IgG Rheumatoid Factor Research Articles

Rheumatoid factors in systemic lupus erythematosus: association with clinical and laboratory parameters. SLE study group.

The prevalence and clinical and laboratory associations of IgM, IgG and IgA rheumatoid factors (RF) were determined in 352 patients with systemic lupus erythematosus (SLE). IgM, IgG, and IgA class RF were detected in 17.9%, 20.5%, and 20.5% of the sera, respectively. RF were associated with sicca syndrome, hypergammaglobulinemia, high titer of antinuclear antibodies, anemia, SSA- and SSB-antibodies, and with the presence of HLA-DR3. RF correlated negatively with nephritis and livedo racemosa. Moreover, we observed an association of RF and parameters of inflammatory activity such as elevated erythrocyte sedimentation rate (ESR) and leukopenia. Analysis of immunoglobulin classes revealed that laboratory parameters of inflammatory activity, SSA- and SSB-antibodies and HLA-DR3 correlated with IgA RF only. IgA RF define a subgroup of SLE patients characterized by distinct autoimmune phenomena and high disease activity in the absence of nephritis.

Induction of tumor necrosis factor alpha production by adhered human monocytes: a key role for Fcgamma receptor type IIIa in rheumatoid arthritis.

Small IgG rheumatoid factor immune complexes may provide the trigger for macrophage-derived tumor necrosis factor alpha (TNFalpha) production in rheumatoid arthritis. Immune complexes may bind to any of 3 IgG Fc receptors (FcgammaR). Therefore, the ability of monocyte-derived macrophages to produce TNFalpha was examined following ligation of each of the 3 human FcgammaR, using murine monoclonal antibodies (mAb) to each receptor as a model for small immune complexes. Adhered human monocytes expressing all 3 FcgammaR were incubated with murine anti-FcgammaR mAb directed against FcgammaRI, FcgammaRII, or FcgammaRIII. Supernatants were collected at various time points and tested for the presence of TNFalpha and interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay. The anti-FcgammaRIII mAb induced adhered human monocytes to release TNFalpha. However, F(ab)2 and Fab fragments of the anti-FcgammaRIII mAb failed to induce TNFalpha production. TNFalpha was undetectable following incubation with the anti-FcgammaRI or anti-FcgammaRII mAb. Furthermore, blocking FcgammaRI or FcgammaRII had no effect on the levels of TNFalpha released in response to the anti-FcgammaRIII mAb. Of the 3 anti-FcgammaR mAb, only anti-FcgammaRIII induced IL-1alpha production from adhered human monocytes, and this was inhibited by the presence of a neutralizing anti-TNFalpha mAb. This study suggests a dominant role for FcgammaRIIIA in the induction of both TNFalpha and IL-1alpha production by human macrophages in rheumatoid arthritis following receptor ligation by small immune complexes. The signaling of TNFalpha production may require the ligation of either 3 FcgammaRIIIA receptors or only 2 FcgammaRIIIA receptors, where one interaction must involve binding via an Fc domain. In addition, IL-1alpha production following FcgammaRIIIA ligation appears to be dependent on the presence of TNFalpha.

The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs

Sandwich ELISAs have become a widely used method for the quantitative detection of serum proteins. However, they can be biased by a variety of interfering substances. As reported recently, we observed false-positive levels of interferon (IFN)-α and -β in up to 27% of sera from healthy blood donors using commercial ELISAs. We now demonstrate that two different groups of naturally occurring heterophilic antibodies (IgG-type) are responsible for these titers. Group I (representing 85% of positive samples) binds to the Fab region of IgG from goat, mouse, rat, horse, and bovidae (but not rabbit). Group II (15%) recognizes an epitope in the Fc region of mouse, horse, bovine, and rabbit (but not goat or rat) immunoglobulins. The antibodies did not crossreact with human IgG subclasses but contributed to false-positive IgG rheumatoid factor levels obtained using a commercially available ELISA. To investigate the susceptibility of assays to these artifacts, various combinations of capture and detection antibodies have been tested. On this basis, we defined the relative risks that standard ELISAs might be influenced by heterophilic anti-immunoglobulin antibodies. In general, assays that use monoclonal antibodies for both capture and detection are less susceptible than others which include at least one polyclonal antiserum. However, only systems utilizing rabbit F(ab′) 2 fragments have been found to be immune to this interference.

