2019 Volume 44 Issue 3 Pages 213-224
The human cell line activation test (h-CLAT) is a skin sensitization test that measures the expression of cell surface proteins CD86 and CD54 to evaluate the skin sensitization potential of test chemicals. However, some skin irritants have been reported to induce dramatically high CD54 expression leading to false-positive h-CLAT results. Furthermore, CD54 expression is strongly induced by cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, or danger signals that activate its signaling pathways. In this study, we focused on the relationship between CD54 expression and the Nucleotide binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome, a protein complex that plays a pivotal role in intra-cellular inflammation. We observed the activation of caspase-1 and production of IL-1β after exposure of THP-1 cells to 2,4-dinitrochlorobenzene (DNCB, sensitizer), octanoic acid (OA, non-sensitizer), and salicylic acid (SA, non-sensitizer), implying NLRP3 activation. These observations confirmed the activation of the inflammasome by CD54-only positive chemicals. CD54 expression, induced by OA and SA, was suppressed by potassium chloride, a typical inhibitor of NLRP3 inflammasome activation. These results suggested that the NLRP3 inflammasome may be activated in THP-1 cells resulting in the expression of CD54, and subsequently leading to false-positive results.
In recent years, several in vitro assay methods have been developed to ensure the safety of cosmetic ingredients. Peiser et al., reviewed the epidemiology, molecular mechanisms and regulatory aspects of allergic contact dermatitis caused by exposure to agents such as 2,4-dinitrochlorobenzene and oxazolone (Peiser et al., 2012). Accurate safety evaluation of skin sensitization potential of ingredients is a key focus area in the cosmetic industry. Currently, several in vitro skin sensitization test methods are being developed following the Adverse Outcome Pathway (AOP) and the OECD testing guidelines (OECD, 2014). The h-CLAT, adopted as OECD TG442E, is a test method to identify skin sensitizers by evaluating the activation of dendritic and human acute monocyte leukemia (THP-1) cells. Exposure of dendritic cells to skin sensitizing substances increases the expression of cell surface molecules such as CD40, CD54, CD83, CD86, and HLA-DR (Aiba et al., 1997; Arrighi et al., 2001; Coutant et al., 1999) and induces inflammatory cytokines such as TNF-α, IL-8, IL-1β, and IL-6 (Aiba et al., 2003; Cumberbatch et al., 1996; Enk and Katz, 1992). Based on these findings, h-CLAT is used to evaluate the increase of CD86 and CD54 expression in THP-1 cells after exposure to chemical agents to identify the skin sensitizer (Ashikaga et al., 2002, 2006; Yoshida et al., 2003; Sakaguchi et al., 2006). The high coincidence rates with LLNA (85%) (Ashikaga et al., 2010) and humans (83%) suggest that h-CLAT is a useful in vitro skin sensitization test method for cosmetic products.
Accurate evaluation is a critical performance parameter for test methods. However, in case of h-CLAT, 15 of 39 non-sensitizing substances tested, gave false-positive results (Ashikaga et al., 2010; EC EURL-ECVAM, 2012; Takenouchi et al., 2013). In the safety assessment tests for skin sensitization, minimizing false-negative results while identifying the skin sensitizers is prioritized so that any potentially unsafe chemical ingredient may be detected (Narita et al., 2018). However, the methods to identify false-positive results are still insufficient, and an active and useful ingredient in a new product under development may be eliminated due to it being a false-positive by current test methods. Such errors are not desirable in commercial development and manufacture of cosmetic products making the investigation for false positive results equally important as the detection of false-negatives.
In several false-positive cases, CD54 expression was exclusively over the cut-off value of 200%. Previous studies have shown that CD54 is activated downstream of IL-1β production which is induced by the activation of the NLRP3 inflammasome (Chen et al., 2000; Roebuck and Finnegan, 1999) and IL-1β stimulated the expression level of CD54. In addition, lipopolysaccharide and nickel are chemical substances known to induce NLRP3 inflammasome activation (Schmidt et al., 2010; Trompette et al., 2009), and the CD54 expression is significantly elevated in response to these agents (Narita et al., 2018; Tsukumo et al., 2018). Furthermore, of the 14 substances which yielded false-positive results in h-CLAT, 11 caused increase in CD54 expression (EC EURL-ECVAM 2012; Takenouchi et al., 2013).
