Adrenomedullin in Cultured Human Retinal Pigment Epithelial Cells

Abstract

purpose. To determine whether adrenomedullin (ADM), a vasorelaxant peptide is produced and secreted by human retinal pigment epithelial (RPE) cells, whether ADM expression is regulated by inflammatory cytokines and a growth factor, and whether ADM has proliferative effects on these cells.

methods. Production and secretion of ADM by cultured human RPE cells were examined by Northern blot analysis and radioimmunoassay. Regulation of the ADM expression by basic fibroblast growth factor, interferon (IFN)-γ, tumor necrosis factor-α, interleukin (IL)-1β, or all-trans-retinoic acid was studied. In addition, proliferative effects of ADM on human RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

results. ADM mRNA was expressed constitutively in all three human RPE cell lines (F-0202, D407, and ARPE-19) examined. Immunoreactive ADM was detected in the cultured media by radioimmunoassay. Sephadex G-50 column chromatography of the cultured medium showed a single peak eluting in the position of ADM-(1-52). Treatment with IFN-γ or IL-1β increased ADM mRNA levels and immunoreactive-ADM levels in the medium in dose- and time-dependent manners in ARPE-19 cells. Exogenously added ADM increased the number of F-0202 cells and ARPE-19 cells, and the treatment with ADM antibody or ADM-(22-52) (an ADM antagonist) decreased it.

conclusions. Human RPE cells produced and secreted ADM. IFN-γ and IL-1β induced ADM expression in ARPE-19 cells. Furthermore, ADM stimulated proliferation of RPE cells. These results raise the possibility that ADM is related to the pathophysiology of some inflammatory and proliferative ocular diseases.

Adrenomedullin (ADM) is a vasorelaxant peptide originally isolated from human pheochromocytoma. ¹ ADM consists of 52 amino acids and has approximately 20% similarity to calcitonin gene–related peptide (CGRP). Immunoreactive ADM (IR-ADM) and ADM mRNA are detectable, not only in pheochromocytoma but also in multiple human tissues and cells, including adrenal medulla, heart, lung, aorta, kidney, gastrointestinal tract, vascular endothelial cells, and vascular smooth muscle cells. ^{2 3 4} ADM mRNA expression and ADM binding sites are also found in the brain, ^{5 6 7 8 9} suggesting that ADM is a novel neuromodulator. In addition to its potent vasodilator action, ADM has been found to have a broad range of biologic actions. It can stimulate or inhibit cell proliferation, depending on the cell type and the experimental conditions. For example, ADM stimulates DNA synthesis and cell proliferation in Swiss 3T3 fibroblasts ¹⁰ and has mitogenic effects on human oral keratinocytes. ¹¹ In contrast, it has inhibitory effects on rat mesangial cell mitogenesis ¹² and antiproliferative effects on rat cultured vascular smooth muscle cells. ^{13 14} Furthermore, ADM inhibits water drinking, supporting a role of ADM as a neuromodulator. ¹⁵ We have found the production and secretion of ADM from brain tumor cell lines, such as cultured choroid plexus carcinoma cells ¹⁶ and T98G glioblastoma cells. ¹⁷  

Retinal pigment epithelial (RPE) cells are located between the neural retina and the choroid of the eye and form one component of the blood–retinal barrier. ¹⁸ RPE cells play an essential role in the function and survival of photoreceptors, including phagocytosis of shed outer segments of rods and cones and synthesis and transportation of many substances, such as vitamin A metabolites. RPE cells, as well as differentiated melanocytes, produce melanin, which absorbs light, to reduce scattering and to improve image sharpness. ¹⁹ Normally, RPE cells do not grow during adult life, but under some pathologic conditions, such as proliferative vitreoretinopathy (PVR), they migrate into the vitreous and proliferate on the surface of the retina and within the vitreous. ²⁰  

