Zika Virus Infection of Human Iris Pigment Epithelial Cells.

During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals.

Generation and Characterization of Anti-Glucosepane Antibodies Enabling Direct Detection of Glucosepane in Retinal Tissue.

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

Calf melanin immunomodulates RPE cell attachment to extracellular matrix protein.

PURPOSE: It is widely accepted that RPE melanin has a protective effect against oxidative damage in RPE cells. It is possible that an additional protective characteristic of melanin is the ability to modulate RPE cell immune response. In this study, in vitro modeling was used to probe the relationship between RPE pigmentation and immune response by monitoring IL-6 expression and secretion in calf melanin pigmented ARPE-19 cells seeded onto glycated extracellular matrix as a stressor. METHODS: ARPE-19 cells were left unpigmented or were pigmented with either calf melanin or latex beads, and were then seeded onto RPE-derived extracellular matrix (ECM) or tissue culture-treated plates (no ECM). ECMs were modified by glycation. IL-6 expression was measured using qPCR and IL-6 secretion was determined using an ELISA, both at 30 min and 24 h after seeding. MTT assay was used to quantify cell attachment to glycated matrices 30 min after seeding. In unpigmented ARPE-19 cells, rate of cell attachment to substrate was monitored for 60 min after seeding using a hemacytometer to count unattached cells. Additionally, cell viability was evaluated using the Neutral Red assay 24 h after seeding. RESULTS: A significant increase in IL-6 expression was observed in calf melanin pigmented cells versus latex bead and unpigmented controls (p < 0.0001) 30 min after seeding onto ECM. Twenty-four hours after seeding, a significant decrease in IL-6 expression was observed in calf melanin pigmented cells (p < 0.0001) versus controls, implicating down-regulation of the cytokine. Additionally, calf melanin pigmented cell populations showed significant increase in attachment compared to unpigmented controls on either no ECM or unmodified ECM. CONCLUSIONS: Pigmentation of RPE cells with calf melanin resulted in significant changes in IL-6 expression regardless of ECM modification, in vitro. These findings suggest that melanin in the RPE may participate in immune response modulation in the retina with particular regard to cell attachment to protein substrates. The results of this study further implicate the role of chemical changes to melanin in regulating inflammation in retinal disease.

Immune Privilege and Eye-Derived T-Regulatory Cells.

Certain cellular components of the eye, such as neural retina, are unable to regenerate and replicate after destructive inflammation. Ocular immune privilege provides the eye with immune protection against intraocular inflammation in order to minimize the risk to vision integrity. The eye and immune system use strategies to maintain the ocular immune privilege by regulating the innate and adaptive immune response, which includes immunological ignorance, peripheral tolerance to eye-derived antigens, and intraocular immunosuppressive microenvironment. In this review, we summarize current knowledge regarding the molecular mechanism responsible for the development and maintenance of ocular immune privilege via regulatory T cells (Tregs), which are generated by the anterior chamber-associated immune deviation (ACAID), and ocular resident cells including corneal endothelial (CE) cells, ocular pigment epithelial (PE) cells, and aqueous humor. Furthermore, we examined the therapeutic potential of Tregs generated by RPE cells that express transforming growth factor beta (TGF-ß), cytotoxic T lymphocyte-associated antigen-2 alpha (CTLA-2α), and retinoic acid for autoimmune uveoretinitis and evaluated a new strategy using human RPE-induced Tregs for clinical application in inflammatory ocular disease. We believe that a better understanding of the ocular immune privilege associated with Tregs might offer a new approach with regard to therapeutic interventions for ocular autoimmunity.

A pilot study on expression of toll like receptors (TLRs) in response to herpes simplex virus (HSV) infection in acute retinal pigment epithelial cells (ARPE) cells.

