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C N Nagineni

Publications and source records attributed to C N Nagineni.

At least 19 recordsLinked to original sources
PubMed2002

Transforming growth factor-beta expression in human retinal pigment epithelial cells is enhanced by Toxoplasma gondii: a possible role in the immunopathogenesis of retinochoroiditis.

Retinochoroiditis caused by Toxoplasma gondii infection results in inflammation and necrosis of the retina. We have used human retinal pigment epithelial cultures (HRPE) as an in vitro model to investigate the role of TGF-beta in T. gondii-induced retinochoroiditis. RT-PCR analyses showed enhanced steady state levels of TGF-beta1 and TGF-beta2 mRNA in T. gondii-infected HRPE. Uninfected HRPE secrete TGF-beta1 in a latent form while 10-30% of the secreted TGF-beta2 was in the active form. T. gondii infection induced a significant increase (P < 0.01) in total TGF-beta1 and TGF-beta2 secretion by HRPE. In addition, soluble extracts of T. gondii (ST) stimulated secretion of both TGF-beta1 and TGF-beta2 significantly (P < 0.01). Interestingly, T. gondii infection as well as ST of the parasites completely inhibited secretion of the active form of TGF-beta2. Studies evaluating the effect of TGF-beta on T. gondii replication in HRPE revealed that TGF-beta enhanced parasite replication. The interactions between host retinal cells and T. gondii may play an active role in the pathogenesis of retinochoroiditis.

Animals↗
PubMed2001

Inhibition of human cytomegalovirus replication in a human retinal epithelial cell model by antisense oligonucleotides.

PURPOSE: The antiviral activity of first and second generation antisense oligonucleotides on human cytomegalovirus (CMV) replication was evaluated in two cell systems, the traditional system on human fibroblasts and on human retinal pigment epithelial (HRPE) cell culture system. METHODS: To evaluate CMV replication strategies within the retina, an HRPE cell system permissive to CMV replication was developed. In this study, the antiviral activity of the antisense oligonucleotides, ISIS 2922 (Vitraven) and ISIS 13312, was evaluated in the traditional fibroblast antiviral assay and in the HRPE cell system. Antiviral activity was measured by evaluating inhibition of virus induced cytopathic effect, virus plaque formation, and virus gene expression. RESULTS: Both oligonucleotides produced concentration-dependent inhibition of CMV cytopathic effect and CMV plaque formation in both human RPE cells and a human fibroblast cell line, MRC-5. The oligonucleotide, ISIS 2922, demonstrated a mean 50% inhibitory concentration (IC(50)) of 0.04 and 0.24 microM in HRPE and MRC-5 cells, respectively. The second-generation oligonucleotide, ISIS 13312, yielded similar results with IC(50) levels of 0.05 and 0.3 microM in HRPE and MRC-5 cells, respectively. Similar findings were obtained with a CMV clinical isolate. In addition, initiation of effective oligonucleotide treatment could be introduced 6 days after CMV infection in HRPE cells, whereas, in the fibroblast cell line, oligonucleotide treatment was only effective up to 3 days after infection. Semiquantitative RT-PCR analysis demonstrated significant inhibition of CMV intermediate early and late mRNAs by both oligonucleotides. CONCLUSIONS: These studies demonstrate that HRPE cells were significantly more sensitive than fibroblasts to the antiviral actions of ISIS 2922 and ISIS 13312. Moreover, the data indicate that the anti-CMV potency of the two oligonucleotides was similar. The enhanced potency of these oligonucleotides in HRPE cells may be associated with a delay in viral gene transcription and slow viral replication and spread in these cells.

Antiviral Agents↗
PubMed2001

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.

Blotting, Western↗
PubMed2000

Molecular characterization and developmental expression of NORPEG, a novel gene induced by retinoic acid.

We have characterized NORPEG, a novel gene from human retinal pigment epithelial cells (ARPE-19), in which its expression is induced by all-trans-retinoic acid. Two transcripts ( approximately 3 and approximately 5 kilobases in size) have been detected for this gene, which is localized to chromosome band 5p13.2-13.3. Placenta and testis showed the highest level of expression among various human tissues tested. Six ankyrin repeats and a long coiled-coil domain are present in the predicted sequence of the NORPEG protein, which contains 980 amino acid residues. This approximately 110-kDa protein was transiently expressed in COS-7 cells as a FLAG fusion protein and immunolocalized to the cytoplasm. Confocal microscopic analysis of the NORPEG protein in ARPE-19 cells showed threadlike projections in the cytoplasm reminiscent of the cytoskeleton. Consistent with this localization, the expressed NORPEG protein showed resistance to solubilization by Triton X-100 and KCl. An ortholog of NORPEG characterized from mouse encoded a protein that showed 91% sequence similarity to the human NORPEG protein. The expression of Norpeg mRNA was detected in mouse embryo at embryonic day 9.5 by in situ hybridization, and the expression appears to be developmentally regulated. In adult mouse, the highest level of expression was detected in the seminiferous tubules of testis.

