Lingli Yang1, Minoru Fujimoto2, Hiroyuki Murota2, Satoshi Serada2, Manabu Fujimoto2, Hiromi Honda2, Kohji Yamada1, Katsuya Suzuki2, Ayumi Nishikawa2, Yuji Hosono2, Yoshihiro Yoneda2, Kazuhiko Takehara2, Yoshitaka Imura2, Tsuneyo Mimori2, Tsutomu Takeuchi2, Ichiro Katayama2, Tetsuji Naka3. 1. Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan. Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan. 2. Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan. 3. Department of Dermatology, Osaka University Graduate School of Medicine, Laboratory of Immune Signal, National Institute of Biomedical Innovation, Department of Dermatology, Kanazawa University, Kanazawa, Biomolecular Dynamics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto and National Institute of Biomedical Innovation, Osaka, Japan. tnaka@nibio.go.jp.
Abstract
OBJECTIVE: The aim of this study was to identify cold-associated autoantibodies in patients with RP secondary to CTDs. METHODS: Indirect immunofluorescence staining was performed on non-permeabilized cold-stimulated normal human dermal microvascular endothelial cells (dHMVECs), using patients' sera. Cold-induced alterations in cell surface proteomes were analysed by isobaric tag for relative and absolute quantitation (iTRAQ) analysis. Serological proteome analysis (SERPA) was applied to screen cold-associated autoantigens. The prevalence of the candidate autoantibody was determined by ELISA in 290 patients with RP secondary to CTDs (SSc, SLE or MCTD), 10 patients with primary RP and 27 healthy controls. RESULTS: Enhanced cell surface immunoreactivity was detected in cold-stimulated dHMVECs when incubated with sera from patients with secondary RP. By iTRAQ analysis, many proteins, including heterogeneous nuclear ribonucleoprotein K (hnRNP-K), were found to be increased on the cell surface of dHMVECs after cold stimulation. By the SERPA approach, hnRNP-K was identified as a candidate autoantigen in patients with secondary RP. Cold-induced translocation of hnRNP-K to the cell surface was confirmed by immunoblotting and flow cytometry. By ELISA analysis, patients with secondary RP show a significantly higher prevalence of anti-hnRNP-K autoantibody (30.0%, 61/203) than patients without RP (9.2%, 8/87, P = 0.0001), patients with primary RP (0%, 0/10, P = 0.0314) or healthy controls (0%, 0/27, P = 0.0001). CONCLUSION: By comprehensive proteomics, we identified hnRNP-K as a novel cold-associated autoantigen in patients with secondary RP. Anti-hnRNP-K autoantibody may potentially serve as a biomarker for RP secondary to various CTDs.
OBJECTIVE: The aim of this study was to identify cold-associated autoantibodies in patients with RP secondary to CTDs. METHODS: Indirect immunofluorescence staining was performed on non-permeabilized cold-stimulated normal human dermal microvascular endothelial cells (dHMVECs), using patients' sera. Cold-induced alterations in cell surface proteomes were analysed by isobaric tag for relative and absolute quantitation (iTRAQ) analysis. Serological proteome analysis (SERPA) was applied to screen cold-associated autoantigens. The prevalence of the candidate autoantibody was determined by ELISA in 290 patients with RP secondary to CTDs (SSc, SLE or MCTD), 10 patients with primary RP and 27 healthy controls. RESULTS: Enhanced cell surface immunoreactivity was detected in cold-stimulated dHMVECs when incubated with sera from patients with secondary RP. By iTRAQ analysis, many proteins, including heterogeneous nuclear ribonucleoprotein K (hnRNP-K), were found to be increased on the cell surface of dHMVECs after cold stimulation. By the SERPA approach, hnRNP-K was identified as a candidate autoantigen in patients with secondary RP. Cold-induced translocation of hnRNP-K to the cell surface was confirmed by immunoblotting and flow cytometry. By ELISA analysis, patients with secondary RP show a significantly higher prevalence of anti-hnRNP-K autoantibody (30.0%, 61/203) than patients without RP (9.2%, 8/87, P = 0.0001), patients with primary RP (0%, 0/10, P = 0.0314) or healthy controls (0%, 0/27, P = 0.0001). CONCLUSION: By comprehensive proteomics, we identified hnRNP-K as a novel cold-associated autoantigen in patients with secondary RP. Anti-hnRNP-K autoantibody may potentially serve as a biomarker for RP secondary to various CTDs.