Therefore, in this study, we aimed to elucidate the relationship between NLRP3 inflammasome activation and CD54 expression. First, we analyzed the activation of caspase-1 and production of IL-1β as indicators of NLRP3 inflammasome activation used as sensitizers and false positive chemicals. We next examined whether the inhibitor of NLRP3 inflammasome activation (NAC or KCl) influenced CD86 and CD54 expression in THP-1 cells.
THP-1 cells from the American Type Culture Collection (ATCC; Manassas, VA, USA) were cultured in RPMI1640 medium (Wako Pure Chemical Industries Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 units/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2-mercaptoethanol (all obtained from Wako Pure Chemical Industries Ltd.) at 37°C with 5% CO2. THP-1 cells were passaged by the addition of fresh medium twice weekly and maintained at 0.1-0.6 × 106 cells/mL.
Table 1 summarizes the test chemicals used in this study. Benzaldehyde (BA), 1-bromobutane (BB), chlorobenzene (CB), 2,4-dinitrochlorobenzene (DNCB), N- acetylcysteine (NAC), nickel sulfate (Ni), 4-nitrobenzyl bromide (NB), salicylic acid (SA), lipopolysaccharide (LPS), monosodium urate crystal (MSU), and globulin Cohn fraction II, III human were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Diethyl phthalate (DP), octanoic acid (OA), hydrogen peroxide (H2O2), dimethyl sulfoxide (DMSO), potassium chloride (KCl), 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). FITC-conjugated 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 protein expression analysis. Propidium iodide (PI, 1 mg/mL) was purchased from Dojindo Laboratories, Kumamoto, Japan.
DNCB and NB were used as representative sensitizers; BA and BB, CB, DP, OA, SA were used as false-positive chemicals. THP-1 cells were plated at 2 × 106 cells/500 µL in 24-well flat-bottomed plates and co-cultured with sensitizer (2.7-4 µg/mL of DNCB, 3.4-5.0 µg/mL of NB) or false-positive chemicals (416.7-600.0 µg/mL of BA, 500.0-720.0 µg/mL of CB, 500.0-720.0 µg/mL of DP, 300.0-432.0 µg/mL of OA, 578.8-833.3 µg/mL of SA) in a total volume of 500 µL for 24 hr. The concentrations of test chemicals were decided based on the chemical’s solubility and CD86/CD54 expression in accordance with OECD test guidelines (TG 442E) and Tsukumo et al., 2018. CD86 and CD54 expression was analyzed based on our previous publication (Mitachi et al., 2018). To investigate the effect of inhibitor (NAC or KCl), THP-1 cells were plated with inhibitor (5 mM of NAC or 50, 75 mM of KCl) at 1 × 106 cells/1 mL in a 24-well flat-bottomed plate for 1 hr. After centrifugation, the cells were re-plated at 2 × 106 cells/500 µL and incubated with 500 µL of the test chemicals. Then, CD86 and CD54 expression was analyzed.
THP-1 cells (1 × 106 cells/mL) were plated in a 12-well flat-bottomed plate and exposed to test chemicals for 12 hr. The protein level of caspase-1 and phosphorylated caspase-1 was analyzed by western blotting, as described in our previous study (Mitachi et al., 2018). Primary antibodies against caspase-1 (Catalog number: 14F468) and phosphorylated caspase-1 p10 subunit (sc-56036) were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA). Both antibodies were used at 1:1000 dilutions for 60 min at RT. β-actin, used as an internal loading control, was purchased from Wako Pure Chemical Industries Ltd. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies at 1:2000 dilutions for 45 min at RT. Immunoreactive bands were detected using LAS-400mini (GE Healthcare UK Ltd, Little Chalfont, BH, England).
