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Andrew W Taylor, Robert Shannon, Issac Benque, Tat Fong Ng; Regulation of antigen processing in macrophages by RPE. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5741. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous work demonstrated that healthy retinal pigment epithelial cell (RPE) monolayers promote suppressor cell functions in macrophages, and regulate the process of phagocytosis. The neuropeptides alpha-melanocyte stimulating hormone (α-MSH) and Neuropeptide Y (NYP) produced by healthy (RPE) mediate the suppression of phagolysosome activation in macrophages that have phagocytized opsonized-material. This suggested that the RPE neuropeptides are potentially regulating antigen processing in resident macrophages and microglial cells by altering phagosome maturation. Therefore, we assayed the content of vesicles with phagocytized materials for markers of phagosome/endocytic maturation.
The macrophage cell line RAW 264.7 were treated with the neuropeptides (1 ng/ml each), and fed opsonized-Ovalbumin (OVA)-coated magnetic Dynabeads under serum-free conditions. After 24 hours the cells were lysed in 250 mM Sucrose, 3mM Imidazole lysing buffer to preserve the intracellular vesicles. The magnetic bead containing vesicles were isolated using a magnet. The isolated vesicles were assayed by immunoblotting for OVA protein, Rab5 (early phagosome marker), Rab7 (late phagosome marker), Lamp1 (marker of phagolysosme formation), and MHC class II-beta chain (an indicator of fusion with the MHC class II compartment for peptide loading onto the MHC II molecule). Band intensity was measured, and the relative intensities of Rab7, Lamp1, and MHC class IIβ to Rab5 were calculated. For OVA analysis the relative levels of whole verses fragmented OVA was measured.
The intracellular vesicles containing phagocytized magnetic beads from α-MSH/NPY treated macrophages had significantly more intact OVA than the vesicles from untreated phagocytizing macrophages. The immunoblotting showed that there was a high expression of Rab5 from the treated macrophages relative to the expression of Rab7, Lamp1, and MHC class IIβ. In contrast, there was significantly higher relative expression of Rab7, Lamp1 and MHC class IIβ in the vesicles from the untreated macrophages.
The results demonstrate that RPE neuropeptides α-MSH and NPY suppress the maturation of phagosomes in macrophages. This suggests that the mechanisms of retinal immune privilege include a mechanism to alter the handling of phagocytized materials in macrophages and microglial cells to help prevent presentation of autoantigen peptides and induction of autoimmune disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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