Amino Acid Sequence↗
PubMed2000

Toxoplasma gondii infection induces gene expression and secretion of interleukin 1 (IL-1), IL-6, granulocyte-macrophage colony-stimulating factor, and intercellular adhesion molecule 1 by human retinal pigment epithelial cells.

We have used human retinal pigment epithelial (HRPE) cultures to investigate the primary cellular responses of retinal resident cells to intracellular Toxoplasma gondii replication. At 4 days postinoculation, when all of the cells were infected, the secretion of interleukin 1beta (IL-1beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and intercellular adhesion molecule 1 (ICAM-1) was augmented by 23-, 10-, 8-, and 5-fold, respectively, over the control. Northern and reverse transcriptase PCR analyses showed significant upregulation of steady-state levels of mRNA for IL-1beta, IL-6, GM-CSF, and ICAM-1. The secretion of these molecules by HRPE cells may play a critical immunoregulatory role in the pathophysiological processes associated with T. gondii-induced retinochoroiditis.

Animals↗
PubMed1999

Interferon regulatory transcription factors are constitutively expressed and spatially regulated in the mouse lens.

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulation of cell growth and immunological responses. Nine IRFs have been described and they are expressed in a variety of cells, except for ICSBP and LSIRF/Pip, which are thought to be expressed exclusively in immune cells. Here, we show that IRF-1, IRF-2, ICSBP, and LSIRF/Pip are constitutively expressed in the mouse lens. These IRFs are present in both the cytoplasm and the nuclei of lens cells. However, the nuclear and cytoplasmic proteins exhibit distinct mobilities on SDS/PAGE. We further show that in the developing mouse lens, IRF-1 and IRF-2 are expressed at high levels in differentiated lens fiber cells with very low and barely detectable levels in undifferentiated lens epithelial cells. Although the level of ICSBP expression is very low in the normal mouse lens, in transgenic mice with constitutive expression of interferon gamma in the lens, its level is markedly elevated and ICSBP expression is detected exclusively in the nuclei of undifferentiated lens cells. Taken together, our data suggest that expression of IRF transcription factors is spatially regulated in the lens and that distinct IRFs may contribute to differential gene regulation in the epithelial and fiber compartments of the vertebrate lens.

Animals↗
PubMed1999

Interferon consensus sequence-binding protein is constitutively expressed and differentially regulated in the ocular lens.

Interferon signaling is mediated by STATs and interferon regulatory factor (IRF) families of transcription factors. Ten distinct IRFs have been described and most are expressed in a variety of cells except for interferon consensus sequence-binding protein (ICSBP) and lymphoid-specific IRF/Pip that are thought to be exclusively expressed in lymphoid cells. We show here for the first time that ICSBP is constitutively and inducibly expressed in the mouse lens. In contrast to lymphoid cells with exclusive expression of ICSBP in the nucleus, ICSBP is present in both the cytoplasm and nucleus of the lens cell. However, ICSBP in the nucleus is of lower apparent molecular weight. We further show that the ICSBP promoter is constitutively bound by lens nuclear factors and that its activation requires binding of additional factors including STAT1. Furthermore, transcriptional activation of ICSBP gene by interferon gamma is accompanied by selective nuclear localization of ICSBP in proliferating epithelial cells but not in the nuclei of nondividing cells in the lens fiber compartment. Constitutive and inducible expression of ICSBP in the ocular lens and differential regulation of its subcellular localization in the developing lens suggest that ICSBP may have nonimmunity related functions and that the commonly held view that it is lymphoid-specific be modified.

Animals↗
PubMed1999

Interferon-gamma signaling in human retinal pigment epithelial cells mediated by STAT1, ICSBP, and IRF-1 transcription factors.

