Reduced chemotaxis of polymorphonuclear leukocytes in sera from patients with rheumatoid arthritis

The chemotaxis of normal leukocytes washed and suspended in Hanks solution is inhibited by addition of serum from patients with definite rheumatoid arthritis as was shown by experiments in which an extract from Proteus mirabilis served as attractant. From a suspension of normal leukocytes to which normal serum had been added, significantly more leukocytes actively moved through a millipore filter toward the attractant than from a suspension of similar leukocytes in serum from patients.     

Identification of auto-antibodies in the sera of patients with rheumatoid arthritis

Serological analysis of a recombinant cDNA expression library was carried out and a number of auto-antibodies were found that were highly prevalent in the sera of such patients.     

Identification of multiple Epstein-Barr virus-induced nuclear antigens with sera from patients with rheumatoid arthritis.

By means of the protein immunoblot technique, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) could be identified in a variety of EBV-transformed cell lines with anti-EBNA-positive sera from normal donors. The molecular weight of EBNA expressed in each of the cell lines varied between 70,000 and 75,000 and was dependent upon the strain of infecting virus. In contrast, 15 of 21 sera from patients with rheumatoid arthritis identified antigens in addition to EBNA. The most prominent of these antigens had molecular weights of 110,000 to 115,000 and 92,000. All of the EBV genome-positive cell lines except for QIMR-GOR and cell lines containing the P3HR-1 virus expressed these antigens. The antigens were not present in the EBV genome-negative Ramos and BJAB cell lines, nor were they identified with EBV seronegative sera, indicating that they were EBV related. There was no direct correlation between the presence of antibodies in sera to EBNA, viral capsid antigen or early antigen, and reaction with the 92,000-molecular-weight antigen in immunoblots, indicating that this antigen was distinct from previously described EBV-related antigens.     

Natural autoantibodies reactive with glycosaminoglycans in rheumatoid arthritis

Introduction

Although natural autoantibodies make up the majority of circulating immunoglobulins and are also present in high numbers in therapeutically used intravenous immunoglobulin preparations, they have received little attention and their precise role remains largely unknown. An increasing awareness of the importance of posttranslational autoantigen modifications and glycobiology led us to explore carbohydrate-reactive natural autoantibodies in patients with rheumatoid arthritis. This study examined systematic antibodies reactive to glycosaminoglycans (GAGs), the carbohydrate components of proteoglycans that are released in large amounts from degrading cartilage.

Methods

To measure antibodies reactive to six different types of GAGs, a specialised ELISA was used in which the carbohydrates were covalently linked to the plastic surface through a 2 nm spacer. Sera from rheumatoid arthritis patients (n = 66), umbilical cord serum samples (n = 11) and adult controls (n = 54) were studied. In order to explore cross-reactivity with microbial antigens, bacterial peptidoglycans and fungal polysaccharides were used. Sera and synovial fluid samples were also tested using a GlycoChip carbohydrate array to characterise individual carbohydrate recognition patterns. We followed a multistep statistical screening strategy for screening GAG-reactive antibodies as predictive disease markers.

Results

While anti-GAG antibodies were absent in the umbilical cord sera, they were readily detectable in adult controls and were significantly elevated in patients with rheumatoid arthritis (p < 0.001). Anti-GAG antibodies showed significant cross-reactivity among different types of GAGs. They also reacted with bacterial peptidoglycans and fungal polysaccharides. Interestingly, anti-chondroitin sulphate C IgM antibody levels showed inverse correlation both with the Disease Activity Score (DAS) 28 scores and C-reactive protein (CRP) levels in rheumatoid arthritis.

Conclusion

The highly abundant and cross-reactive, GAG-specific natural autoantibodies in serum may serve as novel disease-state markers in patients with rheumatoid arthritis.     

New autoantibodies in early rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease causing articular cartilage and bone destruction. Since irreversible joint destruction can be prevented by intervention at the early stages of disease, early diagnosis of RA is important. In this study, we identified new autoantibodies in the sera of patients with early (less than one year) RA.

