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     

IgG and IgA antibodies to the collagen-like region of C1q in rheumatoid vasculitis

We investigated the presence of IgG and IgA antibodies to C1q in serum samples from 80 patients with rheumatoid arthritis (RA), 31 patients with rheumatoid vasculitis, and 80 healthy controls. IgG and IgA antibodies to C1q, as measured by enzyme-linked immunosorbent assay, were found in less than 5% of the sera from RA patients and from healthy controls. In contrast, IgG and IgA antibodies to C1q were found in 29% and 61%, respectively, of the sera from patients with rheumatoid vasculitis. The occurrence of IgA antibodies to C1q has not been previously demonstrated. These results also demonstrate that IgG antibodies to C1q do not occur exclusively in systemic lupus erythematosus patients: Sera of patients with rheumatoid vasculitis frequently contain IgG or IgA antibodies to C1q, which contribute to immune complex formation.     

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     

Correlation between rheumatoid factor and IL-6 activity in synovial fluids from patients with rheumatoid arthritis

Synovial fluids (SF) and sera (S) from patients with rheumatoid arthritis (RA) were examined for IgM, IgM-rheumatoid factor (IgM-RF), albumin and interleukin-6 (IL-6) activity. The quotient of SF/S IgM-RF was elevated compared with that of SF/S albumin in 7 patients with seropositive RA, although the quotient of SF/S IgM was lower than that of SF/S albumin. SF IL-6 activity was much higher than serum IL-6 activity in all the 7 RA patients. In synovial fluids from 22 seropositive RA patients, SF IL-6 activity was significantly correlated with the SF IgM-RF, IgG-RF and IgA- less than RF, but not with SF IgM, IgG or IgA. Moreover, SF IgM-RF as well as SF IL-6 activity was significantly correlated with the Westergren erythrocyte sedimentation rate (ESR) or the Lansbury articular index. These results indicate that IL-6 and RF might be produced within the rheumatoid joints as a result of abnormal immune system activation, which is associated with the disease activity of RA. Three of the 4 seronegative RA patients, however, showed high SF IL-6 without detectable levels of SF IgM-RF, indicating that IL-6 alone is not sufficient for IgM-RF production.     

IgG and IgA antibody titers against human heat-shock protein (hsp60) in sera of rheumatoid arthritis and osteoarthritis patients

Abstract

To learn whether heat-shock proteins (HSP) are involved in the pathogenesis of rheumatoid arthritis (RA), antirecombinant human heat-shock protein 60 (hsp60) IgG and IgA in sera of RA and osteoarthritis (OA) patients were investigated. Only the anti-hsp60 IgG titer of seropositive (RF-positive) patients was found to be elevated. Although RF titers of the sera of seropositive RA patients were increased, there was no correlation between the individual anti-hsp60 IgG titer and the corresponding RF titer. In contrast, all the anti-hsp60 IgA titers of the sera of OA, seronegative RA, and seropositive RA patients were found to be elevated. Among them, only the serum IgA concentration of seropositive RA patients was increased. Thus, it was suggested that the increased anti-hsp60 IgG reflects the pathogenesis of RA and its activity. It was also suggested that the increased anti-hsp60 IgA response reflects an involvement of hsp60 in the pathogenesis of arthritides rather than the pathogenesis of RA.     

Serum antiimmunoglobulins reactive with human and rabbit IgG in rheumatoid arthritis and other conditions

IgG, IgA, and IgM antiimmunoglobulins reactive with human and rabbit IgG were measured in patients with rheumatoid arthritis (RA), patients with rheumatic fever or osteoarthritis, and normal individuals. Values of all antiimmunoglobulins were significantly evaluated in RA patients as compared with other groups and depended upon activity and stage of the disease. IgM antibodies with specificity for human IgG predominated quantitatively over others in sera of RA patients with high titers of RF, whereas most of those reactive with rabbit IgG in latex negative or positive RA patients belonged to IgG class. The reaction with human IgG included thermostable and thermolabile IgM antiimmunoglobulins but in that with rabbit IgG only thermostable antibodies were active.     

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.     

