Interferon-γ exacerbates dry eye-induced apoptosis in conjunctiva through dual apoptotic pathways

Abstract

Purpose: To investigate the role of interferon (IFN)-γ in dry eye-associated conjunctival apoptosis.

Methods: Desiccating stress (DS) was created in C57BL/6 (B6) and C57BL/6 IFN-γ-knockout (B6γKO) mice. A separate group of mice of both strains also received subconjunctival injections of exogenous IFN-γ or vehicle control (BSA) at days 0, +2, and +4 after DS. Immunoreactivity to active (Ac)-caspase-3, -8, and -9 and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) were evaluated in cryosections. Goblet cell apoptosis was assessed by MUC5AC and TUNEL double staining. Levels of caspase-3, -8, -9, Fas, and Fas-associated protein with Death Domain (FADD) mRNA in conjunctiva were measured by real-time PCR. The activity of caspase-3, -8, or -9 was measured using fluorometric assay.

Results: Increased Ac-caspase-3 and -8 and TUNEL immunoreactivity were noted in conjunctival epithelia in B6 mice compared with B6γKO mice after DS, and exogenous IFN-γ administration further increased these parameters. DS-induced conjunctival apoptosis was greatest in the goblet cell area and was accompanied by a decrease in MUC5AC expression in the B6 and B6-IFN-γ-injected groups compared with the B6γKO and B6-BSA-injected groups. B6γKO mice were resistant to DS-induced apoptosis; however, B6γKO receiving IFN-γ yielded results similar to those for B6 wild-type. Caspase-9 production and activity were not increased with DS in B6 or B6γKO mice; however, the administration of IFN-γ significantly increased caspase-9 production and activity in both strains compared with vehicle-injected mice.

Conclusions: IFN-γ plays a pivotal role in exacerbating conjunctival apoptosis through dual apoptotic pathways with DS.

Figure 1.

Merged images of IFN-γR (green) immunofluorescence staining for conjunctiva with PI counterstaining (red) in conjunctiva (A). IFN-γRα mRNA transcripts in the conjunctiva after DS and exogenous IFN-γ treatment (B).

Figure 2.

Merged images of Ac-caspase-3, -8, and -9 (green) immunofluorescence staining for conjunctiva with PI counterstaining (red).

Figure 3.

Merged images of TUNEL (green) immunofluorescence staining for conjunctiva with MUC5AC (blue) and PI (red) counterstaining.

Figure 4.

Ac-caspase-3 (A), -8 (C), and (D) immunofluorescence intensity and the ratio of TUNEL-positive cells (B) in conjunctival epithelia. Data were shown in mean ± SEM.

Figure 5.

Relative levels of Fas (A), FADD (B), caspase-3 (C), caspase-8 (D), and caspase-9 (E) mRNA transcripts evaluated by real-time PCR as well as caspase-3 (F), caspase-8 (G), and caspase-9 (H) activities. Data were shown in mean ± SEM.