We have recently reported elevated IR-ADM levels in vitreous fluid of patients with PVR that may be due to the increased secretion of ADM by RPE cells migrating into the vitreous cavity. ²¹ RPE cells produce various cytokines and growth factors, such as platelet-derived growth factor, ²² interleukin (IL)-1β, ^{23 24} IL-6, ²⁵ IL-8, ²⁶ tumor necrosis factor (TNF)-α, ²⁷ transforming growth factor (TGF)-β, ²⁸ insulin-like growth factors I and II, ²⁹ and vascular endothelial growth factor. ³⁰ However, it has not been reported that RPE cells produce neuropeptides or vasoactive peptides. Montuenga et al. ³¹ reported that ADM and ADM mRNA were detected in the outer neuroblastic layer of the retina on embryonic days 14 to 15 in mice by immunocytochemistry and in situ hybridization. 

Because ADM is known to influence cell migration and proliferation in a cell-type–specific manner, it is important to identify its production by RPE cells and its effects on proliferation of RPE cells. Inflammatory cytokines including IL-1β ^{32 33 34 35} and interferon (IFN)-γ ^{32 33} are known to be involved in the pathophysiology of inflammatory ocular diseases. Some cytokines such as TNF-α increase ADM expression in vascular smooth muscle cells. ⁴ We therefore sought to determine whether ADM is produced and secreted by human RPE cells, whether the ADM expression is regulated by inflammatory cytokines and a growth factor, and whether ADM has proliferative effects on human RPE cells. 

Materials and Methods

Materials

Human ADM-(1-52); ADM-(22-52), an ADM antagonist; and CGRP-(8-37), a CGRP antagonist, were obtained from the Peptide Institute (Osaka, Japan). ADM-Gly ³⁶ was kindly supplied by Kazuo Kitamura (Miyazaki Medical College). Dulbecco’s modified Eagle’s medium (DMEM), a 1:1 mixture of DMEM and nutrient mixture Ham’s F12, minimum essential medium (MEM), and penicillin-streptomycin were obtained from Life Technologies (Rockville, MD). Fetal bovine serum (FBS) was obtained from CSL (Parkville, Victoria, Australia), basic fibroblast growth factor (bFGF) from Pepro Tech EC (London, UK), TNF-α from Genzyme (Boston, MA), and all-trans-retinoic acid (RA) from Wako (Osaka, Japan). IL-1β and IFN-γ were gifts from Otsuka Pharmaceutical (Tokusima, Japan) and Shionogi (Osaka, Japan), respectively.[α -³²P]-dCTP was obtained from Amersham Pharmacia Biotech (Tokyo, Japan) and ¹²⁵INa from Daiichi Kagaku (Tokyo, Japan). Restriction endonucleases were purchased from Takara (Otsu, Japan) and New England BioLabs (Beverly, MA). Cell-Counting Kit was purchased from Dojindo (Kumamoto, Japan), and a 48-well plate was purchased from Sumitomo Bakelite (Tokyo, Japan). 

Cell Culture

The human RPE cell lines ARPE-19 and D407 used in this study were kindly given by Leonard M. Hjelmeland (Department of Biological Chemistry, University of California, Davis, CA) and Richard C. Hunt (Department of Microbiology, University of South Carolina Medical School, Columbia, SC), respectively. ARPE-19 cells ³⁷ were cultured in 1:1 mixture of DMEM and nutrient mixture F12 containing 10% FBS, 56 mM sodium bicarbonate, 2 mM l-glutamine, and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin) at 37°C in 10% CO₂ and 90% room air. D407 cells ³⁸ were cultured in DMEM, containing 10% FBS, 2 mM l-glutamine, 4500 mg/l glucose, and antibiotics at 37°C in 5% CO₂ and 95% room air. F-0202 human RPE cells were established from human fetal eyes, as described previously. ³⁹ Cells from the seventh to ninth passages were used in the experiments. F-0202 cells were cultured in MEM containing 10% FBS, 10 ng/ml bFGF, and antibiotics at 37°C in 5% CO₂ and 95% room air. 

