Differentiation of skin sensitizers from irritant chemicals by interleukin-1α and macrophage inflammatory protein-2 in murine keratinocytes

Toxicology Letters 216 (2013) 65–71

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Differentiation of skin sensitizers from irritant chemicals by interleukin-1␣ and macrophage inflammatory protein-2 in murine keratinocytes Dain Son a , Yirang Na a , Wan-Seob Cho b , Byoung-Hee Lee c , Yong Heo d , Jae-Hak Park e , Seung Hyeok Seok a,∗ a

Department of Microbiology and Immunology, Institute of Endemic Disease, College of Medicine, Seoul National University, Seoul 110-799, South Korea Department of Medicinal Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, South Korea c Division of Biological Resources Coordination, National Institute of Biological Resources, Incheon 404-708, South Korea d Department of Occupational Health, College of Natural Sciences, Catholic University of Daegu, Daegu 712-702, South Korea e Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 121-742, South Korea b

h i g h l i g h t s  In vitro alternative method for identifying skin sensitizers is proposed.  Skin sensitizer can be distinguished by IL-1␣ and MIP-2 level.  HEL30 is a reliable “in vitro” cell system for detecting skin sensitizers.

a r t i c l e

i n f o

Article history: Received 6 April 2012 Received in revised form 23 October 2012 Accepted 25 October 2012 Available online 22 November 2012 Keywords: HEL-30 Interleukin-1␣ Irritant Macrophage inflammatory protein-2 Murine keratinocyte Skin sensitizer

a b s t r a c t The development of novel alternative testing methods is required to identify the sensitizing capacity of chemicals as a replacement for animal experimentation. We aimed to evaluate in vitro assays as screening tools for detecting skin sensitizers. The murine epidermal keratinocyte cell line HEL-30 was exposed to 16 relevant skin sensitizers and 6 skin irritants. The dose causing 75% cell viability (CV75 ) measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was chosen as a highest dose and three more doses (0.5×, 0.1×, and 0.01× of CV75 ) were tested. As parameters, levels of interleukin 1␣ (IL-1␣), macrophage inflammatory protein 2 (MIP-2), IL-6, and IL-18 production were measured using 4 different doses. The accuracy of detecting sensitizers or irritants by IL-1␣ or MIP-2 alone was exactly same: 75% (12 out of 16) for sensitizers, 83% (5 out of 6) for irritants, and overall 77% (17 out of 22). However, combination of IL-1␣ and MIP-2 showed better accuracy: 94% (15 out of 16), 67% (4 out of 6), and overall 86% (19 out of 22). IL-6 and IL-18 could not differentiate sensitizers from irritants. This study suggests that the combination of pro-inflammatory cytokines IL-1␣ and MIP-2 in murine HEL-30 cells can be a reliable in vitro method for identifying chemicals that may act as skin sensitizers. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Identification or categorization of sensitizing chemicals to skin is an important process in chemical risk assessment. Current classifications of skin sensitizers have mostly been achieved using the mouse local lymph node assay (LLNA) and guinea pig tests, including the Buehler test or guinea pig maximization test (GPMT) (Kimber et al., 1995; Kimber and Weisenberger, 1989;

∗ Corresponding author at: Department of Microbiology and Immunology, Institute of Endemic Disease, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul 110-799, South Korea. Tel.: +82 2 740 8302; fax: +82 2 743 0881. E-mail address: [email protected] (S.H. Seok). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.10.017

Magnusson and Kligman, 1969). Meanwhile, the European Union Council announced that animal experimentations for the toxicity testing of cosmetic ingredient would be completely banned in Europe beginning in 2013 (Taylor et al., 2011). Thus, an increasing effort exists to develop alternative in vitro skin sensitization tests to replace animal experimentations. Currently, reconstituted human skin models are considered to be the most attractive alternative methods, but still have some economic and reliability problems (Aardema et al., 2010; Berna et al., 1998; Kandarova et al., 2009). Therefore, more convenient and cost-effective in vitro alternative methods for identifying skin sensitizers must be developed as large-scale preliminary screening tests in a weight of evidence. Skin is the largest immune organ in the human body. This integumentary system guards the body from various microbes as

