Complement-mediated inhibition of immune precipitation in rheumatoid vasculitis

Complement-mediated inhibition of immune precipitation (CMIP) was measured in patients with rheumatoid arthritis (RA), rheumatoid vasculitis (RA VASC), patients with skin vasculitis not associated with a systemic connective tissue disease and normal healthy controls. CMIP was impaired in 100% (14/14) of the RA vasculitic patients, 60% (12/20) of the RA patients and 22% (2/9) of the dermovasculitic patients. The degree of impairment of CMIP was significantly greater in the RA vasculitic patients compared to the non-vasculitic patients. This difference was due to the significantly lower complement levels and the presence of higher concentrations of an inhibitor of CMIP in the RA vasculitic sera. The levels of this inhibitory activity correlated significantly with IgM rheumatoid factor concentration. Serial studies in three patients with RA vasculitis treated with corticosteroids and immunosuppressive drugs showed significant clinical improvement in two patients, which was associated with improvement in CMIP, reduction in circulating immune complex levels and reduction in IgM rheumatoid factor concentrations.     

Inhibition of C1q binding to antigen-antibody complexes by a factor in rheumatoid arthritis serum

Summary The sera and synovial fluids of patients with rheumatoid arthritis (RA) contain a factor which decreases the binding of C1q to antigen-antibody complex (IC). Several lines of evidence suggest that this factor is distinct from the documented C1q inhibitor which is a chondroitin sulphate. (1) It binds to IC rather than to C1q. (2) It is resistant to digestion with chondroitinase ABC. (3) The addition of chondroitin sulphate to serum does not inhibit the binding of IC to C1q. The observation that three purified IgM and IgG rheumatoid factors (RF) did not reduce C1q binding to IC indicates that the factor is not RF. The ability of RA sera to reduce IC binding to C1q was inversely correlated with their ability to prevent immune precipitation (PIP), and directly with levels of an inhibitor of PIP. These data suggest that the factor which binds to IC and reduces C1q binding may be responsible for the excessive immune precipitation which occurs in RA sera.     

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.     

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.     

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.     

Inhibitory effect of IgM rheumatoid factor on immune complex solubilization capacity and inhibition of immune precipitation

Purified IgM rheumatoid factors (RF; 3 monoclonal and 2 polyclonal) were shown to inhibit, in a dose-dependent manner, 2 complement-mediated functions, i.e., the immune complex solubilization capacity and the inhibition of immune precipitation. Inhibition of immune complex solubilization capacity occurred only if RF was added at the same time as, but not after, addition of the complement source. Experimental evidence suggests that the effects of RFs were not related to their anticomplementary activity, but rather required the attachment of RF to the Fc region of the IgG molecule. Although no clinical data are available so far, it might be plausible that these newly described properties of RF have biologic relevance.     

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.     

An inhibitor of complement-mediated prevention of immune precipitation in rheumatoid arthritis — relationship to disease activity and systemic manifestations

Summary In normal serum complement prevents precipitation of antigen-antibody complexes (PIP). However rheumatoid arthritis (RA) serum contains an inhibitor of this complement-mediated function. We have undertaken two prospective studies in order to look for any relationship between the presence and levels of inhibitory activity in sera and synovial fluids (SF) of patients with RA and disease activity (study A), and the presence of systemic manifestations (nodules and vasculitis) of RA (study B). In study A, levels of inhibitory activity were highest in the sera and synovial fluids of patients with seropositive RA. However there was no correlation between the inhibitory levels and indices of generalised disease activity (articular index, erythrocyte sedimentation rate (ESR), haemoglobin, white cell and platelet counts). Local joint tenderness score correlated weakly with the inhibitory level in SF (P<0.05). There was no correlation, however, with either the SF protein concentration or white cell count. In study B, PIP was shown to be lower in patients with the systemic manifestations of RA than in those with purely articular manifestations. PIP was particularly low in those patients with vasculitis compared to those with subcutaneous nodules. Serum levels of inhibitory activity were highest in patients with vasculitis and lowest in those with articular disease only, whereas patients with nodules had intermediate levels. Our conclusion is that inhibition of immune precipitation is not associated with disease activity, but is associated with the extra-articular manifestations of RA. The inhibitory factor may play a role in the pathogenesis of RA.     