PURPOSE: Studies have shown that interferon (IFN)-gamma stimulates expression of intercellular adhesion molecule-1 (ICAM-1), major histocompatibility complex (MHC) class II, interleukin (IL)-6, and inducible nitric oxide synthase and inhibits replication of Toxoplasma gondii in human retinal pigment epithelial (HRPE) cells. The present study was undertaken to investigate the molecular mechanisms of IFN-gamma action. METHODS: RNA, whole-cell extracts, and nuclear extracts were prepared from HRPE cells cultured in the presence or absence of IFN-gamma. Activation of IFN-gamma-responsive genes was analyzed by electrophoretic mobility shift assay, reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis, and immunoprecipitation. RESULTS: HRPE cells constitutively expressed two members of the IFN regulatory factor (IRF) family of transcription factors, IRF-1 and IRF-2. After exposure to IFN-gamma, transcription of IRF-1 and IFN consensus sequence binding protein (ICSBP) genes were induced; IRF-2 gene transcription was not upregulated. Activation of IFN-gamma-responsive genes was mediated by tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)-1 factor. CONCLUSIONS: This study characterized the IFN-gamma signaling pathway in HRPE cells and identified IRF-1, ICSBP, and tyrosine-phosphorylated STAT1 as mediators of IFN-gamma action in these cells. ICSBP is thought to be exclusively used in immunologic responses and has previously been detected only in lymphoid cells. However, the current study shows that ICSBP expression is inducible in HRPE cells, suggesting that it may regulate gene transcription in RPE cells and possibly in other nonimmunologic cell types.

Blotting, Western↗
PubMed1996

Virus RNA persists within the retina in coronavirus-induced retinopathy.

The murine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces a biphasic retinal disease in adult BALB/c mice. In the early phase, Day 1 to Day 7, a retinal vasculitis is noted which is associated with the presence of viral proteins and infectious virus. In the late phase, Day 10 to Day 140, a retinal degeneration is associated with the absence of viral proteins, infectious virus, and inflammatory cells. The purpose of this study was to determine if viral RNA persists within the retina during the retinal degenerative phase of the disease. BALB/c mice were inoculated by the intravitreal route with 10(4.0) TCID50/5 microliters of virus. The presence of viral RNA was detected by in situ hybridization with a viral cDNA probe and viral proteins were identified by immunocytochemical staining. During the acute phase of the infection, viral RNA was found in the retina, RPE, ciliary body epithelium, and the iris epithelium. During the late phase of the infection, viral RNA was almost exclusively found within the retina and RPE and not in the anterior segment of the eye. Within the retina, viral RNA was detected in the ganglion cell layer, the inner retina, the outer retina, and the RPE cell. Immunocytochemical staining identified viral protein within the retina only from Day 1 to Day 8. This ocular disease was also associated with a persistent systemic infection. Both viral RNA and viral proteins were identified within the liver during the first 8 days. However, only viral RNA was detected in the liver from Day 8 to Day 60. These studies demonstrate that MHV established an acute infection (Day 1-8) where infectious virus and viral proteins were identified. This was followed by a persistent infection within the retina and liver where only viral RNA were detected by in situ hybridization.

Animals↗
PubMed1996

Inflammatory cytokines induce intercellular adhesion molecule-1 (ICAM-1) mRNA synthesis and protein secretion by human retinal pigment epithelial cell cultures.

Retinal inflammatory diseases in man are associated with an upregulation in the expression of intercellular adhesion molecule-1 (ICAM-1) in cells within the retina and with an increase in soluble ICAM-1 within the vitreous. These studies suggest that this protein may contribute to immunopathological processes within the eye. The effects of inflammatory mediators on the regulation of the expression and secretion of ICAM-1 by human retinal pigment epithelial cell cultures (HRPE) were investigated in order to identify the possible source of soluble ICAM-1 and the conditions which enhance its production. Immunofluorescence studies on TNF-alpha and/or IFN-gamma treated HRPE cells demonstrated cellular expression of ICAM-1 which was predominantly localized to intercellular junctions. Moreover, treatment of HRPE for 24 h with tumour necrosis factor alpha (TNF-alpha) (10 ng/ml), interferon gamma (IFN-gamma) (500 u/ml), interleukin 1 alpha (IL-1 alpha) (10 ng/ml) and IL-1 beta (10 ng/ml) results in the secretion of ICAM-1, ranging from 9 to 13 ng per 10(6) cells. IFN-gamma acts synergistically with (TNF-alpha) and IL-1 in the secretion of ICAM-1 by HRPE. Only 1.75 ng of soluble ICAM-1 was detected in untreated HRPE cells. In contrast, lipopolysaccharide (LPS), IL-6, IFN-alpha or TGF-beta did not exhibit any influence on ICAM-1 secretion by these cells. Northern blot analysis reveals an increased expression of ICAM-1 mRNA in HRPE stimulated with IFN-gamma, TNF-alpha or IL-1 for 24 h. In untreated cells, ICAM-1 mRNA is not detectable. There is a progressive increase in ICAM-1 mRNA levels in cytokine-treated HRPE, that reaches steady state by 12 h. Furthermore, a close correlation is noted between ICAM-1 mRNA levels and the secretion of ICAM-1 protein, suggesting regulation at the level of gene transcription. ICAM-1 secretion by RPE might actively participate in the immune reactions in the retina, by recruiting and activating lymphocytes, and contribute to the immunopathological processes in inflammatory diseases.

