2016 年 39 巻 2 号 p. 272-277
Di-n-butyl phthalate (DBP), a phthalate ester, has been shown to have an adjuvant effect on fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse models. Di-n-butyl maleate (DBM), widely used as a plasticizer for industrial application, has been reported to cause dermatitis in humans. DBM is a butyl alcohol ester of di-carboxylic acid that represents a part of the DBP structure, while di-n-butyl fumarate (DBF) is a trans isomer of DBM. We examined whether DBM or DBF exhibits an adjuvant effect like DBP does. When BALB/c mice were epicutaneously sensitized with FITC in the presence of DBM or DBF, the FITC-specific CHS response was enhanced, as we have observed for DBP. As to underlying mechanisms, DBM and DBF facilitated the trafficking of FITC-presenting CD11c+ dendritic cells (DCs) from skin to draining lymph nodes and increased the cytokine production by draining lymph nodes. In conclusion, DBM and DBF may have an effect that aggravates contact dermatitis through a skin sensitization process.
In 1985, a dermatitis outbreak occurred in an envelope-making factory where di-n-butyl maleate (DBM)-containing polyvinyl acetate glue was used.1) In 2009, a case of allergic dermatitis due to occupational exposure to DBM was reported.2) A 47-year-old woman working as a batch mixer in a factory producing gelcoats and specialty paints developed eczematous lesions until she was withdrawn from work. Interestingly, among the North American Contact Dermatitis Group standard series, plastics, glues, adhesives as well as products in the workplace, DBM was the only chemical that gave a positive reaction in the patch test. DBM is a colorless, clear, and oily liquid used for industrial applications. DBM consists of an alkenyl di-carboxylic acid with two butyl alcohol esters of cis configuration.
Di-n-butyl fumarate (DBF) is an isomer of DBM with butyl alcohol esters of trans configuration. There has been no report on dermatitis caused by DBF. However, dimethyl fumarate (DMF) has been banned for the use in consumer products in the European Union (EU), because it caused an unprecedented large-scale dermatitis outbreak.3) DBM and DBF have been used as plasticizers, raw materials for polymer syntheses, and preservatives for oils and fats, but the risk of their use for dermatitis development has not been established.4)
DBM represents a part of the structure of di-n-butyl phthalate (DBP), an aromatic dibutyl ester. DBP is widely used as a plasticizer for consumer products such as paints, varnishes, paper coatings and cosmetics.5,6) Our previous studies demonstrated that DBP contributed to contact hypersensitivity (CHS) to isothiocyanate haptens as adjuvant chemicals.7) Thus, we hypothesized that DBM might exhibit an enhancing effect through a similar mechanism as observed for DBP. We also examined whether or not DBF, an isomer of DBM, produced such an enhancing effect.
In this study, we investigated whether DBM and DBF could have an enhancing effect on a fluorescein isothiocyanate (FITC)-induced mouse CHS model. As underlying mechanisms, we examined the trafficking of FITC-presenting dendritic cells (DCs) from skin to draining lymph nodes as well as cytokine production by lymph nodes. The results showed that not only DBM but also DBF had an enhancing effect, which might contribute to the development of contact dermatitis.
Acetone, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), DBP (CAS No: 84-74-2), hydrogen peroxide, polyoxyethylene (20) sorbitan monolaurate (Tween 20), kanamycin sulfate, and RPMI 1640 were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); DBF (CAS No: 105-75-9) and DBM (CAS No: 105-76-0) from Tokyo Chemical Industry (Tokyo, Japan); bovine serum albumin (BSA; fraction V) from Sigma-Aldrich (St. Louis, MO, U.S.A.); Dulbecco’s modified Eagle’s medium (DMEM) from Nissui Pharmaceuticals (Tokyo, Japan); fetal bovine serum (FBS) from Hyclone (South Logan, UT, U.S.A.); FITC from Dojindo Laboratories (Kumamoto, Japan); and pentobarbital from Kyoritsu Seiyaku Corporation (Tokyo, Japan). Phycoerythrin (PE)-conjugated hamster anti-CD11c monoclonal antibodies (mAb) (clone HL3; immunoglobulin G1 (IgG1)) and a PE-conjugated hamster IgG1 isotype control (clone G235-2356) were purchased from BD Biosciences (San Jose, CA, U.S.A.); purified rat anti-mouse interferon-γ (IFN-γ) mAb (clone AN-18), biotin-conjugated rat anti-mouse IFN-γ mAb (clone R4-6A2), and recombinant mouse IFN-γ from BioLegend (San Diego, CA, U.S.A.); purified rat anti-mouse interleukin-4 (IL-4) mAb (clone 11B11), biotin-conjugated rat anti-mouse IL-4 mAb (clone BVD6-24G2), and recombinant mouse IL-4 from eBioscience (San Diego, CA, U.S.A.). Horseradish peroxidase (HRP)–avidin was purchased from Zymed Laboratories Inc. (South San Francisco, CA, U.S.A.).
