988

IgG RHEUMATOID FACTOR Relationship to Seropositive Rheumatoid Arthritis and Absence in Seronegative Disorders RICHARD M. POPE and SANDRA J. McDUFFY

IgC rheumatoid factor was detected in the sera of the majority of patients with seropositive rheumatoid arthritis. Values suggestive of IgG rheumatoid factor were noted in one-quarter of patients with seronegative inflammatory arthropathies. These determinations were always low and correlated with elevated IgC concentrations, suggesting nonspecific adherence of IgG rather than a true antigen-antibody reaction. In support of this conclusion, nonrheumatoid factor IgC was capable of concentrationdependent nonspecific adsorption to the solid phase. IgC, but not IgM, rheumatoid factor corresponded with disease activity in patients with seropositive rheumatoid arthritis, suggesting that IgC rheumatoid factor may be important in the pathogenesis. IgG autoantibodies to IgG (IgG rheumatoid factors) may be of considerable importance in the pathogenesis of rheumatoid arthritis. Immune complexes containing IgG rheumatoid factors (RF) have been detected in the serum ( l a ) , synovium (5,6), and synovial fluids (7-9) of patients with rheumatoid arthritis. Analysis of complexes obtained from patient sera (3,4) From the Division of Rheumatology, Department of Medicine, University of Texas Health Science Center at San Antonio, and Audie Murphy Veterans AdministrationHospital. Supported by an Arthritis.Clinical Research Center Grant from the Arthritis Foundation and by grants from the South Central Texas Chapter of the Arthritis Foundation and by General Research Support Grant RR-05643 from the National Institutes of Health. Richard M. Pope, M D Assistant Professor, Department of Medicine; Sandra J. McDuffy, MS Research Assistant. Address reprint requests to Richard M. Pope, MD, University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Rheumatology, 7703 Floyd Curl Drive, San Antonio, Texas 78284. Submitted for publication December 26, 1978; accepted in revised form April 16, 1979. Arthritis and Rheumatism, Vol. 22, No. 9 (September 1979)

and synovial fluids (9) demonstrated that certain complexes were formed predominantly by the self-association of IgG RF. Examination of patient synovium has also revealed self-associating IgG RF within plasma cells (6). The self-association of IgG RF occurs because each molecule can serve as both antigen and antibody. Therefore, complexes may be formed even without continued foreign antigenic stimulus. Once production is initiated in genetically predisposed individuals (10) selfassociating IgG RF, together with IgM RF, may be responsible for the self-perpetuation of rheumatoid arthritis. The significance of IgG RF in rheumatoid arthritis has been questioned because it has been described not only in seropositive and seronegative rheumatoid arthritis, but also in the seronegative spondylarthropathies, juvenile rheumatoid arthritis, osteoarthritis, gout, and even normal individuals (1 116). However, technical considerations raise questions concerning many of these associations. The techniques employed to detect and quantitate IgG RF have generally been laborious, insensitive, and not specific. These have included the agglutination of IgG-coated cells or latex particles following the removal of IgM RF by chromatography (17,18) or by concanavalin A (19), or after destruction of IgM RF by reduction and alkylation (20). Agglutination of uncoated latex particles has also been described (21). Quantitation of immunoglobulins thought to be R F following elution from solid phase IgG immunoadsorbants has been widely applied (1 114,16). With this technique, immunoglobulins lacking rheumatoid factor activity will be adsorbed to the immunoadsorbant and eluted together with rheumatoid factors (22,23) resulting in false ascertainment. It is not

IgG RHEUMATOID FACTOR

surprising that large quantities of supposed IgG R F have been found by this method in a wide variety of diseases other than rheumatoid arthritis as well as in normal individuals. Recently, radioimmunoassays have been developed to detect IgG RF (15,24). While these assays were sensitive and quantitative, the potential interference by non-RF IgG has not been clearly defined. Additionally, the effect of IgM RF on the detection of IgG RF has only recently been appreciated (24). IgM RF may bind simultaneously both patient IgG and the solid phase IgG used to detect the IgG RF, resulting in the false identification of non-RF IgG as IgG RF. Destruction of IgM RF prior to analysis abrogated this interference (24,25). In order to determine the significance of IgG RF, a sensitive radioimmunoassay was used to examine the sera of patients with seropositive rheumatoid arthritis, as well as those with a variety of seronegative disorders. The potential contributions of non-RF IgG and of IgM RF were carefully monitored.