INTRODUCTION: Toll like receptors (TLRs) have been proven to play an important role in mounting the innate immune response in an infected host. The expression of TLRs against herpes simplex virus (HSV) have not been studied in retinitis. Therefore, the current study was undertaken to determine the same using the retinal pigment epithelial (ARPE-19) cell line. MATERIALS AND METHODS: APRE cells cultured in vitro were challenged with HSV 1 and 2 standard strains and 20 other clinical isolates. The cells were observed for cytopathic changes. The cell culture harvest was subjected to RNA extraction using a Total RNA mini kit. The RNA was subjected to reverse transcriptase polymerase chain reaction (PCR) for the amplification of TLRs 3, 4 and 9 and GAPDH housekeeping gene. The amplified products were subjected to electrophoresis on a 2% agarose gel and viewed under a transilluminator. RESULTS: TLR 3 and 4 were expressed by ARPE treated with all the 22 isolates. TLR 9 expression was seen in 16 of the 22 isolates. Bacterial contamination was ruled out by subjecting the harvests to PCR amplification of 16sRNA gene amplification of the eubacterial genome. CONCLUSIONS: The expression of TLR 4 has been reported for the first time in HSV infection. TLR 4 along with TLR 3 and 9 is responsible for the antiviral response in HSV infections.

C5a contributes to intraocular inflammation by affecting retinal pigment epithelial cells and immune cells.

BACKGROUND: The complement activation molecule C5a has been found in the eye and is implicated in the pathogenesis of ocular inflammatory diseases. In this study, the authors sought to investigate C5a's effects on human retinal pigment epithelial (RPE) cells and peripheral blood mononuclear cells (PBMCs), and on the interaction between RPE cells and PBMCs. METHODS: Arising retinal pigment epithelia cell line-19 and PBMCs isolated from healthy donors were used in this study. Western blot, real-time PCR and cell surface receptor staining were used to detect C5a receptor expression. Real-time PCR was used to detect cytokine mRNA expression. A thiazolyl blue tetrazolium bromide assay was used to detect cell viability. Cells were stained with Annexin V and 7-aminoactinomycin D for an apoptosis assay. Cell proliferation was measured using a tritiated thymidine incorporation assay. RESULTS: C5a receptors were present on RPE cells, and receptor expression was increased by pro-inflammatory cytokines. C5a suppressed RPE cells' production of transforming growth factor ß2, an important immunosuppressive agent in the eye. In addition, the viability of RPE cells was decreased in the presence of C5a, and this effect was not due to apoptosis. C5a increased proliferation of PBMCs and upregulated their production of pro-inflammatory cytokines. Finally, C5a decreased RPE cells' ability to suppress immune cell proliferation. CONCLUSION: The results provide a direct link between complement activation and intraocular inflammation. This line of information may help to understand the mechanism of the pathogenesis of intraocular inflammatory diseases. Moreover, the authors show that a close, reciprocal interaction between the innate immune system and the adaptive immune system may be involved in the development of such diseases.

Celastrol regulates innate immunity response via NF-κB and Hsp70 in human retinal pigment epithelial cells.

Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA. Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB.

Immune regulation in the retina.

Immune reactivity in the retina can be critically important in inflammation and infections, but regulation of this response is essential. The retinal pigment epithelial (RPE), a unique retinal cell, displays a number of essential functions to support the health of the retina. In this review, we highlight how the RPE cell plays a pivotal role in immune defense. The RPE cell orchestrates both innate and adaptive immunity since it expresses TLRs, complement components, MHC class I and II molecules, and serves as an antigen presenting cell. Moreover, both of these immune responses result in the production of a plethora of cytokines, mainly proinflammatory. In order to counteract these inflammatory factors and silence unwanted immune reactivity, the RPE cell also generates suppressive molecules. Recently, chronic immune reactivity has been implicated in a number of retinal diseases, such as age-related macular degeneration (AMD). Current evidence suggests that the generation of excessive retinal inflammation may be the consequence of a loss of RPE immunosuppressive factors. Herein, we summarize the varied interactions of the RPE cell with the immune response and highlight how the RPE cell survives and participates in this dynamic environment.

