Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells

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1 The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.43, No.5, , Original Article Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells Takafumi Mitachi 1, Minori Mezaki 1, Kunihiko Yamashita 2 and Hiroshi Itagaki 1 1 Department of Chemical and Energy Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama, Kanagawa , Japan 2 Corporate Research Center, Daicel Corporation, 1239 Shinzaike, Aboshi-ku, Himeji, Hyogo , Japan (Received December 7, 2017; Accepted February 14, 2018) ABSTRACT To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular ph and the expression of the skin sensitization test human cell line activation test (h-clat) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular ph (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular ph was confirmed. Furthermore, we observed that Na + / H + exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular ph, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular ph has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future. Key words: Sensitization, ph, CD86, CD54, Allergen INTRODUCTION The development of non-animal tests to evaluate the safety of chemicals in cosmetics has recently accelerated. For example, many tests have been developed to evaluate skin sensitization. Allergic contact dermatitis (ACD) is a common health problem characterized by sensitivity to allergens (Peiser et al., 2012). ACD occurs in two phases: induction and elicitation. In the induction phase, a chemical, called a hapten, penetrates the skin and forms a conjugate with nucleophilic skin proteins. Hapten binding proteins are recognized by and activate dendritic cells (DCs) and Langerhans cells. DC activation induces the expression of the cell surface molecules CD40, CD54, CD83, CD86, and HLA-DR (Aiba et al., 1997; Arrighi et al., 2001; Coutant et al., 1999), as well as the production of the cytokines TNF-α, IL-8, IL-1β, and IL-6 (Aiba et al., 2003; Cumberbatch et al., 1996; Enk and Katz, 1992). Activated DCs migrate to skin-draining lymph nodes and present antigens to antigen-specific T lymphocytes. Finally, T lymphocytes proliferate and disseminate into the peripheral circulation. In the elicitation phase, repeated exposure to the same allergen after the induction of sensitization triggers the release of inflammatory cytokines, leading to ACD. In vitro, in chemico, and in silico skin sensitization tests focus on key events in the adverse outcome pathways for skin sensitization (OECD, 2012a, 2012b). Among these tests, the KeratinoSens TM assay based on the keratinocyte response (Emter et al., 2013) and the human cell line activation test (h-clat) based on the activation of DCs (Ashikaga et al., 2010) were adopted as OECD test guidelines (OECD, 2015, 2017). Additionally, LuSenS based on the keratinocyte response (Ramirez et al., 2014), the IL-8 Luc assay (Takahashi et al., 2011; OECD, 2017), and U-SENS based on the acti- Correspondence: Hiroshi Itagaki ( itagaki-hiroshi-dw@ynu.ac.jp)

2 300 T. Mitachi et al. vation of DCs (Piroird et al., 2015; OECD, 2017) have been reported. In these assays, a cellular indicator is measured to evaluate the skin sensitization potential. When a particular assay focused on DC activation from among key events, it has reported that an acidic extracellular ph affects the release of various cytokines. According to a previous study, lactate enhances the release of IL-6 and IL-8 in human leukemic monocytes U937 and human monocytes (Samuvel et al., 2009). Moreover, acidic extracellular ph induces the augmentation of CD80 and CD86 expression and the production of IL-1β, IL-18, and TNF-α (Kong et al., 2013; Rajamäki et al., 2013). Therefore, the skin sensitization markers are affected by acidic extracellular ph. However, the mechanism underlying this relationship remains unclear. In this study, we investigated whether an acidic ph affects the expression of CD86 and CD54, predictive makers of the skin sensitization potential in h-clat. These results provide significant insights into the improvement of current in vitro assays. MATERIALS AND METHODS Cell culture THP-1 cells from the American Type Culture Collection (ATCC; Manassas, VA, USA) were cultured in RPMI1640 (Wako Pure Chemical Industries Ltd., Osaka, Japan) containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 units/ml penicillin (Wako Pure Chemical Industries Ltd.), 100 μg/ml streptomycin (Wako Pure Chemical Industries Ltd.), and 0.05 mm 2-mercaptoethanol (Wako Pure Chemical Industries Ltd.) at 37 C with 5% CO 2. THP-1 cells were passaged by the addition of fresh medium twice a week and maintained at cells/ml. Reagents Table 1 summarizes the test chemicals used in this study. 