Effect of TGF-beta on interferon-gamma-induced HLA-DR expression in human retinal pigment epithelial cells.

PURPOSE: Retinal pigment epithelial (RPE) cells express human leukocyte antigen (HLA)-DR (class II) antigens when stimulated with interferon gamma (IFN-gamma) and may be capable of local antigen presentation. The authors examined the effect of transforming growth factor-beta (TGF-beta), a cytokine normally found in the eye, on the expression of these immunoregulatory molecules in vitro and attempted to determine the mechanism by which this cytokine acts. METHODS: Human RPE cells were cultured in the presence of IFN-gamma and then stained immunohistochemically for HLA-DR antigens. TGF-beta 1 or TGF-beta 2 was added simultaneously with IFN-gamma or after 3 days of IFN-gamma treatment. In parallel experiments, RPE cells were pretreated with 4-phorbol-12 myristate-13 acetate (PMA), staurosporine, or calphostin C before stimulation with IFN-gamma or TGF-beta. Quantitative analysis was performed by fluorescence-activated cell sorting. RESULTS: IFN-gamma induced HLA-DR expression on RPE cells. Both TGF-beta 1 and TGF-beta 2 were able to inhibit this effect. These inhibitory effects of TGF-beta were augmented by pretreatment with either PMA or calphostin C. Pretreatment of the cells with PMA before stimulation with IFN-gamma downregulated HLA-DR expression. Staurosporine pretreatment suppressed HLA-DR expression by IFN-gamma-stimulated RPE cells, but this was not additive with TGF-beta. CONCLUSIONS: The authors conclude that TGF-beta 1 and TGF-beta 2 strongly inhibit the IFN-gamma-induced upregulation of class II antigens on human RPE cells. The modulation of these IFN-gamma and TGF-beta effects by calphostin C, staurosporine, and PMA treatment suggests involvement of the protein kinase C pathway.

Intercellular gap formation induced by thrombin in confluent cultured bovine retinal pigment epithelial cells.

PURPOSE: Thrombin is formed at the site of intraocular hemorrhage and may be important in the development of progressive retinal damage. The authors observed that thrombin-treated bovine retinal pigment epithelial (RPE) cell cultures develop intercellular gaps and initiated this study to examine in detail the effects of thrombin on RPE cell morphology, adhesion, and cytoskeleton. METHODS: Confluent cultures of bovine RPE cells were incubated for various times (0 to 24 hours) with alpha-thrombin (0.1 to 100 U/ml) or enzymatically inactive thrombin. Intercellular gaps were quantitated by light microscopy in ten representative fields (magnification X400) as number of gaps per field (gaps/f). RPE cytoskeleton was studied using immunofluorescent staining for vinculin and F-actin. The mechanism of thrombin-induced RPE cell gap formation was studied by preincubation with specific drugs, including a protein kinase inhibitor (staurosporine), protein kinase C inhibitors (H-7 and calphostin C), cyclic adenosine monophosphate (cAMP) inducer (forskolin), and cytoskeleton-disrupting agents (cytochalasin B or colchicine). RESULTS: Intercellular gaps (20 to 80 microns in diameter) were markedly increased in number in thrombin-treated cultures in a dose-dependent and time-dependent manner and were associated with an alteration in the distribution of F-actin and vinculin. Whereas control cultures showed 3.3 +/- 2.4 gaps/f, incubation with 8 U/ml of alpha-thrombin for 3 hours resulted in 44.8 +/- 15.3 gaps/f. These changes were most prominent shortly after the 3-hour coincubation, but the cultures did return to their original confluent state within 24 hours. Cultures treated with an enzymatically inactive thrombin showed fewer intercellular gaps than those treated with enzymatically active thrombin but had significantly more intercellular gaps than control cultures. Thrombin-induced intercellular gap formation was blocked by preincubation with forskolin (14.6 +/- 7.1 gaps/f), staurosporine (10.2 +/- 5.0 gaps/f), or H-7 (24.5 +/- 9.8 gaps/f). CONCLUSIONS: Exposure to an enzymatically active thrombin results in formation of intercellular gaps between cultured RPE cells. Inhibition of this phenomenon by protein kinase inhibitors and by a cAMP inducer suggests that this effect is mediated, at least in part, through protein kinase C- and cAMP-dependent pathways. Thrombin generation associated with intraocular hemorrhage may thus result in direct damage to the RPE monolayer, possibly via the same pathway(s).

