Proteomic analysis of peripheral blood mononuclear cells: selective protein processing observed in patients with rheumatoid arthritis

In a comparative proteome analysis of peripheral blood mononuclear cells (PBMCs), we analyzed 130 two-dimensional gels obtained from 33 healthy control individuals and 32 patients diagnosed with rheumatoid arthritis (RA). We found 16 protein spots that are deregulated in patients with RA and, using peptide mass fingerprinting and Western blot analyses, identified these spots as belonging to 9 distinct proteins. A hierarchical clustering procedure organizes the study subjects into two main clusters based on the expression of these 16 protein spots, one that contains mostly healthy control individuals and the other mostly RA patients. The majority of the proteins differentially expressed in RA patients when compared with healthy controls can be detected as protein fragments in PBMCs obtained from RA patients. This set of deregulated proteins includes several factors that have been shown to be autoantigens in autoimmune diseases.     

Microarray analyses of peripheral blood cells identifies unique gene expression signature in psoriatic arthritis

Psoriatic arthritis (PsA) is a chronic and erosive form of arthritis of unknown cause. We aimed to characterize the PsA phenotype using gene expression profiling and comparing it with healthy control subjects and patients rheumatoid arthritis (RA). Peripheral blood cells (PBCs) of 19 patients with active PsA and 19 age- and sex-matched control subjects were used in the analyses of PsA, with blood samples collected in PaxGene tubes. A significant alteration in the pattern of expression of 313 genes was noted in the PBCs of PsA patients on Affymetrix U133A arrays: 257 genes were expressed at reduced levels in PsA, and 56 genes were expressed at increased levels, compared with controls. Downregulated genes tended to cluster to certain chromosomal regions, including those containing the psoriasis susceptibility loci PSORS1 and PSORS2. Among the genes with the most significantly reduced expression were those involved in downregulation or suppression of innate and acquired immune responses, such as SIGIRR, STAT3, SHP1, IKBKB, IL-11RA, and TCF7, suggesting inappropriate control that favors proin-flammatory responses. Several members of the MAPK signaling pathway and tumor suppressor genes showed reduced expression. Three proinflammatory genes--S100A8, S100A12, and thioredoxin--showed increased expression. Logistic regression and recursive partitioning analysis determined that one gene, nucleoporin 62 kDa, could correctly classify all controls and 94.7% of the PsA patients. Using a dataset of 48 RA samples for comparison, the combination of two genes, MAP3K3 followed by CACNA1S, was enough to correctly classify all RA and PsA patients. Thus, PBC gene expression profiling identified a gene expression signature that differentiated PsA from RA, and PsA from controls. Several novel genes were differentially expressed in PsA and may prove to be diagnostic biomarkers or serve as new targets for the development of therapies.     

Detection of multiple autoantibodies in patients with ankylosing spondylitis using nucleic acid programmable protein arrays

Ankylosing spondylitis (AS) is a common, inflammatory rheumatic disease that primarily affects the axial skeleton and is associated with sacroiliitis, uveitis, and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine whether plasma from patients with AS contained autoantibodies and, if so, characterize and quantify this response in comparison to patients with rheumatoid arthritis (RA) and healthy controls. Two high density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA, and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis to determine the patterns of signaling cascades or tissue origin. 44% of patients with ankylosing spondylitis demonstrated a broad autoantibody response, as compared with 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The autoantibody responses in the AS patients were targeted toward connective, skeletal, and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic acid programmable protein arrays constitute a powerful tool to study autoimmune diseases.     

Altered expression and glycosylation of plasma proteins in rheumatoid arthritis

Altered glycosylation of plasma proteins has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). The present study investigated the changes in the Concanavalin-A (Con-A)-bound plasma proteins in the RA patients in comparison to that of the healthy controls. Two proteins (MW ∼32 kDa and ∼62 kDa) showed an alteration in expression while an altered monosaccharide profile (high mannose) was observed in the ∼62 kDa protein in the samples collected from RA patients. The 2-dimensional polyacrylamide gel electrophoresis analysis of the Con-A-bound plasma samples showed a large number of protein spots, a few of which were differentially expressed in the RA patients. Some unidentified proteins were detected in the RA patients which were absent in the control samples. The present study, therefore, enunciates the role of carbohydrates as well as that of the acute phase response in the disease pathogenesis.     