MATERIALS AND METHODS Preparation of antigens and antibodies. Pooled normal human IgG was isolated from Cohn Fraction I1 (Sigma Chemical Co., St. Louis, Missouri) by employing a DEAE cellulose ion exchange column (3.14 cm2 x 40 cm) equilibrated in 0.01Msodium phosphate, pH 8.0 (3). To obtain Fc and Fab fragments, IgG was papain digested according to the method of Porter (26). Fc fragments were isolated by ion exchange chromatography employing the method of Franklin (27). Any non-digested IgG was removed by chromatography with a Sephadex G-200 (Pharmacia Fine Chemicals, Piscataway, New Jersey) column (5.3 cm2 X 90 cm). The Fc fragments were finally passed over an immunoadsorbant column of Sepharose4B (Pharmacia Fine Chemicals) to which goat antihuman X and K had been covalently coupled utilizing the cyanogen bromide method (28). Fab fragments were obtained from the papain digested IgG by ion exchange chromatography by using the method of Carson et a1 (24). Intact IgG was separated by molecular sieve chromatography with Sephadex G-200. The Fc and Fab preparations were pure as determined by double diffusion in agarose and by immunoelectrophoresis with monospecific antisera and an antiserum to whole human serum. IgM X and IgM K macroglobulins were isolated from defibrinated plasma (kindly provided by Dr. Mart Mannik, Seattle, Washington) by ammonium sulfate and euglobulin precipitation as well as column chromatography with Sepharose 6B and Sephadex G-200. One preparation was reduced and alkylated (29) prior to the final gel filtration. IgA K was obtained by ammonium sulfate precipitation, DEAE cellulose chromatography employing a gradient from 0.01M to 0.3M sodium phosphate, pH 8.0, and sieve chromatography with Sephadex G-200. All three preparations were pure by immu-

989

noelectrophoresis and double diffusion in agarose employing monospecificantisera and an anti-whole human serum. Rabbits were immunized subcutaneously and in the foot pads with 20-25 mg of purified human IgG in complete Freund’s adjuvant. Three and 6 weeks later the rabbits were boosted with 10 mg IgG in incomplete Freund’s adjuvant subcutaneously and bled on the seventh week. The gammaglobulin fraction was precipitated three times with ammonium sulfate, dialyzed exhaustively against 0.1M sodium acetate, pH 4.5, and then pepsin-digested with 1.0 mg pepsin per 50 mg protein (30). After dialysis against 0.02M sodium borate, 0.5M sodium chloride, pH 8.0 (0.02M borate), the sample was eluted over a human Fc immunoadsorbant column (28). The F(ab’), anti-human Fc antibodies were eluted with 0.01N HCl, 0.15M NaCl (0.01N HC1) and immediately dialyzed against 0.02M borate. F(ab’), antibody fragments were employed to decrease potential binding through the Fc portion of the antibody. The anti-Fc was further purified by elution over separate Sepharose 4B columns to which were covalently coupled IgM X and IgA K proteins. The rabbit F(ab’)2 anti-human Fc (anti-Fc) was specific by radioimmunoassay (see Results section). Antibodies specific for human IgM were prepared by immunizing a goat with 7 mg of purified IgM X in complete Freund’s adjuvant at multiple sites subcutaneously. The animal was boosted 6 times subcutaneously with 1 to 7 mg of the IgM X or the p chains obtained from this protein over the next 4 months. Serum was obtained 10 days following the last immunization and precipitated with ammonium sulfate. The gammaglobulin fraction was purified by elution over immunoadsorbant columns prepared with cord serum, human serum albumin, and pooled normal human IgG. Specific antibodies were adsorbed with a Sepharose 4B-IgM K column in 0.02M sodium borate and recovered by elution with 0.01N HCl. The data reported are those obtained by utilizing the intact antibody. For some experiments the antibodies were pepsin digested and the F(ab’), fragments employed with similar results. The anti-p was specific for human IgM by radioimmunoassay (see Results section). Radioiodination of isolated proteins and fragments was performed by the iodine monochloride method using an IC1:protein molar ratio of 8: 1 (31). Specific activity ranged from 0.10 to 0.16 pCi/jg. Rabbit IgG was obtained from pooled normal rabbit serum by ammonium sulfate precipitation followed by DEAE and G-200 column chromatography. It gave a single line on immunoelectrophoresiswith anti-normal rabbit serum. Selfassociating IgG RF was isolated from a patient’s serum by Sepharose 6B column chromatography as previously described by us (3). A monoclonal IgM RF was isolated from serum (kindly provided by Dr. F. C. McDuffie, Rochester, Minnesota) by column chromatography over a Sephadex G200 column in a 0.05M sodium acetate, 0.15M sodium chloride, pH 3.5, buffer as described for this protein by others (32). Sera and synovial fluids. Sera were obtained from patients followed in the Rheumatology Clinics at the Bexar County Hospital and Audie Murphy Veterans Administration Hospital in San Antonio, Texas. Sera of patients with the following diagnoses were employed: seropositive (SSCA) and seronegative (SSCA and latex agglutination) rheumatoid ar-