Suppression of bystander T helper 1 cells by iris pigment epithelium-inducing regulatory T cells via negative costimulatory signals.

PURPOSE: To determine whether iris pigment epithelium (IPE)-induced T regulatory (Treg) cells can suppress the activation of bystander T cells with cell contact via costimulatory interactions. METHODS: CD8(+) T cells were cocultured with IPE, x-irradiated, and then used as regulators (IPE-induced Treg cells). The target CD4(+) T cells from wild-type control or knockout donors were used for the assay. T-cell activation was assessed for proliferation by examining both [(3)H]-thymidine incorporation and cytokine production. Expression of costimulatory molecules on IPE-induced Treg cells was evaluated using RT-PCR, immunostaining, and flow cytometry. Expression of costimulatory receptors on target T cells or Treg cells was evaluated by flow cytometry. Neutralizing antibodies were then used to abolish regulatory function. RESULTS: CD8(+) IPE-induced Treg cells significantly suppressed the activation of effector target T cells, e.g., T-cell proliferation and cytokine production such as Th1, Th2, and Th17 cytokines. Although IPE-induced Treg cells expressed various costimulatory molecules, including programmed cell death 1 ligand 1 (PD-L1), only PD-L1 on the Treg cells was actually delivered to target Th1 cells using cell-to-cell interaction (T-T interaction). If neutralizing antibodies for PD-L1 were cocultured with Treg cells, Th1 suppression was impaired. Moreover, Treg cells failed to suppress IFNgamma production by target CD4(+) T cells from programmed cell death 1 (PD-1) knockout donors. Th1-specific inhibition was exclusively achieved with direct cell contact. CONCLUSIONS: T cells exposed to IPE in the eye that acquires full regulatory capacity express negative costimulators and suppress bystander Th1-type effector cells.

[Intracameral moxifloxacin: a safe option for endophthalmitis prophylaxis? In vitro safety profile for intraocular application]. / Moxifloxacin intrakameral: Eine sichere Option zur Endophthalmitisprophylaxe? In-vitro-Sicherheitsprofil zur intraokularen Anwendung.

BACKGROUND: Moxifloxacin (Vigamox), a 4th-generation fluoroquinolone, covers most isolates causing endophthalmitis. It is safe and effective for systemic and topical use; however, only very limited data are available on prophylactic intracameral administration to prevent endophthalmitis. This study investigated the safety of Vigamox for intracameral application in a cell-culture model. METHODS: The endothelial toxicity of moxifloxacin (Vigamox) was evaluated in cultured human corneas. Primary human retinal pigment epithelium cells (RPEs), trabecular meshwork cells (TMCs), lens epithelium cells (LECs), and corneal endothelial cells (CECs) were treated with concentrations of Vigamox. Toxic effects were evaluated after 24 h (MTT assay and live-dead assay). By treating TMC, CEC, and RPE cells either with oxidative stress or tumor necrosis factor-alpha (TNF-a), lipopolysaccharide (LPS), and interleukin-6 (IL-6), the effects of moxifloxacin on cellular viability under conditions of inflammation were investigated. RESULTS: No corneal endothelial toxicity could be detected after 30 days of treatment with moxifloxacin 500 microg/ml. Primary RPEs, TMCs, LECs, and CECs showed adverse effects on proliferation and viability only at concentrations higher than 150 microg/ml moxifloxacin. After preincubation with TNF-a, LPS, and IL-6 for 24 h and subsequent treatment with moxifloxacin at concentrations of 10-150 microg/ml for 24 h, no significant decrease in proliferation or viability was observed. H2O2 exposure did not increase cellular toxicity CONCLUSION: Vigamox did not show significant toxicity on primary RPEs, TMCs, LECs, CECs, or human corneal endothelium at concentrations up to 150 microg/ml. The MIC90 of moxifloxacin for pathogens commonly encountered in endophthalmitis is known to be in the range of 0.25-2.5 microg/ml. Therefore, intracameral use of Vigamox at concentrations up to 150 microg/ml may be safe and effective for preventing endophthalmitis after intraocular surgery.