Activation of the classical pathway of complement by rheumatoid factors

A simple, sensitive solid-phase radioimmunoassay to quantitate the activation of the classical pathway of complement by rheumatoid factor (RF) is described. RF (purified, in serum or synovial fluid) was bound to reduced and alkylated IgG adsorbed to polyvinyl chloride microtiter plates and reacted with diluted normal human serum (complement). The activation and binding of C4 were quantitated with ¹²⁵I-Fab'₂-anti-C4. Purified, polyclonal IgM–RF was 100- to 1,000-fold more effective than purified IgG–RF in activating complement. The amount of complement activation produced by RF in each of the 57 sera and 2 synovial fluid samples correlated directly with the amount of IgM–RF present. The complement activating abilities of polyclonal IgM–RF in the sera of 15 rheumatoid arthritis patients were homogeneous. This novel technique is readily applicable to the investigation of complement activation by RF in disease.     

Complement mediated vascular endothelial injury in rheumatoid nodules: a histopathological and immunohistochemical study

OBJECTIVE: To evaluate morphologically and immunohistochemically the role of IgM rheumatoid factor (RF) immune complexes and complement activation in rheumatoid nodule vascular injury, a typical extraarticular manifestation of rheumatoid arthritis. METHODS: Histological features such as cellular infiltration, endothelial alteration, fibrinoid degeneration, and basement membrane alterations were observed in the small vessels in rheumatoid nodules. An immunohistochemical study was also carried out. RESULTS: Distinct colocalization of IgM RF and terminal complement complexes (TCC: C5b-9) was observed on the luminal surface in some of the damaged endothelial cells. Immuno-electron microscopy revealed endothelial vesiculation, typical of the in vitro protective mechanism against complement attack, with deposition of not only TCC but also IgM RF. Most TCC positive endothelial cells simultaneously expressed the major complement regulatory factor, CD59. CONCLUSION: These data suggest that, in rheumatoid nodules, vascular injury mediated by complement activation involves the assembly of IgM RF on the endothelial cell surface.     

Measurement of rheumatoid factors by an enzyme-linked immunosorbent assay (ELISA) and comparison with other methods.

IgM rheumatoid factor (RF) was measured in the sera of 48 rheumatoid patients and of 48 age and sex-matched normal controls by the Rose-Waaler and latex agglutination tests, a rate nephelometer, and an enzyme-linked immunosorbent assay (ELISA). Good correlation was obtained between all assays. The rate nephelometer assay was the easiest and quickest to perform and gave results in international units/ml. The Rose-Waaler was the least sensitive assay and the most difficult to perform and interpret. Both the latex agglutination and the ELISA were sensitive, though some overlap of patient and control sera was seen with all the assays. In addition to IgM RF the ELISA was used to measure IgG RF and IgA RF in both rheumatoid and control sera. Although some normal sera had detectable amounts of IgG and IgA RF, the levels of both were significantly raised in the rheumatoid sera. IgG RF levels were lower after pepsin digestion of the sera, suggesting that IgM RF interfered with the assay for IgG RF unless this treatment was included.     

Enhancement of Clq binding activity by IgM rheumatoid factor

Clq binding activity (ClqBA) averaged 18.1 +/- 14.5% (1 SD) in 28 rheumatoid arthritis (RA) sera (normal sera = 3.9 +/- 0.4%). Further analysis indicated that rheumatoid factor (RF) positive [RA (+)] sera averaged 30.4% ClqBA, significantly greater than the 3.9% ClqBA in RA RF negative [RA(-)] sera (p less than 0.01). In the RA(+) sera, RF titer correlated with ClqBA (r = +0.73). Addition of IgM RF to sera of normal, SLE, and RA(-) patients, as well as to aggregated IgG and reduced and alkylated aggregated IgG, resulted in significant increases in ClqBA, up to 14% in the latter group (p less than 0.01). Control IgM added to these same systems had no effect on ClqBA. IgM RF only slightly increased Clq binding of monomeric IgG.     

IgM rheumatoid factor estimation by ELISA in seronegative rheumatoid arthritis before and after IgM fractionation: does seronegative RA exist?

We looked for the presence of IgM rheumatoid factor (RF) using an enzyme-linked immunosorbent assay (ELISA) in 25 patients with active seronegative rheumatoid arthritis. In unfractionated sera, 12 patients (48%) were positive for IgM RF (classical), but after IgM fractionation of 23 samples (2 samples were not available) using high performance liquid chromatography for fractionating IgM, 12 patients were positive for RF by ELISA indicatingt the presence of hidden RF. Finally, three patients were labelled as truly seronegative for IgM RF. Classical IgM RF as deteced by ELISA correlated significantly with erosive disease. Hidden RF did not correlate with disease activity or severity in this cross-sectional study, and though its presence was associated with shorter disease duration. this did not reach statistical significance.     