2,4-Dinitrochlorobenzene (DNCB), imidazolidinyl urea (IU), lactic acid (LA), and globulin Cohn fraction II, III human were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Hydrochloric acid (HCl), citric acid (CA), glycerol (GC), isopropanol (IP), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and protease-free bovine serum albumin (BSA) were purchased from Wako Pure Chemical Industries Ltd. Physiological saline was purchased from Otsuka Pharmaceutical Co. Ltd. (Tokyo, Japan). An FITCconjugated anti-cd86 antibody (Isotype: IgG1κ) (BD Bioscience, Franklin Lakes, NJ, USA), FITC-conjugated anti-cd54 antibody (DAKO, Glostrup, Denmark), and isotype control antibody (Mouse IgG1) (DAKO) were used for the protein expression analysis. phrodo TM Green AM Intracellular ph Indicator (Thermo Fisher Scientific), valinomycin (Sigma-Aldrich Co.), nigericin (Merck, Darmstadt, Germany), and HEPES (4-(2-hydroxyethyl)-1- piperazineethane-sulfonic acid) (Dojindo Laboratory Co. Ltd., Kumamoto, Japan) were used to measure intracellular ph. Propidium iodide (PI, 1 mg/ml) was purchased from Dojindo. Cariporide, an NHE-1 inhibitor, was purchased from Cayman Chemical (Ann Arbor, MI, USA). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) (phosphor-p38 MAP kinase, #9211; p38 MAPK, #9212). Sensitization test HCl, LA, and CA were used as acidic test chemicals; GC and IP were used as non-acidic test chemicals; DNCB and IU were used as representative sensitizers. THP-1 cells were plated at cells/500 μl in a 24-well flatbottomed plate and co-cultured with 250 μl of the sensitizer (3.6 μg/ml of DNCB or 37.6 μg/ml of IU) and 250 μl of acidic/non-acidic test chemicals (15-22 mm of HCl, mm of LA, mm of CA, 3,500-5,000 μg/ml of GC, 3,500-5,000 μg/ml of IP) for 24 hr. The concentrations of DNCB and IU were adjusted to obtain a cell viability of approximately 85%. The Table 1. Summary of chemicals used to investigate the relationship between the skin sensitization and acidity. Chemical Abbreviation CAS RN Source LLNA Vehicle CD86 CD54 2,4-Dinitrochlorobenzene DNCB Sigma-Aldrich Positive DMSO Positive Positive Imidazolidinyl urea IU Sigma-Aldrich Positive Saline Positive Positive Hydrochloric acid HCl Wako - Saline - - Lactic acid LA Wako Negative Saline Negative Negative Citric acid CA Wako - Saline - - Glycerol GC Wako Negative DMSO Negative Negative Isopropanol IP Wako Negative DMSO Negative Negative Abbreviations: CAS RN = Chemical Abstracts Service Number. The LLNA data are based on Gerberick et al. (2005) and the CD86/ CD54 data are based on Ashikaga et al. (2010).

3 301 Acidic conditions induce the suppression of CD86 and CD54 expression concentrations of HCl, LA, and CA were adjusted to prepare a solution with ph between 7 and 6. The concentrations of GC and IP were decided based on the chemical s solubility, according to OECD test guidelines (TG 442E). Then, CD86 and CD54 expression was analyzed. Analysis of CD86 and CD54 expression Measurements of CD86 and CD54 expression were performed according to OECD test guidelines (TG 442E). After treatment with the chemicals for 24 hr ± 30 min, cells were washed with staining buffer (PBS with 0.1% BSA). Then, blocking solution (staining buffer containing 0.01% (w/v) globulin (Cohn fraction II, III)) was added for 10 min at 4 C. After centrifugation, the cells were stained with anti-cd86 (dilution, 3:25), anti-cd54 (dilution, 3:50), and IgG1 antibodies (dilution, 3:50) for 30 min at 4 C. After they were washed with staining buffer three times, the cells were resuspended in staining buffer, and PI solution (0.625 μg/ml) was added. CD86 and CD54 expression levels and cell viability were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA). Dead cells were gated out, and a total of 10,000 living cells were analyzed. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for positive control cells and chemical-treated cells was calculated according to the following equation: Measurements of intracellular ph THP-1 cells ( cells/ml) were washed with 1 ml HEPES buffer (ph 7.4) and incubated with 10 μm phrodo TM Green AM for 30 min at 37 C. After centrifugation, the cells were re-suspended in 500 μl serumfree medium and treated with 250 μl DNCB and 250 μl acidic/non-acidic test chemicals for 10 min. The concentrations of the test chemicals were the same as those in the sensitization test. After treatment, the intracellular ph, indicated as phrodo TM Green AM fluorescence intensity, was assessed using a microplate reader (EnVision, PerkinElmer, Inc., Waltham, MA, USA). The intracellular ph was calculated based on the calibration curve. To create a calibration curve, five concentrations of HEPES buffer (ph 8, 7.5, 7, 6.5, and 6) were tested. THP-1 cells ( cells/ml) were washed with 1 ml HEPES buffer (ph 7.4) and incubated with 10 μm phrodo TM Green AM for 30 min at 37 C. After centrifugation, the cells were re-suspended in the five other concentrations of 1 ml HEPES buffer (ph 8, 7.5, 7, 6.5, and 6), each containing 10 μm valinomycin and 10 μm nigericin, for 5 min at 37 C to reach different intracellular ph. After incubation, each intracellular ph was also assessed using a microplate reader. Western blot analysis of p38mapk THP-1 cells were incubated with the test chemicals for 2 hr in 24-well plates at cells/ml. Cells were then washed with PBS, mixed with SDS sample buffer, boiled, and subjected to SDS-polyacrylamide gel electrophoresis, followed by blotting onto a polyvinylidene difluoride membrane (ATTO Co., Tokyo, Japan). The membranes were blocked for 1 hr with EzBlock Chemi (ATTO) and probed with primary antibodies overnight at a 1:1000 dilution for p38 MAPK. After washing with Tris-buffered saline (ph 7.6) containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies at a 1:2000 dilution for 45 min. Immunoreactive bands were treated with EzWestLumi plus (ATTO) and detected using LAS-4000mini (GE Healthcare UK Ltd, Little Chalfont, BH, England). Antibodies for total p38 MAPK were used as loading controls to analyze p38 MAPK expression. Statistical analysis Each experiment was performed at least three times. Results are expressed as means ± standard deviation (S.D.). One-way analysis of variance followed by Dunnett s post-hoc tests were used to evaluate statistical significance using EZR, a graphical user interface for R (The R Foundation for Statistical Computing, version 3.3.0). A p-value of less than 0.05 was considered statistically significant. RESULTS Acidic conditions decreased the augmentation of CD86 and CD54 by a sensitizer First, to investigate the relationship between CD86/ CD54 expression and acidic ph, acidic medium was prepared by adding HCl. In acidic conditions, THP-1 cells were exposed to the representative contact allergen DNCB (Fig. 1). The augmentation of CD86/CD54 by DNCB decreased significantly in acidic conditions (Fig. 1-A, B). Cell viability was > 75% in each sample (Fig. 1-C). To confirm that these results were not specific to DNCB, THP-1 cells were similarly exposed to another contact allergen, IU (Fig. 2). The expression of CD86/ CD54 in cells treated with IU was statistically significantly lower in acidic conditions than in control conditions. These results indicate that the expressions of both CD86 and CD54 with the sensitizer were decreased with HCl.

4 302 T. Mitachi et al. Fig. 1. The suppression of CD86 and CD54 expression in THP-1 cells treated with the representative contact allergen DNCB and the effect of HCl. THP-1 cells exposed to DMSO (vehicle), DNCB (3.6 μg/ml), and HCl (15, 18, 22 mm: 1.2-fold) for 24 hr ± 30 min. A) CD86, B) CD54, C) cell viability. Results are presented as means ± S.D. of three independent experiments. Statistical significance was calculated using Dunnett s post-hoc tests (*p < 0.05, **p < 0.01).

5 303 Acidic conditions induce the suppression of CD86 and CD54 expression Fig. 2. The suppression of CD86 and CD54 expression in THP-1 cells treated with the representative contact allergen IU and the effect of HCl. THP-1 cells were exposed to DMSO (vehicle), IU (37.6 μg/ml), and HCl (15, 18, 22 mm: 1.2-fold) for 24 hr ± 30 min. A) CD86, B) CD54, C) cell viability. Results are expressed as means ± S.D. of three independent experiments. Statistical significance was calculated using Dunnett s post-hoc tests (*p < 0.05, **p < 0.01).

