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 IgM rheumatoid factors reacting with IgG subclasses

Summary To estimate the complement-activating property (CAP) of IgM rheumatoid factor (RF), which was purified from synovial fluids of patients with rheumatoid arthritis, in a reaction with each IgG subclass, the activation and binding of C4 in the classical pathway of complement by IgM RF was measured in an enzyme-linked immunosorbent assay using biotinylated F(ab')₂ antibody to human C4. The CAP of IgM RF reacting with IgG3 was significantly higher than that of IgM RFs bound to the other IgG subclasses (P<0.01). These results suggest that IgM RF reacting with IgG3 in synovial fluid could induce a greater degree of complement-dependent inflammation in RA synovium than IgM RF reacting with other IgG subclasses.     

IgG and IgM rheumatoid factor in active seronegative nodular rheumatoid arthritis and in children with benign rheumatoid nodules

Summary Rheumatoid nodules in rheumatoid arthritis are usually associated with high levels of IgM rheumatoid factors and aggressive disease. IgM, IgG, and C3 have been identified in tissue sections of rheumatoid nodules, suggesting pathogenetic importance. The role of IgM and IgG rheumatoid factors in sero-negative RA is poorly understood. Seven patients with active seronegative RA and rheumatoid nodules and 10 seronegative children with the syndrome of benign rheumatoid nodules were studied for the presence of IgG and IgM rheumatoid factors by radioimmunoassay and for complement-fixing IgM rheumatoid factor by haemolytic assay. Elevated titres of hidden complement fixing IgM rheumatoid factor were found in 60 % of the patients with benign rheumatoid nodules studied but in none of the patients with active seronegative nodular RA. Serum IgG and IgM rheumatoid factor levels by radioimmunoassay and circulating immune complex levels in both groups were not significantly different from normal controls. These data suggest IgG and IgM rheumatoid factors do not participate in the pathogenesis of active seronegative rheumatoid arthritis with rheumatoid nodules.     

IgG and IgM anti-endothelial cell antibodies in patients with collagen-vascular disorders

Summary A cellular enzyme-linked immunosorbent assay (ELISA) was used to detect circulating anti-endothelial cell antibodies (AECA) in sera of patients suffering from rheumatoid arthritis (RA), Felty's syndrome (FS), systemic lupus erythemtosus (SLE), progressive systemic sclerosis (PSS) and those with a lupus anticoagulant (LA). IgG AECA were detected in RA, FS, SLE and LA sera, while IgM AECA were only detected in RA and FS. AECA were not specific for endothelial cells. However, IgG binding to endothelial cells and dermal fibroblasts was F(ab) mediated while in all other cell types tested, nonspecific binding most likely via the Fc region occurred. In RA and FS rheumatoid factor was shown to augment immunoglobulin binding to endothelial cells. Additional studies revealed that some of these pathological sera also contained cytotoxic IgG autoantibodies which fixed complement and damaged human umbilical vein endothelial cells in vitro. These studies suggest that a group of autoantibodies, present in a variety of collagen vascular disorders, react with endothelial cells and thus may be important aetiopathogenic factors in the vasculopathies associated with these disorders.     

Importance of the IgG isotype, not the state of glycosylation, in determining human rheumatoid factor binding

We investigated the influence of carbohydrate on the binding of human rheumatoid factors (RF) to the Fc fragment of IgG. The monoclonal RF studied were derived from the serum of patients with mixed cryoglobulinemia or from hybridomas generated from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus. Polyclonal RF were derived from patients with RA. The carbohydrate located on the Fc fragment, regardless of whether it contained different amounts of mannose or reduced amounts of galactose, or was removed, did not affect the binding of the RF. In contrast, the isotype of the Fc was found to be critical. Two groups of hybridoma RF could be delineated. One group bound preferentially to IgG1 and/or IgG2, and a second group (primarily from patients with RA) bound preferentially to IgG3 and/or IgG4. Our results indicate that the isotype of the Fc fragment, and not the extent of galactosylation, influences the binding of the RF.     