Metabolism of c4 and factor b in rheumatoid arthritis

Metabolic turnover determined by radioiodide labeled C4 and Factor B was studied in 18 patients with rheumatoid arthritis (RA) and 19 normal control subjects as a means of estimating the relative ratio of consumption of components in the classical and alternative pathways of complement activation. Predominance of fractional catabolic rate (FCR) of C4 over Factor B was demonstrated with differentially labeled C4 and Factor B. The hypercatabolism occurred in the extravascular space. C4 FCR correlated significantly with rheumatoid factor (RF) determined in a hemolytic assay (r_s = 0.72), measured as IgG RF (r_s = 0.57), and as IgM RF (r_s = 0.45). There were no significant correlations with several other antibodies measured. These results are consistent with the hypothesis that RA is a systemic, extravascular immune complex disease, in which RF immune complexes play a significant pathogenetic role principally via activation of the classical pathway of complement.     

Characterisation of the size and composition of circulating immune complexes in patients with rheumatoid arthritis.

The size and composition of circulating immune complexes in the sera of patients with rheumatoid arthritis (RA) were studied in relation to different manifestations of the disease. Circulating immune complexes from the sera of 94 patients (50 with extra-articular disease) and 10 matched controls were fractionated by sucrose density gradient ultracentrifugation. The composition, immunoglobulin and rheumatoid factor (RF) concentrations within each of the fractions were determined by a sensitive enzyme linked immunosorbent assay (ELISA). Intermediate size (14S-21S) IgG complexes containing RF activity and 22S IgG-IgM RF complexes were found in the sera of 40 patients with RA, while intermediate size complexes of self associated IgG RF and larger size complexes (greater than 22S) of IgG RF and IgM RF were associated with extra-articular features of RA (50% of extra-articular disease). Complexes containing IgA were found in the sera of many patients with RA, and dimeric IgA RF mainly in patients with extra-articular disease. These results support the view that whereas small size circulating immune complexes are of no primary pathogenic importance in synovitis, large size (greater than 22S) circulating immune complexes may play a role in extra-articular disease in RA. Current understanding of the formation of large complexes provides a biological explanation for their occurrence and effects.     

Monoclonal antibody 6b6.6 defines a cross-reactive kappa light chain idiotope on human monoclonal and polyclonal rheumatoid factors

Mouse monoclonal antibody (MAb) 6B6.6 was raised against a cross-reactive idiotope (CRI) present on the light chains of 2 human IgM paraproteins with rheumatoid factor (RF) activity. The MAb inhibited the IgG-binding activity of these proteins, and thus appears to react with an epitope located at or near the RF-binding site. Enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting studies indicate that the 6B6.6 CRI is associated with kIIIa sub-subgroup light chains, is not related to the Wa, Po, and Bla RF cross-idiotypic specificities, and is clearly distinct from the kIIIb-associated CRI detected by MAb 17.109. Using an ELISA, we detected 6B6.6 CRI in 59% of 107 sera and 48% of 50 synovial fluids from patients with seropositive rheumatoid arthritis (RA). However, the quantities of CRI-positive RF were small, and the amount of CRI-positive RF did not correlate with the amount of IgM-RF. The 6B6.6 CRI was shown to occur primarily in the IgM fraction of RA sera by both chromatographic studies and isotype-specific ELISA, although small quantities appeared to be associated with IgA and IgG in some sera. The presence of 6B6.6 CRI on both monoclonal and polyclonal RF is consistent with the view that both are derived, at least in part, from a common gene pool. However, its occurrence in relatively low levels suggests that the number of germline genes encoding for RF is large or that extensive mutation occurs in the course of RF expression in RA.     

Complement-activating rheumatoid-factor-containing complexes in patients with rheumatoid vasculitis.