POPE AND McDUFFY

990

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Figure 1. Specificity of F(ab'), rabbit anti-human Fc. Plastic beads were coated for I hour with 50 &ml human IgG (M20 ), &ml Fc (W), 50 pg/ml IgM (O), 50 &ml IgA 0, or 20 pg/ml Fab (€3).After washing and blocking the remaining sites with Tris-BSA, beads were incubated with "'I anti-Fc as described in Materials and Methods.

thritis, juvenile rheumatoid arthritis, systemic lupus erythematosus; and the seronegative spondylarthropathies including psoriatic arthritis, Reiter's syndrome, and ankylosing spondylitis. Normal sera were obtained from university employees. All samples were stored at -20°C. Synovial fluids were obtained from patients with rheumatoid arthritis, acute rheumatic fever, and degenerative joint disease. Fluids were clarified by centrifugation and stored at -70°C. Prior to analysis synovial fluids were incubated with hyaluronidase (25 IU/ml) at 37°C for 1 hour. Reduction and alkylation. Samples to be reduced and alkylated prior to the radioimmunoassay were diluted 1 : 10 in 0.55M Tris HCl, 0.1 1M 2-mercaptoethanol, pH 8.2, and incubated for 1 hour at room temperature and then 15 minutes in an ice bath. The samples were rendered 0.11M with iodoacetamide in 0.55M Tris, pH 8.2, and incubated for 1 hour on ice. Tris-BSA was then added to a final dilution of 1 :200 and the assay performed. Column chromatography. Serum samples (0.5 ml) were subjected to molecular sieve chromatography employing

either an AcA 22 (LKB, Rockville, Maryland) or a Sepharose 6B column (5.3 cm2 X 90 cm) in 0.20M sodium borate, 0.15M NaCl, pH 8.0. Four-milliliter fractions were collected. Optical density at 280 nm and IgG and IgM RF radioimmunoassays were performed on these fractions. Some sera were reduced and alkylated (29) before and others after chromatography. Miscellaneous. Erythrocyte sedimentation rates were performed by the Westergren method (33). Serum IgG concentration was performed by radial immunodiffusion employing a commercially available kit (Hyland Laboratories, Costa Mesa, California). Statistical analyses were performed by the following methods: the Pearson product moment (34) to determine exact correlation coefficients, and the procedure of Student-Newman-Keuls (35) and the Student's t test to determine the significance of differences between the means of groups. Radioimmunoassay. The technique employed for the detection of IgG RF was similar to the solid phase radioimmunoassay described by Nineham et a1 (36). We used 6 mm plastic beads (Grieger's Inc., Pasadena, California) as the solid support system. In our hands the plastic beads were more specific and reproducible; additionally, they were technically simpler, since the bead is totally immersed in the reactants and inadvertent contact with the side of the tube does not affect the results. Plastic beads were placed individually into 12 X 75 mm glass tubes. One milliliter of rabbit IgG at 50 pg/ml in 0.1M Tris, 0.15M sodium chloride, pH 7.5 (Tris buffer), was added to each tube for one hour at 4°C. Following aspiration and washing with Tris buffer to which 1% bovine serum albumin was added (Tris-BSA), tubes were incubated with Tris-BSA for one hour at room temperature to block the remaining available sites and washed with Tris-BSA once again. One milliliter of serum diluted 1 :200 in Tris-BSA was added and incubated at 37°C for 1 hour and then at 4°C overnight. Heat-inactivation of sera at 56" for 30 minutes prior to testing did not change the results and was not routinely employed. The samples were then aspirated, the tubes washed twice and 1.0 ml of I2' anti-Fc (2 pg/ml) was added to each tube and incubated for 30 minutes first at 37°C and then at 4°C. Tubes were aspirated and washed two more times. Additional washings were examined but did not improve the results and were not routinely used. The plastic beads were transferred to clean 12 X 75 mm glass tubes by suction at the tip of a Pasteur capillary pipette attached to a vacuum system and were counted in an automatic well type gamma counter (Beckman Corp., Palo Alto, California). Results were reported as the mean "'I cpm or ng anti-Fc bound/bead. All samples were run in duplicate. The IgM RF radioimmunoassay was performed in exactly the same fashion except that specific '''I anti-human p was employed.