Keratocyte loss and repopulation of anterior corneal stroma after de-epithelialization.

OBJECTIVE: To quantitate corneal keratocyte loss and repopulation of anterior stroma after de-epithelialization. METHODS: Fourteen white New Zealand rabbits, each weighing 2.3 to 3.2 kg, were divided into seven groups. Each rabbit underwent a bilateral 6-mm mechanical deepithelialization procedure. The rabbits were killed two at a time after 1, 3, 6, 8, 10, 14, and 28 days. The corneas were labeled for mitosis using 5-bromo-2-deoxyuridine and stained for keratocyte quantification. Three untreated rabbits were added as controls. RESULTS: Corneas that were not operated on showed a higher density of keratocyte nuclei within the anterior corneal stroma than in the posterior stroma. Following de-epithelialization, there was a decrease in the ratio of anterior-posterior keratocytes, with maximal decrease on the third postoperative day. Keratocyte repopulation was completed by day 14. Mitotic activity was seen on days 1, 3, and 6 in the anterior half of the de-epithelialized stroma. CONCLUSIONS: Anterior stromal keratocytes are lost after epithelial removal, but repopulation is complete within 2 weeks. These findings demonstrate a loss of keratocytes and their recovery after corneal epithelial removal, as well as an interaction between corneal epithelium and stroma in rabbits.

A new model of retinal pigment epithelium transplantation with microspheres.

OBJECTIVES: To develop a 3-dimensional carrier system for subretinal transplantation of human fetal retinal pigment epithelial (HFRPE) cells and to assess their growth pattern in the rabbit subretinal space. METHODS: After a standard 3-port vitrectomy, HFRPE cells grown as microspheres on cross-linked fibrinogen were introduced into the subretinal space of rabbits. The eyes were studied at 7, 14, and 30 days after surgery by ophthalmoscopy and light microscopy. RESULTS: Ophthalmoscopically, at day 7, 11 (61%) of the 18 eyes showed radiating hyperpigmentation around the transplanted HFRPE microspheres. The results of a histological examination revealed a monolayer outgrowth of HFRPE cells, overlying host retinal pigment epithelium. The control eyes revealed a patch of chorioretinal atrophy with lymphocytic infiltration around the microspheres. CONCLUSIONS: Human fetal retinal pigment epithelial cells grown as microspheres on cross-linked fibrinogen can be successfully transplanted into the subretinal space. Cells can survive for at least 1 month and form a monolayer over the host retinal pigment epithelium cells, with a mild local inflammatory response. The difference in inflammatory responses between the eyes that underwent transplantation and the control eyes may suggest a modulating effect of the HFRPE cells on inflammation, immunity, or both. This new xenogenic model may have importance in the study of subretinal transplant cell biology and the associated immune response. CLINICAL RELEVANCE: The results of this study may be important for better understanding of the mechanisms of retinal pigment epithelium cell behavior after transplantation. The proposed model may be applicable for future clinical and experimental investigations in the area of retinal pigment epithelium transplantation.

Minoxidil-induced alteration of corneal topography after radial keratotomy.

