Martin B. Wax

Plasma and Aqueous Humor Endothelin Levels in Primary Open-Angle Glaucoma

Purpose:Endothelins (ET) have some effects on the regulation of aqueous humor dynamics. To investigate their possible role in glaucoma, we measured plasma and aqueous humor ET levels in patients with and without primary open-angle glaucoma. Methods:Plasma and aqueous humor samples were obtained from 31 patients with primary open-angle glaucoma and 24 patients without glaucoma. Measurements were made by radioimmunoassay (RIA) for ETs with the following crossreactivities: ET-1, ET-2, and big ET-1, 100%; and ET-3, 70%. Results:The ages (mean ± SD) of the patients with primary open-angle glaucoma (72.3 ± 10 years) and normal subjects (72.8 ± 8 years) were similar (p = 0.92). There was no significant difference between plasma ET levels of the two groups (p = 0.07). However, aqueous humor ET levels (mean ± SD) were higher in the primary open-angle glaucoma group (44.26 ± 2.6 pg ml-1) than in normal subjects (42.17 ± 1.6 pg ml-1) (p = 0.001). The ratios of corresponding aqueous humor to plasma levels of ETs were ∼10% higher in the primary open-angle glaucoma group (3.76) than in normal subjects (3.41) (p = 0.0002). Conclusions:The small increase in aqueous humor endothelin levels in patients with primary open-angle glaucoma versus controls of similar age may be relevant to the understanding of the various roles of ETs in aqueous humor dynamics in these patients.

Regulation of Aquaporin-4 Water Channels by Phorbol Ester-dependent Protein Phosphorylation

The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidney, the eye contains multiple water channel proteins (aquaporins) that transport water through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. However, because humans with defective AQP1 are phenotypically normal and because the ocular application of phorbol esters reduce intraocular pressure, we postulated that the water channel activity of AQP4 may be regulated by these agents. We now report that protein kinase C activators, phorbol 12,13-dibutyrate, and phorbol 12-myristate 13-acetate strongly stimulate the phosphorylation of AQP4 and inhibit its activity in a dose-dependent manner. Phorbol 12,13-dibutyrate (10 μm) and phorbol 12-myristate 13-acetate (10 nm) reduced the rate of AQP4-expressing oocyte swelling by 87 and 92%, respectively. Further, phorbol 12,13-dibutyrate significantly increased the amount of phosphorylated AQP4. These results demonstrate that protein kinase C can regulate the activity of AQP4 through a mechanism involving protein phosphorylation. Moreover, they suggest important potential roles for AQP4 in several clinical disorders involving rapid water transport such as glaucoma, brain edema, and swelling of premature infant lungs.

Anti-Ro/SS-a positivity and heat shock protein antibodies in patients with normal-pressure glaucoma

PURPOSE To describe the clinical and laboratory findings in 10 patients with normal-pressure glaucoma and anti-Ro/SS-A positivity by enzyme-linked immunosorbent assay (ELISA) and to determine whether that positivity may be related to an autoimmune mechanism for the optic neuropathy. METHODS In this prospective study, we evaluated ocular and systemic clinical findings of 10 patients with normal-pressure glaucoma and anti-Ro/SS-A positivity by ELISA, including sicca complex features. Oüchterlony immunodiffusion was performed to confirm the presence of antibodies for Ro/SS-A, and the presence of other serum antibodies and their possible cross-reactivities with Ro/SS-A were investigated. RESULTS None of the 10 patients with normal-pressure glaucoma and anti-Ro/SS-A positivity (by ELISA) had clinical or laboratory signs of Sjögren syndrome or other connective tissue diseases. Only one of 10 patients had evidence of anti-Ro/SS-A antibodies by Oüchterlony immunodiffusion. All patients demonstrated serum immunoreactivity to bacterial heat shock protein 60 (hsp60) by Western blotting. Cross-reactivity between bacterial hsp60 and Ro/SS-A was demonstrated by Western blotting. Immunoreactivity to bacterial hsp60 by ELISA was significantly elevated in the sera of patients with normal-pressure glaucoma. Furthermore, patients with either normal-pressure or primary open-angle glaucoma had increased serum immunoreactivity to human hsp60. CONCLUSIONS Anti-Ro/SS-A positivity by ELISA in 10 patients with normal-pressure glaucoma was associated with a high level of serum immunoreactivity to bacterial hsp60, which may indicate that their glaucomatous optic neuropathy involves an as yet unidentified autoimmune mechanism. The identification of autoantibodies that react with human hsp60 in patients with normal-pressure and primary open-angle glaucoma may signify a common finding associated with the glaucomatous optic neuropathy process in some patients and appears to be unrelated to intraocular pressure levels.

