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.

Distribution of ferritin chains in canine lenses with and without age-related nuclear cataracts.

PURPOSE: It was determined in an earlier study that ferritin-heavy (H) and -light (L) chains in lens fiber cells are modified in comparison to those in lens epithelial cells. The purpose of the present study was to determine whether changes in ferritin chain characteristics are developmental, age-related, or associated with cataractogenesis, by analyzing the distribution of modified chains throughout the lens fiber mass. METHODS: After removing the capsule, noncataractous and cataractous lenses were separated into six layers of fiber cells. The content of ferritin H and L chains in each layer was determined by western blotting with chain-specific antibodies. The level of ferritin complex (450 kDa protein made up of assembled L and H chains) was determined using the enzyme-linked immunosorbent assay. The ability of ferritin complex to bind iron was assessed by in vitro labeling with (59)Fe. RESULTS: Fiber cell ferritin L chains were 30 kDa (modified from the normal 19 kDa), and were present at the highest level in the outermost layers of both cataractous and non-cataractous lenses. The amount of modified L chains decreased gradually in the inner layers of the fiber mass, and was undetectable in the inner two layers of cataractous lenses. The ferritin H chains were also modified to 12 kDa (perhaps truncated from the normal 21 kDa size) in both cataractous and non-cataractous lenses. Similar levels of this modified H chain were found throughout the normal lens. Interestingly, in cataractous lenses, the modified H chains were found in decreasing amounts towards the interior of the lens, and were undetectable in the nucleus. However, in these cataractous lenses, the normal-sized ferritin H chains (21 kDA) appear in small quantities in the outer fiber layers, and increase in quantity and size (to 29 kDa) in the inner layers. This observation was best seen and demonstrated in advanced cataracts. Ferritin, which can bind iron, was found mainly in the outer layers of the lens fiber mass of normal lenses, but was more evenly distributed in fiber layers from cataractous lenses. CONCLUSIONS: Both ferritin H and L chains were modified in lens fiber cells from normal and cataractous canine lenses. These modifications were not age-related, and most likely occur during the differentiation of epithelial cells to fiber cells, since only normal-sized chains have been found in lens epithelial cells. In addition, there was a specific and distinct distribution of these modified chains throughout the lens fiber mass. The most striking differences between normal and cataractous lenses fiber cells were the appearance of normal-sized ferritin H chains and the relatively even distribution of iron binding capacity throughout the fiber mass of the cataractous lenses. These differences may reflect a response of the lens to increased oxidative stress during cataractogenesis.

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.

Lens epithelial cells synthesize and secrete ceruloplasmin: effects of ceruloplasmin and transferrin on iron efflux and intracellular iron dynamics.

Although an essential nutrient, iron can catalyze damaging free radical reactions. Therefore elaborate mechanisms have evolved to carefully regulate iron metabolism. Ceruloplasmin, a protein with ferroxidase activity, and transferrin, an iron binding protein have important roles in maintaining iron homeostasis in cells. Since oxidative damage is a hallmark of cataractogenesis, it is essential to determine iron's role in lenticular physiology and pathology. In the current study of lens epithelial cells, the effects of ceruloplasmin and transferrin on intracellular distribution and efflux of iron were determined. Both ceruloplasmin and transferrin increased iron efflux from these cells and their effects were additive. Ceruloplasmin had significant effects on extracellular iron distribution only in cases of iron overload. Surprisingly, both transferrin and ceruloplasmin had significant effects on intracellular iron distribution. Under physiological conditions, ceruloplasmin increased iron incorporation into the storage protein, ferritin. Under conditions of iron overload, it decreased iron incorporation into ferritin, which is consistent with increased efflux of iron. Measurements of an intracellular chelatable iron pool indicated that both transferrin and ceruloplasmin increased the size of this pool at 24 h, but these increases had different downstream effects. Finally, lens epithelial cells made and secreted transferrin and ceruloplasmin. These results indicate an important role for these proteins in iron metabolism in the lens.

Rabbit pigmented ciliary epithelium produces interleukin-6 in response to inflammatory cytokines.

