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Involvement of P2Y11 receptor in silica nanoparticles 30-induced IL-6 production by human keratinocytes

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Chihiro Nagakura a , Yusuke Negishi a , Mitsutoshi Tsukimoto a , Satomi Itou b , Takeshi Kondo b , Ken Takeda c , Shuji Kojima a,∗

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Department of Radiation Biosciences, Tokyo University of Science (TUS), 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan Research Institute for Science and Technology, Tokyo University of Science (TUS), 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan c Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science (TUS), 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan b

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Article history: Received 12 February 2014 Received in revised form 12 March 2014 Accepted 30 March 2014 Available online xxx

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Keywords: SNP30 IL-6 production ATP P2Y11 receptor Skin inflammation

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1. Introduction

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We have previously reported that P2Y11 receptor mediates IFN-␥-induced IL-6 production in human keratinocytes, suggesting the importance of purinergic signaling in skin inflammatory diseases. In this study, the involvement of various P2 receptors in IL-6 production induced by silica nanoparticle 30 (SNP30) was examined in a human keratinocyte cell line, HaCaT. Exposure to SNP30 increased IL-6 production in the cells. Ecto-nucleotidase (apyrase), a non-selective antagonist of P2Y receptors (suramin), and a selective P2Y11 receptor antagonist (NF157) all inhibited IL-6 production. Nucleotides such as ATP and UTP themselves also significantly increased IL-6 production in the cells. It was further confirmed that ATP was released from HaCaT cells exposed to SNP30. These results strongly support the possible role of ATP in SNP30-induced IL-6 production by HaCaT cells. In conclusion, these data demonstrate that P2Y11 receptor also mediates SNP30-induced IL-6 production in human keratinocytes, confirming that the ATP-P2Y11 purinergic signaling is a common pathway leading to induction of skin inflammatory diseases. © 2014 Elsevier Ireland Ltd. All rights reserved.

Various types of nanoparticles (NP) with novel electrical, catalytic, magnetic, mechanical, photonic and thermal properties have been developed and are already being used or tested in a wide range of consumer products, such as sunscreens, composites, and medical and electric devices. Specific properties of NP, such as their small size, shape, high surface area, and special structure, make these compounds promising candidates in both industries and bio´ logical applications (Luo et al., 2006; Yang et al., 2008; Uskokovic, 2013; Zhang et al., 2013). However, in recent years, there has been increasing evidence of the adverse effects of NP (Aillon et al., 2009; Crosera et al., 2009; Donaldson et al., 2005; Medina et al., 2007; Xia et al., 2006). In the face of using NP, assessment of human health and environmental safety is now urgently required. On commercial available NP, such as silica, titanium oxide (TiO2 ), silver, chrysotile asbestos, carbon nanotubes, and some magnetic particles, their

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∗ Corresponding author. Tel.: +81 47121 3613; fax: +81 47121 3613. E-mail address: [email protected] (S. Kojima).

biosafety and cytotoxicity have been now extensively investigated in vitro and in vivo experimental systems. Silica NP (SNP), non-metal oxides, have been found extensive applications as additives such as cosmetics, medical supplies, the toner of the printer, the food, in various fields. Meanwhile, it is also concerned that a particle is uptaken by the living body in a process to use as a manufacturing process and a product, and unfavorable influence to the human body (Napierska et al., 2010). The skin is composed of epidermis, dermis, and subcutaneous tissue, and serves to protect internal organs from potentially hazardous external factors, including mechanical stimuli, chemical stimuli, ultraviolet (UV) radiation, bacteria and so on. In the outer epidermal layer, about 95% of the cells are keratinocytes which produce various cytokines, such as interleukin-1 (IL-1), IL-6, IL-8, tumor necrosis factor-alpha (TNF-␣), transforming growth factorbeta (TGF-␤), platelet-derived growth factor, and chemokines (Ansel et al., 1990; Elder et al., 1989; Kock et al., 1990; Neuner et al., 1991; Schwarz and Luger, 1989). Production of these cytokines increases in response to environmental stimuli and injury (Kirnbauer et al., 1991; Kondo et al., 1993; Neuner et al., 1991). IL-6, a well-studied inflammatory cytokine that plays a central role in immune responses, is produced by T cells, macrophages,

http://dx.doi.org/10.1016/j.tox.2014.03.010 0300-483X/© 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Nagakura, C., et al., Involvement of P2Y11 receptor in silica nanoparticles 30-induced IL-6 production by human keratinocytes. Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.03.010

