Cultured Pigment Epithelial Cells Research Articles

Expression of a chemokine by ciliary body epithelium in horses with naturally occurring recurrent uveitis and in cultured ciliary body epithelial cells.

To determine whether a chemokine (RANTES)-like protein expressed by ciliary epithelium plays a role in uveitis. 3 clinically normal horses intradermal, 5 eyes from 5 horses with recurrent uveitis, and 10 normal eyes from 5 age- and sex-matched horses. Cross-reactivity and sensitivity of recombinant human (rh)-regulated upon activation, normal T-cell expressed and secreted (RANTES) protein were evaluated in horses by use of intradermal hypersensitivity reactions and a chemotaxis assay. Aqueous humor and ciliary body of eyes from clinically normal horses and horses with uveitis were examined for RANTES expression by use of an ELISA and reverse transcription-polymerase chain reaction (RT-PCR). Expression of RANTES mRNA and protein content of primary cultures of equine ciliary pigmented epithelial cells (RT-PCR) and culture supernatant (ELISA) were measured 6 or 24 hours, respectively, after cultures were stimulated with interleukin-1beta and tumor necrosis factor-alpha. Strong reactions to intradermal hypersensitivity testing and significant chemotaxis of equine leukocytes to rh-RANTES wereas observed. Aqueous humor of eyes from horses with uveitis contained increased concentrations of rh-RANTES-like protein (mean +/- SD, 45.9+/-31.7 pg/ml), compared with aqueous humor from clinically normal horses (0 pg/ml). Ciliary body from horses with uveitis expressed RANTES mRNA, whereas ciliary body from clinically normal horses had low mRNA expression. Stimulated ciliary pigmented epithelial cells expressed increased amounts of rh-RANTES-like protein (506.1+/-298.3 pg/ml) and mRNA, compared with unstimulated samples. Ciliary epithelium may play a role in recruitment and activation of leukocytes through expression of RANTES.

Synthesis of the All-trans-retinal Chromophore of Retinal G Protein-coupled Receptor Opsin in Cultured Pigment Epithelial Cells

Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cis isomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[(3)H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[(3)H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[(3)H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis.

Neurogenin2 elicits the genesis of retinal neurons from cultures of nonneural cells.

neurogenin2 (ngn2) encodes a basic helix-loop-helix transcription factor and plays an important role in neurogenesis from migratory neural crest cells. Its role in retinal development is poorly understood. We observed that in the developing chick retina, ngn2 was expressed in a subpopulation of proliferating progenitor cells. Ectopic expression of ngn2 in nonneural, retinal pigment epithelial cell culture triggered de novo generation of cells that expressed neural-specific markers and exhibited neuronal morphologies. Further molecular and morphological analyses showed that the main products of the induced neurogenesis were cells resembling young photoreceptor cells and cells resembling retinal ganglion cells. The generation of multiple cell types suggests that ngn2 induces various retinal pathways. Thus, unlike in the peripheral nervous system where ngn2 specifies one type of sensory neuron, ngn2 in the retina is likely involved in a common step leading to different cellular pathways. Our finding that ngn2 can instruct nonneural retinal pigment epithelial cells to differentiate toward retinal neurons demonstrates one possible way to induce de novo retinal neurogenesis.

Upregulation of pigment epithelium-derived factor after laser photocoagulation

PURPOSE: To determine the changes in the expression of pigment epithelium-derived factor in cultured human retinal pigment epithelial cells and rat retinas after laser photocoagulation. METHODS: Experimental study of laser photocoagulation on human retinal pigment epithelial cells in culture and on adult rats. Reverse transcription-polymerase chain reaction and semiquantitative polymerase chain reaction analysis were used. RESULTS: After photocoagulation, the mRNA expression of pigment epithelium-derived factor was upregulated in human retinal pigment epithelial cells at 6 hours and then gradually decreased. Compared with controls, significantly higher levels of pigment epithelium-derived factor were observed in rat retinas from 6 to 24 hours after laser photocoagulation ( P < .005), and they were still higher than before photocoagulation at 2 weeks. CONCLUSION: An upregulation of pigment epithelium-derived factor in retinal pigment epithelial cells and in the retina after photocoagulation suggests that pigment epithelium-derived factor plays a role in inhibiting neovascularization by its antiangiogenic activity.

Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress.

To explore the role of lipid peroxidation (LPO) products in the initial phase of stress mediated signaling, we studied the effect of mild, transient oxidative or heat stress on parameters that regulate the cellular concentration of 4-hydroxynonenal (4-HNE). When K562 cells were exposed to mild heat shock (42 degrees C, 30 min) or oxidative stress (50 microM H2O2, 20 min) and allowed to recover for 2 h, there was a severalfold induction of hGST5.8, which catalyzes the formation of glutathione-4-HNE conjugate (GS-HNE), and RLIP76, which mediates the transport of GS-HNE from cells (Awasthi, S., Cheng, J., Singhal, S. S., Saini, M. K., Pandya, U., Pikula, S., Bandorowicz-Pikula, J., Singh, S. V., Zimniak, P., and Awasthi, Y. C. (2000) Biochemistry 39, 9327-9334). Enhanced LPO was observed in stressed cells, but the major antioxidant enzymes and HSP70 remained unaffected. The stressed cells showed higher GS-HNE-conjugating activity and increased efflux of GS-HNE. Stress-pre-conditioned cells with induced hGST5.8 and RLIP76 acquired resistance to 4-HNE and H2O2-mediated apoptosis by suppressing a sustained activation of c-Jun N-terminal kinase and caspase 3. The protective effect of stress pre-conditioning against apoptosis was abrogated by coating the cells with anti-RLIP76 IgG, which inhibited the efflux of GS-HNE from cells, indicating that the cells acquired resistance to apoptosis by metabolizing and excluding 4-HNE at a higher rate. Induction of hGST5.8 and RLIP76 by mild, transient stress and the resulting resistance of stress-pre-conditioned cells to apoptosis appears to be a general phenomenon since it was not limited to K562 cells but was also evident in lung cancer cells, H-69, H-226, human leukemia cells, HL-60, and human retinal pigmented epithelial cells. These results strongly suggest a role of LPO products, particularly 4-HNE, in the initial phase of stress mediated signaling.

The phagocytosis of rod outer segments is inhibited by selected drugs in retinal pigment epithelial cell cultures.

The effects of tamoxifen, toremifene and chloroquine on the phagocytosis of rod outer segments by retinal pigment epithelium were evaluated in human retinal pigment epithelial cell line D407 and pig retinal pigment epithelial cell culture. Retinal pigment epithelial cells were exposed to different concentrations of tamoxifen (1-20 microM), toremifene (1-20 microM) and chloroquine (1-1000 microM), and challenged with FITC-labeled rod outer segments for 24 hr. The phagocytized (bound and ingested) rod outer segments were measured fluorometrically, and the effect of the drugs on the phagocytosis was determined. The cytotoxicity of the drugs was evaluated by measuring their effects on mitochondrial enzyme activities (WST-1-test). The results showed that the test compounds inhibited the phagocytosis of rod outer segments in both D407 and pig retinal pigment epithelial cells. The phagocytic activity was more sensitive to tamoxifen (EC(50) 7.2 microM for D407 cells and 3.6 microM for pig retinal pigment epithelial cells) and toremifene (EC(50) 6.2 microM and 3.1 microM respectively) than to chloroquine (EC(50) 77.2 microM for D407 cells). The inhibition of rod outer segment phagocytosis in both cell cultures started at lower dose levels of test compounds than the cytotoxicity indicated by the WST-1-test. The experiments were carried out both in serum-free medium and serum-containing medium. Serum seemed to be a critical factor in the medium and caused difficulties in the interpretation of the results.

A novel immunoassay for the evaluation of rod outer segment digestion in cultured retinal pigment epithelial cells.