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well as exogenous chemicals, including irritants and sensitizers. The major constituent of skin is the keratinocyte, which makes up almost 95% of the total cell mass of the human epidermis (Barker et al., 1991). Keratinocytes are not only the first targets for stimuli, but also act as a local signal transducer, producing systemic cytokine and chemokine responses (Corsini and Galli, 2000; Enk and Katz, 1992; Grone, 2002; Kupper et al., 1986). In response to exogenous stimuli, keratinocytes produce pro-inflammatory cytokines such as interleukin 1 (IL-1), IL-6, IL-8, IL-18, and tumor necrosis factor (TNF) (Ansel et al., 1990; Grone, 2002; Luger and Schwarz, 1990; McKenzie and Sauder, 1990). The anatomical location of keratinocytes and their ability to produce pro-inflammatory cytokines justifies the use of these cells to assess the sensitizing potency of chemicals (Van Och et al., 2005). Recently, the use of murine and human keratinocytes in combination with proinflammatory cytokines release such as IL-1␣, IL-18, and TNF-␣ have been suggested as a preliminary screen method for identification of chemical allergens or irritants (Ansel et al., 1990; Corsini and Galli, 2000; Grone, 2002; Haas et al., 1992; Luger and Schwarz, 1990; McKenzie and Sauder, 1990; Van Och et al., 2005). Among cytokine markers, the selective induction of intracellular IL-1␣ in response to chemical allergens has been well studied using murine epithermal keratinocyte cell line HEL-30 (Corsini et al., 1998). These results are partially consistent with in vivo studies using BALB/c mice that have examined total epidermal IL-1␣, IL-1␤, and IFNinduced protein 10 (IP-10) mRNA expression levels. The primary source of these cytokines was also revealed to be keratinocytes in the mouse epidermis (Corsini and Galli, 2000; Enk and Katz, 1992; Haas et al., 1992). Macrophage inflammatory protein-2 (MIP-2), a murine functional equivalent of human IL-8, effects the activation and movement of epidermal Langerhans cells and inflammatory cells during the induction phase of allergic contact dermatitis (Enk and Katz, 1992). In the present study, we focused on evaluating

the combination of IL-1␣ and MIP-2 in the murine epidermal keratinocyte cell line HEL-30 as a reliable in vitro skin sensitization test that can replace animal experimentation. 2. Materials and methods 2.1. Chemicals Total 16 relevant skin sensitizers and 6 skin irritants were tested and the information of each chemical was indicated in Table 1.

2.2. Cell culture The C3H mouse-derived keratinocytes cell line HEL-30 was kindly provided by the German Cancer Institute (Heidelberg, Germany). HEL-30 cells were cultured in Dulbecco’s modified Eagle medium, supplemented with 10% heated-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% penicillin–streptomycin (Gibco, Life Technologies, Carlsbad, CA, USA), 1% non-essential amino acids (Gibco, Life Technologies). Cells were cultured at 37 ◦ C in a humidified atmosphere with 5% CO2 in air. For the in vitro experiment, chemicals were dissolved in dimethyl sulfoxide (DMSO; the maximum concentration of DMSO in the culture medium was 0.0128%) and phosphate-buffered saline (PBS). All in vitro tests were performed according to the Organization for Economic Cooperation and Development Test Guideline 429 (OECD TG 429).

