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Immunopathologic studies of rheumatoid arthritis. I. Absence of complement-dependent cytotoxicity of rheumatoid sera for rheumatoid synovial cell cultures.

A sensitive complement-dependent chromium release cytotoxicity assay was used to determine whether sera from rheumatoid arthritis (RA) patients contain antibody specific for an antigen on rheumatoid synovial cell cultures. Two hundred eight RA sera-RA synovial culture combinations were studied employing 21 sera and 16 synovial membranes; control combinations were derived from 5 normal sera and 10 degenerative joint disease synovial membranes. Anticomplementary activity of some rheumatoid sera was overcome using an increased complement concentration. The percent cytotoxicity of RA serum-RA culture combinations, both homologous and autologous, was not significantly greater than that of RA serum-control culture combinations. No correlation between duration of disease or duration of cell culture and percent cytotoxicity was found. Thus a unique antigen on cultured rheumatoid synovial cells was not recognized by rheumatoid serum antibody by use of this cytotoxicity assay.

Adolescent

The major rheumatoid factor crossreactive idiotype and IgA rheumatoid factor in juvenile rheumatoid arthritis.

One third of patients with juvenile rheumatoid arthritis (JRA) are seronegative for classic or hidden IgM rheumatoid factor (RF) yet express the major RF crossreactive idiotype (RCRI). We studied 60 children with JRA and 57 pediatric controls for IgA RF and RCRI expression to determine whether RCRI is associated with IgA RF in JRA. Twenty-one patients had IgA RF using rabbit or human IgG as substrate. Twenty-seven patients with JRA expressed high concentrations of RCRI in their sera. Only 9 of these had IgA RF. Eleven of the RCRI+ sera contained neither IgA RF nor classic or hidden IgM RF, and IgA RF was found in patients with all JRA onset subtypes. In JRA, RCRI may be expressed on either IgG RF or on parallel set antibodies without RF activity.

Adolescent

IgG rheumatoid factor in dental periapical lesions of patients with rheumatoid disease.

To detect "hidden" IgG rheumatoid factor in tissues from dental periapical lesions, 26 rheumatoid and 14 control patients were examined by the direct immunofluorescence technique for binding of FITC-labelled IgG before and after pepsin digestion. Whereas free rheumatoid factor was detected in only 1 rheumatoid tissue before digestion with pepsin, this tissue as well as 8 other rheumatoid tissues strongly bound heat-aggregated IgG after the digestion procedure. Six of these 9 tissues also bound native IgG. In no control tissue was free rheumatoid factor detected and in only 1 was "hidden" rheumatoid factor revealed after pepsin digestion. Hence, presence of IgG rheumatoid factor correlated positively with the presence of rheumatoid disease, and evidence was established that certain features of rheumatoid inflammation occur in dental periapical lesions of many patients with rheumatoid disease.

Adult

Free rheumatoid factor in dental periapical lesions and gingivae of patients with rheumatoid disease.

To determine whether evidence of rheumatoid inflammation, in the form of free rheumatoid factor, might be found in the teeth-supporting tissues of patients with known rheumatoid disease, tissues from the dental periapical lesions of one group of 50 rheumatoid and 23 control patients, and from the marginal gingivae of a second group of 58 rheumatoid patients were examined by the direct immunofluorescence technique that employed fluroesceinisothiocyanate (FITC)-labelled aggregated human IgG. The gingival tissues contained no free rheumatoid factor. Free rheumatoid factor-producing plasma cells were, however, detected in the dental periapical lesions of 3 of the 50 rheumatoid patients, i.e. in 6%, and in 1 of the control patients i.e., in 4%. This control patient had suffered from nephritis 10 months prior to the investigation. Because free rheumatoid factor did occur, albeit infrequently, in the dental periapical lesions of rheumatoid patients, a search for IgG rheumatoid factor, known to occur in greater abundance than the IgM type although "hidden", was indicated.

Adult

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.

Antigen-Antibody Complex

Changes of the lung in rheumatoid arthritis--rheumatoid pneumonia. A clinicopathological study.

