Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immuneprivileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Mü ller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis. Molecular & Cellular Proteomics 9: 2292-2305, 2010.

Identification of biomarkers is of critical relevance toward improving diagnosis and therapy of autoimmune disorders. Serum markers are a desirable choice as sera are easily accessible and the development of assays for routine clinical detection prompts feasible. Autoimmune uveitis, a recurrent disease affecting the eye, is characterized by returning inflammatory attacks of the inner eye followed by variable periods of quiescent stages. Spontaneous equine recurrent uveitis (ERU) is the equine equivalent and serves as a model for the human disease. To identify potential biomarker candidates, we first systematically compared the proteomes of individual ERU cases with healthy controls by proteomic profiling using 2-D difference-gel-electrophoresis (2-D DIGE) followed by tandem mass spectrometry. A total of seven differentially expressed proteins were identified. Besides the upregulation of IgG and the significant lower expression of albumin, Antithrombin III, and Vitamin D binding protein, we found complement components C1q and C4, to be downregulated in uveitic state. Interestingly, Pigment epithelium-derived factor (PEDF), a marker already detected by 2DE differential proteome analysis in ERU target tissues, vitreous and retina, was found to be also significantly downregulated in sera. The lower expression of PEDF in sera of horses with uveitis could be verified in a cohort of 116 ERU cases and 115 healthy controls. Our findings of a significant lower PEDF expression in ERU cases also in the periphery of the eye proves PEDF as a promising uveitis biomarker.

Since neovascularization plays a major role in the pathogenesis of uveitis, the identification of a proangiogenic factor in the retina presents an important finding and may contribute to elucidating the pathogenesis of uveitis.

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