Studies of the third component of complement in synovial fluid from arthritic patients. II. Conversion and its relation to total complement

Plasma and synovial fluid from arthritic patients were studied with antigen–antibody crossed electrophoresis for the conversion of C3. When present, C3 conversion was estimated planimetrically. The material included patients with rheumatoid arthritis and systemic lupus erythematosus as well as patients with non-rheumatoid arthritis.

C3 conversion was not found in plasma from any of the patients studied.

In non-rheumatoid synovial fluids there was no conversion in five and less than 10% in four of the samples. In rheumatoid synovial fluids C3 conversion proved significantly (P<0·01) more pronounced, the degree of conversion exceeding 10% in fourteen out of twenty-three cases. An inverse relationship was found in synovial fluid between the degree of C3 conversion on the one hand, and the total complement activity or the C3 concentration on the other.

     

Gamma globulin complexes in synovial fluids of patients with rheumatoid arthritis. Partial characterization and relationship to lowered complement levels

Gamma globulin complexes were demonstrable in certain joint fluids from patients with rheumatoid arthritis by analytic and density gradient centrifugation. They form a continuum of high molecular weight components ranging from 7S to 30S and were dissociable primarily to 7S γG globulin. The larger complexes were also detectable by precipitation reactions with C1q and with γM rheumatoid factor. This permitted the isolation and partial characterization of the complexes. Non-immunoglobulin constituents were not detectable. Evidence was obtained that 7S γG globulin rheumatoid factors represented an important constituent of the complexes.

A relationship was encountered between the amount of γ globulin complex present in the joint fluids and diminution in total haemolytic complement activity. All fluids with abundant γ globulin complexes contained markedly lowered complement levels. A decrease in levels of C1q and β_1A was found to correlate with the amount of γG globulin complexes. Although patients who had diminished complement levels in their joint fluid have serum γM rheumatoid factor, its titre does not correlate well with the extent of complement depression.

Joint fluids with abundant γ globulin complexes manifested an anticomplementary effect. This activity was most apparent at 37°C when fresh rheumatoid serum was used as a source of complement. Evidence indicating the participation of γM rheumatoid factor in this anticomplementary effect was obtained. All fluids with significant anticomplementary activity formed precipitin bands with C1q on agarose plates. γM rheumatoid factor was not necessary for this reaction.

     

Relationship between leukotriene B4 and immunological parameters in rheumatoid synovial fluids

Leukotriene B₄ (LTB₄) was measured in synovial fluid from 20 patients with rheumatoid arthritis and 15 patients with osteoarthritis. The level of LTB₄ was significantly higher in synovial fluid from rheumatoid arthritis patients as compared with synovial fluid from osteoarthritis patients. LTB₄ levels also significantly correlated with cell numbers, rheumatoid factor, and immune complexes in synovial fluid from rheumatoid arthritis patients. There was an inverse correlation between LTB₄ levels and complement components. The high-pressure liquid chromatography peak of immunoreactivity extracted from the synovial fluid occurred at a retention volume identical to that of authentic LTB₄. These results suggest that the increased level of this mediator in synovial fluid may contribute to perpetuation of inflammation and tissue destruction in rheumatoid arthritis.     

Immunopathological changes in rheumatoid arthritis and other joint diseases.

A comparative study of the distribution of immunoglobulins G, M, and A and C3 in the synovium and inside synovial fluid leucocytes and of the relative levels of IgG, IgM, AND C3 in paired samples of serum and synovial fluid from both seropositive and seronegative patients with rheumatoid arthritis and other types of non-infective synovitis shows that although there is no distinctive immunopathological feature of rheumatoid arthritis, the incidence of immune complexes containing IgG and IgM with and without detectable C3 in the affected synovium or inside synovial fluid granulocytes is higher in rheumatoid arthritis and especially so in seropositive cases. The mean level of C3 in synovial fluid from patients with rheumatoid arthritis is lower than that from the group without rheumatoid arthritis. In contrast to previous reports, extracellular clumps of IgA could be detected in the affected synovium of a number of affected patients. Aggretated human IgG could be bound by some of the synovial biopsies and synovial fluid leucocytes from both seropositive and seronegative rheumatoid arthritis patients. Antinuclear factor and rheumatoid factor could be detected in the synovial fluid but not in the serum of several patients suggesting either selective sequestration or local synthesis of antinuclear and rheumatoid factors in the affected joints.     