Methods

We screened the sera of 20 RA patients with disease duration less than one year, 19 RA patients with disease duration more than five years and 23 controls on 8,268 human protein arrays. We confirmed the validity of protein array detection by ELISA assays. We then performed epitope mapping with overlapping 15-mers to analyze RA sera reactivity.

Results

WIBG (within BGCN homolog (Drosophila)), GABARAPL2 (GABA(A) receptor associated protein like 2) and ZNF706 (zinc finger protein 706) proteins are preferentially recognized by autoantibodies from early RA patients. Of interest, autoantibodies to WIBG are very specific for early RA. Indeed, 33% of early RA patients'' sera recognize WIBG versus 5% of RA patients with disease duration more than 5 years and 2% of controls. We identified three linear peptides on WIBG GABARAPL2 and ZNF706 that are preferentially recognized by sera of early RA patients.

Conclusions

We identified new autoantibodies associated with RA with disease duration less than one year. These autoantibodies could be used as diagnosis markers in RA patients.     

Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.     

Tissue autoantibodies during therapy with levamisole for rheumatoid arthritis

In 20 patients affected with rheumatoid arthritis, tissue autoantibodies were studied during a year of treatment with levamisole. Before therapy, antinuclear antibodies were present in 11 cases, anti-thyroid microsomes in 1 and anti-smooth muscle in 6. During therapy, autoantibodies remained almost unchanged in all patients, thus indicating that levamisole does not influence this aspect of humoral immunity in rheumatoid arthritis. Furthermore, no correlation was found between autoantibody pattern before treatment and effects of levamisole on clinical course of disease.     

C1q-containing immune complexes purified from sera of juvenile rheumatoid arthritis patients mediate IL-8 production by human synoviocytes: role of C1q receptors.

Immune complexes that vary in size and composition are present in the sera and synovial fluid of juvenile rheumatoid arthritis (JRA) patients. They are believed to be potent inducers of the ongoing inflammatory process in JRA. However, the precise composition and role of these complexes in the pathophysiology of JRA remain unclear. We hypothesized that circulating ICs have the potential to interact with resident joint synovial fibroblasts (synoviocytes) and induce the expression of inflammatory cytokines. To test this hypothesis, cultures of synoviocytes from healthy individuals were treated with ICs isolated from the sera of JRA patients. Studies reported in this work demonstrate that IgM affinity-purified ICs from the sera of JRA patients contain IgM, C1q, IgG, and C3 to a variable extent. These ICs induce IL-8 mRNA and protein production in normal synoviocytes. Our data indicate that C1q in these ICs mediates, in part, IL-8 induction in synoviocytes. This is based on our findings of C1q-binding proteins for collagen stalks (cC1qR) and globular heads (gC1q-binding protein) of C1q in synoviocytes. In addition, collagen stalk and to some extent globular head fragments of C1q inhibit IC-mediated IL-8 induction in synoviocytes. Together, these findings provide evidence for a novel mechanism of IL-8 production by synoviocytes, which could play a key role in inflammation by recruiting leukocytes to synovial tissue and fluid-and subsequently contributing to joint disease.     

Synovial stromal cells from rheumatoid arthritis patients attract monocytes by producing MCP-1 and IL-8

Macrophages that accumulate in the synovium of rheumatoid arthritis patients play an important role in the pathogenesis of this inflammatory disease. However, the mechanism by which macrophages are attracted into the inflamed synovium and accumulate there has not been completely delineated. The results of this study show that rheumatoid arthritis synovial stromal cells produce the chemokines monocyte chemotactic protein-1 and IL-8, and these have the capacity to attract peripheral monocytes. These results suggest that one of the mechanisms by which macrophages accumulate in the inflamed synovium is by responding to the chemokines produced locally.     

