IgM and IgA rheumatoid factors purified from rheumatoid arthritis sera boost the Fc receptor- and complement-dependent effector functions of the disease-specific anti-citrullinated protein autoantibodies.

Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1ß ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.

Dietary fish and fish oil and the risk of rheumatoid arthritis.

BACKGROUND: Intake of fish oil and oily fish has been reported to improve clinical symptoms in people who have rheumatoid arthritis. Whether the intake of oily fish and fish oil might also protect against the development of rheumatoid arthritis is not known. OBJECTIVE: We investigated the association between intake of oily fish and fish oil supplements and the risk of rheumatoid arthritis in a population-based case-control study. METHODS: The study comprised 1889 incident cases of rheumatoid arthritis and 2145 randomly selected controls recruited from a geographically defined area of Sweden during 1996-2005. Data on the consumption of oily fish and fish oil supplements 5 years preceding enrollment had been obtained through a questionnaire. We calculated odds ratios (ORs) for the development of rheumatoid arthritis, using logistic regression to adjust for age, residential area, body mass index, smoking, and alcohol consumption. RESULTS: Compared with subjects who never or seldom consumed oily fish, the OR for developing rheumatoid arthritis was 0.8 (95% confidence interval = 0.6-1.0) for subjects who consumed oily fish 1-7 times a week. The results did not change notably when stratifying the cases for rheumatoid factor or for antibodies to citrullinated peptide antigens. Similar results were seen for subjects consuming oily fish 1-3 times a month. Cases and controls did not differ in their consumption of fish oil supplements. CONCLUSION: Intake of oily fish was associated with a modestly decreased risk of developing rheumatoid arthritis.

Label-free electrochemical impedimetric immunosensor for sensitive detection of IgM rheumatoid factor in human serum.

The authors report a label-free and direct detection of rheumatoid factor- Immunoglobulin M (IgM-RF) based on an impedimetric-interdigitated wave type microelectrode array (IDWµE). IDWµE was functionalized with a self-assembled monolayer (SAM) of thioctic acid for antigen immobilization. The SAM functionalized IDWµE were characterized by atomic force microscopy, Energy Dispersive X-Ray Spectroscopy, and X-ray photoelectron spectroscopy. The covalent immobilization of IgG-Fc onto the SAM modified electrode arrays was characterized morphologically via AFM and electrochemically via cyclic voltammetry and electrochemical impedance spectroscopy. Impedimetric measurements in the presence of redox probe (Potassium ferrocyanide and potassium ferricyanide) showed a significant change in both the impedance spectrum and charge transfer resistance upon IgM-RF binding. Impedance measurements were target specific and linear with an increase in IgM-RF concentrations between 1 and 200 IU mL-1 in redox probe and human serum, respectively. The sensor showed detection limits of 0.6 IU mL-1 in the presence of redox probe and 0.22 IU mL-1 in the human serum. The biosensor exhibited good reproducibility (relative standard deviation (RSD), 4.96%) and repeatability (RSD, 2.31%) with an acceptable selectivity towards IgM-RF detection. The sensor also showed a good stability for 3 weeks at 4 °C in 1X PBS. Therefore, the sensitive and stable immunosensor exhibiting IDWµE features can be integrated with a miniaturized potentiostat to develop a sensing system that detects IgM-RF for point-of-care applications.

Immune complexes in sera and synovial fluids of patients with rheumatoid arthritis. Radioimmunoassay with monocylonal rheumatoid factor.

Evidence for the presence of immune complexes in blood, synovial fluid, and tisues of patients with rheumatoid arthritis (RA) includes low complement levels in blood and effusions, deposition of immunoreactants in tissues and vessel walls, precipitate formation after addition of monoclonal rheumatoid factor (mRF) to serum or synovial fluid. To quantitate immune complex-like material in RA patients, we developed a radioimmunoassay based on inhibition by test samples of the interaction of (125I)aggregated IgG (agg IgG) and mRF coupled to cellulose. This method could measure immune complexes of human antibody with hemocyanine prepared in vitro. The assay was not influenced by presence of polyclonal RF in test samples, nor by freezing and thawing. Normal levels of immune complex-like material in serum were less than 25 mug agg IgG EQ/ML. 12 of 51 RA sera examined (26%) contained more than 25 mug/ml. The presence of this material in RA sera was found to correlate with severity of disease, as measured by anatomical stage and functional class. There was an inverse correlation of the material with serum C4 level. Rheumatoid synovial fluids generally contained higher levels than serum, and five of 23 contained very much higher levels. The frequency of elevated levels of immune complex-like material in sera of patients with systemic lupus erythematosus (2 of 29) and with miscellaneous vasculitides (2 of 21 was much lower than in RA, suggesting that mRF exhibits a specificity for only certain kinds of immune complexes. The reason for this apparent specificity may explain such distinctive features of RA as the high frequency of polyclonal RF, the lack of immune complex nephritis, and the generally normal levels of serum complement.

