M.C. McGahan

OCULAR INFLAMMATORY EFFECTS OF INTRAVITREALLY INJECTED TUMOR NECROSIS FACTOR-ALPHA AND ENDOTOXIN

Intravitreal injection of human recombinant tumor necrosis factor-alpha (TNF) induced inflammation in the rabbit eye characterized by dilation of blood vessels in the iris, disruption of the blood-ocular barriers, infiltration of inflammatory cells into the anterior chamber, and accumulation of prostaglandin E in intraocular fluids. Inflammation first appeared on day 1, increased on day 2, and remained elevated on day 7. The inflammatory eell infiltrate in the anterior segment of the eye was largely monocytic on days 1 and 2; by day 7 large numbers of lymphocytes were also present. TNF-induced ocular inflammation therefore differed from that reported for intravitreally injected endotoxin in terms of time course and the types of inflammatory cells in the aqueous humor. In a series of experiments in which combinations of TNF and endotoxin were used, intravitreal injection of TNF, 24 h after a low dose ofEscherichia coli endotoxin, produced no more inflammation than that produced by TNF following an injection of endotoxin vehicle. However, if TNF was injected 24 h before endotoxin, the resulting inflammation was greater than that observed in animals given TNF followed by endotoxin vehicle.

Iron metabolism in the eye: A review

This review article covers all aspects of iron metabolism, which include studies of iron levels within the eye and the processes used to maintain normal levels of iron in ocular tissues. In addition, the involvement of iron in ocular pathology is explored. In each section there is a short introduction to a specific metabolic process responsible for iron homeostasis, which for the most part has been studied in non-ocular tissues. This is followed by a summary of our current knowledge of the process in ocular tissues.

Transferrin secretion by lens epithelial cells in culture

Transferrin (Tf), the plasma iron transport protein which supports cell proliferation and differentiation and has bacteriostatic, antioxidant and anti-inflammatory activity, has been found in relatively high concentrations in the intraocular fluids. Intraocular synthesis of Tf has recently been demonstrated, although the intraocular tissue(s) responsible have not been identified. We designed this study to determine whether certain ocular tissues can make and secrete transferrin. Transferrin content of aqueous and vitreous humors and whole lenses was determined by ELISA. Transferrin secretion by cultured epithelia from lens and ciliary body was also measured. In addition, Northern blots of RNA from cultured lens epithelial cells, ciliary body pigmented and non-pigmented epithelial cells, and from whole iris, ciliary body and retina were probed with riboprobes for Tf mRNA and 18S rRNA. Transferrin made up 23% and 16% of total canine aqueous and vitreous protein. All ocular tissues and cultured cells tested contained mRNA for Tf, however Tf was secreted into the bathing medium from lens epithelial cell cultures, but not from either the pigmented or non-pigmented epithelial cells of the ciliary body cultures, but not from either the pigmented or non-pigmented epithelial cells of the ciliary body Cycloheximide inhibited secretion of Tf from the lens epithelial cells. Lenses from inflamed eyes contained higher levels of Tf than their contralateral controls. This is the first experimental demonstration that an intraocular tissue can make and secrete Tf. Transferrin secretion by the lens may contribute significantly to the IOF content of this important intraocular protein.

Selenium concentration in ocular tissues and fluids.

A simple, precise method for the analysis of selenium (Se) in ocular fluids and tissues using electrothermal atomic absorption spectroscopy is presented. Se concentrations ranged from 0.23 to 0.41 microgram/g wet weight in the cornea, iris, lens, and retina. The Se concentration in aqueous humor was 0.008 microgram/ml, thus much lower than that of plasma (0.21 microgram/ml). The concentration of protein in aqueous humor is about 1.0% of plasma. Since in plasma, Se is entirely bound to proteins, it is likely that the difference in Se concentration between plasma and aqueous humor reflects the relative distribution of protein between these fluids.

Ferroxidase activity increases in the aqueous humor during the ocular inflammatory response.

Ferroxidase activity was increased in the aqueous humor from inflamed eyes compared to their uninflamed contralateral controls 24 h after intravitreal injection of 10 ng of endotoxin. Changes in ferroxidase activity and copper concentration paralleled each other indicating that the plasma copper transport protein ceruloplasmin (plasma ferroxidase) entered the inflamed aqueous humor from plasma through disrupted blood ocular barriers. The presence of ferroxidase activity would facilitate the removal of potentially damaging, free radical generating Fe+2. Therefore, plasma proteins may perform important protective functions in the inflamed intraocular fluids.

