection of immune complexes in sera and synovial fluids of rheumatoid arthritis patients by a radioimmunoassay based on reaction with monoclonal rheumatoid factor and gel diffusion procedures that use monoclonal rheumatoid factor and Clq. Immune complexes were detected at similar high frequencies in rheumatoid synovial fluids by precipitin methods and the radioimmunoassay; in contrast, immune complexes were detected with a much higher frequency in rheumatoid sera by the radioimmunoassay than by the precipitin methods. Levels of immune complexes noted by the radioimmunoassay in the sera were generally lower than those found in the synovial fluids. In addition, preliminary data from this and another laboratory have been obtained on the comparison of certain radioassays in various rheumatic diseases including rheumatoid arthritis (4-6). Different patterns of reactivity were observed for various diseases indicating differences in the biologic properties of the immune complexes detected. The frequency and levels of immune complexes by three radioassays (monoclonal rheumatoid factor radioimmunoassay, C 1 q binding assay, and Raji cell radioassay) in rheumatoid arthritis sera and synovial fluids are examined in the present study. Further, the
44 I
IMMUNE COMPLEXES IN RA
relationship between the results o f each assay and various clinical parameters was studied. T h e three radioassays employed in this study recognize different biologic properties of immune complexes. Detection of immune complexes by C 1q is dependent on reaction with classic complement pathway binding sites on either IgG or IgM molecules (7). Paji cell reactivity depends on the presence o f C3 fragments on the surface of immune complexes (8); in addition, the presence of staphylococcal protein-A-reactive immunoglobulin molecules is also required for detection. Monoclonal rheumatoid factors detect immune complexes by reacting with structures on the Fc portion of IgG (9).
MATERIALS AND METHODS Sera and synovial fluids from 88 patients with definite or classic rheumatoid arthritis (RA) were studied for the presence of immune complexes. Paired samples were available in 82 patients. Determination of clinical stages was performed according to Steinbrocker et al(10); joint count was based on the number of tender and swollen joints; radiographic review was performed independently by two rheumatologists on hand films obtained at the time sera were collected; duration of morning stiffness, disease duration, and the presence of extraarticular features were determined by clinical assessment and chart review. Clinical and radiographic review was performed without prior knowledge of results of the immune complex assays. Forty-four (50%) patients were female, 76 (86%) were seropositive, 32 (36%) had antinuclear antibodies, and 39 (44%)had subcutaneous nodules. Fourteen (16%) had extraarticular manifestations of RA (excluding nodules) including pulmonary involvement in 6, vasculitis in 7, and severe constitutional symptoms with Still's type onset in 1. Forty-five non-RA patients were also studied for comparison. Paired sera and synovial fluids were examined in 32. Diagnosis in these patients included osteoarthritis in 27, Reiter's in 6, traumatic synovitis in 5, gout in 3, septic arthritis in 2, osteochondritis in 1, and juvenile rheumatoid arthritis in I . Erythrocyte sedimentation rate was measured by the Westergren method (1 I), rheumatoid factor titers by the latex fixation method (12), and antinuclear antibodies (ANA) by indirect immunofluorescence. After consent, sera and synovial fluids were obtained and aliquots were stored at -70°C. Each specimen was analyzed for the presence of immune complexes by a monoclonal rheumatoid factor radioimmunoassay (MRF-RIA), the C Iq binding assay (Clq-BA), and the Raji cell radioassay (RCRA). The MRF-RIA was based on the inhibition of the binding of '251-labeled heat aggregated human IgG to a monoclonal rheumatoid factor-latex immunoadsorbent (3). The Clq-BA was performed as described by Zubler et a1 (7) by using human C l q purified by affinity chromatography on concanavalin A-Sepharose (13) and labeled with "'1 by the lactoperoxidase method (14). The RC-RA was performed as described by Theofilopoulos et a1 (15) except that 12SI-labeled staphylococcal protein-A (SpA) was used in place of I2'I-la-
beled anti-IgG globulin (16). Analysis of control sera from 28 healthy laboratory and hospital personnel gave 4.4 f 2.3 pg heat aggregated human IgG (HAG) equivalents/ml (mean f SD) for the MRF-RIA, 10.9 f 1.5 percent C l q bound for the Clq-BA, and 1.7 f 3.0 pg HAG equivalents/ml for the RCRA. The upper limit of normal for sera was taken as the mean + 2 SD. The coefficient of variation for the Clq-BA as determined on two pathologic sera assayed nine times each was 11%. The coefficient of variation for the RC-RA determined on 5 pathologic sera assayed 10 times each was IW' at high levels of activity (500 pg HAG equivalents/ml) and 100% at low levels of activity (9 pg HAG equivalents/ml). The average variation for 7 pathologic specimens analyzed on three occasions was 25% for the MRF-RIA. Density gradient ultracentrifugation was performed in a Beckman L2 ultracentrifuge using an SW-50 rotor. Samples (0.2 ml) were applied to linear 12-30% sucrose gradients prepared in pH 7.4 phosphate buffered saline (PBS) and centrifuged at 33,000 rpm and 2°C for 5 hours. Serial fractions were collected by flotation from the top of the tube, and the remaining pellet was dissolved in 0.2 ml of 1% BSA in PBS. The positions of 7 s and 19s proteins were determined by double agar diffusion with anti-IgG and anti-IgM antisera, respectively. Each fraction was tested for immune complexes by the RC-RA as described. The presence of immune complexes by the Clq-BA was evaluated after the addition of an equal volume of normal human serum (NHS). Immune complexes reacting with monoclonal rheumatoid factor were detected by a sandwich-type radioassay (monoclonal rheumatoid factor binding assay on MRF-BA). The latter procedure was based on detecting immune complexes bound to the monoclonal rheumatoid factor-latex immunoabsorbent by reaction with 12'I-MRF. A standard curve was prepared by using heat aggregated human IgG, and results reported as pg HAG equivalents/ml. An unpaired Student's t test was used to compare mean values in the RA and non-RA groups. The difference between the frequency of positive results for each radioassay was analyzed by the chi-square method. Correlation coefficients were calculated by using Spearman's rank correlation test.
RESULTS Individual values given by each assay for sera from R A and non-RA patients are indicated in Figure 1. Elevated values by the M R F - R I A were found in 62/ 88 (70%) R A sera and in 7/36 (20%) non-RA sera. Similarly, 74/88 (85%) R A sera and 11/35 (31%) non-RA sera gave elevated values by the Clq-BA. W h e n analyzed by the RC-RA, 26/88 (30%) R A sera and only l / 36 (3%) non-RA sera gave elevated values. T h e difference in the frequency of positive results in R A and nonR A sera was significant for each assay (P < 0.001 for M R F - R I A and Clq-BA and P = 0.01 for RC-RA). The levels by the M R F - R I A and C l q - B A were generally higher in RA patients, and the mean levels by both as-
HALLA ET AL
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