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fibroblasts, keratinocytes, and endothelial cells. In keratinocytes, IL-6 induces proliferation and migration, thereby promoting skin repair (Bowman et al., 1997) and leading to skin diseases, such as psoriasis (Kawakami et al., 1997). UV is one of the hazardous external factors and produces IL-6 in the keratinocytes, resulting in thermal damage (Gallucci et al., 2004; Urbanski et al., 1990). ATP is used as an energy source in cells, but extracellular ATP also acts as an intercellular signaling factor (Burnstock, 1997). Extracellular ATP is degraded to ADP, AMP, and adenosine by ectonucleotidases (Lazarowski et al., 2000). Purinergic receptors are receptors of these nucleotides, and are expressed on the membrane of cells. Activation of purinergic receptors induces various physiological effects (Dubyak and el-Moatassim, 1993). Purinergic receptors are categorized into adenosine receptor P1 receptors and ATP or ADP receptor P2 receptors. P2 receptors are classified into the ionotropic P2X1-7 receptor subtypes and the G-protein coupled P2Y1-14 receptor subtypes (Abbracchio and Burnstock, 1994). Purinergic receptors are expressed in human keratinocytes (Pastore et al., 2006), though the expression pattern of purinergic receptor subtypes depends on the grade of keratinocyte differentiation (Burrell et al., 2003; Dixon et al., 1999; Greig et al., 2003; Koegel and Alzheimer, 2001). Cells, including keratinocytes, release ATP in response to various stimuli, such as mechanical stimuli, hypoosmotic stimuli, and radiation (Azorin et al., 2011; Koizumi et al., 2004; Mizumoto et al., 2003). The mechanisms of this release include a maxi-anion channel, P2X7 receptor/pore, a volume-sensitive outwardly rectifying chloride channel, a member of the ATP-binding cassette protein family, a gap junction hemichannel, and vesicular exocytosis (Fitz, 2007; Hisadome et al., 2002; Ohshima et al., 2010; Pankratov et al., 2006; Pellegatti et al., 2005; Sabirov et al., 2001; Sprague et al., 1998; Stout et al., 2002). ATP released from cells via these pathways activates P2 receptors in an autocrine or a paracrine manner (Corriden and Insel, 2010). Recently, it has been found that extracellular ATP induces IL-6 production via P2Y receptors (Inoue et al., 2007; Ishimaru et al., 2013; Yoshida et al., 2006). Moreover, it has already been reported that SNP produces IL-6 by keratinocytes under UV exposure, but not in the dark condition (Koizumi et al., 2004). However, the mechanism of cytokine-induced IL-6 production by keratinocytes is not fully understood. In this study, we investigated the involvement of extracellular ATP and various P2 receptors in IL-6 production induced by 30-nanometer silica nanoparticle (SNP30) in a human keratinocyte cell line, HaCaT.

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2. Materials and methods

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2.1. Reagents

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SNP of diameters with 30, 70, and 300 nm (designated as SNP30, SNP70 and SNP300) were purchased from Micromod Partikeltechnologie GmbH). Dulbecco’s modified Eagle’s medium containing 1.0 g glucose/l (low-glucose type DMEM), penicillin, streptomycin, dithiothreitol (DTT), and polyoxyethylene sorbitan monolaurate (equivalent of Tween-20) were purchased from Wako Pure Chemical Industries (Osaka, Japan). ATP, UTP, and apyrase were from Sigma–Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was from Biowest (Nuaillé, France). The broadspectrum P2X antagonist pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), the P2Y12 antagonist clopidogrel and the broad-spectrum P2Y antagonist suramin sodium salt (suramin) were obtained from Sigma–Aldrich (St. Louis, MO). The P2X7 antagonist N-[2-[[2-[(2-hydroxyethyl)amino]ethyl]amino]-5-quinolinyl]-2-tricyclo-[3.3.1.13,7]-dec-1-ylacetamide dihydro-chloride (AZ10606120), the P2Y1 antagonist 2 -deoxy-N-6-methyladenosine 3 ,5 -bisphosphate tetrasodium salt (MRS2179), the P2Y6 antagonist N,N -1,4-butanediylbis[N -(3-isothioc yanatophenyl)thiourea (MRS2578), the P2Y13 antagonist 2-[(2-chloro-5-nitrophenyl)azo]-5-h ydroxy-6-methyl-3[(phosphonooxy)-methyl]-4-pyridine carboxaldehyde disodium salt (MRS2211), and the P2Y11 antagonist 8,8 -[carbonylbis[imino-3,1-phenylene carbonylimino (4-fluoro-3,1-phenylene) carbonyl-imino]] bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF157) were from Tocris Bioscience (Ellisville, MO). All other chemicals used were of the highest purity available.