Interleukin-6 is a multifunctional cytokine that is found in high concentrations in intraocular fluids during the uveitic response. Although monocytic cells are a major source of interleukin-6, resident intraocular cells may also contribute to its accumulation in intraocular fluids during uveitis. The purpose of this study was to determine whether interleukin-6 is produced by pigmented ciliary epithelial cells and whether agents known to stimulate interleukin-6 production, such as interleukin-1beta, tumor necrosis factor-alpha, bacterial endotoxin, and stimulators of the adenylyl cyclase/adenosine 3',5'-cyclic monophosphate system, increase interleukin-6 production by these cells. Primary and first-passage cultures of nontransformed rabbit pigmented ciliary epithelial cells were incubated with the test agents for varying periods of time in serum-free medium and interleukin-6 levels in the cell-conditioned medium were measured by bioassay.Little, if any interleukin-6 was released from pigmented ciliary epithelial cells incubated for up to 18 hr in serum-free medium. Interleukin-1betastimulated interleukin-6 release in a time- and concentration-dependent manner. Tumor necrosis factor-alpha, although ineffective alone, increased interleukin-1beta-induced interleukin-6 release in a concentration-dependent manner when co-incubated with interleukin-1betafor 18 hr. However, tumor necrosis factor-alphadid not enhance interleukin-1beta-induced interleukin-6 release if co-incubated with interleukin-1betafor a shorter time (6 hr). A 6 hr exposure to bacterial endotoxin did not stimulate interleukin-6 release from pigmented ciliary epithelial cells. Co-incubation of pigmented ciliary epithelial cells with interleukin-1betaand agents that stimulate the adenyl cyclase/adenosine 3',5'-cyclic monophosphate system through cell surface G-protein transduced receptors, i.e. isoproterenol, vasoactive intestinal peptide or prostaglandin E(2), significantly enhanced the ability of interleukin-1betato stimulate interleukin-6 release. However, neither the adenyl cyclase activator, forskolin or the adenosine 3', 5'-cyclic monophosphate-mimetic, dibutyryl 3',5'-cyclic monophosphate enhanced interleukin-1beta-induced release of interleukin-6. These results indicate that the pigmented ciliary epithelium is one potential source of interleukin-6 and may contribute to the elevation in intraocular fluid interleukin-6 levels observed during various intraocular inflammatory episodes. Although agents that activate the adenyl cyclase/adenosine 3', 5'-cyclic monophosphate system through cell surface G-protein transduced receptors increased interleukin-1beta-induced release of interleukin-6, the ineffectiveness of forskolin and dibutryl 3', 5'-cyclic monophosphate suggest that simply increasing intracellular 3',5'-cyclic monophosphate is not sufficient to augment interleukin-1beta-induced release of interleukin-6. The significance of interleukin-6 in the intraocular inflammatory response is discussed in terms of its proposed role in an endogenous antiinflammatory system acting through induction of interleukin-1 receptor antagonist, soluble tumor necrosis factor receptor, acute-phase proteins and corticosteroids.

The effect of ascorbic acid and ferric ammonium citrate on iron uptake and storage in lens epithelial cells.