IL-1β production was analyzed based on a previous study (Pétrilli et al., 2007). THP-1 cells (1 × 106 cells/mL) were plated in a 12-well flat-bottomed plate containing 60 nM phorbol 12-myristate 13-acetate (PMA) for 24 hr. After re-suspension with fresh medium, the cells were cultured for an additional 48 hr to confirm differentiation. The cells were then checked for adhesion and morphological changes under a microscope. After a 6 hr exposure to test chemicals, IL-1β production was assessed by an enzyme-linked immunosorbent assay (ELISA) kit (R&D System, Inc., Minneapolis, MN, USA) following the manufacturer’s instructions.
THP-1 cells (1 × 106 cells/mL) were labeled with ROS probe, 5-(and-6)-carboxy-2’, 7’-difluorodihydrofluorescein diacetate (CM-H2DCFDA) (Thermo Fisher Scientific) at a final concentration of 2 µM at 37°C for 15 min in the dark. After resuspension in serum-free RPMI medium, 2 × 105 cells/200 µL THP-1 cells were challenged with 200 µL test chemicals for 30 min at 37°C, at five concentrations described previously (Saito et al., 2013). PI solution (0.625 µg/mL) was added after 30 min of incubation with the chemicals, and ROS production and cell viability were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA). Based on the mean fluorescence intensity (MFI), ROS production was calculated according to the following equation:
ROS production = MFI of chemical-treated cells / MFI of vehicle-treated cells
All experiments were performed at least three times. Results are expressed as mean ± standard deviation (S.D.). Differences in means were considered statistically significant when p < 0.05 and 0.01, as tested by Student’s t-test (Microsoft Excel, USA).
Activation of NLRP3 inflammasome was first tested by assessing caspase-1 (p10 subunit) phosphorylation in THP-1 cells challenged with the sensitizer DNCB. Total and phosphorylated caspase-1 were analyzed by western blot. OA and SA were used as CD54-only false-positive chemicals while LPS and MSU was used as a positive control of inflammasome activation (Fig. 1A). We observed that caspase-1 p10 was activated (phosphorylated/total caspase 1) in THP-1 when challenged with any of the four agents DNCB, LPS, OA, or SA. Moreover, to evaluate IL-1β production as an additional indicator of NLRP3 inflammasome activation, we treated differentiated THP-1 cells with DNCB and Ni, LPS, OA, or SA. We observed increased production of IL-1β in cells treated with either the sensitizers or CD54-only false-positive chemicals, implying that the NLRP3 inflammasome was induced not only by the sensitizer, but also the CD54 positive chemicals in THP-1 cells (Fig. 1B).
Induction of caspase-1 activation and IL-1β production in THP-1 cells treated with the sensitizer and false-positive chemicals. A) Expression of phosphor-p10 caspase-1, caspase-1, and β-actin were analyzed by western blot analysis. THP-1 cells were exposed to control (DMSO), LPS (10 ng/mL), DNCB (4.2 μg/mL), OA (360 μg/mL), SA (833 μg/mL), or MSU (400 μg/mL) for 12 hr. B) IL-1β production was assessed by ELISA. THP-1 cells were exposed to control (DMSO), LPS (10 ng/mL), Ni (200 mg/mL), DNCB (4.2 μg/mL), OA (360 μg/mL), or SA (833 μg/mL) for 6 hr.
We investigated ROS production in THP-1 cells, as ROS are considered to be an activator of the NLRP3 inflammasome. THP-1 cells were pre-loaded with CM-H2DCFDA for 15 min and then exposed to 7 test chemicals (H2O2; positive control, DNCB, Ni; sensitizers, BB;CD86/CD54-double false-positive chemical, DP, OA, SA; CD54-only false-positive chemicals) or vehicle for 30 min. ROS accumulation in the cells was evaluated based on methods described previously (Saito et al., 2013). We verified that the THP-1 cell viability was not decreased significantly by these treatments. H2O2 (0.5%) induced approximately 20-fold ROS production in THP-1 cells compared to that in the vehicle-treated control (Fig. 2A). DNCB (53.5 µg/mL) induced over 4-fold ROS production in THP-1 cells (Fig. 2B) while over 2-fold ROS production was observed in DP, OA, and SA-treated cells (Fig. 2D-F). However, neither the sensitizer Ni nor the false-positive chemical BB induced ROS production in THP-1 (Fig. 2C, G).