The frequency and patho-histological characteristics of pulmonary changes were studied on the autopsy material of 100 patients with rheumatoid arthritis (RA). The formal pathogenesis and different stages of vasculitis, rheumatoid nodule, interstitial pneumonia, rheumatoid pleuritis, obliterative bronchiolitis, amyloidosis and the so-called rheumatoid pneumonia in the lungs is discussed. The rheumatoid pneumonia is a disseminating inflammatory lobular-sublobular process, not described previously. The frequency of rheumatoid pneumonia was 4%. The rheumatoid pneumonia is characterized by the necrotic vasculitis, fibrinoid necrosis or thrombovasculitis of the pulmonary and bronchial arterioles, and of small arteries. Because of the diminished blood supply distal to the vascular changes inflammatory foci may develop, more or less respecting the anatomic borders of pulmonary tissue. The lobular-sublobular inflammation is basically of non haemorrhagic character. Because of the recurrent nature of vasculitis, foci of inflammation in different stages can be observed in the lungs simultaneously side by side. Clinically the rheumatoid pneumonia was accompanied by severe RA, according to the frequency and severity of acute exacerbations. In all 4 cases of rheumatoid pneumonia the pulmonary process had been proven clinically and radiologically. Rheumatoid pneumonia occurred subsequent to recurrent arthritis following steroid withdrawal, and it was resistant to antibiotics.

Adult

[Stimulation of immunoglobin synthesis and rheumatoid-factor production by rheumatoid synovial effusions and by supernatants of synovial tissue cultures].

Factors capable of stimulating the synthesis of immunoglobulin by circulating human lymphocytes, as measured by a radioactive immune coprecipitation method, have been demonstrated in rheumatoid synovial eeffusions but not in effusions from patients with other arthritides. The activity of the ffusions was localized in two fractions eluted from an agarose A 5m column: fraction II eluting with IgM and fraction E eluting with albumin. Fraction II enhanced immunoglobulin synthesis only in circulating rheumatoid lymphocytes, while fraction V enhanced immunoglobulin synthesis in both rheumatoid and normal lymphocytes. Similar active fractions were found in culture supernatants of rheumatoid synovial tissue. Enhanced immunoglobulin synthesis was also observed following addition of aggregated IgG to rheumatoid lymphocytes. Finally, increased synthesis of anti-IgG (rheumatoid factor) was observed following addition of fractions II and V to rheumatoid but not to normal lymphocytes. The possible relationship of the active material in fraction II to IgG-containing immune complexes and the active material in fraction V to a T cell produced lymphokine-like mediator of T cell function are discussed. The findings presented provide evidence that in addition to antigenic stimulation, the action of one or more enhancing factors liberated by antigen stimulated mononuclear cells, possibly T lymphocytes, plays a role in the immunoglobulin synthesis observed in rheumatoid synovial tissue.

Arthritis, Rheumatoid

Heteroclitic polyclonal and monoclonal anti-Gm(a) and anti-Gm(g) human rheumatoid factors react with epitopes induced in Gm(a-), Gm(g-) IgG by interaction with antigen or by nonspecific aggregation. A possible mechanism for the in vivo generation of rheumatoid factors.

Heteroclitic rheumatoid factors (RF) are specific for allotypic determinants, e.g., Gm(a) or Gm(g) on allogeneic, but not autologous IgG. All polyclonal RF we isolated from nine rheumatoid arthritis patients with circulating Gm(a-), (b+), (g-), (f+) IgG displayed dual heteroclitic activity for the Gm(a) and Gm(g) allotypes, as shown by using appropriate RBC agglutination assays and affinity columns bearing Gm(a+) or Gm(g+) IgG. To investigate possible mechanisms underlying the in vivo generation of heteroclitic RF, we tested the ability of nonspecifically and immune-specifically aggregated Gm(a-), (g-) IgG to function as targets for RF from Gm(a-), (g-) patients with rheumatoid arthritis. Heat aggregation (63 degrees C for 20 min) or binding to Ag (as in tetanus toxoid-antitetanus toxoid complexes) induced a "functional" Gm(a+) and/or (g+) phenotype in Gm(a-), (g-) IgG from five healthy subjects and five rheumatoid patients, as suggested by the ability of these altered IgG to function as efficient targets for six heteroclitic RF in direct binding and competitive inhibition experiments. That heterocliticity and dual Gm(a), Gm(g) specificity can be features of a single antibody molecule was formally demonstrated by analysis of a monoclonal RF (IgM mAb 61) generated from a Gm(a-), (g-) rheumatoid patient. RF mAb 61 displayed a high affinity (Kd, 10(-7) M) for IgG Fc fragment of Gm(a+) and (g+) IgG or aggregated autologous Gm(a-), (g-) IgG but did not bind to native autologous IgG. To investigate the molecular basis of the acquired Gm(a) phenotype, PBMC from five Gm(a-) patients with rheumatoid arthritis and two Gm(a-) normal subjects arthritis and two Gm(a-) normal subjects were cultured in vitro after activation with PWM. In most instances, these PBMC produced IgG that behaved as Gm(a+) in sensitive ELISA. Application of the polymerase chain reaction (PCR), using probes specific for the nucleotide sequence coding for the Gm(a) tetrapeptide, to the amplification of DNA from the in vitro-stimulated Gm(a-) normals or rheumatoid patients' PBMC provided no evidence for Gm(a) nucleotide sequences. The present data suggest that acquisition of the Gm(a) determinant by Gm(a-) IgG may result from subtle changes in the CH2-CH3 RF-binding region. Such changes would occur when Gm(a) IgG are complexed with Ag or nonspecifically altered, thereby providing a possible explanation for the induction of heteroclitic RF in Gm(a-) rheumatoid arthritis patients.

