Ultraviolet A irradiation inhibits thymus- and activation-regulated chemokine (TARC/CCL17) production by a human keratinocyte

ARTICLE

Auteur(s) : Xueyi ZHENG, Koichiro NAKAMURA, Michiko TOJO, Hitoshi AKIBA, Noritaka OYAMA, Akiko NISHIBU, Fumio KANEKO, Yuichiro TSUNEMI*, Takashi KAKINUMA*, Hidehisa SAEKI*, Kunihiko TAMAKI*

Department of Dermatology, Fukushima Medical University School of Medicine, Hikarigaoka 1, Fukushima 960-1295, Japan.
* Department of Dermatology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Article accepted on 8/04/2003

Abbreviations: IFN-γ (interferon-γ), TARC/CCL17 (thymus and activation-regulated chemokine), TNF-α (tumor necrosis factor-α), UVA (ultraviolet A).

Thymus- and activation-regulated chemokine (TARC/CCL17) is a CC chemokine secreted by dendritic cells, monocytes and by keratinocytes as well [1]. TARC functions as a selective chemoattractant for activated T cells which belong to the Th2 subset and express CC chemokine receptor (CCR4), the receptor for TARC [2]. TARC can be produced locally and recruit CCR4 + T cells and it may play an important role in the immune responses in which CCR4 + Th2 cells participate [3]. Recently, it has been shown that high levels of TARC were expressed in various allergic diseases [4-6]. In particular, TARC levels were markedly elevated in serum from patients with atopic dermatitis and TARC proteins were expressed strongly by their epidermal keratinocytes [6]. The concentration of TARC in the serum was also related to the disease activity of atopic dermatitis [6]. It has been suggested that TARC could be a useful target for therapeutic approaches to allergic disease in which TARC and CCR4 are involved [1, 7].

UV irradiation has been shown to modulate the immune function. [8, 9]. One of the possible mechanisms might be the direct and/or indirect effect of UV irradiation on the modulation of cytokines produced by immune cells [9, 10]. However, to our knowledge, the effects of UV irradiation on TARC expression by keratinocytes have not been examined. In this study, we examined the effects of UVA irradiation on the expression of TARC mRNA and the release of TARC protein by HaCaT cells, a human keratinocyte cell line.

Materials and methods

Reagents and cell culture

The cytokines used were recombinant human (rh) tumor necrosis factor (TNF)-α (Peprotech Inc. London, UK), and rh interferon (IFN)-γ (R&D Systems Inc. Minneapolis, MN). HaCaT cells which were kindly provided by Prof. NDEDFusenig (German Cancer Research Center, Heidelberg, Gemany) were cultured at 37°C, 5% CO₂ in RPMI 1640 containing 10% fetal calf serum.

UV irradiation

The UV source was a DERMARAY Medical Ultraviolet Irradiation Apparatus (Clinical Supply, Tokyo, Japan). This machine can be adjusted to radiate UVA or UVB specifically. The emission spectrum of the lamp used for UVA irradiation is 310-410nm, and the peak emission is at 360nm. The irradiation dose was measured with a UV radiometer (Clinical Supply, Tokyo, Japan). The UVA irradiation doses used were 1, 4 and 7J/cm². Cultures were fed fresh culture medium, with or without TNF-α (10ng/ml) and IFN-γ (10ng/ml), 24 hours before UVA irradiation. Just before UVA exposure, the medium was collected. The cells were washed twice with PBS (37°C) and then irradiated in the presence of PBS without the plastic lid. For a 6-cm diameter plate 3-ml PBS were added. After UV irradiation, the PBS was removed, the collected medium was added and the cells were incubated for the indicated times. Sham-irradiated controls were placed under aluminum foil while irradiation was being carried out. The irradiation distance was 40 cm.

