Effect of berberine on lipopolysaccharide-induced monocyte chemotactic protein-1 and interleukin-8 expression in a human retinal pigment epithelial cell line.

PURPOSE: In this study, we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in a human retinal pigment epithelial cell line (ARPE-19) caused by lipopolysaccharide (LPS) stimulation. METHODS: ARPE-19 cells were cultured to confluence. Berberine and LPS were added to the medium. MCP-1 and IL-8 mRNA were measured by real-time polymerase chain reaction. MCP-1 and IL-8 protein concentrations in the media were measured using enzyme-linked immunosorbent assay. RESULTS: After stimulation with LPS, MCP-1 and IL-8 mRNA in ARPE-19 cells reached maximum levels at 3 h, and MCP-1 and IL-8 protein in the culture media reached maximum levels at 24 h. Berberine dose-dependently inhibited MCP-1 and IL-8 mRNA expression of the cells and protein levels in the media stimulated with LPS. CONCLUSIONS: These findings indicate that berberine inhibited the expression of MCP-1 and IL-8 induced by LPS.

First identification of Gordonia sputi in a post-traumatic endophthalmitis patient - a case report and literatures review.

BACKGROUND: We present a case of post-traumatic endophthalmitis with relatively good prognosis caused by Gordonia sputi, which, to our knowledge is the first case in the literature. CASE PRESENTATION: A 24 year old man, who underwent an intraocular foreign body extraction half a month before presentation in the left eye, was referred to us complaining of blurred vision and slight pain for 5 days. His first presentation showed moderate intracameral and intravitreous purulent inflammation with a best corrected vision of counting fingers. After gram staining of the intravitreous samples revealed a gram-positive bacilli infection, a combination of amikacin and vancomycin was initially injected intravitreously. The left eye kept stable for three days but deteriorated on the 4th day. On the 5th day after presentation conventional culture characterized the bacterium as an Actinomyces sp. while 16S ribosomal RNA gene sequencing confirmed it as Gordonia sputi. Thereby a complete pars plana vitrectomy combined with lensectomy and silicone oil tamponade was performed. During the surgery an intraocular irrigation with penicillin G was adopted, followed by administration of intravenous penicillin G twice one day for a week. A relatively normal fundus with slight intracameral inflammation was observed a week after the operation, and the best corrected vision recovered to 0.15. One year later his vision remained 0.1. CONCLUSION: Gordonia sputi should be taken into consideration in patients with post-traumatic endophthalmitis especially due to foreign body penetration. Compared to conventional laboratories, molecular methods are recommended for an accurate diagnosis. A comprehensive strategy of antimicrobial agents and vitrectomy may render a satisfactory result.

[Clinical results of ultrasound biometry in silicone oil-filled eye].