AnimalsFemale BALB/c mice were purchased from Japan SLC Inc. (Shizuoka, Japan) at 7 weeks of age and held for 1 week before use. They were housed at 22 to 24°C with 50 to 60% humidity under artificial lighting conditions with a 12-h light/dark cycle. They had access to food (Oriental Yeast Co., Tokyo, Japan) and water ad libitum. Animal care and experiments were performed humanely in accordance with the Law Concerning the Protection and Control of Animals, and with the guidelines for the care and use of laboratory animals of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and those of the University of Shizuoka. The plans of animal studies were reviewed and approved (approval numbers: 136031 and 146135) by the Institutional Animal Care and Use Committee of the University of Shizuoka.
FITC-Induced CHSFITC-induced contact hypersensitivity experiments were performed as described previously.8) Briefly, on days 0 and 7, mice were topically sensitized on shaved forelimb skin with an FITC solution (0.5% [w/v]) in one of the following solvents in a volume of 160 µL under anesthesia. The solvents were acetone alone, DBM (2% [v/v] in acetone), DBF (2% [v/v] in acetone), and DBP (2% [v/v] in acetone). The concentration of 2% DBP has been determined as a minimum level of concentration that produces stable and reproducible results in the FITC-induced CHS. We chose the same concentration for other chemicals as well. On day 14, mice were challenged by painting with 20 µL of an FITC solution (0.5% [w/v] in acetone–DBP [1 : 1]) on the right auricle. The left auricle was painted with 20 µL of acetone–DBP (1 : 1) alone as a control. Ear thickness was measured with a dial thickness gauge (Mitutoyo, Kanagawa, Japan) before elicitation (0 h), and 24, 48, and 72 h after elicitation. Ear swelling at Xh is defined as follows: [thickness of the right ear−thickness of the left ear] at Xh−[thickness of the right ear−thickness of the left ear] at 0 h.
DC-TraffickingTrafficking of FITC-presenting DC from the skin to draining lymph nodes was examined as described previously with some modifications.8) In brief, mice were epicutaneously treated with 160 µL of an FITC solution in acetone alone, or in acetone containing 2% DBM, 2% DBF or 2% DBP on forelimbs. Twenty-four hours after sensitization, brachial lymph nodes were collected and pooled for each condition to prepare single-cell suspensions. After washing in phosphate-buffered saline containing 0.1% BSA and 0.1% NaN3, the cells were adjusted to 2×106 cells/100 µL for staining with PE-conjugated anti-CD11c mAb (2 µg/mL) or isotype control mAb for 15 min at 4°C as described previously.8) After washing with the same buffer and filtering through a cell strainer (70-µm pore, nylon; Corning, Union City, CA, U.S.A.), a total of 5×105 cells were analyzed with a flow cytometer (BD FACS Canto II; BD Biosciences, San Jose, CA, U.S.A.) using gates for forward and side scatters to collect signals of cell-associated fluorescence. An isotype control was used to monitor non-specific binding of antibodies. Data were analyzed with FACSDiva software. The threshold for FITC-positive cells was set in such a way that no more than 0.01% of total lymph node cells exceed this threshold when lymph nodes were obtained from mice that had been treated with 2% DBP in acetone without FITC.
Cytokine Production by Draining Lymph NodesCytokine production by lymph nodes upon skin sensitization to FITC was determined as described previously.9,10) In brief, on days 0 and 7, BALB/c mice were sensitized on shaved forelimbs with 160 µL of an FITC solution under anesthesia. The solvents were acetone alone, or acetone containing 2% DBM, 2% DBF or 2% DBP. Twenty-four hours after the second sensitization, lymph node cells were collected, single-cell suspensions were prepared, and then the cells were cultured (2.5×106 cells/mL) for 72 h in RPMI 1640 supplemented with 10% FBS and 60 µg/mL kanamycin at 37°C under a humidified atmosphere of 5% CO2/95% air. Culture supernatants were collected at 24, 48, and 72 h to evaluate the production of IL-4 and IFN-γ.
