Cyclooxygenase-2 gene expression and regulation in human retinal pigment epithelial cells

PURPOSE: Cyclooxygenases (COX) orchestrate a variety of homeostatic processes and participate in various pathophysiological conditions. The retinal pigment epithelium (RPE) cell performs a variety of regulatory functions within the retina. The conditions under which COX-1 and COX-2 are expressed and upregulated in human RPE (HRPE) cells were determined. METHODS: COX gene expression was examined using RT-PCR analysis of untreated HRPE cultures or cultures exposed to bacterial lipopolysaccharide or various cytokines. COX proteins were detected by immunohistochemistry and Western blot analysis. Prostaglandin (PG) production was analyzed by EIA. RESULTS: Examination of untreated RPE cells revealed the presence of COX-2 mRNA and the absence of COX-1 mRNA. Moreover, cytokine stimulation more readily enhanced COX-2 gene expression than COX-1 gene expression. IL-1 beta, the most potent inducer of COX-2, also resulted in detection of COX-2 protein by immunocytochemical staining and Western blot analysis. There was a direct relationship between both the appearance and amount of COX-2 mRNA and protein synthesis and the degree of PG synthesis by RPE cells. Furthermore, COX inhibitors significantly decreased PG production. Pretreatment of RPE cells with a NF-kappa B inhibitor, PDTC, resulted in dose-dependent decrease in IL-1 beta-induced COX-2 gene expression and PG production. CONCLUSIONS: COX-2 was the predominant isoform of cyclooxygenase in untreated HRPE cells. When HRPE cells were treated with proinflammatory cytokines, COX-2 gene expression and synthesis of PGs were enhanced. NF-kappa B mediated the induction of COX-2 gene expression in HRPE cells. These studies indicate that RPE cells may participate in normal and pathologic retinal conditions through the induction of COX-2.     

TLR4 mediates human retinal pigment epithelial endotoxin binding and cytokine expression

PURPOSE: It was previously demonstrated that toll-like receptor 4 (TLR4) is involved in species-specific human retinal pigment epithelial (HRPE) photoreceptor outer segment recognition and oxidant production. This study was performed to demonstrate the classical role of TLR4 in HRPE endotoxin (lipopolysaccharide; LPS) binding leading to HRPE proinflammatory cytokine secretion. METHODS: Cultured HRPE cells were examined for TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. HRPE cells labeled with fluorescent monoclonal antibodies (mAbs) to TLR4 and its associated adhesion molecule, CD14, were analyzed by real-time microscopy and resonance energy transfer (RET), determining associations of fluorescent LPS, TLR4, and CD14. LPS-induced HRPE secretion of interleukin (IL)-8 was measured with and without specific blocking mAb to TLR4 and CD14. HRPE TLR4 expression was measured after LPS exposure in the presence and absence of blocking antibodies to TLR4 and CD14. RESULTS: All three HRPE cell lines demonstrated constitutive TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. Examination of HRPE cells labeled with fluorescent mAb to TLR4, CD14, and LPS demonstrated RET among the three molecules, indicating that LPS-CD14 binding preceded LPS-TLR4 binding and the close association of CD14 and TLR4. LPS-induced IL-8 was significantly reduced (P < 0.05) in the presence of blocking mAb to TLR4 or CD14. Upregulation of HRPE TLR4 gene and protein expression occurred in response to LPS exposure and was inhibited by mAb to TLR4 or CD14. CONCLUSIONS: HRPE TLR4 is a multifunctional molecule that participates in photoreceptor outer segment membrane recognition, oxidant production, LPS recognition, and cytokine production. These roles indicate potential involvement in retinal degenerative and inflammatory processes.     

