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M.B. Wax, G. Tezel, J. Yang, R. Patil, R. Sappington, D. Calkins; Induced Autoimmunity to Heat Shock Proteins Elicits Glaucomatous Loss of Retinal Ganglion Cells via a FAS/FAS–Ligand Pathway . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1828.
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© ARVO (1962-2015); The Authors (2016-present)
Few autoimmune diseases have fulfilled one of Koch’s key postulates; namely, that the disease can be induced by the suspect agent. In the case of autoimmunity, the agent is antigen innoculation. Here we sought to elicit experimental glaucoma in rats immunized by heat shock protein (HSP) and identify a mechanism by which RGCs die in the presence of HSP–sensitized T–cells in vitro.
We immunized Lewis rats with HSP60 or HSP27 and quantified RGC loss in flat–mount preparations of the retina and of RGC axons in cross–sections of the ONH. We also performed in vitro co–culture studies using a rat RGC cell line (RGC–5) and T–cells isolated from HSP–immunized rats to determine cell fate resulting from their interactions. We measured sFasL levels in conditioned media from activated T–cells using ELISA and performed experiments to validate that sFasL was a causative mediator of the RGC toxicity.
HSP immunization resulted in the glaucomatous loss of RGCs and their axons. Utilizing Brn3a immunoreactivity as an indicator of RGC loss, we found that HSP60 or HSP27 immunization significantly (p<.01) reduced the number of RGCs in a spatially specific manner in vivo in a pattern consistent with human glaucoma. In comparison to control animals, a 25% reduction in RGCs occurred for HSP60–immunized animals at 2–4 mm from the ONH. This loss corresponded to a loss nearly 15,000 axons in the HSP60–immunized animals. We further identified the mechanism in vitro, by which T–cells activated by HSP–immunization induce RGC apoptosis; namely, via the release of the inflammatory cytokine Fas–L. In the presence of T–cells activated by HSP60 or HSP27 immunization, RGC apoptosis as measured by flow cytometry was 24.9 + 1.8 % and 21.8 % + 2.1, respectively. In control co–cultures virtually no apoptosis was detectable (p<.01). Conditioned medium obtained from HSP60–activated T–cells had a ∼20 higher level of sFasL compared to the medium obtained from HSP27–activated or control T–cells (p<.01). Incubation of RGC–5 in the presence of increasing concentrations of recombinant human FasL (25–200 ng/ml) for 5 h resulted in RGC death in a dose–dependent manner.
We propose that the RGC death in selected patients with glaucoma likely involves failed immunoregulation of the microglia/T–cell/RGC axis and is thus a disturbance of both pro–apoptotic and protective pathways.
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