Upregulation of lipocalin-2 in the retina of experimental autoimmune uveitis

Abstract

Lipocalin-2, a siderophore-binding protein, is an antimicrobial, exerting both pro-inflammatory and anti-inflammatory actions. Lipocalin-2 is also involved in glial activation, matrix metalloproteinase stabilization, and cellular iron flux, all of which play roles in autoimmune diseases. The present study aimed to determine the expression of lipocalin-2 in the eyes of Lewis rats with interphotoreceptor-binding protein-induced experimental autoimmune uveitis (EAU). Significantly elevated serum lipocalin-2 levels were also detected in rats by enzyme-linked immunosorbent assay (ELISA). Lipocalin-2 immunostaining was detected predominantly in activated glial cells, including glutamine synthase-positive Müller cells, and ionized calcium-binding adaptor molecule 1-positive microglia and macrophages in EAU rats. Taken together, the results presented herein show that lipocalin-2 is a potential diagnostic marker for uveitis.

Keywords: ciliary body; experimental autoimmune uveitis; lipocalin-2; retina.

Fig. 1.

Serum lipocalin-2 levels in normal control and IRBP-immunized rats. The levels of lipocalin-2 in EAU rats on days 9 and 14 post-immunization were significantly higher than those in normal controls and declined by day 21. Values are shown as means ± SEM (n=4 for each group). **P<0.01. EAU: experimental autoimmune uveitis; D9PI, day 9 post-immunization; D14PI, day 14 post-immunization; D21PI, day 21 post-immunization; EAU, experimental autoimmune uveitis; IRBP, interphotoreceptor retinoid-binding protein; SEM, standard error of the mean.

Fig. 2.

Histopathological findings of the uvea and retina of normal (A and C) and IRBP-immunized rats (B and D). No inflammation was observed in the uvea (C) or retina of normal rats (A). On day 9 post-immunization, infiltration of inflammatory cells was observed in the ciliary body (D) and retina of EAU rats (B). The arrows indicate infiltrating inflammatory cells. Scale bars in A–D: 20 µm. D9PI, day 9 post-immunization; EAU, experimental autoimmune uveitis; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IRBP, interphotoreceptor retinoid-binding protein; IS/OS, inner segment/outer segment junction; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

Fig. 3.

Immunohistochemical localization of lipocalin-2 in the eyes of normal (A and C) and EAU-induced rats on days 9 (B and D). Lipocalin-2 immunostaining is observed in the GCL and RPE of the eyes (A) and in the ciliary body of normal rats (C). Lipocalin-2-positive immunoreaction was also enhanced in the GCL, IPL, and ONL of the retina (B). Lipocalin-2 was also detected in inflammatory cells in the ciliary region of EAU rats on day 9 post-immunization (D). The arrows denote inflammatory cells. Scale bars in A–D: 20 µm. D9PI, day 9 post-immunization; EAU, experimental autoimmune uveitis; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IRBP, interphotoreceptor retinoid-binding protein; IS/OS, inner segment/outer segment junction; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

Fig. 4.

Double-labeling immunofluorescence analysis of lipocalin-2 and GS in the retina of EAU rats. Double-labeling immunofluorescence for lipocalin-2 (A) and the Müller cell marker GS in the retina of EAU rats on day 9 post-immunization (B). Lipocalin-2 is localized in GS-positive Müller cells (C). Arrowheads, Müller cells with lipocalin-2-positive and GS-positive immunoreactions. Scale bars in A–C: 20 µm. EAU, experimental autoimmune uveitis; GS, glutamine synthase.

Fig. 5.

Double-labeling immunofluorescence analysis of lipocalin-2 and Iba1 in the retina and ciliary body of EAU-induced rats. Double-labeling immunofluorescence of lipocalin-2 and the microglial marker Iba1 in the retina of EAU rats on day 9 post-immunization. Lipocalin-2 (green: A and D) was occasionally localized in some Iba1-positive microglia in the retina (red: B) and in macrophages (red: E) in the ciliary bodies of EAU-induced rats (yellow: C and F). Arrowheads, microglia, and/or macrophages showing lipocalin-2-positive and Iba1-positive immunoreactions. Scale bars in A–F: 20 µm. EAU, experimental autoimmune uveitis; Iba1, ionized calcium-binding adaptor molecule 1.