Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Notes
An Aliphatic Ester Diisopropyl Sebacate Exhibited an Adjuvant Effect on Fluorescein Isothiocyanate-Induced Contact Hypersensitivity Mouse Models
Kohta KurohaneAyako KimuraRie TerasawaKamiyu KobayashiWakana SuzukiTakeshi MatsuokaYasuyuki Imai
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2018 年 41 巻 1 号 p. 147-150

詳細
Abstract

Alternative plasticizers have become more popular due to health concerns about phthalate esters. We demonstrated that phthalate esters enhanced skin sensitization to fluorescein isothiocyanate (FITC) in mouse contact hypersensitivity models. Alternative plasticizers have not been well studied as to their effect on the immune system. We previously found that diisopropyl adipate (DIPA), an aliphatic dicarboxylic acid ester, enhanced skin sensitization to FITC. Sebacate esters are also widely used as alternative plasticizers. Here we tested diisopropyl sebacate (DIPS), which has the same alcohol with an aliphatic dicarboxylic acid of longer chain, using BALB/c mice. The results showed that DIPS facilitated skin sensitization to FITC and increased FITC-presenting dendritic cell trafficking from the skin to draining lymph nodes. Furthermore, DIPS activated transient receptor potential ankyrin 1 (TRPA1). The latter feature has been commonly observed for phthalate esters and DIPA, which have adjuvant effects. In summary, the adjuvant effect of a sebacate ester was demonstrated in a mouse model.

Due to health concerns about phthalate esters, expectation of the use of alternative plasticizers in consumer products has been increasing.1) Phthalate esters are made up of an aromatic dicarboxylic acid and two alcohols. One group of alternative plasticizers is the adipate esters, which consist of an aliphatic dicarboxylic acid. Another type of aliphatic plasticizers is sebacate esters, which are applied for medical devices as well as drug formulation.24)

We have been investigating phthalate esters from an immuno-toxicological point of view. We showed that contact sensitization to fluorescein isothiocyanate (FITC) is enhanced in the presence of certain types of phthalate esters in mouse models.5,6) Thus, phthalate esters with short chain alcohols, such as dibutyl phthalate (DBP) and di-n-propyl phthalate (DPP), exhibited strong activities among phthalate esters.6) Those phthalate esters were shown to exhibit agonist activity toward transient receptor potential ankyrin 1 (TRPA1) cation channels, TRPA1 being used as a nociceptor by sensory neurons.7) The results may suggest a close relationship between the adjuvant activity on skin sensitization and TRPA1 agonistic activity.

As to alternative plasticizers, we previously showed that diisopropyl adipate (DIPA) facilitated skin sensitization to FITC in vivo. TRPA1 agonist activity of DIPA was also shown in vitro.8) In the present study, we investigated the activity of diisopropyl sebacate (DIPS), in which isopropyl alcohol is a shared component with DIPA. As a positive control, we employed DBP to check each experimental system. The results clearly demonstrated that DIPS also activated TRPA1 and enhanced FITC-induced contact hypersensitivity (CHS).

MATERIALS AND METHODS

Chemicals and Reagents

Acetone, dibutyl phthalate (DBP; CAS No.: 84-74-2), and DIPS (CAS No.: 7491-02-3) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), FITC and Fluo 4-AM from Dojindo Laboratories (Kumamoto, Japan), dimethyl sulfoxide (DMSO) from Nacalai Tesque (Kyoto, Japan), and ionomycin from Life Technologies (Carlsbad, CA, U.S.A.). Phycoerythrin (PE)-conjugated hamster anti-mouse 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.).

Cells

Establishment and maintenance of Chinese hamster ovary (CHO) cells expressing human TRPA1 (TRPA1-CHO) were performed as described previously.79) T-REx CHO cells that do not express TRPA1 were used as a specificity control.

Animals

Specific pathogen-free female BALB/c mice were purchased from Japan SLC Inc. (Shizuoka, Japan) at 7 weeks of age. Animal care and experiments were performed as described previously8) in accordance with the guidelines published by the Ministry of Education, Culture, Sports, Science and Technology of Japan, and those of the University of Shizuoka. The procedures for animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Shizuoka (approval numbers: 146135 and 166199).

TRPA1 Activation in Vitro

TRPA1 activation was detected using TRPA1-CHO cells loaded with a Ca2+ sensitive fluorescent probe, Fluo 4-AM, as described previously.79) After addition of a test chemical (at 30 s), the intracellular Ca2+ level was automatically measured over time for 120 s with a fluorometric imaging plate reader, FLEXstation II (Molecular Devices, Sunnyvale, CA, U.S.A.). During the 120 s period with a test sample, the highest Ca2+ level was considered as a response for each sample. To calculate the % of maximal response, the maximal Ca2+ level was determined with 5 µM ionomycin added at 150 s.

