FIBRONECTIN BINDING WITH IMMUNOGLOBULIN AGGREGATES AND ITS ASSOCIATION WITH RHEUMATIC DISORDERS

Fibronectin was shown to bind heat-aggregated, but not monomeric,human IgG, suggesting that fibronectin may bind directly toIgG immune complexes. The presence of material both bindingand already containing fibronectin was demonstrated in polyethyleneglycol precipitates of sera and synovial fluids from patientswith rheumatoid arthritis (RA) and gout, but not normal sera.By contrast, complement-fixing complexes contained fibronectinin RA synovial fluids and sera, but not in sera and synovialfluids from other rheumatic disorders. It is considered thatfibronectin binds to immune complexes in RA synovial fluidsand sera but that some other, as yet unidentified material,is effective in binding fibronectin in sera and synovial fluidsfrom patients with osteoarthritis and crystal synovitis.     

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.     

Ia specific antilymphocyte antibodies in rheumatoid arthritis

Antilymphocyte antibodies (ALA) in rheumatoid arthritis (RA) have increased reactivity with phytohemagglutinin (PHA) activated lymphoblasts which are known to have increased expression of Ia antigen. The present experiments suggest that part of this reactivity represents an Ia specificity of ALA. Fifteen of 18 RA sera tested were able to inhibit the binding of monoclonal anti-Ia antibodies as measured by a rosette method. RA sera did not inhibit the binding of other monoclonal antibodies: anti-OKT3, anti-OKT4, and anti-OKT8. The ability of RA sera to inhibit the binding of anti-Ia antibody was eliminated after absorption of the RA sera with an Ia positive human cell line (B35M) but not by an Ia negative line (MOLT4). Blocking of anti-Ia binding was greater in the IgG fraction of the RA sera but also occurred in the IgM fraction. Experiments including ultracentrifugation, pepsin digestion of RA sera, and preincubation of lymphoblasts with aggregated IgG demonstrated that Fc binding by RA sera was not a factor. Both monoclonal anti-Ia and anti-Ia heteroantiserum also had increased reactivity with lymphoblast target cells. Pepsin digested Fab fragments of the anti-Ia heteroantiserum were able to block the activity of cytotoxic RA serum. However, ALA cytotoxic to lymphoblasts did not correlate with anti-Ia by rosette method. Ia-specific ALA by rosette method was associated with donor variability but did not appear to be HLA-DR restricted. Ia-specific ALA did correlate with disease activity. These data suggest that anti-Ia activity is present in RA sera and may play an immunoregulatory role in this disease.     

Characteristics of antinuclear antibodies in rheumatoid. arthritis

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

RHEUMATOID ARTHRITIS IN A POPULATION OF PERSONS AGED 85 YEARS AND OVER

A Dutch urban population of 977 persons aged 85 years and overwas examined for the presence of rheumatoid arthritis (RA).Prevalence rates for definite RA, past polyarthritis with jointdeformation and past polyarthritis without joint deformationwere 0.3%, 0.3% and 0.7%, respectively. The polyarthritis patientsdid not differ from age-matched controls with respect to crudescores of physical disability, the presence of serum rheumatoidfactor or the presence of HLA-DR4. Although these data shouldbe interpreted with caution because of the small patient groups,we conclude that in persons aged 85 years and over the prevalenceof RA is low, and the disease is relatively mild. KEY WORDS: Rheumatoid arthritis, Population study, Aged, Oldest old, HLA-DR4 antigen     

KNEE LAXITY IN PATIENTS WITH OSTEOARTHRTTIS AND RHEUMATOID ARTHRITIS

Thirty-four patients with osteoarthritis (OA) and 32 patientswith rheumatoid arthritis (RA) were studied to determine theeffects of OA and RA on the laxity of the knee joints. Laxitywas measured with the Genucom Knee Analysis System. The antero-posteriorlaxity of the OA and RA knees was greater than the control,normal knees in the early stage, and decreased with the severityof disease in OA, but not in RA. Severe OA and RA were associatedwith a restricted internal-external rotation at the knee jointcompared with the control. Internal-external rotation decreasedwith worsening of both diseases. Varus-valgus laxity tendedto increase slightly with the severity of disease. While themorphological changes of the cruciate ligaments in advancedOA and RA were not statistically different, the laxity of OA-afflictedknees was affected slightly by the severity of the damage tothe cruciate ligaments. KEY WORDS: Osteoarthritis, Rheumatoid arthritis, Knee joint, Laxity, Cruciate ligament     

Lymphocytes bearing Fc gamma receptors in rheumatoid arthritis. I. An increased subpopulation of cells in rheumatoid arthritis detected with Facb rosettes.

