Circulating immune complexes and rheumatoid arthritis

Circulating immune complexes (CIC), as detected by the Clq binding assay (ClqBA) in sera from patients with rheumatoid arthritis (RA) were not demonstrable on analysis by ultracentrifugation on sucrose gradients. This discrepancy could be explained by the finding that polyethylene glycol 6000(PEG), used in the ClqBA to separate free radiolabelled Clq from complex bound Clq, increased the avidity of rheumatoid factor (RF), resulting in the formation of Clq binding RF IgM IgG complexes. Addition of purified RF IgM to normal human serum generated a positive ClqBA in a dose dependent way. The increased complex formation between RF IgM and IgG by PEG was also demonstrated in an enzyme linked immunoabsorbent assay and with sucrose gradients, where complexes became detectable when PEG was present. On the other hand RF IgM IgG Clq complexes obtained from the ClqBA dissociated upon removal of PEG. We conclude that high amounts of immune complexes, detected in RA sera by the ClqBA, are at least partly the result of in vitro complex formation between RF IgM and IgG. Therefore the results of this assay do not reflect the situation in the circulation in vivo.     

Relation of clinical activity of rheumatoid arthritis to immune complexes,complement components and anti-immunoglobulins

Summary Circulating immune complexes by fluid phase Clq binding assay, complement components and anti-immunoglobulin levels were studied in sera of 35 patients with rheumatoid arthritis (RA). In 23 of the 35 sera (65.7%), circulating immune complexes were positive, and the mean±SD of Clq binding activity (ClqBA), 44.5±19.4%, was significantly high compared to that of healthy persons, 17.4±8.2%. Antigenic determination of complement components revealed that Clq, C3, C5, C9, factor B and Cl esterase inhibitor (CIINH) were significantly high in sera of RA, but C4 and properdin were not. The disease activity correlated with ClqBA, IgG- and IgM-anti-immunoglobulins, C9 and serum IgG. On the other hand, ClqBA correlated with both IgG- and IgM-anti-immunoglobulin levels but not with complement components.     

Effect of circulating immune complexes on the binding of rheumatoid factor to histones

OBJECTIVE: To determine whether the reaction of rheumatoid factor (RF) with solid phase histone is due to the simultaneous presence of circulating immune complexes (CICs) or aggregated IgG. METHODS: Serum samples from 56 patients with seropositive rheumatoid arthritis (RA) and 50 random blood bank donors were used. Binding of immunoglobulins to histone was determined by enzyme linked immunosorbent assay (ELISA) and by western blots. Aggregated IgG was obtained by heating at 61(o)C for 30 minutes. RESULTS: Among the RA sera tested by ELISA, 54% were positive for histone binding by IgM, IgG, or IgA and 20% by IgM only. Heating of normal sera caused a significant enhancement in the binding of IgG to histone (p<0.001). This binding had a non-cognate behaviour-that is, it was destroyed by pepsin treatment of serum and was not significantly inhibited by competition with free histone. The same behaviour was seen for IgM, IgG, and IgA binding from RA sera. However, cognate IgG antibody binding to histone was inhibited by free histone and was resistant to pepsin digestion. Addition of heat aggregated IgG to RA sera or pretreatment of histone with aggregated IgG caused a significant increase in IgM binding to histone. CONCLUSION: IgM, IgG, and IgA RF bind to solid phase histone as a result of attachment to histone of immune complexes or aggregated IgG and not as a result of a cognate reaction with histone.     

IgA containing immune complexes in rheumatoid vasculitis and in active rheumatoid disease

Serum and synovial fluid (SF) from 68 patients with rheumatoid arthritis (RA) were studied for the presence of immune complexes (IC) and the results correlated with extraarticular features and/or disease activity. IC were measured by the 125I Clq binding assay (ClqBA) and with one detecting IgG, IgA, C3 or C4 in IC. Disease activity correlated significantly with IgG or IgA containing and Clq binding IC. The IgA containing IC were found only in 25% of the patients, including all but one case of rheumatoid vasculitis, but otherwise only in seropositive active RA. C3 and C4 IC did not correlated with disease activity, seropositivity or vasculitis. IC in serum did not correlate with SF levels, but C4 containing IC were more frequent in SF (60%) than in serum (30%). Thus serum IC did not reflect SF levels. Patients with vasculitis showed more IC in the sera than in SF.     

Complement mediated inhibition of immune precipitation in rheumatoid arthritis: studies on interaction of heat aggregated IgG with IgM rheumatoid factor.

