Soluble Low-density Lipoprotein Receptor-related Protein 1 in Juvenile Idiopathic Arthritis.

OBJECTIVES: This study aimed to expand knowledge about soluble low-density lipoprotein receptor-related protein 1 (sLRP1) in juvenile idiopathic arthritis (JIA) by determining associations of sLRP1 levels in nonsystemic JIA patients with clinical and inflammatory biomarker indicators of disease activity. METHODS: Plasma sLRP1 and 44 inflammation-related biomarkers were measured at enrollment and 6 months later in a cohort of 96 newly diagnosed Canadian patients with nonsystemic JIA. Relationships between sLRP1 levels and indicators of disease activity and biomarker levels were analyzed at both visits. RESULTS: At enrollment, sLRP1 levels correlated negatively with age and active joint counts. Children showed significantly higher levels of sLRP1 than adolescents (mean ranks: 55.4 and 41.9, respectively; P = 0.02). Participants with 4 or fewer active joints, compared to those with 5 or more active joints, had significantly higher sLRP1 levels (mean ranks: 56.2 and 40.7, respectively; P = 0.006). At enrollment, considering the entire cohort, sLRP1 correlated negatively with the number of active joints (r = -0.235, P = 0.017). In the entire cohort, sLRP1 levels at enrollment and 6 months later correlated with 13 and 6 pro- and antiinflammatory biomarkers, respectively. In JIA categories, sLRP1 correlations with inflammatory markers were significant in rheumatoid factor-negative polyarticular JIA, oligoarticular JIA, enthesitis-related arthritis, and psoriatic arthritis at enrollment. Higher sLRP1 levels at enrollment increased the likelihood of absence of active joints 6 months later. CONCLUSION: Plasma sLRP1 levels correlate with clinical and biomarker indicators of short-term improvement in JIA disease activity, supporting sLRP1 as an upstream biomarker of potential utility for assessing JIA disease activity and outcome prediction.

Prevalence of anti-peptidylarginine deiminase type 4 antibodies in rheumatoid arthritis and unaffected first-degree relatives in indigenous North American Populations.

OBJECTIVE: To determine whether anti-peptidylarginine deiminase type 4 (PAD4) antibodies were present in first-degree relatives (FDR) of patients with rheumatoid arthritis (RA) in 2 indigenous North American populations with high prevalence of RA. METHODS: Participants were recruited from 2 indigenous populations in Canada and the United States, including patients with RA (probands), their unaffected FDR, and healthy unrelated controls. Sera were tested for the presence of anti-PAD4 antibodies, anticyclic citrullinated peptide (anti-CCP) antibodies, and rheumatoid factor (RF). HLA-DRB1 subtyping was performed and participants were classified according to number of shared-epitope alleles present. RESULTS: Antibodies to PAD4 were detected in 24 of 82 (29.3%) probands; 2 of 147 (1.4%) relatives; and no controls (p < 0.0001). Anti-CCP was present in 39/144 (27.1%) of the relatives, and there was no overlap between positivity for anti-CCP and PAD4 in the relatives. In RA patients, anti-PAD4 antibodies were associated with disease duration (p = 0.0082) and anti-CCP antibodies (p = 0.008), but not smoking or shared-epitope alleles. CONCLUSION: Despite a significant prevalence of anti-CCP in FDR, anti-PAD4 antibodies were almost exclusively found in established RA. The prevalence of anti-PAD4 antibodies in RA is similar to the prevalence described in other populations and these autoantibodies are associated with disease duration and anti-CCP in RA.

Cross-linking of IgGs bound on circulating neutrophils leads to an activation of endothelial cells: possible role of rheumatoid factors in rheumatoid arthritis-associated vascular dysfunction.

