Lloyd N. Fleisher

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.

The lens influences aqueous humor levels of transforming growth factor-β2

Abstract · Background: Transforming growth factor-beta 2 (TGF-β2) is a pluripotent cytokine which has been suggested to play a number of roles in ocular physiologic and pathologic states. Intraocular fluid (IOF) levels of TGF-β2 are quite high. Although the sources of ocular TGF-β are not completely defined, the retinal pigment epithelium, the epithelium of the ciliary body and trabecular meshwork cells all secrete it. In this study we utilized canine lens and rabbit ciliary pigmented epithelial cell cultures to quantitate the in vitro secretion of TGF-β2. In addition, the effects of aphakia or the presence of cataractous lenses on IOF TGF-β2 levels were determined. · Methods: Lens and ciliary body epithelial cell culture supernatants and aqueous humors were assayed for total TGF-β2 levels by ELISA and bioassay. · Results: TGF-β2 accumulated in the media bathing lens epithelial cell cultures (0.7 ± 0.03 ng/ml at day 2) and ciliary pigmented epithelial cell cultures (0.8 ± 0.06 ng/ml at day 2) in a time-dependent manner. Surprisingly, aqueous humor from aphakic rabbit eyes contained significantly higher levels of TGF-β2 than their contralateral phakic controls. Furthermore, aqueous humor from canine eyes with cataracts also contained significantly higher levels of TGF-β2 than normal eyes. · Conclusions: These results suggest that the lens secretes TGF-β2 and that the presence and status of the lens may influence IOF TGF-β2 levels.

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.

Iron Regulates L-Cystine Uptake and Glutathione Levels in Lens Epithelial and Retinal Pigment Epithelial Cells by Its Effect on Cytosolic Aconitase

PURPOSE The authors previously published the novel finding that iron regulates L-glutamate synthesis and accumulation in the cell-conditioned medium (CCM) by increasing cytosolic aconitase activity in cultured lens epithelial cells (LECs), retinal pigment epithelial (RPE) cells, and neurons. The present study was designed to determine whether iron-induced L-glutamate accumulation in the CCM regulates L-cystine uptake and glutathione (GSH) levels through the aconitase pathway in LECs and RPE cells. METHODS The presence of xCT, the light chain of X(c)(-), a glutamate/cystine antiporter, was analyzed by RT-PCR, immunoblotting, and immunocytochemistry. Uptake of L-[(35)S]cystine and L-[(3)H]glutamate was measured in the presence or absence of transporter inhibitors. L-cystine uptake and intracellular GSH concentration were measured in the presence or absence of iron-saturated transferrin, the iron chelator dipyridyl (DP), or oxalomalic acid (OMA), an aconitase inhibitor. RESULTS LECs and RPE cells express xCT, as evidenced by RT-PCR analysis and immunoblotting. xCT was localized by immunocytochemistry. The authors found that the iron-induced increase in L-glutamate availability increased L-cystine uptake, with subsequent increases in GSH levels. In addition, L-glutamate production, L-cystine uptake, and GSH concentration were inhibited by OMA and DP, indicating a central role for iron-regulated aconitase activity in GSH synthesis in LECs and RPE cells. CONCLUSIONS These results demonstrate for the first time that iron regulates L-cystine uptake and the downstream production of GSH in two mammalian cell types. It is possible that the increase in intracellular antioxidant concentration induced by iron serves as a protective mechanism against the well-established capacity of iron to induce oxidative damage.

Antioxidant activity of aqueous and vitreous humor from the inflamed rabbit eye

The effects of aqueous and vitreous humors and plasma on the rate of auto-oxidation of a rabbit brain homogenate were measured. Both aqueous and vitreous humors from normal eyes increased, while plasma decreased the rate of oxidation in the homogenate. During endotoxin-induced ocular inflammation the copper (Cu) and iron (Fe) concentrations of both the aqueous and vitreous humors increased, most likely due to the influx of their plasma binding proteins, ceruloplasmin (Cu) and transferrin (Fe). As both proteins are known to be antioxidants, it was not surprising to find that the aqueous and vitreous humor from the inflamed eyes had significant antioxidant activity. This antioxidant activity correlated well with the concentrations of Cu and Fe in aqueous humor and Cu but not Fe in the vitreous humor throughout the time course of the inflammatory response. Thus, entry of plasma proteins through disrupted blood ocular barriers may function in protecting ocular tissues against the increased oxidation which occurs during inflammation.

A micromethod for the determination of iron and total iron-binding capacity in intraocular fluids and plasma using electrothermal atomic absorption spectroscopy.