Antibodies, Monoclonal

IgG-, IgM- and IgA-rheumatoid factors in healthy adults and rheumatoid patients determined by an indirect immunofluorescence method.

Sera from 173 healthy adults and 55 rheumatoid patients were studied for IgG-, IgM- and IgA-rheumatoid factors (RFs) by a modification of Este's indirect immunofluorescence method. Rabbit IgG bound to smeared sheep red cells was used as antigen. With each serum tested a smear on non-sensitized cells was used as control antigen. Anti-IgG of the sera studied, binding to the antigen, was demonstrated by fluorescein-conjugated antisera, monospecific for gamma, mu and alpha chains, and not containing antibodies to sheep erythrocytes or rabbit IgG. Positive reactions were obtained with IgG as antigen, but not with the F(ab')2 fragment. The sera tested were treated with dithiothreitol before they were assayed for IgG-RF, in order to abolish false-positive reactions due to IgM-RF activity. The detection limit for IgM-RF was 1 IU per ml. IgM-RF titres of 9 occurred in 7% of healthy adults and 73% of rheumatoid patients, titres greater than or equal to 18 in 3.5% and 67% respectively. IgG-RF titres of 9 occurred in 9% of healthy adults, 21% of seronegative and 24% of seropositive rheumatoid patients. Titres of 18 occurred in 3% of healthy adults and in 14% of seronegative rheumatoid patients. Titres of greater than or equal to 18 occurred in 22% of seropositive rheumatoid patients. IgG-RF was correlated with an involvement of more than 20 joints. IgA-RF was found in 83% of seropositive, 11% of seronegative rheumatoid patients and in none of the healthy adults (serum dilution 1:9).

Adult

Cathepsin D agglutinators and neutral protease agglutinators in rheumatoid arthritis. II. Production of CDA and NPA by rheumatoid synovium and phagocytosis of CDA by synovial phagocytic cells.

Tissue culture methods demonstrated the production of agglutinators against the cathepsin D site in IgG (CDA) and a neutral protease site in IgG (NPA) by rheumatoid synovial tissue. Seven of 11 specimens from seropositive and 2 of 7 specimens from seronegative RA patients were positive for CDA, whereas 2 of 11 specimens from seropositive and 1 of 7 specimens from seronegative patients were positive for NPA. None of the 6 control specimens was positive for both types of agglutinators. Chromatography of two synovial tissue incubates showed that the CDA were of the IgG type. By the immunofluorescence technique, plasma cells containing CDA were demonstrated in rheumatoid synovial tissue and draining lymph nodes of rheumatoid joints. The staining for CDA in the synovium was different from the staining for pepsin agglutinators in adjacent sections. Phagolysosomes containing CDA were found in synovial exudate cells from rheumatoid patients as well as in phagocytosing lining cells and macrophages of the sublining layer of rheumatoid synovial tissue. These findings suggest that antibodies directed at hidden antigenic sites in IgG revealed by endogenous proteolysis take part in the immune reaction and in the inflammation of rheumatoid joints.

Agglutinins

IgG rheumatoid factor. Relationship to seropositive rheumatoid arthritis and absence in seronegative disorders.

IgG rheumatoid factor was detected in the sera of the majority of patients with seropositive rheumatoid arthritis. Values suggestive of IgG rheumatoid factor were noted in one-quarter of patients with seronegative inflammatory arthropathies. These determinations were always low and correlated with elevated IgG concentrations, suggesting nonspecific adherence of IgG rather than a true antigen-antibody reaction. In support of this conclusion, nonrheumatoid factor IgG was capable of concentration-dependent nonspecific adsorption to the solid phase. IgG, but not IgM, rheumatoid factor corresponded with disease activity in patients with seropositive rheumatoid arthritis, suggesting that IgG rheumatoid factor may be important in the pathogenesis.

Arthritis, Juvenile

Molecular analysis of V kappa III variable regions of polyclonal rheumatoid factors during rheumatoid arthritis.