FITC-Induced CHS

CHS experiments were carried out as described previously.6,8) BALB/c mice were epicutaneously sensitized with 0.5% FITC on shaved forelimbs on days 0 and 7. FITC was dissolved in one of the following solvents: acetone alone, acetone containing 2% (v/v) DBP, or 20% DIPS. On day 14, a 0.5% FITC solution in acetone–DBP (1 : 1) was applied on the right auricle to elicit inflammation, while acetone–DBP alone was applied on the left auricle as a control. To distinguish the effect on the sensitization phase from that on the elicitation phase, all mice were challenged with FITC dissolved in acetone–DBP (1 : 1) as described before. Ear thickness was measured before (0 h), and 24, 48 and 72 h after elicitation by means of a dial thickness gauge (Mitutoyo, Kanagawa, Japan). Ear swelling at X h is defined as follows: (thickness of the right ear−thickness of the left ear) at X h−(thickness of the right ear−thickness of the left ear) at 0 h.

Dendritic Cell (DC) Trafficking

Trafficking of FITC-presenting DC from the skin to draining lymph nodes was examined as described before.10) In brief, mice were epicutaneously treated with 0.5% FITC in acetone alone or in acetone containing 20% DIPS or 50% DBP (positive control). Twenty-four hours after treatment, single-cell suspensions were prepared from brachial lymph nodes. After staining with PE-conjugated anti-CD11c mAb (2 µg/mL) or isotype control mAb, 5×105 cells were analyzed with a flow cytometer (BD FACS Canto II; BD Biosciences, San Jose, CA, U.S.A.). An isotype control was used to monitor non-specific binding of antibodies. Data were analyzed with FACSDiva software.

Statistics

Multiple comparisons were performed using one-way ANOVA followed by Dunnett’s test with Graphpad Prism 5 (version 5.02; Graphpad Software, San Diego, CA, U.S.A.).

RESULTS

As an ester made of an aliphatic dicarboxylic acid, we previously demonstrated that DIPA increased the cytoplasmic Ca2+ level in TRPA1-CHO cells but not in T-REx CHO cells without TRPA1 expression.8) We examined the effect of DIPS on TRPA1 activation. DIPS dose-dependently increased the cytoplasmic Ca2+ level in TRPA1-CHO cells but not in T-REx CHO cells (Fig. 1). The 50% effective concentration (EC50) value for DIPS was 5.8 µM.

Fig. 1. Increase of Intracellular Ca2+ in TRPA1-Expressing Cells Caused by DIPS

Various concentrations of DIPS (abscissa) were added to TRPA1-CHO (closed circles) or control T-REx CHO (open circles) cells, and the intracellular Ca2+ level was recorded. The percentages of the maximal calcium level (ionomycin) are shown on the ordinate. Each datum is the mean±standard error (n=4). Error bars underneath the symbols are not visible.

We then examined the effect of DIPS on the sensitization phase of FITC using a mouse CHS model. When FITC was epicutaneously applied in acetone, a very weak ear-swelling response to FITC was observed. When sensitized in the presence of 20% DIPS, mice exhibited much stronger immunity against FITC (Fig. 2). As we have already demonstrated,8) 2% DBP exhibited enhanced sensitization to FITC as well.

Fig. 2. Adjuvant Effect of DIPS on Skin Sensitization to FITC

BALB/c mice were sensitized with 0.5% FITC dissolved in acetone alone (open circles; n=6, negative control), or acetone in the presence of 2% DBP (closed circles; n=6, positive control) or 20% DIPS (closed triangles; n=7), on days 0 and 7. On day 14, the mice were challenged on an ear auricle with 0.5% FITC in acetone–DBP (1 : 1). The ear-swelling responses at 24, 48 and 72 h after challenge in an individual mouse (each point) and means (horizontal bars) are shown. * p<0.05, ** p<0.01, *** p<0.001 compared with acetone alone conditions.

As to the mechanism underlying the enhanced sensitization, we examined DCs in draining lymph nodes, which represent migrants from skin sites, by flow cytometry 24 h after FITC application. The FITC-presenting DC are recognized as FITC+ and CD11c+, the latter being a DC marker. Under the acetone alone conditions, a very small proportion of draining lymph node DC was FITC positive (Fig. 3). As a positive control, we used 50% DBP to recognize FITC positive DC more clearly. Under the DBP conditions, FITC-presenting DC increased in the draining lymph nodes. In the presence of DIPS, DC-trafficking was significantly elevated as compared with under the acetone alone conditions. The percentages of FITC+CD11c+ cells relative to total CD11c+ cells were calculated from three independent experiments as means and standard errors. The values are 0.6±0.1, 25.0±2.0 and 21.9±0.1 under acetone alone, 50% DBP and 20% DIPS conditions, respectively. These values under the DBP and DIPS conditions are significantly higher than that under the acetone alone one (p<0.001, Dunnett’s test).