Human iris pigment epithelium suppresses activation of bystander T cells via TGFbeta-TGFbeta receptor interaction.

Iris pigment epithelial (IPE) cells from the anterior segment in the eye are able to suppress activation of bystander responder T cells in vitro. The cultured IPE cells fully suppress proliferation and cytokine production by responder T cells via direct cell-to-cell contact. We have now investigated whether primary cultured human iris pigment epithelial (h-IPE) cells that were established from fresh iris tissues can also inhibit the activation of T cells in vitro. We found that cultured h-IPE cells significantly inhibited T cell proliferation and the IFN-gamma production by the target T cells from both the allogeneic and autogeneic peripheral blood mononuclear cells (PBMCs). The h-IPE cells also inhibited the activation of CD4(+) T cells from patients with active uveitis. The suppression by h-IPE occurred in a completely contact-dependent manner. The h-IPE constitutively expressed transforming growth factor beta (TGFbeta) and the receptors, and the T cells exposed to h-IPE greatly expressed Smad transcripts. In addition, TGFbeta2-siRNA transfected h-IPE failed to inhibit activation of responder T cells. Similarly, h-IPE cells in the presence of anti-TGFbeta neutralizing antibodies or recombinant TGFbeta receptor blocking proteins failed to inhibit the T-cell activation. In conclusion, cultured human iris pigment epithelium fully inhibits T cell activation in vitro. Our data support the hypothesis that the ocular resident cells play a critical role in immunosuppression in the eye.

Human iris pigment epithelial cells suppress T-cell activation via direct cell contact.

The purposes of the present study were to investigate whether cultured human iris pigment epithelial (hIPE) cells acquire the ability to modify T-cell activation, and if so, to identify the mechanism. Human IPE cells were prepared from patients who underwent glaucoma surgery, and were cultured in RPMI 1640 medium containing 10% fetal calf serum for 4-7 days. Expression of MHC molecules and co-stimulatory molecules on cultured hIPE cells either unstimulated or stimulated with IFN-gamma was examined by FACS. In addition, peripheral blood T cells were incubated with cultured hIPE cells prepared from the same patients and anti-CD3 antibody in a transwell culture system, or in the presence of anti-PD-L1 and PD-L2 antibodies, and T cell proliferation was assessed by [3H]-thymidine incorporation. The hIPE cells inhibited anti-CD3-driven T-cell activation but the inhibition was diminished when tested in the transwell culture system, indicating that a contact-dependent mechanism is important in the immunoregulatory roles of hIPE. Although cultured hIPE cells expressed Class I and PD-L1 but not Class II or PD-L2, all these molecules were observed on hIPE cells cultured in the presence of IFN-gamma. Blocking antibodies against both PD-L1 and PD-L2 reduced the immunoregulatory activity of hIPE cells. Our data indicates that cultured hIPE cells inhibit T-cell activation by T-cell receptor ligation, which is mediated by cell-to-cell contact in part via the PD-L1 and PD-L2 pathways.

Immunoproteasome responds to injury in the retina and brain.

It is well known that immunoproteasome generates peptides for MHC Class I occupancy and recognition by cytotoxic T lymphocytes (CTL). The present study focused on evidence for alternative roles for immunoproteasome. Retina and brain were analyzed for expression of immunoproteasome subunits using immunohistochemistry and western blotting under normal conditions and after injury/stress induced by CTL attack on glia (brain) or neurons (retina). Normal retina expressed substantial levels of immunoproteasome in glia, neurons, and retinal pigment epithelium. The basal level of immunoproteasome in retina was two-fold higher than in brain; CTL-induced retinal injury further up-regulated immunoproteasome expression. Immunoproteasome up-regulation was also observed in injured brain and corresponded with expression in Purkinje cells, microglia, astrocytes, and oligodendrocytes. These results suggest that the normal environment of the retina is sufficiently challenging to require on-going expression of immunoproteasome. Further, immunoproteasome up-regulation with retinal and brain injury implies a role in neuronal protection and/or repair of damage.

