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Purpose: FLVCR, BCRP, and PCFT/HCP-1 represent the three heme transporters identified thus far in mammalian cells, but there is very little known about their expression and regulation in the retina. In this study, the expression of these transporters in mouse retina and retinal pigment epithelium (RPE) and their regulation in the iron-overload disease hemochromatosis were examined., Methods: The expression of FLVCR, BCRP, and PCFT in mouse retina and primary mouse RPE cells was studied by RT-PCR and immunofluorescence. Polarized localization of the transporters in RPE was studied by co-localization using a specific marker of the RPE apical membrane. Uptake of heme in primary RPE cells was determined using zinc-mesoporphyrin, a fluorescent heme analogue. The regulation of heme transporters by iron overload was studied in two genetic models of hemochromatosis (HFE-null mouse and HJV-null mouse) and in two nongenetic models of iron overload (cytomegalovirus infection and treatment with ferric ammonium citrate)., Results: All three heme transporters were expressed in the retina and RPE. In the RPE, the expression of FLVCR was restricted to the apical membrane, and the expression of BCRP and PCFT was restricted to the basolateral membrane. In all cases of iron overload, the expression of FLVCR and PCFT was upregulated and that of BCRP was downregulated., Conclusions: Hemochromatosis is associated not only with excessive accumulation of free iron in the retina and RPE but also with excessive accumulation of heme. Since heme is toxic at high levels, as is free iron, heme-induced oxidative damage may also play a role in hemochromatosis-associated retinal pathology.

Purpose: GPR91, a succinate receptor, is expressed in retinal ganglion cells and induces vascular endothelial growth factor (VEGF) expression. RPE also expresses VEGF, but whether this cell expresses GPR91 is not known. Excessive iron is also proangiogenic, and hemochromatosis is associated with iron overload. Therefore, we examined the expression and iron-dependent regulation of GPR91 in the RPE., Methods: GPR91 expression was examined by RT-PCR and immunohistochemistry. Hemochromatosis mice, cytomegalovirus (CMV) infection of retina, expression of CMV-US2 in RPE, and exposure of RPE to ferric ammonium citrate (FAC) were used to examine the iron-dependent regulation of GPR91 expression. VEGF expression was quantified by qPCR. Knockdown of GPR91 in ARPE-19 cells was achieved with shRNA., Results: GPR91 was expressed in RPE but only in the apical membrane. Retinal expression of GPR91 was higher in hemochromatosis (Hfe(-/-)) mice than in wild-type (WT) mice. Primary RPE cells from Hfe(-/-) mice had increased GPR91 expression compared with WT RPE cells. Iron accumulation in cells induced by CMV infection, expression of CMV-US2, or treatment with FAC increased GPR91 expression. VEGF expression in the Hfe(-/-) mouse retina was increased at ages younger than 18 months, but the expression was downregulated at older ages. The involvement of GPR91 in succinate-induced expression of VEGF in RPE cells was confirmed with GPR91-specific shRNA., Conclusions: GPR91 is expressed in the RPE with specific localization to the apical membrane, indicating that succinate in the subretinal space serves as the GPR91 agonist. Excessive iron in the retina and RPE enhances GPR91 expression; however, VEGF expression does not always parallel GPR91 expression.

Purpose: Both caspase-dependent and caspase-independent apoptosis contribute to retinal damage during murine cytomegalovirus (MCMV) retinitis, and TNF-α is among the inducers of apoptosis. The aim of this study was to determine the contribution of TNF-α by studying virus replication and apoptosis in immunosuppressed (IS) TNF-α(-/-) mice., Methods: IS TNF-α(-/-) mice or wild-type mice were inoculated with MCMV by the supraciliary route. Injected eyes were examined by plaque assay, electron microscopy, Western blot analysis (caspase-3, caspase-8, caspase-12, Bid, NF-κB, cFlip, XIAP), staining for MCMV early antigen, and TUNEL assay., Results: Although the titer of MCMV was similar in both groups, significantly more apoptotic cells were observed in the retinas of IS TNF-α(-/-) mice than in those of wild-type mice. The level of active caspase-3 was similar in both groups; however, more activated proteins for genes involved in the mitochondrial pathway (cleaved caspase-8, tBid) and endoplasmic reticulum (ER) stress (cleaved caspase-12) and, though less active, NF-κB subunits and antiapoptotic proteins (XIAP and cFlip) were detected in the TNF-α(-/-) eyes compared with wild-type mice., Conclusions: Although TNF-α is an inducer of apoptosis, the results of this study suggest that TNF-α is also antiapoptotic by the following mechanism: TNF-α activation of NF-κB promotes the production of the antiapoptosis genes, c-flip or XIAP, which, in turn, inhibit the activation of caspase-8 and the mitochondrial pathway or the activation of caspase-12 and ER stress.