RESULTS Specificity of antisera. In order to evaluate the specificity of the anti-Fc antiserum, beads were coated with human IgG, Fc, Fab, IgA, and IgM. As seen in Figure 1, the antiserum binds to both human IgG and Fc but not to human IgA, IgM, or the Fab fragments

IgG RHEUMATOID FACTOR

24

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than IgG RF in most sera, the degree of interference with the assay by normal IgG was examined. Isolated pooled normal human IgG examined at concentrations from 500 to 2,500 mg/dl was substituted for patient sera and the assay was performed in the usual fashion. Figure 2 demonstrates that normal IgG in high concentrations may be detected as IgG RF. This experiment was repeated numerous times with similar results. Blocking the beads for up to 24 hours with Tris-BSA prior to the addition of IgG did not affect the results. Importantly, IgG RF was detected over two hundred times more readily than the normal pooled IgG. The observations with the normal IgG could reflect either nonspecific binding of non-RF IgG or low levels of IgG RF in the pooled human IgG. To study this, Fc and Fab fragments were isolated from the pooled normal human IgG. These fragments were radiolabeled with '251and incubated separately overnight with beads coated with rabbit IgG in the usual fashion. 16

12

obtained from IgG. Additionally, there was no binding of the antiserum to rabbit IgG. Therefore, the antiserum is specific for the Fc portion of normal human IgG. The slight differences noted with the IgG and the Fc coatings may be due to possible variation in the extent of coating by these molecules and their exposure or accessibility on the bead surface. The purity of the radiolabeled anti-human p (at 2 pg/ml) was examined by employing plastic beads coated with purified human IgM, IgA, and IgG at 50 &ml. The mean cpm bound were 5.37 X lo', 199, and 137 respectively, while the BSA background control was 100. Therefore, the anti-human p was specific for IgM. Sensitivity and specificity of the radioimmunoassay. The sensitivity of the radioimmunoassay for the detection of IgG RF was examined with isolated IgG RF in concentrations ranging from 0.5 to 25 mg/dl. Samples were diluted 1 :200 and the radioimmunoassay was performed as usual. Figure 2 demonstrates that as little as 10 pg/ml of isolated IgG RF were detectable. Since IgG without RF activity is likely to be more abundant

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p g Added Figure 3. Adherence of Fc and Fab fragments of normal human IgG to beads coated with rabbit IgG. lZ5I Fc (M and ) lz5I Fab (M) were diluted in Tris-BSA and added to the beads coated with rabbit IgG in the concentrations indicated. Fragments were incubated for 24 hours, washed, and counted without the addition of antiserum.