Idiotypic and subgroup analysis of human monoclonal rheumatoid factors. Implications for structural and genetic basis of autoantibodies in humans.

Rheumatoid factors (RFs) in humans have been studied intensively because of their association with autoimmune and lymphoproliferative diseases. Many human IgM-RFs express cross-reactive idiotypes (CRIs) and have homologous light chains, some of which are encoded by a single V kappa gene, termed V kappa 325. However, although antibody activity generally requires the interaction between heavy and light chain variable regions, much less is known about structural relationships among RF heavy chains. To delineate further the structural and genetic basis of RF autoantibody synthesis, we generated "sequence-dependent" reagents specific for the human heavy and kappa light chain subgroups, and used them to analyze a panel of 27 monoclonal RFs. In addition, these proteins were tested for the expression of a heavy chain-associated CRI (G6), and a light chain-associated CRI (17.109). The results showed that most 17.109-reactive RFs contain heavy chains of the VHI subgroup, which bear the G6 idiotypic marker. However, among the 14 17.109-reactive RFs, two have heavy chains of the VHII subgroup, and another two contain heavy chains of the VHIII subgroup. Previously, we have shown that 17.109 is a phenotypic marker of the human V kappa 325 gene. Accordingly, these results demonstrate that the same human V kappa gene can combine with several VH genes from different VH gene subgroups to generate RF activity.

Rheumatoid factors from the peripheral blood of two patients with rheumatoid arthritis are genetically heterogeneous and somatically mutated.

We report the DNA sequences of the heavy and light chain immunoglobulin genes of 11 monoclonal rheumatoid factor (RF)-secreting lines derived from the peripheral blood of two patients with rheumatoid arthritis (RA). It is evident from immunogenetic analysis of these lines that RA-associated RF activity can arise from a wide variety of heavy and light chain genes and gene combinations. Although the RF response from our two patients shows a bias in gene usage toward those genes used to encode monoclonal RF, particularly VkIII, relatively few of these RFs are reactive with the monoclonal antiidiotypes 6B6.6 and 17.109 that define VkIII germline-encoded light chains and the loss of this idiotypic reactivity is clearly related to somatic mutation. Finally, RFs derived from peripheral blood of RA patients show a similar heterogeneity of epitope binding to Fc as that seen for synovium-derived RF and some are clearly different in binding specificity from the restricted RF population found in patients with B cell malignancies. Somatic mutations as well as different VH/VL combinations contribute to the heterogeneity in the binding patterns of these RA-derived RF.

Rheumatoid arthritis associated with hyperviscosity syndrome and intermediate complex formation.

A patient with long-standing rheumatoid arthritis developed hyperviscosity syndrome that correlated with the presence of abundant intermediate complexes in plasma. The intermediate complexes were isolated, and evidence is presented that these complexes are composed predominantly of igG-rheumatoid factor.

Isolation and characterization of a monoclonal rheumatoid factor specific for the hinge region of rat IgG2b.

The isolation and characterization of an isotype-specific autoantibody-secreting hybridoma NET/2/3 from rats bearing the syngeneic tumour HSN is described. This rheumatoid factor of the IgM class recognizes an epitope within the hinge region of rat immunoglobulins of the IgG2b subclass which is destroyed by reduction of disulphide bonds. The specificity of NET/2/3, although not allotype-restricted, is highly isotype-restricted, as it does not bind to rat Ig other than IgG2b, nor does it react with the majority of mouse IgG, although some reactivity occurs with mouse IgG3. One remarkable feature of NET 2/3 is that it binds more strongly to F(ab')2 and Fab' fragments of rat IgG2b, obtained by digestion with pepsin, than to the whole molecule. This anti-isotype response is not peculiar to the HSN tumour model as NET/2/3-like antibodies have been found in the sera of rats immunized with various protein and cellular antigens. The possible biological role of this anti-isotype antibody is discussed.

The use of indirect immunofluorescence to evaluate the gel filtration method of fractionating human immunoglobulins.

