title: Interferon-γ exacerbates dry eye-induced apoptosis in conjunctiva through dual apoptotic pathways.
abstract: OBJECTIVE
  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.
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Cintia S
authors.initials: CS
authors.lastname: De Paiva
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Corrales
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Volpe
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: McClellan
authors.forename: William J
authors.initials: WJ
authors.lastname: Farley
authors.forename: De-Quan
authors.initials: DQ
authors.lastname: Li
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Pflugfelder
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002352:Carrier Proteins / Q000235:genetics; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D056970:Co-Repressor Proteins; D003228:Conjunctiva / Q000378:metabolism / Q000473:pathology*; D004195:Disease Models, Animal; D015352:Dry Eye Syndromes / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D005260:Female; D020397:Goblet Cells / Q000378:metabolism / Q000473:pathology; D020287:In Situ Nick-End Labeling; D007371:Interferon-gamma / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018832:Molecular Chaperones; D055271:Mucin 5AC / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D017471:Receptors, Interferon / Q000378:metabolism; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D013312:Stress, Physiological; D019014:fas Receptor / Q000235:genetics; D000096743:Interferon gamma Receptor
chemical_list: D002352:Carrier Proteins; D020169:Caspases; D056970:Co-Repressor Proteins; D007371:Interferon-gamma; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D055271:Mucin 5AC; D009687:Nuclear Proteins; D012333:RNA, Messenger; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D019014:fas Receptor; D000096743:Interferon gamma Receptor; C491473:Daxx protein, mouse; C524679:Muc5ac protein, mouse