Neutrophils compromise retinal pigment epithelial barrier integrity

Abstract

Abstract: : Purpose:: Neutrophils are the most abundant leukocyte population in the circulation. The capacity of neutrophils to compromise retinal pigment epithelial (RPE) barrier integrity is investigated in vitro. Methods: Murine neutropils were isolated after induction of glycogen–induced peritonitis. The ability of neutrophils to compromise RPE barrier integrity of bovine RPE–Choroid explants was evaluated in a modified Ussing chamber. The effect of neutrophils on RPE tight junction integrity was assessed by immunofluorescent staining of zonula occludin–1 (ZO–1), a tight junction associated protein. Confocal immunostaining was conducted to determine whether neutrophils express hepatocyte growth factor (HGF) and matrix metalloproteinase–9 (MMP–9) which decrease RPE barrier integrity as well as vascular endothelial growth factor–A (VEGF– A) and angiopoietin–2 (Ang–2), potent angiogenic factors. Western blot and zymography were also employed to confirm the expression of HGF and MMP–9 respectively. Results: Neutrophils dramatically decreased ZO–1 expression and increased the permeability of bovine RPE–Choroid explants. Confocal immunostaining demonstrated the expression of HGF, MMP–9, VEGF– A, and Ang–2 in neutrophils. Moreover, a colocalization of HGF and MMP–9 was revealed by confocal immunostaining. Western blot analysis confirmed HGF expression in the neutrophil lysates. Zymography also demonstrated pro–MMP–9 enzymatic activity inside the granules of neutrophils. Conclusions: These data demonstrate neutrophils possess the machinery and ability to induce the loss of RPE–Choroidal barrier integrity, suggesting that they could play an important role in ocular disorders such as choroidal neovascularization.

Keywords: retinal pigment epithelium • inflammation • neovascularization