CFH Y402H polymorphism is associated with elevated vitreal GM-CSF and choroidal macrophages in the postmortem human eye

Abstract

Purpose: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people 50 years of age or older in developed countries. The homozygous CC genotype in the complement factor H (CFH) Y402H single nucleotide polymorphism (SNP; rs1061170) is widely recognized as a risk factor for the development of AMD. In this study, we examined vitreal levels of granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic cytokine, and macrophages in the choroid of postmortem human eyes genotyped for the CFH Y402H SNP.

Methods: Twenty-two pairs of postmortem, non-diseased, human donor eyes were obtained. The vitreous and retinal tissues of the left eyes were collected for GM-CSF level measurement and CFH Y402H genotyping, respectively. The right eyes were paraffin-embedded and sectioned for immunohistochemistry using a macrophage and microglia marker, CD68. Cell cultures of RPE cells were stimulated with complement C3a, C5a, 4-hydroxynonenal (HNE), or tumor necrosis factor alpha (TNF-α), and GM-CSF expression was measured with a suspension assay or quantitative PCR.

Results: Eyes genotyped with the CC or the CT risk variant of the CFH Y402H SNP showed significantly increased levels of GM-CSF in the vitreous compared to eyes with the protective TT variant (mean ± standard error of mean, 607.54±85.83 pg/ml or 656.32±15.20 pg/ml versus 286.69±81.96 pg/ml, p<0.05). The choroid of eye tissues genotyped with the CC variant showed higher levels of CD68 immunoreactivity than the tissues genotyped with the TT variant (p<0.05). The GM-CSF levels detected in the supernatant of RPE cells in culture treated with HNE or TNF-α were significantly higher compared to the non-treated control (145.88±5.06 pg/ml and 149.32±3.76 pg/ml versus 123.27±4.05 pg/ml, p<0.05). Furthermore, the gene expression of GM-CSF detected in the lysate of RPE cells stimulated with complement C3a or C5a showed significantly increased fold changes compared to the non-treated control (C3a: 2.38±0.31 fold, p<0.05; C5a: 2.84±0.54 fold, p<0.01).

Conclusions: Our data showed a relationship between the CFH Y402H polymorphism and GM-CSF levels in the vitreous and accumulation of choroidal macrophages in the postmortem eye. These data suggest that the at-risk variant of the CFH gene may contribute to the dysregulation of proinflammatory cytokines locally in the eye.

Figure 1

Increased GM-CSF in vitreous was associated with the at-risk variants of the CFH Y402H polymorphism. The GM-CSF level differed significantly among donors with CC, CT, or TT variants of the CFH Y402H polymorphism (one-way ANOVA, p<0.01). The GM-CSF level was significantly higher in the vitreous from donors with CC and CT variants compared to that of donors with the protective TT variant (mean ± SEM, 607.54±85.83 pg/ml, n=4, 656.32±15.20 pg/ml, n=10 versus 286.69±81.96 pg/ml, n=8, respectively, post hoc Bonferroni multiple comparison test, *p<0.05).

Figure 2

The increased number of macrophages in the choroid was associated with the at-risk variants of the CFH Y402H polymorphism. The macrophages in the postmortem human eye sections were immunoreacted with CD68, a known marker for macrophages. The immunoreactivity for CD68 was developed with AEC (red) and counterstained with Mayer’s hematoxylin (blue; scale bar: 20 μm). A: A representative picture of CD68 immunoreactivity in the outer retina from a donor eye with the at-risk CC variant. B: A representative picture of CD68 immunoreactivity from a donor eye with the heterozygous CT variant. C: A representative picture of CD68 immunoreactivity from a donor eye with the protective TT variant. D: Negative control by primary antibody omission. E: The number of CD68-positive cells per 0.1 mm² choroid area differed significantly among donors with CC, CT, or TT variants of the CFH Y402H polymorphism (mean ± SEM, one-way ANOVA, p<0.05). There are more CD68-immunoreactive cells in the choroid of the CC eyes (3.44±0.77 per 0.1 mm², n=4) compared to the TT eyes (1.12±0.20 per 0.1 mm², n=6; post hoc Bonferroni multiple comparison test, *p<0.05).

Figure 3

Stimulation with complement activation products upregulated the expression of GM-CSF in the RPE. The expression of GM-CSF in ARPE-19 cells was upregulated 2.84- and 2.38-fold by C5a and C3a at 5 µg/ml after 24 h incubation, respectively (C5a: mean ± SEM, 2.84±0.54 fold, n=3, Student t test, **p<0.01; C3a: 2.38±0.31 fold, n=3, Student t test, *p<0.05).

Figure 4

The RPE secreted higher levels of GM-CSF after stimulation with HNE and TNF-α. The GM-CSF secreted into the culture supernatant was increased when primary RPE cells were exposed to 4-hydroxynonenal (HNE), an agent that promotes oxidative stress, at 10 µM for 6 h (mean ± SEM, 145.88±5.06 pg/ml versus 123.27±4.05 pg/ml, n=3, Student t test, *p<0.05). In addition, RPE cells stimulated with 20 ng/ml tumor necrosis factor alpha (TNF-α) for 6 h also resulted in increased levels of GM-CSF secreted into the culture supernatant (149.32±3.76 pg/ml versus 123.27±4.05 pg/ml, n=3, Student t test, *p<0.05).