Infiltration of Proinflammatory M1 Macrophages into the Outer Retina Precedes Damage in a Mouse Model of Age-Related Macular Degeneration
Figure 4
CEP-MSA immunization leads to M1 (TNF-α, IL-12 producing) macrophage recruitment and activation in the subretinal space. (a) Frozen sections followed by immunostaining and confocal microscopy. Surface marker stains were used to identify macrophages as the infiltrate cells. F4/80+ and CD68+ cells are absent from the RPE of naive mice, but they are found in CEP-MSA-immunized BALB/c and B6 mice. (b) Intracellular stains for TNF-α, IL-12, and IL-10 production were used to determine the phenotype of the activated macrophages observed in CEP-MSA BALB/c mice at the intermediate recovery time. IgG2b was used as isotype control. Representative images are shown (from 3–5 mice per group per experiment; two or three independent experiments were performed for each strain). INL: inner nuclear layer; ONL: outer nuclear layer; ROS: rod outer segment; RPE: retinal pigment epithelium; CHO: choroid. The scale marker represents a 20 m length. (c) Infiltrating macrophages from B6 mice were isolated by laser microdissection, and RNA was obtained for qPCR analysis of gene expression. Relative mRNA (in arbitrary units) was calculated using the method with Actin as the calibrator gene. Transcripts for M1 marker genes (IL-1β, TNF, and Ccl2) were detectable in 3 out of 5 CEP-MSA-immunized mice but were not present in CFA age-matched controls (). M2 marker expression did not correlate with CEP immunization: IL-10 was completely absent, whereas the levels of Arg-1 did not increase. Results are representative of at least two independent laser capture experiments.