Ferritin is the major intracellular iron storage protein which has been shown to protect cells against oxidative damage. Recent reports that an inherited abnormality in human ferritin synthesis is associated with early bilateral cataracts underscore the importance of understanding ferritin synthesis and iron storage in lens epithelial cells. We previously demonstrated that ascorbic acid greatly increases de novo synthesis of ferritin in lens epithelial cells. The objectives of the present study were to determine: (1) the effects of ascorbic acid and ferric ammonium citrate on iron uptake by canine lens epithelial cells from iron bound to transferrin and from ferric chloride and (2) the incorporation of this element into ferritin. Iron uptake by lens epithelial cells from 59ferric chloride was 20 times higher than from 59iron-transferrin and iron deposition into ferritin was 8-fold higher when 59ferric chloride was the source. Ascorbic acid had a stimulatory effect on iron uptake from transferrin and on incorporation of this element into ferritin. The ascorbic acid-induced increase of iron uptake required de novo protein synthesis but not specifically de novo ferritin biosynthesis. Although ferritin is not directly involved in iron uptake, the level of ferritin protein could control the pool of intracellular iron. The present results indicate that iron homeostasis in lens epithelial cells is affected mainly by changes in apoferritin synthesis, which is greatly stimulated by ascorbic acid, rather than by altering the rate of protein degradation, which is very slow in these cells under all circumstances. Ferric ammonium citrate activates iron uptake from transferrin in a wide range of cell lines by generation of free radicals. Ferric ammonium citrate also increased iron uptake from Tf in lens epithelial cells. Ferric ammonium citrate treated cells incorporated 5 times more iron and deposited 2 times more iron into ferritin than control cells. Increased incorporation of iron into ferritin was due to ferric ammonium citrate-induced stimulation of de novo ferritin synthesis rather than an increased rate of iron deposition into pre-existing ferritin. Ferric ammonium citrate had a different effect on iron uptake from ferric chloride; total iron uptake was not significantly increased while deposition into ferritin was significantly decreased. These results demonstrate that iron homeostasis in lens epithelial cells is regulated by ascorbic acid and by changes in the rate of de novo ferritin synthesis. In addition, the differences in iron uptake from transferrin and ferric chloride and its subsequent incorporation into ferritin suggests that the mechanisms by which iron is incorporated into ferritin are source dependent.

The lens influences aqueous humor levels of transforming growth factor-beta 2.

BACKGROUND: Transforming growth factor-beta 2 (TGF-beta 2) is a pluripotent cytokine which has been suggested to play a number of roles in ocular physiologic and pathologic states. Intraocular fluid (i.o.f.) levels of TGF-beta 2 are quite high. Although the sources of ocular TGF-beta 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-beta 2. In addition, the effects of aphakia or the presence of cataractous lenses on IOF TGF-beta 2 levels were determined. METHODS: Lens and ciliary body epithelial cell culture supernatants and aqueous humors were assayed for total TGF-beta 2 levels by ELISA and bioassay. RESULTS: TGF-beta 2 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-beta 2 than their contralateral phakic controls. Furthermore, aqueous humor from canine eyes with cataracts also contained significantly higher levels of TGF-beta 2 than normal eyes. CONCLUSIONS: These results suggest that the lens secretes TGF-beta 2 and that the presence and status of the lens may influence IOF TGF-beta 2 levels.

Mechanisms by which ascorbic acid increases ferritin levels in cultured lens epithelial cells.

A previous study demonstrated that ascorbic acid increased the concentration of the iron storage protein, ferritin. In cultured lens epithelial cells. The current study was designed to determine the mechanism by which ascorbic acid exerts this effect. Ascorbic acid increased both ferritin mRNA levels (by about 30%) and translation of ferritin (de novo synthesis was increased up to 15-fold) within 6 hr. Cycloheximide completely abolished the ability of ascorbic acid to increase ferritin levels, whereas actinomycin D only decreased it by about 30%. Therefore, the ascorbic-acid induced increase in ferritin concentration is due mainly to an increase in ferritin synthesis at the translational levels. This is a novel role for ascorbic acid. Addition of iron with ascorbic acid further increased de novo synthesis of ferritin, but this additive effect was only noted at a later time point (20 hr). Factors which decrease ferritin mRNA translation, such as the reducing agent dithiothreitol or the iron chelator desferrioxamine, reduced the ascorbic acid effect on de novo ferritin synthesis. The effects of ascorbic acid on ferritin mRNA levels may be mediated by its oxidation product, H2O2, since, like ascorbic acid, H2O2 increased ferritin mRNA levels by 30%. However, in contrast to the ascorbic acid-induced increase in translation of ferritin, H2O2 substantially decreased de novo ferritin synthesis. This effect of H2O2 could have physiological significance in eyes where concentrations of H2O2 in the aqueous humor are elevated. High levels of H2O2 could decrease the concentration of ferritin within the lens. Since ferritin sequesters iron and has been shown to decrease oxidative damage by limiting the availability of iron to catalyse free radical reactions, H2O2-induced reduction in ferritin concentration in the lens could have deleterious effects. The ability of ascorbic acid to increase ferritin concentration in lens epithelial cells could provide an additional protective mechanism for this antioxidant vitamin. The importance of ferritin to normal lens functioning is underscored by the recent finding that humans with a dominantly inherited abnormality in ferritin synthesis exhibit early bilateral cataracts.