Cells, Cultured↗
PubMed1996

Mechanisms of interferon-induced inhibition of Toxoplasma gondii replication in human retinal pigment epithelial cells.

Inflammation associated with retinochoroiditis is a major complication of ocular toxoplasmosis in infants and immunocompetent individuals. Moreover, Toxoplasma gondii-induced retinal disease causes serious complications in patients with AIDS and transplant patients. The retinal pigment epithelial (RPE) cell is an important regulatory cell within the retina and is one of the cells infected with T. gondii in in vivo. We have developed a human RPE (HRPE) cell in vitro model system to evaluate T. gondii replication and the regulation of this replication by cytokines. T. gondii replication was quantitated by counting the foci of infection (plaque formation) and the numbers of tachyzoites released into the supernatant fluids. Pretreatment of cultures with recombinant human tumor necrosis factor alpha, alpha interferon (IFN-alpha), IFN-beta, or IFN-gamma for 24 h prior to inoculation inhibited T. gondii replication in a dose-dependent manner. Of these cytokines, IFN-gamma was the most potent, and T. gondii replication was completely inhibited at a concentration of 100 U/ml. The anti-toxoplasmotic activity of IFN-gamma was significantly blocked by monoclonal antibody to IFN-gamma. Treatment of the cultures with IFN-gamma from day 1 or 2 postinoculation with T. gondii also offered protection against the parasite. The anti-toxoplasmotic activity of tumor necrosis factor alpha or IFN-alpha, -beta, or -gamma in these cultures was found to be independent of the nitric oxide (NO) pathway, since NO production was not found in HRPE cells treated with these cytokines. However, addition of tryptophan to IFN-gamma-treated cells significantly reversed the inhibitory effects of IFN-gamma, suggesting that IFN-gamma acts by depleting cellular tryptophan. This effect was further confirmed by reverse transcription-PCR and Northern (RNA) blot analysis, which indicated induction of indoleamine 2,3-dioxygenase (IDO), an enzyme that converts tryptophan to kynurenine. These results indicated that interferons inhibited T. gondii replication in HRPE by NO-independent but IDO-dependent mechanisms. This in vitro model of T. gondii replication in HRPE may be useful in evaluating the effects of cytokines and drugs on T. gondii replication within the retina.

Animals↗
PubMed1996

Cytomegalovirus replication in human retinal pigment epithelial cells. Altered expression of viral early proteins.