Enzyme-linked immunosorbent assay (ELISA)

The concentration of TARC in culture supernatants was measured using a commercially available kit (Genzyme, Minneapolis, USA). In brief, samples were added to wells onto which a monoclonal antibody specific for TARC had been pre-coated. After washing, an enzyme-linked polyclonal antibody conjugated to horseradish peroxidase was added. Following washing, color was developed. The optical density (OD) was measured using an ELISA reader (Spectra and Rainbow Readers). The minimum detectable concentration was less than 7pg/ml. The protein level in the supernatant was normalized to total cell number. Each supernatant from four different experiments was analyzed in duplicate.

Northern hybridization [11]

Total mRNA was extracted from the cells using a lysis buffer (Invitrogen Co., CA, USA). Total mRNA (20μg) was fractionated on a 1.5% agarose-formaldehyde gel and transferred to a nylon membrane (Bio-Rad, Hercules, CA, USA). The membrane was UV cross-linked, incubated in 10ml of pre-hybridization solution (0.25M Na₂HPO₄ pH 7.2 and 7% SDS) for 2 hours at 65°C and then hybridized for 20 hours with a cDNA probe labeled with digoxigenin(DIG)-dUTP using PCR amplification [9]. After washing stringently, the cDNA-mRNA hybrids were visualized using a DIG nucleic acid detection kit (Roche, Germany). The probe was a 511bp TARC cDNA fragment.
For cDNA probe synthesis, polyA⁺ mRNA was extracted using a Micro-FastTrack^TM 2.0 kit (Invitrogen Co., CA, USA). Approximately 1μg of polyA⁺ mRNA was reverse-transcripted using a First cDNA kit (Invitrogen Co., CA, USA). The PCR labeling mixture included cDNA, each primer (25-50pmol), MgCl₂, dNTP (the ratio of dTTP to DIG-dUTP was 19) and Taq polymerase (TaKaRa, Otsu, Japan). The cycling condition consisted of 40 cycles of denaturation at 95°C, annealing at 57°C, and extension at 72°C. After PCR, the probe was purified. The relative blot intensities of the TARC mRNA were quantified by densitometric scanning and analyzed using NIH Image computer software. Because UV irradiation might influence the expression of GAPDH mRNA expression [12] and our preliminary results also indicated that β – actin mRNA expression may be affected by UVA irradiation, the amounts of RNA loaded were normalized to 28S RNA.

Cell viability [13]

After UVA irradiation (1, 4 and 7J/cm²), cell viability was evaluated by trypan blue dye exclusion [13]. Cells floating in the supernatant and cells adhering to the dish were collected 24 hours or 48 hours after UVA irradiation together using 0.02% EDTA and 0.25% trypsin. The viable and dead cells were counted.

Statistical analysis

ANOVA was used for comparing the differences in TARC mRNA or protein levels between the UVA-irradiated and sham-irradiated groups. Non-parametric Spearman’s correlation test was used to evaluate the correlation between the doses of UVA irradiation and their effects on TARC mRNA or protein levels. Student’s t-test was used to compare the differences in TARC mRNA or protein levels at different time points between the UVA-irradiated and sham-irradiated groups. The values shown are means ± standard deviation (SD). Differences were considered significant at p < 0.05.

Results

Effects of UVA irradiation dose on cell viability

The percentages of viable cells, based on three counts per plate, and three plates per treatment, were not significantly different for UVA doses of 0, 1, 4 and 7J/cm² at either the 24 or 48 hours post-UVA irradiation. There was no difference between the UVA irradiated and non-irradiated groups (P > 0.2) (Data not shown).

Effects of UVA irradiation on the baseline level of TARC mRNA expression and TARC protein secretion in non-stimulated HaCaT cells