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2.2. Characterization of silica NPs

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The particle size distribution of SNP30, SNP70, and SNP300 in DMEM was evaluated by dynamic light-scattering (DLS), using NICOMP 370 (Particle Sizing System, Co., Tokyo Japan). Briefly, 2.5 mg of each SNP was suspended in 1 mL low-glucose type DMEM, mixed thoroughly with vortex mixture, and immediately subjected to DLS analysis at room temperature. Morphology of SNP30 prepared by the same method was assessed at room temperature by transmission electron microscopy (TEM), using a JEOL JEM 2010 (JEOL Ltd., Tokyo, Japan). SNP30 suspended in low-glucose type DMEM was dropped on the 400 mesh grid coated with carbon membrane, kept for 2 h at 37 ◦ C, and then dried. The dried SNP30 was observed by TEM.

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2.3. Reverse transcription polymerase chain reaction (RT-PCR)

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Total RNA was isolated from HaCaT cells using a Relia Pre RNA cell Minipre System kit (Promega Co., WI). The first-strand cDNA was synthesized from total RNA with PrimeScript Reverse Transcriptase (Takara Bio). Specific primers were designed with PrimerQuestTM (Integrated DNA Technologies, Inc., Coralville, IA) and synthesized by Sigma Genosys. The sequences of specific primers used in this study are shown in Table 1. GAPDH mRNA was determined as a positive control. PCR was carried out by incubating each cDNA sample with the primers (0.5 M each), PrimeSTAR HS DNA Polymerase (1.25 units, Takara Bio), and a deoxynucleotide mixture (0.2 mM each, Takara Bio). After the samples were incubated at 95 ◦ C for 2 min, amplification was carried out for 35 cycles (each cycle: 95 ◦ C for 30 s, annealing at 65 ◦ C for 1 min) and incubated at 72 ◦ C for 10 min. The products were then subjected to 2% agarose gel electrophoresis. Bands were stained with ethidium bromide and photographed.

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2.4. Assay of extracellular ATP

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Cells were exposed to SNP30 and the extracellular ATP concentration was measured using ENLITEN rLuciferase/Luciferin Reagent (Promega, Madison, WI). At the indicated time points, each sample was centrifuged at 600 × g for 1 min and 10 ␮L of the supernatant was collected for ATP determination. The concentration of ATP was determined by measuring chemiluminescence with a Wallac 1420 ARVO fluoroscan (Wallac, Turku, Finland) after adding 100 ␮L of luciferin-luciferase reagent to 10 ␮L of sample solution.

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2.5. Assay of IL-6, TNF-˛, and IFN-

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HaCat cells were stimulated with SNPs and incubated. The culture supernatants were collected and measured the concentrations of IL-6 and TNF-␣ by enzymelinked immunosorbent assay as mentioned below. The cells were exposed to various doses of SNP30 for 24 h. The culture supernatants were collected and the concentration of TNF-␣ was measured by enzyme-linked immunosorbent assay. In another experiment, the cells were pre-incubated with apyrase (5 U/ml) for 10 min, and PPADS (100 ␮M), AZ10606120 (10 ␮M), suramin (100 ␮M), MRS2179 (100 ␮M), MRS2578 (10 ␮M), NF157 (50 ␮M), clopidogrel (30 ␮M), or MRS2211 (100 ␮M) for 30 min, and further incubated for 24 h, and the concentration of IL-6 in supernatants was assayed. A 96-well plate was coated with purified anti-human IL-6 or TNF-␣ mAb (1:500) (eBioscience) and incubated overnight at 4 ◦ C. The plate was washed with PBS containing 0.05% Tween 20. Phosphate-buffered saline containing 1% BSA was added to the plate and the plate was incubated at room temperature to block the non-specific binding. The plated was washed, and added the supernatants were added for 2 h at room temperature. After washing, biotin-conjugated anti-human IL-6 mAb (1:1000) or TNF-␣ mAb (1:500) (eBioscience) was added for 1 h room temperature. The plate was washed, and added avidin-horseradish peroxidase (Sigma) for 30 min at room temperature. The plate was further washed, and added 3,3 ,5,5 -tetramethylbenzidine for a few minutes to stain. The reaction was stopped by adding 5 N H2 SO4 . The absorbance at 450 nm was measured with a Wallac 1420 ARVO fluoroscan (Wallac, Turku, Finland). The concentration of IFN␥ was measured using Quantikine ELISA kit for human IFN-␥ (R&D Systems, Inc., MN, USA) following manufacturer’s instructions. A standard curve was established with recombinant human IL-6 (15.6–1000 pg/mL), IFN-␥ (15.6–1000 pg/mL) or TNF␣ (15.6–1000 pg/mL) (eBioscience), and the concentration of IL-6 or TNF-␣ was estimated from the standard curve.