Identification of citrullinated alpha-enolase as a candidate autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as alpha-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated alpha-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated alpha-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. Alpha-enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated alpha-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.     

Identification of citrullinated α-enolase as a candidate autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.     

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (p<0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.     

Identification of citrullinated eukaryotic translation initiation factor 4G1 as novel autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis. We previously reported that functional variants of the gene encoding peptidylarginine deiminase type 4 were closely associated with RA. The purpose of this study was to investigate the citrullinated autoantigens recognized by serum samples from patients with RA. The human chondrocyte cDNA expression library was citrullinated by PADI4 and was immunoscreened with anti-modified citrulline antibodies and sera from patients with rheumatoid arthritis. One immunoreactive cDNA clone containing a 2480-base pair insert was isolated and sequence analysis revealed that the cDNA included a part of the eukaryotic translation initiation factor 4G1. Immunoreactivity against a recombinant citrullinated eIF4G1 fragment was observed with high specificity in 50.0% of RA patients. The levels of antibodies against citrullinated eIF4G1 were correlated with those of anti-CCP antibodies. Citrullinated eIF4G1 was identified as a candidate citrullinated autoantigen in RA patients. Citrullination of eIF4G1 may thus be involved in the pathogenesis of RA.     

Chronically inflamed synovium from spondyloarthropathy and rheumatoid arthritis investigated by protein expression profiling followed by tandem mass spectrometry

We investigated the cytosolic proteome of inflamed synovial tissue by hierarchical clustering analysis and validated the feasibility of this proteome analysis by identifying proteins that were differentially expressed between rheumatoid arthritis (RA), spondyloarthropathy (SpA), and osteoarthritis (OA). Synovial biopsy samples were obtained from 18 patients undergoing needle arthroscopy for knee synovitis associated with RA (n = 6) and SpA (n = 6), and for joint effusion of the knee associated with OA (n = 6). Cytosolic proteins were extracted from the tissue and subjected to two-dimensional gel electrophoresis. Protein expression patterns were statistically analyzed and used for hierarchical cluster analysis. Proteins of interest were independently identified by matrix-assisted laser desorption/ionization- and electrospray ionization-mass spectrometry. Hierarchical cluster analysis of the complete match set, containing 640 spots, remarkably segregated SpA from RA and OA. Next, we used a subset of spots that was statistically, differentially expressed (P < 0.01), between RA and SpA, SpA and OA, or RA and OA, in both Student's t-test and Mann-Whitney U-test. The dendrograms revealed distinct clustering of RA versus SpA and RA versus OA. Spots that were differentially expressed between the groups were identified by tandem mass spectrometry. Fructose bisphosphate aldolase A and alpha-enolase showed higher expression levels in SpA than in OA (P < 0.01). Calgranulin A myeloid related protein-8 (MRP-8) was markedly up-regulated in RA and SpA patients in comparison to OA patients where this spot was below detection limit. The analysis of the cytosolic proteome of synovial tissue is a useful approach to identify disease-associated proteins in chronic inflammatory arthritis.     

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.     

