Ann Dermatol.  2012 Aug;24(3):324-336. 10.5021/ad.2012.24.3.324.

Effect of Spa Spring Water on Cytokine Expression in Human Keratinocyte HaCaT Cells and on Differentiation of CD4+ T Cells

• Lee HP
• Choi YJ
• Cho KA
• Woo SY
• Yun ST
• Lee JT
• Kim HJ
• Lee KH
• Kim JW

• KMID: 2265306
• DOI: http://doi.org/10.5021/ad.2012.24.3.324

Abstract

BACKGROUND
Skin acts as the first line of defense against any foreign materials outside of our body. In inflammatory skin disease, the pathogenesis is due to an immune reaction in the keratinocytes, immune cells and soluble mediators. Balneotherapy is widely used for the treatment of inflammatory skin disease, but the mechanisms are only partly understood by immune regulation. Balneotherapy in dermatologic disease can affect the secretion of pro-inflammatory cytokines, IL-1alpha and tumor necrosis factor from keratinocytes, and possibly affect the T cell differentiation.
OBJECTIVE
In this study, we evaluated the effect of spa spring water from Yong-gung oncheon on the cells, and investigated the skin immune reaction.
METHODS
We investigated the immunomodulatory or anti-inflammatory effect of thermal spring water on the expression of pro-inflammatory cytokines in the HaCaT cells under Toll-like receptor (TLR) stimulation, as well as the effect on the differentiation of CD4+ T cells under spring water.
RESULTS
The treatment of spa spring water from Yong-gung oncheon decreased the expression of proinflammatory cytokines under TLR stimulation to the HaCaT cells and antigen presenting cells. In addition, spa spring water attenuated the differentiation process of subsets of CD4+ T cells, i.e., Th1, Th2 and Th17 cells. All these immune parameters can be used to evaluate the efficacy of spa spring water in Korea, in terms of the immune modulatory effect.
CONCLUSION
Spa spring water treatment suppressed the inflammatory cytokines production and also modulated the differentiation of CD4+ T cells into Th1, Th2, and Th17 cells, but not the Tregs cells.

Keyword

Balneotherapy; CD4+ T cells; Cytokines; HaCaT cells; Skin inflammation

MeSH Terms

Antigen-Presenting Cells
Balneology
Cytokines
Humans
Keratinocytes
Korea
Skin
Skin Diseases
T-Lymphocytes
Th17 Cells
Toll-Like Receptors
Tumor Necrosis Factor-alpha
Fresh Water
Water Purification
Cytokines
Toll-Like Receptors
Tumor Necrosis Factor-alpha
Fresh Water

Figure

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  Fig. 1 Cell culture with spa spring water led to detach HaCaT cells. The HaCaT cells cultured in Dulbeco's Modified Eagle Medium (DMEM, Gibco-BRL, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Gibco-BRL) (A) and DMEM prepared from undiluted spa spring water with 10% FBS (B). Original magnification ×200. Tx: Treatment.

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  Fig. 2 Viability of HaCaT cells was not affected by spa spring water in the 1:32 fold of dilution. The HaCaT cells were plated at a density of 3×104/200 µl in fresh medium and treated with spa spring water as indicated. After 1, 4, 10, and 24 hours of culture, 20 µl of MTT (5 mg/ml in PBS) was added to each of the wells. After 4 hours, dark blue formazan crystals were dissolved, and the plates were read at a wavelength of 570 nm. 100% viability was determined based on the result of MTT assay of normally cultured HaCaT cells. PBS: peripheral blood smear examination.

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  Fig. 3 Schematic diagram of experimental design. (A) The HaCaT cells were pre-incubated for 2 hours with spa spring water at a concentration of 1 : 30 fold dilution, and then stimulated with TLR1 to TLR6 agonist as indicated time. (B) The HaCaT cells were simultaneously exposed to spa spring water and TLR agonist and incubated as indicated time. TLR: toll-like receptor.

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  Fig. 4 Spa spring water impairs pro-inflammatory cytokine production on HaCaT cells under TLR stimulation. The HaCaT cells were treated with TLR1 through TLR6 agonist for 1, 4, 10, and 24 hours, respectively. Spa spring water diluted 1 : 30 fold was added prior to adding TLR agonists or simultaneously. After the indicated time, the supernatants were collected to measure secreted level of IL-6 (A), IL-8 (B), TNF-α (C), IL-1α (D), and GM-CSF (E) via ELISA. The data are expressed as the means±SEM (*p<0.05) of three independent experiments. TLR: Toll-like receptor, IL: interleukin, HKLM: heat-killed Listeria monocytogenes, LPS: lipopolysaccharide, FSL1: Pam2CGDPKHPKSF, TNF: tumor necrosis factor, GM-CSF: granulocyte macrophage-colony stimulating factor, SEM: standard error of the mean.

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  Fig. 5 Spa spring water inhibited the differentiation and proliferation of effector T cells and promoted Treg proliferation. The CD4+ naïve T cells were isolated, labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE, CellTrace™ CFSE Cell Prolifetration kit, Invitrogen, Paisley, UK), and cultured under skewing conditions for Th1, Th2, Th17, and Treg cells, respectively, in the presence or absence of spa spring water. Serially diluted (1 : 3) anti-CD3 antibodies were treated at a concentration starting from 3 µg/ml to monitor dose-dependent proliferation. On day 3, cells were harvested and proliferation was measured by flow cytometry. CD4+ IFN-γ+ Th1 cells (A), CD4+ IL-4+ Th2 cells (B), and CD4+ IL-17+ Th17 cells (C) were proliferated along with the increase of anti-CD3 stimulation, whereas they were inhibited under spa spring water. In contrast, spa spring water promoted proliferation of CD4+Foxp3+ Tregs (D) relative to those of the HaCaT cells cultured alone. Percentages of proliferative cells were represented as precursor frequency via analysis using ModFit LT software (Verity Software House, Topsham, ME, USA) based on the reduction of CFSE positive cells. The data are expressed as the means±SEM (*p<0.05) from three independent experiments. Tx: treatment, IFN: interferon, IL: interleukin, TGF: transforming growth factor, SEM: standard error of the mean.

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  Fig. 6 Spa spring water decreased class II MHC expression and impaired pro-inflammatory cytokine production on APCs under TLR3 stimulation. APCs were isolated and stimulated with 20 µg/ml of poly (I : C) for 24 hours. (A) Class II MHC expression was increased under poly (I : C) treatment and significantly reduced by adding spa spring water. Expressed levels were represented by histogram (left) and quantified as MFI (right). Poly (I : C) induced IL-6 (B) and TNF-α (C) were attenuated under spa spring water treatment. The data are expressed as the means±SEM (*p<0.05) from three independent experiments. APC: antigen presenting cell, TLR: Toll-like receptor, Tx: treatment, MFI: mean fluorescence intensity, IL: interleukin, IFN: interferon, SEM: standard error of the mean.

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