OBJECTIVE: To evaluate a method of ultrasound biometry in silicone oil-filled eye and its clinical results. METHODS: This was a series case study. According to the principle of measuring a distance with ultrasound, we compared the measured distance between a space filed with balanced salt solution and silicone oil at same height, to calculate a conversion factor (0.674) between them. A formula for corrective axial length in silicone oil-filled eye was established. The formula = ab + 0.674 x bc (a, b and c standing for the apex of the cornea, the posterior pole of the lens or the center of the capsular membrane and the anterior surface of the macular, respectively). The axial lengths of 150 silicone oil-filled eyes in 150 cases were then measured before and after silicone oil removal with Vivid 7 Dimension ultrasound. According to the axial length, they were divided into two groups, namely group 1 (the length < 25 mm) and group 2 (the length > or = 25 mm). In 76/150 eyes, before combined silicone oil removal and intraocular lens (IOL) implantation, the SRKT formula was used for intraocular lens calculation; the post-operative actual refraction was compared with the pre-operative predicted refraction and statistics analysis was made. RESULTS: The retinal condition of 150 silicone oil-filled eyes in 150 cases after 3 months' follow-up was stable after surgery. The results of the biometry were as follows. In the first group, the mean corrective axial lengths of 111 silicone oil-filled eyes before silicone oil removal was (22.77 +/- 1.00) mm (ranging from 21.10 to 24.90 mm); the mean axial lengths after silicone oil removal was (22.76 +/- 0.99) mm (ranging from 21.00 to 24.70 mm). The difference between them was not statistically significant (t = 0.518, P > 0.05). The vitreous cavity depth before and after silicone oil removal was (26.57 +/- 2.14) mm and (17.90 +/- 1.38) mm, respectively. The ratio of the latter to the former was 0.673 78. In the second group, the mean corrective axial lengths of 39 silicone oil-filled eyes before silicone oil removal was (26.52 +/- 1.31) mm (ranging from 25.00 to 30.58 mm); the mean axial lengths after silicone oil removal was (26.53 +/- 1.29) mm (ranging from 25.00 to 30.59 mm). The difference between them was not statistically significant (t = 0.109, P > 0.05). The vitreous cavity depth before and after silicone oil removal was (32.01 +/- 2.90) mm and (21.57 +/- 2.04) mm, respectively. The ratio of the latter to the former was 0.673 95. In 76 eyes with IOL, the post-operative actual refraction after at least 3 months follow-up was compared with the pre-operative predicted refraction (-1.50 DS) in both groups. The differences between them were not statistically significant (t(1) = 0.253, P(1) > 0.05; t(2) = 0.209, P(2) > 0.05) in each group. CONCLUSION: Ultrasound biometry in silicone oil-filled eye is accurate and simple, and has good results in clinical measurement.

A cholinergic agonist attenuates endotoxin-induced uveitis in rats.

PURPOSE: Investigation of physiological anti-inflammatory mechanisms can contribute to the treatment of inflammatory disorders. The purpose of the present study was to investigate the effect of nicotine, a selective cholinergic agonist, on endotoxin-induced uveitis (EIU) in rats and the underlying molecular mechanism. METHODS: Lipopolysaccharide (LPS; endotoxin) and nicotine were injected intraperitoneally. Clinical scores were evaluated by slit lamp. Intracameral protein content and the number of cells were determined. Immunohistochemical reactivity of alpha7 nicotine acetylcholine receptor (alpha7nAChR) was examined in the iris and ciliary body (ICB). mRNA and protein levels of cytokines and chemokines were measured by real-time PCR and enzyme-linked immunosorbent assay. RESULTS: After LPS injection, clinical scores, as well as protein content and number of cells in the aqueous humor increased during 18 to 36 hours. Nicotine inhibited the endotoxin-induced elevation of these levels. mRNA and protein of alpha7nAChR expression levels were significantly increased by LPS and/or nicotine injection. Nicotine showed no effects on endotoxin-induced elevation of mRNA levels in ICB. However, nicotine decreased the endotoxin-induced elevation of interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, cytokine-induced neutrophil chemoattractant (CINC)-1, and monocyte chemotactic protein (MCP)-1, but did not affect IL-10 in the serum and aqueous humor. CONCLUSIONS: Nicotine attenuated endotoxin-induced uveitis through directly decreasing the levels of multiple cytokines and chemokines in the aqueous humor, but did not affect the mRNA levels of these factors. The findings suggest that the nicotinic anti-inflammatory pathway may be involved in the pathogenesis of EIU.

Effect of N-acetylcysteine on lipopolysaccharide-induced uveitis in rats.

PURPOSE: In this study we investigated the in vivo effect of N-acetylcysteine (NAC) on lipopolysaccharide (LPS)-induced uveitis in rats. METHODS: To induce uveitis, LPS (100 microg) was injected into subcutaneous tissue of Wistar rats (170-190 g). NAC was injected intraperitoneally. Intracameral levels of protein, cells, nitrite, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 were determined by spectrophotometry, hemocytometry, and enzyme-linked immunosorbent assay. Expression of TNF-alpha, IL-6, endothelial leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and inducible nitric oxide synthase (iNOS) mRNA was examined by real-time polymerase chain reaction. RESULTS: LPS injection elevated intracameral protein and cells, and the elevation was inhibited by NAC. LPS injection induced expression of TNF-alpha, IL-6, E-selectin, and ICAM-1 mRNA in the iris/ciliary body at 3 h, and iNOS mRNA at 6 h. The LPS-induced elevation of the mRNA levels was inhibited by NAC. NAC inhibited LPS-induced intracameral elevation of TNF-alpha, IL-6, and nitrite. CONCLUSION: NAC decreased LPS-induced uveitis in vivo by reducing the expression of proinflammatory cytokines and adhesion molecules.

