Interferon-γ Contributes to HLA-B27-associated Unfolded Protein Response in Spondyloarthropathies

Objective. HLA-B27 positivity strongly influences the susceptibility to and phenotype of spondyloarthropathies (SpA). This study was designed to screen factors that activate the promoter of HLA-B27 in U937 cells, and to assess whether these promoter-activating factors induce the unfolded protein response (UPR) in HLA-B27-expressing cells. Methods. Cytometric Bead Array, flow cytometry, and real-time polymerase chain reaction were used to detect the expression of cytokines and UPR-associated proteins in peripheral blood and synovial fluid of patients with SpA. The HLA-B27 promotor transfectant was incubated separately with cytokines and Toll-like receptor ligands. After interferon-g (IFN-g) stimulation, expressions of GRP78, CHOP, and XBP-1 were tested in HLA-B27-expressing U937 cells and peripheral blood mononuclear cell (PBMC) of patients with ankylosing spondylitis (AS). (Clinical trial registration no. ChiCTR-OCC-11001565) Results. Expressions of GRP78, CHOP, and XBP-1 in monocytes/macrophages of SpA peripheral blood and synovial fluid were higher than those in healthy controls and patients with osteoarthritis (OA) (p < 0.05). Tumor necrosis factor-α (TNF-α) and IFN-α, IFN-ß, and IFN-g were found to have activated the HLA-B27 promoter in the U937 cell line (p < 0.05). Following stimulation with IFN-g, the expressions of GRP78, CHOP and XBP-1 in HLA-B27-transfected U937 cells and PBMC of HLA-B27-positive AS patients were more intense than those in A2-U937 cells, HLA-B27-negative AS patients, or healthy controls (p < 0.05). Conclusion. Expressions of GRP78, CHOP, and XBP-1 were higher in monocytes/macrophages of patients with SpA than those in both OA patients and healthy controls, suggesting that UPR may participate in the pathogenesis of SpA. TNF-α and IFN-α, IFN-ß, and IFN-g significantly activated HLA-B27 promoter in the U937 cell line, and IFN-g, the strongest activating factor, may induce the UPR in HLA-B27-expressing cells. (First Release Jan 15 2012; J Rheumatol 2012;39:574–82; doi:10.3899/ jrheum.101257) Key Indexing Terms: HLA-B27 SPONDYLOARTHROPATHY TUMOR NECROSIS FACTOR-α INTERFERON-g UNFOLDED PROTEIN RESPONSE From the Department of Clinical Immunology, State Key Discipline of Cell Biology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China. Y. Feng, PhD; J. Ding, PhD; C.M. Fan, BA; P. Zhu, MD, Professor, Department of Clinical Immunology, State Key Discipline of Cell Biology, Xijing Hospital, Fourth Military Medical University. Dr. Feng and Dr. Ding contributed equally to this work. Address correspondence to Dr. P. Zhu, Department of Clinical Immunology, State Key Discipline of Cell Biology, Xijing Hospital, Fourth Military Medical University, 15 Changlexi Road, Xi’an, Shaanxi Province, China. E-mail: zhuping@fmmu.edu.cn Accepted for publication October 17, 2011. Spondyloarthropathies (SpA) are a cluster of interrelated and overlapping chronic inflammatory rheumatic diseases that primarily include ankylosing spondylitis (AS), reactive arthritis (ReA), psoriatic arthritis (PsA), and inflammatory bowel disease arthritis. SpA commonly cause inflammation of the spine, eye, bowel, genital tract, or skin. SpA present striking familial aggregations and are typically associated with HLA genes, particularly HLA-B271,2. But it remains unclear how HLA-B27 contributes to arthritis. Different hypotheses have been formulated to explain this association: among them, the presentation of exogenous or self-peptides to HLA-B27+CD8+ T cells, the antigen presentation by HLA-B27 free heavy-chain homodimers, and HLA-B27 misfolding and the endoplasmic reticulum unfolded protein response (ER UPR)3. In addition, other mechanisms such as interaction of cell-surface dimers with killer cell Ig-like receptors (KIR) have been shown by Bowness, et al4. These hypotheses are all based on the activities of HLA-B27 protein. It is believed that in addition to certain other genes and/or environmental factors, the degree of expression of the HLA-B27 gene is also responsible for the susceptibility to SpA. Taurog, et al have shown that in HLA-B27*05 transgenic rats, the degree of susceptibility of various lines to the development of an SpA-like spectrum of lesions correlates with the level of HLA-B27 transgene expression at the mRNA and protein levels5. Cauli, et al reported that the expression of HLA-B27 molecule on the surface of peripheral blood mononuclear cells (PBMC) was higher in patients with AS than in HLA-B27-positive healthy volunteers6. However, very little research has been conducted to investigate HLA-B27 gene regulation. www.jrheum.org Downloaded on June 2, 2022 from 575 Feng, et al: IFN-g and UPR in SpA The findings that misfolded HLA-B277 can form aberrant disulfide-linked homodimers8 have brought forth some hypotheses concerning the role of HLA-B27 in the pathogenesis of SpA. One