Diagnostic value of antiagalactosyl IgG antibodies in rheumatoid arthritis

Our objective in this study was to explore the diagnostic value of antiagalactosyl IgG antibodies in rheumatoid arthritis (RA). The study comprised 266 Japanese patients with systemic autoimmune diseases, including 60 with RA. Human agalactosyl IgG was prepared enzymatically, and the serum levels of antiagalactosyl IgG antibodies were determined using a lectin enzyme immunoassay. Serum IgG and IgM rheumatoid factors (RF) were measured using laser nephelometry for IgM (LN-RF) and an enzyme-linked immunosorbent assay for IgG (IgG-RF). Antiagalactosyl IgG antibodies were significantly more common in patients with RA than in those without (78% vs. 18%, odds ratio (OR) 16.51, 95% confidence interval (CI) 8.12–33.58, p<0.0001). Patients with RA also had a higher frequency of LN-RF than those without RA (75% vs. 28%, OR 7.81, 95% CI 3.91–15.58, p< 0.001). The specificity of antiagalactosyl IgG antibodies for RA was significantly higher than that of LN-RF (82% vs. 72%, p<0.0011). There was a significant correlation between titers of antiagalactosyl IgG antibodies and C-reactive protein levels. Antiagalactosyl IgG antibodies are more specific markers for RA than conventional LN-RF, and may provide useful information for the diagnosis of RA.Abbreviations AKA Antikeratin antibodies - RA Rheumatoid arthritis - RF Rheumatoid factor - SLE Systemic lupus erythematosus     

Effect of aggregated IgG on mononuclear and polymorphonuclear cell cooperation

Summary The leukocyte adherence inhibition (LAI) test was used to determine the response of different leukocyte subpopulations in rheumatoid arthritis (RA) patients and normal donors to heat-aggregated gammaglobulins (HAGGs). The adherence of polymorphonuclear cells of (PMNs) could be inhibited either directly by high concentrations of HAGGs or indirectly by soluble factors secreted by RA mononuclear cells incubated with low amounts of HAGGs. The stimulatory site of the IgG was located on the Fc fragment. Since the LAI reaction was also observed with material recovered from rheumatoid synovial fluid, tissue, and nodules, these results could have clinical relevance in RA.     

Differences in the relationship of specificity to titre and functional affinity between circulating Ga- and pan-reactive IgM rheumatoid factors in rheumatoid arthritis

OBJECTIVE: To determine if there are the differences in titre and functional affinity for immunoglobulin (Ig) G subclasses and glycoforms between the Ga- and pan-specific IgM rheumatoid factors (RFs) present in the sera of patients with rheumatoid arthritis (RA), and to determine whether these two broad specificities have different functional roles in RA. METHODS: We used direct ELISA and modified ELISA to study the binding of IgM RF in the sera of 32 patients with RA with a range of RF titres to a panel of 14 IgG paraproteins of all four subclasses, some allotypes and different glycosylation patterns. RESULTS: Pan-specific RFs were mostly found in RA sera with high RF titres, and these RFs generally had higher avidity. A trend towards higher avidity of RFs with higher titre was observed for pan-specific, but not for Ga-specific RFs. With increasing titre, pan-specific RFs tended to react strongly with fucosylated and bisected variants of hypogalactosylated IgG3 of G3m(b1) allotype and hypergalactosylated IgG4 of 4a allotype. CONCLUSION: Among high-titred pan-specific IgM RFs, there is a subpopulation responsible for strong anti-IgG activity in RA. The possible mechanisms of production of pan- and Ga-specific RFs are discussed.     

Isotypes of rheumatoid factors in rheumatoid arthritis and chronic liver diseases