新規の自然発症自己免疫モデルマウスの自己抗体価測定キット

IgG-rheumatoid factor (RF), IgM-RF, antidsDNA, anti-ssDNA, and anti-Sm antibody have been detected and have been thought to play an important role on pathogenesis of systemic lupus erythematosus (SLE) . MRL/MP-lpr/lpr (MRL/lpr) mice have been studied widely as experimental animal model for autoimmune disease. IgG-RF, IgM-RF and anti-ds DNA antibody, anti-ss DNA antibody and anti-Sm antibody were titrated by a one-step sandwich enzyme-linked immuno-sorbent assay (ELISA) system in the serum of MRL/ lpr mice. Titer of the autoantibodies was ranged from 4 to 1000 mU.These results disclosed evidences that the new ELISA kits were useful to titrate IgG-RF, IgM-RF and anti-ds DNA antibody, anti-ss DNA antibody and anti-Sm antibody. The assay system could be employed to study on efficacy of therapeutic agents and to analyse pathoetiology of the autoimmune disease in mice.

Recombinant cytomegalovirus glycoprotein gB (UL55) induces an autoantibody response to the U1-70 kDa small nuclear ribonucleoprotein.

Human cytomegalovirus (CMV) infection can be life threatening in the immune compromised and is associated with congenital defects and / or mental retardation in the neonate. The demonstrated association between CMV infection and rheumatoid factor (RF) raised the possibility of an induction of an autoimmune response upon vaccination with a candidate CMV vaccine, glycoprotein gB (UL55). The antibody responses generated after injections of an adenovirus-gB construct (Ad-gB) were studied in autoimmune-prone (MRL/mpj) and normal (BALB.k, C3H, and BALB/c) mice. Enzyme-linked immunosorbent assay and immunoblot analyses were done to identify the autoantibodies produced following immunization. Immunization with Ad-gB induced a significant IgG anti-viral response in all strains tested (p < 0.0001) compared to phosphate-buffered saline or HeLa controls. Ad-gB induced a significant IgG autoantibody response (p > 0.005) to the U1-70 kDa spliceosome protein in both autoimmune and normal strains whereas immunization with recombinant human La/SS-B did not. Autoantibodies to U1-70 kDa are part of the anti-ribonucleoprotein response seen in systemic lupus erythematosus and mixed connective tissue disease. Low levels of IgG RF and anti-double-stranded DNA antibodies were also induced. This study raises concern that immunization with CMV gB in individuals genetically predisposed to autoimmunity could trigger the development or acceleration of an autoimmune disease.

Role of the Costimulatory Molecule CD28 in the Development of Lupus in MRL/lprMice

AbstractMRL/Mpj-lpr/lpr (MRL/lpr) mice develop autoimmune disorders, including lymphoproliferation, glomerulonephritis, autoantibody production, and hypergammaglobulinemia. To investigate the role of the costimulatory molecule CD28 in the development of these disorders, MRL/lpr mice lacking CD28 were generated by gene targeting. Compared with CD28+/+ MRL/lpr mice, CD28−/− MRL/lpr mice showed decreased lymphadenopathy but increased splenomegaly associated with the expansion of abnormal B220+ TCRαβ+ T cells. Although levels of IgM Abs were unchanged in CD28−/− MRL/lpr mice, the production of anti-DNA IgG Abs and IgG rheumatoid factors were suppressed. IgG deposition in the glomeruli was markedly decreased, and the development of glomerulonephritis was significantly retarded. Furthermore, renal vasculitis and arthritis were absent in CD28−/− MRL/lpr mice. These results indicate that, although CD28 is not required for the generation of the abnormal T cell population in MRL/lpr mice, it does play an important role in the development of autoimmune disease in these animals.