POPE AND McDUFFY

992

I

30

to the bead itself. Nonspecific Fc-Fc interactions (22) or minute quantities of IgG RF could have accounted for the remainder of the binding by the normal IgG. Potential effect of IgM RF on IgG RF radioimmunoassay. In order to evaluate the effect of IgM RF on the radioimmunoassay for IgG RF, an isolated monoclonal IgM RF was added at a final concentration of 50 mg/dl to monomeric pooled normal human IgG at concentrations ranging from 100 to 2,500 mg/dl. The resulting preparations were diluted and incubated with the beads in the place of serum. At all concentrations of IgG examined, the IgM RF resulted in an increase in the detection of normal IgG as IgG RF. The mean increase was 87 11% (SEM). Additionally, this IgM RF was added to five normal sera at a final concentration of 50 mg/dl. This resulted in a mean 97 f 7% increase in the ng '*'I anti-Fc bound per bead. IgM RF in patient sera. IgM RF by radioimmunoassay was detected in all seropositive sera. There was a strong correlation (r = 0.6105, P = 0.001) between the radioimmunoassay value and the SSCA titer. Thirteen percent of the 23 seronegative rheumatoid arthritis patients also had a positive IgM RF by radioimmunoassav. The values were low and less than those seen with SSCA positive sera (Table 1). IgG R F in patient sera. The distribution of valpatient ues for I ~ GRF is described for the groups in Figure 4. The distribution of values for patients with seropositive rheumatoid arthritis is different from that of all seronegative groups examined. IgG RF was detected in 88% of seropositive rheumatoid arthritis sera. Twenty-two percent of seronegative rheumatoid arthritis sera and 29% of the seronegative samples from the other patient groups also possessed IgG RF determinations which exceeded the control mean plus two standard deviations. However, these values were always relatively low. As seen in Table 1, the mean values and binding ratios for the IgG RF of these groups were not statistically different from the normal controls but were different from the seropositive patients (P< 0.01). Since non-RF IgG may nonspecifically adhere to the solid phase and be detected as low levels of IgG RF, the IgG concentration of patient sera was determined. For the seronegative patients a significant correlation (r = 0.5530, P = 0.001) existed between the IgG RF value and the IgG concentration. With two exceptions every seronegative patient with an increased IgG RF also had an increased concentration of IgG. A similar correlation did not exist for those with seropositive rheumatoid arthritis (r = 0.1262).

*

I

*

Normals RF+ RA

RFRA

AS

PA

Reiters PSS

SLE ARF

1

JRA

Figure 4. Distribution of IgG RF in the sera of patients and normal controls. The patient groups respresented are: RF + RA = seropositive rheumatoid arthritis; RF - RA = seronegative rheumatoid arthritis; AS = ankylosing spondylitis; PA = psoriatic arthritis; Reiters = Reiters syndrome; PSS = progressive systemic sclerosis; SLE = systemic lupus erythematosus; ARF = acute rheumatic fever; JRA = juvenile rheumatoid arthritis. Each symbol represents the mean of duplicate determinations reported as ng 1251 anti-Fc bound per bead. The dashed line represents two standard deviations above the mean of the normal controls.

Following washing without the addition of antiserum, both the Fc and the Fab bound similarly to the beads (Figure 3). If binding by low levels of IgG RF was responsible, enhanced binding by Fab would be expected. To further examine the adherence of normal IgG, beads were coated with either rabbit IgG or BSA. After blocking with BSA in the usual fashion, beads were incubated with either isolated IgG RF (5, 10, and 20 mg/ dl) or pooled normal human IgG (500, 1,000, and 2,000 mg/dl). Following washing and addition of antiserum in the usual manner, the binding of IgG RF to the BSA coated beads was diminished 100 & 0% (SEM) compared to the rabbit IgG coated beads. With the normal IgG, only a 33 & 5% decrease was observed, indicating that the majority of the adherence by normal IgG was

993

IgG RHEUMATOID FACTOR

Table 1. Detection of IgG and IgM RF by radioimmunoassay Patient group

Number

IgGRF*

Number

IgMRFt

Normal controls Seropositive rheumatoid arthritis

28 26

12 26

Seronegative rheumatoid arthritis

23

2.29 f 0.09 15.3 f 3.03$ (6.68)§ 2.11 f 0.28 (1.2) 2.95 f 0.41 (1.3) 2.19 f 0.22 ( 1.2) 2.41 f 0.28 (1.1) 3.22 f 0.57 (1.4)

0.21 f 0.05 3.04 jz 0.33$ (13.8) 0.30 f 0.04 (1.4) ND#

Juvenile rheumatoid arthritis

6

Seronegativespondylarthropathy

10

Systemic lupus erythematosus

9

Othed

6

* IgG RF expressed as ng anti-Fc bound/bead

23

ND ND ND

f SEM.

t IgM RF expressed as ng anti-p bound/bead f SEM.

$ Significantly different from all other groups, P < 0.01. 8 Mean binding ratio, in parenthesis, obtained by dividing the patient values by the controls. # ND = not done. 1Progressive systemic sclerosis (4) and acute rheumatic fever (2).