2.3. Determination of the 75% cell viability (CV75 ) for HEL-30 cells To determine the treatment doses, the 75% of cell viability (CV75 ) was assessed using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay (Gerlier and Thomasset, 1986). HEL-30 cells were seeded in a 96-well plate at a concentration of 1.5 × 104 cells/well and incubated overnight. Cell medium was then replaced with fresh medium containing increasing concentrations of test chemicals or DMSO (0.01%) as a vehicle control. At 24 h after incubation, 10 ␮l of MTT solution (5 mg/ml) in culture medium was added. Cells were then incubated further 4 h at 37 ◦ C. Thereafter, medium was discarded and cells were lysed in 100 ␮l DMSO. The absorbance of the resulting solutions was read at a wavelength of 560 nm in a VICTORTM X3 micro-plate reader (Perkin-Elmer, Waltham, MA, USA). The CV75 was calculated for each chemical by five-parameter non-linear regression analysis of data.

Table 1 Concentration of chemicals that induced 75% cell viability (CV75 ) of HEL-30 cells and their classification.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Chemical

CV75 (␮g/ml)

CAS No.a

Supplier

Vehicle

LLNAb

GPMT/BTc

Humand

CMI/MIe DNCB 4-Phenylenediamine Cobalt chloride Isoeugenol 2-Mercaptobenzothiazole Citral HCA Eugenol Phenyl benzoate Cinnamic alcohol Imidazolidinyl urea Methyl methacrylate Chlorobenzene Isopropanol Lactic acid Methyl salicylate Salicylic acid Sodium lauryl sulfate Ethylene glycol dimethacrylate Xylene Nickel chloride

0.00015% 1.7 ± 148.0 ± 73.0 ± 318.1 ± 370.9 ± 4.9 ± 135.0 ± 76.6 ± 3429.8 ± 606.7 ± 85.7 ± 5000 2031.6 ± 5000 705.9 ± 5000 2656.2 ± 640.5 ± 116.9 ± 5000 138.2 ±

26172-55-4/2682-20-4 97-00-7 106-50-3 7791-13-1 97-54-1 149-30-4 5392-40-5 101-86-0 97-53-0 93-99-2 104-54-1 39236-46-9 80-62-6 108-90-7 67-63-0 50-21-5 119-36-8 69-72-7 151-21-3 97-90-5 1330-20-7 7718-54-9

DOW Aldrich Sigma Sigma Aldrich Aldrich Aldrich Aldrich Fluka Aldrich Aldrich Aldrich Aldrich Sigma–Aldrich Amresco Aldrich Sigma–Aldrich Sigma Sigma Aldrich Sigma–Aldrich Aldrich

PBS DMSO DMSO PBS DMSO DMSO DMSO DMSO DMSO DMSO PBS PBS DMSO DMSO PBS PBS DMSO DMSO DMSO DMSO DMSO DMSO

+ + + + + + + + + + + + + − − − − − + + + −

+ + + + + + + + + + + + + − − − − − − −

+ + + + + + + + + + + + +

0.3 56.4 15.0 161.2 231.4 4.1 54.4 10.5 330.4 85.3 4.9 408.5 215.7 806.8 88.7 50.0 15.1

f

+

g

+ g

− − − + − +

Confluent cells were treated for 24 h with different chemicals or DMSO as the vehicle control. Results are expressed as means ± SD (␮g/ml) of the concentration of chemical resulting in 75% cell viability, as assessed by MTT reduction. CV75 was calculated by five-parameter non-regression analysis data. DMSO, dimethyl sulfoxide; CMI/MI, 5-chloro-2-methyl 4-isothiazolin-3-one (CMI)/2-methyl-4-isothiazolin-3-one (MI). DNCB, 2,4-dinitrochlorobenzene; HCA, hexyl cinnamic aldehyde. a CAS No. (chemical abstracts service number). b Testing results using the LLNA (local lymph node assay) and data taken from OECD test guideline (TG406, TG429). c Testing results using the GPMT (guinea pig maximized test) and data taken from OECD test guideline (TG406, TG429). d Testing results using the BT (Buehler test) and data taken from OECD test guideline (TG406, TG429). e Commercially available as Kathon CG, which is a 3:1 mixture of CMI and MI. f GP data not available. g Presumed to be a non-sensitizer in humans based on OECD TG429.

