Cytokine modulation of costimulatory molecules on human fetal retinal pigment epithelial cells.

PURPOSE: Experiments were performed to evaluate the effect of various pro- and anti-inflammatory cytokines on the human fetal retinal pigment epithelium's (HFRPE) expression of major histocompatibility complex (MHC) and costimulatory molecules. METHODS: Pure cultures of HFRPE cells were isolated. HFRPE cells were incubated in the presence of Interferon-gamma (IFN-gamma), IFN-beta, Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta), Tumor Growth Factor-beta (TGF-beta), and a combination of IFN-gamma and TGF-beta (pre-incubation and simultaneously incubated). The expression of MHC class I and class II, Intercellular cell adhesion molecule (ICAM-1), B7-1 (CD80), and B7-2 (CD86) molecules was quantitatively analyzed by flow cytometry. RESULTS: The cultured HFRPE cells expressed high levels of MHC class I and low levels of MHC class II and ICAM-1 molecules. After culture with the above mentioned cytokines, IFN-gamma up-regulated the HFRPE's expression of MHC class II and ICAM-1. IFN-beta and IL-beta1 only up-regulated the expression of ICAM-1. TGF-beta was unable to suppress the up-regulatory effect of IFN-gamma in HFRPE cells (pre-incubated and simultaneously incubated). The other cytokines did not have any significant effect on HFRPE's expression of MHC I and II or the selected costimulatory molecules. CONCLUSIONS: Our findings indicate that TGF-beta cannot suppress up-regulating effects of IFNgamma- on HFRPE's expression of MHC and costimulatory molecules. Overall, the weak or lack of expression of costimulatory molecules after stimulation with various cytokines further confirms that HFRPE cells are weak antigen presenting cells.

Human fetal retinal pigment epithelium-induced cell cycle arrest, loss of mitochondrial membrane potential and apoptosis.

PURPOSE: To investigate the mechanism of action of the soluble immune suppressive product secreted by human fetal retinal pigment epithelial (HFRPE) cells in a model system using the human T-cell line Jurkat (Jkt). METHODS: Pure HFRPE cells were isolated and cultured. The supernatants of both nonactivated and IFN-gamma-activated HFRPE cells were isolated. Cells from the human T-cell line Jkt were incubated either in standard culture medium or in the supernatant isolated from HFRPE cells. In the first assay Jkt cell proliferation was measured by [(3)H]thymidine incorporation. In the second assay Jkt cell apoptosis was examined for annexin V staining by flow cytometry. In the third assay Jkt cell division was evaluated with carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye. In the last assay the mitochondrial transmembrane potential of Jkt cells was measured with the cationic lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)]. In all the assays the effect of supernatants isolated from both nonactivated and IFN-gamma-activated HFRPE cells were compared with standard culture medium. The involvement of antiapoptotic human gene bcl-x(L:)was determined by using a Jkt cell line that was stably transfected with bcl-x(L:). RESULTS: The supernatant isolated from HFRPE cells significantly suppressed the cell division in Jkt cells and induced apoptosis. These effects were stronger when the supernatant was isolated from IFN-gamma-activated HFRPE cells. The apoptosis pathway induced by the secreted product of HFRPE cells involved the early disruption of mitochondrial transmembrane potential. Although the overexpression of bcl-x(L) gene rescued the Jkt cells from supernatant-induced apoptosis, it could not restore the proliferation of Jkt cells. CONCLUSIONS: These data suggest that HFRPE cells secrete a product that initiates an early cell cycle arrest in the human T-cell line Jkt, which is followed by the activation of an apoptotic pathway that involves the loss of mitochondrial membrane potential. The latter could be prevented by bcl-x(L) overexpression. Also these data suggest that the HFRPE-induced T-cell apoptosis may play a significant role in maintaining the immune privilege in the subretinal space.

Iris pigment epithelial cell translocation in exudative age-related macular degeneration. A pilot study in patients.