Hemoglobin exacerbates the ocular inflammatory response to endotoxin.

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.

Interleukin-1 beta increases prostaglandin E2-stimulated adenosine 3',5'-cyclic monophosphate production in rabbit pigmented ciliary epithelium.

This study was designed to determine the effects of interleukin-1 on basal and prostaglandin E2-stimulated adenosine 3',5'-cyclic monophosphate production by primary and first passage cultures of non-transformed rabbit pigmented and non-pigmented ciliary epithelial cells. Confluent cultures of rabbit pigmented and non-pigmented ciliary epithelial cells were incubated for varying periods of time in serum-free medium with or without interleukin-1 beta, tumor necrosis factor-alpha, bacterial lipopolysaccharide, transforming growth factor-beta 2, cycloheximide, indomethacin and combinations of these agents. Cells were then preincubated for 10 min with serum-free medium plus the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (for basal adenosine 3',5'-cyclic monophosphate production) or serum-free medium containing several concentrations of prostaglandin E2 and 3-isobutyl-1-methylxanthine. In certain experiments isoproterenol, vasoactive intestinal peptide, or forskolin was substituted for prostaglandin E2. Adenosine 3',5'-cyclic monophosphate was then extracted into ice-cold absolute ethanol and measured by radioimmunoassay. Prostaglandin E2 stimulated adenosine 3',5'-cyclic monophosphate production in pigmented and non-pigmented ciliary epithelial cells in a dose-dependent manner. Incubation with interleukin-1 beta (150 U ml-1) increased prostaglandin E2-stimulated, but not basal adenosine 3',5'-cyclic monophosphate production in pigmented ciliary epithelial cells. This interleukin-1 beta-induced enhancement of prostaglandin E2-stimulated adenosine 3',5'-cyclic monophosphate production, called the interleukin-1 effect, was not seen with non-pigmented ciliary epithelial cells. The interleukin-1 effect was dependent upon interleukin-1 beta concentration, time and de novo protein synthesis. The interleukin 1 effect could not be reproduced by replacing interleukin-1 beta with tumor necrosis factor-alpha or bacterial lipopolysaccharide and was specific for prostaglandin E2, since interleukin-1 beta did not enhance isoproterenol-, vasoactive intestinal peptide-, or forskolin-induced adenosine 3',5'-cyclic monophosphate production. Chronic exposure to prostaglandin E2 (during the 3 hr incubation period), with or without interleukin-1 beta in the incubation medium, reduced subsequent prostaglandin E2-stimulated adenosine 3',5'-cyclic monophosphate production. Inhibition of de novo prostaglandin synthesis with indomethacin increased the interleukin-1 effect. The interleukin-1 effect was inhibited by the immunosuppressive cytokine, transforming growth factor-beta 2, in a dose-dependent manner. This is the first report of prostaglandin E2-induced stimulation of adenosine 3',5'-cyclic monophosphate production by pigmented ciliary epithelial cells and of the unique ability of interleukin-1 to increase this effect. The results are consistent with interleukin-1-induced upregulation of prostaglandin E receptors. Since transforming growth factor-beta 2 inhibited this interleukin-1 effect, this immunosuppressive cytokine may exert negative feedback and thus regulate the physiological consequences of the interleukin-1 effect.

Intravitreal transforming growth factor-beta 2 decreases cellular infiltration in endotoxin-induced ocular inflammation in rabbits.