6 304 T. Mitachi et al. Acidic chemicals specifically suppressed CD86 and CD54 expression in h-clat We next investigated whether acidic test chemicals induced the suppression of CD86/CD54 expression with a sensitizer. THP-1 cells with DNCB were exposed to LA, CA, GC, and IP (Table 2). As shown in Table 2, both LA- and CA-treated cells exhibited significantly suppressed CD86/CD54 expression compared with that of DNCB-treated cells. In contrast, the non-acidic chemicals GC and IP did not have a substantial influence on CD86/ CD54 expression. Moreover, the decreases in CD86/CD54 expression by LA and CA were similarly observed in cells treated with IU (Table 3). Extracellular ph was decreased for both LA- and CA-treated cells, but not changed for GC- and IP-treated cells (data not shown). These results indicate that an acidic extracellular ph induces the suppression of CD86/CD54 expression in THP-1 cells. Acidic test chemicals alter the intracellular ph Our results demonstrated that an acidic extracellular chemical is one of the key factors determining CD86/ CD54 expression. However, the pathway by which marker expression is suppressed by the extracellular acidic chemical is unclear. Therefore, we examined the suppression of CD86/CD54 with respect to intracellular ph. Using phrodo TM Green AM, we produced a calibration curve (R = ) and calculated intracellular ph (Fig. 3). These results showed that all acidic chemicals Table 2. RFI values for cells treated with DNCB and four test chemicals (LA, CA, GC, and IP). Sample RFI 1 2 CD86 CD54 Cell viability (%) DMSO ± 0.3 DNCB 3.6 μg/ml ± ± ± 4.2 DNCB 3.6 μg/ml LA 21 mm ± 27.5** ± 12.9** 86.0 ± 3.3 DNCB 3.6 μg/ml LA 25 mm ± 11.7** ± 43.0** 86.2 ± 1.7 DNCB 3.6 μg/ml LA 31 mm 52.9 ± 17.7** 52.7 ± 26.3** 76.6 ± LA 21 mm 72.7 ± ± ± LA 25 mm 67.5 ± ± ± LA 31 mm 61.2 ± ± ± 1.7 DMSO ± 0.6 DNCB 3.6 μg/ml ± ± ± 0.4 DNCB 3.6 μg/ml CA 3.7 mm ± ± ± 1.1 DNCB 3.6 μg/ml CA 5.2 mm ± 59.8** ± 18.4** 80.5 ± 1.4 DNCB 3.6 μg/ml CA 7.3 mm ± 33.1** ± 32.5** 81.6 ± CA 3.7 mm ± ± ± CA 5.2 mm ± ± ± CA 7.3 mm 78.3 ± ± ± 0.7 DMSO ± 1.3 DNCB 3.6 μg/ml ± ± ± 1.4 DNCB 3.6 μg/ml GC 3500 μg/ml ± ± ± 1.4 DNCB 3.6 μg/ml GC 4200 μg/ml ± ± ± 0.5 DNCB 3.6 μg/ml GC 5000 μg/ml ± ± ± GC 3500 μg/ml 88.5 ± ± ± GC 4200 μg/ml 94.1 ± ± ± GC 5000 μg/ml ± ± ± 0.7 DMSO ± 1.0 DNCB 3.6 μg/ml ± ± ± 2.5 DNCB 3.6 μg/ml IP 3500 μg/ml ± ± ± 4.4 DNCB 3.6 μg/ml IP 4200 μg/ml ± ± ± 2.5 DNCB 3.6 μg/ml IP 5000 μg/ml ± ± ± IP 3500 μg/ml ± ± ± IP 4200 μg/ml 91.2 ± ± ± IP 5000 μg/ml 87.3 ± ± ± 0.02 Means ± S.D. of three independent experiments. Statistical significance was calculated using Dunnett s post-hoc tests (*p < 0.05, **p < 0.01).