BACKGROUND: This prospective, non-controlled pilot study investigates the practicability of IPE translocation and functional outcome in ARMD patients. Removal of submacular choroidal neovascularization (CNV) in age-related macular degeneration (ARMD) is usually associated with RPE damage and poor visual prognosis. Homologous RPE transplants fail to preserve macular function, possibly due to immune rejection. Instead of homologous RPE, we suggest translocating autologous iris pigment epithelium (IPE), building on earlier evidence from animal and in vitro investigations that IPE can substitute RPE functions in the experimental animal. Immunological cell rejection is avoided. METHODS: Four eyes with well-defined and eight eyes with ill-defined subfoveal CNV were submitted to operation and followed up for a minimum of 6 months. IPE cells were harvested from a peripheral iridectomy. A vitrectomy was performed. Submacular membranes were removed, and isolated IPE cells were injected into the subretinal space. Examinations included ETDRS visual acuity, fluorescein angiography, and SLO microperimetry. RESULTS: All patients underwent successful surgical removal of CNV and subretinal IPE injection. Compared to preoperative visual acuity (20/400-20/100) no significant change was observed after 6 months (20/320-16/80). A change of more than two ETDRS chart lines was defined as significant. One eye with preoperative ill-defined CNV developed a recurrence, leading to reduced visual acuity. In all patients, postoperative fluorescence angiography revealed early hyperfluorescence (window defect) in the surgically denuded area. Central fixation was demonstrated in 50% of eyes. CONCLUSIONS: Preliminary data suggests that IPE translocation in submacular surgery for ARMD can preserve but not improve preoperative visual acuity over 6 months. Functional results are promising compared to submacular membrane extraction alone and RPE transplantation. Continued research on improvement of IPE translocation seems justified.

A model for xenogenic immune response.

PURPOSE: To develop a model for analyzing the immune response after xenogenic human fetal retinal pigment epithelium (HFRPE) transplantation. MATERIALS AND METHODS: Pure sheets of HFRPE cells were isolated and attached to poly (DL-lactide-co-glycolide) polymer films and HFRPE spheroids were formed. The spheroids were transplanted into the subretinal space of New Zealand albino rabbits and were observed for 5 months. Bare polymer films were transplanted into the subretinal space of Dutch Belted pigmented rabbits, serving as control. RESULTS: The polymer film was biodegraded within 3 weeks in the subretinal space. No signs of inflammation in the retina or choroid were observed. The HFRPE spheroids were easily transplanted into the subretinal space. The immune response was followed with ophthalmoscopy. Light microscopy indicated a localized immune response in the transplanted area in which the retina and the choroid were infiltrated with immune cells. This infiltration was denser in the choroid. CONCLUSIONS: HFRPE spheroid transplantation may be utilized as a model for studying the xenogenic immune response after HFRPE transplantation. This model may also have applications in evaluating the role of immune suppressive agents in preventing rejection after HFRPE transplantation.

Human fetal retinal pigment epithelium suppresses the activation of CD4(+) and CD8(+) T-cells.

BACKGROUND: The suppressive effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human CD4(+) and CD8(+) T-cells was evaluated in vitro. METHODS: Pure populations of CD4(+) and CD8(+) T-cells were isolated from human peripheral blood-derived buffy coats by negative immunomagnetic selection. The purity of the cells was examined by flow cytometry using anti-CD3-FITC, anti-CD4-FITC, anti-CD8-PE, and anti-CD20-PE mAbs. HFRPE cells were isolated from fetal eyes and pure cultures were obtained. The effect of normal or IFN-gamma-activated HFRPE cells at early (P3) or late (P6) passages on the activation of CD4(+) and CD8(+) T-cells was assessed in two different T-cell activation assays. In both activation models anti-CD3 mAb (OKT3) provided the antigen-specific signal. The secondary signal for the activation of CD4(+) and CD8(+) T-cells was provided with anti-CD18 mAb (TS1/18) and anti-CD28 mAb (9.3) in the first and the second assay respectively. Cross-linking of these soluble mAbs was performed with sheep-anti-mouse IgG-coated latex beads. The T-cell activation was determined by cell proliferation measured by [(3)H]thymidine incorporation. In each activation assay T-cells were incubated with HFRPE cells in a ratio of T-cells to HFRPE of 1:1 or 1:4. RESULTS: CD4(+) and CD8(+) T-cells were activated by cross-linking CD3 and CD18 in the first assay (CD3/CD18) and CD3 and CD28 in the second assay (CD3/CD28). In both assays HFRPE inhibited the activation of CD4(+) and CD8(+) T-cells. IFN-gamma-activated HFRPE cells totally suppressed the T-cell activation at a 1:1 ratio. This suppressive effect was weaker at lower cell ratios. Some donor variation was observed in the inhibition at the lower cell ratios, especially for the inhibition of CD8(+) T-cell activation with anti-CD3/CD18. The passaging of HFRPE cells did not alter their suppressive effect on CD4(+) and CD8(+) T-cells. CONCLUSIONS: HFRPE cells suppressed the activation of both CD4(+) and CD8(+) T-cells in vitro. These findings suggest that RPE-induced immune suppression may play a significant role in maintaining immune privilege in the subretinal space.

