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Darren J. Lee, Andrew W. Taylor; Identification of a MC5r+ Gr-1low F4/80+ CD11b+ APC Associated with EAU Recovery. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6309.
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Mice that have recovered from experimental autoimmune uveoretinitis (EAU) have developed IRBP-specific regulatory immunity in the spleen. This post-EAU regulatory immunity is mediated by tolerogenic APC, and requires melanocortin 5 receptor (MC5r) expression. Also, treating EAU mice with an adenosine 2A receptor (A2Ar) agonist promotes induction of regulatory immunity. Therefore, we further characterized the MC5r-dependent tolerogenic APC, and determined the role of A2Ar expression in mediating the presence of tolerogenic APC in the post-EAU spleen.
C57BL/6 (WT) and A2Ar(-/-) mice were immunized with interphotoreceptor retinoid binding protein (IRBP) in adjuvant to induce EAU. At resolution of EAU, spleen APC were collected and stained for CD11b, Gr-1, and F4/80. Flow cytometry analysis, and sorting was done. The sorted cells were pulsed with IRBP and used as APC to stimulate IFN-γ production by syngeneic IRBP-specific Th1 cells. Primary peritoneal macrophages from WT and MC5r(-/-) mice were stained for CD11b, Gr-1, and F4/80, and analyzed by flow cytometry.
The spleen had Gr-1low and Gr-1high F4/80+ CD11b+ cells with a defined increase in Gr-1low cells and corresponding decrease of Gr-1high cells in the post-EAU WT spleen. The T cells stimulated by the sorted Gr-1low F4/80+ CD11b+ APC were greatly diminished in IFN-γ. While the course of EAU was no different in comparison to EAU WT mice, the post-EAU A2Ar(-/-) mice had no regulatory immunity in the spleen. There was no change in the percentage of spleen Gr-1low F4/80+ CD11b+ cells in the post-EAU A2Ar(-/-) mice compared to post-EAU WT mice; however, there was an increase in Gr-1high F4/80+ CD11b+ cells. Primary macrophages from MC5r(-/-) mice were highly deficient in Gr-1+ and F4/80+ CD11b+.
The development of post-EAU regulatory immunity in the spleen is associated with the expansion of Gr-1low F4/80+ CD11b+ APC in the spleen that suppresses effector T cell activation. The APC presence, and possibly development, is dependent on expressing MC5r, but not on A2Ar. Therefore, the post-EAU regulatory immunity is mediated by a MC5r+ Gr-1low F4/80+ CD11b+ APC, and the A2Ar mediated suppression must be through other cells involved in immune regulation such as Treg cells.
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