HeLa human cervical cancer cells were cultured in MEM containing 10% FBS and antibiotics at 37°C in 5% CO₂ and 95% room air. Human umbilical vein endothelial cells (HUVECs) were used as a positive control, because vascular endothelial cells were reported to produce and secrete a large amount of ADM. ³ HUVECs were obtained from Kurabo (Osaka, Japan). EGM-2 medium and cell growth supplement (EGM-2-MV SingleQuots) were purchased from Takara (Otsu, Japan). HUVECs were cultured in EGM-2 medium supplemented with 2% FBS and other cell growth supplements at 37°C in 5% CO₂ and 95% room air. 

To examine effects of inflammatory cytokines and a growth factor on the expression of ADM, ARPE-19 cells were exposed for 24 hours to bFGF (10 ng/ml), IFN-γ (100 U/ml), TNF-α (20 ng/ml), IL-1β (10 ng/ml), or RA (1 nmol/ml). Dose response effects (1–100 U/ml IFN-γ, or 0.1–10 ng/ml IL-1β,) and time course effects of IFN-γ or IL-1β (6, 12, and 24 hours) were studied in ARPE-19 cells, because the induction of ADM expression was observed in 24-hour treatment with IFN-γ or IL-1β. Furthermore, ARPE-19 cells were treated for 24 hours with combinations of three cytokines (100 U/ml IFN-γ, 1 ng/ml IL-1β, and 20 ng/ml TNF-α). The experiments were performed in five dishes per each cytokine treatment. The culture media were collected for the measurement of IR-ADM. The cells were harvested and pooled from five dishes per each treatment for RNA extraction. 

RNA Extraction and Northern Blot Analysis

Total RNA was extracted from cultured cells by the guanidium thiocyanate-cesium chloride method. Total RNA (15 μg/lane) was fractionated by electrophoresis through a 1.0% agarose gel containing 2 M formaldehyde, transferred to a nylon membrane filter (Zeta-Probe membrane; Bio-Rad, Richmond, CA), and fixed with a UV linker (Stratalinker 1800; Stratagene, La Jolla, CA). A hybridization probe for ADM was the HindIII-EcoRI fragment of pBS-hADM2. ¹⁶ Expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was examined as an internal control. The probe for G3PDH mRNA was the NcoI-PstI fragment derived from rat 720-bp G3PDH cDNA fragment (nucleotides 5 to 104 and 368 to 987) subcloned into pGEM-T vector (Promega, Madison, WI), ⁴⁰ kindly provided by Kazuhito Totsune (The Second Department of Internal Medicine, Tohoku University School of Medicine). These probes were labeled with [α-³²P]-dCTP by the random priming method. The RNA blot was prehybridized at 42°C in a solution consisting of 5× SSC (0.75 M sodium chloride and 0.075 M sodium citrate), 1% sodium dodecyl sulfate (SDS), 50% formamide, 5× Denhardt’s solution, and 0.2 mg/ml salmon testis DNA, for at least 3 hours, and then hybridized overnight at 42°C. The hybridized filter was washed at 65°C with 0.1× SSC and 0.1% SDS. Radioactive signals were detected by exposing the filters to x-ray film (X-AR5; Eastman Kodak, Rochester, NY) or with an image analyzer (BAS 1500; Fuji Film, Tokyo, Japan). The intensity of hybridization signals was determined by photo-stimulated luminescence with the image analyzer. 

Peptide Extraction and Radioimmunoassay

Peptides in the medium were extracted with a Sep-Pak C18 cartridge (Waters, Milford, MA). ¹⁶ IR-ADM in the extract was measured by radioimmunoassay, as previously reported, ^{5 16} using the antiserum against human ADM-(1-52) raised in a rabbit (No. 102). ¹⁶ The assay showed a 40% cross reaction with ADM-Gly but a less than 0.001% cross reaction withα -CGRP, neuropeptide Y, somatostatin, growth hormone–releasing hormone, corticotropin-releasing hormone, arginine vasopressin, vasoactive intestinal polypeptide, endothelin-1, and atrial natriuretic peptide. 