We first examined the effects of UVA irradiation on the basal level of the expression of TARC mRNA and the secretion of TARC proteins. Non-stimulated HaCaT cells expressed a low level of TARC mRNA and secreted a very small amount of TARC protein, as has been reported [14]. Under our experimental conditions, per 10⁶ cells secreted 64 ± 12.9 pg/ml TARC proteins. Culture supernatants and /or total mRNAs were harvested at 24 hours after UVA irradiation or sham-irradiation. The concentrations of TARC protein in the culture supernatants were 53.8 ± 11.8, 24.7 ± 10.5, and 8.63 ± 4.2 pg/ml at UVA irradiation doses of 1, 4, and 7 J/cm², respectively (Fig. 1). The level of TARC protein secretion was significantly inhibited at doses of 4 and 7 J/cm², but not at 1 J/cm², when the TARC protein concentration was measured at 24 hours after UVA irradiation. Because the minimum detectable dose was about 7pg/ml, we think that the basal levels of TARC protein secretion were almost completely inhibited by UVA irradiation at a dose of 7J/cm². There was a significant correlation between the inhibitory effects of UVA on TARC secretion and the UVA irradiation doses (r = 0.57, P = 0.003 < 0.01). Northern hybridization showed the same pattern of suppression of TARC mRNA expression. At UVA irradiation doses of 1, 4 and 7 J/cm², TARC mRNA expression was suppressed by 22%, 58% and 79%, respectively compared to controls (0 J/cm²) (Fig. 2).

Effects of UVA irradiation on the levels of TARC mRNA expression and TARC protein secretion by HaCaT cells co-stimulated with IFN-γ and TNF-α

High levels of TARC production can be induced in HaCaT cells by co-stimulating them with IFN-γ and TNF-α [14]. Moreover, it has been shown the both IFN-γ and TNF-α were involved in the pathogenesis of inflammatory skin disease such as atopic dermatitis [15]. Thus co-stimulating HaCaT cells with IFN-γ and TNF-α mimics the inflammatory condition. Further research was conducted to examine whether UVA irradiation could regulate IFN-γ and TNF-α stimulation induced TARC mRNA expression and TARC protein secretion by HaCaT cells. Fig. 3 shows the inhibitory effects of UVA irradiation on IFN-γ and TNF-α stimulation induced TARC protein secretion by HaCaT cells. The TARC protein levels in the culture supernatants which were collected at 24 hours after UVA irradiation were 825.1 ± 92.8, 755.5 ± 79.6, 685.3 ± 68.3, and 623.2 ± 76.2 pg/ml at UVA irradiation doses of 0, 1, 4 and 7 J/cm² respectively. There was significant suppression after exposure to UVA irradiation dose of 1, 4 and 7 J/cm². There was a significant correlation between the inhibitory effects of UVA on TARC secretion and the UVA irradiation doses (r = 0.59, P = 0.003 < 0.01). Northern hybridization showed that, compared to sham-irradiated controls, UVA irradiation inhibited IFN-γ and TNF-α stimulation induced TARC mRNA expression by 24%, 49% and 74% at dose of 1, 4 and 7 J/cm² respectively (Fig. 4).

Time course of the effects of UVA irradiation on IFN-γ and TNF-α stimulation induced TACR mRNA expression and TARC protein secretion by HaCaT cells

Confluent HaCaT cells was co-stimulated with IFN-γ-and TNF-α 24 hours before UVA irradiation as stated at materials and methods. After one exposure to UVA irradiation (7 J/cm²), total RNA was harvested at different time points and was subjected to Northern hybridization (Fig. 5). Significant inhibitory effect on TARC mRNA expression was detected 8 hours after UVA irradiation. The inhibitory effect on TARC mRNA expression was the most effective at 16-24 hours after UVA irradiation. At about 36 hours after UVA irradiation, the inhibitory effect of UVA irradiation on TARC mRNA expression became less effective at 36-hour time point than that of 24-hour time point. This suggests that the inhibitory effects of UVA irradiation on IFN-γ and TNF-α stimulation induced TARC mRNA expression began to reduce. Fig. 6 shows the inhibitory effects of UVA irradiation on TARC protein secretion. Culture supernatant was collected at indicated time points. UVA irradiation partially inhibited IFN-γ-and TNF-α co-stimulation induced TARC protein secretion by HaCaT cells under our experimental conditions (Table I). TARC protein concentration in the culture supernatant after 24-hour time point was significantly lower than that of controls. The most effective inhibition of UVA irradiation on TARC protein secretion occurred during the first 8 hours after UVA irradiation because the rate of TARC protein secretion was 43 ± 4.9% of the control which was the smallest compared to that of any other periods after UVA irradiation (not shown). During the period of from 36 hours to 48 hours after UVA irradiation, the rate of TARC protein secretion was 83 ± 9.4% of controls. This secretion rate was not significantly different to the control groups, even through the TARC concentrations at 36-hour and 48-hour time point were significantly lower than control. Our results also shows the inhibitory effect of UVA irradiation on TARC secretion gradually became weaker and at about 36 hours after UVA irradiation the rate of TARC secretion is not significantly different from the controls. Thus, 36 hours after UVA irradiation, the inhibitory effects of UVA irradiation on TARC protein secretion could not countercheck the IFN-γ-and TNF-α-induced TARC secretion by HaCaT cells nor it is likely that the inhibitory effect of UVA irradiation ceased because of the cellular self-repair function. Another interesting finding is that the inhibitory effects of UVA irradiation on TARC mRNA expression and the inhibitory effects of UVA irradiation protein secretion did not parallel each other. This may suggest that multiple points might be affected by UVA irradiation in the process of TARC production.

