Abstract
Purpose: :
To determine the uveitogenic potential of malate dehydrogenase (MDH), a novel autoantigen detected in equine recurrent uveitis (ERU) in Lewis rats and in horses in order to investigate the pathological relevance of this autoantigen.
Methods: :
We have identified MDH as a novel autoantigen in spontaneous equine recurrent uveitis (ERU) by 2DE Western blots. Frequency of anti-MDH autoantibodies in ERU cases and healthy controls was measured with an enzyme linked immunosorbent assay, using 5µg/ml MDH (Calbiochem) for coating of the plates (Nunc Maxisorp). Sera were diluted 1:4000 in PBS-T. A polyclonal, chicken anti-MDH antibody (self-made) was used as a positive control. Binding of sera to MDH was visualized with an anti-horse-IgG-POD antibody and TMB detection. T cell assays were performed using 5 µg MDH per well as stimulatory antigen in in vitro proliferation assays. Cells were harvested after 5 days of stimulation and pulsed for the last 18 hours with 3H thymidine. To complete the characterization of MDH as an uveitis autoantigen, we tested the uveitogenic potential of 100 µg MDH per rat in 14 Lewis rats and 500 µg MDH per horse in 4 horses.
Results: :
In a large scale study, no difference in the autoantibody frequency to MDH was detectable between ERU cases and controls. 6.1% of tested ERU cases were anti-MDH-autoantibody positive (17 out of 277) and 6.5% of healthy controls (5 out of 77). Lymphocytes of 43% tested ERU cases were positive to in vitro stimulation with MDH, whereas none of the healthy controls reacted positive. Uveitis incidence was 10 out of 14 MDH-immunized rats. MDH-induced uveitis in the rat was characterized by focal destruction of photoreceptor outer segments and massive infiltration of the retina, mainly driven by T-cells. In contrast, injections with MDH failed to induce uveitis in all four tested horses despite formation of considerable anti-MDH autoantibody titers and a positive T cell response to MDH stimulation in vitro after immunization with MDH.
Conclusions: :
Our findings suggest that the proteomic approach for autoantibody profiling of various autoimmune diseases is a convenient and effective tool for detecting autoantigens. Further investigations are needed to explain the discrepancy of the pathological potential of MDH in Lewis rats and horses.
Keywords: uveitis-clinical/animal model • autoimmune disease