DOI: 10.1111/exd.12268
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
Galectin-7, induced by cis-urocanic acid and ultraviolet B irradiation, down-modulates cytokine production by T lymphocytes Takashi Yamaguchi, Kana Hiromasa, Rieko Kabashima-Kubo, Manabu Yoshioka and Motonobu Nakamura Department of Dermatology, University of Occupational and Environmental Health, Kitakyushu, Japan Correspondence: Motonobu Nakamura, Department of Dermatology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan, Tel.: +81-93-691-7445, Fax: +81-93-691-0907, e-mail: [email protected] Abstract: Urocanic acid (UCA) is an epidermal chromophore that undergoes trans to cis isomerization after UVB irradiation. cisUCA is a potent inhibitor of cutaneous acquired immunity. The aim of this study was to explore the genes, which are upregulated by cis-UCA in normal human epidermal keratinocytes (NHEK) and investigated its role in vitro using human T-lymphocyte cell line, Jurkat cells. DNA microarray analysis and real-time PCR investigation revealed that cis-UCA, not trans-UCA, increased the expression of a gene encoding a b-galactoside-binding lectin, galectin-7, LGALS7B. Immunohistochemical study demonstrated that galectin-7 was highly expressed in the epidermis in the
Background Urocanic acid (UCA) is produced by the degradation of filaggrin and other proteins in the epidermis (1,2). UCA is an epidermal chromophore that undergoes trans to cis isomerization after UVB irradiation. cis-UCA is a potent inhibitor of cutaneous acquired immunity. Precise mechanisms of immunomodulatory effects by cis-UCA remained to be clear. Galectins are a family of soluble lectins sharing a unique carbohydrate recognition domain (CRD) that confers specificity for b-galactoside derivatives (3–5). Some of the 15 galectin members have been reported to play important roles in immunomodulation; however, the effect of Galectin-7 on cutaneous immunity remained to be unclear.
Questions addressed Here, we address a question what is a signalling pathway of cutaneous immunomodulatory effect of cis-UCA.
Experimental design NHEK (Neonatal foreskin from Kurabo, Osaka, Japan) were cultured in HuMedia-KG2 medium (Kurabo). After incubation with or without 100 lg/ml cis-UCA (Sigma, St. Louis, MO, USA) for 24 h, total RNA was extracted from NHEK using Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). DNA microarray for RNA from NHEK was performed with a hybridization chamber (Takara, Otsu, Japan) and 3D-Gene Scanner 3000 (Toray, Kamakura, Japan). Total RNA was retrotranscribed into cDNA with 1st strand cDNA synthesis kit for RT-PCR (AMV) (Roche Diagnostics, Mannheim, Germany) (6). Real-time PCR analysis of galectin-7, IL2, IFNG, IL4 and control glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA expression levels in NHEKs was performed using 209 Assays-on-Demand Gene Expression Assay Mix (Hs00221068_m1, Hs00174114_m1, Hs00989291_m1, Hs0017 4112_m1 and Hs00266705_g1, respectively, Applied Biosystems, Foster City, CA, USA) with a 7000 system (Applied Biosystems).
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patients with actinic keratosis. Galectin-7 administration upregulated apoptosis and inhibited the expression of interleukin2 (IL2) and interferon-c (IFNG) mRNA in Jurkat cells. Taken together, galectin-7 may play important roles in downregulating the functions of T lymphocytes after UVB irradiation and can be developed into novel immunosuppressive therapies for inflammatory skin diseases. Key words: actinic keratosis – apoptosis – cis-urocanic acid – galectin-7 – UVB
Accepted for publication 15 October 2013
The concentration of human IL-2, IFN-c, IL-4 and galectin-7 were measured by enzyme-linked immunosorbent assay (ELISA) using Qunatikine Immunoassay and DuoSet ELISA Development Kit (R&D systems, Minneapolis, MN, USA) with iMark Microplant Reader (BIO-RAD, Hercules, CA, USA). Paraffin sections of the five actinic keratosis patients (three females and two males, aged 66–80) and five age-matched samples without actinic keratosis were deparafinized and stained with antigalectin 7 antibody (Epitomics, Burlingame, CA, USA) at 1:50 concentration. Human T lymphocyte line Jurkat cells were cultured at 37°C (5% CO2) in a RPMI1640 medium (Sigma), containing 10% foetal bovine serum (Invitrogen, Grand Island, NY, USA). TUNEL assay was performed using Apoptosis in situ Detection Kit (Wako, Kyoto, Japan). Jurkat cells were cultured (5 9 105 cells/each in 24 well dish) with 1 lM ionomycin (Sigma) and 10 ng/ml phorbol 12 myristate-B acetate (PMA) (Sigma) with or without recombinant human galectin 7 (Cell Sciences, Canton, MA, USA) for 24 h.
