Cntf in the Aqueous Humor of Enucleated Eyes and Mechanismof Its Release by Corneal Endothelium in Organ Culture

Abstract

Abstract: : Purpose: To demonstrate the presence of ciliary neurotrophic factor (CNTF) in the intra-ocular fluid, the aqueous humor(AQ), of eyes with extra-ocular injury in enucleated eyes and the mechanism of its release by corneal endothelium (CE) in organ culture. Methods: AQ was collected from donor human eyes from Maryland Eye Bank and enucleated bovine eyes from a local abattoir, concentrated by centrifugation in a filter unit, and analyzed by Western blot (WB) and immunoprecipitation (IP) using two anti-human CNTF antibodies and an anti-human CNTF receptor α subunit (CNTFRα) antibody. Viability of the AQ-bathed tissues was examined using a LIVE/DEAD Viability kit. Corneoscleral explant cultures were established from fresh bovine eyes and cornea cups were metabolically labeled with 0.1 mCi/ml ₃₅S-methionine for 8 h after 20 h of methionine deprivation. WB and IP were used to identify CNTF and CNTFRα in the cornea cup-conditioned medium and CE cells, following centrifugal concentration and cell homogenization, respectively. Results: Both anti-CNTF antibodies used in WP of AQ demonstrated the molecule with the expected CNTF M.W.(25 kDa). Cells in the CE, trabecular meshwork, ciliary body, iris, and lens remained nearly 100% alive, ruling out the possibility that CNTF originated from cell lysis in tissues bathed in the AQ. Non-reducing SDS-PAGE of the immunoprecipitated-AQ in WB demonstrated co-precipitation of CNTF and CNTFRα in a series of complexes with M.W. of 61, 100, 150, 175, and 210 kDa. The major ₃₅S-methionine-labeled- CNTF-immunoreactive molecules released to the cornea-cup conditioned medium showed M.W. of 25 and 61 kDa, while lesser amounts of the higher M.W. complexes were also detected. Except the 25-kDa, all CNTF-immunoprecipitated molecules was recognized by the anti-CNTFRα antibody in WB. Whereas CE cells in fresh corneas express the 25 kDa CNTF (submitted), those in ₃₅S-methionine-labelled cornea cups in organ cultures synthesized the 61 kDa CNTF. Conclusion: CNTF, a well-known neurotrophic factor, which lacks the signal sequence for secretion, is released by viable intra-ocular tissues in eyes with extra-ocular injury. The mechanism of CNTF release involves the formation and release of CNTF and CNTFRα complexes.

Keywords: 324 aqueous • 371 cornea: endothelium • 380 cytokines/chemokines