Immunological protein expression profile in Ccl2/Cx3cr1 deficient mice with lesions similar to age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Ccl2 knock-out (KO) mice sporadically develop the cardinal features of AMD in their senescent stage. Humans bearing a loss of function variant or single nucleotide polymorphism (SNP) in CX3CR1 are at increased risk of developing AMD. We recently developed Ccl2(-/-)/Cx3cr1(-/-) mice, which consistently develop retinal degeneration with many AMD features. Since there is strong evidence for an immunological role in AMD pathogenesis, we examined ocular immune protein expression levels in Ccl2(-/-)/Cx3cr1(-/-), Ccl2(-/-), Cx3cr1(-/-), and age-matched wild-type (WT) mice. Immunohistochemistry revealed increased complement C3d in Bruch's membrane, retinal pigment epithelium (RPE), choroidal capillaries, and particularly drusen of the Ccl2(-/-)/Cx3cr1(-/-) mice relative to the WT controls. No change was detected in single KO mice. Real-time RT-PCR revealed a 2.5-fold increase in C3 expression in the Ccl2(-/-)/Cx3cr1(-/-). While the retinas of four month old WT and Ccl2(-/-) showed minimal immunoreactivity for markers of macrophages and microglia, infiltrates of these mononuclear phagocytic cells were detected in the Ccl2(-/-)/Cx3cr1(-/-)retinal lesions and a few foci in the Cx3cr1(-/-) retina. The Ccl2(-/-)/Cx3cr1(-/-) had reduced toll-like receptor 4 (TLR4) expression in the RPE. Following LPS injection, the Ccl2(-/-)/Cx3cr1(-/-) had significantly reduced endotoxin-induced uveitis scores and showed a diminished increase in Tlr4 mRNA expression. No changes in TLR4 expression were detected in either single KO. Autoantibodies against the retina and photoreceptors were also detected in the Ccl2(-/-)/Cx3cr1(-/-) serum. Real-time RT-PCR revealed significant increases in Ccl5 transcript in the Ccl2(-/-)/Cx3cr1(-/-) relative to the WT. These results suggest that innate immunity and possibly adaptive immunity play an important role in Ccl2(-/-)/Cx3cr1(-/-) retinal degeneration. Moreover, since human AMD patients show similar immunopathological profiles, these results support the Ccl2(-/-)/Cx3cr1(-/-) as a suitable model for human AMD.

IFN-beta provides immuno-protection in the retina by inhibiting ICAM-1 and CXCL9 in retinal pigment epithelial cells.

The retinal pigment epithelial (RPE) cell is a potent regulatory cell that facilitates normal physiologic processes and plays a critical role in a variety of retinal diseases. We evaluated IFN-beta production in human RPE cells through TLR signaling and investigated the effects of IFN-beta on RPE cells. RPE cells treated with poly(I:C) or infected with an RNA virus produce IFN-beta. Kinetic studies revealed that IFN-beta levels continue to increase over a 48-h period and this was associated with the up-regulation of IRF-7 gene expression, a known positive feedback molecule for IFN-beta production. Microarray analysis revealed that in IFN-beta treated cells, 480 genes of 22,283 genes were up or down-regulated by >2-fold. We hypothesize that IFN-beta induction during TLR signaling in the retina is an immunosuppressive factor produced to limit immunopathologic damage. Cytokine activation of RPE cells results in the production of the chemokines, CXCL9 and CXCL10, and the adhesion molecule, ICAM-1. Pretreatment of RPE cells with IFN-beta resulted in inhibition of ICAM-1 production and elimination of CXCL9 production. This treatment did not alter CXCL10 production. Anti-IFN-beta Ab blocked the inhibitory action of IFN-beta. Real time PCR analysis revealed that IFN-beta treatment inhibited gene expression of sICAM-1 and CXCL9. The results indicate a critical role for RPE cell derived IFN-beta in the down-regulation of CXCL9 and ICAM-1 expression in the retina and suggest that the inhibition of CXCL9 is an immuno-suppressive mechanism that protects the retina from excessive inflammation.