Osteonectin/SPARC secreted by RPE and localized to the outer plexiform layer of the monkey retina

PURPOSE: Osteonectin/SPARC is a secreted protein that has been implicated in ocular disease. Deletion of osteonectin/SPARC causes age-onset cataract in mice and the cataractous human lens has increased expression of osteonectin/SPARC. In this study, the expression and localization of osteonectin/SPARC in the monkey retina were determined as was secretion by cultured human retinal pigment epithelial (RPE) cells. METHODS: Adult Rhesus monkey eyes (Macaca mulatta) were dissected, and 5-mm macula and peripheral retina punches were obtained. Supernatants were collected from cultured human RPE cells. Subcellular fractionation of whole monkey retina was also performed. Osteonectin/SPARC expression and/or secretion was monitored by Northern and Western blot analyses, and localization was determined by immunocytochemistry. RESULTS: Outside of the retina osteonectin/SPARC mRNA is broadly expressed in many human tissues. Northern blot analysis shows that in the retina osteonectin/SPARC is expressed almost exclusively by the macular RPE/choroid. Western blot analysis revealed osteonectin/SPARC in both the macula and the peripheral neural retina but only in trace amounts in the RPE/choroid. In subcellular fractions of the whole retina, osteonectin/SPARC was detected, mainly in the soluble fraction but also in the membrane and nuclear fractions. Immunohistochemical analysis localized osteonectin/SPARC specifically to the outer plexiform layer. Western blot analysis of conditioned medium from human RPE cells cultured on porous substrates indicated that osteonectin/SPARC is secreted in large amounts from both the apical and basal sides of the RPE. CONCLUSIONS: Collectively these data provide evidence that osteonectin/SPARC is synthesized in the macular RPE, secreted, and subsequently transported to the outer plexiform layer. The expression pattern of osteonectin/SPARC in the subcellular retinal fractions is consistent with a soluble protein that is transported and internalized.     

Differential expression of chemokines by human retinal pigment epithelial cells infected with cytomegalovirus

PURPOSE: To evaluate the effects of human cytomegalovirus (HCMV) infection on chemokine gene expression and secretion by human retinal pigment epithelial (HRPE) cell cultures. METHODS: HRPE cells were infected with HCMV (strain AD169) at an MOI of 5. Culture supernatants, collected at various postinoculation days, were used for the analyses of chemokines by ELISA. The steady state levels of chemokine and chemokine receptor mRNA were analyzed by RT-PCR. Effects of interferon and MCP-1 on HCMV replication in HRPE cells were evaluated by plaque assays. RESULTS: HRPE cells infected with HCMV exhibited characteristic cytopathic effects. The reduction in the levels of monocyte chemotactic protein (MCP)-1 and -3 mRNA in HCMV-infected HRPE cells was observed in comparison to uninfected HRPE cells. In contrast, HCMV infection enhanced IL-8 mRNA levels, whereas regulated on activation normal T-cell expressed and secreted (RANTES) mRNA was not detectable in either control or infected HRPE cells. A significant decrease in MCP-1 (P < 0.01) and MCP-3 (P < 0.05), but a significant increase in IL-8 (P < 0.05), protein secretion was observed. Expression of the chemokine receptors CCR2, specific for MCP-1, and CXCR1 and CXCR2, specific for IL-8, were not altered by HCMV infection. Treatment of HRPE cultures with MCP-1 had no significant effect on HCMV replication in HRPE cells. CONCLUSIONS: HCMV infection in HRPE cells resulted in the modulation of MCP-1, MCP-3, and IL-8. Because chemokines facilitate the activation of leukocytes and their migration to the sites of inflammation, the modulation of chemokine production by the virus suggests a role for chemokines in immune evasion and/or immunopathogenesis of CMV retinitis.     

Maspin: synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix.

PURPOSE: Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS: Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. RESULTS: Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce maspin, responded to exogenous recombinant maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin. CONCLUSIONS: The corneal stromal cell is the first nonepithelial cell type shown to synthesize maspin. Loss of maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous maspin suggests a role for maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing.     

Induction of interleukin-8 in human retinal pigment epithelial cells after denuding injury

AIM: To determine interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) expression in response to mechanical injury in human retinal pigment epithelial (HRPE) cells. METHODS: Enzyme linked immunosorbent assay (ELISA) was performed to determine IL-8 and MCP-1 secretion by HRPE cells after mechanical denudation. IL-8 and MCP-1 mRNA expression by HRPE cells was assessed using semiquantitative RT-PCR. The effects of immunosuppressive drugs, dexamethasone (DEX) and cyclosporin A (CSA), as well as immunosuppressive cytokines, interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 13 (IL-13), on chemokine expression in HRPE cells after denuding injury were analysed. RESULTS: Mechanical injury induced HRPE IL-8 mRNA and IL-8 secretion. Although MCP-1 mRNA was enhanced slightly after denuding injury, MCP-1 secretion was not increased. DEX and CSA inhibited HRPE chemokine expression after injury. IL-4 and IL-13 enhanced IL-8 and MCP-1 production by HRPE cells after injury while IL-10 had no effect. CONCLUSIONS: These results suggest that IL-8 may be involved in retinal inflammatory responses to injury and that DEX and/or CSA treatment may help control the inflammatory components of retinal diseases such as proliferative vitreoretinopathy.     

Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system

PURPOSE: To investigate the effect of transcorneal electrical stimulation (TES) on the survival of axotomized RGCs and the mechanism underlying the TES-induced neuroprotection in vivo. METHODS: Adult male Wistar rats received TES after optic nerve (ON) transection. Seven days after the ON transection, the density of the surviving RGCs was determined, to evaluate the neuroprotective effect of TES. The levels of the mRNA and protein of insulin-like growth factor (IGF)-1 in the retina after TES were determined by RT-PCR and Northern and Western blot analyses. The localization of IGF-1 protein in the retina was examined by immunohistochemistry. RESULTS: TES after ON transection increased the survival of axotomized RGCs in vivo, and the degree of rescue depended on the strength of the electric charge. RT-PCR and Northern and Western blot analyses revealed a gradual upregulation of intrinsic IGF-1 in the retina after TES. Immunohistochemical analysis showed that IGF-1 immunoreactivity was localized initially in the endfeet of Muller cells and then diffused into the inner retina. CONCLUSIONS: TES can rescue the axotomized RGCs by increasing the level of IGF-1 production by Muller cells. These findings provide a new therapeutic approach to prevent or delay the degeneration of retinal neurons without the administration of exogenous neurotrophic factors.     

TGF-beta receptor expression and smad2 localization are cell density dependent in fibroblasts

PURPOSE: In nonconfluent cultures, TGF-beta induces differentiation of corneal fibroblasts to myofibroblasts. However, in confluent cultures, few fibroblasts differentiate to myofibroblasts after TGF-beta1 addition. This study investigated the hypothesis that functional TGF-beta receptor expression is greater in low-density cultures and is decreased in confluent cultures. METHODS: Northern and western blot analyses were used to detect smooth muscle (SM) a-actin message and protein. 125I-labeled TGF-beta1 was used in a radioreceptor-binding assay as an index of functional receptors on the cell surface of rabbit corneal fibroblast cultures prepared either at high density (cell-cell contact) or low density (absence of contact). Cell lysates were analyzed by SDS-PAGE and autoradiography. Total TGF-beta receptor expression was evaluated in western blot analysis. Smad2, a downstream effector of TGF-beta receptor activation, was immunodetected. RESULTS: Low-density cultures expressed more SM alpha-actin mRNA and protein than high-density cultures, indicating that the low-density cells were differentiating into myofibroblasts. When 125I-TGF-beta1 was added to low- and high-density fibroblasts, fibroblasts plated at low density bound more than fibroblasts in high density, confluent cultures. Furthermore, after the cells differentiated into myofibroblasts, they continued to bind 125I-TGF-beta1. Specificity of 125I-TGF-beta1 binding was demonstrated by complete inhibition by excess nonradioactive TGF-beta1. Localization of Smad2 was correlated with SM alpha-actin induction: Smad was nuclear in low-density cells and cytoplasmic in high-density cells. After TGF-beta1 treatment, Smad2 remained cytoplasmic in high-density cells but was localized to nuclei in cells that were nonconfluent. CONCLUSIONS: Low cell density is correlated with increased functional expression of TGF-beta receptors and promotion of signal transmission from these receptors. Thus, conditions that decrease cell density such as wounding favor myofibroblast differentiation in response to TGF-beta.     