Serum samples from patients with seropositive rheumatoid arthritis contain an inhibitor of complement mediated inhibition of immune precipitation (CMIP). This inhibitory effect can be produced by the addition of either purified monoclonal or polyclonal IgM rheumatoid factor (RF) to human serum. The specificity of the rheumatoid factor influences the degree of inhibition, and when precipitation occurs the rheumatoid factor coprecipitates with the antigen-antibody complex. In rheumatoid sera there was a significant positive correlation between IgM RF concentration and inhibitory activity, though the range of inhibitory activity seen for the same concentration of rheumatoid factor was considerable. Small quantities of heat aggregated IgG (HAGG) had a much greater effect on the measurement in an enzyme linked immunosorbent assay (ELISA) of IgM RF than they did on the inhibitory activity of IgM RF in the CMIP assay. Larger quantities of HAGG initiated complement activation and increased the precipitation of immune complexes. IgM RF reduced the complement activating properties of HAGG by reducing the amount of Clq which bound to the aggregate. The mechanisms by which IgM RF overcomes CMIP in rheumatoid sera may involve its inhibitory effects on the binding of Cl to the antigen-antibody complex.     

Prevalence of IgE rheumatoid factor (IgE RF) in mixed cryoglobulinemia and rheumatoid arthritis

IgE RF was measured by ELISA assay using aggregated IgG as a solid phase immunosorbent and alkaline phosphatase-conjugated Fc epsilon-specific monoclonal and polyclonal antibodies as indicators. The presence of IgE RF was defined in this assay as binding of the conjugate greater than 2.33 SD above the mean for control sera (N = 27). Total IgE was elevated in 25% (13/52) of sera of patients with seropositive Rheumatoid Arthritis (RA), yet was normal in the sera of 17/19 Mixed Cryoglobulinemia (MC) patients. IgE RF was present in 33% (21/63; p less than 0.05) RA sera, and none of sera from 19 MC patients tested. It did not correlate with IgM RF titer or total IgE, and was not detected in separated IgM and IgG fractions of 7 purified mixed cryoglobulins from patients with MC. These findings suggest that IgE RF may not be an important pathogenic factor in the clinical manifestations of MC. Its potential significance in RA is discussed.     

The quantitative determination of the IgM rheumatoid factor using a solid-phase radioimmunoassay. Comparison with the agglutination test and the radioimmuno-polyethyleneglycol precipitation test

A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor using a monoclonal anti-mu-chain antibody is described. Human IgG did not interfere with the detection of IgM RF by this method. The small nonspecific binding of nonRF IgM to the human IgG coated tubes utilized in the assay must be corrected for by assaying samples in parallel bovine serum albumin coated control tubes only in cases of deviation of IgM from normal range. 69 coded and randomly arranged sera from patients with rheumatoid arthritis (RA), nonrheumatic joint diseases and healthy adult control subjects were investigated by this method, agglutination techniques as well as RIPEGA. A good correlation between solid-phase radioimmunoassay and agglutination techniques was found. Patients with seropositive RA had significantly higher concentrations of IgM RF than seronegative RA patients or control subjects (mean +/- 1 SD = 133,3 +/- 187,2 micrograms/ml versus 4,7 +/- 6,5 micrograms/ml and 2,2 +/- 4,0 micrograms/ml; resp.).     

Enzyme immunoassay of complement-binding rheumatoid factors

Summary We describe an enzyme immunoassay for the determination of complement-binding rheumatoid factors. Polystyrene tubes are coated with heat aggregated human IgG. The rheumatoid factors (RFs) of patients heat inactivated sera are allowed to bind to aggregated IgG and thereafter saturated with fresh human complement. The amount of C 3 complement bound is measured by indirect enzyme immunoassay. The levels of complement binding RFs were measured in 30 patients with seropositive rheumatoid arthritis (RA), in 19 patients with systemic lupus erythematosus (SLE), and in 30 healthy control subjects. Compared to the controls high levels of complement-binding RFs were found both in RA and in SLE (P<0.0005). The mean level of the complement binding RFs was higher (P<0.05) in active than in inactive SLE. Even though the 19 S IgM RF bound complement, in RA no correlation was found between the level of complement binding RFs and Waaler-Rose titre, but the level of complement binding RF correlated with the levels of nonagglutinating IgM RF (r=0.56, P<0.01) and IgG RF (r=0.70, P<0.001) that were obtained by enzyme immunoassay.     