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2.6. Cell viability

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A colorimetric assay with WST-1 reagent was used to evaluate cell viability in accordance with the manufacturer’s directions. HaCaT cells (1 × 105 /mL) were exposed to 5 ␮g/cm2 of SNP30 for 24 h. Then 10 ␮l of WST-1 reagent, diluted 4fold in phosphate buffer, was added to each well and incubation was continued for another 4 h at 37 ◦ C. The absorbance of the wells at 450 nm was measured with a plate reader. Relative cell viability was calculated as a percentage of the control group, to which SNP30 had not been added.

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Please cite this article in press as: Nagakura, C., et al., Involvement of P2Y11 receptor in silica nanoparticles 30-induced IL-6 production by human keratinocytes. Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.03.010

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Table 1 Primers of P2 receptors. Gene

Primers (forward and reverse)

Accession number in GenBank

Product size (bp)

P2X1

5 -CCAGCTTGGCTACGTGGTGCAAGA-3 5 -ACGGTAGTTGGTCCCGTTCTCCACAA-3 5 -CCCGAGAGCATAAGGGTCCACAAC-3 5 -AATTTGGGGCCATCGTACCCAGAA-3 5 -CCCCTCTTCAACTTTGAGAAGGGA-3 5 -GTGAAGGAGTATTTGGGGATGCAC-3 5 -CCTTCCCAACATCACCACTACTTACC-3 5 -AGGAGATACGTTGTGCTCAACGTC-3 5 -AGCACGTGAATTGCCTCTGCTTAC-3 5 -ATCAGACGTGGAGGTCACTTTGCTC-3 5 -GGTTTCCGTCACTCAGATCAAGG-3 5 -GGCACCAACTCCAGATCTCAC-3 5 -CTGCTCTCTTGAACAGTGCCGAAA-3 5 -AGTGATGGAACCAACGGTCTAGGT-3 5 -ACCTCAGACGAGTACCTGCGAAGT-3 5 -AGAATGGGGTCCACACAACTGTTGAG-3 5 -GTGTCTGGGCGTCTTACGACCTCT-3 5 -AGAATGGGGTCCACACAACTGTTGAG-3 5 -GTGTCCTTTTCCTCACCTGCATCA-3 5 -ACGAGCCATGAGTCCATAGCAAAC-3 5 -TTCAGGCTGAGGAGATGGGT-3 5 -GCCAGAGCAAGGTTTAGGGT-3 5 -AGAAGCTGCGTGTGGCAGCGTTGGT-3 5 -ACGGTTTAGGGGCGGCTGTGGCATT-3 5 -GGAACAGGACCACTGAGAAC-3 5 -TCATGCCAGACTAGACCGAA-3 5 -CCTTTCAAAATCCTCTCTGACTC-3 5 -TCCTTGTTGCTCAAGATCGT-3 5 -CTCTGCCGTGCTCTTCTACGTCAA-3 5 -TTAATGCTTTGTGCCACTTCCGT-3 5 -GAAGGTGAAGGTCGGAGTC-3 5 -GAAGATGGTGATGGGATTTC-3

U45448

226

AF190823

208

NM002559

245

U85975

256

AF016709

183

AF065385

290

Y09561

270

NM002563

353

NM176072

215

NM002565

311

HSU52464

286

AY449733

369

AF313449

302

NM023914

266

NM014879

275

NM002046

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P2X2 P2X3 P2X4 P2X5 P2X6 P2X7 P2Y1 P2Y2 P2Y4 P2P6 P2Y11 P2Y12 P2Y13 P2Y14 GAPDH

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2.7. Statistics

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Results are expressed as the mean ± S.E.M. The statistical significance of differences between two groups was calculated by using the unpaired Student’s t-test. The statistical significance of differences between control and other groups was calculated by using Dunnett’s test. The criterion of significance was P < 0.05 as determined with the Instat version 3.0 statistical package (Graph Pad Software, San Diego, CA).

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3. Results

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3.1. Characterization of SNPs

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Since NPs often agglomerates in culture medium, the average sizes of SNP30, SNP70, and SNP300, suspended in low-glucose type DMEM, were analyzed by DSL. Furthermore, the form of SNP30 in the low-glucose type DMEM were observed TEM. As shown in Fig. 1A–C, the average particle diameters of SNP30, SNP70 and SNP300 were 35.1, 65.6 and 278.7 nm, respectively, in the medium. TEM micrographs revealed that the majority of SNP30 had the primary particle size of less than