Effect of berberine on barrier function in a human retinal pigment epithelial cell line.

PURPOSE: To examine the effects of berberine, an alkaloid isolated from some medicinal herbs, on the disruption of the barrier function in a human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin-1beta (IL-1beta). METHODS: ARPE-19 cells were cultured to confluence. Berberine and IL-1beta were added to the medium. Barrier functions were evaluated by measuring transepithelial electrical resistance (TER) and the permeability to horseradish peroxidase (HRP) and sodium fluorescein (SF). RESULTS: Berberine dose-dependently inhibited decreased TER and increased the permeability to HRP and SF in the cells stimulated with IL-1beta. CONCLUSIONS: Berberine dose-dependently inhibited the disruption of the barrier function in the ARPE-19 cell line induced by IL-1beta.

Effect of berberrubine on interleukin-8 and monocyte chemotactic protein-1 expression in human retinal pigment epithelial cell line.

We examined the effects of berberrubine, a protoberberine alkaloid, on interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNF-alpha). ARPE-19 cells were cultured to confluence. Berberrubine and IL-1beta or TNF-alpha were added to the medium. IL-8 and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. IL-8 and MCP-1 mRNA were measured by real time polymerase chain reaction. Nuclear factor kappaB (NF-kappaB) translocation was examined by immunofluorescent staining/microscopy. Berberrubine dose-dependently inhibited IL-8 and MCP-1 protein levels in the media and mRNA expression of the cells stimulated with IL-1beta or TNF-alpha. Immunofluorescent staining/microscopy of NF-kappaB in the nucleus of unstimulated cells was faint (51+/-14 arbitrary units). Fluorescein was dense (215+/-42 or 170+/-24 arbitrary units, respectively) 30 min after stimulation with IL-1beta or TNF-alpha and was decreased to 62+/-18 or 47+/-16 arbitrary units, respectively, by berberrubine. Berberrubine dose-dependently inhibited IL-8 and MCP-1 expression and protein secretion induced by IL-1beta or TNF-alpha. Possibly, the effect on chemotactic factors may be via suppression of NF-kappaB translocation.

Vitreous Chemokines and Sho (Zheng in Chinese) of Chinese-Korean-Japanese medicine in patients with diabetic vitreoretinopathy.

We examined the levels of vitreous chemokines and Sho (Zheng in Chinese) of Chinese-Korean-Japanese medicine in diabetic patients. Patients undergoing vitrectomy were classified into Group 1 (no diabetic retinopathy), Group 2 (diabetic retinopathy with no or a few new vessels), and Group 3 (diabetic retinopathy with many new vessels). The levels of IL-8, MCP-1, MIP-1alpha, MIP-1beta, and RANTES in the vitreous fluid were measured using cytometric bead array method. Sho was determined by the standard diagnostic method of Chinese-Korean-Japanese medicine. Vitreous levels of IL-8 and MCP-1 in Groups 2 and 3 were higher than those in Group 1. MIP-1alpha, MIP-1beta, and RANTES levels in Groups 2 and 3 were almost the same as those in Group 1. The percentage of patients with Keishibukuryo-gan (Guizhifuling-wan in Chinese) sho in Group 3 was higher than that in Group 1. In conclusion, vitreous levels of IL-8 and MCP-1 were high in patients with diabetic vitreoretinopathy. Keishibukuryo-gan sho may be associated with diabetic vitreoretinopathy.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on IL-6, IL-8, and MCP-1 expression in human retinal pigment epithelial cell line.