The chronic production of IgM and IgG antiglobulins is a major feature of rheumatoid arthritis. This implies an abnormal interaction between rheumatoid leucocytes and IgG. A novel rosette assay employing rabbit Facb-coated calf red blood cells has been developed to study receptors for IgG on peripheral blood lymphocytes. Cells were obtained from groups of patients with rheumatoid arthritis (RA), osteoarthritis (OA), ankylosing spondylitis (AS), and healthy control subjects. Receptors for Facb were found on an increased proportion of lymphocytes from RA patients compared with the other groups tested. It has been shown that the Facb rosette assay detects a subpopulation of lymphocytes bearing receptors for the Fc region of IgG. This receptor is clearly capable of recognising and binding only the C gamma 2 domain within the Fc region. As such it shows different specificity from some other Fc receptors detected on mononuclear cells. The number of Facb rosette-forming lymphocytes in an individual sample correlated well with the number of cells bearing 'high avidity' Fc receptors. However, the incidence of these cells in RA patients could not be correlated with disease activity, disease duration, or levels of IgM and IgG rheumatoid factor. Thus increased Facb rosette cells may represent a fundamental imbalance of the immune response in patients with rheumatoid arthritis.     

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.     

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.     

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.     

DETECTION OF MYCOBACTERIUM TUBERCULOSIS ANTIGEN IN SYNOVIAL FLUID OF PATIENTS WITH RHEUMATOID ARTHRITIS

By use of antimycobacterial saline extract antibodies, Mycobacteriumtuberculosis (MT) antigens have been detected in synovial fluid(SF) of patients with rheumatoid arthritis (RA) by a highlyspecific and sensitive double-antibody sandwich enzyme-linkedimmunosorbent assay (ELISA). Absorbance for 15 gout, 14 osteoarthritis(OA) patients, 12 patients with spondylarthropathies (SA) andeight patients with other inflammatory disorders (OID) rangedfrom 0.001 to 0.025 with the mean values of 0.0086 ±0.0078, 0.0077 ± 0.0051, 0.0069 ± 0.0059 and 0.0113± 0.0059, respectively. Among 65 SF of patients withRA examined, 34 were found to be negative for MT antigen withabsorbance ranging from 0.002 to 0.024 and a mean value of 0.0114± 0.0070. For 31 (47.7%) MT antigen-positive specimensof RA, optical density ranged from 0.052 to 2.446 with a meanvalue of 0.5564 ± 0.7354. Significant statistical difference(P < 0.05) was found when MT antigen positives were comparedwith MT antigen negatives, gout, OA, SA and OID groups. Ourresults indicate that MT may be relevant to the pathogenesisof RA. KEY WORDS: MT antigen and RA, Synovial fluid, Antimycobacterial saline extract antibody, ELISA     

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.     

Epitope-specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen-induced arthritis in the mouse

OBJECTIVE: To analyze the fine specificity of IgG autoantibodies in sera from rheumatoid arthritis (RA) patients for type II collagen (CII) epitopes that are arthritogenic in collagen-induced arthritis (CIA), a relevant murine model of RA. METHODS: For enzyme-linked immunosorbent assay (ELISA) analysis of conformation-dependent autoantibody binding, recombinant chimeric collagens that harbor the respective CII epitopes as an insertion within the frame of a constant type X collagen triple helix were constructed. In addition, synthetic peptides mimicking the native collagen structures were applied for the first time in the ELISA assessment of humoral CII autoimmunity. RESULTS: The pathogenicity of IgG responses to certain CII determinants in CIA was demonstrated by arthritis development in BALB/c mice upon the combined transfer of 2 mouse monoclonal antibodies specific for precisely mapped conformational CII epitopes (amino acid residues 359-369 [C1(III)] and 551-564 [J1]), whereas antibodies to another epitope (F4) were not arthritogenic. To test whether human autoimmune responses are similarly directed to these conserved CII determinants, serum IgG was analyzed. The prevalence of sera with increased IgG binding to the C1(III) epitope was significantly higher in RA compared with sera from healthy donors or from patients with other rheumatic conditions, e.g., osteoarthritis (OA), systemic lupus erythematosus (SLE), or relapsing polychondritis (RP), whereas levels of antibodies specific for the nonarthritogenic F4 epitope were associated with OA rather than RA. CONCLUSION: Autoimmunity to CII, although detectable in different rheumatic conditions, differs in fine specificity between distinct disease entities. In RA, in contrast to degenerative joint disease, RP, and SLE, autoantibody responses are directed to an evolutionary conserved CII structure that is also targeted by pathogenic autoimmune responses in murine models of arthritis.     

