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. 2011 Aug 9;52(9):6279-85.
doi: 10.1167/iovs.10-7081.

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

Xiaobo Zhang  1 , Wei Chen, Cintia S De Paiva, Rosa M Corrales, Eugene A Volpe, Andrew J McClellan, William J Farley, De-Quan Li, Stephen C Pflugfelder
Affiliations

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

Xiaobo Zhang et al. Invest Ophthalmol Vis Sci. .

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.

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Figures

Figure 1.
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.
Figure 2.
Merged images of Ac-caspase-3, -8, and -9 (green) immunofluorescence staining for conjunctiva with PI counterstaining (red).
Figure 3.
Figure 3.
Merged images of TUNEL (green) immunofluorescence staining for conjunctiva with MUC5AC (blue) and PI (red) counterstaining.
Figure 4.
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.
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.
See this image and copyright information in PMC

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