Table I. The time course results of TARC protein concentration (pg/ml)

Discussion

Keratinocytes are considered an important component of the skin immune system and actively participate in various kinds of immune responses [16]. In humans, because of their anatomical location, keratinocytes are the natural targets of UV irradiation. Therefore, keratinocytes have been used as a model in studies on the effects of UV irradiation [17]. In this study, we showed that UVA irradiation inhibited both the basal level and the IFN-γ and TNF-α stimulation-induced TARC mRNA expression and TARC protein secretion by HaCaT cells. The UVA irradiation doses (1, 4 and 7J/cm²) we used, did not affect cell viability, in agreement with what others have reported [18]. This excluded the possibility that the inhibition of TARC mRNA expression and TARC protein secretion was caused by UVA irradiation-induced cytotoxicity. To our knowledge, this is the first report showing that UVA irradiation can inhibit TARC mRNA expression and TARC protein secretion by a human keratinocyte line, HaCaT cells. We believe that this finding is important to understand the underlying mechanism of UVA irradiation in the process of modulating immune responses.
High levels of TARC production can be induced in HaCaT cells through co-stimulating with IFN-γ and TNF-α [14]. Because it has been shown that both IFN-γ and TNF-α were involved in the pathogenesis of many inflammatory diseases [15], the stimulation of HaCaT cells with IFN-γ and TNF-α mimics the inflammatory condition, as mentioned before [19]. We found that UVA irradiation could inhibit the IFN-γ- and TNF-α-induced TARC mRNA expression and TARC protein secretion by HaCaT cells, indicating that UVA irradiation might suppress the IFN-γ- and TNF-α-induced TARC-involved inflammation. Our time course experiments also show that the inhibitory effect of UVA irradiation on TARC secretion gradually became weaker and that at about 36 hours after UVA irradiation, the rate of TARC secretion is not significantly different from the controls. Thus, 36 hours after UVA irradiation, the inhibitory effects of UVA irradiation on TARC protein secretion could not countercheck the IFN-γ-and TNF-α-induced TARC secretion by HaCaT cells nor it is likely that the inhibitory effect of UVA irradiation ceased because of the cellular self-repair function.
It has been proven that UVA irradiation affects the immune function [20-23]. One proposed mechanism was the modulation of immuno-modulatory cytokines of keratinocytes [9, 10, 23]. Substantial evidence suggests that UVA irradiation is selective in its modulating of the induction of cytokines, such as interleukin-12, that promotes Th1 responses and inhibits Th2 responses [20, 21]. TARC acts specifically on Th2 lymphocytes and promotes Th2 response. Our findings that UVA irradiation inhibited TARC mRNA expression and TARC protein secretion by HaCaT cells are consistent with previous findings that UVA irradiation modulates Th2 response [21]. Based on our results, we suggest that UVA irradiation affects the human cutaneous immune function, at least in part, by modulating the capacity of keratinocytes to produce TARC. It has been shown that high production of TARC was detected by lesional keratinocytes in atopic dermatitis and that a high level of serum TARC concentration is correlated with the disease severity of atopic dermatitis [6]. In this study, we found that UVA irradiation inhibits TARC mRNA expression and TARC protein secretion by human HaCaT keratinocyte cell lines. Thus, we consider that the inhibitory effects of UVA irradiation on TARC production might be an underlying mechanism responsible for the effectiveness of UVA irradiation on immune modulation.
The mechanism by which UVA irradiation inhibited TARC mRNA expression and TARC protein secretion needs to be investigated. Berlin et al. showed that nuclear factor (NF)-kB participated in epithelial cell TARC mRNA expression [24]. Djavaheri-Mergny M et al reported that UVA could induce a decrease in NF-kB activity in human keratinocytes [25]. Whether UVA inhibited TARC mRNA expression and TARC protein secretion through regulating NF-kB remains to be clarified. Also, we found that the inhibitory effect of UVA irradiation on TARC mRNA expression and the inhibitory effect of UVA irradiation protein secretion which were induced by co-stimulation with IFN-γ-and TNF-α did not parallel each other. This may suggest that multiple points might be affected by UVA irradiation in the process of TARC production. Further research on the underlying mechanism may lead to important findings.
In summary, our data provide the first evidence that UVA inhibits TARC mRNA expression and its protein production by HaCaT cells in a dose-dependent manner. This observation indicates that UVA-irradiation might modulate the skin immune function through regulating TARC production by keratinocytes. < 