In this work a novel enzyme-linked immunoabsorbent assay quantifying residual rod outer segments in the medium of rod outer segment-challenged retinal pigment epithelial cells is described. A retinal pigment epithelial cell line (D407) that produces low level of cathepsin D, and a primary human retinal pigment epithelial cell culture (HRPE51) that has normal cathepsin D levels, were challenged with bovine rod outer segments. At 3 days post-challenge, the amount of undigested or residual bovine rod outer segments left in the culture medium was quantified by an enzyme-linked immunoabsorbent assay. An antibody raised against bovine rod outer segments, which had been purified and labelled with nitroiodophenyl haptens, was used in the assay. The sensitivity of the immunoassay was less than 10(2) bovine rod outer segments per mL and the signal followed a linear curve, saturating around 10(6) bovine rod outer segments per mL. HRPE51 cells had no residual bovine rod outer segments present in the medium following a challenge with 10(4) bovine rod outer segments per mL. In the medium of D407 cells, residual bovine rod outer segment levels were higher at all bovine rod outer segment concentrations when compared to the residual bovine rod outer segment levels in HRPE51 cells, suggesting that D407 cells have a lower digestive capacity. These results demonstrated that the immunoassay for detecting bovine rod outer segments is a sensitive and reliable technique that can be used to quantify the amount of residual bovine rod outer segments, following bovine rod outer segment challenge of retinal pigment epithelial cells.

Human Immunodeficiency Virus Type 1 Can Infect Human Retinal Pigment Epithelial Cells in Culture and Alter the Ability of the Cells to Phagocytose Rod Outer Segment Membranes

Human immunodeficiency virus type 1 (HIV-1) has been found in the vitreous of persons with AIDS. Here we investigated the susceptibility of human retinal pigment epithelial (RPE) cells to HIV-1 infection in culture and the effects of HIV-1 on the phagocytic function of the RPE. We found that 10 of 11 populations of RPE cells isolated from different fetal or adult eyes were susceptible to low-level replication of HIV-1/NL4-3 as determined by the detection of viral DNA and spliced viral RNA encoding envelope. HIV-1 infection was not inhibited by recombinant soluble CD4, suggesting that CD4 is not required for virus entry into RPE cells. RPE cells fused with target cells constitutively expressing HIV-1 envelope glycoproteins, indicating that HIV-1 enters cells by receptor-mediated fusion. Exposure to HIV-1 or recombinant gp120 caused a two- to four-fold increase in the binding and uptake of isolated rod outer segments by RPE cells. These findings introduce a new cell target of HIV-1 replication in the eye and indicate that RPE cells function aberrantly when exposed to HIV-1 or its envelope glycoprotein.

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-1β, tumor necrosis factor-α, 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-1βstimulated interleukin-6 release in a time- and concentration-dependent manner. Tumor necrosis factor-α, although ineffective alone, increased interleukin-1β-induced interleukin-6 release in a concentration-dependent manner when co-incubated with interleukin-1βfor 18 hr. However, tumor necrosis factor-αdid not enhance interleukin-1β-induced interleukin-6 release if co-incubated with interleukin-1βfor 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-1βand 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 E2, significantly enhanced the ability of interleukin-1βto 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-1β-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-1β-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-1β-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.

Air-interface condition promotes the formation of tight corneal epithelial cell layers for drug transport studies.

To identify the growth conditions that would favor the development of a functional primary culture of pigmented rabbit corneal epithelial cells on a permeable support comparable to the intact tissue in bioelectric properties. Rabbit corneal epithelial cells were isolated and cultured on precoated fibronectin/collagen/laminin permeable filters. Cells were grown at an air-interface with supplemented DMEM/F12 medium. Immunofluorescence and electron microscopy techniques, respectively, were used to confirm cornea-specific marker and morphological features. Permeability of the cell layers to model polar compounds was evaluated using 14C-mannitol, fluorescein isothiocyanate (FITC) and fluorescein isothiocyanate-dextran of 4,000 molecular weight (FD4). We found that culturing the epithelial cells at an air-interface (AIC) was a critical factor in the formation of tight cell layer and that omitting fetal bovine serum and keeping the concentration of epidermal growth factor at 1 ng/ml were equally important. Phenotypically, the AIC cell layers were found to express cornea-specific 64 kD keratin. Compared with cells cultured under the liquid-covered (LCC) condition, those cultured under AIC exhibited a significantly higher peak transepithelial electrical resistance (TEER) of up to 5 kOhm x cm2, a higher potential difference (PD) of up to 26 mV, and an estimated short-circuit current (Ieq) of 5 microA/cm2 after 7-8 days of culture. These values were comparable to those in the excised cornea. Consistent with the TEER, the AIC cell layers were 4-40 times less permeable to paracellular markers than their LCC counterpart. The AIC model merits further characterization of drug transport mechanisms as well as drug, formulation, physiological, and pathological factors influencing corneal epithelial drug transport.