We studied isotype-specific rheumatoid factors (RFs) to clarify their significance in rheumatoid arthritis (RA) and to verify the difference in RF isotypes between RA and chronic liver diseases (CLD). Isotype-specific RFs in RA and in CLD were measured by enzyme-linked immunosorbent assay (ELISA). Most sera (n = 51, 94.1%) from RA patients contained some kind of RF isotypes (92.1% for IgM RF, 76.4% for IgG RF, and 43.1% for IgA RF), and seronegative RA by ELISA was seen in only 11.8% (n = 6). The most characteristic combination of RF isotypes in active RA was IgG, IgA, and IgM. This combination of RF isotypes changed to IgG plus IgM, according to the diminution of RA activity; then, we found only IgM RF in inactive RA. The titers of each RF isotype also decreased in parallel with the activity of RA. IgA RF seemed to be the most sensitive factor for evaluating the activity of RA. In CLD, almost the same high frequency (n = 49, 89.8% for IgM RF, 59.2% for IgG RF), with the same titer levels seen in RA, was observed. On the other hand, IgA RF was significantly lower in frequency (n = 9, 18.4%) and in titer, compared with the finding in RA. Surprisingly, even in CLD, true seronegativity by ELISA was also found in very few patients (n = 4, 8.1%). In CLD, positive RFs detected by agglutination assay were seen more often in chronic hepatitis than in liver cirrhosis. In RA patients, significant associations of IgA RF and the serum concentration of IgA, and IgG RF and the serum concentration of IgG, were observed. On the other hand, in CLD patients, significant associations of IgG RF and the serum IgG concentration, and of IgM RF and the serum IgM concentration, were observed. These results indicated that IgA RF in active RA is the most characteristic RF isotype distinguishing it from other nonrheumatic diseases, as well as from inactive RA. RF isotypes reflected the background polyclonal B-cell activation in different manners in both diseases. In CLD, RF isotypes seemed to be disease-related immunological disorders reflecting disease progression. Received: February 17, 2000 / Accepted: July 5, 2001     

Spontaneous and aggregated IgG induced rheumatoid factor producing cells in rheumatoid arthritis

Summary Seropositive rheumatoid arthritis (RA) patients were found to have high numbers of spontaneously occurring cells making rheumatoid factor (RF) reactive with human IgG as measured by a RF plaque forming cell (RF-PFC) assay. There was a significant positive correlation between the number of RF-PFC and both disease activity measured by the sedimentation rate and RF titer measured by the RA latex test. Aggregated IgG and pokeweed mitogen were equally effective stimulators of RF-PFC in cultures of RA peripheral blood mononuclear leukocytes. The rheumatoid ratio of helper (T4): suppressor (T8) T lymphocytes was also significantly increased over the ratio of normal controls, but this ratio did not correlate with the number of RF-PFC. Aggregated IgG or immune complexes may be responsible for stimulating RA RF-PFC in vivo.     

Raised serum IgG and IgA antibodies to mycobacterial antigens in rheumatoid arthritis.

Autoantigens cross reactive with mycobacteria are implicated in the pathogenesis of adjuvant arthritis in the rat, and there are reports of changes in the immune response to mycobacteria in human rheumatoid arthritis (RA). We have therefore examined the IgM, IgG, and IgA antibody levels to crude mycobacterial antigens and to two recombinant mycobacterial heat shock/stress proteins (65 kD and 71 kD) in sera from patients with RA, systemic lupus erythematosus (SLE), and Crohn's disease, and from healthy controls. IgA binding to the crude mycobacterial antigens was significantly raised in RA sera, though IgG and IgM binding tended to be lower than in controls. Both IgA and IgG binding to the heat shock proteins were significantly raised in the RA sera. Smaller significant rises in both classes were seen in sera from patients with SLE, and in the IgA class only to the 65 kD protein in Crohn's disease. The rises in IgG and IgA antibodies to the 65 kD protein in RA were significantly higher than in the other diseases, however. It is interesting that this protein is the one responsible for adjuvant arthritis in the rat.     

Sera from patients with systemic lupus erythematosus demonstrate enhanced IgG binding to endothelial cells pretreated with tumour necrosis factor alpha

Fluorescence flow cytometry and indirect immunofluorescence were used to detect circulating IgG antiendothelial cell antibodies (IgG-AECA) in the sera of patients suffering from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS). Pretreatment of endothelial cells with tumour necrosis factor alpha (TNF), but not with interferon gamma (IFN), increased the IgG binding from sera of some patients with active SLE. In contrast, no change in binding activity to cytokine-stimulated endothelial cells was observed in the RA and PSS sera. The results of this study suggested that the enhanced binding of IgG from the sera of patients with SLE to endothelial cells stimulated with TNF may be due to the ability of this cytokine to increase the expression of potential antigens on the surface of these cells. Hence, TNF may play a role in the immune-mediated vascular damage associated with SLE.     