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2.4. Basal cytokine production

2.6. Statistical analysis

To assess basal IL-1␣ and MIP-2 production, cells were seeded in 24-well plates at concentrations of 0.5 × 105 , 1 × 105 , 2.5 × 105 , and 5 × 105 cells/ml (500 ␮l total volume/well), and were then treated with chemicals for 24, 48, 72, and 96 h. The reference sensitizer, 2,4-dinitrochlorobenzene (DNCB, 1.7 ␮g), was treated to identify optimal experimental condition. After treatment, supernatants were carefully collected and stored at −80 ◦ C for extracellular cytokine release. The attached cells were lysed in lysis buffer (Cell Signaling, Danvers, MA, USA) and stored at −80 ◦ C for intracellular cytokine release. The cytokine levels were measured using a Duoset enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA).

All experiments were performed at least three times and the data presented are expressed as the mean ± standard error of the mean (SEM). Differences between the average values of three different duplicated experiments were assessed by a Student’s t-test as indicated in the figure legends. P < 0.05 was considered to be statistically significant.

2.5. Cytokine measurement To assess IL-1␣ and MIP-2 production, cells were seeded in 24-well plates at a concentration of 1 × 105 cells/ml (500 ␮l total volume/well). At 24 h post incubation, culture medium was discarded and cells were treated with 1 ml of 0.01, 0.1, 0.5, and 1× CV75 concentration of each chemical and 0.01% DMSO as vehicle control (the highest DMSO concentration was 0.0128% in 1× CV75 HCA). After 24 h of incubation, cytokines were measured in supernatants or cell lysates using a Duoset ELISA kit (R&D Systems). The protein content of the cell lysates was measured using the BCA protein assay method (Thermo Scientific, Rockford, IL, USA). The stimulation index (SI) was calculated as follows:

SI =

cytokine produced in chemical-treated cells cytokine produced in vehicle control

3. Results 3.1. Determination of 75% cell viability (CV75 ) The CV75 data for the sensitizer and irritants were shown in Table 1. Methyl methacrylate, isopropanol, methyl salicylate and xylene showed no cytotoxicity at 5000 ␮g/ml. 3.2. Determination of cell density and adhesion time for basal cytokine production in HEL-30 cells IL-1␣ was detected in cell lysates at 3–37-fold higher concentrations than in supernatants (Fig. 1A). By contrast, MIP-2 was detected in supernatants at 31–125-fold higher concentrations than in lysates (Fig. 1B). Cells showed an optimal 90–95% confluency at 24 h after seeding at 1 × 105 cells/ml and the expression

Fig. 1. Effect of cell density and incubation time on intracellular and extracellular cytokine production. Interleukin 1␣ (IL-1␣; A) and macrophage inflammatory protein 2 (MIP-2; B). HEL-30 cells were seeded in 24-well plates at different densities (0.5–5 × 105 cells/ml) and different times (24–96 h). 2,4-Dinitrochlorobenzene (DNCB, 1.7 ␮g), the reference sensitizer was treated to identify optimal experimental condition; IL-1␣ (C) and MIP-2 (D). Results are expressed as means ± SEM of three duplicated independent experiments.

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Table 2 The relative interleukin-1␣ (IL-1␣) production rate by HEL-30 cells at 24 h after the initiation of chemical exposure. Chemicals

IL-1␣ production rate (means ± SEM)a 0.01 × CV75

CMI/MI DNCB 4-Phenylenediamine Cobalt chloride Isoeugenol 2-Mercaptobenzothiazole Citral HCA Eugenol Phenyl benzoate Cinnamic alcohol Imidazolidinyl urea Methyl methacrylate Chlorobenzene Isopropanol Lactic acid Methyl salicylate Salicylic acid Sodium lauryl sulfate Ethylene glycol dimethacrylate Xylene Nickel chloride