Complement-activating properties of immune complexes are suppressed by IgM rheumatoid factor and enhanced by IgG rheumatoid factor

Summary The effect of rheumatoid factor (RF) on complement-activating capacity of aggregated IgG was investigated. The degree of complement activation induced by the addition of specific amounts of aggregated IgG to patients' sera and normal sera was demonstrated by the inhibition of hemolytic activity (%IHA). The %IHA was significantly lower in rheumatoid arthritis (RA) sera and higher in systemic lupus erythematosus (SLE) sera, compared with normal sera. There was a negative correlation between %IHA and IgMRF/IgGRF ratio in RA and SLE sera, and RA synovial fluid. The %IHA and IgGRF were positively correlated in RA sera. The IgMRF/IgGRF ratio was significantly lower in SLE sera than in RA sera and systemic sclerosis sera, and was significantly lower in RA synovial fluid than in osteoarthritis synovial fluid.Isolated RF, consisting of mostly IgMRF class, inhibited complement-activating properties of aggregated IgG, depending on the concentration of RF. Isolated RF was further purified by the fractionation using high pressure liquid chromatography, and IgGRF and IgMRF were obtained. IgMRF significantly suppressed the complement-activating capacity of aggregated IgG, whereas IgGRF promoted it. These observations suggest that IgMRF acts protectively, while IgGRF induces inflammation.Thus, the expression of the biological activity of RF with special reference to immune complex interaction mainly depends on the IgMRF/IgGRF ratio.     

Studies of serum immunoglobulin binding to synovial fibroblast cell cultures from patients with rheumatoid arthritis

Using a sensitive 125I-protein A (PrA) binding assay to detect cell surface IgG, we have studied seven different synovial fibroblast cell cultures from patients with rheumatoid arthritis (RA). When these cultures were incubated in the presence of serum from 18 autologous and allogeneic RA patients (all seropositive), we were unable to detect significant IgG binding. Since IgM rheumatoid factor (RF) can block PrA binding, sera were absorbed with aggregated IgG to remove RF without affecting the results. Similar studies on three cell lines with seven rheumatoid sera were performed by antibody-dependent cell-mediated cytotoxicity. No significant cytotoxicity was observed. Since antibodies to collagen are present in rheumatoid sera, several cultures were incubated with ascorbic acid (12.5 microgram/ml) to optimize synthesis of cell surface collagen. These culture conditions did not affect serum immunoglobulin binding by the 125I-PrA assay. Thus, we can find no evidence for a direct humoral immune mediation of synovial proliferation in rheumatoid arthritis. These data do not support the hypothesis that the inflammatory process within the synovium of RA patients is an immunologic response to a fibroblast-associated antigen in the synovial membrane.     

Differences in immunochemical characteristics of cryoglobulins in rheumatoid arthritis and systemic lupus erythematosus and their complement binding properties.

Cryoglobulins isolated from sera of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) were analysed for their immunoglobulin, antibody, and complement components. In both disease categories the cryoglobulins contained predominantly IgG with lesser amounts of IgM and IgA, but relative to serum more IgM was concentrated in the cryoglobulins. IgM rheumatoid factor was found in 65% of RA cryoglobulins but in only 17% of SLE cryoglobulins (p less than 0.02), whereas SLE cryoglobulins contained more DNA binding activity than RA cryoglobulins (p less than 0.01). C1q binding activity was detectable in the majority of SLE and RA sera and SLE cryoglobulins. Paradoxically only two out of 34 RA cryoglobulins bound C1q, although rheumatoid factor activity was present in both cryoglobulins and sera. When isolated from serum the rheumatoid factor fraction strongly bound C1q. Both RA and SLE cryoglobulins contained similar small amounts of C3 and C4. Differences in antibody composition and complement binding activity of cryoglobulins from RA and SLE sera may reflect properties of immune complexes which affect their tissue localisation and pathogenicity.     

A sensitive radioimmunoassay for quantitation of igm rheumatoid factor

A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor (RF) in biologic fluids has been developed. Binding curves for monoclonal IgM RF and polyclonal IgM rheumatoid factors were similar under the conditions utilized for the assay. Human IgG did not interfere with the detection of IgM RF by this method. Small quantities (≤ 0.2%) of nonspecific binding by nonRF IgM to the human IgG coated tubes utilized in the assay were corrected for by assaying samples in parallel bovine serum albumin coated control tubes. As expected, patients with seropositive rheumatoid arthritis (RA) had significantly higher concentrations of IgM RF than seronegative RA patients (mean ± 1 SD = 652 ± 553 μg/ml versus 11.3 ± 13.3 μg/ml, P < 0.001). In contrast, all normal control sera assayed to date contained < 0.1 μg/ml of IgM RF. The capacity of the assay to detect nanogram quantities of IgM RF should permit investigation of the cellular mechanisms underlying RF production.     