表皮生长因子对人视网膜色素上皮细胞Cx43mRNA表达的影响

目的:观察表皮生长因子(epidermalgrowthfactor,EGF)对培养的人视网膜色素上皮(retinapigmentepitheliumcell,RPE)细胞中缝隙连接蛋白Cx43蛋白及mRNA的表达影响。方法:不同浓度的EGF作用于培养的第4代人的RPE细胞24h,采用Westernblot的方法测定Cx43的蛋白;原位杂交观察Cx43mRNA在RPE细胞中的表达特点;RT-PCR实验对EGF影响后的RPE细胞的mRNA进行半定量观察。结果:Westernblot试验证实不同浓度的EGF均可使Cx43蛋白表达量明显减少,并且与EGF的浓度呈正相关,原位杂交显示所有细胞均表达Cx43mRNA,其表达部位主要在细胞质和核;RT-PCR实验对mRNA进行半定量观察显示表皮生长因子使RPE细胞的mRNA表达量明显减少。结论:EGF可以下调Cx43蛋白及Cx43mRNA在人RPE细胞中的表达。     

Differential expression of retinal pigment epithelium (RPE) IP-10 and interleukin-8

Interferon-gamma induced protein of 10 kDa (IP-10) is a C-X-C chemokine that attracts T lymphocytes and inhibits angiogenesis. In this study, we investigated the expression of IP-10 by human retinal pigment epithelial cells (HRPE) and compared IP-10 expression to that of interleukin-8 (IL-8), which is a leukocytic chemoattractant and pro-angiogenic factor. Cultured HRPE cells were incubated with either IL-1 beta (0.2-20 ng/ml) or tumor necrosis factor (TNF)-alpha (0.2-20 ng/ml) alone or in combination with interferon-gamma (IFN-gamma) (1000 U/ml). HRPE cells were also incubated with: (1) media conditioned by activated human T lymphocytes (CM), or (2) the same CM treated with neutralizing antibodies to IL-1, TNF, and/or IFN-gamma. IL-8 and IP-10 protein levels were measured by ELISA and mRNA levels by Northern blot analysis of HRPE cells. HRPE cells produced very high levels of IP-10 in response to either IL-1 beta/IFN-gamma, TNF-alpha/IFN-gamma or CD3-activated T-lymphocyte CM. The levels of IP-10 were at least tenfold higher (p<.001) than IL-8 measured in the same samples. Neutralizing antibodies to TNF and IFN-gamma, but not to IL-1, abrogated the ability of the CD3-activated T lymphocytes CM to induce HRPE IP-10 (p<.001). HRPE cells produce differential levels of IP-10 and IL-8 in response to various combinations of recombinant and T-lymphocyte-secreted pro-inflammatory cytokines. This may be important in evolving inflammatory and angiogenic ocular responses.     

Presence of adipose differentiation-related protein in rat meibomian gland cells

Adipose differentiation-related protein (ADRP) is an intrinsic lipid storage protein found in lipid droplets of different type of cells. ADRP has been recognized to be a specific marker of lipid accumulation and a marker of differentiated adipocytes. The purpose of this study was to determine whether ADRP was present in the cells of the meibomian gland. The expression of the mRNA of ADRP was determined by RT-PCR and Northern blot analysis of the meibomian gland and other rat tissues. A newly generated polyclonal antibody against rat ADRP was used for Western blot analysis and immunohistochemical staining to determine whether ADRP was expressed in the rat meibomian gland. Meibomian gland acinar cells were isolated to determine when ADRP was expressed during cell differentiation in vitro. Northern blot analysis and Western analysis showed that ADRP was expressed in the meibomian gland. Immunoreactivity to ADRP was observed in the lobules of acinar cells in the meibomian gland, and was preferentially located adjacent the vacuolated cytoplasm. In culture, the meibocytes began to store lipid droplets in the cytoplasm as they became confluent, and the immunoreactivity for ADRP was found at the margins of the oil droplets. Our results suggest that ADRP can serve as a new marker for the identification of differentiated meibocytes containing lipid droplets.     

In vitro phenotypic and functional characterization of human pigment epithelial cell lines

We have characterized human retinal pigment epithelium (HRPE) for the expression of cell surface antigens. Primary HRPE cultures, established cell lines, and freshly brushed pigment epithelial cells all express HLA-ABC but not HLA-DR antigens. However, both primary cultures and established cell lines can be induced by gamma interferon stimulation to express HLA-DR in a dose dependent manner. Only freshly brushed HRPE cells express Fc, and no cells demonstrated the presence of C3b. Our results show that HRPE cells change in culture, as reflected by the loss of Fc receptors, but retain the ability to synthesize HLA-ABC spontaneously and HLA-DR upon stimulation.