Retinal Pigment Epithelial Cells Suppress Phagolysosome Activation in Macrophages

Abstract

Purpose: The eye is an immune-privileged microenvironment that has adapted several mechanisms of immune regulation to prevent inflammation. One of these potential mechanisms is retinal pigment epithelial cells (RPE) altering phagocytosis in macrophages.

Methods: The conditioned media of RPE eyecups from eyes of healthy mice and mice with experimental autoimmune uveitis (EAU) were used to treat primary macrophage phagocytizing pHrodo bacterial bioparticles. In addition, the neuropeptides were depleted from the conditioned media of healthy RPE eyecups and used to treat phagocytizing macrophages. The conditioned media from healthy and EAU RPE eyecups were assayed for IL-6, and IL-6 was added to the healthy conditioned media, and neutralized in the EAU conditioned media. The macrophages were treated with the conditioned media and assayed for fluorescence. The macrophages were imaged, and the fluorescence intensity, relative to active phagolysosomes, was measured. Also, the macrophages were assayed using fluorescent viability dye staining.

Results: The conditioned media from healthy, but not from EAU RPE eyecups suppressed phagolysosome activation. Depletion of the neuropeptides alpha-melanocyte-stimulating hormone and neuropeptide Y from the healthy RPE eyecup conditioned media resulted in macrophage death. In the EAU RPE eyecup conditioned media was 0.96 ± 0.18 ng/mL of IL-6, and when neutralized the conditioned media suppressed phagolysosome activation.

Conclusions: The healthy RPE through soluble molecules, including alpha-melanocyte-stimulating hormone and neuropeptide Y, suppresses the activation of the phagolysosome in macrophages. In EAU, the IL-6 produced by the RPE promotes the activation of phagolysosomes in macrophages. These results demonstrate that under healthy conditions, RPE promotes an altered pathway of phagocytized material in macrophages with implications on antigen processing and clearance.

Figure 1

The effects of RPE CM on phagolysosome activation. Conditioned media were collected from 24-hour cultures of healthy RPE eyecups (RPE CM). Primary resting macrophages were treated with the RPE CM, and fed opsonized pHrodo-bioparticles. The cells were incubated for 24 hours, imaged by microscopy, and florescent intensity measured. (A) Presented are representative images of the pHrodo-bioparticle expression in the macrophages. (B) The relative fluorescent intensity was calculated versus untreated macrophage cultures, and presented are the mean ± SEM of three independent experiments. Significant suppression, *P ≤ 0.001, of phagolysosome activation was seen in cells treated with RPE CM.

Figure 2

The effects of depleting α-MSH and NPY from RPE CM on phagolysosome activation. Conditioned media from healthy RPE eyecup cultures were treated with antibodies to α-MSH and NPY, and absorbed with Protein A/G–coated beads. An irrelevant rabbit IgG (irr IgG) was used as a control. The primary resting macrophages were treated with the absorbed CM and opsonized pHrodo-bioparticles. After 24 hours, the cells were imaged and fluorescent intensity was measured. The presented results are (A) representative images of the treated macrophages, and (B) the mean ± SEM of the relative fluorescent intensity of three independent experiments. Depletion of α-MSH and NPY had no effect on detecting suppressed phagolysosome activation by the RPE CM. *Significantly different P ≤ 0.001 to untreated macrophages, and †P ≤ 0.001 with RPE CM (irr IgG)-treated macrophages.

Figure 3

The effects of depleting α-MSH and NPY from RPE CM on macrophage viability. Conditioned media from healthy RPE eyecups were depleted of α-MSH and NPY and macrophages treated as in Figure 2. In addition, cell tracker viability dye was added in the last hour of the 24-hour incubation. The cells were imaged (A) and green fluorescent intensity was measured (B). The presented results are representative images of the treated macrophages, and the mean ± SEM of the relative fluorescent intensity of three independent experiments. Depletion of α-MSH and NPY had a significant effect on macrophage viability. *Statistical differences, P ≤ 0.001, to untreated macrophages. No statistical differences were seen between RPE CM with or without irr IgG treatment.

Figure 4

The effects of RPE CM from uveitic eyes on phagolysosome activation. Conditioned media were collected from 24-hour cultures of RPE eyecups (RPE CM) from mice with active EAU. Primary resting macrophages were treated with the RPE CM and fed opsonized pHrodo-bioparticles. The cells were incubated for 24 hours, imaged by microscopy, and the fluorescent intensity measured. (A) Presented are representative images of the treated macrophages. (B) The relative fluorescent intensity was calculated versus untreated macrophage cultures and presented as the mean ± SEM of three independent experiments. Significant suppression, *P ≤ 0.001, of phagolysosome activation was seen in cells treated with healthy RPE CM, but not (NS) with RPE CM from EAU mice.

Figure 5

Interleukin-6 in EAU RPE eyecup CM. Conditioned media of RPE CM from healthy eyes and EAU eyes were assayed for IL-6 by ELISA. No IL-6 was detected in the healthy RPE CM, and 0.96 ± 0.18 ng/mL was detected in the EAU RPE CM. This is the mean ± SEM ng/mL of IL-6 from four RPE CM.

Figure 6

Effects of IL-6 on RPE CM regulation of phagolysosome activation. Added to healthy RPE CM 1 ng/mL of IL-6, and to EAU RPE CM was added neutralizing IL-6 antibody. Macrophages were treated with CM and fed opsonized pHrodo-bioparticles. After 24 hours, the cells were imaged and fluorescent intensity was measured. The presented results are the mean ± SEM of the relative fluorescent intensity of three independent experiments. Although neutralization of IL-6 recovered significant suppression of phagolysosome activation, the addition of IL-6 into healthy RPE CM did not enhance phagolysosome activation. *Statistical differences, *P ≤ 0.001 to untreated macrophages, and †P ≤ 0.001 with EAU RPE CM-treated macrophages. NS, not statistically significant.