Reaction of rheumatoid factors with IgG3 monoclonal anti-Rh(D) antibodies: more frequent reactivity to a monoclonal antibody of the Gm allotype G3m(5) in rheumatoid patients negative for G3m(5).

Human monoclonal anti-Rh(D) antibodies of known IgG isotype and Gm allotype were bound to erythrocytes and then used as the target IgG antigens for rheumatoid factors (RFs) in a direct haemagglutination test. When serum samples from patients with rheumatoid arthritis (RA) were tested for RF specificity towards these IgG monoclonal anti-D antibodies the incidence and titre of reactivity towards an IgG3 monoclonal anti-D antibody was considerably greater than for a polyclonal anti-D antibody of the same Gm allotype, G3m(5). This difference was not explained by the amount of each anti-D antibody which bound to erythrocytes. Furthermore, when patients with RA were divided into groups according to their Gm phenotype, sera from a greater proportion of patients negative for the phenotype G3m(5) reacted to the G3m(5) monoclonal anti-D antibodies than sera from those patients positive for this allotype. Analysis of RF reactivities towards two IgG3 and three IgG1 monoclonal anti-D antibodies, each with different Gm allotypic epitopes, indicated, however, that individual serum samples contained RFs with a spectrum of specificities; some sera appeared to react to a single set of Gm alleles, whereas others also reacted to isotypic or iso-allotypic epitopes, or both. Our data suggest that RFs with specificity for Gm allotypes do not arise in patients who carry that particular allotype owing to tolerance induced in fetal-neonatal life. Conversely, RFs with apparent specificity for a Gm allotype formed in patients negative for that allotype may be reacting to a closely related but different epitope. Final proof requires precise specificities for each RF formed, and IgG3 monoclonal anti-D antibodies would be useful reagents for this purpose.     

Serum IgG4 anti-Fab antibodies in rheumatoid arthritis are constitutively expressed

Summary IgG4 comprises a significant proportion of the total anti-Fab antibody (aFABA) response in many but not all patients with rheumatoid arthritis (RA). Analyses of the dynamics of IgG aFABA subclass expression in 11 RA patients for periods of up to 11 months demonstrated that IgG4 aFABA was restricted to 6 of the 11 RA patients' sera initially studied and comprised approximately 25% (or more) of the total IgG aFABA response. Quantities of IgG4 aFABA in subsequent, serially obtained serum samples from these patients remained stable throughout the study period, whereas the remaining RA patients whose initial sera possessed small quantities of serum IgG4 aFABA failed to generate any augmented IgG4 aFABA response during the study. Elevated expression of IgG4 aFABA did not appear to be a consequence of a generalized polyclonal gammopathy or a generalized increase in autoantibody expression, though patients with higher total IgG4 serum levels expressed significantly greater quantities of IgG4 aFABA. These results indicate that the differential expression of IgG4 aFABA among RA patients reflects constitutive production within a subset of RA patients in whom IgG4 appears to comprise a significant proportion of the total IgG aFABA response.     

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.     

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.     

Monoclonal IgM rheumatoid factors bind IgG at a discontinuous epitope comprised of amino acid loops from heavy-chain constant-region domains 2 and 3.

A combination of site-directed mutagenesis and exon exchange has been used to further define the structure on IgG recognized by monoclonal IgM rheumatoid factors (RFs) from patients with Waldenstrom macroglobulinemia. Most of these RFs bound IgG1, -2, and -4 but not IgG3. For these RFs, His-435 is a critical residue for binding and replacing it with arginine, the residue present in IgG3, destroys or reduces RF binding. However, additional polymorphic sequences in both the heavy-chain constant-region domains (CH) 2 and 3 are important for RF binding. Among the important residues in CH2 are amino acids 252-254 and 309-311, which are conserved among IgG isotypes and comprise two loops of amino acids on the surface of the domain. Therefore, at least three regions, two from CH2 and one from CH3, contribute significantly to the epitope recognized by the RFs. Although this epitope contains many of the same residues as the staphylococcal protein A binding site on IgG, the binding specificities of staphylococcal protein A and monoclonal RFs are not identical. Sera from patients with rheumatoid arthritis contain antibodies directed not only at this epitope but also at other sites on IgG.     