A new micromethod (requiring only 30 microliters of sample) for the determination of iron (Fe) concentration and total iron-binding capacity (TIBC) in intraocular fluids (IOFs; aqueous and vitreous humors) and plasma has been developed using electrothermal atomic absorption spectrophotometry. Values for Fe concentration in the IOFs were similar between species and were generally higher in the vitreous (0.031-0.060 mg/liter) than aqueous (0.014-0.038 mg/liter) humors obtained from the same eye. Plasma Fe concentration and TIBC fell within established limits for all species studied. The iron binding capacity of the aqueous and vitreous humors from the same species is similar. While the TIBC of the aqueous humor is saturated to the same extent as plasma, vitreous TIBC is more highly saturated in all species. Using this method considerably less species variation in IOF Fe concentration was found as compared to previous studies. Some possible methodological reasons for this difference are discussed.

Inflammation-induced changes in the iron concentration and total iron-binding capacity of the intraocular fluids of rabbits

Changes in iron (Fe) concentration and total-iron-binding capacity (TIBC) of the intraocular fluids were measured during endotoxin-induced ocular inflammation in rabbits over a 3-week time course. In the aqueous humor, both Fe and TIBC increased to peak levels 24 h after intravitreal injection of endotoxin (10 ng) and gradually decreased to baseline levels by 3 weeks. In the uninflamed eye, the TIBC of the aqueous was only 23% saturated. During inflammation the TIBC became more highly saturated over time, reaching 50% at 3 weeks. In the vitreous humor the picture was more complicated due to the presence of slight hemorrhage. Noncellular Fe and TIBC increased to peak levels by 7 days, while TIBC approached 100% saturation. Both returned to baseline by 21 days. The influx of the partially saturated plasma protein transferrin through disrupted blood-ocular barriers most likely accounts for the increased TIBC in the inflamed eye and could provide some protection against the potentially harmful effects of Fe arising from tissue necrosis and hemolysis subsequent to hemorrhage. Under conditions of the model of inflammation studied here, the TIBC was not exceeded at any time during the 3 weeks. However, with more severe and long-lasting inflammation or when there is greater hemorrhage, the TIBC could be exceeded. This could lead to greater, and perhaps irreversible, damage to ocular tissues.

Endotoxin-induced ocular inflammation increases prostaglandin E2 synthesis by rabbit lens

Twenty-four hours after the intravitreal injection of 0.1-100 ng of Escherichia coli endotoxin into one eye of the New Zealand white rabbit, lenses from the inflamed eyes synthesized significantly more prostaglandin E2 (PGE2) than their contralateral, control lenses at all doses of endotoxin greater than 0.1 ng. PGE2 elevations were also seen in the aqueous and vitreous humors from inflamed eyes. Lenses did not synthesize 6-keto-PGF1alpha (a stable metabolite of PGI2). Incubation of untreated lenses with 1 microgram ml-1 of endotoxin for 24 h did not increase PGE2 production. These results indicate that rabbit lens can synthesize PGE2, that this synthesis is significantly increased 24 hr after the intravitreal infusion of E. coli endotoxin, and that this increased PGE2 synthesis is most likely not due to a direct action of endotoxin on the lens.

Hemoglobin exacerbates the ocular inflammatory response to endotoxin

Abstract• Background: There is a clinical impression that bleeding into sites of inflammation exacerbates the inflammatory response. It has been hypothesized that hemoglobinic iron (Fe) contributes to this response by catalyzing free radical reactions. In the present study, the effects of autologous hemoglobin on the inflammatory response to endotoxin was determined. In addition, the possible contributions of Fe to this response was assessed by co-injection of either transferrin or desferrioxamine. • Methods: A mild ocular inflammation was induced in rabbits by intravitreal injection of 0.25 ng endotoxin. In some animals apotransferrin, hemoglobin, hemoglobin + apotransferrin or hemoglobin + desferrioxamine were co-injected. Twenty-four hours later, anterior uveitis was quantified by slit-lamp examination and determination of protein concentration and infiltration of white cells into the aqueous humor. • Results: Co-injection of autologous hemoglobin with endotoxin greatly exacerbated the ocular inflammatory response to endotoxin, especially the infiltration of white cells, which was increased 15-fold. Both apotransferrin, which binds Fe at high affinity, and desferrioxamine, which chelates Fe, greatly decreased the cellular response to the co-injection. • Conclusions: It is likely that hemoglobinic Fe is responsible for the increased infiltration of white cells caused by the co-injection of autologous hemaglobin and endotoxin.