Identification of Novel Autoantigen in the Synovial Fluid of Rheumatoid Arthritis Patients Using an Immunoproteomics Approach

Abstract

Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.

Clinical and immunological characteristics of the spectrum of GFAP autoimmunity: a case series of 22 patients

“…The recent identification of GFAP autoantibodies in the synovial fluid of patients with rheumatoid arthritis may support the hypothesis22 of a possible role of the GFAP autoimmunity in rheumatoid meningitis.…”

Synovial tissue research: a state-of-the-art review

“…72 Studies performed using this strategy first identified proteins that may be of interest in the synovial fluid, and then searched for antibodies to these proteins in the plasma. 73 It is possible that this methodology may be useful in future experiments of synovial tissue, and the results of such research may be more easily translated into clinical practice.…”

Identification of Novel Chondroprotective Mediators in Resolving Inflammatory Exudates

“…Herein, we progressed our investigation by utilizing a dual translational remit: 1) testing the identified factors against human arthritic synovial fluids, which allowed for assessment of their effects against the numerous catabolic and proinflammatory mediators, contained in the complex arthritic synovial fluids, which drive cartilage destruction in arthritis (21), and 2) in vivo administration of those proteins in a model of inflammatory arthritis. Of interest, and in support of our approach, recent proteomic analysis of human arthritic synovial fluids identified these proteins though without postulating relevant bioactions (33). AAT, GSN, and HX are acute phase proteins released by the liver in settings of systemic stress.…”

“…AAT is a 52-kDa glycoprotein functioning as the major natural inhibitor of serine proteases (34) with potent effects on neutrophil elastase (35,36). It is produced mainly by hepatocytes, yielding circulating steady-state levels of 1-3 mg/ml; these levels rise during infection and inflammation (33) and remain elevated for 7-10 d (37). Local inflammationdriven production of AAT by endothelial and myeloid cells is also reported (37).…”

“…In analogy to ATT, abundant GSN has been identified in human inflammatory fluids (33,52,55). HX (60 kDa) is primarily produced by hepatocytes, with moderate amounts also synthesized by the nervous system, skeletal muscle, and kidney (56).…”