Effect of alpha-melanocyte-stimulating hormone on interleukin 8 and monocyte chemotactic protein 1 expression in a human retinal pigment epithelial cell line.
OBJECTIVE
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.
Cui
Hu-Shan
HS
Department of Ophthalmology, Toyama Medical and Pharmaceutical University, Toyama, Japan. ophthal@ms.toyama-mpu.ac.jp
Hayasaka
Seiji
S
Zhang
Xue-Yun
XY
Chi
Zai-Long
ZL
Hayasaka
Yoriko
Y
D002460:Cell Line; D018932:Chemokine CCL2 / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D044101:Receptors, Melanocortin / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000521:alpha-MSH / Q000494:pharmacology*
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D007375:Interleukin-1; D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger; D044101:Receptors, Melanocortin; D014409:Tumor Necrosis Factor-alpha; D000521:alpha-MSH