183.3 315.8 214.0 151.3 226.6 223.4 67.0 253.3 75.4 444.1 108.9 184.5 188.0 192.3 85.6 178.4 373.0 72.6 86.8 167.1 125.3 63.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

37.5 141.4 27.1* 28.4 35.1* 43.6* 3.7 52.7* 17.2 101.9* 21.0 28.5* 6.9*** 12.1** 10.2 27.9* 72.2* 14.7 18.0 29.9 1.3*** 10.4*

0.1 × CV75 445.2 238.6 270.6 271.2 216.3 240.6 303.7 254.0 435.9 444.5 108.1 184.5 193.6 193.2 94.5 141.0 329.3 217.7 83.0 211.9 372.9 81.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

91.8* 75.7 37.1* 62 12.3*** 29.6** 62.3* 59.3* 98.7* 95.3* 13.8 28.1* 11.2** 11.1** 13.4 18.4* 28.6** 43.2 17.2 3.9*** 2.9*** 9.1

0.5 × CV75 86.6 288.4 253.6 358.3 230.5 326.3 75.4 939.5 438.8 349.8 200.1 174.6 224.3 223.2 47.9 186.2 398.6 292.2 355.5 228.0 114.9 65.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

17.9 88.3 20.5** 89.8* 41.7* 4.1*** 15.4 460.7 87.9* 138.0 39.1 23.7* 12.6*** 17.6** 11.2* 40.9 141.7 50.4* 137.5 21.4** 38.6 15.3

1 × CV75 791.2 308.6 146.7 127.8 346.3 209.1 562.6 403.7 120.9 81.5 398.5 199.6 195.0 246.3 46.0 213.2 701.2 289.8 474.0 215.5 200.4 71.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

167.2* 91.4* 14.9* 34.9 107.1 31.1* 110.4* 114.8* 22.9 4.9*** 183.6 79.5 10.4*** 38.5* 5.1* 13.5*** 258.3 66.3* 174.3 10.9*** 69.4 17.9

CMI/MI, 5-chloro-2-methyl 4-isothiazolin-3-one (CMI)/2-methyl-4-isothiazolin-3-one (MI); DNCB, 2,4-dinitrochlorobenzene; HCA, hexyl cinnamic aldehyde. Vehicle control – 5% cDMEM or 0.1% DNCB. a The total (intracellular and extracellular) IL-1␣ production rate, expressed as the percentage relative to control cells, was determined by dividing the amount of IL-1␣ (pg/ml) produced by chemically treated cells by the amount of IL-1␣ produced by vehicle control-treated cells and multiplying by 100 for the relative percent expression. Data are expressed as the means ± SEM of three duplicated independent experiments. * Significantly different from the vehicle control: p < 0.05. ** Significantly different from the vehicle control: p < 0.01. *** Significantly different from the vehicle control: p < 0.001.

level of IL-1␣ and MIP-2 was the highest at 48 h after treatment of the reference sensitizer, 1.7 ␮g DNCB (Fig. 1C and D).

of 22). However, combination of IL-1␣ and MIP-2 showed better accuracy: 94% (15 out of 16), 67% (4 out of 6), and overall 86% (19 out of 22) based on LLNA and GPMT results (Table 4).

3.3. IL-1˛ production following chemical treatment Intracellular or extracellular IL-1␣ production by HEL-30 cells following incubation with chemicals were investigated. 12 out of 16 chemicals were categorized as skin sensitizers by the increased total IL-1␣ production (Table 2). However, 4-phenylene diamine (p-PDA), imidazolidinyl urea, methyl methacrylate (MMA), ethylene glycol dimethacrylate (EGDMA) failed to be categorized as skin sensitizers (Table 2). 3.4. MIP-2 production following chemical treatment 12 out of 16 sensitizers categorized as skin sensitizers by the significantly increased total MIP-2 production (Table 3). However, cobalt chloride, isoeugenol, MMA and sodium lauryl sulfate failed to be categorized as skin sensitizers (Table 3). Additionally, neither IL-18 nor IL-6 did discriminate sensitizers from irritants (data not shown). 3.5. Classification of tested chemicals as sensitizers or non-sensitizers based on the SI The SI represents the threshold for determining whether tested chemicals have sensitizing abilities. According to the OECD TG 429, chemicals are considered to be sensitizers when SI is over 3. To assess chemicals as skin sensitizers in the present study, we selected the highest production rates of IL-1␣ and MIP-2 among the four different CV75 points and the data were graphed as shown in Fig. 2. The accuracy of detecting sensitizers or irritants by IL-1␣ or MIP-2 alone was exactly same: 75% (12 out of 16) for sensitizers, 83% (5 out of 6) for irritants, and overall 77% (17 out