PURPOSE: To examine pituitary adenylate cyclase-activating polypeptide (PACAP) receptors (PAC1, VPAC1, and VPAC2) mRNA and the effect of PACAP on interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) expression in human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin-1beta (IL-1beta). METHODS: Expression of PACAP receptor mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). PACAP and IL-1beta were added to serum-free medium. IL-6, IL-8, and MCP-1 mRNA were measured by real-time PCR. IL-6, IL-8, and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. Nuclear factor kappaB (NF-kappaB) translocation was examined by immunofluorescence. RESULTS: PAC1 and VCAP1 receptors mRNA were expressed in unstimulated cells. VCAP2 mRNA was expressed in cells stimulated with IL-1beta. IL-1beta stimulated IL-6, IL-8, and MCP-1 mRNA expression and protein levels. PACAP (10(- 7) to 10(- 6) M) inhibited IL-1beta -stimulated IL-6, IL-8, and MCP-1 mRNA and protein levels. Immunofluorescence of NF-kappaB in the nucleus was dense 30 min after stimulation with IL-1beta, and it was decreased by PACAP. CONCLUSIONS: ARPE-19 cells had PACAP receptors mRNA. PACAP inhibited IL-6, IL-8, and MCP-1 expression and protein secretion. Possibly, the effect on cytokines may be via suppression of NF-kappaB translocation.

Changes in vitreous VEGF, bFGF and fibrosis in proliferative diabetic retinopathy after intravitreal bevacizumab.

AIM: To evaluate the relationship between intravitreal bevacizumab (IVB) treatment and the levels of vitreous vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and vitreous-retina surface fibrosis in patients with proliferative diabetic retinopathy (PDR). METHODS: This study was a prospective, open-label, controlled, randomized clinical trial. Sixty-eight eyes of PDR patients (n=53) and macular hole patients (n=15) were enrolled in this study. Thirty-four eyes of the PDR patients received IVB before vitrectomy. Twenty-three of the 34 PDR patients received IVB treatment 5d before vitrectomy (subgroup a), and 11 of the 34 PDR patients received IVB treatment greater than 2wk prior to vitrectomy (subgroup b). Nineteen of the PDR patients did not receive IVB treatment at any time prior to vitrectomy. The levels of bFGF and VEGF in vitreous samples were measured using enzyme-linked immunosorbent assay (ELISA) and the degree of vitreoretinal fibrosis was characterized using clinical data and data obtained intra-operatively. RESULTS: In PDR patients, VEGF and bFGF levels were significantly increased compared to non-PDR (control) subject's eyes (P<0.01). In PDR patients, vitreous VEGF levels were significantly decreased following IVB treatment compared to PDR patients that did not receive IVB treatment (P<0.01). The degree of vitreoretinal fibrosis was significantly increased in subgroup b compared to subgroup a(P<0.05) and to patients that did not receive IVB (P<0.05). Vitreous bFGF levels were significantly greater in subgroup b than subgroup a (P<0.01) or in patients who did not receive IVB treatment (P<0.05). A Spearman's rank correlation test indicated that higher levels of vitreous bFGF, but not VEGF, correlated with the degree of vitreoretinal fibrosis. CONCLUSION: We found that bFGF levels increase in PDR patient's vitreous after IVB treatment longer than two weeks prior to vitrectomy and correlated with the degree of fibrosis after IVB treatment. These findings suggest vitreous fibrosis is increased in PDR patients after IVB treatment may be due to increased levels of bFGF.

Effects of rolipram, a selective inhibitor of type 4 phosphodiesterase, on lipopolysaccharide-induced uveitis in rats.