The immune system and glaucoma.

Purpose of review Compelling evidence obtained from studies over the last decade strongly suggests the involvement of immune system regulation in cell fate decisions in glia and retinal ganglion cells that lead to glaucomatous optic nerve degeneration. Recent findings Recent studies reveal seemingly conflicting roles of the immune system in glaucomatous optic nerve degeneration. T cells directed against specific antigens may have a beneficial effect to protect neurons from the consequences of axonal injury. However, the immune response in glaucoma also has the capacity to cause neuronal injury. The balance between the benefit of protective immunity and the risk of inducing an autoimmune neurodegenerative disease is critical. The immunoregulatory function of glial cells and the presence of tissue stress seem to be important factors that determine the balance between diverse roles of the immune system in glaucomatous optic nerve degeneration. Thus, the net effect of immunoregulation may be either neuroprotective or neurodestructive. Summary Despite the neuroprotective features of the immune system, an autoimmune component, resulting from a failure to properly control aberrant, stress-induced immune response, likely accompanies the progression of neurodegeneration in glaucoma in some patients. A better understanding of the diverse roles of the immune system in all forms of glaucomatous optic nerve degeneration will facilitate the development of effective neuroprotective strategies in glaucoma. The basis of a sustainable neuroprotective strategy is to harness immunoregulation of glial and retinal ganglion cell fate to maximize beneficial effects, while minimizing negative sequelae, for therapeutic gain.

Induced Autoimmunity to Heat Shock Proteins Elicits Glaucomatous Loss of Retinal Ganglion Cell Neurons via Activated T Cell-Derived Fas-Ligand

Glaucomatous optic neuropathy causes blindness through the degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. Glaucoma traditionally is associated with elevated intraocular pressure, but often occurs or may progress with intraocular pressure in the normal range. Like other diseases of the CNS, a subset of glaucoma has been proposed to involve an autoimmune component to help explain the loss of RGCs in the absence of elevated intraocular pressure. One hypothesis involves heat shock proteins (HSPs), because increased serum levels of HSP autoantibodies are prominent in some glaucoma patients with normal pressures. In the first direct support of this hypothesis, we found that HSP27 and HSP60 immunization in the Lewis rat induced RGC degeneration and axon loss 1–4 months later in vivo in a pattern with similarities to human glaucoma, including topographic specificity of cell loss. Infiltration of increased numbers of T-cells in the retina occurred much earlier, 14–21 d after HSP immunization, and appeared to be transient. In vitro studies found that T-cells activated by HSP immunization induced RGC apoptosis via the release of the inflammatory cytokine FasL, whereas HSP immunization induced activation of microglia cells and upregulation of the FasL receptor in RGCs. In summary, our results suggest that RGC degeneration in glaucoma for selected individuals likely involves failed immunoregulation of the T-cell-RGC axis and is thus a disturbance of both proapoptotic and protective pathways.

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

Increased Incidence of Paraproteinemia and Autoantibodies in Patients With Normal-pressure Glaucoma

We assessed the incidence of paraproteins and autoantibodies in 44 patients (mean age, 65.3 +/- 1.9 years) with normal-pressure glaucoma and 41 patients (mean age, 68.8 +/- 1.8 years) monoclonal proteins occurred more often (P = .0047) in patients with normal-pressure glaucoma (eight of 44, 18%) than in control subjects with primary open-angle glaucoma (zero of 41, 0%). Autoantibodies to extractable nuclear antigens, most often Sjögrens syndrome A antigen (SSA[Ro]), were also found more frequently (P = .0022) in patients with normal-pressure glaucoma (13 of 44, 30%) than in control subjects (one of 41, 2%). Paraproteins and autoantibodies to extractable nuclear antigens generally occurred in different patients with an overall incidence of 20 of 44 (45%) patients with normal-pressure glaucoma. In contrast, no significant difference in the incidence of antinuclear antibodies existed between the groups. These findings suggest that humoral immune mechanisms may have a role in the pathogenesis of optic neuropathy in patients with normal-pressure glaucoma.

Serum autoantibodies to heat shock proteins in glaucoma patients from Japan and the United States.

