Cis-urocanic acid suppresses UV-B-induced interleukin-6 and -8 secretion and cytotoxicity in human corneal and conjunctival epithelial cells in vitro

Abstract

Purpose: Urocanic acid (UCA) is a major ultraviolet (UV)-absorbing endogenous chromophore in the epidermis and is also an efficacious immunosuppressant. The anti-inflammatory and cytoprotective effects of cis-UCA were studied in ocular surface cell cultures exposed to UV-B irradiation.

Methods: Human corneal epithelial cells (HCE-2) and human conjunctival epithelial cells (HCECs) were incubated with 10, 100, 1,000, and 5,000 microg/ml cis-UCA with and without a single UV-B irradiation dose. The concentrations of IL-1beta, IL-6, IL-8, and TNF-alpha in the culture medium and caspase-3 activity in the cell extract sampled were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was measured by the colorimetric MTT (3-(4,5-dimethyldiazol- 2-yl)-2,5-diphenyltetrazolium bromide) assay.

Results: UV-B irradiation multiplied interleukin IL-6 and IL-8 secretion levels in HCE-2 cells and HCECs as analyzed with ELISA. Cell viability as measured by the MTT assay declined by 30%-50% in HCE-2 cells and by 20%-40% in HCECs after UV-B irradiation. Moreover, UV-B increased caspase-3 activity in both cell types as analyzed with ELISA. Treatment with 100 microg/ml cis-UCA completely suppressed IL-6 and IL-8 secretion, decreased caspase-3 activity, and improved cell viability against UV-B irradiation. No significant effects on IL-6 or IL-8 secretion, caspase-3 activity, or viability of the non-irradiated cells were observed with 100 microg/ml cis-UCA in both cell types. The 5,000 microg/ml concentration was toxic.

Conclusions: These findings indicate that cis-UCA may represent a promising anti-inflammatory and cytoprotective treatment option to suppress UV-B-induced inflammation and cellular damage in human corneal and conjunctival epithelial cells.

Figure 1

Effect of cis-UCA on IL-6 secretion. The HCE-2 cells (A) and HCECs (B) were either untreated (C) or exposed to different concentrations of cis-UCA for 24, 48, or 72 h. For statistical analysis, cis-UCA treated samples were compared with C samples. An asterisk indicates p<0.05, and a double asterisk denotes p<0.001 (n=6 dishes).

Figure 2

Effect of cis-UCA on UV-irradiation-induced IL-6 secretion. The HCE-2 cells (A) and HCECs (B) were non-irradiated (C) or UV-irradiated (UV; lower panels), or exposed to different concentrations of cis-UCA or UV irradiated and treated with cis-UCA for 24, 48, or 72 h (upper panels). For statistical analysis, cis-UCA+UV samples were compared with UV samples. An asterisk indicates p<0.05, and a double asterisk denotes p<0.001 (n=6 dishes).

Figure 3

Effects of cis-UCA and UV irradiation on viability. The non-irradiated (C) and UV-irradiated (UV) HCE-2 cells (A) and HCECs (B) were exposed to different concentrations of cis-UCA for up to 72 h. For statistical analysis, cis-UCA samples and cis-UCA+UV samples were compared with C samples. An asterisk indicates p<0.05, and a double asterisk denotes p<0.001 (n=6 dishes).

Figure 4

Photoisomerization of UCA. UV excitation of one isomer leads to the formation of the other isomer (A). The concentrations of UCA isomers is measured in the cell culture medium of HCE-2 cells (B) and HCECs (C) treated with 100 µg/ml cis-UCA for 24 h with or without UV irradiation.

Figure 5

UV absorption effect of the culture medium on IL-6 secretion. The HCE-2 cells (A) and HCECs (B) were irradiated in a colorless buffer solution or in a normal culture medium prior to change of medium without or with 100 μg/ml cis-UCA and were then followed for 24 h. An asterisk indicates p<0.05, and a double asterisk denotes p<0.001 (n=6 dishes). Abbreviations: C, control; m, medium; PBS, phosphate buffered saline; UCA, urocanic acid; UV, ultraviolet.

Figure 6

Effect of cis-UCA on UV-irradiation-induced IL-8 secretion. The HCE-2 cells (A) and HCECs (B) were non-irradiated (C), UV-irradiated (UV), treated with 100 mg/ml cis-UCA (c-UCA), or UV-irradiated and treated with cis-UCA (100 mg/ml c-UCA+UV) for 24, 48, or 72 h. Statistical significance is shown by an asterisk (p<0.05) or a double asterisk (p<0.001; n=6 dishes). Compared samples are shown by the horizontal lines.

Figure 7

Effect of cis-UCA on UV-irradiation-induced caspase-3 activity. The HCE-2 cells (A) and HCECs (B) were non-irradiated (C), UV-irradiated (UV), treated with 100 mg/ml cis-UCA (c-UCA), or UV-irradiated and treated with cis-UCA (100 mg/ml c-UCA+UV) for 24, 48, or 72 h. Statistical significance is shown by an asterisk (p<0.05) or a double asterisk (p<0.001; n=6 dishes). Note that there is no significant difference (ns) between the control sample and the c-UCA-treated sample after the 72 h follow-up.