IgG rheumatoid factor in early rheumatoid arthritis as a predictor of radiological changes and disease activity

Our objective was to confirm whether IgG rheumatoid factor (IgG RF) assessed in early rheumatoid arthritis (RA) could be a prognostic factor of disease activity and articular destruction. The IgG RF index was assessed by a kit (Eitest IgG RF) in 46 patients with early RA (disease duration less than 1 year). Damage score (DS), carpal height ratio (CHR), clinical variables, and conventional RF values were evaluated at the initial visit and 2–3 years (average 2.5 years) after the initial visit. The incidence of IgG RF was 23.9% at the initial visit. Patients with positive IgG RF showed higher DS and higher Lansbury’s index in the final observation. They also showed a greater increase of DS and a greater decrease of CHR. The IgG RF index correlated with the final DS and final Lansbury’s index. We conclude that although the incidence was low, the IgG RF index in early RA could be a prognostic factor in radiographic changes and disease activity 2–3 years after the initial visit.

IgG rheumatoid factor in early rheumatoid arthritis as a predictor of radiological changes and disease activity

Our objective was to confirm whether IgG rheumatoid factor (IgG RF) assessed in early rheumatoid arthritis (RA) could be a prognostic factor of disease activity and articular destruction. The IgG RF index was assessed by a kit (Eitest IgG RF) in 46 patients with early RA (disease duration less than 1 year). Damage score (DS), carpal height ratio (CHR), clinical variables, and conventional RF values were evaluated at the initial visit and 2–3 years (average 2.5 years) after the initial visit. The incidence of IgG RF was 23.9% at the initial visit. Patients with positive IgG RF showed higher DS and higher Lansbury’s index in the final observation. They also showed a greater increase of DS and a greater decrease of CHR. The IgG RF index correlated with the final DS and final Lansbury’s index. We conclude that although the incidence was low, the IgG RF index in early RA could be a prognostic factor in radiographic changes and disease activity 2–3 years after the initial visit.

Fibroblast-Like Synoviocytes from Rheumatoid Arthritis Patients Have Intrinsic Properties of Follicular Dendritic Cells

The production of IgG rheumatoid factors in the inflamed synovium of many patients with rheumatoid arthritis (RA) implies that local sites exist where plasma cell precursors undergo isotype switching and affinity maturation by somatic mutation and selection. Lymphonodular infiltrates of the synovium-containing germinal centers (GCs), are candidates to fulfill such function in the rheumatoid patient. It has been suggested that these GCs are organized around, obviously ectopic, follicular dendritic cells (FDCs). The present study attempts to find out whether these putative FDCs 1) are specific for RA, 2) have the same phenotype and functional capacity as FDCs in lymphoid organs, and 3) may locally differentiate from fibroblast-like synoviocytes (FLS). Synovial biopsies from patients with RA versus non-RA, yet arthritic backgrounds, were compared. Cells with the FDC phenotype were found in both RA and non-RA tissues as well as in single cell suspensions thereof. When FLS were cultured in vitro, part of these cell lines could be induced with IL-1beta and TNF-alpha to express the FDC phenotype, irrespective of their RA or non-RA background. By contrast, the FDC function, i.e., stable binding of GC B cells and switching off the apoptotic machinery in B cells, appeared to be the prerogative of RA-derived FLS only. The present data indicate that FDC function of FLS in RA patients is intrinsic and support the idea that synovial fibroblast-like cells have undergone some differentiation process that is unique for this disease.

Prevalence of IgM-, IgA- and IgG-rheumatoid factors in seronegative polyarticular disease compared to pauciarticular disease in juvenile chronic arthritis as measured by ELISA.