Rheumatoid factor in rheumatoid arthritis associated renal disease and in lupus nephritis.

To test the hypothesis that rheumatoid factor (RF) protects against (immune complex mediated) renal disease, patients with rheumatoid arthritis (RA; 48 with nephropathy of various types, 35 without renal disease) and systemic lupus erythematosus (SLE; 35 with and 17 without nephritis) were evaluated for the presence and titre of RF. There was no correlation between RF and nephropathy in RA, whereas in SLE RFs were almost exclusively seen in patients without nephropathy. This result supports the above hypothesis for lupus nephropathy but not for RA associated renal disease, and it may be explained by a more pronounced role for immune complexes in SLE and interference of RFs with the complexes.     

A comparative study of IgG second- and third-generation anti-cyclic citrullinated peptide (CCP) ELISAs and their combination with IgA third-generation CCP ELISA for the diagnosis of rheumatoid arthritis

To compare IgG anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assays (ELISAs) of second (anti-CCP2) and third generations (anti-CCP3) for the diagnosis of rheumatoid arthritis (RA), an IgA CCP3 ELISA was also evaluated. Combinations of the use of the three tests were evaluated. Anti-CCP2 IgG, anti-CCP3 IgG, and anti-CCP3 IgA antibody levels were determined by ELISAs in the serum of 70 patients with rheumatoid arthritis, 34 patients with systemic lupus erythematosus (SLE), and 54 normal subjects. We evaluated the serum levels, diagnostic performance, and the use of a combination of tests for RA diagnosis. Statistical analyses include receiver operating curves (ROCs) and others. The serum levels were higher in RA patients. Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio (LR), and negative LR were, respectively, 78.6%, 94.3%, 91.7%, 84.7%, 13.8, and 0.23 for anti- CCP2 IgG and 82.9%, 93.2%, 90.6%, 87.2%, 12.2, and 0.18 for anti-CCP3 IgG. These values were better than the same statistical tests for anti-CCP3 IgA. ROC analysis showed that anti-CCP2 IgG and anti-CCP3 IgG had good performance and similar areas. Measuring both IgG and IgA anti-CCP tests increases the specificity if both tests were positive and increases the sensitivity if either test were positive. In our population, anti-CCP2 IgG and anti-CCP3 IgG had good diagnostic performance. Anti-CCP3 IgG had 4.3% more sensitivity than the anti-CCP2 IgG test while sustaining high specificity. This and other studies suggest the development of a dual test—CCP3 IgG and IgA that may be a potential diagnostic tool.     

Profiles of antibodies to histones, DNA and IgG in patients with systemic rheumatic diseases determined by ELISA

The occurrence of antibodies against the total histone complex and the histone fraction H1, antibodies against denatured (ss) DNA and the synthetic double stranded polynucleotide poly dAT, as well as rheumatoid factors (RF) was determined in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Sj?gren's syndrome (SS) using enzyme linked immunosorbent assays (ELISA). Antihistone antibodies could be demonstrated at a frequency of about 17% in the patients with systemic rheumatic disease with no differences between the groups, even if there was a tendency for anti-H1 antibodies to occur more often in the SLE and SS patients than in the RA patients. Some of the antihistone antibody activity seen in the RA patients seems to be due to crossreactive RF. All patient groups showed significant IgG anti-ssDNA antibody activity compared to the controls, but the highest antibody levels were seen in the SLE patients. IgG antipoly dAT antibodies occurred significantly more often and at higher levels in the SLE patients than in the other patient groups. Although the individual tests did not readily distinguish the 3 diseases from each other, the antibody profiles were different. Patients with SS had the broadest reactivity, and the SLE patients had antibodies predominantly restricted to polynucleotides.