BACKGROUND: Rheumatoid arthritis is characterized by the presence of circulating auto-antibodies, including rheumatoid factors, which recognize the Fc portion of IgGs. The neutrophil is the most abundant circulating leukocyte and it expresses high levels of FcγRs on its surface. The aim of the present study was to examine the capacity of circulating human neutrophils to be activated by rheumatoid factors and the consequences of these events on endothelium. METHODS: Neutrophil-bound IgGs were cross-linked with anti-human IgGs to mimick the presence of circulating rheumatoid factors and FcγRs-dependent signalling events and functions were examined. The IgG and IgM composition of rheumatoid factors isolated from the serum of RA patients was characterized. Adhesion of neutrophils to endothelial cells was quantified in response to the addition of rheumatoid factors. RESULTS: Cross-linking of IgGs bound on neutrophils leads to FcγRs-dependent tyrosine phosphorylation, mobilisation of intracellular calcium and the extracellular release of superoxide anions and lysozyme. Incubation of endothelial cells with the supernatant of activated neutrophils increases ICAM-1 expression and IL-8 production by endothelial cells. Finally, rheumatoid factors enhance neutrophil adhesion to endothelial cells. CONCLUSIONS: Our results show that activation of neutrophils' FcγRs by rheumatoid factors could participate in rheumatoid arthritis-associated vascular damage.

A rheumatoid factor paradox: inhibition of rituximab effector function.

INTRODUCTION: Rituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC). METHODS: We developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated. RESULTS: Using human sera, addition of RTX resulted in rapid and profound (>50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis. CONCLUSIONS: Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.

Chronic smoke exposure induces rheumatoid factor and anti-heat shock protein 70 autoantibodies in susceptible mice and humans with lung disease.

The impact of cigarette smoke (CS), a risk factor for rheumatoid arthritis (RA), on sauto-antibody production was studied in humans and mice with and without chronic lung disease (LD). Rheumatoid factor (RF), anti-cyclic citrullinated peptides (CCPs), and anti-HSP70 autoantibodies were measured in several mouse strains and in cohorts of smokers and nonsmokers with and without autoimmune disease. Chronic smoking-induced RFs in AKR/J mice, which are most susceptible to LD. RFs were identified in human smokers, preferentially in those with LD. Anti-HSP70 auto-antibodies were identified in CS-exposed AKR/J mice but not in ambient air exposed AKR/J controls. Whereas inflammation could induce anti-HSP70 IgM, smoke exposure promoted the switch to anti-HSP70 IgG autoantibodies. Elevated anti-CCP autoantibodies were not detected in CS-exposed mice or smokers. AKR/J splenocytes stimulated in vitro by immune complexes (ICs) of HSP70/anti-HSP70 antibodies produced RFs. The CD91 scavenger pathway was required as anti-CD91 blocked the HSP70-IC-induced RF response. Blocking Toll-like receptors did not influence the HSP70-IC-induced RFs. These studies identify both anti-HSP70 and RFs as serological markers of smoke-related LD in humans and mice. Identification of these autoantibodies could suggest a common environmental insult, namely CS, in a number of different disease settings.

Familial clustering of the serum cytokine profile in the relatives of rheumatoid arthritis patients.

OBJECTIVE: Rheumatoid arthritis (RA) is prevalent in North American Native populations, with a high frequency of multicase families and seropositivity in first-degree relatives. This study was undertaken to determine whether the serum cytokine profile of first-degree relatives of North American Native patients with RA differed from that of individuals with no family history of autoimmunity and whether there was an association with RA autoantibodies. METHODS: North American Native patients with RA (n = 105), their first-degree relatives (n = 273), healthy North American Native controls (n = 200), and Caucasian controls (n = 150) were studied. Serum levels of 42 cytokines were tested using a multiplex laser bead assay. Rheumatoid factor (RF), anti-cyclic citrullinated peptide 2 (anti-CCP-2), monocyte chemotactic protein 1 (MCP-l), and high-sensitivity C-reactive protein (hsCRP) were tested by enzyme-linked immunosorbent assay, and HLA-DRB1 alleles by specific primers. Discriminant analysis and logistic regression classified individuals based on their cytokine profile. RESULTS: The prevalence of RF (cutoff level predetermined to include 5% of Caucasian controls) and anti-CCP (cutoff level of ≥40 units) was, respectively, 88% and 81% in the RA patients, 34% and 9% in first-degree relatives, and 9% and 4% in North American Native controls; the prevalence of anti-CCP was 0% in Caucasian controls. Levels of most cytokines were highest in RA patients; 17 of 40 cytokines (43%) were significantly higher in first-degree relatives than in controls, including multiple proinflammatory cytokines. Discriminant analysis showed a notable distinction between the groups, with 85% classification accuracy. First-degree relatives had markedly higher MCP-1 and hsCRP levels than North American Native controls, but there was no consistent association with RA autoantibodies. CONCLUSION: Our findings indicate that levels of multiple cytokines and hsCRP are higher in first-degree relatives of North American Native patients with RA compared to individuals from a nonautoimmune background. These data suggest that elevated baseline cytokine levels may be part of the risk profile for developing RA.