We report the first molecular characterization of V kappa regions of the main human autoantibodies occurring during rheumatoid arthritis, the polyclonal rheumatoid factors. Using two sets of polymerase chain reactions in order to amplify the cDNA derived from both peripheral blood and synovial fluid rheumatoid factor-secreting cells, nucleotide analysis of the V kappa III family usage shows the following: (a) at least three different V kappa III genes are used to encode polyclonal rheumatoid factors in a single patient, (b) each one of these genes seems more or less somatically mutated (from 1 to 14 mutations), (c) the mutation process preferentially affects the complementarity determining regions suggesting a selective pressure of antigen and (d) there is no clear difference between the mutation rates affecting the synovial fluid and peripheral blood rheumatoid factor-secreting cells. These results are able to explain some of the known idiotypic differences between monoclonal and polyclonal rheumatoid factors in humans. They also provide evidence that polyclonal autoantibodies arising during an autoimmune disease can be the products of multiple somatically mutated genes and suggest that this process is antigen driven, whether this antigen is the Fc piece of IgG or another unknown antigen.

Amino Acid Sequence

[Levels of IgA rheumatoid factors in seropositive rheumatoid polyarthritis. Absence of correlation with disease activity or pejorative course].

Rheumatoid factors of the IgA isotype directed to human IgG Fc fragment were assayed, using an Elisa test, in the serum of 30 patients with seropositive rheumatoid arthritis and in the synovial fluid of 9 of them. A high incidence was found in the serum (90%) and synovial fluids (77%). Clinical, radiological and biological parameters of each patients were recorded at the time of the assay, and two years later. There was no statistically significant association between IgA rheumatoid factors levels and other parameters, nor with a pejorative evolution. However, a significant negative correlation was observed between IgA rheumatoid factors levels and the duration of the disease, suggesting that IgA rheumatoid factors are predominantly produced at the earliest stages of rheumatoid arthritis.

Adult

[The experimental induction of rheumatoid factors of low affinity and cross reactivity with nonhistone nucleoproteins by means of anti-idiotypes directed at rheumatoid factors of high affinity and cross reactivity with histones H1 and H2b].

The study of the anti-nuclear cross reactivity of rheumatoid factors has shown differences between patients with rheumatoid arthritis and healthy seropositive subjects. This paper describes the finding, of a subpopulation of rheumatoid factor molecules which show a cross reactive idiotype and show high affinity for IgG and cross reactivity against histones in rheumatoid arthritis patients but not in healthy subjects. These molecules, in turn, were able to induce, experimentally, heterogeneous rheumatoid factor molecules that show a low affinity for IgG and cross-reactivity against nucleoproteins other than histones, most likely through idiotypic networks. The mechanism could be responsible for the diversification of serum autoantibodies found in rheumatoid arthritis.

Animals

Viruses and lymphocytes in rheumatoid arthritis. II. Examination of lymphocytes and sera from patients with rheumatoid arthritis for evidence of retrovirus infection.

The possible involvement of retroviruses in the aetiology of rheumatoid arthritis (RA) was investigated. Retrovirus antigens were not expressed on rheumatoid synovial and peripheral blood lymphocytes as judged by membrane immunofluorescence, radioimmunoassay, and complement-mediated cytotoxicity. The specific antiretroviral (anti-RD-144 and anti-SSAV) sera used in this study were produced in rabbits immunised with viral antigens grown in a homologous system (rabbit cells and medium supplemented with normal rabbit serum), avoiding non-specific immunofluorescence previously detected with donated antiretroviral sera. Immune complexes lodged in the rheumatoid synovial membranes did not contain, and other cells within the membranes did not express, retroviral antigens. Antibodies cross-reacting with primate retrovirus antigens were sought in sera from patients with 'autoimmune' diseases by means of solid phase radioimmunoassay. There were no retrovirus antibodies in the 3 groups of patients studied, that is, those with rheumatoid arthritis, systemic lupus erythematosus, and with non-RA conditions. Absorption of rheumatoid factor did not alter this conclusion. These results give little support to the hypothesis that activation of endogenous human retroviruses or an infection with horizontally transmitted retroviruses is associated with the rheumatoid process.

Antigens, Surface

Nucleotide sequence analysis of rheumatoid factors and polyreactive antibodies derived from patients with rheumatoid arthritis reveals diverse use of VH and VL gene segments and extensive variability in CDR-3.

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of VH and VL gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain 'restrictions' do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of VH3 genes beyond that one would expect based on random utilization.

Amino Acid Sequence