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Stress Proteins and Immunostimulatory Signaling Through Toll-Like Receptors in Glaucoma

^AOphthalmology & Visual Sciences, ^BBiochemistry & Molecular Biology, ^CMedicine, ^DAnatomical Sciences & Neurobiology, ¹University of Louisville, Louisville, Kentucky

Commercial Relationships: C. Luo, None; X. Yang, None; D.W. Powell, None; J.B. Klein, None; G. Tezel, None.

Support: NEI grants, 2R01EY013813, 1R01EY017131, and R24EY015636, and RPB

Abstract

Purpose:Our ongoing studies aim to elucidate mechanisms of immune dysregulation in glaucoma. Based on known immunoregulatory abilities of heat shock proteins (HSPs) via toll-like receptors (TLRs), this study aimed to determine the expression and regulation of these molecules during glaucomatous neurodegeneration.

Methods:Intraocular pressure elevation was induced in rats by hypertonic saline injections into episcleral veins. Alterations in protein expression were repeatedly determined in tryptic digests of retinal protein samples through 2-dimensional capillary liquid chromatography coupled with tandem mass spectrometry. Bioinformatic analysis using ARIADNE Pathway Studio and Ingenuity Pathways Analysis systems defined functional pathways. Quantitative Western blot analysis was utilized to validate proteomic findings and immunohistochemical analysis determined cellular localization of TLRs (TLR2, 3, and 4) and HSPs (HSP27, 60, and 72) in histological sections of the retina and optic nerve head obtained from human donor eyes with (n:34) or without glaucoma (n:20). Furthermore, in vitro experiments determined retinal microglial TLR expression and immunoregulatory function after exposure to exogenous HSPs.

Results:Mass spectrometric analyses and tissue immunolabeling consistently detected that parallel to prominent up-regulation of HSPs (over two-fold), expression of TLRs (TLR2, TLR3, and TLR4) is increased in ocular hypertensive rat eyes and glaucomatous human donor eyes relative to controls. Bioinformatic pathway analysis of the high-throughput comparative mass spectral data linked many adaptor proteins and kinases identified in the retinal proteome to TLR signaling leading to cytokine production, cell death, and immune response during glaucomatous neurodegeneration. Findings of in vitro experiments provided supportive evidence that microglial TLR and MHC class II expression and cytokine production is regulated by HSPs, and glial antigen presentation is stimulated by exposure to exogenous HSPs as assessed by increased proliferation and cytokine production of co-cultured T cells.

Conclusions:Findings of this study support that TLR signaling is involved in the activation of innate immune defense and antigen-specific adaptive immunity in glaucoma. Besides neuroprotective abilities of intracellular HSPs, membrane-bound HSPs or extracellular HSPs upon active secretion by stressed cells and/or passive release from dying neurons in glaucoma, may also serve as an immunostimulatory signal through TLRs.

Keywords: proteomics • glia • immunomodulation/immunoregulation

© 2009, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.