PURPOSE: To determine the antiproliferative effect of minoxidil on human corneal epithelium (hCE) proliferation in vitro and to assess whether topical minoxidil can significantly alter corneal topography after radial keratotomy (RK) by inhibiting myofibroblast activity in the keratotomy wound. SETTING: Corneal Research Laboratory, University of Chicago, Illinois, USA. METHODS: In the in vitro evaluation, proliferating hCE was exposed to minoxidil (0.1 to 2.0 mM) for 96 hours to determine the minimum inhibitory dose. Human corneal epithelium cell proliferation was assessed by the incorporation of bromodeoxyuridine (BRDU) into DNA. In the in vivo analysis, eight New Zealand albino rabbits had an eight-incision bidirectional RK on one eye and were divided into two groups. The control eyes (n = 3) received tobramycin and dexamethasone (TobraDex), ciprofloxacin hydrochloride (Ciloxan), and balanced salt solution (BSS) drops four times a day for 3 weeks, while the treatment eyes (n = 5) received TobraDex, Ciloxan, and minoxidil 1.0 mM drops four times daily for 3 weeks. The net change in corneal curvature at 3 weeks was analyzed with corneal topography. Myofibroblast activity in the keratotomy wound was assessed using alpha smooth muscle actin staining techniques. RESULTS: At concentrations of 1.0 mM and above, minoxidil caused a statistically significant, dose-dependent reduction in hCE cellular proliferation ranging from 29 to 44% (P < .05). Minoxidil (1.0 mM) caused a statistically significant central corneal flattening effect of 4.66 diopters (D) after RK in the treatment eyes compared with 1.11 D in the control eyes (P = .05). Histologically, minoxidil-treated keratotomy wounds lacked cells with contractile elements consistent with myofibroblast differentiation. Corneal epithelial wound healing was similar in both groups. CONCLUSION: At the appropriate dose, topical minoxidil may be a useful adjunctive treatment that can reduce the number of undercorrections after mini-RK without apparent toxicity to the corneal epithelium.

Immunochemistry with 5-bromo-2-deoxyuridine for visualization of mitotic cells in the corneal epithelium.

The purpose of this study was to determine whether the DNA of mitotic cells in the corneal and conjunctival epithelium can be labeled with 5-bromo-2-deoxyuridine (BrdU) immunostaining. Both corneas of four New Zealand white rabbits were deepithelialized in the center and the regenerated epithelium was evaluated for mitosis at 1, 3, 6, and 10 days. Unwounded corneas of three rabbits were labeled for baseline measurements. We administered the marker intravenously to all seven rabbits 15 h before scheduled killing. Immediately after killing, all of the globes were enucleated and histologic sections were prepared. In unwounded corneas, labeled cells were quantitated and the fraction of mitotic cells in the center of the cornea, in the periphery of the cornea, and in the conjunctiva were compared. In deepithelialized corneas, increase in mitosis in the central epithelium was quantitated. All of the unwounded eyes showed mitosis in the basal layer of both the corneal and conjunctival epithelium. In the center of the cornea 4.1 +/- 2.9% of the epithelial cells were labeled, in the corneal periphery 4.3 +/- 1.7% of the cells were labeled, and in the conjunctiva 4.1 +/- 1.9% of the epithelial cells were labeled, with a p value ranging from 0.84 to 0.99. In wounded corneas, when compared with cell counts in unwounded eyes, 52.6% of the epithelial cells were in mitosis on day 3, 13.9% were in mitosis on day 6, and by day 10 baseline values of 4.2% were obtained. We conclude that BrdU immunostaining is a safe, efficient, and less costly alternative to autoradiography for visualization of dividing corneal and conjunctival epithelium.

In vitro stimulation of retinal pigment epithelium proliferation by taurine.

Taurine is an amino acid that is essential for retinal integrity and function. Although it has been suggested that the ratio of melatonin to taurine in the interphotoreceptor matrix may regulate the phagocytosis of outer segments by retinal pigment epithelial (RPE) cells, the effect of taurine on the RPE has not been studied. Using cultured RPE cells, we found that in vitro taurine specifically stimulated proliferation of human and rabbit RPE, but had only minimal effect on cultured scleral fibroblasts. The RPE proliferation was due to more cells entering into S-phase and thus an increase in DNA synthesis, was not dependent upon cell density, and was most pronounced in the presence of a low concentration of fetal bovine serum.

Effect of leukopenia on experimental post-traumatic retinal detachment.

Macrophages are invariably present in the intraocular membranes of patients with traumatic proliferative vitreoretinopathy (PVR). There are two sources from which these macrophages could be recruited: adjacent tissues and the systemic circulation. In the study described herein, the role of circulating white blood cells and monocytes in experimental, traumatic PVR was studied. The circulating white blood cells of 20 rabbits were depleted by intravenous injection of strontium-89. Posterior perforating eye injury with subsequent intravitreal injection of autologous whole blood or autologous activated macrophages was then performed on these leukopenic animals. The experiments demonstrated that severe bone marrow depression reduced significantly the incidence of retinal detachments in eyes receiving whole blood, and reduced the severity of retinal detachments in eyes injected with activated macrophages. An association between the degree of leukopenia, monocytopenia, and protection from retinal detachment was demonstrated. These results support the hypothesis that macrophage infiltration is an important component of intraocular cellular proliferation, but does not exclude the role of other types of white blood cells in the pathogenesis of PVR.