4. Discussion Enormous efforts have been made to develop an alternative in vitro method for the skin-sensitizing test, which is consistent with the 3Rs (Replacement, Reduction, and Refinement) for animal experimentation. The present study introduced a very reliable in vitro method using a combination of the pro-inflammatory cytokines IL-1␣ and MIP-2 on the well-established murine keratinocyte cell line HEL-30. The experimental conditions were optimized to clarify the cell density and attaching time issue. Because the secretion pattern was different from those cytokines, the total (intracellular and extracellular) IL-1␣ and MIP-2 production levels were investigated in murine keratinocytes after exposure to 16 skin sensitizers and 6 irritants. The HEL-30 cell line has several advantages as compared with animal and human experimentation, such as its application in the GPMT, LLNA, patch test of Buehler, human repeated insult patch test (HRIPT), 4 h human patch test (HPT), and mouse ear swelling test (MEST), which have been internationally adopted as an in vivo method for the evaluation of skin sensitizers (Basketter et al., 1994, 2004, 2005; Dunn et al., 1990; Gafner et al., 1988; Garrigue et al., 1994; Haneke et al., 2001; Jirova et al., 2010; Kligman, 1966; Robinson et al., 2005; York et al., 1996; Zaghi and Maibach, 2009). As an alternative non-animal based in vitro system, HEL-30 cells may replace and reduce animal use in various scientific experiments for the characterization and identification of chemical functions. Additionally, the murine HEL-30 cell line is more comparable with already published animal in vivo results as compared with results from human-originated cell lines. The allergenic potency of chemicals has been categorized based on in vivo experiments (Basketter

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Table 3 The relative macrophage inflammatory protein 2 (MIP-2) production rates by HEL-30 cells at 24 h after the initiation of chemical exposure. MIP-2 production rate (means ± SEM)a Chemicals

0.01 × CV75

CMI/MI DNCB 4-Phenylenediamine Cobalt chloride Isoeugenol 2-Mercaptobenzothiazole Citral HCA Eugenol Phenyl benzoate Cinnamic alcohol Imidazolidinyl urea Methyl methacrylate Chlorobenzene Isopropanol Lactic acid Methyl salicylate Salicylic acid Sodium lauryl sulfate Ethylene glycol dimethacrylate Xylene Nickel chloride

200.6 178200.0 294.4 45.48 157.9 216.5 76.5 67.8 92.4 681.8 233.5 145.6 266.1 285.0 85.6 47.0 311.2 99.5 142.1 280.3 123.1 75.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 × CV75 29.7* 112700.0 27.2** 11*** 3.7*** 14.1** 9.1 13.8* 4.2 57.9*** 45.5* 21.2 72.3 73.6 10.2 11.8* 68.5* 13.4 12.9* 94.7 15.2 9.4

421.8 135300.0 619.1 47.0 114.0 274.8 363.6 73.6 443.7 697.6 148.5 133.4 185.3 283.2 94.5 50.6 202.1 230.4 185.7 240.1 341.2 81.2

0.5 × CV75 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

68.4** 86250.0 113.2* 12.4** 15.7 50.3* 14.6*** 11.6 50.5** 181.8* 18.0 13.3 16.8** 67.2 13.4 12.8* 58.7 40.1* 17.6*** 22.4** 40.9** 13.1

