Glaucomatous tissue stress and the regulation of immune response through glial Toll-like receptor signaling.

PURPOSE: To determine the regulation of immune system activity associated with Toll-like receptor (TLR) signaling in glaucoma. METHODS: Retinal protein samples obtained from human donor eyes with (n = 10) or without (n = 10) glaucoma were analyzed by a quantitative proteomic approach involving mass spectrometry. Cellular localization of TLR2, -3, and -4 was also determined by immunohistochemical analysis of an additional group of human donor eyes with glaucoma (n = 34) and control eyes (n = 20). In addition, in vitro experiments were performed in rat retinal microglia and astrocytes to determine glial TLR expression and immunoregulatory function after exposure to exogenous heat shock proteins (HSPs) and H(2)O(2)-induced oxidative stress. RESULTS: Proteomic analyses of the human retina detected expression and differential regulation of different TLRs in glaucomatous samples. Parallel to the upregulation of TLR signaling, proteomic findings were also consistent with a prominent increase in the expression of HSPs in glaucoma. Immunohistochemical analysis supported upregulated expression of TLRs on both microglia and astrocytes in the glaucomatous retina. In vitro experiments provided additional evidence that HSPs and oxidative stress upregulate glial TLR and MHC class II expression and cytokine production through TLR signaling and stimulate proliferation and cytokine secretion of co-cultured T cells during antigen presentation. CONCLUSIONS: The findings of this study support the upregulation of TLR signaling in human glaucoma, which may be associated with innate and adaptive immune responses. In vitro findings showed that components of glaucomatous tissue stress, including upregulated HSPs and oxidative stress, may initiate the immunostimulatory signaling through glial TLRs.

Oxidative stress and the regulation of complement activation in human glaucoma.

PURPOSE: As part of ongoing studies on proteomic alterations during glaucomatous neurodegeneration, this study focused on the complement system. METHODS: Human retinal protein samples obtained from donor eyes with (n = 10) or without (n = 10) glaucoma were analyzed by a quantitative proteomic approach using mass spectrometry. Cellular localization of protein expression for different complement components and regulators were also determined by immunohistochemical analysis of an additional group of human donor eyes with glaucoma (n = 34) compared with age-matched control eyes without glaucoma (n = 20). In addition, to determine the regulation of complement factor H (CFH) by oxidative stress, in vitro experiments were performed using rat retinal cell cultures incubated in the presence and absence of an oxidant treatment. RESULTS: Proteomic analysis detected the expression and differential regulation of several complement components in glaucomatous samples, which included proteins involved in the classical and the lectin pathways of complement activation. In addition, several complement regulatory proteins were detected in the human retinal proteome, and glaucomatous samples exhibited a trend toward downregulation of CFH expression. In vitro experiments revealed that oxidative stress, which was also prominently detectable in the glaucomatous human retinas, downregulated CFH expression in retinal cells. CONCLUSIONS: These findings expand the current knowledge of complement activation by presenting new evidence in human glaucoma and support that despite important roles in tissue cleaning and healing, a potential deficiency in intrinsic regulation of complement activation, as is evident in the presence of oxidative stress, may lead to uncontrolled complement attack with neurodestructive consequences.

Hemoglobin expression and regulation in glaucoma: insights into retinal ganglion cell oxygenation.

PURPOSE: To determine expression, cellular distribution, and regulation of hemoglobin (Hb) in normal and glaucomatous tissues. METHODS: Proteomic analysis of Hb expression was conducted on protein samples from ocular hypertensive and control rat eyes and human donor eyes with or without glaucoma. Proteomic findings were validated by quantitative (q)RT-PCR, Western blot analysis, immunohistochemistry, and the analysis of new Hb synthesis in culture. Hypoxic regulation of Hb expression was also studied in primary cultures of rat RGCs and macroglia and after transfer of the glia-conditioned medium to RGCs. The role of erythropoietin (EPO) signaling in Hb induction and cell survival was determined by applying recombinant (r)EPO treatment and performing EPO neutralization experiments by using soluble EPO receptor treatment of hypoxic cultures. RESULTS: In vivo findings revealed Hb expression in the retina and optic nerve head macroglia and RGCs, suggesting an approximately two-fold upregulation in ocular hypertensive rat eyes and glaucomatous human donor eyes relative to the control eyes. In vitro findings collectively supported that hypoxia boosts glial Hb expression through hypoxia-inducible EPO signaling in an autocrine manner. Based on passive transfer experiments, hypoxia-induced production of glial EPO was also found to upregulate Hb expression in RGCs in a paracrine manner, thereby increasing the hypoxic survival of these neurons. CONCLUSIONS: Findings of this study provide new insights into tissue oxygen transport in the inner retina and optic nerve head through the regulated expression of Hb in macroglia and RGCs. Upregulation of Hb expression appears to be an intrinsic protective mechanism to facilitate cellular oxygenation and may also provide free radical scavenging.