Results and discussion cis-UCA, not trans-UCA, induced expression of galectin-7 in NHEKs To explore the genes that are upregulated by cis-UCA in NHEKs, we added 100 lg/ml cis-UCA into the culture medium for NHEKs for 24 h and isolated RNA from the cells. We compared RNA expression level in NHEKs with or without 100 lg/ml cis-UCA addition by performing DNA microarray (Table 1). Out of the upregulated genes shown in Table 1, we focused on galectin-7, because galectin families have recently been reported to modulate immune reactions (3,4). To ascertain the DNA microarray data, we performed real-time PCR analysis. Both in 50 and 100 lg/ml, cis-UCA significantly increased the expression of galectin-7 mRNA in NHEKs, while trans-UCA gave no significant change in the galectin-7 mRNA expression level (Fig. 1a). ELISA of culture supernatant revealed
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2013, 22, 832–849
Letter to the Editor
(a)
Table 1. The list of genes upregulated by cis-UCA in NHEKs
Galectin-7/GAPDH
Galectin-7
(b)
ng/ml
Description
Gene symbol
Fold change
SLCO4A1
4.3
0.0014
9
P = 0.008
P
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
Galectin-7, induced by cis-urocanic acid and ultraviolet B irradiation, down-modulates cytokine production by T lymphocytes Takashi Yamaguchi, Kana Hiromasa, Rieko Kabashima-Kubo, Manabu Yoshioka and Motonobu Nakamura Department of Dermatology, University of Occupational and Environmental Health, Kitakyushu, Japan Correspondence: Motonobu Nakamura, Department of Dermatology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan, Tel.: +81-93-691-7445, Fax: +81-93-691-0907, e-mail: [email protected] Abstract: Urocanic acid (UCA) is an epidermal chromophore that undergoes trans to cis isomerization after UVB irradiation. cisUCA is a potent inhibitor of cutaneous acquired immunity. The aim of this study was to explore the genes, which are upregulated by cis-UCA in normal human epidermal keratinocytes (NHEK) and investigated its role in vitro using human T-lymphocyte cell line, Jurkat cells. DNA microarray analysis and real-time PCR investigation revealed that cis-UCA, not trans-UCA, increased the expression of a gene encoding a b-galactoside-binding lectin, galectin-7, LGALS7B. Immunohistochemical study demonstrated that galectin-7 was highly expressed in the epidermis in the
Background Urocanic acid (UCA) is produced by the degradation of filaggrin and other proteins in the epidermis (1,2). UCA is an epidermal chromophore that undergoes trans to cis isomerization after UVB irradiation. cis-UCA is a potent inhibitor of cutaneous acquired immunity. Precise mechanisms of immunomodulatory effects by cis-UCA remained to be clear. Galectins are a family of soluble lectins sharing a unique carbohydrate recognition domain (CRD) that confers specificity for b-galactoside derivatives (3–5). Some of the 15 galectin members have been reported to play important roles in immunomodulation; however, the effect of Galectin-7 on cutaneous immunity remained to be unclear.
Questions addressed Here, we address a question what is a signalling pathway of cutaneous immunomodulatory effect of cis-UCA.