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

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

SPECIFICITY OF ANTIPERINUCLEAR FACTOR FOR RHEUMATOID ARTHRITIS IN RHEUMATOID FACTOR-POSITIVE SERA

Although antiperinuclear factor (APF) has the same specificityfor rheumatoid arthritis (RA) as rheumatoid factor (RF), thereis no evidence that this specificity is maintained in patientswith positive RF-agglutination tests. Thus, we evaluated thespecificity and usefulness of APF for RA diagnosis, regardlessof RF titre. APF was tested (1:100 threshold) on 214 sera sentfor RF evaluation over a 9-month period. These sera were previouslydetermined to have latex or Rose-Waaler (RW) titres     

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.     

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.     

Interleukin-1–inhibitory IgG in sera from some patients with rheumatoid arthritis

Inhibition of interleukin-1°aL (IL-1°aL) activity was detected in 7 of 41 serum samples from patients with rheumatoid arthritis (RA). These 7 sera inhibited not only IL-1°aL–induced endothelial cell adherence to neutrophils, but also IL-1°bT–induced endothelial cell adherence, although to a lesser extent. These sera showed no influence on tumor necrosis factor–induced endothelial cell adherence. No inhibitory activity was found in 40 sera from normal control subjects. Studies to further examine these effects included gel filtration analysis of 2 of the RA sera. The inhibitory activity was eluted near M_r 158 kd and above M_r 250 kd. Analysis by protein A affinity chromatography showed that IL-1–inhibitory activity was present in protein A–binding fractions. Purified IgG (by DE-52 column chromatography) from RA patients was found to be as potent an inhibitor as the protein A–binding fractions, which suggests that the major inhibitory activity in RA sera is attributable to IgG molecules. These purified IgG molecules also inhibited IL-1–induced proliferation of mouse thymocytes but did not influence IL-2–dependent proliferation of the CTLL-2 murine T cell line. The 7 patients whose sera showed IL-1–inhibitory activity had mild RA and low titers of rheumatoid factor. The findings, taken together, suggest a possible regulatory role of IL-1–inhibitory IgG in RA disease activity.     

High diagnostic value of anticalpastatin autoantibodies in rheumatoid arthritis detected by ELISA using human erythrocyte calpastatin as antigen

OBJECTIVE: To develop a quantitative method of measuring autoantibodies against human calpastatin in rheumatoid arthritis (RA) and to determine their diagnostic value compared with other autoimmune and articular diseases. METHODS: We performed a highly sensitive ELISA for IgG and IgM anticalpastatin autoantibodies in human sera using human erythrocyte calpastatin as an antigen. Samples were diluted 1:2000 for the measurement of IgG and 1:400 for IgM. RESULTS: IgG anticalpastatin antibodies were found in the sera of 48 of 58 patients (82.8%) with RA. In contrast, IgG anticalpastatin antibodies were found in the sera of only 2 of 11 (8.3%) patients with osteoarthritis (OA). Compared to sera from patients with other autoimmune diseases, anticalpastatin antibody sensitivity for RA was better than that of systemic lupus erythematosus (5.6%), systemic sclerosis (0%), mixed connective tissue disease (0%), and Sj?gren's syndrome (20%). IgG anticalpastatin antibodies also showed high specificity (96.1%) for RA. Almost 90% of patients with RA were positive for IgG or IgM anticalpastatin antibodies. CONCLUSION: We have developed a simple, sensitive, specific, and quantitative ELISA for anticalpastatin antibodies that may have a high diagnostic value for RA.