ABSTRACT
Autoimmune uveitis is characterized by crossing of blood-retinal barrier (BRB) by autoaggressive immune cells. Equine recurrent uveitis (ERU) is a valuable spontaneous model for autoimmune uveitis and analyses of differentially expressed proteins in ERU unraveled changed protein clusters in target tissues and immune system. Healthy eyes are devoid of leukocytes. In ERU, however, leukocytes enter the inner eye and subsequently destroy it. Molecular mechanisms enabling cell migration through BRB still remain elusive. Previously, we detected decreased talin 1 expression in blood-derived granulocytes of ERU cases, linking the innate immune system to ERU. Because changes in leukocyte protein expression pattern may play a role in pathological abnormalities leading to migration ability, we aimed at identifying interactors of talin 1 in leukocytes with immunoprecipitation, followed by LC-MS/MS for candidate identification. This enabled us to identify CD90 (Thy1) as novel interactor of talin 1 besides several other interactors. In blood-derived granulocytes from healthy individuals, CD90 was highly abundant and significantly reduced in ERU, especially in effector cells. Connection between talin 1 and CD90 and their expression differences in inflammation is an interesting novel finding allowing deeper insight into immune response of innate immune system and granulocyte migration ability in this organ-specific autoimmune disease.
Subject(s)
Granulocytes/metabolism , Horse Diseases/genetics , Talin/genetics , Thy-1 Antigens/genetics , Uvea/metabolism , Uveitis/veterinary , Animals , Autoantibodies/biosynthesis , Autoimmune Diseases , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Blood-Retinal Barrier , Case-Control Studies , Cell Movement , Chromatography, Liquid , Gene Expression Regulation , Granulocytes/immunology , Granulocytes/pathology , Horse Diseases/immunology , Horse Diseases/metabolism , Horse Diseases/pathology , Horses , Immunoprecipitation , Mass Spectrometry , Molecular Sequence Annotation , Protein Binding , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Talin/immunology , Talin/metabolism , Thy-1 Antigens/immunology , Thy-1 Antigens/metabolism , Uvea/immunology , Uvea/pathology , Uveitis/immunology , Uveitis/metabolism , Uveitis/pathologyABSTRACT
The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.