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Figure 5. Relationship of IgG RF to IgM R F by radioimmunoassay. The assays were performed on patient and control sera as described in Materials and Methods. The groups represented are: normal controls (O), SSCA positive (O), and negative (A)rheumatoid arthritis. The dashed lines represent the normal mean plus two standard deviations for the respective assays.

The relationship of IgM RF to IgG RF in patients with seropositive rheumatoid arthritis was examined. While no correlation (r = 0.1740) existed between IgG RF and the SSCA, a relationship (r = 0.4440,P = 0.012) was present between IgG RF and IgM RF by radioimmunoassay (Figure 5). Since IgM RF is capable of binding both the solid phase rabbit IgG as well as normal IgG resulting in the false detection of normal IgG as IgG RF, patient and control sera were examined following reduction and alkylation to eliminate the interference by IgM RF. As anticipated, this treatment essentially abolished IgM RF activity (Figure 6). In contrast, IgG RF activity was only modestly affected (Figure 7). The mean IgG RF of a larger group of 32 seropositive rheumatoid arthritis patients was decreased by 34%. Similar treatment of control sera resulted in a two-fold increase, which was most likely due to nonspecific adherence. The IgG R F values of 8 of the 32 (25%) patients fell within the 95% confidence limits of the reduced and akylated normal controls. The sera of all but 1 of these 8 patients had a relatively low IgG RF (7 ng anti-Fc bound/bead or less) prior to treatment with 2-mercaptoethanol. No correlation (r = 0.2686) existed between the IgM R F prior to this treatment and the IgG RF determined following reduction and alkylation. Nevertheless, a very strong correlation (r = 0.8940, P = 0.001) existed between individual serum IgG RF values determined prior to and following reduction and alkylation. To further examine the effects of IgM RF on IgG

POPE AND McDUFFY

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ng anti-Fc bound/bead and two others with vasculitis had values of 19.1 and 25.9. The erythrocyte sedimentation rate (ESR), while not specific for rheumatoid arthritis, is perhaps the laboratory test that corresponds most closely with disease activity (37). To examine the relationship between the IgG RF and the clinical activity, the IgG RF and the ESR were compared in 24 patients with seropositive rheumatoid arthritis and 25 patients with other rheumatic disorders. The IgG RF correlated with the ESR in patients with seropositive rheumatic arthritis (r = 0.4093, P = 0.024). In those with acute rheumatic fever, juvenile rheumatoid arthritis, systemic lupus erythematosus, and the seronegative spondylarthropathies, no correlation of IgG RF with ESR was observed. Neither SSCA titer (r = 0.1097) nor IgM RF by radioimmuno-

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Figure 6. Effect of reduction and alkylation on IgM RF. Patient (solid lines) and normal control (dashed lines) sera were examined for IgM RF before and after reduction and alkylation. The techniques are described in Materials and Methods. 0

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RF detection, 7 sera were subjected to molecular sieve chromatography (not shown). The observed patterns were similar to those previously described by Carson et a1 (24). Sera from a normal control and 2 patients with seronegative rheumatoid arthritis with increased IgG RF values revealed only small peaks of IgG RF-like activity in the 6.6s IgG regiop and no IgM RF activity. We attributed these peaks to nonspecific IgG adherence. Importantly, no IgG RF activity was observed in the fractions eluting between the IgM and IgG markers. Sera from 5 patients with seropositive rheumatoid arthritis demonstrated IgM and IgG RF activity in the fractions eluting at and ahead of 19s IgM as well as IgG RF activity in the intermediate complex region between IgM and IgG. Following reduction and alkylation, no IgM RF peaks were detectable and only the IgG RF activity eluting between the 19s and 6.6s markers was observed. Relationship of circulatiqg IgG RF to disease activity. Of our patients with rheumatoid arthritis, 4 had extraarticular manifestations. Two with the hyperviscosity syndrome had IgG RF values of 30.8 and 75.9

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Figure 7. Effect of reduction and alkylation on IgG RF. IgG RF radioimmunoassay was performed on patient (solid lines)and and normal (dashed lines) sera prior to and following reduction and alkylation.