Production of intracellular ROS in cells treated with test chemicals. THP-1 cells were exposed to A) medium (vehicle), H2O2 (0.125, 0.25, 0.5, 1.0, 2.0%: 2-fold), B) DMSO (vehicle), DNCB (53.5, 107, 214, 428, 856 μg/mL: 2-fold), C) saline (vehicle), Ni (91, 182, 364, 729, 1457 μg/mL: 2-fold), D) DMSO (vehicle), DP (91, 182, 364, 728, 1456 µg/mL: 2-fold), E) OA (156, 313, 625, 1250, 2500 µg/mL: 2-fold), (F) SA (302, 604, 1208, 2417, 4834 µg/mL: 2-fold), or (G) BB (90.4, 180.8, 361.5, 723, 1446 µg/mL: 2-fold) for 30 min.
Since both sensitizers and false-positive chemicals induced intracellular ROS production, we analyzed whether the ROS quencher NAC suppresses CD86 and CD54 expression in THP-1 cells treated with sensitizers DNCB and NB or CD54-only false-positive chemicals DP and OA. The concentration of test chemicals used in this experiment were based on the h-CLAT protocol while NAC treatment conditions were based on previously reported methods (Nukada et al., 2011). We observed a significant reduction in CD54 induction by DNCB (Fig. 3C) or by NB (Fig. 3D) when cells were treated with NAC. However, NAC treatment suppressed CD86 expression when THP-1 cells were cultured with low concentrations of DNCB (2.7 μg/mL), whereas higher concentrations, 3.3 and 4.0 μg/mL, and NB did not have a noticeable effect on CD86 induction (Figs. 3A, B). Additionally, treatment with NAC did not attenuate an increase in CD86/CD54 expression in THP-1 cells treated with the false-positive chemicals DP or OA (Fig. 4).
Effect of NAC for CD86 and CD54 expression in THP-1 cells treated with DNCB or NB. THP-1 cells were exposed to DMSO (vehicle), DNCB (2.7, 3.3, 4.0 µg/mL: 1.2-fold), or NB (3.4, 4.1, 5.0 µg/mL: 1.2-fold) for 24 hr. A, B) CD86, C, D) CD54, E, F) cell viability. Results are expressed as mean ± S.D. of three independent experiments. Statistical significance was calculated using Student’s t-test (*p < 0.05, **p < 0.01).
Effect of NAC for CD54 expression in THP-1 cells treated with false-positive chemicals DP or OA. THP-1 cells were exposed to DMSO (vehicle), DP (500, 600, 720 µg/mL: 1.2-fold), or OA (300, 360, 432 µg/mL: 1.2-fold) for 24 hr. A, B) CD54, C, D) cell viability. Results are expressed as mean ± S.D. of three independent experiments. Statistical significance was calculated using Student’s t-test.
We next examined whether the cytosolic K+ concentration, which is known to affect NLRP3 inflammasome activation, played a role in the regulation of CD86 and CD54 expression in THP-1 cells. THP-1 cells treated with MSU showed lower caspase-1 activation and IL-1β production, implying inhibition by rising intracellular K+ levels (Fernandes-Alnemri et al., 2007; Pétrilli et al., 2007; Rajamäki et al., 2013). The concentration of KCl (sensitizer: 75 mM, false-positive substance: 50 mM) was decided based on statistically insignificant cytotoxicity to THP-1 cells. THP-1 cells were pretreated with KCl for 1 hr. Results showed that the induction of CD54 expression by LPS and DNCB decreased significantly when cells were pretreated with KCl (Figs. 5C, D). In contrast, KCl pretreatment showed no substantial effect on CD86 expression in cells exposed to LPS or DNCB (Figs. 5A, B).