To determine the prevalence of the rheumatoid factor isotypes measured by enzyme-linked immunosorbent assay (ELISA) in polyarticular and pauciarticular juvenile chronic arthritis (JCA), and evaluate the diagnostic test qualities. 53 patients with JCA (20 with seronegative polyarticular disease at onset, 21 with pauciarticular onset and course of disease and 12 with extended pauciarticular disease), as well as 125 control children (58 healthy controls and 67 patients with other diseases) were tested. ELISA for the detection of IgM-, IgA- and IgG-isotypes of RFs was used. The diagnostic characteristics of the tests were evaluated by means of clinical epidemiology methods. The prevalence of the ELISA for IgG-, IgA-, and IgM-RF for JCA patients vs all controls at optimal cut-off titres was 13%, 29%, and 32%, respectively. The test for IgG-RF was established to be of no significance. IgA-RF had higher prevalence in the polyarticular and extended pauciarticular form, 40% and 33%, respectively. IgM-RF showed a prevalence of 55% for the polyarticular and 42% for the extended pauciarticular form. No significant prevalence has been found in the pauciarticular form. Our results indicate that ELISA for IgG-RF is of no diagnostic value for JCA. The ELISAs for IgM- and IgA-RFs demonstrated a diagnostic significance for the polyarticular and extended pauciarticular form. Juvenile chronic arthritis (JCA) is a heterogeneous disease which encompasses different forms defined by the type of onset. There is evidence, supported by immunogenetic studies that the various subgroups may represent distinct disease entities [1, 2]. Numerous immunological abnormalities have been detected in JCA, but the most characteristic serological findings are ANA and IgM-rheumatoid factor, thought to be useful in the classification of patients and their management. Antinuclear antibodies are universal in JCA, most commonly found in children with early onset pauciarthritis and late onset seropositive polyarthritis [1, 2, 3]. In contrast, the IgM-rheumatoid factor, measured by conventional agglutination techniques, is a hallmark only of polyarthritis with late onset resembling adult rheumatoid arthritis. This group of patients with "seropositive" disease represents less than 20% of all JCA children. Of those patients with "seronegative" disease 20-30% have a systemic onset and the remainder have either a pauciarticular or polyarticular form [2, 3]. Following the introduction of more sensitive techniques, it has already been established that rheumatoid arthritis (RA) patients' sera contain not only the "classical" 19S IgM-RF, but also other isotypes of the rheumatoid factor (RF). A number of studies have emphasized the presence of IgG-, IgA-, IgM- and even IgE- RF in patients with "seronegative" RA [4, 5, 6]. The aim of this study is to determine the prevalence of IgG, IgM and IgA RFs and to attempt at evaluating the diagnostic and prognostic qualities of the ELISA-tests for rheumatoid factor isotypes in polyarticular and pauciarticular forms at onset of JCA.

Role of neutrophils in murine cryoglobulinemia.

Murine IgG3 anti-IgG2a rheumatoid factor (RF) monoclonal antibodies (mAb) with cryoglobulin activity, are able to induce, in normal mice, skin leukocytoclastic vasculitis and lupus-like glomerulonephritis resembling 'wire-loop' lesions (subendothelial immune deposits). The development of glomerular, but not skin, lesions in immunoglobulin-deficient mice (lacking the corresponding IgG2a autoantigen) receiving IgG3 RF cryoglobulins indicates that the RF activity of IgG3 monoclonal cryoglobulins and subsequent formation of IgG3-IgG2a immune complexes play a critical role in the development of skin vasculitis. In contrast, nephritogenic activity is solely contributed by IgG3-associated cryoglobulin activity. Polymorphonuclear leukocyte (PMN) infiltration is one of the major pathologic changes observed in both types of lesions. Treatment with mAbs against the adhesion molecules leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) (both known for their involvement in PMN-endothelial cell interaction) inhibits the development of skin vascular lesions. However, it has no effect on the generation of glomerulonephritis. Apparently, adhesion molecule requirements for PMN interaction with glomerular capillary endothelial cells are different from those for PMN infiltration of the skin. However, the PMN depletion experiment has clearly shown that PMNs play an active role in the development of 'wire-loop' glomerular lesions. In the absence of the glomerular PMN infiltration, IgG3 RF cryoglobulins induce a different type of glomerular lesion, characterized by voluminous intracapillary thrombi and mesangial deposits, yet lacking subendothelial deposits. This is consistent with the fact that the latter lesions can be induced by certain IgG3 mAbs, which are unable to provoke glomerular PMN infiltration. Finally, the activation of the complement system does not appear to play a major role in either skin or glomerular lesions induced by IgG3 RF cryoglobulins.