活动期类风湿关节炎患者血清及关节液白细胞介素-17水平变化及与有关实验室指标的关系

目的:分析类风湿关节炎(RA)患者外周血和关节液中T辅助细胞17(Thl7)相关细胞因子白介素-17(IL-17)水平,探讨其在RA中的变化特点及与实验室指标的相关性.方法:用ELISA检测50例活动期RA患者血清和其中15例关节液以及30例正常人血清IL-17水平.同时测定RA患者血清超敏C反应蛋白(hs-CRP)、...     

Terminal complement complexes and C1/C1 inhibitor complexes in rheumatoid arthritis and other arthritic conditions.

Terminal complement complex (TCC) and C1r-C1s-C1 inhibitor complex (C1/C1 INH) concentrations were measured in plasma and synovial fluid from patients with arthritis and related to other measures of disease activity. Both TCC and C1/C1 INH concentrations were significantly increased in patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis (plasma and synovial fluid, P less than 0.05) and normal subjects (plasma only, P less than 0.001). In the patients with RA, there was no correlation between plasma or synovial fluid TCC concentrations and IgM rheumatoid factor, immune complex or C1/C1 INH levels. However, in 10 patients with seronegative RA, C1/C1 INH and immune complex levels correlated significantly in synovial fluid (r = 0.69, P less than 0.05) although not in plasma (r = 0.52). Plasma and synovial fluid TCC and C1/C1 INH concentrations did not differ in rheumatoid patients with severe compared with mild joint disease (categorized by the Ritchie score). These results confirm a role for complement activation in RA but suggest that several mechanisms are involved in its pathogenesis.     

Specific binding of immunoconglutinin in tissues and synovial fluids from patients with rheumatoid arthritis

In eluates from rheumatoid synovial tissue, immunoconglutinin was present in only one of fifteen cases. After pepsin digestion, immunoconglutinin activity was increased or revealed in almost all eluates examined. Natural antibodies, present in the corresponding sera, could not be detected in the eluates before or after pepsin digestion. Neither was any immunoconglutinin present or revealed in eluates from non-rheumatoid synovial tissue. The experiments indicated that this antibody is specifically fixed in synovial tissue of patients with rheumatoid arthritis. The immunoconglutinin activity in the eluates was probably due to an IgG antibody. Density gradient ultracentrifugation and treatment with 2-ME indicated that both IgG and IgM immunoconglutinin were present in sera from these patients.

In rheumatoid synovial fluids, immunoconglutinin was present in an amount equal to, or somewhat lower than in serum. By density gradient ultracentrifugation at pH 3·0, IgG immunoconglutinin activity seemed to be revealed or increased in most cases, indicating a fixation of this antibody also in synovial fluids.

Immunoconglutinin in human sera could be absorbed out with immune precipitates exposed to fresh human serum, and this reaction seemed to increase the complement fixing ability of the precipitates. If binding of immunoconglutinin in rheumatoid synovial tissue and synovial fluids increases complement fixation and activation in a similar way, it might be a pathogenetic factor in the rheumatoid inflammation.

     

The concetrations of the fourth component of complement and of the C[unk] inactivator in synovial fluid from arthritic patients

The concentration of the fourth component (C4) of complement (C) in synovial fluid was immunochemically determined in forty-nine cases of arthritis. The lowest C4 values were found in the patients with rheumatoid arthritis (RA), in particular those with depressed synovial fluid C values (and positive rheumatoid factor tests), whereas the highest were obtained in cases of non-rheumatoid arthritis (pelvospondylites ossificans, Reiter's disease and psoriasis arthropathica) and in the majority of the sero-negative RA patients. Low C4 values also proved closely associated with low values for the third component (C3), and with pronounced conversion of this component. The C[unk] inactivator (immunochemically determined) of the synovial fluid which was correlated with the albumin concentration, did not vary with total C, C4 or C3.     