PURPOSE: To investigate effects of rolipram, an inhibitor of type 4 phosphodiesterase, on lipopolysaccharide (LPS)-induced uveitis in Wistar rats. METHODS: A total of 100 microg LPS was injected into the rat footpad. Rolipram (Wako Pure Chemical, Osaka, Japan) was injected intraperitoneally 30 minutes before administration of LPS. Levels of intracameral protein, cells, E-selectin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and nitrite were determined. E-selectin, TNF-alpha, IL-6, and inducible nitric oxide synthase (iNOS) mRNAs and immunohistochemical reactivity of nuclear factor (NF)-kappa B and TNF-alpha were also examined in the iris-ciliary body. RESULTS: After LPS injection, intracameral protein and cells increased from 18 to 30 hours later. Rolipram, however, inhibited elevation of protein and cells. After LPS injection, mRNA levels of E-selectin, TNF-alpha, and IL-6 in the iris-ciliary body increased 3 hours later, and iNOS mRNA increased 6 hours later. Elevation of mRNA levels for E-selectin, TNF-alpha, and IL-6 was inhibited by rolipram. After LPS injection, intracameral TNF-alpha and IL-6 levels increased 4 to 6 hours later, and nitrite levels increased 14 to 20 hours later. Elevation of TNF-alpha and IL-6 levels was decreased by rolipram. Rolipram did not affect iNOS mRNA and nitrite levels. Immunoreactivity of NF-kappa B was strong 1 hour after LPS injection, and was decreased by rolipram. Immunoreactivity of TNF-alpha was strong 4 hours after LPS injection and was decreased by rolipram. CONCLUSIONS: NF-kappa B translocation and expression of E-selectin, TNF-alpha, and IL-6 are involved in the pathogenesis of LPS-induced uveitis and are inhibited by rolipram. The inhibitory effect of rolipram in uveitis may be independent of iNOS synthesis.

Berberine exerts neuroprotective actions against in vitro ischemia-induced neuronal cell damage in organotypic hippocampal slice cultures: involvement of B-cell lymphoma 2 phosphorylation suppression.

In this study we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on ischemic neuronal damage in mouse organotypic hippocampal slice cultures (OHSCs) caused by oxygen and glucose deprivation (OGD) and N-methyl-D-aspartate (NMDA) -type glutamate receptor stimulation. Hippocampal slices obtained from 7-d-old ICR mice were cultured for 10 d before the experiments. Ischemia-related damage was induced by OGD (5, 15, 45 min) or NMDA (10 microM) treatment, and was evaluated by measuring propidium iodide (PI) uptake. Levels of apoptotic marker proteins, B-cell lymphoma 2 (Bcl-2) and phosphorylated-Bcl-2 (p-Bcl-2), in the OHSCs were measured as indices of biochemical neuronal cell damage by Western blotting. Berberine (5, 25 microM) or the NMDA antagonist MK-801 (25 microM) was added to the medium 30 min before OGD or NMDA treatment. OGD time-dependently increased PI uptake of the OHSCs. Both berberine (5, 25 microM) and MK-801 (25 microM) significantly inhibited PI uptake at 24 h after 45-min OGD treatment and PI uptake in OHSCs exposed to NMDA for 24 h. OGD treatment also significantly increased the level of p-Bcl-2 but not that of Bcl-2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in OHSCs. Berberine (5-25 microM) significantly suppressed the OGD-induced increase of p-Bcl-2 level in OHSCs when tissue was exposed to the alkaloid prior to OGD or simultaneously with OGD. These findings suggest that berberine has protective effects against ischemic damage in mouse OHSCs and that the effects are at least partly mediated by suppression of Bcl-2 phosphorylation.

S100A9-positive granulocytes and monocytes in lipopolysaccharide-induced anterior ocular inflammation.

S100A9 is a pro-inflammatory protein expressed in infiltrating granulocytes and monocytes. We determined role of S100A9 in endotoxin (LPS)-induced uveitis (EIU) and keratitis in Wistar rats. Anti-S100A9 antibody decreased partially clinical scores, protein, and cells in the aqueous humor at 18-36 h, compared with the LPS group. S100A9-positive cells were expressed in the iris-ciliary body (ICB) and cornea at 24-48 h. Activated caspase-3 (related to apoptosis) and S100A9 co-expressed in ICB at 18-48 h after LPS injection. S100A9 was not expressed in ED2-positive cells in ICB. Dexamethasone (DEX) increased S100A9 mRNA and protein levels in the circulating blood leukocytes, but reduced S100A9 mRNA and protein levels in ICB after LPS injection. BAY 11-7085 (an inhibitor of I-kappaB phosphorylation) suppressed S100A9 mRNA in leukocytes (43.5%) and ICB (68.5%), respectively, after LPS injection. It is possible that S100A9-positive granulocytes and monocyte/macrophages may play a role in the late phase of EIU and keratitis that DEX may inhibit the migration of S100A9-positive granulocytes and monocytes from the blood into the extravascular tissues, and that nuclear factor (NF)-kappaB pathway may be involved in S100A9 expression. S100A9 could play a role in the clearance of inflammatory cells at the late phase of EIU.