Transforming growth factor-beta (TGF-beta), a multifunctional cytokine which has been identified in normal and inflamed ocular fluids, may play a role in the evolution of inflammatory ocular lesions. In this study we utilized a rabbit model of LPS-induced uveitis to determine if exogenous TGF-beta 2 could alter its course. Recombinant TGF-beta 2 (1-2000 ng), LPS (10 or 20 ng), or TGF-beta 2 (100 ng) plus LPS (10 ng) were injected intravitreally in one eye of a New Zealand white rabbit and the contralateral eye served as a paired control which received an equal volume of vehicle. The uveitic response was assessed by biomicroscopic examination of the anterior uvea and analysis of protein and cells in the aqueous humor. Ocular tissues were processed for histologic, immunohistochemical and in situ hybridization analyses. Rabbits injected with doses of TGF-beta 2 > or = 500 ng developed a mild uveitic response, compared to LPS alone, accompanied by expression of IL-1 beta mRNA and protein in the anterior uvea. Interestingly, rabbits coinjected with LPS (10 ng) and a nonuveitic dose (100 ng) of TGF-beta 2 exhibited a similar increase in ocular vascular permeability, but a decrease in inflammatory cell infiltration into the anterior uvea and aqueous humor (1185 +/- 117 versus 2465 +/- 176; p < 0.05). No evidence of inflammation was observed in eyes injected with 100 ng TGF-beta 2 alone. Similar to other models of inflammation, TGF-beta may interrupt the cascade of events leading to ocular inflammation, thereby suggesting therapeutic potential.

Nitric oxide synthase inhibitors exert differential time-dependent effects on LPS-induced uveitis.

Nitric oxide (NO) is a highly reactive radical which plays an integral role in physiological and pathophysiological processes. NO is produced endogenously in small amounts by a constitutive NO synthase (cNOS) as a regulator of vascular tone and neurotransmission. NO can also be produced in large amounts by an inducible NOS (iNOS) in response to endotoxin and cytokines, and has been reported to be a mediator of lipopolysaccharide (LPS)-induced uveitis in rats. The purpose of the present study was to investigate the effects of NOS inhibitors with different NOS isoform specificities in the rabbit model of endotoxin-induced ocular inflammation. LPS and/or inhibitors of NOS. NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG), were injected intravitreally and the eyes observed by slit lamp for 24 hr. Coinjection of LPS with L-NAME inhibited anterior inflammation in rabbits. Iridal hyperemia (IH) and aqueous flare (AF) were completely abolished in eight out of nine rabbits in a dose-dependent manner. In addition, total cell counts were significantly suppressed (7393 +/- 697 vs. 325 +/- 188, P < 0.05) and aqueous protein levels were reduced to near control levels (25 +/- 0.75 vs. 1.72 +/- 0.36, P < 0.05). Similar suppression was seen with AG (cell counts = 351 +/- 246 and proteins = 3.1 +/- 1.2). Administration of L-NAME 0.5 hr after LPS injection suppressed inflammation to a lesser extent than coinjection. In contrast, administration of L-NAME 6 hr after LPS injection was not inhibitory, and in fact significantly increased cellular infiltration. However, AG given 6 hr after LPS had a remarkably different effect, since it significantly decreased both protein extravasation and cellular infiltration into the aqueous humor. In fact, our results suggest that cNOS may play a greater role in the earlier stages of this developing inflammatory response. These results extend others' observations that NO is a key mediator in uveitis, that induction of iNOS plays a critical role in experimental uveitis, and suggest that NO has a complex role in the ocular inflammatory process. Inhibitors of NOS can abort the LPS-induced inflammatory response if administered early enough, but could potentially exacerbate an established inflammatory episode.

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 uptake by cultured lens epithelial cells.

BACKGROUND: Transferrin and Fe concentrations increase in the intraocular fluids in pathological conditions and the lens accumulates Fe during ocular inflammation. Tissues take up Fe from transferrin by two mechanisms, receptor-medicated endocytosis of diferric transferrin and a process occurring at the cell membrane which may be mediated by an oxido-reductase. However, Fe metabolism, transport and storage have not been previously investigated in the lens. This study was designed to characterize the uptake of Fe from transferrin by lens epithelial cells in culture. METHODS: Primary, secondary and tertiary cultures of canine lens epithelial cells and cultures obtained from cataractous lenses were studied. Uptake of 59Fe from transferrin by these cultured cells was measured. Transferrin receptor populations were determined in receptor-binding assays. RESULTS: There was a distinct relationship between the amount of Fe-transferrin added and the amount of Fe taken up, which was linear for the primary cultures but significantly reduced for the secondary, tertiary and cataract cultures (252 +/- 21, 169 +/- 14, 153 +/- 14 and 96 +/- 2 ng Fe/mg protein, respectively). Transferring receptor expression in lens cell cultures was reduced 10-fold within 2 days of addition of serum to cells grown in low-Fe, serum-free medium for 1 week. CONCLUSIONS: The reduction of Fe uptake by the subcultured and cataract cell lines probably reflects a decrease in transferrin receptor expression and in the activity of an alternative pathway for Fe transferrin uptake occurring over time. This reduced Fe uptake may result from long-term exposure to relatively high Fe concentration in the media. A reduction in the expression of the transferrin receptor after incubation with high concentrations of Fe supports this conclusion.