Chromatographic characterization of the culture medium extracts of F0202 RPE cells and ARPE-19 RPE cells was performed by Sephadex G-50 (superfine) column chromatography and reversed-phase high-performance liquid chromatography (HPLC) using a C18 column (3.9 mm × 300 mm,μ Bondapak; Waters). The extract was reconstituted in 1 M acetic acid containing 0.5% (wt/vol) bovine serum albumin and loaded onto the Sephadex G-50 column (10 × 560 mm). Peptides on the column were eluted with 1 M acetic acid containing 0.5% (wt/vol) bovine serum albumin at a flow rate of 6 ml/h. Fractions (0.8 ml/fraction) were collected, dried by air, reconstituted in assay buffer, and assayed. 

For the reversed-phase HPLC analysis, the extract was reconstituted in 0.1% (vol/vol) trifluoroacetic acid and loaded onto the column. Peptides were eluted with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid from 10% to 60% at a flow rate of 1 ml/min per fraction over 50 minutes. Each fraction (1 ml) was collected, dried by air, reconstituted with assay buffer, and assayed. 

Cell Proliferation Assay

Effects of ADM on the proliferation of F-0202 or ARPE-19 cells were examined using the cell-counting kit (modified3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide[ MTT] assay). ⁴¹ 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8), which produces a highly water-soluble formazan dye was used instead of MTT. F-0202 or ARPE-19 cells were seeded in the well of a 48-well plate at a density of approximately 1.5 × 10⁴ cells/well. F-0202 cells were grown in MEM containing 10% FBS, and ARPE-19 cells were in 1:1 mixture of DMEM and nutrient mixture F12 containing 10% FBS for 12 hours, respectively. Medium was then replaced by fresh medium containing human ADM-(1-52); normal rabbit serum (NRS); polyclonal anti-human ADM antibody (ADM Ab; No. 102) ^{6 16} ; ADM-(22-52), an ADM antagonist, or CGRP-(8-37), a CGRP antagonist, and the cells were incubated for 24 hours. WST-8 solution was added, and the reaction was stopped after 1 hour’s incubation by adding SDS solution (final 0.1%). The optical density of 450 nm was determined by spectrophotometer. Six wells were measured per treatment. 

Statistical Analysis

Data are means ± SEM, unless otherwise stated. Statistical analysis was performed by one-way analysis of variance followed by the Scheffé multiple comparison test. 

Results

Basal Expression and Secretion of ADM in Human RPE Cell Lines

Northern blot analysis showed that ADM mRNA was expressed in all three human RPE cell lines (F-0202, D407, and ARPE-19; Fig. 1 ). The size of the major hybridization signals was approximately 1.6 kb, which is consistent with the reported size of human ADM mRNA. ⁴² The expression levels of ADM mRNA in these human RPE cell lines were comparable to those in human pheochromocytomas, which were included as positive controls, ¹ and 70% to 260% of the levels in HUVECs (Table 1) . 

IR-ADM was detectable in the cultured media of all three human RPE cell lines, HUVECs, and HeLa cells by radioimmunoassay (Table 1) . IR-ADM concentrations in the cultured media of F-0202, D407, and ARPE-19 cells were 1.8 to 5.8 times higher than those in HUVECs (Table 1) . 

Analysis by Chromatography

Sephadex G-50 column chromatography of the culture medium extracts of F-0202 cells and ARPE-19 cells showed a single peak eluting in the position of ADM, indicating that IR materials secreted by these RPE cells were identical with or similar to ADM-(1-52) (Figs. 2A 2C ). 

