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.     

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.     

Circulating immune complexes and rheumatoid arthritis: the induction of immune complex formation between rheumatoid factor and IgG by polyethylene glycol

Circulating immune complexes (CIC) as detected by the C1q binding assay (C1qBA) in sera from patients with rheumatoid arthritis (RA) were not demonstrable in these sera with the indirect granulocyte phagocytosis test (IGPT). This discrepancy could be explained by the finding that polyethylene glycol (PEG), used in the C1qBA, enhanced the binding of rheumatoid factor IgM (RFIgM) and IgG resulting in immune complex (IC) formation. The addition of PEG to RA sera and subsequent testing of these sera in the IGPT revealed increased uptake of IC by these cells, dependent on the PEG dose. Addition of purified RFIgM to a normal human serum generated a positive IGPT in a dose dependent way. We conclude that in RA sera PEG induces IC between RFIgM and IgG. Therefore, assays devised to measure CIC in RA sera which are based on PEG (like the C1qBA) overestimate the amounts of IC present in the circulation in vivo.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparison of immune complexes and complement components in arterial and venous blood of patients with rheumatoid arthritis

Immune complexes (IC) are frequently found in the venous blood and synovial fluid of patients with rheumatoid arthritis (RA). Although IC are claimed to have a pathogenetic role in RA, there is generally poor correlation between different IC tests and between individual tests and clinical features. We have therefore sought differences in the levels of IC detected by the Clq binding assay (ClqBA) and the indirect polymorphonuclear phagocytosis test (IPPT) in the arterial and venous blood of 16 patients with RA and 6 disease control subjects to determine which IC are pathogenetically important. Complement components, IgA, IgG, and rheumatoid factor were also measured. Eight of 10 patients with clinically active RA had higher ClqBA results in arterial blood while IgA IC in the IPPT and most complement components were higher in venous blood. No such differences were seen in patients with inactive RA or controls. These results suggest that IC other than those containing IgA are not formed in limb tissues including the synovium and may explain the variable results previously seen in patients with RA.     

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.     

Immunopathological abnormalities in the normal skin of patients with rheumatoid arthritis in relation to clinical and serological findings: a one year follow up study.

Fifty two patients with seropositive rheumatoid arthritis (RA) were studied over a period of one year to investigate possible relationships among changes of circulating immune complexes (CIC), deposits of immunoglobulins and complement around the cutaneous blood vessels, clinical activity of the disease, and the presence of extra-articular manifestations (EAM). The presence or absence of IgM and C3 in and around the cutaneous blood vessels correlated significantly with the presence or absence of extra-articular features in cross sectional and longitudinal studies. Patients with evidence of these cutaneous immune deposits also had a greater prevalence of CIC as determined by the Clq binding assay (ClqBA) or polyethylene glycol (PEG) assay for IC containing IgM (IgM IC). Although the degree of perivascular mononuclear cell infiltration around the blood vessels in the papillary dermis was related to the patients' clinical state at the initial assessment, it did not correlate with the later changes in the activity of the joint disease or the occurrence of EAM. Thus the deposition of immunoglobulin or complement, or both, seems to be independent of cellular infiltration. The meaning of these cellular infiltrates is not yet fully understood. Our study has shown that many patients with RA who appeared to have only joint disease in fact had subclinical systemic disease as reflected by a positive skin biopsy or CIC. Moreover, the disappearance of IgM deposits from the skin correlated with the disappearance of EAM and improvement of joint disease.     

Serum osteocalcin in chronic polyarthritis in stage III

Osteocalcin, a major noncollagenous protein component of bone matrix which reflects the level of new bone formation, was measured in serum by radioimmunoassay, in 45 patients with rheumatoid arthritis (RA) (Steinbrocker stage III) and in 45 age- and sex-matched healthy subjects. According to the literature, serum osteocalcin concentrations were slightly decreased in the RA group but this was not statistically significant. Only RA patients on corticosteroid therapy had statistically significant decreased osteocalcin levels, compared with normal controls. No correlation was determined in the RA patients between serum osteocalcin levels and levels of C-reactive protein, erythrocyte sedimentation rate, circulating immune complexes (Clq binding assay) and rheumatoid factors of the IgA, IgG or IgM class.     

RHEUMATOID FACTOR HAS INCREASED REACTIVITY WITH IgG FROM SYNOVIAL FLUIDS OF PATIENTS WITH RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS

Synovial fluid IgG may be altered in rheumatoid arthritis (RA)and promote the formation of immune complexes with rheumatoidfactor. To investigate this possibility, monomeric IgG was preparedfrom synovial fluids from a range of arthritides for use asthe antigen in a rheumatoid factor microplate radioimmunoassay.In comparisons with normal serum IgG antigen, increased rheumatoidfactor binding was shown to IgG antigens prepared from synovialfluids from patients with RA and osteoarthritis (OA). Increasedbinding was also shown to RA sera IgG, but not to OA sera IgG.This increased binding was not due to increased IgG antigenbinding to the plate or to IgG rheumatoid factor in the antigenpreparations. It was considered that the cause was a structuralalteration of the IgG as a result of inflammation within therheumatoid and OA joint. KEY WORDS: Antigenicity, Immune complex, Arthritis, IgM     