Experimental design NHEK (Neonatal foreskin from Kurabo, Osaka, Japan) were cultured in HuMedia-KG2 medium (Kurabo). After incubation with or without 100 lg/ml cis-UCA (Sigma, St. Louis, MO, USA) for 24 h, total RNA was extracted from NHEK using Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). DNA microarray for RNA from NHEK was performed with a hybridization chamber (Takara, Otsu, Japan) and 3D-Gene Scanner 3000 (Toray, Kamakura, Japan). Total RNA was retrotranscribed into cDNA with 1st strand cDNA synthesis kit for RT-PCR (AMV) (Roche Diagnostics, Mannheim, Germany) (6). Real-time PCR analysis of galectin-7, IL2, IFNG, IL4 and control glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA expression levels in NHEKs was performed using 209 Assays-on-Demand Gene Expression Assay Mix (Hs00221068_m1, Hs00174114_m1, Hs00989291_m1, Hs0017 4112_m1 and Hs00266705_g1, respectively, Applied Biosystems, Foster City, CA, USA) with a 7000 system (Applied Biosystems).
840
patients with actinic keratosis. Galectin-7 administration upregulated apoptosis and inhibited the expression of interleukin2 (IL2) and interferon-c (IFNG) mRNA in Jurkat cells. Taken together, galectin-7 may play important roles in downregulating the functions of T lymphocytes after UVB irradiation and can be developed into novel immunosuppressive therapies for inflammatory skin diseases. Key words: actinic keratosis – apoptosis – cis-urocanic acid – galectin-7 – UVB
Accepted for publication 15 October 2013
The concentration of human IL-2, IFN-c, IL-4 and galectin-7 were measured by enzyme-linked immunosorbent assay (ELISA) using Qunatikine Immunoassay and DuoSet ELISA Development Kit (R&D systems, Minneapolis, MN, USA) with iMark Microplant Reader (BIO-RAD, Hercules, CA, USA). Paraffin sections of the five actinic keratosis patients (three females and two males, aged 66–80) and five age-matched samples without actinic keratosis were deparafinized and stained with antigalectin 7 antibody (Epitomics, Burlingame, CA, USA) at 1:50 concentration. Human T lymphocyte line Jurkat cells were cultured at 37°C (5% CO2) in a RPMI1640 medium (Sigma), containing 10% foetal bovine serum (Invitrogen, Grand Island, NY, USA). TUNEL assay was performed using Apoptosis in situ Detection Kit (Wako, Kyoto, Japan). Jurkat cells were cultured (5 9 105 cells/each in 24 well dish) with 1 lM ionomycin (Sigma) and 10 ng/ml phorbol 12 myristate-B acetate (PMA) (Sigma) with or without recombinant human galectin 7 (Cell Sciences, Canton, MA, USA) for 24 h.
Results and discussion cis-UCA, not trans-UCA, induced expression of galectin-7 in NHEKs To explore the genes that are upregulated by cis-UCA in NHEKs, we added 100 lg/ml cis-UCA into the culture medium for NHEKs for 24 h and isolated RNA from the cells. We compared RNA expression level in NHEKs with or without 100 lg/ml cis-UCA addition by performing DNA microarray (Table 1). Out of the upregulated genes shown in Table 1, we focused on galectin-7, because galectin families have recently been reported to modulate immune reactions (3,4). To ascertain the DNA microarray data, we performed real-time PCR analysis. Both in 50 and 100 lg/ml, cis-UCA significantly increased the expression of galectin-7 mRNA in NHEKs, while trans-UCA gave no significant change in the galectin-7 mRNA expression level (Fig. 1a). ELISA of culture supernatant revealed
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2013, 22, 832–849
Letter to the Editor
(a)
Table 1. The list of genes upregulated by cis-UCA in NHEKs
Galectin-7/GAPDH
Galectin-7
(b)
ng/ml
Description
Gene symbol
Fold change
SLCO4A1
4.3
0.0014
9
P = 0.008
P