74.7 123200.0 237.2 51.9 237.5 138.4 99.9 363.1 591.8 635.1 157.6 650.7 120.3 367.7 47.9 105.8 34.9 125.7 26.5 315.1 92.2 39.6

1 × CV75 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12.5 81440.0 61.9 14.9** 68.0 31.8 4.7 75.7* 86.8** 247.0 30.6 175.2* 25.8 137.2 11.2* 24.19 8.4** 39.3 6.6*** 46.2** 43.8 8.8**

623.3 13120.0 139.9 79.9 77.7 394.9 689.8 227.4 196.8 39.5 473.2 139.4 67.24 308.9 46.0 104.2 49.8 83.8 38.1 312.9 98.7 60.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

147.3* 8292.0* 54.9 21.3 17.3 67.6* 53.6*** 96.7 42.9 18.9* 111.7* 36.7 16.8 78.0 5.1* 18.9 11.7* 22.3 9.6** 88.7 44.9 11.3*

CMI/MI, 5-chloro-2-methyl 4-isothiazolin-3-one (CMI)/2-methyl-4-isothiazolin-3-one (MI); DNCB, 2,4-dinitrochlorobenzene; HCA, hexyl cinnamic aldehyde. Vehicle control – 5% cDMEM or 0.1% DNCB. a The total (intracellular and extracellular) MIP-2 production rate, expressed as the percentage relative to control cells, was determined by dividing the amount of MIP-2 (pg/ml) produced by chemically treated cells by the amount of MIP-2 produced by vehicle control-treated cells, and then multiplying by 100 for relative percent expression. Data are expressed as the means ± SEM of three duplicated independent experiments. * Significantly different from the vehicle control: p < 0.05. ** Significantly different from the vehicle control: p < 0.01. *** Significantly different from the vehicle control: p < 0.001.

et al., 1994, 2005; Dunn et al., 1990; Gafner et al., 1988; Garrigue et al., 1994; Haneke et al., 2001; Kligman, 1966; Zaghi and Maibach, 2009). However, limitations exist regarding direct application on humans and chemical tests (Van Och et al., 2005). To overcome this problem, a human cell-based in vitro method is required for assessing the allergenic potency of chemicals. Additionally, for more a thorough understanding of animal in vivo and human in vitro

results, an animal cell-based in vitro method is necessary to mediate these two systems. As a major component of the epidermis, keratinocytes have the ability to produce various pro-inflammatory cytokines that can represent direct impact on skin and immune reactions against chemical exposure. Most of all, this in vitro test is a simple and cost-effective method as compared with animal experiments and reconstructed human epidermis in vitro studies

Fig. 2. Comparison of the cytokine production rates calculated in Tables 2 and 3. The highest cytokine production rate for each chemical is presented. a The small dashed line indicates the stimulation index (SI) 3. b Positive (+) or negative (−) skin-sensitizing activity, refers to previously published data obtained using the LLNA (local lymph node assay) (Basketter and Scholes, 1992; Basketter et al., 1995; Haneke et al., 2001). Classification: the chemical is classified based on the stimulation index (SI) value of our results. Positive (+) ≥3, negative (−) <3. CMI/MI, 5-chloro-2-methyl 4-isothiazolin-3-one (CMI)/2-methyl-4-isothiazolin-3-one (MI); DNCB, 2,4-dinitrochlorobenzene; p-PDA, 4-phenylenediamine; 2-MBT, 2-mercaptobenzothiazole; HCA, hexyl cinnamic aldehyde; MMA, methyl methacrylate; SLS, sodium lauryl sulfate; EGDMA, ethylene glycol dimethacrylate. Results are expressed as means ± SEM of three duplicated independent experiments.