Cytokine Concentration MeasurementThe concentrations of IL-4 and IFN-γ in each culture supernatant were determined by means of a sandwich enzyme-linked immunosorbent assay (ELISA) as described previously.9,10) In brief, cytokines in the supernatants were captured with immobilized purified anti-IL-4 or anti-IFN-γ antibodies. To detect the captured IL-4 or IFN-γ, biotin-conjugated anti-IL-4 or anti-IFN-γ was applied followed by HRP-avidin. ABTS and H2O2 were used as enzyme substrates. Recombinant IL-4 and IFN-γ were used to generate standard curves. The cytokine level in each sample was determined from the respective standard curve.
StatisticsDifferences between the group treated with FITC in acetone and each experimental group were analyzed using one-way ANOVA followed by Dunnett’s test with Graphpad Prism 5 (Version 5.02; Graphpad Software, San Diego, CA, U.S.A.).
The structures of DBM, DBF and DBP are shown in Fig. 1. To determine whether DBM and DBF enhance skin sensitization to FITC, mice were epicutaneously sensitized with FITC in the presence of 2% DBM, 2% DBF, or 2% DBP in acetone, or in acetone alone, followed by challenge with FITC in 50% DBP. Compared with sensitization with FITC in acetone alone, sensitization with FITC in the presence of DBP (positive control), DBM or DBF caused an enhanced ear-swelling response (Fig. 2). The ear-swelling peaked at 24 h and then decreased with time. For each time point, the responses of the DBP, DBM and DBF groups were significantly higher than that of the acetone alone group.
Di-n-butyl phthalate (DBP), di-n-butyl maleate (DBM) and di-n-butyl fumarate (DBF) were used in this study.
On days 0 and 7, BALB/c mice were sensitized with 0.5% FITC dissolved in acetone containing 2% DBM, 2% DBF or 2% DBP (positive control), or in acetone alone. One week after the second sensitization, mice were challenged on the right auricle with 0.5% FITC dissolved in 50% DBP in acetone, while 50% DBP in acetone was applied to the left auricle. To differentiate the effects of chemicals on the sensitization and elicitation processes, all mice were challenged with FITC in the presence of 50% DBP. The ear-swelling responses at 24 h (A), 48 h (B), and 72 h (C) after challenge were shown for each individual mice that had been sensitized with FITC in acetone alone (circles: n=6), in acetone–DBP (squares: n=7), in acetone–DBF (triangles: n=6), or in acetone–DBM (inverted triangles: n=6). Horizontal bars represent the means for the groups. The p values denote significance compared with the group sensitized with FITC in acetone alone. Reproducibility was confirmed by two independent experiments.
Trafficking of FITC-bearing cutaneous DC to draining lymph nodes is involved in the initial phase of FITC-induced CHS. We have demonstrated that DBP facilitated DC trafficking from skin to draining lymph nodes.8,10) Twenty-four hours after epicutaneous treatment with FITC in the presence of 2% DBP, DBM or DBF, the number of FITC+CD11c+ cells in brachial lymph nodes was increased as compared with the FITC treatment with acetone alone (Fig. 3). The increase in DC trafficking was in the following order: DBP>DBM>DBF conditions.
(A) Representative cytograms showing the presence of FITC-presenting DC in the draining lymph nodes. Twenty-four hours after skin application of 0.5% FITC dissolved in acetone with 2% DBM, 2% DBF or 2% DBP (positive control), or in acetone alone, cells in draining lymph nodes were analyzed for FITC and CD11c by flow cytometry. The numbers above the respective areas are the percentages of cells relative to total lymph node cells. (B) Summary data representing the percentages of FITC+CD11c+ cells relative to total CD11c+ cells (mean±standard error of the mean (S.E.M.), n=3). The p values denote significance compared with the FITC-application in acetone alone. Reproducibility was confirmed by two independent experiments.
The effect of DBP on the FITC-induced CHS has been related to polarization toward T helper type 2 (Th2) responses.11) In fact, we have demonstrated that FITC-sensitization in the presence of DBP enhanced IL-4 production by draining lymph nodes.12) However, our recent studies revealed that enhanced production of IFN-γ rather than IL-4 was involved in the adjuvant effect of an alternative plasticizer, diisopropyl adipate (DIA).10) To evaluate the roles of cytokines on FITC-sensitization in the presence of DBM or DBF, draining lymph nodes were collected 24h after the second sensitization with FITC. At this point, FITC-primed T cells as well as FITC-presenting DC (Fig. 3) are present within the draining lymph nodes. The cytokine levels in the culture supernatants of the draining lymph node cells were compared (Fig. 4). Under the FITC in 2% DBP-acetone condition, IL-4 as well as IFN-γ accumulated over time. In contrast, only a marginal level of IL-4 or IFN-γ was detected under the FITC in acetone condition at any time point. As for the FITC in DBM–acetone condition, the IL-4 level was significantly higher than that under the FITC in acetone condition in the 72 h supernatant. In contrast, the IFN-γ level was significantly higher under the FITC in DBM–acetone condition in both the 48 and 72 h supernatants. As for the FITC in DBF–acetone condition, IL-4 production was not significantly elevated, while IFN-γ was significantly higher in the 72 h supernatant.
