The Interaction Between Macrophages and Retinal Pigment Epithelium in Ccl2/Cx3cr1 Deficient Mice

Abstract

Purpose: : Age-related macular degeneration (AMD) is a multifactorial disease. The retinal pigment epithelium (RPE) serves as the disposal and recycling arm for the constant renewal of photoreceptor outer segment, which is particularly relevant to AMD pathogenesis. Macrophages (MØs) have been demonstrated in the lesions of human AMD eyes, and they have also been implicated in several animal AMD models. This study investigates the interaction between the RPE and MØs isolated from the Ccl2^-/-/Cx3cr1^-/- DKO mouse, a model that spontaneously develops AMD-like features, including RPE and photoreceptor degeneration, elevated A2E levels, and retinal macrophage infiltration.

Methods: : RPE cells were freshly isolated and harvested from 8-week-old DKO and wild type (WT) mice using Dispase digestion as described by Lavail, Hollyfield, and Anderson (2003). Spleen CD11b+ MØs were isolated using magnetic selection from 8-week-old DKO and WT mice. They were collected and treated in a Micro-Boyden-Chamber for 1.5 hrs with no-serum culture medium (NSCM) that had incubated for 24 hrs with either primary DKO or WT RPE cells. For positive and negative controls, MØs were treated with 50 ng/mL RANTES/CCL5 in NSCM or NSCM alone, respectively. The chemotaxis and adhesion capacity of the MØs were evaluated by counting the cells that adhered to the culture plate surface or pass through the membrane of the chamber. TNFα transcript level, a relevant immunological marker for MØ chemotaxis, was also measured.

Results: : DKO MØs showed a lower adhesion capacity compared to WT MØs (24.93±2.43/HPF and 46.67±2.76/HPF, respectively). DKO MØs also exhibited decreased chemotactic movement towards RANTES/CCL5 compared to WT MØs (chemotactic index 2.34±0.20 and 2.60±0.27, respectively). Interestingly, the levels of both DKO and WT MØ migration were greater in DKO-RPE supernatant than in WT-RPE supernatant, though DKO MØs showed slightly more migration than WT MØs. Higher TNFα mRNA levels were detected in the DKO MØs stimulated with DKO-RPE supernatant.

Conclusions: : DKO MØs showed lower chemotactic and adhesion capacities compared to WT MØs. DKO RPE cells may produce specific chemoattractants that can enhance MØ migration and the release of pro-inflammatory cytokines. Data suggest that the interactions between MØs and the RPE may contribute to AMD pathogenesis.

Keywords: age-related macular degeneration • retinal pigment epithelium • cell-cell communication