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Table 4 Comparison of the local lymph node assay (LLNA) versus classified by the highest relative cytokine production rate. Comparison

LLNA vs. IL-1␣ LLNA vs. MIP-2 LLNA vs. IL-1␣ + MIP-2b

Accuracya (%) for Sensitizers

Irritants

75 (12/16) 75 (12/16) 94 (15/16)

83 (5/6) 83 (5/6) 67 (4/6)

reliable model for classifying sensitizing and non-sensitizing chemicals, although additional validation studies should be performed using various chemicals.

Overall accuracy (%)

Conflict of interest 77 (17/22) 77 (17/22) 86 (19/22)

LLNA, local lymph node assay. a Accuracy, the proportion of correct outcomes of a method. Often used interchangeably with concordance. b Chemical classification determined on the basis of the stimulation index. Sensitizer SI ≥ 3 at least in one cytokine production. Irritants SI < 3 in both IL-1␣ and MIP-2 production.

(Aardema et al., 2010; Basketter et al., 1995, 2007; Kandarova et al., 2009; Zaghi and Maibach, 2009). One of the most important functions of keratinocytes is the secretion of pro-inflammatory cytokines. Keratinocytes are the primary source of TNF-␣, IL-1␣, IL-6, IL-18, IP-10, and MIP-2 (Corsini and Galli, 2000; de Vos et al., 1994; Enk and Katz, 1992; Van Och et al., 2005; Williams and Kupper, 1996). Thus, in the present study, the levels of IL-6 and IL-18 were determined, as well as those of IL-1␣ and MIP-2, using the HEL-30 cell line. However, IL-18 production levels did not statistically discriminate between sensitizers and irritants, while IL-6 was not produced by HEL-30. Notably, previous studies using HEL-30 cells showed that chemical rankings based on the intracellular IL-1␣ levels were well correlated with the rankings based on the LLNA (Corsini et al., 1996; Van Och et al., 2005). However, the accuracy of detecting sensitizers or irritants by total IL-1␣ was higher than intracellular IL-1␣ alone: 75% (12 out of 16) vs. 69% (11 out of 16) for sensitizers, 83% (5 out of 6) vs. 67% (4 out of 6) for irritants, and overall 77% (17 out of 22) vs. 68% (15 out of 22) (see Supporting Information, Table S1). Therefore total IL-1␣ showed a more clear discrimination than intracellular IL-1␣ alone in this study. In a animal model, total IL-1␣ and MIP-2 mRNA levels in the epidermis of BALB/c mice was specifically up-regulated by the treatment of allergens (Enk and Katz, 1992). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.toxlet.2012.10.017. IL-1␣ is pleiotropic, pro-inflammatory cytokine and can represent the immune response against epidermal damages in the skin (Apte et al., 1992; Kupper et al., 1988). Although the total IL-1␣ was a more reliable marker in this study, intracellular IL-1␣ levels were higher than extracellular levels. This may account for the activation of cell-associated IL-1␣ forms (intracellular precursor and membrane-bound IL-1␣), which are larger than the secreted forms (Fig. 1). MIP-2, a murine functional equivalent of human IL-8, was originally identified as a heparin-binding protein secreted from a murine macrophage cell line in response to endotoxin stimulation. MIP-2 is a powerful chemo-attractant and activator for neutrophils (Wolpe et al., 1989). In addition, it affects the activation and movement of epidermal Langerhans cells and inflammatory cells during the induction phase of allergic contact sensitivity (Enk and Katz, 1992). Further, MIP-2 is produced by keratinocytes after external stimuli, including arsenic, contact sensitizers, and irritants (Mohamadzadeh et al., 1994; Yen et al., 1996). Taken together, neutrophils may be recruited to the contact dermatitis by MIP-2 and can be activated by IL-1␣ from keratinocytes. Hence, we hypothesized that these two cytokines may be useful for discriminating skin sensitizers. In conclusion, as a non-animal-based alternative chemical sensitizing assay, production of the pro-inflammatory cytokines IL-1␣ and MIP-2 by murine keratinocytes might be a very accurate and

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