The effect of Cytochrome P450 lipid metabolites on leukocyte recruitment in intraocular inflammation.

Abstract

Purpose: We have recently identified long-chain polyunsaturated fatty acids (LCPUFAs) derived from the cytochrome P450 (CYP) family as being important mediators of inflammation and angiogenesis in choroidal neovascularization. Leucocyte recruitment is also known to play a crucial role in inflammation and pathophysiology of a variety of diseases. The purpose of this study is to assess the impact of CYP lipid metabolites on leukocytes recruitment in intraocular inflammation.

Methods: The present studies adhere to ARVO’s Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 mice were treated with laser photocoagulation to induce choroidal neovascularization (CNV). CYP-derived ω-3 LCPUFA metabolites (EDP, EEQ) and CYP-derived ω-6 LCPUFA metabolites (EET) were injected intraperitoneally once a day for 7 days beginning immediately after laser-treatment. Leukocyte rolling velocity was analyzed using the autoperfused microflow chamber assay. FACS analysis was performed to analyze the expression of adhesion molecules, CD18 and CD11b, in peripheral blood leukocytes at day 3. Expression of ICAM-1 and E-selectin in the laser-captured CNV lesion were evaluated by real-time PCR at day 7.

Results: The leukocyte rolling velocity was significantly increased in mice treated with EDP and EEQ CYP lipid metabolites, compared to PBS-treated mice at day 3 after CNV induction. Conversely, the rolling velocity in EET CYP lipid metabolite-treated mice was significantly decreased. FACS analysis revealed that leukocyte CD18 expression in EEQ-treated mice and leukocyte CD11b expression in EDP-treated mice were both significantly decreased at day 3 after CNV induction. In contrast, the leukocyte CD18 expression in EET-treated mice was significantly increased. The mRNA expression of ICAM-1 and E-selectin in the laser-captured CNV lesion were significantly higher in EET-treated mice compared to PBS-treated control animals at day 7 after CNV induction.

Conclusions: We demonstrated that cytochrome P450 lipid metabolites contribute to the leukocyte recruitment by regulating the expression of leukocyte adhesion molecules in both leukocytes and endothelial cells which had a direct effect on their rolling velocity. The mechanism of recruiting inflammatory cells to the site of these lesions likely effects CNV disease severity.