Antibodies to porphyromonas gingivalis are associated with anticitrullinated protein antibodies in patients with rheumatoid arthritis and their relatives.

OBJECTIVE: Anticitrullinated protein antibodies (ACPA) are relatively specific for rheumatoid arthritis (RA), and predate disease. The oral pathogen Porphyromonas gingivalis may play a role in breaking immune tolerance to citrullinated antigens. We studied a cohort of patients with RA and their relatives looking for associations between anti-P. gingivalis antibodies and ACPA. METHODS: Patients with RA (n = 82) and their relatives (n = 205) from a North American Native (NAN) population were studied, along with 47 NAN and 60 non-NAN controls. IgM and IgA rheumatoid factor (RF) were tested by nephelometry and ELISA. Second-generation anticyclic citrullinated peptide (anti-CCP2) isotypes and IgG anti-P. gingivalis lipopolysaccharides were tested by ELISA. HLA-DRB1 typing was performed by sequencing. Oral hygiene and smoking habits were assessed by questionnaires. RESULTS: Autoantibody frequency in patients with RA and relatives: ACPA 91% vs 19%, respectively; IgM RF 82% vs 17%; IgA RF 48% vs 22%. Anti-P. gingivalis levels were higher in patients with RA compared to relatives and controls (p = 0.005) and higher in ACPA-positive patients with RA than in ACPA-negative patients with RA (p = 0.04) and relatives (p < 0.001), but comparable in RF-positive and RF-negative patients and relatives. Poor oral hygiene and smoking were prevalent, but with no clear association with autoantibodies. Relatives with 2 shared-epitope alleles were more likely to be ACPA-positive (OR 2.5, p = 0.02). CONCLUSION: In a genetically predisposed population of NAN patients with RA and their relatives, anti-P. gingivalis antibodies were associated with ACPA. These findings suggest that immune responses to P. gingivalis may be involved in breaking immune tolerance to citrullinated antigens.

Distinct bacterial colonization patterns of Escherichia coli subtypes associate with rheumatoid factor status in early inflammatory arthritis.

OBJECTIVES: The aetiology of RA is unknown; however, bacterial exposure, particularly to Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae, has been linked to disease pathogenesis. The strongest association was observed for RF(+) RA. We compare colonization patterns of these bacteria, and the anti-bacterial antibody levels in early onset RF(+) and RF(-) inflammatory arthritis. METHODS: Bacteria isolated from stool and urine of early-stage RF(+) and RF(-) patients recruited to the Early Arthritis Registry were biochemically identified and genotyped. IgM and IgA anti-bacterial and RF antibodies were assessed by ELISA. RESULTS: Differences in the types of colonizing pathogenic E. coli were identified. RF(+) patients were more commonly colonized with phylogenetic Group D E. coli, whereas RF(-) patients were more commonly colonized with phylogenetic Group B2 E. coli and these individuals also had lower joint scores and inflammatory markers yet higher IgA anti-E. coli antibody responses. CONCLUSIONS: These studies link the type of colonizing bacteria in the gut and urine with the immune response (anti-bacterial and RF) in early-onset inflammatory arthritis and provide evidence for a role of the host-pathogen response in the aetiology of RF.