IgG RHEUMATOID FACTOR

995

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Time (Months) Figure 8. Clinical course of a patient with rheumatoid arthritis. The

dashed lines represent the normal values for each parameter. The patient, who was treated with gold sodium thiomalate injections following the initial evaluation,received a total dose of 1.5 gm.

assay (r = 0.1214) correlated with the clinical activity estimated by the ESR in patients with seropositive rheumatoid arthritis. The clinical course of a patient with severe rheumatoid arthritis is represented in Figure 8. The patient, a 52-year-old woman, had rheumatoid arthritis for 5 years. When first examined by us, the patient had a joint count of 31, 8 hours of morning stiffness, an ESR of 80 mm/hour, an SSCA titer of 1 :224, and an IgG RF of 25.6 ng anti-Fc/bead. The patient was treated with gold sodium thiomalate intramuscularly. Her response was favorable, and after 7 months of therapy she demonstrated little objective or subjective evidence of disease activity and the IgG RF was minimally elevated. After 14 months, the patient became asymptomatic and the IgG RF had returned to within the normal range. However, the SSCA had changed little during this period and was still positive. The ESR, although it had decreased, was still quite elevated. The clinical activity of the rheumatoid arthritis in this patient corresponded with the IgG RF concentration, but not with the SSCA titer and only poorly with the ESR. A second patient who had rheumatoid vasculitis was also followed serially. The patient was a 58-yearold man with a 20-year history of rheumatoid arthritis

and a 5-year history of sicca symptoms. During an episode of vasculitis, the patient’s IgG RF was 25.9 and the ESR was 72 mm/hour. Following therapy with Cytoxan, the vasculitis had resolved, the IgG RF was reduced to 13.9, and the ESR was 37 mm/hour. Synovial fluids of patients with rheumatoid arthritis. Ten patients with rheumatoid arthritis had IgG RF determinations performed on their synovial fluids. The mean value was 14.6 f 5.76 (SEM) ng anti-Fc bound/bead with a range of 1.60 to 6 1.4. The mean IgG RF in synovial fluids obtained from 7 patients with degenerative joint disease was 1.07 f 0.31 (SEM) ng/ bead, while the mean of 6 patients with acute rheumatic fever was 1.29 f 0.71. Four patients with rheumatoid arthritis had normal synovial fluid IgG RF. Two of these were ARA functional class 111, one of whom had a simultaneous Serum determination that was also low, 3.64 ng. The other 2 patients without elevated IgG RF ~ ~ l had mildl disease andl were~functional class I and 11. Of the remaining 6 patients with high IgG RF in the synovial fluid, the ng bound/bead ranged from 8.60 to 61.4. These patients all had evidence of severe disease and demonstrated either prolonged morning stiffness, very inflammatory synovial fluids, or an ARA functional class of 111.

DISCUSSION Our observations reveal that the majority of patients with seropositive rheumatoid arthritis possess circulating IgG RF. The potential effects of IgM RF on the detection of IgG RF have only recently been considered (24,25,38). In our study, the addition of IgM RF with strong avidity for IgG was capable of significantly increasing the amount of normal IgG detected as IgG RF. This most likely occurs because individual IgM RF molecules were capable of simultaneously binding the rabbit IgG coating the bead as well as human IgG in the serum. The human IgG bound to the IgM RF is detected by the anti-Fc antiserum. Carson et al (34) considered such a phenomenon Likely, based on observations following column chromatography and destruction of IgM RF. Others have noted a strong correlation between the IgM and IgG RF values with untreated seropositive sera (36), but have not considered the effects of IgM RF in the interpretation of their data (36,39). The significance of the interference by IgM RF in any individual serum is difficult to determine since IgM RF concentration, specificity, and avidity will all be important factors. Reduction and alkylation destroyed the IgM RF activity and frequently diminished