Rheumatoid factors in psoriatic arthropathy and in Waaler-Rose negative rheumatoid arthritis

Summary Serum levels of IgG, IgA and IgM rheumatoid factor (IgG RF, IgA RF and IgM RF) were determined by means of the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) in 42 Waaler-Rose negative patients with psoriatic arthropathy (PsA) type 1 (arthritis with involvement of distal interphalangeal joints) and type 3 (polyarthritis of rheumatoid type) according to the criteria of Moll and Wright as well as in 53 patients with Waaler-Rose negative rheumatoid arthritis (RA). Elevated levels of RF were found in 22% of patients with PsA type 3 and 45% of patients with Waaler-Rose negative RA. In contrast, none of the patients with PsA type 1 had detectable amounts of RF. It is suggested that the presence of IgG, IgA or IgM RF in patients having psoriasis in conjunction with inflammatory polyarthritis indicates the RA nature of the joint disease and should be considered as exclusion criterion for the diagnosis of PsA.     

The predictive value of rheumatoid factor isotypes, anti-cyclic citrullinated peptide antibodies, and antineutrophil cytoplasmic antibodies for mortality in patients with rheumatoid arthritis

OBJECTIVE: To evaluate the significance of rheumatoid factor (RF) and its isotypes (IgA RF, IgG RF, and IgM RF), anti-cyclic citrullinated peptide antibodies (anti-CCP), and antineutrophil cytoplasmic antibodies (ANCA) in predicting mortality in patients with rheumatoid arthritis (RA). METHODS: The study population comprised 604 patients with RA participating in a cross-sectional study in 1987. Presence of RF (n = 604), RF isotypes (n = 206), anti-CCP (n = 184), and ANCA (n = 200) were determined in these patients from available baseline sera. Vital status was assessed in 1999 and multivariate Cox regression analysis used to compare mortality in RA patients with or without different antibodies. RESULTS: Of the 604 patients with RA, 55% were positive for RF, 66% for anti-CCP, and 14.5% for perinuclear ANCA. Twelve patients (19%) with RF were anti-CCP-negative and 34 (40%) without RF were anti-CCP-positive. Of the total 604 patients, 160 had died by 1999. Positive RF and high IgA and IgM RF levels predicted increased mortality, while positive anti-CCP or ANCA did not. However, high anti-CCP levels were related to an increased mortality risk. CONCLUSION: Patients with RA with positive RF, especially IgA and IgM isotypes, carry a risk of dying earlier than patients without these serological findings.     

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.     

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.     

Prospective study of early rheumatoid arthritis. II. Association of rheumatoid factor isotypes with fluctuations in disease activity.

Thirty-three patients with early peripheral synovitis were followed up for two to four years in order to study the relationship between fluctuations in rheumatoid factor (RF) levels and indices of clinical activity. Twenty-eight of these patients developed classical/definite rheumatoid arthritis (RA). Seventeen patients developed erosive disease of their hands and wrists and thirteen had a positive RF agglutination test. Nineteen patients had raised levels of IgM, RF, IgA, RF, or IgG RF as measured by isotype-specific ELISA techniques. The within-patient fluctuations in IgA RF levels correlated significantly with the corresponding fluctuations in grip strength (p less than 0.05), erythrocyte sedimentation rate (ESR) (p less than 0.01), and a composite index of disease activity (p less than 0.02). IgG RF levels were also associated with changes in ESR and grip strength, but IgM RF showed only a weak association with fluctuations in ESR and not with any other clinical parameters. It is suggested that serum IgA RF may be a useful marker of disease activity in rheumatoid arthritis.     

Prevalence of IgM, IgA and IgG rheumatoid factors in juvenile rheumatoid arthritis

Using an enzyme immunoassay, sera from 50 children with juvenile rheumatoid arthritis (JRA) and 39 controls were tested for IgM, IgA and IgG rheumatoid factors (RF). RF of the IgM and IgA isotypes were present in 11 (22%) patients, but in only one control (p = 0.008). IgG RF was present in the sera of 2 (4%) patients and in none of the controls (p = 0.21). Of the 22 patients with IgM RF or IgA RF, only 3 sera (14%) contained RF of both isotypes. IgM RF was more common in patients with polyarticular disease, while IgA RF was more common in patients with pauciarticular disease. These results indicate that IgM and IgA RF are present in a significant minority of JRA patients and suggest that there is independent expression of the respective RF isotypes.     