7 305 Acidic conditions induce the suppression of CD86 and CD54 expression Table 3. RFI values for cells treated with IU and two acidic test chemicals (LA and CA). Sample RFI 1 2 CD86 CD54 Cell viability (%) Saline ± 0.3 IU 37.6 μg/ml ± ± ± 4.2 IU 37.6 μg/ml LA 21 mm ± 20.7** ± 4.4** 86.0 ± 3.3 IU 37.6 μg/ml LA 25 mm ± 10.4** ± 22.8** 86.2 ± 1.7 IU 37.6 μg/ml LA 31 mm ± 11.1** 81.3 ± 5.2** 76.6 ± LA 21 mm 46.2 ± ± ± LA 25 mm 50.8 ± ± ± LA 31 mm 77.7 ± ± ± 1.7 Saline ± 0.6 IU 37.6 μg/ml ± ± ± 0.4 IU 37.6 μg/ml CA 3.7 mm ± 36.9* ± ± 1.1 IU 37.6 μg/ml CA 5.2 mm ± 26.7** ± ± 1.4 IU 37.6 μg/ml CA 7.3 mm ± 16.8** ± 76.5* 81.6 ± CA 3.7 mm 90.5 ± ± ± CA 5.2 mm 91.2 ± ± ± CA 7.3 mm 69.6 ± ± ± 0.7 Means ± S.D. of three independent experiments. Statistical significance was calculated using Dunnett s post-hoc tests (*p < 0.05, **p < 0.01). (HCl, LA, and CA) resulted in a lower intracellular ph compared with that observed for DNCB treatment alone (Fig. 3A, B, C). In contrast, non-acidic chemicals (GC and IP) did not influence the intracellular ph (Fig. 3D, E). These results indicated that the depression of the intracellular ph, in addition to the suppression of CD86/CD54 expression, was initiated by an acidic test chemical. Na + /H + exchanger 1 (NHE-1) activity influenced the expression of CD86/CD54 and p38 MAPK Our data revealed that the suppression of CD86 and CD54 expression was induced by acidic ph. However, it was unclear whether these phenomena were caused by both acidic extracellular and acidic intracellular ph together or by either one of these conditions separately. To further examine the mechanisms underlying the relationship between ph and marker expression, we examined the effects of NHE-1, a potassium ion transmembrane protein involved in intracellular ph regulation. We measured CD86/CD54 expression after treatment with an NHE-1 inhibitor, cariporide (Fig. 4). Both CD86 and CD54 expression levels were significantly suppressed (Fig. 4A, B), and the intracellular ph was depressed in response to cariporide treatment, whereas the extracellular ph was not affected (data not shown). These results indicate that intracellular acidic ph is one of the key factors influencing CD86 and CD54 expression. Additionally, we analyzed the activation of p38 MAPK, which is known to influence CD86/CD54 expression in THP-1 cells. Fig. 4D shows that p38 MAPK activation treated with DNCB was suppressed by a cariporide. This phenomenon was also observed upon treatment with LA (Fig. 4E). Therefore, NHE-1 inhibitors can induce the reduction of intracellular ph and, therefore, result in the down-regulation of CD86/ CD54 expression by suppressing p38 MAPK activity. DISCUSSION A number of in vitro skin sensitization assays based on predictive markers have been developed. For example, CD86 and CD54 are induced by DNCB and nickel sulfate (NiSO 4 ) in THP-1 cells via the p38 MAPK and NF-κβ pathways (Ade et al., 2007; Aiba et al., 2003; Boislève et al., 2004; Miyazawa et al., 2008). Additionally, many sensitizers induce the production of reactive oxygen species (ROS) via AKT phosphorylation (Miyazawa and Takashima, 2012; Nukada et al., 2011; Saito et al., 2013). Therefore, various factors affect the expression of predictive indicators used for in vitro assays. In this study, we examined the influence of culture conditions on the skin sensitization indicators CD86 and CD54, which are associated with the activation of DCs. In previous studies, the alteration of extracellular and intracellular ph affected the release of cytokines and the activation of signaling pathways (Dietl et al., 2010; Kellum et al., 2004; Riemann et al., 2016; Samuvel et al., 2009). Moreover, CD86 and CD54 expression is affected by acidic ph (Kong et al., 2013; Martínez et al., 2007;

8 306 T. Mitachi et al. Fig. 3. The reduction in intracellular ph in THP-1 cells treated with the representative contact allergen DNCB and the effect of acidic ph. THP-1 cells exposed to DMSO (vehicle), DNCB (3.6 μg/ml), and A) HCl (15, 18, 22 mm: 1.2-fold), B) LA (21, 25, 31 mm: 1.2-fold), C) CA (3.7, 5.2, 7.3 mm: 1.4-fold), D) GC (3500, 4200, 5000 μg/ml: 1.2-fold), and E) IP (3500, 4200, 5000 μg/ml: 1.2-fold) for 10 min. Results are presented as means ± S.D. of three independent experiments. Statistical significance between DNCB-treated cells and DNCB-treated cells with acidic chemicals was calculated using Dunnett s post-hoc tests (*p < 0.05, **p < 0.01). Qadri et al., 2014; Vermeulen et al., 2004). Therefore, we