996

the observed IgG RF values for our patients. Nevertheless, a very strong correlation existed for IgG RF values observed before and after reduction and alkylation. The magnitude of the diminution observed with some sera and the fact that some patients with weakly positive results with untreated sera became negative indicate that a maneuver to destroy IgM RF activity should routinely be performed prior to analysis for IgG RF in seropositive patients in future studies. The data indicate that large quantities of IgG RF are not commonly present in seronegative individuals. Although about one-quarter of our seronegative patients had IgG RF values that were above the 95% confidence limits of the normal controls, we do not believe they truly represent increased levels of IgG RF. Non-RF IgG was capable of nonspecific concentrationdependent adherence to the solid phase. This binding was at least in part nonspecific since Fc adsorbed as readily as Fab. If binding was specific, the Fab would have bound more than the Fc (36). Also, the majority of the normal IgG but not the isolated IgG RF, in the concentrations examined, bound to beads not coated with rabbit IgG. The IgG concentration of the seronegative groups correlated strongly with the IgG RF and all but two of these patients had an increased IgG concentration. Also, the IgG RF values of these patients, although increased, were only marginally elevated. Finally, the mean values for each of our seronegative patient groups were not significantly different from the normal controls. Although less of a problem compared to other techniques, nonspecific adherence of non-RF IgG occurs with the radioimmunoassay and is likely responsible for low IgG RF values in many situations. We conclude that no significantly increased concentrations of IgG RF were present in our seronegative patients. Carson et a1 (24) pointed out that although their seronegative rheumatoid arthritis group had slightly increased IgG RF, the values were not individually of diagnostic significance. Many reports have described increased concentrations of IgG RF in a variety of seronegative disorders including adult and juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, osteoarthritis, gout, and even normal individuals (1 1-16). Our data suggest that the increased values noted in many of these reports were due to nonspecific adherence of non-RF IgG. The absence of low levels of IgG RF cannot be absolutely certain, however, since even the radioimmunoassay is plagued to some extent by nonspecific adherence of IgG. Additionally, we did not examine specifically for hidden RF (40-42) in our

POPE AND McDUFFY

seronegative patients. Reliable quantitation of low levels of IgG RF, particularly in the presence of increased IgG concentrations, requires further evaluation. Our data demonstrate that unequivocally increased concentrations of IgG RF in patients with rheumatoid arthritis are generally found only in those with IgM RF. Exceptions to this observation have been clearly documented for other disorders. Certain patients with hypergammaglobulinemicpurpura (43) as well as patients with an IgG RF producing multiple myeloma (44) may have abundant quantities of IgG RF without detectable IgM RF. These patients differ from those with rheumatoid arthritis since the IgG RF exhibits either very restricted heterogeneity or is of monoclonal origin. Similar findings have not been made in patients with rheumatoid arthritis, although enriched kappa light chain bearing 19s and 7s antiglobulins were detected in one study (45). Although clearly increased levels of IgG RF were present only in those with detectable IgM RF, IgG RF does not appear specific for rheumatoid arthritis. Carson et a1 (25) demonstrated that seropositive patients with subacute bacterial endocarditis possessed IgG RF which was detected following destruction of IgM RF by pepsin digestion. Both IgG and IgM RF decreased following appropriate antibiotic therapy. We have observed detectable levels of IgG RF in the serum of a seropositive patient with Wegener’s granulomatosus (data not presentFd). Further studies will be necessary to determine the frequency of IgG RF in seropositive conditions other than rheumatoid arthritis. The presence of IgM RF in rheumatoid arthritis is associated with a poor prognosis; however, the titer does not correlate well with the disease activity at any given moment (46,47). If IgG RF is important in the pathogenesis, the circulating or synovial fluid concentrations might correlate with the disease activity. In previous studies, IgG RF was present more often and in higher concentrations in patients with rheumatoid vasculitis (24,38). Our data demonstrate that IgG RF may be elevated even in the absence of rheumatoid vasculitis or hyperviscosity syndrome. Circulating IgG RF but not IgM RF corresponded, in the present study, with the clinical activity. Examination of synovial fluids from the seropositive patients also demonstrated an association of more severe disease activity with elevated levels and mild disease activity with low levels. Winchester et a1 (8) demonstrated a correlation between quantity of synovial fluid y-globulin complexes containing IgG RF and the degree of complement consumption, while

IgG RHEUMATOID FACTOR

Bunch et a1 (48) showed that the degree of complement activation corresponded to the level of disease activity. A significant negative correlation was observed in another study (49) between synovial fluid immune complexes and the immunochemical C4 level. These data suggest a relationship between the synovial fluid IgG RF and disease activity in some patients. More detailed clinical studies employing patient sera and synovial fluids are in progress and should be helpful in better clarifying the role of IgG RF in the pathogenesis of rheumatoid arthritis.

ACKNOWLEDGMENT We wish to express our sincere gratitude to Dr. Robert H. Persellin for his constant support and encouragement.

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