Nitric oxide as an inflammatory mediator in autoimmune MRL-lpr/lpr mice.

Nitric oxide (.NO) may exhibit proinflammatory features. .NO synthase type 2 (NOS2) is overexpressed and .NO overproduced in rodent models of induced inflammation. Blockage of .NO production by administration of NOS inhibitors prevents or reduces various types of induced inflammation in mice and rats. We have shown that autoimmune MRL-lpr/lpr mice overexpress NOS2 and overproduce .NO in an age-dependent fashion that parallels expression of arthritis, glomerulonephritis, and vasculitis. Blocking .NO production by oral administration of the NOS inhibitor NG-monomethyl-L-arginine reduced the arthritis, glomerulonephritis, and vasculitis, but it did not modify serum anti-DNA antibody levels or glomerular deposition of immune complexes. When mice with genetically disrupted NOS2 were backcrossed to MRL-lpr/lpr mice, the resultant (-/-) mice expressed no NOS2 and produced no .NO, the wild-type (+/+) mice overexpressed NOS2 and overproduced .NO (in comparison to normal, control mice), and the heterozygous (+/-) mice expressed and produced intermediate levels. Nephritis and arthritis in the (-/-) mice were comparable to that in MRL-lpr/lpr mice, but vasculitis was markedly decreased. Levels of anti-DNA antibodies were comparable in all mice, but IgG rheumatoid factor production was markedly reduced in the (-/-) mice. These results of studies in MRL-lpr/lpr mice with genetically disrupted NOS2 highlight the heterogeneity and complexity of the role of NOS2 and .NO in inflammation.

Does smoking stimulate rheumatoid factor production in non-rheumatic individuals?

Smoking has been associated with increased incidence of rheumatoid arthritis (RA), joint damage and positive rheumatoid factor (RF). Here we report an analysis of the association between smoking and IgM, IgG and IgA RF in a cohort of non-rheumatic individuals participating in a prospective longitudinal study of the incidence and significance of elevated RF. From the initial cohort of nearly 14,000 randomly selected individuals aged 52-80 years, 109 RF-positive and 187 RF-negative non-rheumatic participants were recruited. All participants were tested for RF at least twice at an interval ranging from 4 to 13 years. Of the RF-negative participants 21.9% were active smokers compared to 34.1% of IgM RF-positive (p=0.035), 20.8% of IgG RF-positive (N.S.) and 34.4% of IgA RF-positive participants (p=0.047). Smoking was most prevalent (44.8%) amongst participants with elevation of both IgM and IgA RF (p=0.008), and smokers were also significantly more likely to have a persistent elevation of RF than non-smokers (p=0.024). These findings indicate that smoking may influence the immune system, leading to increased production of IgM and IgA RF.

Induction of IgG Rheumatoid Factor (RF) Production by Antibody–antibody (RF-like) Immune Complexes: the Role of T cells, Complement and Fcγ Receptors

Rheumatoid factors (RF) are autoantibodies with specificity for the constant regions of IgG molecules. They are found in several immunopathological diseases. The mechanism(s) by which these autoantibodies are produced is largely unknown. We have previously shown that a single injection of RF-like immune complexes (ICs) into mice selectively induced an intense IgG1-antibody production with RF activity. This response was sustained for several months and did not resemble a conventional immune response to an antigen or other immune complexes. In the present study, we sought to elucidate the mechanism for the IgG1 RF response to RF-like ICs. Therefore, the roles of CD4+T cells, complement and Fcγ receptors were analysed. In order to characterize the role of CD4+T cells, RF-like induced IgG1-RF production was analysed in NZB mice treated with a monoclonal antibody (mAb) against the CD4 molecule, which resulted in complete abrogation of IgG1 RF production. To evaluate the importance of FcγRs, the effect of RF-like ICs was tested in mice deficient for RFγRI/III. A significant decrease in the numbers of IgG1 antibody secreting cells, as well as in serum IgG1 RF levels, was found in the deficient mice, as compared with their normal outbred littermates. The role of complement in RF-like ICs mediated IgG1 RF was tested in complement depleted NZB mice, using Cobra venom factor. The IgG1 RF response in complement depleted and intact mice was comparable. Thus, our results demonstrate that RF-like immune complexes selectively induce an FcγR-dependent, complement independent antibody response in mice.