BALB/c mice were sensitized to 0.5% FITC dissolved in acetone containing 2% DBM, 2% DBF, 2% DBP (positive control), or in acetone alone on days 0 and 7. Twenty-four hours after the second sensitization, brachial lymph nodes were collected and single cell suspensions were prepared. The lymph node cells were cultured for 24, 48 or 72 h, and then the concentrations of IL-4 (A) and IFN-γ (B) in the supernatants were determined by ELISA. Each data point corresponds to the result for an individual mouse. Data were collected through three experiments and pooled (n=6). Bars represent the mean for the groups. The p values denote significance compared with the FITC-application in acetone alone. ns, not significant.
Phthalate esters are used as plasticizers for industrial applications to impart flexibility to consumer products, especially those made of polyvinyl chloride. One of the phthalate esters, DBP, is ubiquitously present in environments, and its potential toxicity is gaining attention increasingly. Some reports have revealed the risk to reproductive health of the uptake of DBP.13–15) In addition, our previous studies have demonstrated that DBP plays the role of an adjuvant in an FITC-induced CHS mouse model.8,16) We hypothesized that DBM and DBF exposure might cause similar responses for the following reasons. Structurally, DBM and DBF consist of an aliphatic di-carboxylic acid with a single double bond with two butyl alcohol esters of cis and trans configuration, respectively (Fig. 1). The former represents a part of DBP, which is an aromatic dibutyl ester.
To test enhancing effect, BALB/c mice were epicutaneously sensitized with FITC in the absence or presence of 2% DBM, DBF or DBP (positive control), and then FITC-dependent inflammation was measured as the ear-swelling response. In contrast to the low-level of sensitization with FITC in acetone, DBM and DBF enhanced the sensitization to FITC to similar levels of sensitization in the presence of DBP (Fig. 2). The enhanced ear-swelling responses were significantly elevated even on day 3 after elicitation. These results suggested DBM as well as DBF exhibited an enhancing effect on skin sensitization to other immunogenic chemicals such as FITC.
As a mechanism initiating the enhanced sensitization, we investigated the trafficking of FITC-presenting CD11c+ DC from skin to draining lymph nodes. Epicutaneous treatment with FITC in the presence of 2% DBM or 2% DBF facilitated DC-trafficking to a similar level of positive control, 2% DBP (Fig. 3).
In FITC-induced CHS, in which 50% DBP is present in the solvent, production of IL-4 was shown to be required for sensitization by the lack of sensitization in signal transducer and activator of transcription 6 (STAT6) deficient mice.11) On the other hand, an alternative plasticizer, DIA, which has an adjuvant effect on the FITC-induced CHS, augmented the production of IFN-γ while marginal increase in the IL-4 production was observed.10) Thus, it is reasonable to assess the production of IL-4 as well as IFN-γ as markers following the hapten-specific T cell activation.
We epicutaneously sensitized with FITC two times with one week interval. Twenty-four hours after the second treatment, draining lymph nodes are expected to contain both FITC-presenting DC and FITC-specific helper T cells. Thus, cells from draining lymph nodes were cultured and the cytokines secreted into the supernatant were quantified by cytokine-specific ELISA. We have repeatedly demonstrated that 50% DBP enhanced the production of not only IL-4 but also IFN-γ.9,10) In the present experiments, we showed that 2% DBP also enhanced the production of IL-4 as well as IFN-γ (Fig. 4).
Regarding IL-4, FITC-sensitization in the presence of 2% DBM significantly augmented the IL-4 accumulation compared with under the acetone alone condition as seen in the 72 h supernatant. The IL-4 concentrations under the acetone alone condition were marginal, which is consistent with the paucity of FITC-presenting DC in draining lymph nodes and with weak ear-swelling responses. The IL-4 level under the 2% DBF condition was above the detection limit at 72 h, but was not significantly higher than that under the acetone alone condition. On the other hand, the IL-4 accumulation under the 2% DBP condition was evident in 48 and 72 h supernatant. Thus, we think the order of IL-4 production is DBP>DBM>DBF.
Regarding IFN-γ, FITC-sensitization caused an early accumulation of IFN-γ in the supernatant, especially under the 2% DBP condition after 24 h of culture. In the 48 h supernatant, the 2% DBM but not the 2% DBF condition gave rise to significant accumulation of IFN-γ. In the 72 h supernatant, the 2% DBM as well as the 2% DBF condition caused significant production of IFN-γ. Thus, we think the order of IFN-γ production is also DBP>DBM>DBF.
