Effect of Cysteamine on Human Peripheral Blood Mononuclear Cells-Chemically Injured Keratocytes Reaction

Young Bok Lee; Joon Young Hyon; Won Ryang Wee; Tae Young Chung; Eui Sang Chung; Ka Young Yi; Young Joo Shin

doi:10.3341/jkos.2015.56.10.1511

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Lee, Hyon, Wee, Chung, Chung, Yi, and Shin: Effect of Cysteamine on Human Peripheral Blood Mononuclear Cells-Chemically Injured Keratocytes Reaction

Original Article

J Korean Ophthalmol Soc 2015;56(10):1511-1519.

Published online: 29 August 2015

DOI: https://doi.org/10.3341/jkos.2015.56.10.1511

Effect of Cysteamine on Human Peripheral Blood Mononuclear Cells-Chemically Injured Keratocytes Reaction

Young Bok Lee, M.D¹, Joon Young Hyon, M.D, PhD^2,³, Won Ryang Wee, M.D, PhD², Tae Young Chung, M.D, PhD⁴, Eui Sang Chung, M.D, PhD⁵, Ka Young Yi, M.D, PhD¹, Young Joo Shin, M.D, PhD^1,

¹Department of Ophthalmology, Hallym University Kangnam Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea

²Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea

³Department of Ophthalmology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea

⁴Department of Ophthalmology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

⁵Rhee’s Eye Hospital, Daejeon, Korea

Address reprint requests to Young Joo Shin, MD, PhD Department of Ophthalmology, Hallym University Kangnam Sacred Heart Hospital, #1 Singil-ro, Yeongdeungpo-gu, Seoul 07441, Korea Tel: 82-2-829-5193, Fax: 82-2-848-4638 E-mail: schinn@hanmail.net

Received 27 February 201530 May 2015 Accepted 14 August 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

To investigate the effect of cysteamine on mixed peripheral blood mononuclear cells (PBMCs)-chemically injured kera-tocytes reaction (mixed lymphocyte-keratocyte reaction; MLKR).

Methods

PBMC stimulation assay was performed after keratocytes were chemically injured with 0.05 N NaOH for 60 seconds. MLKR was treated with various concentrations of cysteamine (0-10 mM). Intracellular reactive oxygen species (ROS) formation was measured using the oxidation-sensitive fluorescent probe, 2′7′-dichlorofluorescein diacetate (DCF-DA). Proliferation rate of PBMCs stimulated by NaOH-treated keratocytes and secretion profiles of matrix metalloprotease-9 (MMP-9), transforming growth factor-beta1 (TGF-β1), interleukin-6 (IL-6), and macrophage migration inhibitory factor (MIF) were determined using the bromodeoxyuridine proliferation assay and enzyme-linked immunosorbent assay, respectively.

Results

Proliferation rate of PMBCs was suppressed by cysteamine in a dose-dependent manner ( p = 0.019). Fluorescence of DCF-DA decreased depending on cysteamine concentration ( p < 0.001). MMP-9, IL-6 and TGF-β1 levels were suppressed by cyste-amine in a dose-dependent manner ( p < 0.05), whereas MIF levels increased with cysteamine concentration of 0.5-10 mM ( p = 0.008).

Conclusions

These study results indicate that cysteamine induced the ROS-mediated inhibition of inflammatory cytokine re-lease and proliferation of PBMCs stimulated by chemically injured keratocytes. Thus, cysteamine can be used in the treatment of chemical corneal burns.

Keywords: Cysteamine, Macrophage migration inhibitory factor, Mixed peripheral lymphocyte-keratocyte reaction, Reactive oxygen species, Transforming growth factor-beta1

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Figure 1.

Inverted phase-contrast image and immunofluorescent vimentin staining of human keratocytes. (A) Keratocytes were ob-served growing parallel to the etched lines. Magnification, ×200. (B) Immunofluorescence with anti-vimentin antibody, a general marker of keratocyte, revealed the morphology. The nuclei were stained using Hoechst 33342 (blue). Magnification, ×400.

Figure 2.