The indirect immunofluorescent technique has been shown to be a more sensitive method than either immunoelectrophoresis or gel filtration for testing the efficacy of the gel filtration method in fractionating human immunoglobulins. It has been confirmed that IgM fractions of some sera from patients with multiple sclerosis, contain antibody which reacts with measles virus-infected tissue culture cells.

The isolation of immune complexes containing IgM rheumatoid factor and recovery of IgG rheumatoid factor from the complexes.

IgG with rheumatoid factor activity has been recovered from immune complexes containing IgM rheumatoid factor. This was done by passing serum from patients with rheumatoid arthritis through an immunoadsorbent column of the F(ab')2 fragment of rabbit anti-human mu chain. The recovered IgM was analysed by radioimmunoassays for IgM and IgG rheumatoid factors before and after sucrose density gradient centrifugation at neutral and acid pH. It is considered that the method may be generally applicable for the isolation of immune complexes containing IgM antibodies.

Non-IgM monoclonal gammopathy in patients with Sjögren's syndrome.

Two Japanese patients with Sjögren's syndrome with non-immunoglobulin M(IgM) class monoclonal gammopathy are described. The monoclonal IgA lambda detected in the serum and saliva was confirmed to possess rheumatoid factor activity in the first patient with a hypergammaglobulinemic purpura and hyperviscosity syndrome. Idiotype specificity was present on the surface membrane of peripheral blood lymphocytes as well as in the cytoplasm of infiltrating cells in the salivary glands. Common idiotypic specificity was found in four of 60 other patients who had rheumatoid factors. In the serum and saliva of the other patient, a monoclonal immunoglobulin G, kappa type (IgG kappa), was detected. Kappa type IgG was found in most of the infiltrating cells in the salivary glands and also in the saline extract from a resected submandibular gland. Our findings indicate that non-IgM class monoclonal gammopathy is also one of the complications of Sjögren's syndrome.

Pericardial disease in rheumatoid arthritis.

Six patients with rheumatoid constrictive pericarditis, five seen in a two and one half year period, are described. All patients were male, all had rheumatoid factor, and all had active arthritis. Diagnosis was suspected from careful physical examination and confirmed in five patients by cardiac catheterization. Pericardiectomy was successful in all five patients on whom it was performed. Rheumatoid constrictive pericarditis should be suspected in any patient with rheumatoid arthritis and unexplained signs of right heart failure.

Systemic involvement and immunologic findings in patients presenting with Raynaud's phenomenon.

Systemic involvement and spectrum of autoantibodies were evaluated in 91 patients presenting with Raynaud's phenomenon. Decreased pulmonary diffusing capacity was observed in 23 percent, esophageal hypomotility in 14 percent and renal involvement in 5 percent of the patients, all without clinical symptoms. Arthralgia or a history of arthritis was present in 27 percent and skin abnormalities in 30 percent. Extent of systemic involvement was correlated with the severity of Raynaud's phenomenon, as measured by photoelectric plethysmography (r = 0.38; p < 0.01). In addition, both the variety of different autoantibodies in the serum of individual patients and the titer of antinuclear antibodies were positively correlated with the number of affected organ systems (r = 0.63; p < 0.01 and r = 0.65; p < 0.01, respectively). Raynaud's phenomenon is an important clinical sign of asymptomatic systemic disease. Measurements of its severity and serologic parameters are helpful in predicting the extent of systemic involvement.

Systemic autoimmunity in LG/J mice.

Humoral and end-organ parameters of autoimmunity were investigated in LG/J mice, which have traditionally been considered normal, non-diseased animals. Surprisingly, LG/J mice were found to possess autoantibodies, including antinuclear antibodies and rheumatoid factor, and to develop renal disease, including glomerulonephritis, interstitial nephritis, and perivasculitis, but not hepatic or cutaneous disease. In contrast, age-matched, identically-housed control animals failed to develop autoantibodies or end-organ disease. These findings have complications for the genetic study of lupus erythematosus in the MRL murine model, which derives heavily from the LG/J background. Thus, the LG/J strain may provide a useful model in the analysis of autoimmunity.

The presence of cryoprecipitable immunoglobulins in normal human sera may reflect specific molecular interactions.