Human fetal retinal pigment epithelial cells induce apoptosis in allogenic T-cells in a Fas ligand and PGE2 independent pathway.

PURPOSE: To evaluate the inhibitory effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human T-cells. METHODS: Pure cultures of HFRPE cells were incubated with purified human T-cells in three different activation assays: 1) allogenic peripheral blood mononuclear cells; 2) OKT3 coated beads in the presence of accessory cells; and 3) stimulation with phorbol ester and phytohemagglutinin. RESULTS: HFRPE cells suppressed the activation of T-cells in all three assays. The mechanism of HFRPE mediated T-cell suppression was apoptosis. The role of Fas ligand(FasL)/Fas-mediated T-cell suppression was excluded, since FasL protein or mRNA could not be detected on HFRPE cells with flow cytometry and by reverse transcriptase polymerase chain reaction, respectively. Additionally, the inhibitory effect of HFRPE cells could not be blocked by anti-Fas ligand or antagonistic anti-Fas antibodies. Moreover, HFRPE cells suppressed the proliferation of anti-CD3 mAb mediated T-cell proliferation of murine splenocytes isolated from lpr mice. The inhibitory effect of HFRPE cells was not PGE2 mediated, since indomethacin could not restore the T-cell activation. Although the HFRPE mediated T-cell apoptosis was cell-cell contact independent, it was not induced by secretion of TNF-alpha, TGF-beta, or IL-10. The ratio between HFRPE and T-cells had a major impact on the HFRPE's inhibitory effect. CONCLUSIONS: HFRPE cells suppressed the activation of human T-cells by induction of T-cell apoptosis through a process that involves the secretion of soluble factors. The HFRPE mediated T-cell suppression was dependent on the ratio between HFRPE and T-cells. This undefined pathway of T-cell apoptosis may play a role in the maintenance of immune privilege in the subretinal space and may reduce the severity of the immune response after HFRPE transplantation.

Cryoprecipitate: An autologous substrate for human fetal retinal pigment epithelium.

PURPOSE: To harvest thin membranes from cryoprecipitates isolated from human blood donors and utilize them as substrates for the adhesion of human fetal retinal pigment epithelial (HFRPE) cells. METHODS: Frozen human cryoprecipitates from anonymous blood donors were obtained from the blood bank. Thin cryo-membranes were harvested by their mixture with riboflavin-5-phosphate (R5P) and overnight exposure to ultra-violet light. Sheets of retinal pigment epithelium (RPE) were isolated from fetal eyes at 17-22 weeks gestational age. The sheets were subsequently attached to cryo-membranes. The morphology of the cells was examined with phase contrast and electron microscope. Cell proliferation was evaluated by their incorporation of 5-bromo-2'-deoxyuridine (BrdU). Functional viability was assessed by rod outer segment (ROS) phagocytosis. RESULTS: Thin membrane films were made from isolated human cryoprecipitates. Isolated sheets of pure HFRPE cells were attached to the membranes. The cells maintained their cuboidal morphology and did not dedifferentiate. The cells subsequently proliferated and migrated onto the culture plate, forming cellular monolayers. The cultured cells phagocytized isolated ROS. CONCLUSIONS: Cryoprecipitate membranes may provide an ideal source for the adhesion, cultivation, and transfer of HFRPE cells. Their autologous isolation from the recipient's blood grants an additional advantage for their application as a carrier for HFRPE transplantation into the subretinal space.