investigated CD86/CD54 expression with respect to medium ph and test chemicals. Our results revealed that extracellular ph affects CD86 and CD54 expression. The augmentation of CD86 and CD54 expression with the representative sensitizers DNCB and IU was suppressed by supplementation with acidic test chemicals (HCl, LA, and CA). Proliferation and morphological changes were not observed in THP-1 cells (data not shown). Furthermore, the intracellular ph decreased. The suppression of CD86/CD54 expression in samples treated with CA appeared to be weaker than that in samples treated with HCl or LA. This could be owing to the differences in ph between each sample. The extracellular ph values of media treated with HCl (15, 18, 22 mm) were 6.5, 6.4, and 6.1, respectively. Additionally, the

9 307 Acidic conditions induce the suppression of CD86 and CD54 expression Fig. 4. The suppression of CD86/CD54 expression and p38 MAPK activation in THP-1 cells treated with the representative contact allergen DNCB and the effect of cariporide. THP-1 cells were exposed to DMSO (vehicle), DNCB (3.6 μg/ml), and cariporide (50, 100, 200 μm: 2-fold) for 24 hr ± 30 min. A) CD86, B) CD54, C) cell viability. Results are expressed as means ± S.D. of three independent experiments. Statistical significance was calculated using Dunnett s post-hoc tests (*p < 0.05, **p < 0.01). The phosphor-p38 MAPK and total p38 were analyzed by western blot analysis. D) THP-1 cells exposed to DMSO (vehicle), DNCB (3.6 μg/ml), and cariporide (200 μm). E) THP-1 cells exposed to DMSO (vehicle), DNCB (3.6 μg/ml), and LA (21, 25, 31 mm: 1.2-fold).

10 308 T. Mitachi et al. ph values with LA treatment (21, 25, 31 mm) were 6.5, 6.2, and 6.0, respectively. However, the ph values with CA treatment (3.7, 5.2, 7.3 mm) were 6.8, 6.6, and 6.3, respectively, making them slightly higher than those of the samples treated with HCl or LA. A similar trend was observed for the intracellular ph of these samples as well. Therefore, it could be suggested that the degree of acidosis influenced CD86/CD54 expression. In contrast, treatment with GC and IP did not have a substantial influence on CD86/CD54 expression or intracellular ph. CD86 and CD54 expression was nonspecifically enhanced when cytotoxicity was increased upon treatment with some of the test chemicals. However, in this study, the acidosis of intracellular ph directly suppressed the CD86/CD54 augmentation, regardless of cytotoxicity. Accordingly, a reduction in intracellular ph affects signaling pathways related to the expression of CD86/CD54. NHE-1, H + -ATPases, and monocarboxylate-h + efflux cotransporters MCT1 and MCT4 regulate extra- and intracellular ph. In particular, cariporide, an inhibitor of NHE-1 activity, decreases CD54 expression in murine microvascular SVEC4-10EE2 endothelial cells (Qadri et al., 2014). In addition, in human chronic myeloid leukemia K562 cells, cariporide induces the suppression of protein kinase C-β (PKC-β) activation (Ma et al., 2015). Other groups have reported that PKC-β activation is associated with CD86 expression and IL-8 production in THP-1 cells (Corsini et al., 2014). Our data showed that inhibition of NHE-1 activity induced the suppression of CD86/CD54 expression and p38 MAPK activation. In this study, we did not analyze NHE-1 activation upon treatment with a sensitizer and acidic/non-acidic chemicals. However, it was observed that p38 MAPK activation was suppressed in LA-treated cells with DNCB, similarly to in cells treated with cariporide. Thus, the reduction in intracellular ph could be attributed to the inhibition of NHE-1 activity. These results suggest that there is a relationship between NHE-1 activity and CD86/CD54 expression, consistent with our findings in THP-1 cells treated with cariporide. In general, consumers are rarely exposed to a single isolated allergen, but are typically exposed to mixtures of allergens (Uter et al., 2013, 2014). Accordingly, to accurately evaluate skin sensitization potential, additional studies are needed to examine mixtures containing multiple test chemicals (Bonefeld et al., 2017; Kienhuis et al., 2015; Lourenço et al., 2015; Morimoto et al., 2014). Moreover, further studies are required to evaluate and improve consumer safety and to clarify the mechanism by which CD86/CD54 suppression was induced by an acidic ph. Conflict of interest---- The authors declare that there is no conflict of interest. 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