Reversed-phase HPLC of the culture medium extract of F-0202 cells showed one major peak eluting in the position of ADM, with two other minor peaks eluting earlier than ADM standard (Fig. 2B) . In contrast, reversed-phase HPLC of the culture medium extract of ARPE-19 cells showed two larger peaks eluting earlier and a peak in the position of ADM (Fig. 2D ). Treatment with IFN-γ (100 U/ml) or IL-1β (10 ng/ml) did not change chromatographic profiles of the culture medium extract of ARPE-19 cells (data not shown). Reversed-phase HPLC of the culture medium extract of D407 cells showed a chromatographic profile similar to that of ARPE-19 cells (data not shown). ADM with an oxidized methionine, which was generated by incubating ADM with 3% (vol/vol) H₂O₂ for 1 hour at room temperature, was eluted in the same position as ADM. ADM-Gly (glycine-extended ADM, an intermediate form processed from proADM) was eluted later than ADM-(1-52). 

Regulation of ADM Gene Expression and ADM Production by Cytokines

To explore the regulation of ADM production, we treated ARPE-19 cells with IFN-γ, TNF-α, IL-1β, bFGF, or RA. Treatment with each of IFN-γ, IL-1β, and bFGF increased IR-ADM concentrations in the culture media by approximately 2.0 (P < 0.0001), 1.7 (P < 0.0001), and 1.3 (P < 0.005) times compared with control, respectively (Fig. 3A ). There were no significant changes in IR-ADM concentrations in the cultured media treated with TNF-α or RA (P > 0.1). Northern blot analysis showed that the expression levels of ADM mRNA in ARPE-19 cells treated with IFN-γ or IL-1β were approximately 2.0 times higher than those in control cells (Fig. 3B) . No notable change in ADM mRNA expression levels was found in ARPE-19 cells treated with bFGF, TNF-α, or RA. The degree of increase in the expression levels of ADM mRNA was almost parallel with the IR-ADM concentrations in the cultured media treated with each cytokine, bFGF or RA. Treatment of F-0202 cells with TNF-α (20 ng/ml) or IL-1β (10 ng/ml) increased IR-ADM concentrations in the cultured media by approximately 1.6 times and approximately 2.8 times, but treatment with IFN-γ (100 U/ml) did not (data not shown). There may be some difference in the regulation of ADM induction by cytokines between these two RPE cell lines. 

IR-ADM in the medium accumulated time dependently up to 24 hours in ARPE-19 cells, either untreated or treated with 100 U/ml IFN-γ or 10 ng/ml IL-1β (Fig. 4A ). Significant increases in the IR-ADM levels were observed at 12 hours and 24 hours by treatment with 100 U/ml IFN-γ and at 24 hours with 10 ng/ml IL-1β, compared with the untreated sample. IR-ADM levels were increased in a dose-dependent manner with IFN-γ (1–100 U/ml) or IL-1β (0.1 and 1 ng/ml; Fig. 4C ). Expression levels of ADM mRNA were increased time dependently up to 24 hours with 100 U/ml IFN-γ or 10 ng/ml IL-1β (Fig. 4B ). Expression levels of ADM mRNA were also augmented in a dose-dependent manner with IFN-γ (1–100 U/ml) or IL-1β (0.1 and 1 ng/ml), consistent with the results in IR-ADM in the medium (Figs. 4C 4D) . IR-ADM levels in the medium and the ADM mRNA expression level were not increased further with 10 ng/ml IL-1β (Figs. 4C 4D) , indicating that the concentration of 1 ng/ml was sufficient for the maximum induction of ADM expression by IL-1β. 

Single TNF-α had no notable effects on ADM expression in ARPE-19 cells (Figs. 3A 3B) . Increases in IR-ADM concentrations in the media and ADM mRNA expression levels by IFN-γ, IL-1β, or IFN-γ+IL-1β were enhanced by the addition of TNF-α in ARPE-19 cells (Figs. 5A 5B ). Treatment with a combination of IFN-γ+IL-1β synergistically increased IR-ADM concentrations in the media (approximately 18 times increase of control) and ADM mRNA expression levels (approximately 20 times). A combination of three cytokines, IFN-γ, IL-1β, and TNF-α, was the strongest stimulus for the ADM induction in ARPE-19 cells among combinations of cytokines examined in the present study. 

