ARTHRITIS & RHEUMATISM Vol. 65, No. 4, April 2013, pp 869–879 DOI 10.1002/art.37881 © 2013, American College of Rheumatology Identification of Citrullinated Hsp90 Isoforms as Novel Autoantigens in Rheumatoid Arthritis–Associated Interstitial Lung Disease Lisa Harlow, 1 Ivan O. Rosas, 2 Bernadette R. Gochuico, 3 Ted R. Mikuls, 4 Paul F. Dellaripa, 2 Chester V. Oddis, 5 and Dana P. Ascherman 1 Objective. Subsets of patients with rheumatoid arthritis (RA) develop extraarticular complications that include interstitial lung disease (ILD). Because stan- dardized algorithms for identification of RA patients at risk of developing clinically significant ILD are lacking, the purpose of this study was to elucidate unique serologic markers of RA-associated ILD (RA-ILD). Methods. Sera from RA patients with ILD and from RA patients without ILD were used to immunopre- cipitate citrullinated and uncitrullinated proteins de- rived from K562 cell extracts. Mass spectrometry was performed to facilitate identification of citrullinated proteins differentially immunoprecipitated by RA-ILD patient sera. These candidate proteins were then used as substrate antigens in custom enzyme-linked immu- nosorbent assays (ELISAs) for high-throughput screen- ing of sera obtained from cohorts of patients with RA, RA-ILD mixed connective tissue disease (MCTD), or idiopathic pulmonary fibrosis (IPF). Results. Differential immunoprecipitation and subsequent mass spectrometric sequencing identified citrullinated Hsp90 and citrullinated Hsp90 as can- didate autoantigens in patients with RA-ILD. ELISAs incorporating uncitrullinated and citrullinated isoforms of Hsp90 as substrate antigens demonstrated that sera from patients with RA-ILD preferentially recognized citrullinated versions of Hsp90 with moderate sensitiv- ity (range 20–30%) and great specificity (>95%) relative to sera derived from patients with RA alone (without ILD), patients with MCTD, or patients with IPF. Conclusion. These studies demonstrate the utility of a novel autoantigen discovery method based on differential immunoprecipitation of citrullinated pro- tein extracts. Application of these techniques identified citrullinated versions of Hsp90 and Hsp90 as auto- antibody targets distinguishing RA-ILD from RA with- out ILD, MCTD, and IPF, suggesting that anti– citrullinated Hsp90/ autoantibodies may serve as effective biomarkers for the potentially devastating pul- monary manifestations of RA-ILD. Rheumatoid arthritis (RA) is the most common systemic autoimmune disease in the United States, affecting 1% of the adult population (1,2). While articular complications represent a dominant feature of this disease, extraarticular manifestations often involve additional tissues that include the skin, eye, vasculature, peripheral nervous system, heart, and lungs. Within the spectrum of pulmonary complications, parenchymal in- volvement in the form of interstitial lung disease (ILD) represents a serious problem that can significantly im- The authors take full responsibility for the contents of this article, which do not represent the views of the Department of Veterans Affairs or the United States Government. Supported in part by the Intramural Research Program of the National Human Genome Research Institute, NIH. Dr. Oddis’ work was supported by a Within Our Reach grant from the Rheuma- tology Research Foundation of the American College of Rheuma- tology. Dr. Ascherman’s work was supported by a Within Our Reach grant from the Rheumatology Research Foundation of the American College of Rheumatology and a VA Merit Review grant (1I01BX0007880). 1 Lisa Harlow, BS, Dana P. Ascherman, MD: University of Miami Miller School of Medicine, Miami, Florida; 2 Ivan O. Rosas, MD, Paul F. Dellaripa, MD: Brigham and Women’s Hospital, Boston, Massachusetts; 3 Bernadette R. Gochuico, MD: National Human Ge- nome Research Institute, NIH, Bethesda, Maryland; 4 Ted R. Mikuls, MD, MSPH: Omaha VA Medical Center, Omaha, Nebraska; 5 Chester V. Oddis, MD: University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. Address correspondence to Dana P. Ascherman, MD, De- partment of Medicine, Division of Rheumatology, University of Miami Miller School of Medicine, RMSB 7152, 1600 NW 10th Avenue, Miami, FL 33136. E-mail: DAscherman@med.miami.edu. Submitted for publication September 5, 2012; accepted in revised form January 17, 2013. 869