PURPOSE: Cytomegalovirus (CMV) infections are frequent complications in patients who have undergone kidney and bone marrow transplant and in patients with acquired immune deficiency syndrome. The mechanism by which CMV is activated and replicated within the retina is unknown. The authors evaluated the ability of human CMV to initiate replication in human retinal pigment epithelial (RPE) cells and compared this system with CMV replication in human fibroblasts (HEL-299, MRC-5) and human amnion epithelial (WISH) cells. METHODS: Human RPE cells were obtained from donor eyes and propagated in vitro. Cells were infected, and CMV replication was evaluated in three ways: the detection of viral antigen by immunofluorescent, flow cytometry, and Western blot assays; the detection of virus-induced cytopathic effect (cpe), and the detection of infectious virus. RESULTS: No evidence of viral replication in the epithelial (WISH) cells was found. Although CMV does not usually replicate in vitro in epithelial cells, CMV replication was detected in RPE cells. There are a number of distinct differences in CMV replication in RPE cells compared to replication in human fibroblasts. Virus-induced cpe and the production of infectious virus by RPE cells were delayed when compared to virus infection in either HEL or MRC 5 cells. At a multiplicity of infection of 0.1 and 1, cpe and infectious virus yield reached maximum levels at days 4 to 5 in fibroblasts and at days 19 to 46 in RPE cells, respectively. Nevertheless, infectious virus produced by RPE cells (10(6.5) TCID50/0.1 ml) significantly surpassed levels produced by HEL cells (10(5.5)TCID50/0.1 ml). The permissive infection in RPE cells consisted of a prolonged period (5 to 6 days) of virus production in the absence of cytopathology. Virus protein expression evaluated by indirect immunofluorescence assays, Western blot analysis, and flow cytometry revealed a delay in viral protein expression in RPE cells compared to viral protein expression in fibroblasts. The pattern of viral protein evaluated by flow cytometry was noticeably different in the two cell types. At the middle phase of CMV replication in RPE cells, a low percentage of cells express immediate early (IE) protein at a time when a high percentage of the cells express early (E) proteins. This IE-1 protein is a stable protein found concurrently with E protein in fibroblasts. This difference in percentage of cells expressing specific CMV proteins is transient, that is, it does not remain apparent at 100% cpe. CONCLUSIONS: Retinal pigment epithelial cells appear to demonstrate a distinct pattern of CMV infection. The low frequency of expression of IE viral protein in RPE cells, the subsequent slow replication of CMV, and the altered expression of IE viral proteins may be critical variables that impact on their relationship to viral persistence and activation within the retina. Alterations in the IE gene product may indicate the existence of positive or negative nuclear transcription factors within infected RPE cells.

Blotting, Western↗
PubMed1995

Transforming growth factor-beta inhibits the cytokine-mediated expression of the inducible nitric oxide synthase mRNA in human retinal pigment epithelial cells.

Human retinal pigment epithelial (RPE) cells in culture respond to a mixture of cytokines (IFN-gamma, IL-1 beta, TNF-alpha) by producing large amounts of nitric oxide. Transforming growth factor-beta, unlike other growth factors, was found to inhibit this response by more than 75%. The expression of mRNA for the inducible form of nitric oxide synthase in RPE cells treated with cytokines was demonstrated by reverse transcription-polymerase chain reaction, sequencing of the PCR product and northern blotting. Transforming growth factor-beta was highly effective in inhibiting (by 75%) the cytokine-induced nitric oxide synthase mRNA expression. This response by RPE may play an important role in the etiology of infectious and inflammatory diseases affecting retina.

Base Sequence↗
PubMed1995

Bidirectional glutathione transport by cultured human retinal pigment epithelial cells.

PURPOSE: To characterize glutathione (GSH) transport by cultured human retinal pigment epithelial (HRPE) cells. METHODS: Cultured HRPE cells were pretreated with acivicin for GSH efflux and with buthionine sulfoximine for GSH uptake to prevent the breakdown and resynthesis of GSH. Efflux was measured by the linear rate of accumulation of GSH in the supernatant; uptake was measured using [35S] GSH plus varying concentrations of GSH. Molecular forms were verified by high-performance liquid chromatography. HRPE cell mRNA was probed for the presence of the two recently cloned rat sinusoidal and canalicular GSH transporters, (RsGshT and RcGshT), by Northern blot analysis. RESULTS: Glutathione efflux was temperature dependent (undetectable at 4 degrees C), and its averaged 23 +/- 3.3 pmol/10(6) cells/minute or 10% of the total GSH effluxed per hour (total cell GSH = 13.6 +/- 1.5 nmol/10(6) cells). Efflux was not influenced by dithiothreitol or sulfobromophthalein-reduced GSH adduct, agents known to affect liver sinusoidal GSH transport. Glutathione uptake was linear up to 45 minutes and was temperature dependent. The difference between 37 degrees C and 4 degrees C uptake values represented true uptake. Glutathione uptake (2 microCi/ml + 1 mM mass) was Na independent and was inhibited significantly by phenol-3,6-dibromphthalein disulfonate. The kinetics of GSH uptake was assessed by measuring uptake with 35S-GSH and 0.05 to 40 mM extracellular GSH for 30 minutes. Uptake was saturable with Vmax = 18.7 +/- 1.7 nmol/10(6) cells/30 minutes, Km = 12.1 +/- 1.9 mM, n (binding site) = 1. On Northern blot analysis, HRPE cells express mRNA for RcGshT but not for RsGshT. CONCLUSIONS: The similarities in functional characteristics of GSH transport and the presence of RcGshT-like mRNA suggest GSH transport in HRPE cells is mediated by a RcGshT homolog. Although the transporter can operate bidirectionally, it is expected to be a net efflux pump under normal physiologic conditions because the intracellular GSH concentration is much higher.