Effect of KCl for CD86 and CD54 expression in THP-1 cells treated with LPS or DNCB.THP-1 cells were exposed to medium or DMSO (vehicle), LPS (0.1, 1, 10 ng/mL: 10-fold), or DNCB (2.41, 2.89, 3.47 µg/mL: 1.2-fold) for 24 hr. A, B) CD86, C, D) CD54, E, F) cell viability. Results are expressed as mean ± S.D. of three independent experiments. Statistical significance was calculated using Student’s t-test (*p < 0.05, **p < 0.01).
Next, we focused on the CD54-only false-positive chemicals (DP, OA, and SA). Induction of CD54 expression by DP and SA were significantly suppressed in cells pretreated with KCl (Figs. 6A, C). Moreover, CD54 expression was suppressed only when KCl pre-treated THP-1 cells were exposed to 250 μg/mL of OA (Fig. 6B). Furthermore, we investigated the effect of KCl pre-treatment of THP-1 cells for the h-CLAT test of CB (CD86-only false-positive chemical) and BA (CD86/CD54 false-positive chemical). In these chemicals, BA had the potential to induce CD86 and CD54 expression and CB induced CD86 expression only in the original h-CLAT test. KCl pre-treatment of THP-1 cells did not affect CD86 expression when applied for the h-CLAT test for CB (Fig.7A), and did not affect both CD86 and CD54 expression in case of BA (Figs. 7C, D).
Suppression of CD54 expression in THP-1 cells treated with CD54 false-positive chemicals DP or OA, SA, and KCl. THP-1 cells were exposed to medium (vehicle), DP (376, 451, 542 µg/mL: 1.2-fold), OA (376, 451, 542 µg/mL: 1.2-fold), or SA (578.7, 694.4, 833.3 µg/mL: 1.2-fold) for 24 hr. A, B, C) CD54, D, E, F) cell viability. Results are expressed as mean ± S.D. of three independent experiments. Statistical significance was calculated using Student’s t-test (*p < 0.05, **p < 0.01).
Effect of CD86 and CD54 expression in THP-1 cells treated with CD86 false-positive chemicals CB or BA and KCl. THP-1 cells were exposed to medium (vehicle), CB (500, 600, 720 µg/mL: 1.2-fold), or BA (416.7, 500, 600 µg/mL: 1.2-fold) for 24 hr. A, C) CD86, D) CD54, B, E) cell viability. Results are expressed as mean ± S.D. of three independent experiments. Statistical significance was calculated using Student’s t-test.
We investigated NLRP3 inflammasome based on previous studies that reported that the inflammasome is induced by a majority of skin sensitizers (Kaplan et al., 2012). The NLRP3 inflammasome is activated by a typical sensitizer, 2, 4-dinitrofluorobenzene (DNFB) (Watanabe et al., 2007; Weber et al., 2010). LPS and sensitizers such as oxazalone and nickel have also been reported to activate the inflammasome via Toll-Like Receptors (TLR) 4 and P2X purinoceptor 7 (P2X7) activation (Schmidt et al., 2010; Trompette et al., 2009; Weber et al., 2010). Therefore, skin sensitization is hypothesized to be associated with NLRP3 inflammasome activation. Furthermore, since CD54 is induced downstream of IL-1β production by the activated inflammasome (Chen et al., 2000). Therefore, we hypothesized that by investigating the effect of NLRP3 inflammasome on CD54 expression in THP-1 cells used in the in vitro skin sensitization test h-CLAT, false positive rates can be improved.
First, we showed a dramatic activation of caspase-1 in cells exposed to LPS and sensitizer DNCB. Furthermore, production of IL-1β was also remarkably increased (Fig. 1). Similar reports have been made by previous studies using different cell lines of keratinocytes and mice (Martin et al., 2008; Watanabe et al., 2007). In this study, THP-1 cells were pretreated with PMA based on a previous study (Petrilli et al., 2007). In addition, our preliminary analyses revealed that IL-1β production in differentiated THP-1 cells is several hundred times higher than that in undifferentiated cells; therefore, we selected the analysis used with differentiation treatment (date not shown). Meanwhile, similar to sensitizers, both activation of caspase-1 and production of IL-1β was confirmed in THP-1 cells, exposed to the false-positive substances OA and SA. These observations confirmed the activation of inflammasome by CD54 positive substances. However, in this study, LPS was used as a positive target that induced NLRP3 inflammasome and also as a CD86/CD54 expression positive test substance under the standards of h-CLAT.