Rheumatoid factor isotypes and circulating immune complexes in rheumatoid arthritis

Solid phase enzyme immunoassays were here used to quantify rheumatoid factors (RF) of the IgM, IgG and IgA classes and the immune complexes (IK) by their ability to bind to C1q or conglutinin in both the serum and synovial fluid of patients with rheumatoid arthritis (RA). Elevated serum levels of any RF isotype could be found in all patients with seropositive RA (IgM: 63%, IgG: 87%, IgA: 90%). Seronegative patients with RA presented to a significantly lesser extent with elevated levels of all the RF isotypes tested (IgM: 0%, IgG: 40%, IgA: 32%). Synovial fluid RF levels were significantly higher in SPRA patients than in SNRA patients with the exception of IgG-RF. All of the RF classes in both RA groups, however, were elevated when compared to RF in the synovial fluid of patients with osteoarthrosis. Both C1q binding and conglutinin binding immune complexes were significantly higher in the synovial fluid than in the serum of RA patients. The erythrocyte sedimentation rate and plasma iron levels were correlated with the levels of C1q binding immune complexes (IC) in the synovial fluid; total iron binding capacity showed an inverse relationship to synovial fluid IgG-RF levels. A radiographic index was also correlated with IgG-RF levels in the synovial fluid. Extraarticular manifestations were significantly more frequent in patients with elevated serum levels of IgM-RF or conglutinin binding IC. These findings indicate that IgG-RF in the synovial fluid and the formation of IC determined by their ability to bind C1q seem to be closely related to clinical features of local disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-IL-8 autoantibodies and complexes in rheumatoid arthritis: polyclonal activation in chronic synovial tissue inflammation

The chemokine interleukin-8 (IL-8) is frequently associated with inflammatory diseases, and autoantibodies against IL-8 are present in the periphery at elevated levels in such conditions as rheumatoid arthritis (RA). Circulating free anti-IL-8 IgG autoantibodies correlate with inflammatory parameters and disease severity in RA. In this study, correlations were sought between these disease parameters and other antibody subclasses. We assayed IgM, IgA and IgG anti-IL-8 antibodies and IL-8 immunoglobulin immune complexes in the serum of 29 healthy controls and 56 patients with defined RA, and compared the results with clinical and humoral disease parameters. IgG and IgM antibodies directed against IL-8 were present in all samples. In the disease groups, all isotypes of free anti-IL-8 antibodies correlated with increasing humoral disease parameters like CRP and CIC and their related anti-IL-8 immune complexes. Samples which contained high titers of anti-IL-8 antibody subclasses and complexes were RF subclass-positive, while IgM RF-negative sera showed low levels of anti-IL-8 and complexes. Detectable levels of IgG and IgA RF were found in all sera. Patients with extra-articular organ manifestation showed significantly increased free IgA and IgA/IL-8 complexes, with no correlation to the IgA RF titer or IgA hypergamma-globulinemia. The highest titers were seen in two RA cases with vasculitis and in one patient with colitis. Polyclonal activation of the humoral antibody system, which normally precedes the resolution of an inflammatory response, can itself lead to secondary stimulation of inflammatory processes via immune complex formation. In the immune pathology of RA, it degenerates into a persistent chronic inflammation accompanied by progressive joint destruction. The presence of elevated IgA subclass anti-IL-8 autoantibodies in RA patients with extra-articular manifestations suggests these autoantibodies as a clinically useful marker of disease severity and extra-articular manifestations. Received: 10 June 1998 / Accepted: 5 October 1998     

Changes in immune function in patients with rheumatoid arthritis following treatment with sodium aurothiomalate.

The mitogenic response of peripheral blood lymphocytes from 21 patients with rheumatoid arthritis to concanavalin-A (con--A), phytohaemagglutinin (PHA), and pokeweed mitogen (PWM) was significantly lower than in 30 normal subjects. After 15--24 weeks' treatment with sodium aurothiomalate (GST) the response to these mitogens rose to within the normal range. Improvement over pretreatment values was significant for con-A and PWM measured as area under the dose response curve but only for con--A if response at optimal mitogen concentration is the sole criterion. The improvement in PHA response was not significant with either method of measurement. There was an improvement in disease activity by 15--24 weeks as measured by a fall in serum C-reactive protein (CRP), IgM rheumatoid factor (RF), Clq binding activity (ClqBA), and Ritchie articular index. Con--A lymphocyte responsiveness was inversely related to serum CRP levels, but measurements of disease activity were otherwise unrelated to lymphocyte mitogen responsiveness. The observed improvement in peripheral blood lymphocyte responsiveness during gold treatment contrasts with the suppressive effect of gold in vitro. We suggest that the improvement in lymphocyte function is due to the lessening of rheumatoid disease activity during gold treatment, and that the low serum gold levels in our patients were insufficient to mask this effect. Sera from some of our patients were capable of suppressing the function of normal lymphocytes, and this was less apparent after treatment. The suppressive effect of sera correlated with ClqBA. Suppressive factors in serum, including possibly immune complexes, may be one factor leading to suppression of lymphocyte function during rheumatoid arthritis. Such an inverse relationship between humoral and cellular immune mechanisms might influence the clinical expression of rheumatoid arthritis.     

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.