Marked differences in fine specificity and isotype usage of the anti-citrullinated protein antibody in health and disease.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) display high association with rheumatoid arthritis (RA) and are implicated in its pathogenesis. The presence of ACPAs is known to precede the onset of RA. In order to identify the features that could confer its pathogenicity, we extensively characterized this antibody response in a unique North American native population of patients with RA and their unaffected relatives. METHODS: The levels of IgA, IgM, and IgG ACPAs, as well as IgM and IgA rheumatoid factor (RF), were measured in serum samples obtained from 81 patients with RA and 195 of their unaffected relatives. The isotype distribution, the fine specificity of the ACPA response, and its association with RF were compared in health and disease. RESULTS: ACPA positivity was observed in 19% of the healthy relatives and approximately 91% of the patients with RA. ACPA isotype usage was strikingly lower in unaffected relatives than in patients with RA (1-2 versus 5-6 isotypes). Fine specificity studies showed that reactivity to citrullinated fibrinogen and vimentin was present in sera from patients with RA, while it was virtually absent in their unaffected relatives. Finally, the ACPA and RF responses were associated in patients with RA but were discordant in their healthy relatives. Extended analyses revealed that the presence of ACPAs was associated with RA irrespective of RF status, while the association of RF with disease relied on its interaction with ACPAs. CONCLUSION: The fine specificity and isotype usage of the ACPA response are qualitatively different in health and disease. Epitope spreading and expansion of the isotype repertoire might be necessary for development of RA, and this could be facilitated by the presence of RF antibodies.

Rheumatoid factors: good or bad for you?

BACKGROUND: Rheumatoid factors (RFs) are autoantibodies associated with rheumatoid arthritis. They can be detected in normal individuals, although transiently. This dichotomy has led to questions about the origins and types of RFs. Recently it has been shown that B cells that produce RFs only do so when activated by two signals, one from engagement of the B-cell receptor and the other from recognition of a pathogen-associated molecular pattern through a Toll-like receptor (TLR). These autoantibodies thus link the innate and acquired immune responses. OBJECTIVE: Through a review of the literature, an examination of the current knowledge of RF induction is presented. The focus is on a discussion of a beneficial or detrimental role for RFs in normal individuals and in those with chronic disease. RESULTS: What makes RF 'good' in some cases and 'bad' in others may reflect the type of RF produced. Low-affinity polyreactive IgM RFs are probably beneficial as they aid in the clearance of immune complexes that are more efficiently cleared, and the RF B cell can act as an antigen-presenting cell and stimulate host defense. However, large amounts of high-affinity RFs found in patients with chronic disease may be harmful by participation in a vicious cycle of autoantibody production by stimulation of self lymphocytes, and/or deposition in blood vessels thus causing vasculitis. CONCLUSIONS: Whether RFs are beneficial or detrimental depends on the context in which they are expressed, the type and amount of RF produced, whether the response is perpetuated by TLR ligation and whether other cells are stimulated either directly or indirectly by RF-positive B cells.

Fragile X-related protein FXR1P regulates proinflammatory cytokine tumor necrosis factor expression at the post-transcriptional level.

Tumor necrosis factor (TNF) is regulated post-transcriptionally by the AU-rich element (ARE) within the 3'-untranslated region of its mRNA. This regulation modulates translational efficacy and mRNA stability. By using a cRNA probe containing the TNF ARE sequence, we screened a macrophage protein expression library and identified FXR1P. Macrophages that we generated from FXR1 knock-out mice had enhanced TNF protein production compared with wild type macrophages following activation. Expression of several other proteins that are regulated by ARE sequences was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3'-untranslated region of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. Finally, we found that the ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes demonstrating the important role of FXR1P in the post-transcriptional regulation of TNF expression. Our data suggest that release of this repression by FXR1P occurs during lipopolysaccharide-induced macrophage activation. Finally, complementation of the knock-out macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation.

Lack of induction of autoantibody responses following immunization with cytomegalovirus (CMV) glycoprotein B (gB) in healthy CMV-seronegative subjects.