Association analysis of IL-4 VNTR polymorphism with rheumatoid arthritis in Iranian patients

Rheumatoid arthritis (RA) is an autoimmune disease characterized by movement disability and pain in the joints. The affected individuals are susceptible to other subsequent diseases, exacerbating the condition. To find out the genetic variability of this disease at the genomic level for the first time in the Iranian population, we carried out an investigation on the VNTR of IL-4 gene within its third intron. For this goal we isolated the genomic DNA from blood samples of 576 rheumatoid arthritis patients and 546 healthy controls and investigated the presence or absence of specific amplicons via polymerase chain reaction (PCR). The size of each amplicon on a 1.5% agarose gel corresponded to a certain number of tandem repeats which indicated a specific allele. Statistical test of χ² Fisher’s exact test and odds ratio (OR) was used to analyze the data. The results showed that RA1/RA1 genotype was the dominant genotype in both healthy controls and patients and the heterozygote genotype of RA1/RA2 was observed more in the healthy controls than patients (108 vs. 66) with significant difference with P value < 0.005 and odds ratio of 0.214. However two genotypes of RA2/RA2 and RA2/RA3 were exclusively observed in the patients’ samples with P value = 0.023 and odds ratio of 0.988. We concluded that IL-4 VNTR polymorphism has a strong association with rheumatoid arthritis and might be a high risk factor for development of rheumatoid arthritis in the investigated Iranian population.     

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.     

Isolation and purification of a Clq-anti Clq precipitin ring enhancing protein from sera of patients with rheumatoid arthritis

We have isolated and purified to apparent homogeneity a serum protein which appears to be a biological marker for active rheumatoid arthritis. The protein has been found in the sera in all 44 active rheumatoid arthritis patients thus far studied and is absent from, or present in undetected amounts, in sera from normal subjects or from patients with other arthritides. The protein has a molecular weight of 135,000 daltons, an isoelectric pH of 5.1-5.3, and it enhances the size of the C1q-anti C1q ring.     

Synovial fibroblasts from patients with rheumatoid arthritis, like fibroblasts from Graves' disease, express high levels of IL-16 when treated with Igs against insulin-like growth factor-1 receptor

We have reported recently that IgG from patients with Graves' disease (GD) can induce the expression of the CD4-specific T lymphocyte chemoattractant, IL-16, and RANTES, a C-C chemokine, in their fibroblasts. This induction is mediated through the insulin-like growth factor-1 receptor (IGF-1R) pathway. We now report that Abs from individuals with active rheumatoid arthritis (RA-IgG) stimulate in their synovial fibroblasts the expression of these same cytokines. IgG from individuals without known autoimmune disease fails to elicit this chemoattractant production. Furthermore, RA-IgG fails to induce IL-16 or RANTES expression in synovial fibroblasts from donors with osteoarthritis. RA-IgG-provoked IL-16 and RANTES production also appears to involve the IGF-1R because receptor-blocking Abs prevent the response. RA fibroblasts transfected with a dominant-negative mutant IGF-1R fail to respond to RA-IgG. IGF-1 and the IGF-1R-specific analog Des(1-3) also induce cytokine production in RA fibroblasts. RA-IgG-provoked IL-16 expression is inhibited by rapamycin, a specific macrolide inhibitor of the Akt/FRAP/mammalian target of rapamycin/p70(s6k) pathway, and by dexamethasone. GD-IgG can also induce IL-16 in RA fibroblasts, and RA-IgG shows similar activity in GD fibroblasts. Thus, IgGs from patients with RA, like those associated with GD, activate IGF-1R, and in so doing provoke T cell chemoattraction expression in fibroblasts, suggesting a potential common pathway in the two diseases. Immune-competent cell trafficking to synovial tissue is integral to the pathogenesis of RA. Recognition of this novel RA-IgG/fibroblast interaction and its functional consequences may help identify therapeutic targets.     

