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Ciliary Neurotrophic Factor Released by Corneal Endothelium Surviving Oxidative Stress Ex Vivo

From the Department of Ophthalmology, University of Maryland at Baltimore, Baltimore, Maryland.

PURPOSE. To demonstrate that corneal endothelial (CE) cells that survive oxidative stress in corneas release ciliary neurotrophic factor (CNTF), which does not possess the secretion signal sequence.

METHODS. CNTF and CNTF receptor subunit (CNTFR) in CE cells and cell-conditioned medium (H₂O₂ and PBS placed in bovine corneal cups for 30 minutes at 37°C) in explant cultures were demonstrated by Western blot (WB) and immunoprecipitation (IP). The number of dead CE cells was determined microscopically with a viability kit, by an observer uninformed of the explants’ identities. CNTF and CNTFR synthesis and release by CE cells in ³⁵S-methionine–labeled (0.1 mCi/mL for 8 hours at 37°C) corneal cups were shown by autoradiography and WB.

RESULTS. CE cells in fresh bovine eyes expressed a 25-kDa CNTF that was recognized by three different antibodies. CE cells expressed a 61-kDa CNTF-immunoreactive molecule (IM), which disappeared from the CE cells in H₂O₂-conditioned corneal cups, concomitant with the appearance of the 25-kDa CNTF in the conditioned medium. Corneal cups containing 0, 0.006, 0.012, 0.023, 0.045, 0.09, 0.18, and 0.35 mM H₂O₂ demonstrated relative levels of CE cell 61-kDa CNTF-IM of 100%, 84%, 77%, 61%, 52%, 39%, 35%, and 35%, respectively, whereas levels of 25-kDa CNTF in the conditioned medium were 23%, 32%, 39%, 63%, 80%, 90%, 100%, and 63%, respectively. CE cells expressed a 53-kDa CNTFR that, along with trace amounts of a 61-kDa CNTFR-IM, appeared concomitantly with the 25-kDa CNTF in the conditioned medium. H₂O₂ (0–0.56 mM) did not affect the viability of CE cells (15 dead cells per 600 cells). CE cells in ³⁵S-methionine–labeled corneal cups synthesized and released a ³⁵S 61-kDa molecule that was both CNTF- and CNTFR-immunoreactive in an H₂O₂-dependent manner, whereas 25-kDa CNTF was detected in the ³⁵S-methionine labeling medium.

CONCLUSIONS. CE cells release autocrine CNTF under sublethal oxidative stress by a mechanism that involves CNTFR and the formation of a 61-kDa CNTF/CNTFR-IM.

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