Acknowledgements. We thank professor N. E. Fusenig for giving us HaCaT cells. This work was supported in part by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 14570817) and by Health Science Research Grants for Research on Immunology and Allergology from Ministry of Health, Labor and Welfare of Japan.

References

1. Kaplan AP. Chemokines, chemokine receptors and allergy. Int Arch Allergy Immunol 2001; 124: 423-31.

2. Imai T, Baba M, Nishimura M, Kakizaki M, Takagi S, Yoshie O. The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4. J Biol Chem 1997; 272: 15036-42.

3. Vestergaard C, Bang K, Gesser B, Yoneyama H, Matsushima K, Larsen CG. A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA⁺ CCR4⁺ lymphocytes into lesional atopic dermatitis skin. J Invest Dermatol 2000; 115: 640-6

4. Vestergaard C, Yoneyama H, Murai M, Nakamura K, Tamaki K, Terashima Y. Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis-like lesions. J Clin Invest 1999; 104: 1097-10.

5. Panina-Bordignon P, Papi A, Mariani M, Di Lucia P, Casoni G, Bellettato C, Buonsanti C, Miotto D, Mapp C, Villa A, Arrigoni G, Fabbri LM, Sinigaglia F. The C-C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics. J Clin Invest 2001; 107: 1357-64.

6. Kakinuma T, Nakamura K, Wakugawa M, Mitsui H, Tada Y, Saeki H, Torii H, Asahina A, Onai N, Matsushima K, Tamaki K. Thymus and activation-regulated chemokine in atopic dermatitis: Serum thymus and activation-regulated chemokine level is closely related with disease activity. J Allergy Clin Immunol 2001; 107: 535-41.

7. Kawasaki S, Takizawa H, Yoneyama H, Nakayama T, Fujisawa R, Izumizaki M, Imai T, Yoshie O, Homma I, Yamamoto K, Matsushima K. Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice. J Immunol 2001; 166: 2055-62.

8. Kondo S. The roles of keratinocyte-derived cytokines in the epidermis and their possible responses to UVA-irradiation. J Investig Dermatol Symp Proc 1999; 4:177-83.

9. Simon JC, Pfieger D, Schopf E. Recent advances in phototherapy. Eur J Dermatol 2000; 10: 642-5.

10. Clingen PH, Berneburg M, Petit-Frere C, Woollons A, Lowe JE, Arlett CF, Green MH. Contrasting effects of an ultraviolet B and an ultraviolet A tanning lamp on interleukin-6, tumour necrosis factor-alpha and intercellular adhesion molecule-1 expression. Br J Dermatol 2001; 145: 54-62.