Serum immune complexes containing IgA appear to predict erosive arthritis in a longitudinal study in rheumatoid arthritis.

Fifty seven patients with rheumatoid arthritis (RA) were studied longitudinally, and the presence of rheumatoid factor (RF) and various types of immune complexes (IC) was correlated with joint activity and the presence of extra-articular features (EAF). In a cross sectional study it was found that the levels of circulating IC and RF correlated significantly with joint disease activity and the presence of EAF. Longitudinally, levels of IC measured by the C1q binding activity and IC containing IgG and IgM correlated significantly with fluctuations in joint disease activity, whereas IC containing IgG and IgA correlated with the occurrence of EAF. RF and IC levels, however, did not predict the clinical course of the disease. IC containing C3 and C4 were found infrequently and were only present in patients with active rheumatoid vasculitis (RV). The continuous presence of these IC appeared to be linked to the recurrence of vasculitis, irrespective of treatment. Significantly more erosions of hands and feet were found after one year follow up in those RA patients who presented early (disease duration less than one year) who initially had a raised serum IgA IC level (r = 0.72; p less than 0.005).     

Spontaneous immunoglobulin synthesis by peripheral mononuclear cells in active rheumatoid arthritis

Summary Spontaneous production of immunoglobulins (Igs) by peripheral blood mononuclear cells (PBMC) in vitro was investigated to assess B cell activity in a group of 24 patients with rheumatoid arthritis (RA) with or without active joint disease and with or without rheumatoid vasculitis (RV) at the time of study. PBMC of patients with active arthritis (Ritchie index above 16) produced significantly more IgG and IgA than those of patients with inactive joint disease or those of 12 healthy controls. Enhanced production of IgG was found mainly among RA patients with concomitant RV, whereas markedly enhanced IgA production could also be found in patients without symptoms of RV. IgM production was only enhanced in two patients who had both active arthritis and RV. High production of IgG and IgA was probably due to increased numbers of Ig-secreting cells among freshly isolated PBMC, since the concentrations of Ig produced in vitro rose steadily, starting on day 0 and persisting throughout the entire culture period. Moreover, IgG and IgA concentrations measured after 7 days of culture showed significant correlations with the numbers of IgG- and IgA-containing plasma cells in PBMC on day 0. Comparison of the spontaneous production of Igs by PBMC with the levels of circulating immune complexes (CIC), showed that CIC levels were also significantly higher in active arthritis and in RV, but that there was no correlation between the CIC levels in individual patients and Ig production by their PBMC in vitro. This indicates that no direct relationship exists between the occurrence of high CIC levels in RA and the presence of a systemic B-cell hyperreactivity which had been suggested by the enhanced Ig production by PBMC.     

Population study of the importance of rheumatoid factor isotypes in adults.

Blood samples collected from 13,858 randomly selected subjects participating in a health survey in Iceland from 1974 to 1983 were tested for rheumatoid factor. Samples that were positive in a sensitive RF screening test were analysed further by the Rose-Waaler technique and an isotype specific enzyme linked immunosorbent assay (ELISA). In 1987 the 173 available participants who were RF positive and 156 matched RF negative controls were evaluated clinically for rheumatoid diseases. RF levels and isotype patterns were more persistent in the patients with rheumatoid arthritis (RA) than in RF positive subjects who did not have overt RA. The prevalence of RA was only 19% in the participants who were RF positive in 1987. Forty per cent of the participants who had a persistent (four to 13 years) increase of IgA RF combined with either IgM or IgG RF were diagnosed as having RA. A positive correlation was found between RF levels and various manifestations of RA. This association was stronger for the IgA and IgG RF isotypes than for IgM RF. Excluding RF positivity as a diagnostic parameter, RA was diagnosed in 33 of the participants and 20 (61%) of these patients had increased levels of IgM and IgA RF. Patients with RA with bone erosions in their hands had higher levels of IgA RF than patients without erosions, but an association was not found between bone erosions and other RF isotypes. None of the RF negative participants who were symptom free when the original blood sample was taken developed RA during the four to 13 year follow up period. In contrast, five symptom free RF positive participants developed RA during this period. These five patients had all had increased levels of at least two RF isotypes before the onset of their symptoms. It is concluded that the IgA and IgG RF isotypes have a closer association with the clinical parameters of RA than IgM RF. Furthermore, increases in RF can precede clinical manifestations of RA and this applies in particular to the IgA and IgG RF isotypes.     

