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D. Li, A.W. Taylor; Transfection With MC1r siRNA Neutralizes Alpha–MSH Suppression of LPS–Stimulated Macrophages . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5147.
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© ARVO (1962-2015); The Authors (2016-present)
The ocular immunosuppressive neuropeptide alpha–melanocyte stimulating hormone (α–MSH) suppresses inflammation mediated by both adaptive and innate immunity. There are three receptors for α–MSH on immune cells, the melanocortin 1, 3, and 5 receptors (MC1, 3, 5r).We recently found that suppression of Th1 cell activation and its induction of CD25+CD4+ regulatory T cells is through MC5r expressed on the CD4+ T cells. We therefore ask which of the MCr on macrophages is important in α–MSH suppression of innate immunity.
A double stranded small interfering RNA (siRNA) was synthesized to target mouse MC1r mRNA. The siRNA was transfected into a mouse macrophage cell line RAW264.7. The MC1r gene silencing (knock–down) was confirmed by quantitative real–time PCR and immunoblot analysis 48 hours after transfection. The cells with or without silenced MC1r were treated with α–MSH and activated with lipopolysaccharide (LPS).The culture supernatant was assayed for nitric oxide (NO) generation with Griess reagent and TNF–α production by ELISA. The LPS–stimulated p38 phosphorylation and NF–ΚB activation were assayed by immunoblotting and by an electrophoretic mobility shift assay (EMSA) on macrophage cytoplasmic and nuclear lysates.
The MC1r siRNA significantly knocked–down MC1r mRNA and protein expression compared to macrophages transfected with an irrelevant siRNA. The MC1r siRNA transfection neutralize α–MSH suppression of NO and TNF–α production in the macrophages. An immunoblot analysis and EMSA showed that α–MSH was unable to effectively neutralize its suppression of LPS–induced p38 and NF–ΚB activation in MC1r siRNA transfected macrophages.
These findings demonstrate that, as MC5r is important in α–MSH mediated suppression of inflammatory activity by CD4+ T Cell, MC1r is the major α–MSH receptor by which α–MSH suppresses LPS–induced inflammatory activity in macrophages. Together with the importance of MC5r in ocular immunity the results imply that functional expression of the MCr on immune cells is as important as the expression of α–MSH within the ocular microenvironment.
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