Maral Tajerian1, Victor Hung2, Hamda Khan2, Lauren J Lahey3, Yuan Sun4, Frank Birklein5, Heidrun H Krämer6, William H Robinson3, Wade S Kingery7, J David Clark4. 1. Veterans Affairs Palo Alto Health Care System Palo Alto, CA, USA; Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA, USA; Palo Alto Veterans Institute for Research, Palo Alto, CA, USA. Electronic address: maral@stanford.edu. 2. Veterans Affairs Palo Alto Health Care System Palo Alto, CA, USA; Palo Alto Veterans Institute for Research, Palo Alto, CA, USA. 3. Veterans Affairs Palo Alto Health Care System Palo Alto, CA, USA; Palo Alto Veterans Institute for Research, Palo Alto, CA, USA; Division of Immunology and Rheumatology, Stanford University, Stanford, CA, USA. 4. Veterans Affairs Palo Alto Health Care System Palo Alto, CA, USA; Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA, USA; Palo Alto Veterans Institute for Research, Palo Alto, CA, USA. 5. Department of Neurology, University Medical Center, Mainz, Germany. 6. Department of Neurology, Justus Liebig University, Giessen, Germany. 7. Veterans Affairs Palo Alto Health Care System Palo Alto, CA, USA; Palo Alto Veterans Institute for Research, Palo Alto, CA, USA; Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA.
Abstract
OBJECTIVE: Using a mouse model of complex regional pain syndrome (CRPS), our goal was to identify autoantigens in the skin of the affected limb. METHODS: A CRPS-like state was induced using the tibia fracture/cast immobilization model. Three weeks after fracture, hindpaw skin was homogenized, run on 2-d gels, and probed by sera from fracture and control mice. Spots of interest were analyzed by liquid chromatography-mass spectroscopy (LC-MS) and the list of targets validated by examining their abundance and subcellular localization. In order to measure the autoantigenicity of selected protein targets, we quantified the binding of IgM in control and fracture mice sera, as well as in control and CRPS human sera, to the recombinant protein. RESULTS: We show unique binding between fracture skin extracts and fracture sera, suggesting the presence of auto-antigens. LC-MS analysis provided us a list of potential targets, some of which were upregulated after fracture (KRT16, eEF1a1, and PRPH), while others showed subcellular-redistribution and increased membrane localization (ANXA2 and ENO3). No changes in protein citrullination or carbamylation were observed. In addition to increased abundance, KRT16 demonstrated autoantigenicity, since sera from both fracture mice and CRPS patients showed increased autoantibody binding to recombinant kRT16 protein. CONCLUSIONS: Pursuing autoimmune contributions to CRPS provides a novel approach to understanding the condition and may allow the development of mechanism-based therapies. The identification of autoantibodies against KRT16 as a biomarker in mice and in humans is a critical step towards these goals, and towards redefining CRPS as having an autoimmune etiology.
OBJECTIVE: Using a mouse model of complex regional pain syndrome (CRPS), our goal was to identify autoantigens in the skin of the affected limb. METHODS: A CRPS-like state was induced using the tibia fracture/cast immobilization model. Three weeks after fracture, hindpaw skin was homogenized, run on 2-d gels, and probed by sera from fracture and control mice. Spots of interest were analyzed by liquid chromatography-mass spectroscopy (LC-MS) and the list of targets validated by examining their abundance and subcellular localization. In order to measure the autoantigenicity of selected protein targets, we quantified the binding of IgM in control and fracturemice sera, as well as in control and CRPShuman sera, to the recombinant protein. RESULTS: We show unique binding between fracture skin extracts and fracture sera, suggesting the presence of auto-antigens. LC-MS analysis provided us a list of potential targets, some of which were upregulated after fracture (KRT16, eEF1a1, and PRPH), while others showed subcellular-redistribution and increased membrane localization (ANXA2 and ENO3). No changes in protein citrullination or carbamylation were observed. In addition to increased abundance, KRT16 demonstrated autoantigenicity, since sera from both fracturemice and CRPSpatients showed increased autoantibody binding to recombinant kRT16 protein. CONCLUSIONS: Pursuing autoimmune contributions to CRPS provides a novel approach to understanding the condition and may allow the development of mechanism-based therapies. The identification of autoantibodies against KRT16 as a biomarker in mice and in humans is a critical step towards these goals, and towards redefining CRPS as having an autoimmune etiology.
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