Reactive Oxygen Species Mediates the Effect of Cysteamine on Cytokine Release by Human Peripheral Mononuclear Cells - Chemically Injured Keratocytes Reaction

Abstract

Purpose: : This study was to investigate if reactive oxygen species (ROS) mediates the effect of cysteamine(CYS) on mixed peripheral blood mononuclear cells (PBMC) - chemically injured keratocytes reaction (mixed lymphocyte-keratocyte reaction; MLKR).

Methods: : The PBMC stimulation assay was then performed using chemically injured keratocytes treated with 0.05 N NaOH for 60 seconds (MLKR). MLKR were treated with a variety of concentrationof CYS (0 - 10 mM). Intracellular ROS formation was measured using the oxidation-sensitive fluorescent probe, 2'7'-dichlorofluorescein diacetate (DCFH-DA).The proliferation rate and secretion profiles of matrix metalloprotease-9 (MMP-9), transforming growth factor-beta 1 (TGF-β1), IL-6 and macrophage migration inhibitory factor (MIF) of PBMCs stimulated by NaOH-treated keratocytes were determined using bromodeoxyuridine proliferation assay and enzyme-linked immunosorbent assay, respectively.

Results: : PBMC proliferation was suppressed by CYS in dose-dependent manner (p = 0.019). DCF fluorescence decreased depending on CYS concentration (p < 0.001). MMP-9 levels and IL-6 levels decreased (p = 0.001 and 0.003, respectively) and MIF levels increased (p = 0.008), while TGF-β1 levels did not differ from those of the negative controls.

Conclusions: : In conclusion, CYS suppressed the proliferation and secretion of inflammatory cytokines in PBMC stimulated by chemically injured keratocytes via ROS reduction although CYS increased MIF levels. These results suggest that ROS mediated CYS-induced inhibition of inflammatory cytokine release from PBMC stimulated by chemically injured keratocytes and cyateamine can be used in treatment of chemical burns via suppressing inflammation.

Keywords: cornea: stroma and keratocytes • cytokines/chemokines • oxidation/oxidative or free radical damage