Targeting immune privilege to prevent pathogenic neovascularization

Abstract

PURPOSE. Current studies suggest that the immune system plays a critical role in blinding eye disorders. The eye is an immune-privileged site, and FasL expression is a major part of that mechanism because Fas/FasL interactions regulate inflammation and neovascularization, preventing damage to delicate ocular structures. These studies were undertaken to test the idea that modulating immune privilege might be an effective therapeutic approach to pathogenic angiogenesis in the eye. METHODS. C57BL/6 mice or FasL-defective B6-gld mice were laser treated to induce choroidal neovascularization (CNV). Mice were injected with cytotoxic FasL in the vitreous cavity or were treated with oral doxycycline in the drinking water. They were evaluated for CNV 7 days later. In some experiments eye tissue was harvested and evaluated for FasL expression, macrophage influx by immunohistochemistry, and release of sFasL. RESULTS. Injection of cytotoxic FasL successfully prevented neovascularization in a mouse model of CNV. Oral doxycycline increased functional FasL in the eye and substantially inhibited neovascularization. Doxycycline treatment increased FasL expression on the RPE cells and reduced circulating and tissue-associated sFasL. Treatment was ineffective in B6-gld mice, demonstrating that CNV inhibition was mediated by FasL. CONCLUSIONS. Targeting immune privilege using cytotoxic molecules or by increasing expression of the proapoptotic protein FasL may be a viable approach to treating neovascular eye disease.

Figure 1.

Intravitreal sFasL inhibits CNV. C57Bl/6 mice were laser treated and on day 2 were injected in the vitreous cavity with PBS, sFasL (300 or 30 ng), or sTRAIL (300 ng). CNV area was evaluated on day 7 by confocal microscopy. (A) CNV lesion volume (expressed as volume of fluorescence) in treated mice. *P < 0.01; significant difference from PBS control. (B–E) Representative CNV lesions from mice treated with PBS, 300 ng sFasL, 30 ng sFasL, or 300 ng sTRAIL, respectively.

Figure 2.

FasL staining on RPE cells. C57BL/6 mice were laser treated and administered water (A–C) or doxycycline (2 mg/mL) (D–F). Sclero-choroidal flatmounts were prepared 1 and 3 days after laser treatment and were stained for surface FasL. Micrographs were taken, centered on the laser lesions. Circles: approximate locations of the laser-induced lesions.

Figure 3.

Doxycycline inhibits CNV. C57BL/6 mice were laser treated and administered water or varying doses of doxycycline. CNV was evaluated on day 7 by confocal microscopy. (A) Volume of CNV lesions (expressed as volume of fluorescence) in treated mice. *P < 0.01; significant difference from water control. Representative CNV lesions from mice treated with water (B); 2 mg/mL doxycycline (C); 1 mg/mL doxycycline (D); 0.5 mg/mL doxycycline (E).

Figure 4.

Doxycycline requires functional FasL to inhibit CNV. C57BL/6 or B6 gld mice were laser treated and administered water or doxycycline (2 mg/mL). (A) CNV volume (expressed as volume of fluorescence) was evaluated on day 7 by confocal microscopy. *P < 0.01; significant decrease from PBS control. **P < 0.01; significant increase from PBS control. (B) C57BL/6 (B6) mice were pre-bled 48 hours before laser treatment. They were then administered water or doxycycline (2 mg/mL) and were laser treated. Mice serially bled at 24 and 48 hours by tail vein and serum concentrations of sFasL were measured with a commercially available ELISA. Levels below the detection limit were coded as 0 pg/mL and were included in the analysis. All assays were conducted in duplicate. NS, not significantly different from the pre-bleed group. (C) C57BL/6 (B6) mice were administered water or doxycycline (2 mg/mL) and then were laser treated. Tissue from the posterior segment of the eye was harvested at 48 and 72 hours, and the soluble proteins were extracted. Concentrations of sFasL were measured with a commercially available ELISA and are expressed as picogram per milligram of protein. Levels below the detection limit were coded as 0 pg/mg protein and were included in the analysis. Each point represents the value from 8 to 10 pooled eyes. NS, not significantly different from the untreated control. (D) C57BL/6 or B6 gld mice were laser treated and administered water or doxycycline (2 mg/mL). On day 3 after laser treatment, eyes were harvested, mRNA was isolated, and qRT-PCR for VEGF was performed.

Figure 5.

Doxycycline does not increase macrophage infiltration or FasL expression. (A) Sclero-choroidal flatmounts were prepared 1, 3, and 7 days after laser treatment and stained for CD11b. Numbers of macrophages (CD11b⁺) were counted per lesion (per 20× field centered on the laser lesion), and average numbers are represented. (B–G) C57BL/6 mice were laser treated and administered water (B–D) or doxycycline (2 mg/mL) (E–G). Sclero-choroidal flatmounts were prepared and costained for FasL (red) (B, E) and CD11b (green) (C, F) and were examined by confocal microscopy. FasL⁺ and CD11b⁺ images were merged to detect coexpression (D, G).