Human fetal retinal pigment epithelial cells induce apoptosis in the T-cell line Jurkat.

PURPOSE: To investigate the mechanism(s) involved in human fetal retinal pigment epithelium (HFRPE)-mediated T-cell death. METHODS: Pure HFRPE cells were isolated and cultured. Normal and interferon (IFN)-gamma-activated HFRPE from early and late in vitro passages were incubated with cells from the human T-cell leukemia line Jurkat (Jkt). Cultures were pulsed with [3H]-thymidine to measure Jkt cell proliferation. Jkt cells were evaluated for apoptosis either by staining with an ethidium bromide/acridine orange mixture (AO/EB) or with Annexin V-phycoerythrin. The role of Fas ligand (FasL) molecule in HFRPE-mediated apoptosis was assessed by using a mutant Jkt cell line (DD3), which is deficient in Fas-mediated signaling. The involvement of the antiapoptotic human gene bcl-xL was determined by using Jkt cells that were stably transfected with bcl-x(L). The role of cell- cell contact in the induction of apoptosis was evaluated in a transwell system in the presence or absence of neutralizing antibodies against IFN-gamma and tumor necrosis factor (TNF)-alpha. RESULTS: HFRPE cells inhibited the proliferation of Jkt cells by inducing apoptosis through a FasL-independent pathway. Passaging and IFN-gamma activation strengthened the inhibitory effect of HFRPE cells on the proliferation of Jkt cells. At lower HFRPE passages (P2), bcl-alphaL, overexpression rescued the HFRPE cell-mediated apoptosis. The separation of the cells by the transwell system did not affect the HFRPE cell-mediated suppression. This suppressive effect was not mediated by the secretion of IFN-gamma or TNF-alpha molecules. CONCLUSIONS: HFRPE cells suppressed the proliferation of Jkt cells by inducing apoptosis. HFRPE cells induced a stronger inhibitory effect on Jkt cells at higher in vitro passages. The HFRPE-induced apoptosis was not mediated through the FasL/Fas pathway or through the secretion of the apoptosis-inducing cytokines IFN-gamma and TNF-alpha. The bcl-xL gene may play a role in preventing HFRPE cell-induced apoptosis in Jkt cells. These combined results suggest that the HFRPE-mediated suppression of primary T cells may also be mediated by the induction of apoptosis. Therefore, the retinal pigment epithelium may play a role in the induction of immune privilege in the subretinal space.

Biodegradable polymer film as a source for formation of human fetal retinal pigment epithelium spheroids.

PURPOSE: To evaluate the attachment of human fetal rctinal pigment epithelial (HFRPE) cells to a biodegradable polymer film with subsequent formation of spheroids in vitro. METHODS: Ten biodegradable polymer films with different compositions were examined for their physical properties and ease of manipulation under a dissecting microscope. The film with the most suitable handling characteristics was chosen, and a purely isolated sheet of HFRPE cells was attached to it. The purity of the cells was assessed by their pigmentation and expression of cytokeratin. Proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdtJ). Cellular structure was analyzed under light and electron microscopes, and the functional capability of the cells was evaluated by rod outer segment (ROS) phagocytosis. RESULTS: The polymer film with composition 50:50 poly (DL-lactide) (PLA)/poly (DL-lactide-co-glycolide) (PLG) with an inherent viscosity of 1.03 dl/g was found to be the most suitable for handling under the microscope. Sheets of HFRPE cells attached to the polymer films within 48 hours and began to form spheroids. All the isolated cells were pigmented and expressed cytokeratin. They possessed a cuboidal morphology, numerous apical microvilli, and no sign of dedifferentiation. HFRPE cells produced extracellular matrix (collagen filaments) on their basal side, filling the cavities of the polymer film. The cells subsequently proliferated, incorporated BrdU, migrated onto the culture plate to form monolayers, and phagocytized ROS. CONCLUSIONS: Biodegradable polymer films can be used as a scaffold for the adhesion of the HFRPE sheet and formation of spheroids. Spheroids represent a source of high density and well-differentiated HFRPE cells that are easy to transfer. Furthermore, the stricture of the membrane makes it suitable for additional applications.

