Abstract
Purpose :
We have previously reported that soluble molecules released by retinal pigment epithelial cells (RPE) suppress the phagocytic process in macrophages. We examined whether retinal microglial cells are also suppressed, and whether their phagocytic activity changes in response to the immunization used to induce experimental autoimmune uveitis (EAU).
Methods :
Neuroretina and RPE eyecup were collected from naive C57BL/6 mice, and mice 3 or 30 days after a subcutaneous injection of complete Freunds adjuvant (CFA). The retinal microglial cells were isolated from neuroretina using a discontinuous Percoll gradient of 35 and 70%. The isolated microglial cells were fed opsonized pHrodo-bioparticles for 24hr. The cells were imaged, and fluorescent intensity was measured for phagocytic activity. The RPE eyecups were cultured in serum-free medium for 24hr. The conditioned medium was collected, and used to treat resting peritoneal macrophages (pMØ). The treated macrophages were fed opsonized pHrodoRed-bioparticles, incubated for 24hr, and then measured fluorescence intensity. Also, the conditioned media was assayed by microarray for expression of cytokines that mediate inflammation.
Results :
Retinal microglia from naive mouse eyes had almost no phagocytic activity with 1 ± 1.8% positive for phagocytosis. In contrast, there was a significant increase in microglial cells with phagocytic activity from eyes of mice 3 (26 ± 9.4%) and 30 days (19 ± 6.8%) after CFA injection. The RPE eyecup conditioned media from naive mouse eyes significantly suppressed phagocytic activity of pMØ to 24 ± 4.8% of the activity in untreated pMØ. The conditioned media from RPE eyecups of mouse eyes 3 days after CFA injection did not significantly suppress pMØ phagocytic activity. The conditioned media from EAU RPE eyecups expressed IL-6 (959 ± 181 pg/ml) that was not in the conditioned media of healthy RPE eyecups, and neutralization of IL-6 recovered suppression of phagocytic activity by the EAU RPE conditioned media.
Conclusions :
In healthy retinas, microglial cells were suppressed in phagocytic activity. This regulation of phagocytosis is lost early in the induction of EAU by the effects of CFA, and IL-6 production by RPE. These results indicate that in the healthy retina the potential is greatly diminished for microglial cells to process, and present autoantigens. In contrast, induction of EAU is associated with microglial cells recovering phagocytic activity.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.