Biological Transport↗
PubMed1995

Intrinsic properties of ACL and MCL cells and their responses to growth factors.

Different intrinsic properties of the constituent cells of the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) have been proposed to be one of the factors in the differential repair mechanisms. We have found that the outgrowth of cells from rabbit ACL explants was slower than from MCL explants after 10 d. Growth curves of ACL and MCL cultures at both Passage numbers 2 and 6 showed a slower rate of proliferation of ACL cells than MCL cells (P < 0.005). The proliferative response of rabbit ACL and MCL cells to b-FGF and TGF-beta was also investigated. Both b-FGF and TGF-beta had no significant effect on cell proliferation of ACL and MCL cultures after 48 h. However, TGF-beta did have an inhibitory effect on thymidine incorporation, especially at concentrations greater than 1 ng.ml-1, while b-FGF stimulated thymidine incorporation in ACL and MCL in a dose-dependent manner. Differences in the cell morphology of the ACL and MCL cells grown in culture were seen also. Cells from Passages 3-6 demonstrated these differences more prominently, and phalloidin staining for actin showed that ACL cells appeared to have more intracellular actin fibers.

Animals↗
PubMed1994

Increased expression of heme oxygenase-1 in human retinal pigment epithelial cells by transforming growth factor-beta.

Antibodies specific for heme oxygenase-1 (HO-1) were produced in rabbits, using the multiple antigen peptide (MAP) technique, and were employed to investigate the ability of transforming growth factor-beta 1 (TGF-beta 1) to induce the HO-1 protein in cultured human retinal pigment epithelial (RPE) cells. Western blot analyses showed that the cytokine induced HO-1 in these cells in a time- and dose-dependent manner. TGF-beta 1 also increased the mRNA for HO-1 in treated cells prior to the increase in HO-1 protein. The induction was effectively blocked by a neutralizing antibody preparation against TGF-beta 1. When tested under similar conditions, other growth factors such as basic fibroblast growth factor-I, platelet-derived growth factor, insulin-like growth factor, transforming growth factor-alpha, and epidermal growth factor did not show appreciable induction of HO-1. Lipopolysaccharide, tumor necrosis factor-alpha, and interferon-gamma were also not inducers, although TGF-beta 2 effectively induced HO-1. Heavy metal ions and thiol reagents were also highly potent inducers of HO-1 in human RPE cells. The induction of HO-1 by TGF-beta 1 was also observed in bovine choroid fibroblasts, but not in HELA, HEL or bovine corneal fibroblasts. Our results demonstrate for the first time that HO-1 can be induced by an important cytokine, TGF-beta 1, causing an increase in the expression of both HO-1 message and protein in specific neuroepithelial and fibroblast cells.

Amino Acid Sequence↗
PubMed1994

Synergistic effects of gamma interferon on inflammatory mediators that induce interleukin-6 gene expression and secretion by human retinal pigment epithelial cells.

The retinal pigment epithelial (RPE) cell is a potent regulatory cell within the retina. It helps to maintain normal retinal activity, and following gamma interferon (IFN-gamma) exposure, it may express major histocompatibility complex class II molecules and function as an antigen-presenting cell. Since interleukin-1 (IL-1) and IL-6 are potent cytokines observed in ocular inflammatory processes, we initiated studies to evaluate conditions which enable RPE cells to produce these cytokines. Cultures of human RPE cells from two eye donors were established and characterized, and enzyme immunoassays were employed to screen for IL-1 and IL-6 production. Treatment of RPE cells with lipopolysaccharide (LPS) or recombinant tumor necrosis factor alpha, IL-1, or IFN-gamma resulted in a significant level of secretion of IL-6. In contrast, treatment with recombinant epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, or transforming growth factor alpha, or LPS can dramatically augment the secretion of IL-6 by RPE cells. Thus, these inflammatory mediators can act alone or synergistically with IFN-gamma to activate RPE cells and dramatically increase the expression and secretion of IL-6. In contrast, IL-1 was not detected following stimulation with any of the above-mentioned cytokines or LPS. Characterization of IL-6 protein production by RPE cells revealed that 98% of the protein is promptly secreted by the cell, its induction is dependent upon the time and concentration of the stimulant, and the continuous presence of the stimulant is required for IL-6 production. Moreover, Western blot (immunoblot) analysis of secreted proteins revealed that IL-6 was produced in multiple molecular forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