Growth of human fetal retinal pigment epithelium as microspheres.

BACKGROUND: The aim was to develop a three-dimensional cell culture system for human fetal retinal pigment epithelial (HFRPE) cells for in vitro cellular studies and for possible application in subretinal transplantation. METHODS: Pieces of freshly isolated HFRPE monolayer tissue were grown on crosslinked fibrinogen (CLF) films. The growth pattern and morphologic characteristics of the implanted tissue were studied using phase-contrast microscopy, photography, and light and electron microscopy. The cells were screened immunohistochemically for HLA-ABC, HLA-DR, ICAM-1, B7, and Cytokeratin. Cell proliferation was studied using 5-bromo-2-deoxyuridine incorporation. RESULTS: After attachment to CLF, HFRPE monolayer tissue formed small tumor-like formations, i.e. microspheres. HFRPE microspheres survived and proliferated in a floating state for at least 4 months. After attachment of the microspheres to the culture dish floor, formation of a confluent HFRPE cell monolayer with high proliferative activity was noted around the microspheres. HFRPE cells stained positive for HLA-ABC, ICAM-1, and cytokeratin and negative for B7 and HLA-DR. The microspheres could be easily detached from the dish and they were able to initiate similar growth after reattachment. CONCLUSION: HFRPE grown on CLF resemble a three-dimensional culture system with high yield of pure cells that can be useful for a wide variety of in vitro studies. Because of their adjustable size, spherical shape, and ability to initiate growth of cells with a high proliferative potential, HFRPE microspheres may be successfully utilized as a source of donor cells for subretinal transplantation.

The immunogenic potential of human fetal retinal pigment epithelium and its relation to transplantation.

PURPOSE: To perform a quantitative analysis of the expression of major histocompatibility molecules (MHC classes I and II) and costimulatory molecules by human fetal retinal pigment epithelial (HFRPE) cells and to evaluate their potential role in providing costimulatory signals for the activation of human T cells in vitro. METHODS: Pure HFRPE cells were isolated and cultured. The ability of HFRPE cells to express MHC class I and II and costimulatory molecules before and after incubation with interferon (IFN)-gamma was quantitatively analyzed by flow cytometry. The potential of HFRPE cells to activate human T cells was assessed in three different lymphocyte activation models. In the first model, anti-CD3 (OKT3)-coated beads were used to provide the T-cell receptor (TcR) signal. In the second model, the allogenic potential of HFRPE cells was assessed, and in the third assay a potent superantigen (SEA) was used to provide the TcR signal. T-cell activation was determined by cell proliferation, measured by [3H]-thymidine incorporation. RESULTS: The cultured HFRPE cells expressed low levels of MHC class I and ICAM-1 molecules. After incubation with IFN-gamma, the expression of MHC class I and ICAM-1 molecules was further upregulated, and the expression of MHC class II and VCAM-1 molecules was induced. The expression of the costimulatory molecules B7-1 and B7-2 was not observed in normal or activated HFRPE cells. In the first T-cell activation model, neither normal nor IFN-gamma-activated HFRPE cells could provide T-cell costimulation for anti-CD3 (OKT3)-coated beads. However, the autologous peripheral blood mononuclear cells (PBMCs; used here as the source of antigen-presenting cells) could provide costimulation for the T cells, inducing their proliferation. In the second T-cell activation assay, normal or IFN-gamma-activated HFRPE cells could not stimulate an alloresponse from the T cells, but they could induce a significant alloimmune T-cell response in the presence of PBMCs. In the third T-cell activation assay, the IFN-gamma-activated HFRPE cells were able to provide T-cell costimulation for the SEA-mediated activation. CONCLUSIONS: In these in vitro experiments, the IFN-gamma-activated HFRPE cells stimulated only the T cells with the potent superantigen SEA. In the absence of antigen-presenting cells, the HFRPE cells did not provide T-cell costimulation in an anti-CD3 mAb-coated bead system or induce significant alloimmune response. These results suggest that in transplantation between donors and recipients with different MHC molecules, the direct MHC peptide presentation by HFRPE cells may not induce a significant allospecific immune response. Nevertheless, an allospecific immune response could occur as a consequence of the indirect presentation, to the host T cells by the host antigen-presenting cells, of the HFRPE cells' derived MHC alloantigens.