Testing of bovine sera by ELISA for IgG, IgM and IgA rheumatoid factors

Sera from 19 colostrum-deprived calves less than 1 week old, 24 colostrum-supplemented calves less than 1 week old, 36 3–5-month-old calves and 200 females greater than 9 months of age were tested by ELISA for the presence of IgM, IgG and IgA rheumatoid factors (RF). An increasing level of IgM- and IgG-RF with age was found. IgG-RF levels in the colostrum-supplemented calves were significantly higher than in the non-supplemented calves ( p<0.001). Individual IgG-RF values correlated with serum IgG levels, as determined by zinc sulphate turbidity testing ( r=0.59, p<0.01). No IgA-RF was detected. The cross-reactivity of IgM-RF with heterologous IgG was found to be greatest with rabbit IgG, followed by mouse and chicken IgG. The significance of rheumatoid factors in relation to diagnostic testing is discussed.

Assessment of enzyme linked immunosorbent assay based diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii in human serum

An enzyme linked immunosorbent assay (ELISA) using penicillinase was developed in the form of diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii. The performance of both the kits was compared with commercially available diagnostic kits, i.e. Enzygnost-Toxoplasmosis/IgG (Behring Co., Germany), TOXOTEK-G (Flow Lab., U.K.) and Toxoplasma IgM Microassay (Diamedix Corp., U.S.A.) by testing toxoplasma-suspected human serum samples. The results indicate a good reliability between these diagnostic kits. Toxokit-G has 86.66 and 96.05% sensitivity and specificity respectively. The main advantage of Toxokit-G is that the end result can be assessed visually without using sophisticated instruments. Toxokit-M has 100% sensitivity and specificity and test results were not affected by the presence of antitoxoplasma IgG antibodies, rheumatoid factor or antinuclear antibodies.

Impaired hepatosplenic elimination of circulating cryoglobulins in patients with essential mixed cryoglobulinaemia and hepatitis C virus (HCV) infection.

The pathogenic mechanisms that lead to renal deposition of the cryoprecipitable IgM rheumatoid factor-IgG complexes in essential mixed cryoglobulinaemia (EMC) are unknown. Defective removal of cryoprecipitable complexes from the circulation has been postulated in EMC-associated nephritis. To test this hypothesis, the kinetics and fate of a trace dose of 123I-radiolabelled autologous cryoglobulins were analysed in 13 patients with EMC grouped according to renal involvement. The time course of radioactivity distribution in the blood and organ uptake were measured by gamma camera scintigraphy. In blood sampled 30-300 s after injection, only a minor fraction (< 15%) of the circulating cryoglobulins bound to the erythrocytes, suggesting the elimination mechanisms are independent of binding to CR1 on erythrocytes. The overall blood disappearance curve showed a fast (< or = 1 min) and slow (> 4 h) biphasic pattern. In patients with quiescent or mild nepthritis, the liver and to a lesser extent the spleen were the major organs that mediated the rapid uptake and processing of the cryoglobulins from the circulation. In contrast, patients with active mesangiocapillary glomerulonephritis showed significantly (P < 0.001) less hepatic uptake, low liver-to-precordium ratio, and slower processing of cryoglobulins, prolonged liver mean transit time, than quiescent patients or mild nephritis patients. To elucidate the role and influence of HCV infection in the pathogenesis of EMC-nephritis, sera and cryoglobulins from all patients were assayed for HCV. None of the control group cases without nephritis showed any evidence of HCV-RNA in serum or cryoglobulin pellet. In contrast, all 10 EMC-nephritis patients' sera, and eight corresponding cryoglobulin pellets contained HCV-RNA. Collectively, these findings suggest an impaired reticuloendothelial system removal of IgM-IgG-HCV complexes may underlie their renal deposition.

Clinical and serologic manifestations of autoimmune disease in MRL-lpr/lpr mice lacking nitric oxide synthase type 2.