Purpose: To develop a technique by which murine cytomegalovirus (MCMV) infection can be confirmed and monitored in vivo in various ocular compartments and to investigate the dynamics and time course of primary ocular CMV infection., Methods: The ability of recombinant MCMV-expressing enhanced green fluorescent protein (eGFP) to serve as a tool to monitor the in vivo dynamics of experimental intraocular CMV infection was examined. Immunocompetent BALB/c mice were infected subretinally with eGFP-MCMV. Confocal scanning laser ophthalmoscopy (SLO) was used to visualize viral spread in vivo on sequential days after infection. Eyes were processed for histology and immunofluorescence microscopy to confirm viral infection and replication by means of GFP signal., Results: Retina was readily permissive to primary infection with eGFP-mCMV, and fluorescent signal was detected by SLO 24 hours after subretinal injection, with scattered foci around the posterior pole of the retina. GFP levels in the retina reached a maximum on day 6. Signal in the iris developed from day 4 and lasted until day 25. Examinations of retinal and iris tissue wholemounts by immunofluorescence revealed signal localized to the outer retina, iris stroma, and anterior lens capsule., Conclusions: The ability to noninvasively monitor infectious agents in the eye may improve current knowledge of the course and pathogenesis of intraocular infections and could lead to further clarification of the mechanisms by which the immune system responds to intraocular pathogens.

Purpose: An organotypic retinal culture model was used to determine the pattern of murine cytomegalovirus (MCMV) infection and whether apoptosis is induced in MCMV-infected cultured retinas., Methods: Retinas harvested from C57BL/6 mice were individually cultured at 37 degrees C on 3-microm filter inserts placed in 24-well plates. Some retinas were infected with MCMV (5 x 10(5) PFU/well). At days 4, 7, and 11 after infection (pi), the culture medium and cultured retinas were collected for examination., Results: Replicating virus was recovered and viral early antigen (EA)- and late antigen (LA)-positive cells were observed in the MCMV-infected retinal cultures. Most MCMV-infected cells were glia and horizontal cells. Infection resulted in atrophy of the photoreceptor cells and cytomegaly. Apoptosis of uninfected bystander cells, including photoreceptor cells and horizontal cells, was observed. TNF-alpha was produced by activated microglia during MCMV infection of the retina. Mouse apoptosis microarray studies, caspase activity studies, and RT-PCR studies showed that the genes involved in both the death receptor-mediated apoptotic pathway and the mitochondrial pathway were upregulated., Conclusions: Many aspects of MCMV infection of retinal cultures parallel those observed during MCMV retinitis in mice. Thus, this in vitro system may be used to explore the role of apoptosis of uninfected retinal cells and the contribution of cytokines and other modulators to the pathogenesis of CMV retinitis.

Purpose: Previous results suggest that apoptosis is involved in the pathogenesis of murine cytomegalovirus (MCMV) retinitis. To explore the mechanism underlying retinal apoptosis in MCMV retinitis, this study was initiated to determine whether the tumor necrosis factor receptor (TNFR)1-TNF pathway is involved in apoptosis during MCMV retinitis., Methods: The left eyes of nonimmunosuppressed (non-IS) BALB/c mice, immunosuppressed (IS) BALB/c mice, TNFR1(-/-) C57BL/6 mice, and wild-type C57BL/6 mice were inoculated with MCMV k181 by way of the supraciliary route. On postinoculation days 3, 7, and 10, injected eyes of non-IS control and IS experimental mice were removed for RT-PCR for TNF-alpha and TNFR1. Protein expression of TNF-alpha, caspase-8, and caspase-3 was determined by staining frozen sections and performing Western blot analysis and quantitative ELISA. Apoptotic cells were identified by TUNEL labeling., Results: In IS BALB/c mice, TNF-alpha mRNA and protein were detected in MCMV-infected eyes throughout the infection. Activation of caspase-3 and caspase-8 was observed. Most of the TNF-alpha-expressing cells were MCMV-infected RPE cells or macrophages derived from RPE cells. TNF-alpha was observed in the area of apoptotic retinal cells, and the level of this cytokine corresponded to the extent of the retinal abnormality and to the number of apoptotic cells. In non-IS MCMV-infected BALB/c mice, TNF-alpha was expressed early in the retinas of MCMV-infected eyes, but its expression was decreased thereafter. TNFR1 mRNA was increased in IS and non-IS BALB/c after MCMV infection. More apoptotic cells were observed in the retinas of non-IS MCMV-infected wild-type C57BL/6 mice than in the retinas of non-IS TNFR(-/-) mice., Conclusions: These results suggest that the TNFR1-TNF pathway is involved in the induction of apoptosis and the exacerbation of retinal abnormality during MCMV retinitis. Furthermore, because TNF-alpha and TNFR1 were present in IS and non-IS mice, TNF-alpha-induced retinal apoptosis during MCMV infection is not T-cell dependent.

