Effects Of Thyroxine And Hydrocortisone On Normal Human Keratocytes And Keratoconic Keratocytes In Vitro

Purpose: To investigate the effects of thyroxine and hydrocortisone on the viability, proliferation, apoptosis, growth factor and interleukin secretion of human keratocytes in vitro in normal and keratoconic corneas. Methods: Seven normal corneas, not meeting the criteria for corneal transplantation, as well as five corneal buttons from patients with keratoconus who underwent keratoplasty were used to isolate normal human primary keratocytes (NHPK) and keratoconus human primary keratocytes (KHPK). NHPK and KHPK were treated with thyroxine and hydrocortisone in concentrations of 0, 0.01, 0.1, 1 and 10 mu g/ml for 24 h. Cell viability was determined by AlamarBlue (R) assay and cell proliferation by the cell proliferation ELISA BrdU kit, while cell apoptosis was evaluated with the APO-DIRECT (TM) kit. Five and 24 hours after hormone treatment, FGFb, HGF, TGF beta 1, VEGF, KGF, IL-1 beta, IL-6 and IL-8 secretion was measured using enzyme-linked-immunosorbent assay (ELISA). Results: Twenty-four hours after hormone treatment, we did not observe any changes neither in cell viability or apoptosis of NHPK and KHPK, nor in proliferation of KHPK. Cell proliferation of NHPK increased significantly 24 h after treatment with thyroxine in all performed concentrations (P<0.01), but there were no changes with hydrocortisone. In NHPK, thyroxine down-regulated IL-6 at 5 h and up-regulated HGF at 24 h, while hydrocortisone down-regulated IL-6, IL-8 and FGFb at 5 h, as well as IL-6, IL-8, EGF and TGF beta 1 at 24 h (P<0.05). In KHPK, thyroxine up-regulated the secretion of IL-8 and TNF alpha at 5 h, as well as HGF at 24 h, whereas hydrocortisone down-regulated IL-6, IL-8 and TNFa at 5 h, down-regulated IL-6 and TGF beta 1 at 24 h and up-regulated HGF at 24 h (P<0.05). Conclusions: Thyroxine and hydrocortisone appear to have no significant impact on cell viability and apoptosis of human keratocytes in vitro. Treatment with thyroxine triggers the proliferation of NHPK, but has no influence on the proliferation of KHPK. Hydrocortisone does not affect significantly cell proliferation of NHPK and KHPK. Thyroxine significantly up-regulates the secretion of certain growth factors and interleukins by KHPK, although it does not affect their secretion by NHPK, while hydrocortisone predominatly down-regulates them by both NHPK and KHPK. It appears that hormonal influences have differential effects on the secretion of these factors by human keratocytes, thereby modulating to some extent the corneal homeostasis.