Clinical significance of the immunological tests in rheumatoid arthritis

Of the many theoretical causes of rheumatoid arthritis(RA), the most widely held theory is the autoimmune mechanism. In order to clarify the clinical significance of the immunological tests in RA, we studied immunoglobulin and complement levels in sera and synovial fluids of 118 RA patients and the following results were obtained. 1) The levels of immunoglobulins were elevated in both serum and synovial fluid and this was more prominent in the seropositive cases than the seronegative ones. 2) The levels of C3 component were decreased in both serum and synovial fluid, while those of C4 were decreased only in synovial fluid. Serum C3 and C4 component levels were more decreased in the seropositive cases than the seronegative ones. 3) The immunoglobulin levels in serum (IgG, IgM and IgA) and synovial fluid (IgG and IgA) and the levels of C3, C4 component in serum were well correlated with the clinical forms of rheumatoid arthritis. 4) The IgA level in serum and IgM level in synovial fluid were more increased in the exacerbated cases than the chronic ones. 5) Serum IgG level was decreased after steroid medication over one month.     

Immune complex detection and complement activity in rheumatoid arthritis: a comparative study of a radioimmunoassay using monoclonal rheumatoid factor, gel diffusion techniques and C4 activity.

Paired sera and synovial fluids from forty-nine patients with rheumatoid arthritis and twenty-five with other forms of arthritis were tested for immune complexes by a radioimmunoassay using monoclonal rheumatoid factor and gel diffusion procedures with monoclonal rheumatoid factor and C1q. Synovial fluid hemolytic C4 and C4 adjusted for IgG concentration were determined in both groups of patients. Immune complexes were detected at similar high frequencies in the rheumatoid synovial fluids by precipitin formation with monoclonal rheumatoid factor (68%) and C1q (71%). In contrast, immune complexes in rheumatoid sera were detected in low frequency by precipitin reactions with monoclonal rheumatoid factor (10%) and C1q (0%). Using the monoclonal rheumatoid factor radioimmunoassay, thirty-one (63%) synovial fluids exceeded the mean non-RA binding activity by one standard deviation. Similarly, twenty-four (49%) rheumatoid sera exceeded the mean non-RA binding activity to one standard deviation. Synovial fluid C4 adjusted for IgG as well as IgG alone distinguished between the two groups of patients whereas the C4 values did not. The C4/IgG value showed a strong negative correlation with the monoclonal rheumatoid factor radioimmunoassay and C1q precipitin formation.     

C1 activation, with C1q in excess of functional C1 in synovial fluid from patients with rheumatoid arthritis

Free Clq, in functionally active form was present in increased amounts in the synovial fluid of patients with rheumatoid arthritis. The presence of free Clq was associated with low concentrations of hemolytic C1, low C4 and raised amounts of C3dg/d fragments in the synovial fluid. The findings suggested intra-articular C1 activation with dissociation of C1 into free C1q and complexes containing C1r, C1s, and C1 inactivator. However, the immunochemical properties of synovial fluid C1r-C1s-C1 inactivator complexes appeared to differ from those of the complexes formed in serum, which hampered quantification with the assay used. Control patients with osteoarthritis or spondylarthritic syndromes did not show evidence of intra-articular complement activation, even though 1 patient with Reiter's disease had unexplained low concentrations of synovial fluid C4 and C3. The concentrations of circulating complement components were largely normal in the patients. Slightly increased concentrations of free C1q and C1r-C1s-C1 inactivator complexes in serum and C3dg/d fragments in EDTA plasma were observed, particularly in the patients with rheumatoid arthritis.     

Immunofluorescence studies for immunoglobulins and complement C3 in synovial joint membranes in psoriatic arthritis.