Purpose: The purposes of this study were to identify iNOS-producing retinal cells and to determine whether lack of iNOS facilitates MCMV spread and replication in the retina., Methods: Immunosuppressed (IS) iNOS(-/-) mice or C57BL/6 (wild-type) mice were inoculated with 5 x 10(4) PFU of MCMV K181 strain (K181) via the supraciliary route. Injected eyes were collected at several times after inoculation and examined by plaque assay for replicating virus, RT-PCR for iNOS RNA, Western blot for iNOS protein and by staining for MCMV early antigen (EA), iNOS, and retinal cell antigens., Results: iNOS mRNA and iNOS proteins were expressed in the MCMV-injected eye of wild-type mice. Most iNOS-producing cells were F4/80-positive, including macrophages, RPE-derived macrophages, and resident microglia. Significantly higher titers of virus were recovered from the injected eyes, and more infected cells were detected in the retina of IS iNOS(-/-) mice than in IS wild-type mice. Retinal necrosis and loss of retinal architecture throughout the retina were noted in IS iNOS(-/-) mice, whereas cytomegalic cells and retinitis were present only in the peripheral retina of IS wild-type mice., Conclusions: iNOS produced by macrophages, especially resident macrophages including microglia and RPE derived macrophages, plays an important role in limiting spread of MCMV in the retina.

Purpose: To determine the role of apoptosis in prevention and/or exacerbation of retinal disease in a mouse model of cytomegalovirus retinitis., Methods: Immunocompetent or T-cell- depleted BALB/c mice were injected with murine cytomegalovirus (MCMV) by supraciliary injection. On sequential days after infection, mice were killed, and eyes were harvested for cryosectioning or for DNA extraction. Ocular sections were stained with monoclonal antibodies specific for MCMV or for T cells or used in the TdT-dUTP terminal nick-end labeling (TUNEL) assay to detect apoptotic cells., Results: In immunocompetent BALB/c mice, TUNEL assays revealed that a large area of the retina was apoptotic in relation to the relatively small number of MCMV-infected cells that were observed in the subjacent choroid and/or retinal pigment epithelium. In infected eyes from T-cell- depleted mice, there were more TUNEL-positive cells, and the areas of apoptosis were more extensive than in immunocompetent mice. These observations correlated with the increased extent of MCMV infection that is observed in the eyes of T-cell- depleted mice. However, irrespective of immune status, TUNEL-positive apoptotic cells were present mainly in areas of the retina overlying areas of MCMV-infected choroid and/or retinal pigment epithelium. More intense DNA laddering, indicative of increased apoptosis, was observed in the posterior segments of the eyes of T-cell- depleted mice after supraciliary inoculation with murine cytomegalovirus compared with less intense DNA laddering in the posterior segments of eyes of immunocompetent MCMV-infected mice., Conclusions: The ability of the mouse's immune system to control MCMV infections in some tissues depends on induction of apoptosis in virus-infected cells. However, in the retina, cells undergoing apoptosis were not virus-infected, a finding that suggests that apoptosis of uninfected retinal cells may play a role in the pathogenesis of MCMV retinitis.

Purpose: Human cytomegalovirus retinitis, the most common ophthalmic infection of AIDS patients, has been modeled in BALB/c mice infected with murine cytomegalovirus by the supraciliary route. A series of depletion and adoptive transfer studies was performed to determine whether adoptive transfer of T cells protects mice from retinitis caused by murine cytomegalovirus infection after supraciliary inoculation and to determine which subset of T cells is responsible for protection., Methods: BALB/c mice were thymectomized and T cell-depleted by injection of monoclonal antibodies to CD4, CD8, or both. Murine cytomegalovirus (9 x 10(2) plaque forming units [pfu]) was injected into the supraciliary space. Experimental animals received murine cytomegalovirus-specific T cells or subsets of T cells 2 hours before virus injection, whereas control animals received herpes simplex virus type 1-specific T cells by tail vein injection. Eight days after virus injection, retinal pathology was scored by histopathologic examination of hematoxylin and eosin-stained ocular sections., Results: CD8+ T cell depletion was sufficient for development of retinitis after supraciliary injection of murine cytomegalovirus. Adoptive transfer of murine cytomegalovirus-specific T cells, but not herpes simplex virus type 1-specific T cells, provided protection from retinitis. Additionally, separation of the murine cytomegalovirus-specific T cells into CD8+ and CD4+ subsets before adoptive transfer showed that the CD8+ fraction of the adoptive T cells was responsible for protection., Conclusions: These results suggest that adoptive transfer of cytomegalovirus-specific T cells or T cell subsets might be used to treat or prevent cytomegalovirus retinitis in immunosuppressed human patients.