Culture in vitro modulation of human leukocyte antigen molecules and costimulatory molecules on human retinal pigment epithelium.

PURPOSE: To determine the potential of culture in vitro to alter the human leukocyte antigen (HLA) molecules and costimulatory molecules on human retinal pigment epithelium (RPE). METHODS: Pure RPE were isolated and cultured. Two sets of RPE (normal and activated) were used. Activated RPE were obtained by incubating primary cultures of RPE with recombinant human interferon-gamma. The expression of HLA molecules and costimulatory molecules on human RPE at different passages after culture in vitro were analyzed quantitatively by flow cytometry. RESULTS: In the process of routine culture on culture flask, the duration of culture in vitro was significantly correlated with the expression of HLA-ABC molecules on the normal RPE and the activated RPE (r = -0.893, p < 0.001 and r = -0.964, p < 0.001 respectively), HLA-DR molecule on the activated RPE (r = -0.901, p < 0.001) and intercellular adhesion molecule-1 on the normal RPE (r = 0.961, p < 0.001). There were no correlations between the duration of culture in vitro and the expression of HLA-DR molecule on the normal RPE, intercellular adhesion molecule-1 on the activated RPE, B7-1 and B7-2 molecules on the normal RPE and the activated RPE. CONCLUSIONS: The chronic rejection is the major immunological rejection following RPE allogeneic graft. Culture in vitro modulation of HLA molecules and costimulatory molecules on RPE may increase lymphocyte infiltration and the activated RPE.

Induction of interleukin-6 in human retinal epithelial cells by an attenuated Herpes simplex virus vector requires viral replication and NFkappaB activation.

Gene delivery has potential for treating ocular disease and a number of delivery systems have been tested in animal models. However, several viral vectors have been shown to trigger undesirable transient inflammatory responses in the eye. Previously, it was shown that an attenuated Herpes simplex virus vector (hrR3) transduced numerous cell types in the anterior and posterior segments of monkey eyes, but this was accompanied by inflammation. In the retina, retinal pigment epithelial cells were the predominant cell type transduced by hrR3. IL-6 is an important pro-inflammatory cytokine and may play a role in the response to the hrR3 vector. Infection of human ARPE-19 cells with hrR3 resulted in increased IL-6 expression and secretion 3-4h post-infection. In the presence of acyclovir (70 microM) or in cells infected with UV-inactivated hrR3, IL-6 was not up-regulated indicating viral replication was required. Expression of the HSV-1 alpha and beta genes may be necessary but was not sufficient for NF-kappaB activation and IL-6 up-regulation. The translocation of NF-kappaB into the nucleus also occurred between 3 and 4h post-infection, coincident with increased IL-6 expression. Inhibition of NF-kappaB translocation by an Adenovirus vector expressing a dominant negative IkappaB (AdIkappaBam) inhibited IL-6 up-regulation, indicating that NF-kappaB plays a role in increasing IL-6 expression in APRE-19 cells. The hrR3 virus lacks viral ribonucleotide reductase (RR) activity, thus RR is not required for NF-kappaB activation or IL-6 up-regulation in ARPE-19 cells.

Functional expression of B7H1 on retinal pigment epithelial cells.