Identification of Autoantibodies against Transthyretin for the Screening and Diagnosis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic and inflammatory rheumatic disease that leads to inflammation of the joints and surrounding tissues. Identification of novel protein(s) associated with severity of RA is a prerequisite for better understanding of pathogenesis of this disease that may also have potential to serve as novel biomarkers in the diagnosis of RA. Present study was undertaken to compare the amount of autoantigens and autoantibodies in the plasma of RA patients in comparison to healthy controls. Plasma samples were collected from the patients suffering from RA, Osteoarthritis (OA), Systemic lupus erythematosus (SLE) and healthy volunteers. The screening of plasma proteins were carried out using 2-dimensional gel electrophoresis followed by identification of differentially expressed protein by MALDI-TOF MS/MS. Among several differentially expressed proteins, transthyretin (TTR) has been identified as one of the protein that showed significantly up regulated expression in the plasma of RA patients. The results were further validated by Western blot analysis and ELISA. In comparison to OA synovium, an exclusive significantly high expression of TTR in RA has been validated through IHC, Western blotting and IEM studies. Most importantly, the increase in expression of TTR with the progression of severity of RA condition has been observed. The autoantibodies against TTR present in the RA plasma were identified using immunoprecipitation-Western methods. The significant production of autoantibodies was validated by ELISA and Western blot analysis using recombinant pure protein of TTR. Hence, these novel observations on increase in TTR expression with the increase in severity of RA conditions and significant production of autoantibodies against TTR clearly suggest that a systematic studies on the role TTR in the pathogenesis of RA is immediately required and TTR may be used as a serum diagnostic marker together with other biochemical parameters and clinical symptoms for RA screening and diagnosis.     

High synovial expression of the inhibitory FcgammaRIIb in rheumatoid arthritis

Activating Fc gamma receptors (FcgammaRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcgammaRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcgammaRIIb isoform. FcgammaRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcgammaRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcgammaRI, FcgammaRII and FcgammaRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcgammaRII, FcgammaRIII but not FcgammaRI, all activating FcgammaRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcgammaRIIb and the activating FcgammaRs. Anti-inflammatory treatment with glucocorticoids reduced FcgammaR expression in arthritic joints, particularly that of FcgammaRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcgammaRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcgammaRIIb and activating FcgammaRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcgammaRs may represent valuable therapeutics in this disease.     

Exosome-encapsulated miR-6089 regulates inflammatory response via targeting TLR4

Exosome-encapsulated microRNAs (miRNAs) have been identified as potential biomarkers in autoimmune diseases. However, little is known about the role of exosome-delivered miRNAs in rheumatoid arthritis (RA). In this study, we investigated the profile of specific exosomal miRNAs by microarray analysis of serum exosomes from three patients with RA and three healthy controls. Quantitative real-time PCR (qRT-PCR) was performed to validate the aberrantly expressed exosomal miRNAs. A total of 20 exosome-encapsulated miRNAs were identified to be differently expressed in the serum of patients with RA compared with controls. Interestingly, we found that exosome-encapsulated miR-6089 was significantly decreased after validation by qRT-PCR in serum exosomes from 76 patients with RA and 20 controls. Besides, miR-6089 could inhibit lipopolysaccharide (LPS)-induced cell proliferation and activation of macrophage-like THP-1 cells. TLR4 was a direct target for miR-6089. MiR-6089 regulated the generation of IL-6, IL-29, and TNF-α by targetedly controlling TLR4 signaling. In conclusion, exosome-encapsulated miR-6089 regulates LPS/TLR4-mediated inflammatory response, which may serve as a novel, promising biomarker in RA.     

Extracellular mitochondrial DNA and oxidatively damaged DNA in synovial fluid of patients with rheumatoid arthritis