Synovial tissues from fifteen patients with psoriatic arthritis were investigated with direct immunofluorescence staining for immunoglobulins (IgG, IgA, and IgM) and from eigth patients for complement component C3. As control groups, there were synovial tissues from seven patients with seropositive rheumatoid arthritis and five patients with meniscal tears. In psoriatic arthritis, immunoglobulins were found in plasma cells in 93% of the cases, always with the presence of IgG (93%) but also with IgA (47%) and IgM (7%). C3 could not be demonstrated. In seropositive rheumatoid arthritis IgG was demonstrated in all patients (100%), often together with IgA (43%) and IgM (57%). C3 was found in all of these patients. In patients with meniscal tears neither immunoglobulins nor C3 could be found. The present findings indicate immunological activity in synovial membranes in psoriatic arthritis. The low amount of IgM and the lack of C3 suggest a difference compared to seropositive rheumatoid arthritis.     

Markers of inflammatory activation: upregulation of complement receptors CR1 and CR3 on synovial fluid neutrophils from patients with inflammatory joint disease.

Expression of the C3 receptors CR1 and CR3 was investigated on neutrophils from paired peripheral blood and synovial fluid samples from 34 patients with inflammatory joint disease (21 patients with rheumatoid arthritis (RA) and 13 patients with other articular diseases (OAD)). Using monoclonal antibodies (anti-CD35, anti-CD11b) and immunofluorescence flow cytometric analyses the percentages of positively labeled cells and the relative fluorescence intensities (as a measure of receptor number) were determined. CR1 and CR3 were found to be present on the majority (> 85%) of circulating neutrophils from normal subjects, RA and OAD patients, and on synovial fluid neutrophils from both patient groups. A strong correlation between neutrophil CR1 and CR3 expression was observed in peripheral blood samples from normal subjects (r = 0.81; P = 0.001), RA (r = 0.79; P = 0.001), and OAD patients (r = 0.83; P = 0.001); in each case the levels of CR3 expression were approximately twice those recorded for CR1. Both CR1 and CR3 expression was upregulated on synovial fluid neutrophils compared with that observed on the corresponding peripheral blood cells. Mean percentage increases observed were: RA patients: CR1, 16.5% (P < 0.001) and CR3, 28.7% (P < 0.001); and OAD patients: CR1, 4.1% and CR3, 26.9% (P = 0.001). Correlation of serum and synovial fluid IL-6, IL-8, and immune complex levels with neutrophil CR1 and CR3 expression failed to demonstrate any significant relationship between the concentrations of these soluble factors and receptor expression. Upregulation of CR1 and CR3 receptors, reflecting neutrophil activation within the inflamed joint, is a consistent finding in patients with inflammatory arthropathies.     

Serum complement levels in patients with rheumatoid arthritis and vasculitis

It is known that serum complement levels are decreased in patients with rheumatoid arthritis associated with vasculitis, also called malignant rheumatoid arthritis (MRA). The complement profiles in patients with MRA were compared with those in uncomplicated rheumatoid arthritis (RA) and in those with systemic lupus erythematosus (SLE). In MRA patients, serum CH50, C4 and C3 levels were all decreased as in SLE patients; and C3d, an activation product of C3, and the C3d/C3 ratio as a C3 activation index were increased. Serum B level was also increased as in RA patients, but AH50 level as the haemolytic activity of the alternative pathway was within the normal range. The regulatory proteins, H and I, were elevated in sera of MRA patients. Circulating immune complexes (CIC) detected by a solid phase C1q binding method were increased, whereas the serum activity to solubilize a performed immune complex (CRA) was decreased in MRA sera. It was suggested that increased consumption of complements, mainly through the classical pathway may be responsible for the complement profiles in MRA patients: a markedly consumed classical pathway and relatively preserved alternative pathway.     