The nature of the minute amounts of cryoprecipitable proteins present in all normal sera has been investigated in particular relation to the physicochemical and functional properties of the precipitated immunoglobulins. Cryoprecipitated material from 48 normal donor sera were first analyzed for their protein, IgG and IgM content. SDS-PAGE analysis revealed many bands in a majority of samples but in about one third a very limited number of bands was observed. IgM, IgG and albumin were identified and a lower molecular weight component (16 KD) was always present. Restricted clonality was demonstrated by IEF in 25-30% of the samples. In 50% of cryoprecipitates IgG was present in higher molecular weight fractions than 7S on sucrose density gradient at neutral pH. Ultracentrifugation at acid pH lead to a dissociation of these IgG fractions in most of the samples. A positive reaction for IgM rheumatoid factor was seen in 63% of the samples and there was a specific enrichment of RF activity in cryoprecipitates as compared to the corresponding serum. These data suggest that the cryoprecipitation of immunoglobulins in normal serum reflects specific interactions between immunoglobulin molecules rather than merely a non-specific precipitation of cold-insoluble molecules.

A subgroup of human VH3 germline genes that encode a high-avidity synovial rheumatoid factor.

We have previously derived and identified a highly avid monoclonal IgM rheumatoid factor (mRF), C6, from unstimulated rheumatoid synovial cells (RSC). At the time, the closet VH germline gene, VH26, demonstrated only 88% homology with C6. To identify the germline counterpart of C6, genomic DNA from the same rheumatoid arthritis (RA) patient from whom C6 was derived was used in the polymerase chain reaction (PCR). Four of the six closely related germline genes that we sequenced had exonic regions that were identical with the VH region of C6 cDNA. These six germline sequences differed in their intronic regions, suggesting that they were distinct, but closely related genomic sequences. To further evaluate the extent of these related genes we identified nine additional germline genes having VH-encoding exons that were 86-97% identical to the C6 cDNA sequence. Furthermore, we examined the polymorphic nature of the C6 VH gene using single strand conformation polymorphism (SSCP), and identified two peaks, confirming the existence of highly homologous genes. The sequence and polymorphism data suggest that: (1) the VH region of the high avidity mRF C6 was derived from an unmutated germline gene; (2) C6 was encoded by a VH gene belonging to a set of homologous genes within the larger VH3 family; and (3) in addition to somatic rearrangements of B-cell genes and antigen-driven somatic mutation, gene duplication and conversion events of germline genes could be important in generating diversity and polyclonality among high-affinity pathogenic autoantibodies.

Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation.

The occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RF activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.

IgM rheumatoid factor and the inhibition of covalent binding of C4b to IgG in immune complexes.

Work by other investigators has shown that IgM-rheumatoid factors (IgM-RF's) can impede complement-mediated inhibition of immune precipitation. We examined the binding of complement component C4b to radiolabelled IgG in model immune complexes and demonstrate that IgM-RF's are capable of reducing the covalent binding of C4b to 125I-IgG in the complexes. Reduced binding to IgG, however, may not be accompanied by binding of C4b to IgM-RF's within the complex, as we also demonstrate that IgM-RF's are relatively poor at C4b capture compared with normal IgM.

Rheumatoid factors bind beta 2 microglobulin. Detection of beta 2 microglobulin-complexes in rheumatoid arthritis.

beta 2 Microglobulin binding rheumatoid factors and occurrence of complexed form of beta 2 microglobulin were found in rheumatoid arthritis (RA) by radioimmunological methods. 1. As related to the appropriate values of control sera and osteoarthritic synovial fluids, one of the six sera and five of the six synovial fluids of RA patients exhibited increased binding activity for 125I-beta 2 microglobulin, 2. Rheumatoid factors (RF) purified by means of immunoadsorbent bound beta 2 microglobulin; 3. Both 19S and 7S components of sera, synovial fluids and purified RF samples had beta 2 microglobulin binding activity, which was inhibited by an excess of unlabeled beta 2 microglobulin; 4. Complexed (3% PEG 6000 insoluble) beta 2 microglobulin was found in three of fourteen sera and in twelve of fourteen synovial fluids of RA patients. Level of complexed beta 2 microglobulin was independent of its total concentration in the original samples; 5. In the fraction of synovial fluids enriched immune complex the amount of beta 2 microglobulin was in positive correlation with quantity of RF. Presumable immunological and pathological significance of "anti-beta 2 microglobulin"-like cross-reactive RF antibodies is discussed.

Incidence and characterization of canine rheumatoid factor.

Rheumatoid factor (RF) was found in titers greater than 1:8 in 72.2% of cases of classical or definite canine rheumatoid arthritis (RA) and in 5.9% or normal sera. Serum fractionation and immunoabsorbant studies that much of the RF present was IgG, although activity was demonstrated in all 3 major immunoglobulin classes. Evidence of involvement of both IgG and IgM to form complexes of varying sizes was obtained.