Inflammation induced changes in adenosine 3',5'-cyclic monophosphate production by ciliary epithelial cell bilayers.

Despite extensive evidence implicating the cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha) in the intraocular inflammatory response, little is known about their effects on signal transduction in anterior uveal tissue. Since these cytokines have been shown to alter the adenylyl cyclase system in nonocular tissues, we tested the hypothesis that IL-1 beta and TNF alpha affect the anterior uvea by altering production of the intracellular second messenger adenosine 3',5'-cyclic monophosphate (cAMP) in ciliary epithelial bilayers. This was accomplished by measuring the levels of cAMP in bilayers ex vivo, following intraocular inflammation induced by intravitreal injection of IL-1 beta, TNF alpha or bacterial endotoxin, and in vitro, following exposure to IL-1 beta, TNF alpha or bacterial endotoxin. Although cAMP production was enhanced in bilayers from IL-1 beta-, TNF alpha- or endotoxin-inflamed eyes, ex vivo, exposure of normal bilayers to IL-1 beta (15 U ml-1), TNF alpha (20 U ml-1), or a low concentration of endotoxin (0.01 microgram ml-1) for 4 hr, in vitro, had no effect on cAMP production. The inability of IL-1 beta, TNF alpha, or the low concentration of endotoxin to increase cAMP production by bilayers, in vitro, suggests that the enhanced cAMP production observed with inflamed bilayers, ex vivo, was not due to a direct action of these inflammatory agonists on the ciliary epithelial bilayer. Although direct exposure to cytokines or endotoxin did not change cAMP production, treatment with IL-1 beta, TNF alpha, or a higher concentration of endotoxin (1 microgram ml-1) did affect signal transduction mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of ferritin levels in cultured lens epithelial cells.

In most eukaryotic cells, synthesis of the iron storage protein, ferritin is regulated by iron levels and redox conditions. Proper iron storage is important to protect against damaging iron-catalysed free radical reactions. Although iron-catalysed reactions are believed to contribute to oxidative damage and cataractogenesis, little is known about iron storage in the lens. In this study, ferritin concentration was measured in cultured canine lens epithelial cells. Baseline ferritin concentration ranged from 76-163 ng (mg protein)-1; cells cultured in low-iron media had significantly lower ferritin levels than cells cultured in iron-supplemented media. Addition of a large excess of iron as hemin resulted in an eight-fold increase in ferritin concentration. The iron chelator, Desferal, significantly decreased ferritin concentration. The reducing agent dithiothreitol decreased the hemin-induced increase in ferritin levels, but not baseline levels. In contrast, ascorbic acid induced a large increase in ferritin content. Other studies have shown that induction of ferritin synthesis can protect against oxidative damage. Regulation of ferritin levels may represent a mechanism by which the lens epithelium is protected from oxidative damage. In vivo, epithelial cells are normally exposed to much lower iron concentrations than the cultured lens epithelial cells in this study. However, in pathological circumstances, the iron content and redox state of the aqueous humor is dramatically altered and may affect the steady state levels of ferritin within the lens. This remains to be determined.

Influence of exposure time on inflammatory response to neodymium:YAG cyclophotocoagulation in rabbits.