Relationship of serum IgG rheumatoid factor to IgM rheumatoid factor and disease activity in rheumatoid arthritis

Rheumatoid factors (RF) constitute the major autoantibodies in rheumatoid arthritis (RA). RF are directed against IgG Fc, are polyclonal, and are predominantly of the IgG and IgM classes. RF may participate in both synovial and extraarticular inflammation in RA, although the precise roles of serum IgG and IgM RF are unclear. The purpose of our study was to correlate serum IgG RF with serum IgM RF levels measured by radioimmunoassay and with clinical disease activity in 42 prospectively evaluated seropositive RA patients. IgM RF correlated with IgG RF levels and articular disease activity. IgG RF correlated with IgM RF but not with articular disease activity when adjusted for IgM RF.     

Comparison of serological markers between ACPA<Superscript>+</Superscript> and ACPA<Superscript>−</Superscript> of RA patients

Anti-citrullinated protein/peptide antibodies (ACPA) have recently emerged as sensitive and specific serological markers of rheumatoid arthritis (RA), providing superior alternative of the rheumatoid factor (RF) test in the laboratory diagnostics of RA. We compare the change of serum RF, CRP, IgG, IgM, IgA, total complement, C3 and C4. The sera sample was collected from 123 patients with RA. ACPA were detected with ELISA, and RF, CRP and total complement (Ct), C3 and C4 were examined by automatic biochemical analyzer. Serum RF and total complement concentrations were significantly higher in ACPA+ than in ACPA−, but there were no correlation between ACPA and RF and Ct. Between ACPA+ and ACPA−, there were no significant difference of CRP, IgG, IgM, IgA, total complement, C3 and C4. While there were significant correlation between the concentration of C3 and IgM and ACPA in ACPA+. Conclusion: This is the first study to show that ACPA concentration in ACPA+ patients with RA is positively related to serum IgM and C3 levels.     

Cryoglobulins in cases of rheumatoid arthritis

Summary Clinically diagnosed cases with different grades of rheumatoid arthritis (RA) were studied for (a) cryoglobulin content (b) constituents of cryoglobulins, and (c) rheumatoid factor (RF) titers in sera and cryoglobulins. Of 60 patients, 28 (46.66%) had significantly high levels of cryoglobulins and were mainly distributed in the severe group. Significant amounts of IgG and IgM were found in the cryoglobulins of the 15 cases of RA studied. Ten of these cases had detectable C₃ in their cryoglobulins. Of 20 cases of RA, 10 had detectable levels of RF in their cryoglobulins, as shown by the latex agglutination method. There was no significant correlation between the RF titers of cryoglobulins and the RF titers in the corresponding sera or the quantity of IgM and IgG components in them. Since the RF titer in cryoglobulin seemed to indicate the severity of the disease, the use of this parameter is proposed for diagnosis and prognosis of RA.     

Expression of the major rheumatoid factor cross-reactive idiotype in juvenile rheumatoid arthritis

The major rheumatoid factor cross-reactive idiotype (RF-CRI), defined by prototypic monoclonal IgM rheumatoid factors, is expressed in high frequency by pokeweed mitogen–derived plasma cells from patients with rheumatoid arthritis who express RF in their sera. Unlike adults with rheumatoid arthritis, most patients with juvenile rheumatoid arthritis are seronegative for RF, as detected by classic IgG binding assays. We report that ∼50% of seronegative patients with juvenile rheumatoid arthritis express the RF-CRI in high frequency among their pokeweed mitogen–derived plasma cells, and that ∼33% of patients express the RF-CRI in high titer in their sera. The possible mechanisms for expression of an idiotypic marker of RF without expression of IgG binding activity by classic assays are discussed.