Effects of Corticosteroid on Porcine Retinal Pigment Epithelial Cells in Culture: 1. Inhibitory Effect on Cell Proliferation

Purpose: Proliferation is an important function of the retinal pigment epithelial (RPE) cells. The effect of a corticosteroid on the proliferation of cultured porcine RPE cells was investigated. Methods: After administration of various concentrations (10–1,000 nM) of betamethasone sodium phosphate (betamethasone), we counted RPE cell numbers at 1, 3, 6, 9, and 14 days. Results: Betamethasone administration resulted in a dose-dependent decrease in RPE cell proliferation. The proliferation of the cultured RPE cells was significantly inhibited by betamethasone, at a dose of 300 nM in 9 days. Ten nanomolar betamethasone neither inhibited nor promoted the RPE cell proliferation in culture. Conclusions: These results suggest that corticosteroids inhibit proliferation of cultured porcine RPE cells. (J Jpn Ophthalmol Soc 103:436–441, 1999)

Retinal pigment epithelial cell cultures as a tool for evaluating retinal toxicity in vitro.

This article reviews in vitro testing of retinal toxicity in retinal pigment epithelium (RPE) cell cultures. It is based on the literature on RPE cell cultures and on our recent studies on the retinal toxicity of selected amphiphilic drugs. The RPE plays a major role in maintaining the homeostasis and health of the retina. Various pharmacological agents are known to cause adverse effects in RPE cells. For example, long-term treatment with chloroquine in patients with rheumatoid arthritis has induced retinopathy, and tamoxifen, a drug that is commonly used in the treatment of advanced breast cancer and in the prevention of breast cancer among high-risk women, has been reported to cause retinal changes and impaired vision. During our research, we have developed novel in vitro methods for evaluating the retinal toxicity of xenobiotics. We have used a pig RPE primary culture and a human RPE cell line (D407), which retain epithelial cell characteristics. They form a layer of hexagonal cells with intercellular junctions, and possess a keratin-containing cytoskeleton. They are both good models for determining the retinal cell toxicity of test compounds. Further studies on phagocytic activity, lysosomal enzyme activity and glutamate uptake might generate new methods for the toxicological evaluation of the retinal side-effects of drugs in vitro.

Pedf (Pigment epithelium-derived Factor) promotes increase and maturation of pigment granules in pigment epithelial cells in neonatal albino rat retinal cultures

Pigment Epithelium-Derived Factor (PEDF), purified from human retinal pigment epithelial (RPE) cell culture medium, is a neurotrophic factor which potentiates the differentiation of human Y-79 retinoblastoma cells and increases the survival of cerebellar granule cells. To investigate the effects of PEDF on non-transformed retinal cells, we used primary cultures of neonatal albino rat retinas, where the three principal cell types of the retinal layers (neuronal, glial and epithelial) were all present and focussed our attention on RPE cells, which are of special relevance for retinal pathophysiology. PEDF had a dramatic effect on these cells. They showed a modified phenotype, with larger dimensions, higher cytoplasmic spreading, presence of phagocytic vacuoles, development of wide intercellular contacts, and increase and maturation of pigment granules. These results suggest that PEDF may have a role in regulating RPE cell differentiation.

