PURPOSE: Macrophages infiltrating an inflamed or injured tissue undergo development of coordinated sets of properties that contribute to tissue damage, repair, and remodeling. The purpose of this study was to determine whether macrophages isolated from normal or inflamed retina are programmed to a distinct set of properties and to examine whether the development of experimental autoimmune uveoretinitis (EAU) affects macrophage function. METHODS: EAU was induced in Lewis rats, and a retina-derived macrophage-enriched population was generated by density centrifugation during the prepeak, peak, and resolution phases of the disease. Cell surface phenotype was assessed by two- and three-color flow cytometry, and function was determined in vitro by nitric oxide (NO) production, with or without further cytokine stimulation or by immunohistochemistry to determine expression of beta-glucuronidase, nitric oxide synthase (NOS)-2, and nitrotyrosine. RESULTS: Myeloid-derived cells from normal retina were programmed similar to TGF-beta-stimulated uncommitted bone-marrow-derived macrophages (BMDMs). Contrary to BMDM behavior, retina-isolated macrophages displayed distinct properties and phenotype at different phases of the disease course and remained resistant throughout, to further cytokine challenge in vitro. During peak disease, retina-isolated macrophages had characteristics of IFN-gamma/TNF-alpha primed cells (nitrotyrosine positive and NO producing). Despite equivalent numbers of macrophages during resolution, their function reverted to characteristics of TGF-beta primed cells (beta-glucuronidase positive). CONCLUSIONS: Resident retinal myeloid-derived cells are primed and are resistant to further cytokine stimulation, and, similar to macrophages derived during EAU recovery, behave operationally as though TGF-beta primed. During peak inflammation, infiltrating macrophages adapt to concurrent hierarchical Th1 T-cell response (IFN-gamma/TNF-alpha), generating NO. The results provide evidence of in vivo programming of macrophages within the retina.