[A model of the biological oscillator as the basis for the coordination of motor activity]. / Model' biologicheskogo ostsilliatora kak osnova koordinatsii dvigatel'noi aktivnosti.

System mechanisms, that underlied motor activity coordinations were investigated on the model of biological oscillator. It has been shown how the value of phase resettings induced by single stimuli sequences depends on the stimulation location in the cycle. The time of reaction to the stimulus has been determined as the function of stimulus location. In addition, the character of transitional process, after which the parameters of initial rhythmic activity completely become normal, has been determined. The data obtained have been interpreted for the model of temporal motor control, which was suggested by A. Wing and A. Kristofferson.

Cellular response in rabbit eyes after human fetal RPE cell transplantation.

BACKGROUND: This study was carried out to study inflammatory and proliferative cellular responses in the rabbit eye after subretinal transplantation of human fetal retinal pigment epithelial (HFRPE) cells. METHODS: 5-Bromo-2-deoxyuridine (BrdU)-labeled HFRPE cells were injected subretinally into rabbit eyes at three different concentrations. Macrophage, glial, and proliferative responses of the eye tissues were studied by immunohistochemistry and light microscopy at different times after the surgery. RESULTS: In transplanted eyes, the HFRPE cells were distributed irregularly either as multilayers or monolayers. In eyes receiving high-density cell suspensions, retinal breaks were seen. No retinal breaks were noted in the eyes receiving low-density HFRPE cell suspensions. The highest intensity of inflammatory response was seen at 4-14 days after surgery, with greater expression in transplanted eyes receiving high-density cell suspensions. The host cellular response was characterized initially by local infiltration of the retina and subretinal space by macrophages and glial cells. After day 14, a decline in the number of donor cells was noted in all eyes. At later stages the host cellular response was characterized mainly by local choroidal thickening and infiltration by inflammatory cells. Proliferative response was expressed mainly by retinal cells. CONCLUSION: Initial inflammatory and proliferative responses after the xenogenic human to rabbit HFRPE cell transplantation were expressed by retinal cells with later involvement of the choroid. Our results showed a decline in the number of donor cells starting from day 14 after the transplantation. This may suggest a possibility of rejection. The initial quantity of injected cells may be critical for the intensity of the immune and inflammatory responses.

Inhibition of experimental proliferative vitreoretinopathy in rabbits by suramin.

Proliferative vitreoretinopathy (PVR) is the most common cause for failure of retinal reattachment surgery. In a search for better pharmacologic treatment of PVR, we investigated the effect of intravenous injections of suramin on an experimental rabbit model of PVR. PVR was induced in rabbits by intravitreal injection of autologous fibroblasts. The experimental group (7 eyes) received intravenous injections of suramin (100 mg/kg body weight) every 3 days for 15 days, beginning 3 days before fibroblast injection. The control group (5 eyes) was treated similarly but received intravenous saline solution in place of suramin. A third group (4 eyes) received suramin according to the protocol above but did not receive intravitreal fibroblasts. The animals were examined by indirect ophthalmoscopy every 3 days and were sacrificed 14 days after the injection of fibroblasts. The serum levels of suramin were evaluated by high-performance liquid chromatography. The PVR was classified as stages I-V, based upon clinical findings. PVR developed in both experimental and control animals but was less severe in those treated with suramin. On day 14, the average stage of PVR in the control group was 3.8; in the suramin-treated group, however, the average stage was 2.4, which was significantly less than in the control group (p < 0.02). None of the rabbits in the third group showed pathologic changes. Serum levels of suramin were maintained at an average of 280.2 micrograms/ml and no apparent toxicity was found in the retina by histologic study.(ABSTRACT TRUNCATED AT 250 WORDS)

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.