Subsequently, to clarify the relationship between upregulated CD54 and NLRP3 inflammasome activation, we analyzed CD54 expression in THP-1 cells after inhibiting the inflammasome. Increased intracellular ROS (Xia et al., 2016), as well as decrease in intracellular K+ concentration (Pétrilli et al., 2007) induces NLRP3 inflammasome. Our quantitative evaluation (Fig. 2) revealed an increase in the amount of ROS production in cells treated with both sensitizer and false-positive chemicals, except for some test substances. Subsequently, we observed that the ROS quencher, NAC was able to suppress the upregulation of CD86 and CD54 in cells treated with sensitizers DNCB and NB (Figs. 3, 4), while having no effect on CD54 upregulation in cells treated by the false-positive substances DP and OA. We believe this can be explained on the basis of different in antioxidant response modalities. NF-E2 related factor 2 (Nrf2) and thioredoxin reductase are key proteins in the antioxidant response pathway (Arnér, 1999; Kaspar et al., 2009). The activation of Nrf2 by exposure to sensitizer DNCB and the suppression of such activation by the addition of NAC have already been reported (Ade et al., 2009). Furthermore, it has been reported that DNCB directly acts on thioredoxin reductase and inhibits the functions of the protein (Arnér et al., 1995; Nordberg et al., 1998). However, in OA and DP, since significant induction of aldo-keto reductase family 1 member C2 (AKRIC2) gene, that acts downstream of Nrf2, was not observed (Natsch et al., 2013), it was assumed that the Nrf2-mediated antioxidant activity was not induced. Failure of NAC to suppress CD54 upregulation in cells treated with false-positive agents led us to conclude that antioxidant treatment may not be a feasible strategy to minimize false-positive evaluation in tests.
Since it has been reported in previous studies that the addition of KCl suppresses the activation of NLRP3 inflammasome by exposure to MSU or asbestos (Dostert et al., 2008), we examined the effect of intracellular K+ concentration on CD54 upregulation in THP-1 cells that were challenged by the various chemicals used in our study (Figs. 5-7). CD54 upregulation was observed in LPS, sensitizer DNCB, false-positive substances DP, OA, and SA substances. Previous reports have also shown KCl to have a superior inhibitory effect on IL-1β production (Pétrilli et al., 2007). Therefore, we hypothesized that manipulation of intracellular K+ concentration may inhibit the NLRP3 inflammasome, which in turn would attenuate the dramatic upregulation of CD54. Induction of CD54 expression by DP and SA were significantly suppressed in cells pretreated with KCl (Figs. 6A, C). Moreover, CD54 expression was suppressed only when KCl-pre-treated THP-1 cells were exposed to 250 μg/mL of OA (Fig. 6B). However, no significant differences were observed in the upregulation of CD86 and CD54 in cells treated with BA or CB and KCl. We believe this may be due to the NLRP3 inflammasome not being involved in the signaling response to these chemicals. These results are summarized in Table 2. Table 2 also shows that NAC or KCl suppressed the CD54 upregulation in presence of sensitizers, but KCl treatment only suppressed the CD54 upregulation in presence of CD54-only positive chemicals. Further studies are required to clarify the underlying mechanism and to improve the CD54-only false positive evaluation.
In conclusion, we suggest for the first time that the induction of CD54 expression is related to the activation of the NLRP3 inflammasome. This study will help to improve existing test methods like h-CLAT while inciting greater interest in designing alternative and robust methods of the skin sensitization test.
This research was partially supported by a Research on Regulatory Science of Pharmaceuticals and Medical Devices grant from the Japan Agency for Medical Research and development (AMED).
The authors declare that there is no conflict of interest.