The Detection of Immune Complexes of Different Immunoglobulin Class

Summary: Immune complexes were detected in 51 sera from patients with a variety of immunological diseases: 14 systemic lupus erythematosus (SLE); 14 infectious mononucleosis (IM); 12 rheumatoid arthritis (RA) and 11 subacute bacterial endocarditis (SBE). Three methods were used to detect complexes: the fluid—phase Clq binding assay (Clq.BA); the solid—phase Clq binding assay (Clq.SP) and the Raji cell radio-immunoassay (RIA). Modification of the Clq.SP and the Raji cell RIA by use of monospecific antisera to immunoglobulins G, A and M enabled the class of antibody in the immune complexes to be determined. Antibodies of all three classes were found in each disease, the predominant ones being IgG and IgM in SLE and SBE, IgM and IgA in RA and IgM in IM.     

Characteristics of antinuclear antibodies in rheumatoid. arthritis

The immunologic specificities of antinuclear antibodies (ANA) in sera from 97 patients with rheumatoid arthritis (RA) were studied. The frequency of ANA detected by immunofluorescence with mouse kidney sections as substrate was 60% (59/97) compared to 13% (7/55) in controls. IgM ANA was positive in 41% (40/97) of RA sera, IgG ANA in 40% (39/97), and IgA ANA in 33% (32/97). Specificities of antibodies in the ANA positive sera were examined by radioimmunoassay for antibodies to DNA and by immunodiffusion for antibodies to Sm antigen, nuclear ribonucleoprotein, and the SS-A and SS-B nuclear antigens. Antibodies to these nuclear antigens were found infrequently (1.7% to 6.8%). Sera were further investigated for antibodies to histones by an immunofluorecent method previously described using acid-extraced and histone reconstituted tissue section. Twinty-four percent of the ANA positive sera (14/55) reacted with a histone-dependent nuclear antigen. The relationship of rheumatoid factor to ANA was studied by isolating rheumatoid factors (RF) from aggregated IgG absorbant columns. Ten of 19 isolated RF showed ANA activity, which did not result from complexes of rheumatoid factor with IgG ANA since IgM rheumatoid factor, dissociated from IgG under acid conditions, continued to show ANA activity. In 8 of 16 RA sera tested, both ANA and rheumatoid factor activity could be inhibited by aggregated IgG. Five of the cross-reacting rheumatoid factors reacted specifically with a histone-dependent antigen in the reconstitution assay. These findings show that approximatley 50% of rheumatoid factors possess cross-reactivity with nuclear components, and histones are involved in a significant number of these reactions.     

Hypergammaglobulinemic purpura of Waldenstr?m: characterization of circulating immune complexes

Circulating immune complexes were detected in the sera of 7 patients with hypergammaglobulinemic purpura of Waldenstr?m by the following methods: KgB-SP, mRF-LIA, Cc test, ClqBA. Analytical ultracentrifugation showed intermediate complexes between 7S and 19S (16S-19S); simple immunodiffusion of the complexes, purified by 2.5% PEG precipitation, revealed the presence of IgG3, IgA, IgM, Clq, C3 and C4. Constant high titers of rheumatoid activity in the sera in toto and after purification, and normal serum complement levels were also detected.     

Precipitation of 125I-labelled IgG aggregates by factors in sera of healthy individuals and of patients with rheumatoid arthritis.

Several factors in human serum are capable of precipitating soluble 125I-labelled heat aggregated IgG (agg IgG*). A study of the nature of these factors resulted in the development of two new methods for the detection and assay of anti-IgG-immunoglobulins (rheumatoid factors) in serum. One method detected rheumatoid factors of the IgG and the IgA classes which are capable of binding and coprecipitating with Clq and agg IgG* in an EDTA milieu. In a second method the serum was first heat inactivated and the assay was then made in a polymeric milieu where rheumatoid factors of the IgM as well as the IgG and IgA classes could be detected. 67 sera from patients with rheumatoid arthritis were tested with this method and rheumatoid factors were detected in all seropositive (as assayed with conventional rheumatoid factor tests) sera and in 58% of the seronegative sera. In the presence of certain anti-IgG-immunoglobulins or polymers, the precipitation of Clq and soluble agg IgG* is greatly enhanced, and we suggest that this can be used as a basis of a sensitive method for the assay of agg-IgG-binding activity of Clq.     