Effect of ADM on Proliferation of Human RPE Cells

The effect of ADM on the proliferation of human RPE cells was analyzed by the modified MTT assay (Fig. 6) . Exogenously added human ADM-(1-52) significantly increased the number of F-0202 at concentrations of 10⁻⁸ and 10⁻⁷ M, and ARPE-19 cells at concentrations of 10⁻⁹ to 10⁻⁷ M (Figs. 6A 6D) . Furthermore, treatment with ADM Ab for 24 hours reduced the number of cells (Figs. 6B 6E) . The number of F-0202 or ARPE-19 cells treated with ADM Ab at a dilution of 1:1000 was 87.2% ± 1.3% or 75.0% ± 9.3% of that treated with NRS (P < 0.005, in both cells). We examined the effect of an ADM receptor antagonist, ADM-(22-52), or a CGRP antagonist, CGRP-(8-37), on the proliferation of F-0202 and ARPE-19 cells. The number of cells was reduced by 10⁻⁷ M ADM-(22-52) significantly in the two cell lines (Figs. 6C 6F) , whereas 10⁻⁷ M CGRP-(8-37) had no effects on the cell number (Figs. 6C 6F) . 

Discussion

The present study showed, for the first time, that human RPE cells produce and secrete ADM. The production and the secretion of ADM by ARPE-19 cells were augmented by treatment with IFN-γ or IL-1β. ADM had proliferative effects on F-0202 cells and ARPE-19 cells. These findings suggest that ADM secreted from RPE cells plays important physiological roles in the eyes and is involved in the pathophysiology of some inflammatory disorders of the eyes, such as PVR. 

PVR is the most common cause of failure in the treatment of retinal detachment and is characterized by the abnormal behavior of RPE cells, including migration into the vitreous or into the retina and abnormal proliferation. ⁴³ These membranes are formed when a number of cell types, including RPE cells, glial cells, macrophages, and fibroblasts, migrate into the vitreous cavity, adhere to the retina and vitreous gel, proliferate, and synthesize extracellular matrix, eventually causing traction. Particularly, RPE cells contribute to the pathogenesis of PVR. ⁴⁴ We have recently reported that IR-ADM levels in the vitreous of patients with PVR are significantly elevated compared with those of other ocular disorders, such as proliferative diabetic retinopathy and age-related macular degeneration. ²¹ It is known that ADM is produced and secreted by various types of cells, including vascular endothelial cells, ³ vascular smooth muscle cells, ⁴ macrophages, ^{45 46 47 48} fibroblasts, ⁴⁹ neurons, ⁶ and astrocytes. ⁵⁰ The findings in the present study raise the possibility that RPE cells are one of the major sources that contribute to the elevated levels of IR-ADM in the vitreous in some inflammatory ocular diseases. It is also noteworthy that two inflammatory cytokines, IFN-γ ^{32 33} and IL-1β, ^{32 33 34 35} which may be involved in the pathophysiology of PVR, induced the expression of ADM in ARPE-19 cells. 

Recent studies have shown that ADM stimulates DNA synthesis and cell proliferation through the cAMP-mediated pathway in Swiss 3T3 cells ¹⁰ and exerts mitogenic effects on human oral keratinocyte through the cyclic adenosine monophosphate (cAMP) cascade. ¹¹ ADM also inhibits proliferation of rat mesangial cells through a cAMP-dependent mechanism. ¹² It has been reported that ADM inhibits growth factor–induced migration of smooth muscle cells ¹³ and mitogenesis of mesangial and vascular smooth muscle cells. ⁵¹ In this study we showed that both exogenously added ADM and endogenously produced ADM stimulated proliferation of RPE cells. 