Roles of IL-1 in the development of rheumatoid arthritis: consideration from mouse models

Expression of inflammatory cytokines is augmented in the joints of patients with rheumatoid arthritis (RA). We found that cytokine levels are also elevated in the joints of a mouse arthritis model, human T-cell leukemia virus type I (HTLV-I) transgenic (Tg) mouse. Depletion of IL-1 by gene targeting greatly reduced the incidence of the disease, indicating the importance of this cytokine in the development of arthritis. Furthermore, IL-1 receptor antagonist (IL-1Ra)-deficient mice develop autoimmunity and arthritis spontaneously. These observations suggest that excess IL-1 signaling the causes autoimmunity. We show that IL-1 activates the immune system non-specifically by inducing CD40L and OX40 co-signaling molecules on T cells. In this review, the roles of IL-1 in the development of autoimmunity and arthritis in mouse models will be discussed.     

Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics

Hybridoma anti-DNA antibodies have been generated from the fusion of the GM 4672 lymphoblastoid line with peripheral blood lymphocytes from four normal subjects, nine patients with rheumatoid arthritis (RA), and 13 patients with systemic lupus erythematosus (SLE). A total of 441 hybridoma clones were obtained, of which 37 secreted anti-DNA autoantibodies. The nucleic acid binding characteristics of the anti-DNA antibodies produced by two hybridomas from normal subjects, nine hybridomas from RA patients, and 18 hybridomas from SLE patients are reported. The hybridoma anti-DNA antibodies from all three groups showed similar antigen-binding characteristics for denatured DNA (dDNA), native DNA (nDNA), poly(I), poly(dT), and cardiolipin, by both direct binding and competitive binding analyses. One difference noted between normal-derived anti-DNA antibodies and autoimmune-derived antibodies was the inability of the former to react with z-DNA. However, this requires further substantiation with larger numbers of normal-derived clones. The broad overlap of reactivity to nucleic acid antigens among individual anti-DNA autoantibodies found in two clinically different autoimmune diseases, namely RA and SLE, suggests that the pathogenicity of anti-DNA autoantibodies may bear no relationship to their nucleic acid antigen-binding characteristics.     

IL-29 enhances Toll-like receptor-mediated IL-6 and IL-8 production by the synovial fibroblasts from rheumatoid arthritis patients

Introduction

We previously reported that IL-29, a newly described member of interferon (IFN) family, was overexpressed in blood and synovium of rheumatoid arthritis (RA) patients and triggered proinflammatory cytokine IL-6 and IL-8 mRNA expression in RA synovial fibroblasts (RA-FLS). This suggests that IL-29 has an important role in synovial inflammation. Toll-like receptors (TLRs) also activate RA-FLS to produce inflammatory mediators including tumor necrosis factor α (TNF-α) and IL-1β in RA-FLS. Since the TLR family plays an early role in the innate immune response and the subsequent induction of the adaptive immune response, we hypothesize that IL-29 interacts with TLRs in RA inflammation. This study aimed to investigate the effect of IL-29 on TLR-mediated proinflammatory cytokine production in RA-FLS.

Methods

The mRNA level of IL-29 receptors (IL-28Rα and IL-10R2) in RA-FLS was determined by semi-quantitative RT- PCR. IL-6 and IL-8 mRNA expressions in RA-FLS were evaluated by real-time PCR after pre-incubation with IL-29 and subsequent stimulation with peptidoglycan (PGN, TLR2 ligand), or polycytidylic acid (poly(I:C), TLR3 ligand), or lipopolysaccharide (LPS, TLR4 ligand) . The production of TLR2, 3, and 4 in RA-FLS after IL-29 stimulation was also assessed by real-time PCR and flow cytometry. IL-29 mRNA and protein expression in RA-FLS after stimulation with PGN, poly(I:C), or LPS were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively.

Results

The IL-29 receptor complex (IL-28Rα and IL-10R2) was identified in RA-FLS. IL-29 enhanced TLR-mediated IL-6 and IL-8 expression in RA-FLS. IL-29 upregulated expression of TLR2, 3 and 4 in RA-FLS. Exposure to PGN, poly(I:C) or LPS triggered IL-29 production by RA-FLS.

Conclusions

We show for the first time that IL-29 enhances TLR-induced proinflammatory cytokine production in RA-FLS via upregulation of TLRs.