Prevention of Glutathione Depletion-Induced Apoptosis in Cultured Human RPE Cells by Flupirtine

We have recently reported that the non-opiate analgesic, flupirtine, counteracts apoptosis in cultures of human retinal pigmented epithelial (RPE) cells induced by deprivation of serum, oxygen and glucose (experimental ischaemia). In the present study, human RPE cells grown on coverslips were treated with buthionine sulphoxamine (BSO), a compound that inhibits glutathione biosynthesis. BSO caused a dose-dependent reduction in culture density and an increase in the number of cell nuclei that were positively labelled by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) procedure. These data show that reduction of glutathione levels causes apoptosis in the RPE cultures. When flupirtine gluconate was co-incubated with BSO, it dose-dependently prevented the induction of apoptosis. The most effective concentration of flupitine found to inhibit cell death caused by BSO (1 µM - 1 mM) was 100 µM. The presence of serum (2% or 10%) in the culture medium did not have any effect on the outcome of apoptosis and overall cell death caused by BSO. Futhermore, melatonin, also known to reduce experimental ischaemia-induced overall cell death and apoptosis of cultured RPE cells had only a mild protective effect at 1 mM. The combined data suggest that flupirtine prevents apoptosis by increasing the cellular levels of reduced glutathione and/or protects the cells against the damaging effects of reactive oxygen species (ROS) that are produced subsequent to inhibition of glutathione production.

Culture of human retinal pigment epithelial cells from peripheral scleral flap biopsies

Purpose. We studied various methods for harvesting retinal pigment epithelium (RPE) biopsies from cadaver human eyes of donors over age 60 years. Our goal was to harvest cells for possible autologous RPE cell transplantation in patients with age-related macular degeneration and to test the viability of the RPE after isolation by evaluating explant growth in culture.Methods. Choroid-RPE biopsies were excised from enucleated human eyes. The RPE was separated from the choroid by treatment with type IV collagenase. RPE patches were cultured. After 100–500 cells had grown out from the explant, the primary cultures were passaged.Results. There was no clear effect of donor age on the ability to establish primary RPE cultures with good morphology from biopsies 2 × 2–10 × 10 mm2 in size. Biopsies 6 × 6 mm2 or larger produced satisfactory primary cultures more than 70% of the time. The number of viable RPE cells (defined as the number of cells adherent to the culture dish 24 h after plating) obtained after enzymatic separation of the RPE and choroid was an important determinant of our ability to establish primary cultures and passage the cells. Primary cultures with good cellular morphology were obtained 100% of the time when RPE explants >4 mm2 in size were obtained from the biopsy specimen. Seventy-three percent of the biopsies yielding explants >4 mm2 in size were successfully passaged.Conclusions. These results suggest that peripheral scleral flap biopsies in aging donors can be used to establish RPE explant primary cultures. These cultures may be suitable as a source for autologous RPE transplantation in patients. Curr. Eye Res. 17: 392–402, 1998.

Mechanism of Tamoxifen's Retinal Toxicity, Studied in Pig Pigment Epithelial Cell Cultures

The anticancer drug tamoxifen is widely used in breast cancer therapy. Tamoxifen has been reported to cause ocular toxicity and impairment of vision in epidemiological studies. To study the possible role of an excitotoxic mechanism in the ocular toxicity of tamoxifen, we investigated the effect of tamoxifen on retinal pigment epithelium (RPE) glutamate uptake in vitro. RPE, a layer of cells between photoreceptors and choroidal capillaries, contributes to the regulation of the concentration of the major excitatory amino acid, glutamate, in the sub-retinal space. Dysfunction in RPE glutamate uptake can lead to accumulation of extracellular glutamate and can cause various excitotoxic effects in the retina. The study was conducted by using cultured pig RPE cells. Six different tamoxifen citrate concentrations, ranging from lμM to 100μM, and [3H]-L-glutamate were added to the culture medium. To specify the glutamate uptake, 1mM dinitrophenol was added and a Na+-free culture was used. Due to the anti-oestrogenic character of tamoxifen, the possible effect of β-oestradiol on the glutamate uptake of RPE was also examined. The results show that glutamate uptake by RPE cells was reduced in the presence of tamoxifen, and that the reduction was dose-dependent. These results suggest that tamoxifen exposure could lead to the extracellular accumulation of glutamate. Disturbances in glutamate uptake can cause eye toxicity via an excitotoxic mechanism. The glutamate uptake of RPE cells was reduced under Na+-free conditions and was also reduced in the presence of dinitrophenol.