Iris pigment epithelium transplantation.

BACKGROUND: Iris pigment epithelium (IPE) cells and retinal pigment epithelium (RPE) cells possess the same embryonic origin. It is also known that the pigmented epithelial cells in the eye have a high transdifferentiation potential. In this study we transplanted IPE cells into the subretinal space of albino Royal College of Surgeons (RCS) rats and evaluated their influence on the degeneration of the photoreceptors. METHODS: IPE cells of Long Evans rats were isolated and pure cultures were obtained. The isolated cells were transplanted into the subretinal space of RCS rats. Light microscopic and morphometric analysis were carried out. RESULTS: The IPE transplants survived in the subretinal space and attached themselves to the Bruch's membrane. The transplanted cells were able to delay the degeneration of the photoreceptors for up to 3 months. CONCLUSION: These results suggest that IPE cells could be successfully transplanted and survive in the subretinal space. In the transplanted eyes the photoreceptors were preserved for a period of 3 months. Further studies are needed to explore the capability of IPE cells to assume the main functions of RPE cells in the subretinal space and their potential in the therapy of selective degenerative diseases of the retina.

A new method of culturing and transferring iris pigment epithelium.

PURPOSE: To optimize a culture technique and transfer iris pigment epithelial (IPE) cells for cellular studies in vitro. METHODS: Porcine iris tissues were obtained, and IPE cells were isolated and cultured at high densities by plating them in the form of drops. Spherically shaped structures containing a high concentration of cells were formed after 7 to 10 days of culture. Cells were subcultured by transferring spheres to new culture dishes without employing enzymatic dissociation. The purity of IPE cells was determined by pigmentation and cytokeratin labeling. Proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine. Cellular structure was analyzed under the light and electron microscopes and function was assayed by rod outer segment phagocytosis. RESULTS: Iris pigment epithelial cells, when cultured at high densities, tended to form elevated spherical structures containing viable cells. The cultured cells were pigmented and showed positive labeling with a monoclonal cytokeratin antibody. The IPE cells proliferated and migrated from the spheres to form monolayers. Cells originating from the transferred spheres also continued to proliferate and to migrate in a similar manner to the originally cultivated cells to form monolayers after 7 to 10 days. These cells were able to phagocytose rod outer segments. CONCLUSIONS: This new method provides a simple method of culturing a large quantity of IPE cells. The high yield of pure IPE cells and the ease of transfer provide an ideal means to study them at the cellular level.

Iris pigment epithelial cells of long evans rats demonstrate phagocytic activity.

The phagocytic activities of iris pigment epithelial (IPE) cells and retinal pigment epithelial (RPE) cells of Long Evans rats towards latex beads and rod outer segments (ROS) were compared in vitro. IPE and RPE cells of Long Evans rats were isolated and pure cultures obtained. The cultures were incubated with latex beads, fixed, and analysed computer morphometrically, IPE and RPE cell cultures were also incubated with isolated ROS and examined using transmission electron microscopy. IPE cells were able to ingest latex beads. There was no significant difference between the number of latex particles phagocytized by IPE and RPE cells. After incubation with isolated ROS, IPE cells also recognized and ingested the ROS particles. However, the specific phagocytic capacity of IPE cells was 76% of that of RPE cells. The autologous IPE cells might have the potential to be used as an alternative to RPE cells for transplantation in the subretinal space.