IgG and IgM rheumatoid factors in rheumatoid arthritis. Quantitative response to penicillamine therapy and relationship to disease activity

Penicillamine treatment of patients with rheumatoid arthritis (RA) leads to falling titers of agglutinating IgM rheumatoid factor (RF), but its effect on IgG RF has not been described. Using specific solid phase radioimmunoassays, we have determined serial levels of IgM RF and IgG RF in 18 patients receiving penicillamine for 1 year, and correlated the results with the change in RA activity. Mean IgM RF levels fell to 76 +/- 10% (mean +/- SEM) after 3 months, and 30 +/- 5% of the pretreatment value after 1 year of penicillamine treatment. This decline was greater than that for total IgM (P less than 0.0001), indicating a selective reduction of RF. Patients receiving maintenance doses of 750 mg/day manifested more rapid and greater decreases than did those given 250 mg/day. In contrast, serial mean IgG RF levels did not change significantly, and actually increased in 6 of 18 cases. At onset, there was a significant correlation with erythrocyte sedimentation rate for both IgM RF (r = 0.535, P = 0.05) and IgG RF levels (r = 0.570, P = 0.02). But changes in RF concentration demonstrated no correlation with changes in either erythrocyte sedimentation rate or joint score over the 1-year period, suggesting that circulating IgM RF or IgG RF levels may be unrelated to the degree of RA activity.     

Immunochemical analyses of components of immune complexes in the sera of patients with autoimmune diseases

Immune complexes were precipitated with polyethylene glycol (PEG) from sera of patients with rheumatoid arthritis (RA), psoriatic arthritis and systemic lupus erythematosus. Precipitates were tested for their capacity to consume complement and for the presence of fibronectin (Fn) and specific autoantibodies rheumatoid factor (RF, anti-DNA). The results showed enrichment of autoantibody activity in the precipitates. In RA, RF was especially enriched in 2.5% PEG precipitates, while IgM anti-DNA activity was more evident in 3.5% precipitates. IgG anti-DNA antibody was detected only in 3.5% precipitates from lupus sera. Complement consumption activity of precipitates was mainly related to IgM autoantibodies. There was a strong correlation between the presence of RF activity and Fn in the PEG precipitates. Nephelometric studies revealed direct interaction between the Fn and IgM RF or heat aggregated gammaglobulin. It may be possible to monitor the serum levels of immune complex material using PEG precipitation.     

HUMAN COMPLEMENT ACTIVATION BY SELF-ASSOCIATED IgG RHEUMATOID FACTORS

IgG rheumatoid factors (IgG–RFs) undergo concentration-dependent self-association into dimers and higher polymers, as previously reported. The interactions of purified IgG–RF from the plasma of 3 patients with rheumatoid arthritis, with guinea pig and human complement were studied. Self-associating IgG–RFs were isolated by affinity columns and gel filtration. These preparations contained no detectable IgM and were composed only of IgG subclasses known to fix complement. Complement utilization of IgG–RF was compared with that of monomeric IgG, heat-aggregated IgG, and soluble rabbit IgG immune complexes. Although incubation of IgG–RF or monomeric IgG with 3 units of guinea pig or human complement resulted in decreased hemolysis of sheep erythrocytes sensitized with IgM hemolysin, these substances were less than 100 times as effective as heat-aggregated IgG or soluble immune complexes. The ability of human or guinea pig complement that had been incubated with IgG–RF to restore hemolytic activity to C4-deficient guinea pig serum served to distinguish Clq binding from complement cascade activation. IgG–RFs and monomeric IgG did not activate guinea pig complement cascade in contrast to aggregated IgG. IgG–RFs, however, activated human complement cascade; monomeric IgG only bound human Clq. These results indicate that self-associated IgG–RFs can activate human complement in fluid phase, but less effectively than aggregated IgG or large-latticed immune complexes.     

Rheumatoid factors in rheumatoid arthritis and vasculitis

Summary To study the occurrence of rheumatoid factors (RF) in relation to the activity of rheumatoid arthritis and the occurrence of vasculitis, RF of IgM, IgA, and IgG classes were measured in sera from 35 patients with definite or classic rheumatoid arthritis (RA) using ELISA. For 26 patients, the RF levels were studied longitudinally and compared with changes in the articular index. Although IgM RF was occasionally found in patients without RA, IgA and/or IgG RF were almost exclusively associated with RA. The titers of IgM, IgA, and IgG RF were significantly higher in sera from patients with clinically diagnosed rheumatoid vasculitis than in sera from patients without vasculitis. No significant correlation between changes in the articular index and changes in titer of any class-specific RF could be found for the group of RA patients as a whole. However, in individual patients, increases or decreases in IgM and IgG RF titer were significantly correlated with an increase or decrease in the articular index.