Ceruloplasmin alters intracellular iron regulated proteins and pathways: Ferritin, transferrin receptor, glutamate and hypoxia-inducible factor-1α

Ceruloplasmin (Cp) is a ferroxidase important to the regulation of both systemic and intracellular iron levels. Cp has a critical role in iron metabolism in the brain and retina as shown in patients with aceruloplasminemia and in Cp-/-hep-/y mice where iron accumulates and neural and retinal degeneration ensue. We have previously shown that cultured lens epithelial cells (LEC) secrete Cp. The purpose of the current study was to determine if cultured retinal pigmented epithelial cells (RPE) also secrete Cp. In addition, the effects of exogenously added Cp on iron regulated proteins and pathways, ferritin, transferrin receptor, glutamate secretion and levels of hypoxia-inducible factor-1α in the nucleus were determined. Like LEC, RPE secrete Cp. Cp was found diffusely distributed within both cultured LEC and RPE, but the cell membranes had more intense staining. Exogenously added Cp caused an increase in ferritin levels in both cell types and increased secretion of glutamate. The Cp-induced increase in glutamate secretion was inhibited by both the aconitase inhibitor oxalomalic acid as well as iron chelators. As predicted by the canonical view of the iron regulatory protein (IRP) as the predominant controller of cellular iron status these results indicate that there is an increase in available iron (called the labile iron pool (LIP)) in the cytoplasm. However, both transferrin receptor (TfR) and nuclear levels of HIF-1α were increased and these results point to a decrease in available iron. Such confounding results have been found in other systems and indicate that there is a much more complex regulation of intracellularly available iron (LIP) and its downstream effects on cell metabolism. Importantly, the Cp increased production and secretion of the neurotransmitter, glutamate, is a substantive finding of clinical relevance because of the neural and retinal degeneration found in aceruloplasminemia patients. This finding and Cp-induced nuclear translocation of the hypoxia-inducible factor-1 (HIF1) subunit HIF-1α adds novel information to the list of critical pathways impacted by Cp.

The Effect of UVB Irradiation on Ferritin Subunit Synthesis, Ferritin Assembly and Fe Metabolism in Cultured Canine Lens Epithelial Cells ¶

Ferritin is a multimeric protein consisting of heavy and light chains assembled in different tissue‐specific ratios, which can protect cells from oxidative stress by storing reactive iron (Fe). Because the lens is constantly exposed to UV irradiation, we studied its effects on ferritin synthesis and Fe metabolism in cultured lens epithelial cells with and without ascorbic acid (Asc). UVB caused a large increase in accumulation of newly synthesized ferritin chains; this increase was additive to that induced by Asc. In contrast to the Asc‐induced increase in Fe storage, Fe storage in ferritin was unaltered by UVB. Although UVB increased accumulation of newly synthesized ferritin chains, total ferritin levels were unaltered. In contrast, Asc, which induced a quantitatively similar increase in accumulation of newly synthesized ferritin chains, doubled the total amount of ferritin. Because UVB did not change Fe storage in ferritin or the size of the labile Fe pool, it was hypothesized and then determined that these newly synthesized chains did not assemble into functional holoferritin. Numerous studies detail the effects of various treatments on de novo ferritin synthesis; however, this study provides a cautionary note regarding the conclusions of such studies in the absence of data indicating assembly of functional ferritin molecules.

Clinical signs of acute ocular inflammatory response to endotoxin are not altered by increasing antioxidant potency of intraocular fluids

Plasma antioxidant activity is due in large part to the ferroxidase activity of the copper (Cu) transport protein, ceruloplasmin. Implantation of osmotic pumps containing copper into rabbits resulted in a doubling of Cu concentration, ferroxidase activity, and antioxidant activity in plasma. Blood-ocular barriers essentially prevent the entry of large molecules such as proteins from plasma into the intraocular fluid compartments. However, during ocular inflammation, when these barriers are disrupted, plasma proteins can enter. Twenty-four hours after the induction of ocular inflammation by intraocular injection of endotoxin, the Cu concentration and antioxidant activity of intraocular fluids from the Cu pump animals was twice that of control groups, reflecting the differences in plasma levels. This is the first direct demonstration that alterations in plasma levels of ceruloplasmin can influence the antioxidant potency of the extracellular fluids. Increased intraocular fluid antioxidant activity did not affect the acute anterior segment response to endotoxin. However, it is possible that the time course and resolution of the response is altered by changes in extracellular fluid antioxidant activity. This possibility is currently under investigation.

Effects of copper depletion andd-penicillamine treatment on the ocular inflammatory response

Rabbits placed on a copper (Cu)-deficient diet for four weeks had lower plasma Cu concentration, ferroxidase activity, and antioxidant activity than rabbits fed an identical Cu-adequate basel diet. Inflamed aqueous humor from rabbits on the Cu deficient diet also had less Cu, ferroxidase and antioxidant activity than inflamed aqueous humor from control rabbits. These differences mirrored antioxidant activity than inflamed aqueous humor from control rabbits. These differences mirrored changes in plasma levels. Furthermore, there was an increase in cellular infiltration into the aqueous humor of Cu-deficient rabbits. It is hypothesized that in Cu-deficient rabbits there is an increase in free radical production and formation of chemotactic substances in the eye due to the decreased aqueous humor concentration of the endogenous antioxidant, ceruloplasmin. It is therefore likely that ceruloplasmin plays a protective role in the intraocular inflammatory response.