Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype

Abstract

The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-kappaB, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eye's aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.

Fig. 1

Immunohistochemical screen for CAMs altered in outflow pathways of glaucomatous eyes. Cross-sections through the angle of the anterior chamber of glaucomatous or normal eyes (left panels), or subcultured TM-cell lines from glaucomatous or normal eyes (right panels) were stained with N-CAM-16 or ELAM-1 antibody probes. Note the strong staining of Schlemm’s canal (arrow) and the surrounding TM (arrowhead) in glaucomatous eyes and TM cells from glaucomatous eyes. Tissue sections were negative for the inflammation marker LFA-1 and the fibrosis marker HPCA-2, and both tissue sections and cultured cells were positive for the TM cell marker HLA class I antigen (data not shown).

Fig. 2

Regulation of ELAM-1 expression by exogenous and endogenous IL-1. Normal and glaucomatous TM cells were plated into replicate cultures and were untreated (−) or treated with IL-1 for 2 h (+). In some experiments, cells were also treated with receptor antagonist (IL-1ra) for 24 h. a, Northern blotting with ELAM-1 cDNA (ref. 42). The GAPDH probe served to determine the equality of RNA loading among lanes. b, RT-PCR analysis for IL-1α, IL-1β and IL-6 mRNA. The cDNA for GAPDH was amplified in parallel reactions to assess cDNA-loading equivalence among samples. c, Indirect immunofluorescent staining with antibody to ELAM-1. NTM: normal TM cells. GTM-A: glaucomatous TM cells. GTM-B: glaucomatous TM cells, specimen-B. GTM-C: glaucomatous TM cells, specimen-C.

Fig. 3

NF-κB activity in normal and glaucomatous TM cells. a, TM cells isolated from normal and glaucomatous eyes were left untreated (−) or treated with IL-1 for 2 h (+). EMSA was performed. Arrowhead indicates the migration of NF-κB protein–DNA complex. Specificity is indicated by supershift analysis with antibodies to the p50 or p65 subunits of NF-κB and competition analysis with ‘cold’ NF-κB probe (1:50). b, EMSA from TM cells left untreated (−), treated with IL-1 for 2 h, or treated overnight with IL-1ra. Specificity was demonstrated using a control probe corresponding to the E-box-like element in the human IL-1α gene promoter. Binding to this probe was unaffected by treatment with either IL-1 or IL-1ra (data not shown). c, Indirect immunofluorescent staining with antibody to ELAM-1.

Fig. 4

Active NF-κB in glaucomatous TM cells in situ. Double-label immunofluorescent localization was performed using antibody that binds ELAM-1 and antibody against the p65 nuclear localization signal, which binds only the activated form of this subunit free of I-κB. Sections were further stained with Hoechst dye to visualize nuclei. ‘Triple’ indicates the overlap of all images. Magnification: ×40.

Fig. 5

Resistance of normal and glaucomatous trabecular meshwork cells to apoptosis in response to an oxidant, and protective role of IL-1 and NF-κB. Normal TM cells or glaucomatous cells were treated with indicated concentrations of the oxidant tBH for 6 hours and stained with fluorescein-TUNEL assay. Groups of cells were pre-treated with IL-1 (10μg/ml), IL-1ra (500 μg/ml) or SN-50 peptide (NF-κB antagonist, 50 μg/ml) then assayed for the apoptotic response. Cells were also stained with propidium iodide to stain the nucleus. Arrows indicate apoptotic cells which stain yellow because of the colocalization of TUNEL staining and propidium iodide.