PURPOSE: The authors have previously shown that apoptosis of stromal cells is downregulated in the lumican-null mouse and that this may be due to disruption of Fas-Fas ligand (FasL) signaling. The present study was undertaken to investigate the role of lumican in regulating Fas and its impact on inflammation and healing of corneal injuries. METHODS: Apoptosis was determined by measuring caspase-3/7 activity in corneal extracts. Protein and RNA levels of Fas were estimated by immunoblot analysis and RT-PCR, respectively. Circular and incisional stromal wounds were exposed to Pseudomonas aeruginosa LPS, and healing was assessed by (1) observing wound closure with fluorescence and bright-field microscopy, (2) histology to quantify inflammatory infiltrates by immunostaining for macrophages (F4/80) and neutrophils (NIMP-R14), (3) measuring myeloperoxidase (MPO) levels by ELISA to quantify neutrophils, and (4) measuring proinflammatory cytokines by ELISA. RESULTS: Lum-/- -injured corneas showed significantly lower caspase-3/7 activity (apoptosis). Lum-/- -wounded corneas showed delayed healing, reduced recruitment of macrophages and neutrophils, lower MPO levels, and no induction of the proinflammatory cytokines TNFalpha and IL1beta. The Fas protein level, before and after wounding, was dramatically lower in Lum-/- - compared with Lum+/+-injured cornea. However, Fas mRNA levels were comparable in both genotypes, suggesting regulation of Fas at the protein level. Moreover, a solid-state binding assay and coimmunoprecipitation of FasL and lumican suggested binding of FasL to lumican. CONCLUSIONS: The data suggest that lumican binds FasL and facilitates induction of Fas. Poor signaling through Fas-FasL in lumican deficiency leads to impaired induction of inflammatory cytokines and corneal healing.
PURPOSE: The authors have previously shown that apoptosis of stromal cells is downregulated in the lumican-null mouse and that this may be due to disruption of Fas-Fas ligand (FasL) signaling. The present study was undertaken to investigate the role of lumican in regulating Fas and its impact on inflammation and healing of corneal injuries. METHODS: Apoptosis was determined by measuring caspase-3/7 activity in corneal extracts. Protein and RNA levels of Fas were estimated by immunoblot analysis and RT-PCR, respectively. Circular and incisional stromal wounds were exposed to Pseudomonas aeruginosa LPS, and healing was assessed by (1) observing wound closure with fluorescence and bright-field microscopy, (2) histology to quantify inflammatory infiltrates by immunostaining for macrophages (F4/80) and neutrophils (NIMP-R14), (3) measuring myeloperoxidase (MPO) levels by ELISA to quantify neutrophils, and (4) measuring proinflammatory cytokines by ELISA. RESULTS:Lum-/- -injured corneas showed significantly lower caspase-3/7 activity (apoptosis). Lum-/- -wounded corneas showed delayed healing, reduced recruitment of macrophages and neutrophils, lower MPO levels, and no induction of the proinflammatory cytokines TNFalpha and IL1beta. The Fas protein level, before and after wounding, was dramatically lower in Lum-/- - compared with Lum+/+-injured cornea. However, Fas mRNA levels were comparable in both genotypes, suggesting regulation of Fas at the protein level. Moreover, a solid-state binding assay and coimmunoprecipitation of FasL and lumican suggested binding of FasL to lumican. CONCLUSIONS: The data suggest that lumican binds FasL and facilitates induction of Fas. Poor signaling through Fas-FasL in lumican deficiency leads to impaired induction of inflammatory cytokines and corneal healing.
Authors: Mary Ann Stepp; James D Zieske; Vickery Trinkaus-Randall; Briana M Kyne; Sonali Pal-Ghosh; Gauri Tadvalkar; Ahdeah Pajoohesh-Ganji Journal: Exp Eye Res Date: 2014-03-04 Impact factor: 3.467
Authors: Holly R Chinnery; Eric C Carlson; Yan Sun; Michelle Lin; Sandra H Burnett; Victor L Perez; Paul G McMenamin; Eric Pearlman Journal: J Immunol Date: 2009-03-01 Impact factor: 5.422