Ocular Immunological Protein Expression in Chemokine Deficient Mice, Potential Models of Age-Related Macular Degeneration

Abstract

Purpose:: Ccl2 knock-out (KO) mice sporadically develop the cardinal features of age-related macular degeneration (AMD) in their senescent stage (Ambati et al, Nature 2003). Humans bearing a loss of function SNP in CX3CR1 are at increased risk of developing AMD (Tuo et al, FASEB 2004). We recently developed a Ccl2/Cx3cr1 double knock-out (DKO) mouse which consistently develops retinal degeneration with many age-related macular degeneration (AMD) features at a young age. Since there is strong evidence for an immunological role in AMD pathogenesis, we determined ocular immune protein expression levels in DKO, Ccl2 KO, Cx3cr1 KO and age-matched wild-type (WT) mice.

Methods:: Eyes from 4 month-old DKO, Ccl2 KO, Cx3cr1 KO, and WT mice were collected, frozen, sectioned and subjected to immunohistochemistry (IHC) and molecular analysis. The avidin-biotin-complex immunoperoxidase method and confocal microscope detection were used to analyze ocular complement C3d, macrophage (F4/80), toll-like receptor 4 (TLR4) and microglia (CD11b). Mouse serum was used for detection of autoantibodies against the retina. SYBER Green real-time RT-PCR was used to evaluate the expression of Cx3cl1 and Ccl5.

Results:: IHC revealed increased complement C3 in Bruch’s membrane, RPE, photoreceptors, choroidal capillaries, and particularly drusen of DKO mice relative to WT. No change was detected in the single KOs. While the retinas of the WT and single KO mice were not infiltrated with macrophages, infiltrations were detected in the DKO retinal lesions. The DKO had reduced microglia in the choroid and reduced TLR4 in the RPE. Autoantibody against the neuroretina was also detected in the DKO. Overall, no changes were detected in either single KO relative to the WT by IHC. Real-time RT-PCR revealed significant increases of Cx3cl1 and Ccl5 transcript in DKO relative to the WT.

Conclusions:: These results suggest innate immunity and possibly adaptive immunity play a critical role in DKO retinal degeneration. Moreover, since human AMD patients show similar immunopathological profiles, the results support DKO as a suitable AMD model. Because the retinal degeneration in Ccl2 KO mice first appears at 9 months of age, further investigation of immunological protein levels in aged single KO mice is needed.

Keywords: age-related macular degeneration • inflammation • cytokines/chemokines