Possible correlations have been proposed between autoimmune diseases, such as systemic lupus erythematosus (SLE), and infection with human cytomegalovirus (CMV). The recent observation that an adenovirus expressing the immunodominant envelope glycoprotein of CMV, glycoprotein B (gB), may be capable of inducing autoantibodies in certain mouse strains has prompted interest in exploring potential relationships between gB immunization and autoimmune disease. We examined whether a recombinant CMV gB vaccine, or a gB canarypox vectored vaccine (ALVAC-CMVgB), administered to a total of 76 CMV-seronegative subjects, was capable of inducing cross-reactive antibodies to Smith antigen (Sm), ribonucleoprotein complex (RNP), and the U1-70 kDa component of the RNP complex. Using immunofluorescence, EIA and immunoblot analyses, we failed to identify induction of autoantibodies following vaccination with gB, whether administered alone as a purified protein subunit with adjuvant, or in combination with expression in a vectored approach using a recombinant canarypox. These data reinforce the favorable safety profile of CMV gB vaccines.

Advanced glycation end-product (AGE)-damaged IgG and IgM autoantibodies to IgG-AGE in patients with early synovitis.

Advanced glycation end-product (AGE)-damaged IgG occurs as a result of hyperglycemia and/or oxidative stress. Autoantibodies to IgG-AGE were previously demonstrated in patients with severe, longstanding rheumatoid arthritis (RA). We investigated whether IgG-AGE and anti-IgG-AGE antibodies were present early in the course of RA and other inflammatory arthropathies. We prospectively followed a cohort of 238 patients with inflammatory arthritis of duration less than 1 year. Patients were evaluated clinically and serologically, and radiographs were obtained at initial and 1-year visits. Sera were assayed for IgG-AGE and anti-IgG-AGE antibodies by enzyme-linked immunosorbent assay (ELISA). Rheumatoid factor (RF) was determined by nephelometry and ELISA. Of all patients, 29% had RF-positive RA, 15% had RF-negative RA, 18% had spondyloarthropathy, and 38% had undifferentiated arthritis. IgG-AGE was present in 19% of patients, and was similar in amount and frequency in all groups. Patients with elevated IgG-AGE levels had significantly higher levels of the inflammatory markers C-reactive protein and erythrocyte sedimentation rate, but there was no correlation with blood glucose levels. Overall, 27% of the patients had IgM anti-IgG-AGE antibodies. These antibodies were highly significantly associated with RFs (P < 0.0001) and with swollen joint count (P < 0.01). In early onset arthritis, IgG damaged by AGE was detected in all patient groups. The ability to make IgM anti-IgG-AGE antibodies, however, was restricted to a subset of RF-positive RA patients with more active disease. The persistence of the anti-IgG-AGE response was more specific to RA, and was transient in the patients with spondyloarthropathy and with undifferentiated arthritis who were initially found to be positive for anti-IgG-AGE antibodies.

Rheumatoid factors: host resistance or autoimmunity?

Rheumatoid factors (RFs), autoantibodies that bind to the Fc portion of IgG, are important in the immune response. RF-committed B-cells exist in the circulating lymphocyte pool in a high frequency (approximately 1-2 %) in normal individuals and in patients with pathological conditions associated with the sustained levels of circulating RF, such as rheumatoid arthritis (RA), Sjogren's syndrome (SS), and mixed cryoglobulinemia, associated with hepatitis C virus infection. RFs are induced by many infectious entities (viruses, bacteria, parasites) as a consequence of a secondary immune response to the pathogen, but usually the response is transient. It is likely that RFs play an important role in the host's defense against infection, both at the cellular level, where the RF B-cell can be an antigen presenting cell which can promote the antipathogen response, and at the humoral level, where RFs can contribute to the mopping up of the IgG antipathogen antibodies by contributing to immune complex formation and clearance. There has been much research on RFs in chronic pathological conditions, and the literature pertaining to their origin, structure, binding specificities, and possible roles in disease are discussed. The importance of the host defense, sometimes at the expense of an autoimmune response, is a balance that needs to be considered in light of a possible outcome of health or disease.