We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) contained extracellular mitochondrial DNA (mtDNA) or the oxidatively damaged DNA adduct 8-hydroxy-2'-deoxyguanosine (8-oxodG). Moreover, we correlated the laboratory findings of the patients with RA with their levels of mtDNA and 8-oxodG. SF and plasma samples from 54 patients with RA, SF from 30 non-arthritic control subjects, and plasma from 22 healthy volunteers were collected. The samples were subjected to polymerase chain reaction (PCR) using mitochondrial genomic primers, and the products were analyzed by SDS–polyacrylamide-gel electrophoresis. The intensities of the PCR-amplified bands were quantified and normalized to a reference sample. Furthermore, the SF samples were assayed by enzyme-linked immunosorbent assay for 8-oxodG. Extracellular PCR-amplifiable mtDNA was detected in the SF of 38 of 54 (70%) patients with RA, but not in any of the SF controls. PCR-amplifiable mtDNA was detected in the plasma of 30 of 54 (56%) of patients with RA and in 6 of 22 (27%) of the healthy volunteers. The levels of mtDNA in the plasma and SF samples of patients with RA were significantly higher (P < 0.0001) than in the respective control samples. The presence of both mtDNA and 8-oxodG in SF was significantly correlated with the presence of rheumatoid factor in the patients with RA. Extracellular mtDNA and oxidized DNA were detected in the SF of the great majority of patients with RA, but were absent or present at low levels in the control SF. These findings indicate that endogenous nucleic acid compounds might participate in joint inflammation by activating immune cells in the joints to produce proinflammatory cytokines.     

Differential expression of CD148 on leukocyte subsets in inflammatory arthritis

Introduction

Monocytic cells play a central role in the aetiology of rheumatoid arthritis, and manipulation of the activation of these cells is an approach currently under investigation to discover new therapies for this and associated diseases. CD148 is a transmembrane tyrosine phosphatase that is highly expressed in monocytes and macrophages and, since this family of molecules plays an important role in the regulation of cell activity, CD148 is a potential target for the manipulation of macrophage activation. For any molecule to be considered a therapeutic target, it is important for it to be increased in activity or expression during disease.

Methods

We have investigated the expression of CD148 in two murine models of arthritis and in joints from rheumatoid arthritis (RA) patients using real-time PCR, immunohistochemistry, and studied the effects of proinflammatory stimuli on CD148 activity using biochemical assays.

Results

We report that CD148 mRNA is upregulated in diseased joints of mice with collagen-induced arthritis. Furthermore, we report that in mice CD148 protein is highly expressed in infiltrating monocytes of diseased joints, with a small fraction of T cells also expressing CD148. In human arthritic joints both T cells and monocytes expressed high levels of CD148, however, we show differential expression of CD148 in T cells and monocytes from normal human peripheral blood compared to peripheral blood from RA and both normal and RA synovial fluid. Finally, we show that synovial fluid from rheumatoid arthritis patients suppresses CD148 phosphatase activity.

Conclusions

CD148 is upregulated in macrophages and T cells in human RA samples, and its activity is enhanced by treatment with tumour necrosis factor alpha (TNFα), and reduced by synovial fluid or oxidising conditions. A greater understanding of the role of CD148 in chronic inflammation may lead to alternative therapeutic approaches to these diseases.     

The major rheumatoid factor cross-reactive idiotype is dominantly expressed by Staphylococcus aureus Cowan strain I-activated CD5+ control B cells.

Some seropositive (RF+) and seronegative (RF-) rheumatoid arthritis (RA) patients selectively express high concentrations of the major RF cross-reactive idiotype (RCRI) in their sera and generate high frequencies of RCRI+ pokeweed mitogen (PWM)-induced plasma cells from their peripheral blood mononuclear cells (PBM). To determine if normal individuals can express RCRI in vitro, B cells from controls were activated with Staphylococcus aureus Cowan strain I (SAC) bacteria to identify RCRI and RF production. In addition, we studied the relationship of RCRI expression with the subset of B cells bearing CD5. Control CD5+ B cells are responsible for RCRI expression following SAC activation. We also observed that RCRI is dominantly expressed by control SAC-induced B cells in frequencies comparable to that expressed by some RA and juvenile rheumatoid arthritis patients' PBM activated by PWM. Therefore, the frequency of RCRI+ B cells in control and arthritis patients' PBL may be similar, or the selection and/or regulation of RCRI+ B-cell expression in vitro and in vivo may be different in arthritis patients compared to normal individuals.     

Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis

Introduction

Anti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.

Methods

We measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.

Results

The serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R² = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.

Conclusions

CitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.