Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

Abstract

The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye.

The Neuropeptides of Ocular Immune Privilege, α-MSH and NPY, Suppress Phagosome Maturation in Macrophages

“…Most of these studies looked at the uptake and not necessarily the phagosome maturation pathway. The results in this manuscript with our previous publication showing suppression of the phagolysosome activation (5, 6, 22) demonstrates that it is not the uptake but the maturation of the phagocytic pathway that is regulated by α-MSH and NPY.…”

“…In addition, microglial cells in the healthy neuroretina have similar alternatively activated myeloid cell characteristics (3). In eyes with wounded RPE, the RPE do not mediate the alternative activation but promote macrophage/microglial cell differentiation into proinflammatory M1 or wound repairing/anti-inflammatory M2 cells (3, 5). The wounded RPE monolayer continues to produce NPY and is greatly diminished in α-MSH production (3).…”

The Role of Retinal Pigment Epithelial Cells in Regulation of Macrophages/Microglial Cells in Retinal Immunobiology

“…Phagocytizing materials is central for an APC to process antigen for presentation on MHC class II molecules (104). Recently we have found that the process of phagocytosis is also altered by the RPE through its release of a-MSH and NPY (105,106). The neuropeptides mediate a differential regulation of phagocytic uptake of gram-negative and gram-positive bacteria (107).…”

“…The RPE from eyes with autoimmune uveitis do not suppress the phagocytic pathway, and this may be associated with high levels of IL-6 expression (105). The regulation of phagolysosome activation is dependent also on the RPE maintaining an intact monolayer (106). Therefore, one potential contribution of the RPE to immune privilege is its suppression of the processing and presentation of self-antigens by retinal APC that would prevent the activation of autoimmune disease-mediating effector T cells.…”

Alpha-Melanocyte-Stimulating Hormone Maintains Retinal Homeostasis after Ischemia/Reperfusion

“…Treatment of human corneal buttons for transplantation retains epithelial cells with less cell death [16]. The neuropeptide prevents apoptosis in cultured macrophages and RPE cells [17,55]. In addition, the effects of augmenting the melanocortin pathways may be associated with which melanocortin receptor (MCr) is stimulated.…”