Peripheral blood monocular cell (PBMC) stimulation test. NaOH-treated human keratocytes served as the stimulators. Cysteamine suppressed the PBMC proliferation in a dose-de-pendent manner ( p = 0.019, Kruskal-Wallis test). PBMC pro-liferation was suppressed with ≥5 mM of cysteamine ( p = 0.034 and 0.021, respectively, Mann-Whitney U-test). PBMC pro-liferation with 0-5 mM of cysteamine was higher than that of neg-ative control ( p = 0.008 for all, Mann- Whitney U-test), whereas it was lower than that of negative control in 10 mM of cysteamine ^* Statistically sig-( p = 0.008). BrdU = bromodeoxyuridine. nificant by Mann-Whitney U-test.

Figure 3.

Relative 2’,7’-dichlorofluorescein diacetate (DCF- DA) fluorescence. Intracellular ROS levels measured by rela-tive DCF fluorescence decreased by 0-10 mM cysteamine in a dose-dependent manner ( p < 0.001; Kruskal-Wallis test). Cyst-eamine concentration of ≥1 mM significantly lowered the intra-cellular ROS levels ( p = 0.014, 0.014 and 0.014, respectively; Mann-Whitney U-test). Intracellular ROS levels with 0-1 mM of cysteamine was higher than that of negative control ( p = 0.016, 0.008, and 0.008, respectively, Mann-Whitney U-test). However, there was no significant difference of intracelluar ROS level between 5-10 mM of cysteamine and negative control. ROS = reactive oxygen species; DCF = 2‘7’-dichl-oroflurescein. ^* Statistically significant by Mann-Whitney U-test.

Figure 4.

MMP-9 levels measured by ELISA. MMP-9 levels decreased in a dose-dependent manner by 0-10 mM cysteamine ( p = 0.001; Kruskal-Wallis test). Cysteamine concentration of ≥5 mM significantly lowered the MMP-9 levels ( p = 0.016, and 0.009, respectively; Mann-Whitney U-test). MMP-9 lev-els with 0-5 mM of cysteamine was higher compared with neg-ative control ( p = 0.016, 0.016, 0.008, and 0.008, respectively, Mann-Whitney U-test). However, there was no significant dif-ference of intracelluar ROS level between 10 mM of cysteamine and negative control. MMP-9 = matrix metalloprotease-9; ELISA = enzyme-linked immunosorbent assay; ROS = reactive oxygen species. ^* Statistically significant by Mann-Whitney U-test.

Figure 5.

IL-6 levels measured by ELISA. Cysteamine decreased IL-6 levels in a dose-dependent manner ( p = 0.003; Kruskal- Wallis test). Cysteamine concentration of ≥1 mM significantly lowered the IL-6 levels ( p = 0.043, 0.021, and 0.021, respectively; Mann-Whitney U-test). IL-6 levels with 0-0.5 mM of cysteamine was higher than that of negative control ( p = 0.016, and 0.032, respectively, Mann-Whitney U-test), whereas IL-6 levels with cysteamine concentration of ≥5 mM were lower than that of neg-ative control ( p = 0.032, and 0.016, respectively; Mann-Whitney U-test). IL-6 = interleukin-6; ELISA = enzyme-linked immunosorbent assay. ^* Statistically significant by Mann- Whitney U-test.

Figure 6.

TGF-β1 levels measured by ELISA. TGF-β1 level was slightly elevated in a low (0.05 mM) cysteamine concen-tration ( p = 0.016; Mann-Whitney U-test). However, it de-creased at high concentrations (5 mM and 10 mM) ( p = 0.009 and 0.009; Mann-Whitney U-test). TGF-β1 levels with 0.5 mM of cysteamine was higher than that of negative control ( p = 0.016, Mann-Whitney U-test), whereas TGF-β1 levels with cysteamine concentration of ≥5 mM were lower than that of negative control ( p = 0.016 for both). TGF-β1 = trans-forming growth factor-beta 1; ELISA = enzyme-linked im-munosorbent assay. ^* Statistically significant by Mann-Whitney U-test.

Figure 7.

MIF levels measured by ELISA. Cysteamine in-creased MIF levels at high concentrations (≥ 1 mM) ( p = 0.014, 0.014, and 0.027, respectively; Mann-Whitney U-test). MIF levels with 0-10 mM of cysteamine was higher than that of negative control ( p = 0.016, 0.008, 0.008, 0.008, and 0.008, respectively). MIF = macrophage migration inhibitory factor; ^* Statistically ELISA = enzyme-linked immunosorbent assay. significant by Mann-Whitney U-test.

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