Altered Cytokine Profiles of Human Retinal Pigment Epithelium: Oxidant Injury and Replicative Senescence

Purpose: Age-related macular degeneration (AMD) is a local, chronic inflammatory disease of the eye that is influenced by oxidative stress and dysregulation of the retinal pigment epithelium (RPE) associated with aging. The purpose of this study is to characterize the effects of oxidative stress and replicative senescence on the secreted cytokine profiles of RPE in vitro.

Methods: We used multiple, serial passages of human RPE cells from primary culture as an in vitro model of aging. Responses of early passage 5 (P5) and late passage 21 (P21) RPE cells were compared. Oxidative stress was induced in RPE cells (P5) by exposure to 75 μM hydroquinone (HQ) for 24 h. The secretome profiles of the RPE cells were measured with a multiplex suspension assay that assayed human cytokine, chemokine, and growth factors. Immunohistochemistry on younger (≤55 years old) and older (≥70 years old) human post-mortem donor eyes was used to verify selected cytokines.

Results: Supernatant of HQ-treated RPE cultures exhibited increased secreted levels of vascular endothelial growth factor (VEGF), interleukin (IL)-12, and IL-10 that reached statistical significance (p<0.05). Supernatant of late passage P21 RPE cultures exhibited decreased secreted levels of stromal cell-derived factor (SDF)-1α, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-8, IL-15, IL-6, and an increased level of IL-1ra compared to early passage P5 RPE cultures that reached statistical significance (p<0.05). Immunohistochemical analysis demonstrated increased expression of IL-1ra in RPE cells from older post-mortem donor eyes (≥70 years old) versus younger eyes (≤55 years old).

Conclusions: Our data demonstrate a unique cytokine secretion profile of primary culture RPE cells at early and late passage. Our in vitro data suggest an age-specific modulation of cytokine secretion in RPE and is consistent with immunohistochemical analysis on post-mortem eyes. The secretion profile associated with RPE under conditions that mimic oxidative stress, another factor associated with the pathogenesis of AMD, emphasizes upregulation of the angiogenic growth factor, vascular endothelial growth factor. Together, these data support the role of advanced age and oxidative stress in inflammatory cytokine modulation in RPE cells.