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A. W. Taylor; Regulation of Apoptotic Pathways by the Ocular Immunoregulating Neuropeptide Alpha-Melanocyte Stimulating Hormone (-MSH). Invest. Ophthalmol. Vis. Sci. 2010;51(13):838. doi: https://doi.org/.
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Our previous discovery that retinal pigmented epithelial cells (RPE) modulate macrophage/microglial cell functionality through alpha-melanocyte stimulating hormone (α-MSH) showed that this neuropeptide was protecting macrophages from apoptosis induced by the RPE under serum free conditions. Since the induction of apoptosis in cultured macrophages can be though caspase dependent and independent pathways, we assayed the apoptotic pathways that α-MSH suppressed in macrophages cultured under serum free conditions.
The mouse leukemic monocyte macrophage cell line RAW 264.7 cells were treated to induce specific apoptotic pathways. A caspase independent apoptotic pathway was induced with alpha-lactalbumin, and a reactive oxygen-dependent pathway was induced with Sulfasalazine. The extrinsic caspase dependent pathway was induced by culturing the cells in serum-free media, and the intrinsic pathway was induced by culturing late passage cells in media with 10%FBS. The cells were incubated for 18 hours and assayed by flow analysis for TUNEL staining, by immunoblotting of their lysate for BAX and Bcl-2 protein, and for Caspase 8 and Caspase 9 specific activity.
There was no observed α-MSH suppression of death in cells treated with alpha-lactalbumin, or with Sulfasalazine. Caspase 8 induced activity in RAW cells cultured in the serum-free media was suppressed by α-MSH in a dose-dependent manner along with a decrease in TUNEL positive cells. Under serum containing conditions, α-MSH suppressed Caspase 9 activity. In addition, RAW cells cultured in serum free media treated with α-MSH had a downward shift of about 20% in the ratio of BAX to Bcl-2 expression.
These results demonstrate that under the same serum-free conditions of our original observation of RPE modulation of macrophage activity α-MSH suppresses the activated extrinsic apoptotic pathway. The implications of these results are that the RPE selectively eliminates monocytes that cannot be immunosuppressed by α-MSH and the other mediators of ocular immune privilege.
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