Nitric oxide (NO) is an important mediator of the inflammatory response. MRL-lpr/lpr mice overexpress inducible nitric oxide synthase (NOS2) and overproduce NO in parallel with the development of an autoimmune syndrome with a variety of inflammatory manifestations. In previous studies, we showed that inhibiting NO production with the nonselective nitric oxide synthase (NOS) inhibitor NG-monomethyl-arginine reduced glomerulonephritis, arthritis, and vasculitis in MRL-lpr/lpr mice. To define further the role of NO and NOS2 in disease in MRL-lpr/lpr mice, mice with targeted disruption of NOS2 were produced by homologous recombination and bred to MRL-lpr/lpr mice to the N4 generation. MRL-lpr/lpr littermates homozygous for disrupted NOS2 (-/-), heterozygous for disrupted NOS2 (+/-), or wildtype (+/+) were derived for this study. Measures of NO production were markedly decreased in the MRL-lpr/lpr (-/-) mice compared with MRL-lpr/lpr (+/+) mice, with intermediate production by the MRL-lpr/lpr (+/-) mice. There was no detectable NOS2 protein by immunoblot analysis of the spleen, liver, kidney, and peritoneal macrophages of the (-/-) animals, whereas that of (+/+) was high and (+/-) intermediate. The (-/-) mice developed glomerular and synovial pathology similar to that of the (+/-) and (+/+) mice. However, (-/-) mice and (+/-) mice had significantly less vasculitis of medium-sized renal vessels than (+/+) mice. IgG rheumatoid factor levels were significantly lower in the (-/-) mice as compared with (+/+) mice, but levels of anti-DNA antibodies were comparable in all groups. Our findings show that NO derived from NOS2 has a variable impact on disease manifestations in MRL-lpr/lpr mice, suggesting heterogeneity in disease mechanisms.

Unique site of IgG2a and rheumatoid factor production in MRL/lpr mice.

MRL/lpr (Fas-deficient) mice develop an autoimmune syndrome associated with excessive production of autoantibodies. A significant portion of these autoantibodies are IgG2a molecules specific for many of the autoantigens recognized by the sera of patients with systemic lupus erythematosus. In addition, MRL/lpr mice make exceedingly high titers of IgG or IgA rheumatoid factors (RF) specific for autologous IgG2a. The microenvironment of the IgG2a-producing B cells as well as the prototypic RF autoantibodies was determined by a combination of immunohistochemical and in situ hybridization techniques. In contrast to the antibody-producing cells present in mice responding to conventional foreign antigens, both IgG2a+ and RF+ B cells were found to be densely clustered in the T-cell-rich inner periarteriolar lymphatic sheath of the spleen. These results suggest that conventional antibody and autoantibody production in MRL/lpr mice may be mechanistically distinct processes.

Rheumatoid Factor Autoantibodies in Health and Diseasea

Recent advances in molecular biological and human cell hybridization technology have significantly advanced the knowledge of mechanisms that underlie human rheumatoid factor (RF) production. These advances have provided insight into the etiopathogenesis of synovial inflammation and lymphocyte recruitment in rheumatoid arthritis (RA) joints. We have examined the mechanisms that lead to RF production in RA patients and those that regulate RF production in normals. The studies revealed structural features that distinguish RF produced in normals from those produced in RA synovial tissue. There are significant differences in the use of VL and VH genes between the two RF populations. Furthermore, IgV genes encoding synovial RF in RA have extensive evidence for nucleotide changes, leading to amino acid replacement in the complementarity determining regions (CDRs). In addition, RF produced in RA synovia show evidence for affinity maturation, isotype switch to IgG RF, and repertoire shift indicative of a continued recruitment of B cells. Together with computer modeling and crystallographic studies, our data suggest that the mechanisms that operate on RF selection in RA synovia are similar to immune responses to exogenous antigens. In contrast, RF established from human immunized donors (HID) are characterized by a very low ratio of replacement to silent (R:S) nucleotide changes in the CDR1+2. In addition, there is little increase in affinity with increasing numbers of mutations. There is thus evidence for regulatory mechanisms that limit affinity maturation of RF in normals.