OBJECTIVE To study serum titers of antibodies against heat shock proteins in glaucoma patients from two different ethnic populations and to examine the relationship between serum antibody titers and glaucomatous damage. STUDY DESIGN Comparative, cross-sectional study. PARTICIPANTS Twenty-seven age-matched patients with primary open-angle glaucoma, 28 patients with normal pressure glaucoma, and a control group of 26 healthy subjects from Japan; and 21 age-matched patients with primary open-angle glaucoma, 40 patients with normal pressure glaucoma, and a control group of 20 healthy subjects from the United States. MAIN OUTCOME MEASURES Measurement of serum antibody titers and examination of optic disc and visual field parameters. METHODS Serum immunoreactivity against heat shock proteins, including hsp27, alphaB-crystallin, and human and bacterial hsp60, was studied by enzyme-linked immunosorbent assay (ELISA). The relationship of serum antibody titers to glaucoma parameters, including mean deviation, neural rim area-to-disc area ratio, and peripapillary atrophy area-to-disc area ratio was examined. RESULTS The serum ELISA titers of antibodies, including hsp27, alphaB-crystallin, human hsp60, and bacterial hsp60 antibodies, were higher in glaucoma patients compared with control subjects in both the Japanese and American groups (P: < 0.05). There was no association between the serum antibody titers and optic disc parameters in either group from Japan or the United States (P: > 0.05). CONCLUSIONS These findings suggest that increased titers of circulating antibodies against heat shock proteins occur in both Japanese and American patients with glaucoma. The lack of a relationship between the level of serum antibody titers and the degree of glaucomatous damage in either ethnic group suggests that increased antibodies to heat shock proteins do not occur as an epiphenomenon of the glaucomatous neurodegeneration process.

T cell subsets and sIL-2R/IL-2 levels in patients with glaucoma.

PURPOSE We hypothesize that cellular immunity may have a previously unrecognized role in glaucomatous optic neuropathy. The purpose of this study is to analyze subsets of T cells and the levels of cytokine IL-2 and the soluble IL-2 receptor in peripheral blood from patients with normal pressure glaucoma (NPG) or primary open angle glaucoma (POAG) in comparison to age-matched control subjects. METHODS In this study, 38 patients (20 NPG; 18 POAG) and 19 controls were included. sIL-2R and IL-2 were assayed by ELISA. T cell subsets were analyzed by flow cytometry and lymphocyte proliferation was used to measure the reactive ability of T cells to phytohemagglutinin (PHA). RESULTS The frequency of CD8(+)HLA-DR(+) lymphocytes were increased in patients with NPG (P = 0.008), and CD3(+)CD8(+) lymphocytes increased in both NPG (P = 0.03) and POAG patients (P = 0.0004). CD5(+) lymphocytes were higher only in POAG patients (P = 0.0012). In comparison to controls, the ratio of CD4(+)/CD8(+) lymphocytes was similar in both groups. The mean concentrations of sIL-2R in NPG (P = 0.011) and POAG (P = 0.0023) patients were higher than that found in control subjects although IL-2 concentrations were similar in these groups. In addition, the reactive ability of T lymphocytes to the non-specific reagent (PHA) was reduced significantly in NPG (P = 0.02) and POAG patients (P=0.04). CONCLUSION The alterations of the cellular immune system in patients with glaucoma support our hypothesis that the immune system may play an important role in the initiation and/or sustainment of glaucomatous optic neuropathy in some patients.

In vitro evaluation of reactive astrocyte migration, a component of tissue remodeling in glaucomatous optic nerve head

In order to improve understanding of remodeling events in the glaucomatous optic nerve head, the migration of optic nerve head astrocytes was studied in vitro. Since elevated intraocular pressure is an important stress factor identified in glaucomatous eyes, optic nerve head astrocytes were incubated under physical stress created by elevated hydrostatic pressure. In addition, they were incubated in the presence of a chemical stimulus, lipolysaccharide (LPS). Migration of reactivated astrocytes in the presence of these stressors was examined using chambers in which cell migration through extracellular matrix‐coated pores is only possible following proteolytic digestion of the matrix. We observed that the migratory ability of optic nerve head astrocytes was approximately 4–6 times greater following exposure to elevated hydrostatic pressure or LPS for up to 48 h. Phosphoinositide 3‐kinase, protein kinase C, and tyrosine kinase were found to be involved in the signal transduction for activated migration of optic nerve head astrocytes in response to elevated hydrostatic pressure or LPS. In addition, we observed that the stress‐induced migration of optic nerve head astrocytes, which is accompanied by proteolytic degradation, resulted in the formation of culture cavities containing mucopolysaccharides. These in vitro findings provide a clearer understanding of the pathophysiologic mechanisms of characteristic tissue remodeling events that occur, in vivo, in the glaucomatous optic nerve head. GLIA 34:178–189, 2001.