Retinal Pigment Epithelium Activation Of Alternative Macrophages

Abstract

Purpose: The retinal pigment epithelium (RPE) through the neuropeptides alpha-melanocyte stimulating hormone (α-MSH) and Neuropeptide Y (NPY) alternatively activate macrophages, with similar characteristics as retinal microglial cells. Recently, we have reported that while α-MSH and NPY do not suppress FcR-mediated phagocytosis they do suppress the acidification of the phagolysosome and the up regulation of FcR-stimulated reactive oxygen species. We examined the potential for α-MSH and NPY to modulate transcription factors associated with differentiation and activation of macrophage functionalities. Also, we examined whether the expression of other FcR-stimulated activation markers are suppressed by the neuropeptides.

Methods: The resting macrophages (RAW 264.7 cells) were co-treated with α-MSH and NPY at 1 ng/ml (the concentration made by an in situ culture of a RPE monolayer in 24 hours). The macrophages were collected and lysed 15 and 30 mins later to assay for phosphorylation of STAT1 (M1 macrophages), STAT6 (M2 macrophages), and STAT3 (myeloid derived suppressor cells). These were assayed by immunoblotting and relative band intensity to beta-actin and to levels in untreated macrophages was calculated. Also, macrophages were given opsonized-E coli-bioparticles, collected at 24 hours, and assayed by flow cytometry for the expression of activation markers CD40, Ia^d, CD80, and CD86.

Results: The FcR-stimulated macrophages significantly up-regulated their expression of Ia^d, CD40, CD80, and CD86; however, FcR-stimulated macrophages co-treated with α-MSH and NPY were suppressed in expressing CD40 and CD80, but not Ia^d and CD86. In resting macrophages 15 mins after co-treatement with α-MSH and NPY there was no change in the levels of phosphorylated STAT1 and STAT6, but there was a significant increase in phosphorylation of STAT3. While STAT3 returned to base levels of phosphorylation 30 min after treatment, the level of phosphorylated STAT1 was significantly suppressed below base-levels.

Conclusions: Resting macrophages are alternatively activated by the RPE produced neuropeptides α-MSH and NPY, and express unique characteristics of suppressor cells. The RPE through α-MSH and NPY have the capacity to mediate activation of retinal macrophage/microglial cells that would function to suppress activation of inflammation and effector T cells.

Keywords: 555 immunomodulation/immunoregulation • 553 immune tolerance/privilege • 701 retinal pigment epithelium