It should be mentioned that the immunogenicity of DBM and DBF to mouse might complicate the interpretation of the enhanced cytokine production. Thus, cells from draining lymph nodes could have produced cytokines due to the DBM or DBF-specific immune responses rather than FITC-specific ones. If this is the case, bystander immune responses in the draining lymph nodes facilitated the FITC-specific response. However, in preliminary experiments, when mice were epicutaneously treated with 2% DBM in acetone twice followed by elicitation with 2% DBM in acetone, we did not observe any ear swelling response (data not shown). This may suggest the lack of immunogenicity of DBM to mouse at least under our current experimental conditions. Immunogenicity of DBM and DBF to mouse should be evaluated in further experiments.
We do not exclude a possibility that physical properties of DBM, DBF and DBP are involved in the enhanced sensitization to FITC through an increase in skin retention and/or in skin permeability. However, our previous results revealed that lipophilicity does not necessarily determine the enhancement. For example, di-n-heptyl phthalate (DHPP) enhanced sensitization to FITC, but di(2-ethylhexyl)phthalate (DEHP) did not.8,17) DEHP has a similar but slightly higher value in the octanol–water partition coefficient (log Pow) than that of DHPP.18,19) Another example is diethyl phthalate (DEP).18,19) DEP enhanced sensitization to FITC as well as IL-4 production upon application to the skin with FITC.8,12) Log Pow for DEP is even lower than that of DIA, suggesting that molecules with relatively low lipophilicity, such as DIA and DEP, can enhance sensitization to FITC.8,10) We have demonstrated a correlation of the adjuvant effect with the ability to activate transient receptor potential ankyrin 1 (TRPA1) cation channels.10,17,20) We are currently under investigation whether DBM and DBF activate TRPA1.
According to several case studies, frequent cases of dermatitis occurred in factories making envelopes where DBM-containing polyvinyl acetate glue was used.1) Another study showed a case of allergic dermatitis reaction to DBM in a factory that produced liquid resins containing DBM.2) Considering a low-grade result in the patch test (+1 positive; light erythma, nonvesicular) recommended by the International Contact Dermatitis Research Group21) upon exposure to 5% DBM, we suspected that DBM may not only function as a hapten by itself but may enhance sensitization to other haptens through a combined effect.
Regarding the combined effects of chemicals, it is important to know how these chemicals are used. DBM was also studied as a promising oxygenating agent for diesel fuel.22) As to transdermal drug delivery, incorporation of DBM into the synthesis of a new type of membrane was shown to be useful to achieve controlled drug release from the membrane.23)
Although 2% DBM may be a high dose as we regularly contact in our daily life, we think it is not out of range for us to contact. A transdermal therapeutic system such as a membrane-stabilizing drug was reported to include DBM monomers in a ratio by weight of 20%.23) Of course it is important to assess the actual concentration of DBM leaching out from such membrane. On the other hand, relatively high dose (5%) of DBM was required to obtain a low-grade positive reaction in a sensitized person with patch test,2) suggesting that a certain level of concentration is required in experimental settings. Another report revealed that DBM was detected inside furniture boxes with a disposable desiccant. The DBM concentrations in the box wall materials were reported to range from 29 to 720 mg/kg.24)
It is not known whether DBF induces contact allergies. However, a similar diester with two methyl groups instead of butyl ones, DMF, recovered from furniture upholstery, was identified as a potent contact sensitizer.25) Thus, DMF was banned from use in consumer products in the EU because of a large-scale outbreak of dermatitis.3)
DBF is supposed to be a good control because it is the trans isomer of DBM. In this study, DBF was also found to enhance FITC-induced CHS. The results may also suggest that other short chain fumarate diesters might enhance sensitization to other haptens.
In conclusion, FITC-induced CHS models revealed that a combined effect of DBM or DBF with other immunogenic haptens might contribute to an aggravation of contact dermatitis through a sensitization process. As underlying mechanisms, facilitated trafficking of FITC-presenting DC as well as cytokine production by draining lymph nodes were shown. Although more detailed quantitative analyses are needed to estimate the relative risk, these results suggest that not only phthalate esters but also alkenyl carboxylic acid diesters may exhibit an enhancing effect regardless of whether of cis or trans configuration.
This work was supported by a Grant-in-Aid for Challenging Exploratory Research (Grant number 15K14990) from Japan Society for the Promotion of Science to YI. We thank Mr. Nicholas J. Halewood for providing language help.
The authors declare no conflict of interest.