Purpose: The purpose of this study was to determine whether adoptive transfer of murine cytomegalovirus (MCMV)-immune lymph node cells prevents retinitis in immunosuppressed mice., Methods: Adult BALB/c mice were thymectomized and T-cell depleted using rat monoclonal antibodies specific for mouse CD4+ and CD8+ T-cells. The level of rat immunoglobulin G in the treated mice was monitored by enzyme-linked immunosorbent assay. Immune cells were labeled with PKH26-GH immediately before adoptive transfer, and flow cytometry was used to determine the percentage of adoptively transferred T-cells (PKH+, fluorescein isothiocyanate [FITC+]) in the spleens of the recipient mice 3 days after transfer. The ability of adoptively transferred cells to protect from retinitis was studied in T-cell-depleted mice injected with MCMV through the supraciliary route. Mice received 4 x 10(7) in vitro-restimulated MCMV-immune cells, 4 x 10(7) freshly isolated MCMV-immune cells, 4 x 10(7) freshly isolated ovalbumin-immune cells, or no cells (control group)., Results: The best time to balance depletion of endogenous T-cells with persistence of transferred cells was 3 weeks after T-cell depletion. Both restimulated and freshly isolated MCMV-immune cells conferred protection from retinitis. Freshly isolated ovalbumin-immune lymph node cells did not prevent retinitis, indicating that protection was virus-specific and merely was not because of transfer of antigen-activated lymph node cells., Conclusions: Adoptive immunotherapy has been used to prevent cytomegalovirus (CMV) infection in patients who have undergone transplantation, and, by extrapolation, the results of these studies suggest that adoptive immunotherapy with human CMV-specific immune cells might be used to prevent or ameliorate CMV retinitis in immunocompromised patients.

Purpose: The aims of this study were to determine whether disruption of the blood-retina barrier (BRB) increases spread of murine cytomegalovirus (MCMV) to the eye after intraperitoneal inoculation and whether systemic immunosuppression influences the location of MCMV in the ocular compartment., Methods: The BRB of the left eye of normal and immunosuppressed mice was disrupted by supraciliary inoculation of tissue culture medium followed 2 hours later by intraperitoneal injection of MCMV. Plaque assay of homogenized ocular tissue was used to determine the frequency of virus-positive eyes and the titer of virus in the eyes. Beta-galactosidase staining of frozen sections was used to locate virus in the eyes., Results: In nonimmunosuppressed mice, the frequency of virus isolation, as well as the titer of virus, were significantly higher in eyes in which the BRB had been disrupted. Although the frequency of virus isolation was the same in both eyes of immunosuppressed mice, the titer of virus was significantly higher in the eye in which the BRB had been disrupted. The most striking result was that the location of virus was different in the nondisrupted eyes of immunosuppressed mice than it was in the disrupted eyes of immunosuppressed mice. In the former, virus was seen only in the outer ocular structures (conjunctiva, sclera, lacrimal gland), whereas in the latter, virus was observed in the retina and anterior segment (iris, ciliary body) as well as the outer ocular structures., Conclusions: The results of these studies suggest that ocular damage followed by increased spread of virus to and within the eye during systemic infection with CMV may be one mechanism by which development of CMV retinitis is facilitated in patients with acquired immune deficiency syndrome.

Purpose: Human cytomegalovirus retinitis is the most common blinding complication of acquired immune deficiency syndrome. However, the pathogenesis of the disease is poorly understood. The authors sought to characterize intraocular viral replication after systemic murine cytomegalovirus (MCMV) infection in the normal and immunosuppressed Balb/c mouse., Methods: Normal or immunosuppressed mice (400 rads radiation plus antilymphocyte serum) were infected intravenously with a recombinant MCMV (RM408) that carries an MCMV IE1 promoter--LacZ insert. In vivo MCMV replication and its tissue distribution were monitored by beta-gal activity with x-gal staining on frozen tissue sections of multiple organs harvested from infected mice at different time points after inoculation., Results: MCMV replication within the eye can be detected in the immunosuppressed Balb/c mouse but not in the normal host. Intraocular viral replication was noted first, and most frequently, in the ciliary body and was mainly restricted to the uveal tract. Intraocular viral replication coincided with the peak of systemic viral replication; however, the neurosensory retina was spared. In contrast, supraciliary inoculation of MCMV in the immunosuppressed Balb/c mouse resulted in massive viral replication and destruction of the neurosensory retina., Conclusions: This study demonstrated that intraocular MCMV replication after systemic infection requires systemic immunosuppression. Furthermore, the ciliary body is the portal of entry for the virus within the eye. MCMV can replicate in the epithelium of the uvea and retinal pigment epithelium, but it does not replicate within the neurosensory retina. The absence of MCMV replication within the neurosensory retina is not caused by either a defect in the recombinant virus or the inability of the host tissue to support viral replication.