11. Kevil CG, Walsh L, Laroux FS, Kalogeris T, Grisham MB, Alexander JS. An improved, rapid Northern protocol. Biochem Biophys Res Commun 1997; 238: 277-9.

12. Garmyn M, Yaar M, Holbrook N, Gilchrest BA. Immediate and delayed molecular response of human keratinocytes to solar-simulated irradiation. Lab Invest 1991; 65: 471-8.

13. Sasaki H, Akamatsu H, Horio T. Protective role of copper, zinc superoxide dismutase against UVB-induced injury of the human keratinocyte cell line HaCaT. J Invest Dermatol 2000; 114: 502-7.

14. Vestergaard C, Kirstejn N, Gesser B, Mortensen JT, Matsushima K, Larsen CG. IL-10 augments the IFN-γamma and TNF-αlpha induced TARC production in HaCaT cells: a possible mechanism in the inflammatory reaction of atopic dermatitis. J Dermatol Sci 2001; 26: 46-54.

15. Farrell AM, Antrobus P, Simpson D, Powell S, Chapel HM, Ferry BL. A rapid flow cytometric assay to detect CD4 + and CD8 + T-helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporin therapy. Br J Dermatol 2001; 144: 24-33.

16. Uchi H, Terao H, Koga T, Furue M. Cytokines and chemokines in the epidermis. J Dermatol Sci 2000; 24: S29-38.

17. Barr RM, Walker SL, Tsang W, Harrison GI, Ettehadi P, Greaves MW, Young AR. Suppressed alloantigen presentation, increased TNF-αlpha, IL-1, IL-1Ra, IL-10, and modulation of TNF-R in UV-irradiated human skin. J Invest Dermatol 1999; 112: 692-8

18. Otto AI, Riou L, Marionnet C, Mori T, Sarasin A, Magnaldo T. Differential behaviors toward ultraviolet A and B radiation of fibroblasts and keratinocytes from normal and DNA-repair-deficient patients. Cancer Res 1999; 59: 1212-8.

19. Rahat MA, Chernichovski I, Lahat N. Increased binding of IFN regulating factor 1 mediates the synergistic induction of CIITA by IFN-γamma and tumor necrosis factor-alpha in human thyroid carcinoma cells. Int Immunol 2001; 13:1423-32.

20. Kondo S, Jimbow K. Dose-dependent induction of IL-12 but not IL-10 from human keratinocytes after exposure to ultraviolet light A. J Cell Physiol 1998; 177: 493-8.

21. Shen J, Bao S, Reeve VE. Modulation of IL-10, IL-12, and IFN-γamma in the epidermis of hairless mice by UVA (320-400 nm) and UVB (280-320 nm) radiation. J Invest Dermatol 1999; 113: 1059-64.

22. Dumay O, Karam A, Vian L, Moyal D, Hourseau C, Stoebner P, Peyron JL, Meynadier J, Cano JP, Meunier L. Ultraviolet A 1 exposure of human skin results in Langerhans cell depletion and reduction of epidermal antigen-presenting cell function: partial protection by a broad-spectrum sunscreen. Br J Dermatol 2001; 144: 1161-8.

23. Grewe M, Gyufko K, Schopf E, Krutmann J. Lesional expression of interferon-gamma in atopic eczema. Lancet 1994;343(8888): 25-6.

24. Berin MC, Eckmann L, Broide DH, Kagnoff MF. Regulated production of the T helper 2-type T-cell chemoattractant TARC by human bronchial epithelial cells in vitro and in human lung xenografts. Am J Respir Cell Mol Biol 2001; 24: 382-9

25. Djavaheri-Mergny M, Gras MP, Mergny JL, Dubertret L. UV-A-induceddecrease in nuclear factor-kappaB activity in human keratinocytes. Biochem J 1999; 338: 607-13.