The analyses of culture medium extracts of F-0202 cells and ARPE-19 cells by Sephadex G-50 column chromatography indicated that these cells secreted IR-ADM identical with or similar to human ADM-(1-52). However, the reversed-phase HPLC analysis showed that F-0202 cells mainly secreted IR-ADM chromatographically identical with human ADM-(1-52), but that ARPE-19 cells secreted mainly IR-ADM eluting in two earlier peaks than human ADM-(1-52). The IR-ADM eluting in these two earlier peaks was not likely to represent ADM with an oxidized methionine or ADM-Gly, and may represent ADM with other types of small modifications. Such modified ADMs were also found in the culture media of DLD-1 human colorectal carcinoma cells, ⁵² SW-13 human adrenocortical adenocarcinoma cells, ⁵³ and human astrocytes. ⁵⁰ In contrast, the number of cells was reduced by the anti-ADM Ab or the ADM receptor antagonist, but not by the CGRP antagonist, in F-0202 cells and ARPE-19 cells. These findings suggest that the “modified ADMs” secreted by ARPE-19 cells stimulated the proliferation, as did the ADM secreted by F-0202 cells, and that these endogenously produced ADMs acted on RPE cells, possibly through ADM-specific receptors. ⁵⁴  

The production of ADM in cultured rat vascular smooth muscle cells and vascular endothelial cells was stimulated by treatment with TNF-α or IL-1β and was inhibited by IFN-γ. ^{3 55 56} In cultured glioblastoma cells, ADM production was stimulated by the treatment with IFN-γ or IL-1β and inhibited by TNF-α. ¹⁷ In the present study, IFN-γ or IL-1β increased the ADM production in ARPE-19 cells, but TNF-α did not. IL-1β or TNF-α increased the ADM production in F-0202 cells, however. There may be some difference in the ADM induction by cytokines among various cell types. Combinations of two or three cytokines synergistically increased the ADM production in ARPE-19 cells. Our findings raise the possibility that in some inflammatory ocular disorders, such as PVR, inflammatory cytokines stimulate the production and secretion of ADM by the RPE cells as well as other types of cells, including vascular endothelial and smooth muscle cells, macrophages, fibroblasts, and glial cells. ADM secreted by these cells in some inflammatory ocular disorders may stimulate the proliferation of RPE cells and other types of cells in an autocrine or paracrine fashion. 

The antiproliferative, ⁵⁷ nonproliferative, ⁵⁸ and proliferative ^{59 60} effects of RA on cultured RPE cells have been reported. RA increased ADM production in a macrophage cell line. ⁴⁶ In this study, however, RA had no significant effects on ADM production. 

In summary, this study has demonstrated that cultured human RPE cells produce and secrete ADM. Proinflammatory cytokines induce the production and secretion of ADM. Furthermore, ADM has proliferative effects on cultured human RPE cells, possibly acting through specific ADM receptors. These findings suggest that ADM secreted by RPE cells is important in the pathophysiology of some inflammatory disorders of the eyes, such as PVR. A better knowledge of ADM in the eye may have therapeutic implications. 

 Supported in part by Grants-in-Aid for Scientific Research (B) (SS), (C) (KT) and on Priority Areas (A) (KT) from the Ministry of Education, Science, Sports and Culture of Japan, by the Nakatomi Foundation (SS), by the Mochida Memorial Foundation for Medical and Pharmaceutical Research (KT), and by the Gonryou Medical Foundation (KT).

 Submitted for publication September 24, 1999; revised December 29, 1999; accepted January 18, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Northern blot analysis of ADM mRNA in three human RPE cells (F-0202, D407, and ARPE-19), HUVECs, and HeLa human cervical cancer cells. Pheo 1, Pheo 2, human pheochromocytomas case 1 and case 2. Each lane contains 15 μg total RNA. Bottom: G3PDH internal control. Human pheochromocytomas and HUVECs were included as positive controls.

Table 1.

 

IR-ADM and Relative Expression Levels of ADM mRNA in Human Cultured Cell Lines

Cell Lines	IR-ADM in Culture Media (fmol/105 Cells per 24 h)	Relative Expression Levels of ADM mRNA*
F-0202	75.30 ± 5.86	2.63
D407	31.72 ± 1.36	0.66
ARPE-19	22.78 ± 1.42	1.14
HUVEC	12.90 ± 1.00	1.00
HeLa	9.54 ± 0.79	0.07

 Each value represents mean ± SEM of five separate dishes.