Complement C4-derived monocyte-directed chemotaxis-inhibitory factor. A molecular mechanism to cause polymorphonuclear leukocyte-predominant infiltration in rheumatoid arthritis synovial cavities

To reveal the mechanism of the lesser infiltration of monocytes in synovial cavities with rheumatoid arthritis despite the presence of chronic inflammation, the synovial fluid from 15 rheumatoid arthritis patients was analyzed with respect to leukocyte chemotaxis. The synovial fluid possessed strong chemotactic activity to polymorphonuclear leukocytes but rather suppressed one to monocytes. The synovial fluid contained two different inhibitory activities in monocyte chemotaxis. One, which also suppressed polymorphonuclear leukocyte chemotaxis, was identified as alpha 1 protease inhibitor. The other, with molecular weight of 8 kd, possessed the specificity to monocytes and shared the antigenicity with complement C4 but not with C3 or C5. A similar inhibitor was generated in normal human plasma when the classical pathway of the complement system was initiated with aggregated human IgG, while it was not when alternative pathway was initiated with zymosan. The small size factor in the synovial fluid, apparently derived from C4, seemed to be a cyto-directed factor that might block an early part of signal transduction system of monocytes in the chemotaxis. After removal of the small-size inhibitor, the synovial fluid exhibited chemotactic ability to monocytes. Therefore the apparent C4-derived factor might play a key role in the polymorphonuclear leukocyte-predominant infiltration in the synovial fluid of rheumatoid arthritis.     

Evidence for immune complexes containing antibody to the pepsin site of IgG in rheumatoid synovial fluids

In seventy cases of rheumatoid arthritis, the synovial fluid to serum ratio for the titre of pepsin agglutinator, i.e. the antibody to the pepsin site of IgG, was studied. This was compared with the corresponding ratio for another antibody of the same immunoglobulin class and for other proteins. By this method there was evidence for local production of pepsin agglutinator in two cases and for local inhibition in two other cases. Density gradient ultracentrifugation, performed at pH 7·4 and 3·0, suggested that pepsin agglutinator in immune complexes was present in most of the synovial fluids containing this antibody. Complexes of pepsin agglutinator and F(ab'')₂ IgG, prepared in vitro, appeared to fix only small amounts of human complement. Neither did fixation of pepsin agglutinator to immune precipitates increase their complement binding activity.     

Antibody-dependent direct cytotoxicity of human lymphocytes. II. Studies on peripheral blood lymphocytes, synovial fluid cells and sera from patients with rheumatoid arthritis.

Antibody-dependent direct cytotoxicity (ADDC) by human lymphocytes was evaluated in patients with rheumatoid arthritis and normal controls. Purified peripheral blood and synovial fluid lymphocytes mediated normal ADDC when compared to control subjects. No correlation could be obtained between the percentages of T, B and null cells in effector cell populations and the degree of 51Cr released from target cells. Sera from 50% of patients with rheumatoid arthritis inhibited ADDC by normal lymphocytes; the degree of inhibition did not correlate with the titre of IgM rheumatoid factor. The pathogenic implications of these findings are discussed.     

IgG with a Deviant Conformation in Serum and Synovial Fluid from Rheumatoid Arthritis Patients

Specific rabbit antisera were prepared against an IgG with a special conformation (IgG spec.) previously detected in some sera from patients with rheumatoid arthritis. The antibodies had no affinity to normal human IgG and were not anti-idiotypic to human rheumatoid factor. The affinity of IgG spec. to the antibodies could not be explained by an antiglobulin activity to rabbit IgG. The amount of protein with affinity to immobilized specific IgG F(ab')2 of the antibodies was determined in serum and synovial fluid from patients with various joint diseases. A relationship between the content of IgG spec. and the diagnosis of seropositive rheumatoid arthritis was found on analysis of serum samples. IgG spec. also occurred in synovial fluid from some individuals with seropositive rheumatoid arthritis. Differences in the serum content of IgG spec. could not be explained by differences in the normal IgG content. Circular dichroism analysis of isolated IgG spec. showed that in the region(s) close to tyrosine residue(s) this polyclonal protein had similarities to heat-aggregated IgG.