The role of complement and rheumatoid factor in immune complexes was examined in patients with a variety of rheumatic diseases. This was done by assessing the amount of rheumatoid factor (RF) bound from sera by F(ab)2 anti-C3 attached to a solid matrix. High levels of RF bound to C3 were detected in patients with rheumatoid arthritis complicated by vasculitis but rarely and in lower levels in patients with synovitis, ankylosing spondylitis, and systemic lupus erythematosus. The activity was bound to anti-C3 through anti-C3 antibodies because little was bound by normal F(ab)2 and was evidently complexed in the sera before in-vitro testing, since it was precipitated by 2 . 5% polyethylene glycol and sedimented with high molecular weight material on sucrose density gradient ultracentrifugation. It is considered that RF-containing complexes are present in vasculitic sera and have the potential to bind complement in vivo.     

Monoclonal antibody 6B6.6 defines a cross-reactive kappa light chain idiotope on human monoclonal and polyclonal rheumatoid factors.

Mouse monoclonal antibody (MAb) 6B6.6 was raised against a cross-reactive idiotope (CRI) present on the light chains of 2 human IgM paraproteins with rheumatoid factor (RF) activity. The MAb inhibited the IgG-binding activity of these proteins, and thus appears to react with an epitope located at or near the RF-binding site. Enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting studies indicate that the 6B6.6 CRI is associated with kappa IIIa sub-subgroup light chains, is not related to the Wa, Po, and Bla RF cross-idiotypic specificities, and is clearly distinct from the kappa IIIb-associated CRI detected by MAb 17.109. Using an ELISA, we detected 6B6.6 CRI in 59% of 107 sera and 48% of 50 synovial fluids from patients with seropositive rheumatoid arthritis (RA). However, the quantities of CRI-positive RF were small, and the amount of CRI-positive RF did not correlate with the amount of IgM-RF. The 6B6.6 CRI was shown to occur primarily in the IgM fraction of RA sera by both chromatographic studies and isotype-specific ELISA, although small quantities appeared to be associated with IgA and IgG in some sera. The presence of 6B6.6 CRI on both monoclonal and polyclonal RF is consistent with the view that both are derived, at least in part, from a common gene pool. However, its occurrence in relatively low levels suggests that the number of germline genes encoding for RF is large or that extensive mutation occurs in the course of RF expression in RA.     

Complement activation by 19S IgM rheumatoid factor: relationship to disease activity in rheumatoid arthritis

19S IgM rheumatoid factor (RF) in rheumatoid arthritis (RA) are polyclonal autoantibodies directed against the Fc piece of IgG. Rheumatoid patients with RF tend to have aggressive synovitis, nodules, and extraarticular manifestations. Although RF titer does not correlate with disease activity, RF activates complement (C) by the classical pathway. Thus, we postulated that selective stimulation of cell clones producing efficient C activating RF molecules might be associated with disease flares, independent of changes in serum RF concentration. To address the question, 42 patients with RA were evaluated prospectively. Serum RF concentration was measured by radioimmunoassay (RIA) and C activating activity by hemolytic assay. We then calculated the mean hemolysis (MH) of sensitized sheep erythrocytes (SRC) produced/ml of RF serum (MH/ml) and MH/microgram of RF as an expression of RF C activating properties (CAP). The following observations were made: RF CAP varied among the patients studied; RF CAP varied over time in individual patients; RF CAP differences varied in both groups independently from RF concentration; RF CAP correlated with both systemic and articular disease activity; and total RF concentration correlated with articular findings and nodules but less well with systemic disease activity.     

Separation and characterization of immune complexes containing 19S IgM rheumatoid factor-IgG in juvenile arthritis

Sera from 10 patients with juvenile arthritis (JA), 2 seropositive and 8 with hidden rheumatoid factor (RF), were subjected to affinity chromatography on a rabbit anti-human IgM column. Material retained by the column was eluted sequentially by 1M NH₃ and 0.1M glycine-HCl buffer, pH 3.0. The affinity fractions contained both 19S IgM RF and IgG, while corresponding fractions from healthy controls contained neither. Sera from 15 patients with JA, 1 seropositive and 11 with hidden RF, were subjected to 4% polyethylene glycol precipitation followed by acid dissociation of the precipitate. Ten of 15 resultant fractions contained both IgM RF and IgG, while corresponding fractions from healthy controls contained only traces of IgG. Sera from 7 of these JA patients were subjected to sucrose density gradient centrifugation and the resultant fractions analyzed for the presence of immune complexes by the Clq solid-phase assay. Immune complexes were detected at and ahead of the IgM marker, as expected for IgM RF-IgG complexes. These combined data show that the majority of JA patients with classic or hidden 19S IgM RF have immune complexes containing IgM RF and IgG in their sera.