OBJECTIVE: To evaluate the influence of duration of exposure on the inflammatory response to transscleral neodymium:YAG cyclophotocoagulation. METHODS: Transscleral cyclophotocoagulation was performed with a contact, Nd:YAG, continuous-wave laser on one eye of 48 Dutch belted rabbits, 10 W and 0.2 second used in half and 1 W and 2 seconds in the other half. One third of each group was evaluated on the operative day and the other thirds on postoperative days 3 and 10. Tissue reaction was inspected grossly and by light microscopy, and inflammatory responses were measured by aqueous leukocyte and erythrocyte counts, aqueous and vitreous protein levels, aqueous prostaglandin levels, and iris and ciliary body myeloperoxidase activity. RESULTS: The shorter-duration protocol was associated with more ciliary epithelial disruption and significantly greater inflammatory responses by one or more of the measures at all times. CONCLUSION: When energy is constant, a shorter duration of exposure with transscleral Nd:YAG cyclophotocoagulation in rabbits is associated with greater tissue disruption and inflammation.

Synergistic uveitic effects of tumor necrosis factor-alpha and interleukin-1 beta.

Tumor necrosis factor (TNF) and interleukin-1 (IL-1), cytokines with multiple, overlapping biologic activities, have been shown to interact synergistically in nonocular tissues. To test the hypothesis that coinjection of TNF and IL-1 interact synergistically in the eye, low, marginally inflammatory doses of human recombinant TNF-alpha (4000 U), IL-1 beta (40 U), and TNF-alpha+IL-1 beta (TNF-alpha/IL-1 beta) were injected into the vitreal chamber of the rabbit eye, and inflammation was assessed at 6, 24, 48, and 168 hr post-cytokine injection. TNF-alpha/IL-1 beta induced an anterior uveitis that was barely detectable at 6 hr, increased at 24 hr, peaked at 48 hr, and largely resolved by 168 hr. Synergy was observed for infiltration of inflammatory leukocytes into aqueous humor at 24 and 48 hr and for protein and prostaglandin E levels in aqueous humor at 48 hr. Based upon protein levels in vitreous humor, TNF-alpha/IL-1 beta also induced a posterior uveitis. This posterior uveitis was not apparent until 48 hr and then increased significantly at 168 hr. At 48 and 168 hr, the effects of TNF-alpha/IL-1 beta on protein levels in vitreous humor were consistent with a synergistic interaction. Results of separate experiments using higher dose combinations of TNF-alpha/IL-1 beta and a longer time course suggested that the effects of TNF-alpha/IL-1 beta on the blood vitreous barrier persisted beyond 168 hr. The results of this study support the hypothesis that TNF-alpha and IL-1 beta interact synergistically when injected into the rabbit eye.(ABSTRACT TRUNCATED AT 250 WORDS)

Inflammatory effects of continuous-wave neodymium: yttrium aluminum garnet laser cyclophotocoagulation.

The uveal inflammatory response was studied in 31 rabbits treated unilaterally with neodymium: yttrium aluminum garnet (Nd:YAG) cyclophotocoagulation. Fifteen applications of 3.5-J energy were delivered to the dorsal and ventral perilimbal sclera using a contact continuous-wave system. On days 1, 3, 8, and 15, the inflammatory effects were assessed. Peak levels of aqueous humor protein (11 +/- 3 mg/ml), prostaglandin E2 (8.9 +/- 3.0 ng/ml), leukocytes (205 +/- 113/microliters), and iris-ciliary body myeloperoxidase activity (6.32 +/- 1.4 U/mg protein) occurred on day 3 and rapidly decreased between days 7 and 15. Vitreal protein levels also peaked at day 3 but remained elevated through day 15 (3.8 +/- 1.3 mg/ml). By contrast, aqueous erythrocytes were most numerous (22,614 +/- 10,517/microliters) on day 8. Levels of leukotriene B4 remained low in all eyes at all intervals. Correlative histologic changes were ciliary coagulation necrosis, severe vascular congestion, and a predominantly mononuclear inflammatory cell infiltrate. These data suggest that Nd:YAG cyclophotocoagulation in rabbits induces a relatively mild inflammatory response that is associated with significant vascular compromise. Although these observations may not be analogous to the situation in the human eye, they may provide a model with which to compare the relative effects of different treatment parameters to help establish the optimum protocol.