Liposome uptake by human retinal pigment epithelial cells in culture

PURPOSE. To examine the internalization of liposomes containing various phospholipids by human retinal pigment epithelial (RPE) cells in culture. The internalization was compared to that exhibited by macrophages and fibroblast cells. METHODS. The uptake into cells was monitored by using a non-degradable, radioactive cholesterol-hexadecyl ether derivative and a pH-sensitive, water-soluble fluorescent dye. Variables tested to determine their effect on uptake included time, phospholipid concentration, the presence or absence of serum in medium and the presence or absence of cholesterol in the liposome bilayer. The most avidly ingested liposome was tested to determine its ability to deliver a liposome-dependent drug. RESULTS. Liposome uptake was time and concentration dependent. Uptake for RPE was maximal in serum-free media, while the opposite was true for the other cells tested. Cell survival following exposure to fluoroorotic acid-containing liposomes correlated with the radioactive cholesterol-hexadecyl ether and fluorescent dye uptake data. CONCLUSIONS. No single liposome carrier or set of conditions tested appeared to be optimum for the delivery of a cytotoxic agent to these three cell types. This bears consideration when designing strategies for the prevention and treatment of intraocular proliferative diseases such as proliferative vitreoretinopathy (PVR).

Retinal pigment epithelial cell culture as a model of the blood-brain barrier

Retinal pigment epithelial cell culture as a model of the blood-brain barrier

Retinal pigment epithelial cell culture as a model of the blood-brain barrier

Retinal pigment epithelial cell culture as a model of the blood-brain barrier

INFLAMMATORY CYTOKINES INDUCE INTERCELLULAR ADHESION MOLECULE-1 (ICAM–1) mRNA SYNTHESIS AND PROTEIN SECRETION BY HUMAN RETINAL PIGMENT EPITHELIAL CELL CULTURES

Retinal inflammatory diseases in man are associated with an upregulation in the expression of intercellular adhesion molecule-1 (ICAM–1) in cells within the retina and with an increase in soluble ICAM–1 within the vitreous. These studies suggest that this protein may contribute to immunopathological processes within the eye. The effects of inflammatory mediators on the regulation of the expression and secretion of ICAM–1 by human retinal pigment epithe-lial cell cultures (HRPE) were investigated in order to identify the possible source of soluble ICAM–1 and the conditions which enhance its production. Immunofluorescence studies on TNF–α and/or IFN–γ treated HRPE cells demonstrated cellular expression of ICAM–1 which was predominantly localized to intercellular junctions. Moreover, treatment of HRPE for 24 h with tumour necrosis factor alpha (TNF–/) (10 ng/ml), interferon gamma (IFN–γ) (500 u/ml), interleukin 1 alpha (IL–1α) (10 ng/ml) and IL–1β (10 ng/ml) results in the secretion of ICAM–1, ranging from 9 to 13 ng per 10 6 cells. IFN–γ acts synergistically with (TNF–α) and IL–1 in the secretion of ICAM–1 by HRPE. Only 1.75 ng of soluble ICAM–1 was detected in untreated HRPE cells. In contrast, lipopolysaccharide (LPS), IL–6, IFN–α or TGF–β did not exhibit any influence on ICAM–1 secretion by these cells. Northern blot analysis reveals an increased expression of ICAM–1 mRNA in HRPE stimulated with IFN–g, TNF–α or IL–1 for 24 h. In untreated cells, ICAM–1 mRNA is not detectable. There is a progressive increase in ICAM–1 mRNA levels in cytokine-treated HRPE, that reaches steady state by 12 h. Furthermore, a close correlation is noted between ICAM–1 mRNA levels and the secretion of ICAM–1 protein, suggesting regulation at the level of gene transcription. ICAM–1 secretion by RPE might actively participate in the immune reactions in the retina, by recruiting and activating lymphocytes, and contribute to the immunopathological processes in inflammatory diseases.