Comparison of tight junction permeability for albumin in iris pigment epithelium and retinal pigment epithelium in vitro.

BACKGROUND: The degenerative retinal diseases are one of the major causes of visual loss in the western world. Although heterologous RPE transplants rescue the photoreceptors in the dystrophic rat model, rejection remains a major limiting factor. Given the common embryonic origin, iris pigment epithelial (IPE) cells might be able to take over the functions of retinal pigment epithelial (RPE) cells, serving as an autologous graft for transplantation and thereby preventing rejection. One of the main functions of RPE cells is the generation of tight junctions which form the outer blood-retinal barrier. In this study we compared the tight junction permeabilities of IPE and RPE cells isolated from Long Evans rats by measuring their albumin clearances. METHODS: IPE and RPE cells were cultured on semipermeable filter supports with and without the addition of 0.02% ethylenediaminetetraacetic acid (EDTA). At selected intervals, the albumin clearances of the IPE and RPE cells were measured spectrophotometrically and compared. The morphology of the cells was compared using electron microscopy and fluorescent labeling. RESULTS: IPE and RPE cells both restricted the passage of albumin in vitro. After the modulation of tight junctions with 0.02% EDTA, the clearance increased in both types of cells in a similar fashion. The morphology of tight junctions was visualized with electron microscopy. CONCLUSION: These results indicate that the functional barrier for macromolecules is similar in IPE and RPE cells in vitro. This raises the possibility that IPE cells would form tight junctions in the subretinal space, thereby substituting for the blood-retinal barrier normally formed by RPE cells.

Posterior drainage of intraocular fluid: an experimental approach on bovine cadaver eyes.

We evaluated the posterior drainage of saline in cadaver bovine eyes using different experimental procedures. The saline perfusion via a pars plana infusion into the vitreous cavity was measured in the following 5 different groups of 20 enucleated bovine eyes: 1, control; 2, vitrectomy; 3, vitrectomy + retinectomy; 4, vitrectomy + retinectomy + removal of retinal pigment epithelium (RPE); and 5, vitrectomy + retinectomy + removal of RPE + choroidectomy. The mean values obtained for the facility of saline outflow were as follows group 1, 0.0458 microliter min-1 mmHg-1; group 2, 0.0393 microliter min-1; group 3, 0.1308 microliter min-1 mmHg-1; group 4, 0.2288 microliter min-1; and group 5, 0.9985 microliter/min-1 mmHg-1. The retina appeared to be the major barrier to fluid movement from the vitreous to the chorioid, perhaps due to the lack of extracellular space and to the tight and impervious structure of the retina. The barrier function of the retina may explain the hypotony seen after rhegmatogenous retinal detachment and the decrease in intraocular pressure observed after the removal of silicone oil in eyes that had undergone retinectomy. Our results suggest that the reduction in intraocular pressure occurring after retinectomy is attributable to the new drainage pathway.

Epidermal dipeptide: a new regulatory factor in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the most severe complication of retinal detachment surgery, occurring approximately in one out of ten operated eyes. The proliferation of retinal pigment epithelial (RPE) cells and fibroblasts is a cornerstone in the pathogenesis of PVR. Since inhibitory peptides may take a part in the feedback mechanism underlying this proliferation, we assessed the effect of synthesized epidermal dipeptide, pyroglu-glyOH (EDP), on the proliferation of RPE cells and fibroblasts in vitro. The maximum inhibitory effect of EDP on the RPE cells was reached at concentrations ranging from 1.07*10(-13) to 1.07*10(-15) M. Its inhibitory effect on fibroblasts followed a similar pattern at all concentrations applied, 1.07*10(-6) to 1.07*10(-15) M. These results enhance the possibility that PVR may be due to an imbalance of inhibition/disinhibition mechanism under participation of several regulatory molecules like EDP. EDP might have the potential for reducing the risk of PVR.