Effect of berberine on monocyte chemotactic protein-1 and cytokine-induced neutrophil chemoattractant-1 expression in rat lipopolysaccharide-induced uveitis.

PURPOSE: The aims of this study were to examine the in vivo effects of berberine, an alkaloid isolated from some medicinal herbs, on monocyte chemotactic protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) expression in rat lipopolysaccharide (LPS)-induced uveitis. METHODS: LPS was injected intraperitoneally. Berberine was orally administered. MCP-1 mRNA and CINC-1 mRNA were measured by semiquantitative reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. MCP-1 and CINC-1 protein concentration in the aqueous humor were measured by enzyme-linked immunosorbent assay. Histopathologic study was performed in the anterior ocular segments. RESULTS: Berberine dose-dependently inhibited LPS-induced MCP-1 mRNA and CINC-1 mRNA expression of the iris-ciliary body. The alkaloid inhibited chemokines, protein and cell levels in the aqueous humor in rats stimulated with LPS. On histopathologic study, the inflammatory cell infiltration was diminished by the berberine treatment. CONCLUSIONS: These findings indicate that berberine dose-dependently inhibited the expression of MCP-1 and CINC-1 induced by LPS and diminished the anterior uveitis.

Effect of berberine on interleukin 8 and monocyte chemotactic protein 1 expression in a human retinal pigment epithelial cell line.

PURPOSE: The aims of this study were to examine the effects of berberine, an alkaloid isolated from some medicinal herbs, on interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin 1beta (IL-1beta) or tumor necrosis factor alpha (TNF-alpha). METHODS: ARPE-19 cells were cultured to confluence. Berberine and IL-1beta or TNF-alpha were added to the medium. IL-8 mRNA and MCP-1 mRNA were measured by semiquantitative reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. IL-8 and MCP-1 protein concentrations in the media were measured using enzyme-linked immunosorbent assay. RESULTS: Berberine dose-dependently inhibited IL-8 mRNA and MCP-1 mRNA expression of the cells and protein levels in the media stimulated with IL-1beta or TNF-alpha. CONCLUSION: These findings indicate that berberine dose-dependently inhibited the expression of IL-8 and MCP-1 induced by IL-1beta or TNF-alpha.

Effect of alpha-melanocyte-stimulating hormone on interleukin 8 and monocyte chemotactic protein 1 expression in a human retinal pigment epithelial cell line.

PURPOSE: To examine melanocortin receptor (from MC-1 to MC-5) mRNA and the effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with IL-1beta or tumor necrosis factor alpha (TNF-alpha). METHODS: Expressions of MC-1 to MC-5 mRNA were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). alpha-MSH and IL-1beta or TNF-alpha were added to serum-free medium. IL-8 and MCP-1 mRNA were measured by real-time PCR. IL-8 and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. Nuclear factor kappaB (NF-kappaB) translocation was examined by immunofluorescent staining/microscopy. RESULTS: MC-1 to MC-5 receptor mRNA was expressed in unstimulated cells. IL-1beta stimulated IL-8 and MCP-1 mRNA at 6 h. TNF-alpha stimulated IL-8 and MCP-1 mRNA expression at 1.5 and 3 h. alpha-MSH (10(-14) to 10(-10)M) inhibited IL-8 and MCP-1 mRNA expression in the cells stimulated with IL-1beta or TNF-alpha. alpha-MSH inhibited IL-1beta or TNF-alpha-stimulated IL-8 and MCP-1 protein levels in the media. Immunofluorescent staining/microscopy of NF-kappaB in the nucleus was dense 30 min after stimulation with IL-1beta or TNF-alpha and was decreased by alpha-MSH. CONCLUSIONS: ARPE-19 cells had MC-1 mRNA. alpha-MSH inhibited IL-8 and MCP-1 expression and protein secretion. Possibly, the effect on chemotactic factors may be via suppression of NF-kappaB translocation.