*  The intensity of hybridization signals representing ADM mRNA in Figure 1 was quantified with an image analyzer, and the intensity of ADM mRNA was normalized with the intensity of G3PDH mRNA in each case. The ratio of each normalized value to that of the HUVECs is shown as the relative expression levels of ADM mRNA.

Figure 2.

 

Sephadex G-50 column chromatography (A, C) and reversed-phase HPLC (B, D) of the culture medium extracts of human RPE cells (A, B F-0202; C, D ARPE-19). The media cultured by F-0202 cells (A, B) and ARPE-19 cells (C, D) for 24 hours were extracted by Sep-Pak C18 cartridges and analyzed by chromatography. Vo, void volume; ADM, the elution position of human ADM-(1-52); ADM-Gly, the elution position of ADM-Gly. Dotted line: gradient of acetonitrile (ACN).

Figure 3.

 

Effects of cytokines on the production of ADM in ARPE-19 cells. (A) The concentrations of IR-ADM in the cultured media of untreated ARPE-19 cells (control) and cells treated with bFGF (10 ng/ml), IFN-γ (100 U/ml), TNF-α (20 ng/ml), IL-1β (10 ng/ml), or RA (10 nmol/ml). The data shown are mean ± SEM (n = 5). Significantly higher than the control: ^** P < 0.005; ^### P < 0.0001. (B) Northern blot analysis of ADM mRNA of untreated or treated ARPE-19 cells. Each lane contains 15 μg total RNA. Bottom: Internal control.

Figure 4.

 

Time course effects (A, B) and dose–response effects (C, D) of IFN-γ and IL-1β on IR-ADM in the medium and ADM mRNA expression in ARPE-19 cells. (A) IR-ADM concentrations in the culture media and (B) Northern blot analysis of ADM mRNA in ARPE-19 cells treated with 100 U/ml IFN-γ or 10 ng/ml IL-1β for 6, 12, and 24 hours. (C) IR-ADM concentrations in the media and (D) ADM mRNA levels in ARPE cells treated for 24 hours with 1, 10, and 100 U/ml IFN-γ, and 0.1, 1.0, and 10 ng/ml IL-1β. (A, C) Data are means ± SEM (n = 5). Significantly higher than the untreated cells: ^# P < 0.01; ^### P < 0.0001. (B, D) Each lane contains 15 μg total RNA. Bottom: Internal control.

Figure 5.

 

Effects of combinations of three proinflammatory cytokines, IFN-γ, IL-1β, and TNF-α on the production of ADM in ARPE-19 cells. (A) IR-ADM concentrations in the cultured media of untreated ARPE-19 cells (control) and cells treated with either IFN-γ (100 U/ml), IL-1β (1 ng/ml), TNF-α (20 ng/ml), or a combination of two or three of these cytokines. Data are means ± SEM (n = 5). Significantly higher than the control: ^### P < 0.0001. (B) Northern blot analysis of ADM mRNA of untreated or treated ARPE-19 cells. Each lane contains 15 μg total RNA. Bottom: Internal control.

Figure 6.

 

The effects of human ADM on the proliferation of human RPE cells. (A, B, and C) F-0202; (D, E, and F) ARPE-19. (A, D) The effects of exogenously added human ADM-(1-52) on the proliferation of human RPE cells. Cells were treated with various concentrations of ADM-(1-52) for 24 hours and counted (modified MTT assay). Data are means ± SEM (n = 6). Significantly higher than the control: ^* P < 0.05; ^** P < 0.005; ^## P < 0.001. (B, E) The effects of anti-ADM antibody (ADM Ab) on the proliferation of human RPE cells. ^**Significantly lower than those treated with NRS, P < 0.005. (C, F) The effects of 10⁻⁷ M ADM receptor antagonist, human ADM-(22-52), and 10⁻⁷ M CGRP antagonist, human CGRP-(8-37), on the proliferation of human RPE cells. Significantly higher or lower than control: ^* P < 0.05; ^# P < 0.01; ^** P < 0.005.