The interaction of programmed death-1 (PD-1) expressed on T cells with its ligands B7H1 (PDL1) and B7DC (PDL2) is known to be a mechanism of T cell inhibition. In the present study, we examined whether human or murine retinal pigment epithelial (RPE) cells express B7H1 and B7DC, and if so, whether these molecules expressed on RPE cells play an inhibitory role via interaction with T cells. The transcriptional levels and surface expression of B7H1 and B7DC on human RPE cell line (ARPE-19), RPE cells freshly isolated from healthy human subjects, and murine RPE cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. In addition, T cells from healthy subjects were cultured with ARPE-19 for 72h in the presence or absence of monoclonal antibody (mAb) to B7H1 or B7DC, and cytokine production (IFN-gamma, IL-8, and MCP-1) was measured. Messenger RNA and cell surface protein expression of B7H1 and B7DC were demonstrated on non-stimulated ARPE-19 and freshly isolated human RPE cells, and the expression of these molecules was predominantly upregulated by treatment with IFN-gamma. In murine RPE cells, B7H1 expression was detected only when stimulated with IFN-gamma. IFN-gamma, IL-8, and MCP-1 production by T cells co-cultured with IFN-gamma-untreated or -treated ARPE-19 was significantly enhanced in the presence of anti-B7H1 mAb. These data suggest that B7H1 expressed on RPE cells plays an immunosuppressive role in ocular inflammation, which may contribute to immune privilege in the posterior segment of the eye.

Ovalbumin serves as a neo-transplantation antigen in retinal pigment epithelial cells.

PURPOSE: Our long-term goal is to determine the optimal methods for inducing allograft tolerance to facilitate transplantation of retinal pigment epithelial cells or stem cells for the treatment of retinal degenerative diseases. These goals have been hampered by the extreme complexity of allograft rejection and the heterogeneity of responding T cells. The current studies were undertaken to develop a simplified transplant model for studying rejection and tolerance in the unique environment of the eye. METHODS: To provide a defined transplantation antigen, transgenic C57BL/6 (B6) mice were produced, which express the exogenous chicken egg ovalbumin (OVA) gene under the regulation of the mouse tyrosinase related protein-1 (TRP-1) promoter that is transcriptionally active in retinal pigmented epithelial (RPE) cells. To determine whether the transgene was expressed as a neo-transplantation antigen, RPE from TRP-1-OVA mice were injected into the subretinal space of B6 mice or B6 mice expressing the OVA-specific (OT1) TCR transgenes and examined for inflammatory cell infiltration. RESULTS: The TRP-1-OVA transgenic mice expressed OVA mRNA in the brain and eye but not the heart or kidney. RPE cells from TRP-1-OVA transgenic mice expressed mRNA and protein encoded by the OVA gene and RPE expressing TRP-1-OVA induced an inflammatory response within the subretinal space of OT1 mice but not in B6 mice. CONCLUSIONS: OVA serves as a defined, neo-transplantation antigen in RPE that is recognized by mice whose CD8+ T cells recognize OVA peptide. These observations provide new tools for future studies of the mechanisms of rejection and prolongation of RPE transplants in the eye.

[Murine ciliary body pigment epithelial cells inhibit activation of T cells].

PURPOSE: Pigment epithelial(PE) cells cultured from the eye possess the novel property of suppressing T cells in vitro. Although cultured iris PE (IPE) and retinal PE (RPE) achieve suppression, the function of ciliary body PE (CBPE) is still unknown. We investigated whether CBPE can suppress the activation of T cells in vitro. METHODS: CBPE, IPE and RPE cells(controls) were established from normal C57BL/6 mice. T-cell activation was assessed by proliferative response and cytokine production, by ELISA. The expression of candidate genes on ocular PE was evaluated with on oligonucleotide microarray, and PE proliferation and PE morphology were also evaluated. RESULTS: CBPE significantly suppressed T cell activation in vitro. Cell division of CBPE was much greater than in other PE, and the cells were confluent from on early phase. Microarray analysis showed that the expression of candidate genes in CBPE is similar to the expression in other PE cells. CONCLUSIONS: CBPE fully suppress T cell activation. Thus, ocular pigment epithelial cells display inhibitory function in the immune privileged site.