hypothesis is that HLA-B27 causes arthritis by inducing an “endoplasmic reticulum unfolded protein response”9. In an HLA-B27 transgenic rat model, it was proven that the UPR is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory diseases. The degree of HLA-B27 upregulation correlates with the magnitude of UPR activation, as shown using cytokines that upregulate HLA class I expression10,11. Gu, et al reported overexpression of a UPR target gene (BiP) in adherent synovial fluid (SF) mononuclear cells from active joints of HLA-B27-positive patients with SpA, in comparison with BiP expression in patients with rheumatoid arthritis12. These findings suggest that under certain circumstances, such as stimulation of some cytokines in SpA monocytes/macrophages, increased expression of HLA-B27 may result in HLA-B27 misfolding in the ER, which may in turn induce activation of the UPR. We investigated the expression of cytokines and UPR-related molecules in monocytes/macrophages of peripheral blood (PB) and SF of patients with active SpA. We wished to identify the factors that might be responsible for the transcription of HLA-B27 in human monocytic cells, and to assess whether any differences in UPR can be detected after incubation with promoter-activating factors in HLA-B27 whole-gene transfectants. MATERIALS AND METHODS Patients. Specimens of PB were obtained from 20 patients with active SpA (16 AS, 4 ReA), 10 patients with OA, and 10 healthy volunteer blood donors. Specimens of synovial fluid (SF) were taken at surgeries from 12 patients with SpA (10 AS, 2 ReA) and 10 with OA. Demographic characteristics of patients and controls are summarized in Table 1. Patients and controls were matched for number, age, sex, and disease duration. Patients were considered to have SpA and OA if they fulfilled the European Spondylarthropathy Study Group and American College of Rheumatology clinical criteria. Patients who received biological and systemic steroid therapy were excluded, and patients treated with dis ease-modifying antirheumatic drugs (DMARD) were included. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Function Index (BASFI) of all patients with SpA were > 4.0. Ethics approval was granted for this study and all subjects provided their informed consent. The clinical trial registration number is ChiCTR-OCC-11001565. Blood and SF specimens. All blood and SF samples were collected into collection tubes containing 0.2 ml sodium heparin. Mononuclear cells were obtained from PB and SF by Ficoll density gradient centrifugation method. Then, mononuclear cells were washed and resuspended in phosphate buffered saline (1% bovine serum albumin) at a concentration of 2 × 109/l. Plasma was obtained after centrifugation and stored at –80 ̊C. Plasmids. The pmaxFP-Yellow-PRL/B27-pro vector containing 432-bp promoter region of HLA-B2705 was kindly provided by D. Yu and colleagues, University of California Los Angeles (UCLA). The plasmid-containing genomic HLA-A2 was a gift from M. Shi, Fourth Military Medical University. The resulting recombinant plasmids were sequenced to ensure that only the intended alteration had been made. Yellow fluorescent proteins were used as reporter genes for pmaxFP-Yellow-PRL. Cell culture and transfectants. U937 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). To create stable cell lines expressing B27-pmaxFP-Yellow-PRL and HLA-A2, cells were transfected with expression vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and selected for with 0.8 mg/ml G418 (Invitrogen). Expression of HLA-A2 by transfectants was detected using the HLA-A2-specific antibody, MA2.1 (US Biological, Salem, MA, USA). Clonal and bulk cultures expressing high amounts of YFP and HLA-A2 were selected by flow cytometry and were used for experiments. The U937 cells transfected with human HLA-B2704 genomic DNA sequence were provided by D. Yu and colleagues, UCLA. Expression of HLA-B27 by transfectants was detected using the HLA-B27-specific antibody, ME1 (gift from Prof. D. Yu). These transfectants were designated the “promoter-B27-U937,” the “A2-U937,” and the “B27-U937.” Transfected cells were cultured in RPMI-1640 medium (Invitrogen) with 10% fetal calf serum at 37 ̊C in a humidified atmosphere containing 5% CO2. Media for the transfectants were supplemented with 0.2 mg/ml G418. 100 ng/ml interferon-g (IFN-g; Sigma, St. Louis, MO, USA) and 20 ng/ml tumor necrosis factor-α (TNF-α; eBioscience, San Diego, CA, USA) were used as stimulators to activate transfected U937 and PBMC of AS patients for 24 h and 8 h, respectively. The concentration and incubation time were optimized by pre-experiments. Cytokine assay. Cytokine profiles were investigated using separated serum samples. Serum was separated by centrifugation and stored in aliquots at –80 ̊C until analysis. Serum cytokine levels were investigated using Cytometric Bead Arrays (BD Biosciences, San Jose, CA, USA) and the levels of infla