Desiccating stress induces CD4+ T-cell-mediated Sjögren's syndrome-like corneal epithelial apoptosis via activation of the extrinsic apoptotic pathway by interferon-γ

Abstract

We investigated the role of CD4(+) T-cell-produced interferon (IFN)-γ on corneal epithelial apoptosis in a murine desiccating stress (DS) model that resembles Sjögren's syndrome. The DS model was generated in C57BL/6 (B6) and B6 IFN-γ-knockout (B6γKO) mice. Adoptive transfer of CD4(+) T cells from DS-exposed donor to recombination activating gene (RAG)-1(-/-) recipient mice and topical neutralization of IFN-γ were performed to determine whether IFN-γ produced by pathogenic CD4(+) T cells promotes corneal epithelial apoptosis. Apoptosis in corneal epithelia was assessed by evaluating the expression and activity of caspases 3, 8, and 9. The activation of caspase-8 mediated increased corneal epithelial apoptosis in B6 mice after DS, and this was exacerbated by subconjunctival IFN-γ injection. B6γKO mice were resistant to DS-induced apoptosis; however, B6γKO mice receiving IFN-γ developed apoptosis similar to that observed in B6 wild-type mice. Adoptive transfer of CD4(+) T cells from donors subjected to DS increased corneal epithelial apoptosis via activation of caspase-8 in recipients, similar to that in the donor mice. Topical neutralization of IFN-γ in adoptive transfer recipients decreased corneal epithelial apoptosis. DS, IFN-γ administration, or CD4(+) T-cell adoptive transfer had no effect on the expression and activation of the intrinsic apoptosis mediator, caspase-9. CD4(+) T-cell-produced IFN-γ plays a pivotal role in DS-induced corneal epithelial apoptosis via activation of the extrinsic apoptotic pathway.

Figure 1

Laser-scanning confocal microscopy of immunofluorescent staining in tissue sections for TUNEL, AC caspases 3, 8, and 9 (green) with propidium iodide (red) nuclear counterstaining in NS control mice, DS5, and DS5 treated with BSA or IFN-γ subconjunctival injection in B6 or B6γKO mice. Increased TUNEL and caspase-3 and caspase-8 immunoreactivity was noted in corneal epithelia in B6 mice after DS, and subconjunctival IFN-γ administration further increased this process. B6γKO mice were resistant to DS-induced apoptosis; however, B6γKO mice receiving IFN-γ showed similar results to B6 wild-type mice. DS and IFN-γ administration had no effect on the immunoreactivity of caspase-9. Scale bars = 50 μm.

Figure 2

The ratio of TUNEL-positive cells (A) and AC caspase-3 (B), AC caspase-8 (C), and AC caspase-9 (D) immunofluorescent intensity in corneal epithelia. DS5+BSA, DS5 treated with BSA subconjunctival injection; DS5+IFN-γ, DS5 treated with an IFN-γ subconjunctival injection. Data are given as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

Relative mRNA levels of caspase-3 (A), caspase-8 (B), and caspase-9 (C), evaluated by real-time PCR; caspase-3 (D), caspase-8 (E), and caspase-9 (F) activities were evaluated by fluorometric assays. DS5+BSA, DS5 treated with BSA subconjunctival injections; DS5+ IFN-γ, DS5 treated with IFN-γ subconjunctival injections. Data are given as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4

A: The purity of the isolated CD4⁺ T cell was almost 90%, whereas the non-CD4⁺ T cell (ie, CD8⁺ T cell, B220⁺ B cell, DX5⁺ NK cell, CD11c⁺ dendritic cell, and CD11b⁺ macrophage) was almost depleted. Clear histograms indicate flow cytometry analysis before isolation, whereas tinted histograms are after CD4⁺ isolation. The percentage of isolated cells in the CD4⁺ T-cell fraction is depicted in the graph. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phosphatidylethanolamine. B: DS increased the production of IFN-γ and IL-17A, whereas it decreased the production of IL-13 by CD4⁺ T cells isolated from the B6 donor mice, as determined by ELISPOT. Data are given as the mean ± SEM. *P < 0.05, and **P < 0.001.

Figure 5

Adoptive transfer of CD4⁺ T cells elicited by DS to RAG-1^−/− mice promoted ocular surface inflammation with increased CD4⁺ T-cell infiltration (A and B), goblet cell loss (C), and increased IFN-γ expression (D) in the ocular surface. NS, RAG-1^−/− mice received CD4⁺ T cells from NS donor mice; DS5, RAG-1^−/− mice received CD4⁺ T cells from DS5 donor mice. *P < 0.05.

Figure 6

A: Laser-scanning confocal microscopy of immunofluorescent staining in tissue sections stained for AC caspases 3, 8, and 9 (green) with propidium iodide (red) nuclear counterstaining in RAG-1^−/− adoptive transfer recipient mice. B: AC caspases 3, 8, and 9 immunofluorescent intensity and relative mRNA levels of caspases 3, 8, and 9 in corneal epithelia in the recipient mice. Data are given as the mean ± SEM. NS, RAG-1^−/− mice received CD4⁺ T cells from NS donor mice; DS5, RAG-1^−/− mice received CD4⁺ T cells from DS5 donor mice; DS5+RatIgG, DS5 RAG-1^−/− adoptive transfer recipients treated with topical application of vehicle control (RatIgG); DS5+anti-IFN-γ, DS5 RAG-1^−/− adoptive transfer recipients treated with topical application of anti-IFN-γ antibody. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars = 50 μm.