Fig. 3. Increased DC-Trafficking Induced by DIPS

Twenty-four hours after skin application of 0.5% FITC in acetone alone, or in acetone containing 50% DBP (positive control) or 20% DIPS, cells in the draining lymph nodes were analyzed as to the levels of FITC (abscissa) and CD11c (ordinate) by flow cytometry. Representative cytograms for three independent experiments are shown. The numbers in the respective areas are the percentages of cells relative to total lymph node cells.

DISCUSSION

Phthalate esters are major plasticizers used for many kinds of consumer products. DBP has become a primary target for the reduction of childrens’ exposure due to the reproductive toxicity, endocrine disruption and neurotoxicity.1,11,12) Reflecting this situation, the use of alternative plasticizers has been increasing.

One of the alternative plasticizers is DIPA. DIPA is used for cosmetics and drug formulations.13,14) DIPA is an ester made of an aliphatic dicarboxylic acid of carbon number 6 (C6). DIPS is another aliphatic ester but has a dicarboxylic acid of C10. One may argue that the difference between DIPA and DIPS is only the difference in the spacer length between the carboxylic acid groups. However, unlike phthalic acid, which has two carboxylic acids with fixed angle on a benzene ring, the spacer of aliphatic dicarboxylic acids allows free rotation due to the sp3 carbon bonds. Therefore it is an important new finding that DIPA8) and DIPS (Fig. 1) both activated TRPA1 despite the potential structural differences due to the spacer length. It is also important that the TRPA1 agonistic activity is associated with the enhanced skin sensitization to FITC.

DIPS induced an increase of intracellular Ca2+ in TRPA1-CHO cells but not in CHO cells without TRPA1 expression. DIPS activates TRPA1 at relatively low concentrations (EC50=5.8 µM). The EC50 values of allyl isothiocyanate (a typical agonist) and DIPA for the activation of TRPA1 were calculated to be 0.2 µM and 37 µM, respectively.8) As to FITC-CHS, the presence of 20% DIPS enhanced sensitization to FITC to a similar level in the presence of 2% DBP. DIPS also caused the trafficking of a comparable number of FITC-positive DC in the draining lymph nodes at 24 h after sensitization compared with under the 50% DBP conditions. Moreover, a clearly distinguishable cell population of FITC+CD11c+ was found in the flow cytometry cytograms, as was the case under the DBP conditions. These results suggested that not only adipate esters but also sebacate esters have the ability to enhance skin sensitization processes against chemical haptens such as FITC potentially through the TRPA1 pathway.

TRPA1 involvement in CHS and neurogenic inflammation in general is still a controversial issue.15) In our experience, the enhanced skin sensitization to FITC in the presence of DBP9) or DIPA8) was inhibited by TRPA1-selective antagonist, HC-030031. We also observed that desensitization by the skin pretreatment with a strong TRPA1 agonist, allyl isothiocyanate, suppressed FITC-CHS.16) As to another mouse model, treatment of wild type mice with HC-030031 or use of TRPA1-deficient mice was reported to result in the diminished response in oxazolone-induced CHS.17)

As to DIPS, we tried to test whether HC-030031 could inhibit TRPA1 activation in vitro. However, when HC-030031 was mixed with DIPS, precipitate was formed in the mixture, so we could not test the effects on TRPA1 activation (unpublished results). Following this result, we did not carry out in vivo experiments using HC-030031 as to the effect on FITC-CHS at present. To demonstrate the involvement of TRPA1 directly, studies using TRPA1-deficient mice or other means of denervation will be considered.

It is not established what type of cells in the skin receive stimuli through TRPA1 leading to the enhancement of FITC-CHS sensitization process. Besides sensory neurons, TRPA1 is expressed on keratinocytes in the skin basal layer.15) Although the expression of transient receptor potential vanilloid 1 channel on DC has been reported,18) expression of TRPA1 on DC has not been established.15) On the other hand, neuropeptides derived from sensory neurons are known to act on DC.15) In the latter possibility, DIPS may stimulate TRPA1 on sensory neurons, which in turn release neuropeptides toward DC. DC activated with neuropeptides may initiate immune responses in the draining lymph nodes.

We do not exclude a possibility that DIPS may contribute to the sensitization process of FITC-CHS by other mechanisms. Due to physicochemical properties, DIPS may enhance the retention and penetration of immunogenic hapten on the skin. Whatever in vivo mechanisms of the adjuvant effect are, TRPA1 activation seems to be a useful sign to predict the adjuvant effect on FITC-CHS.

In this study, we used DIPS to compare the results for DIPA, which has an isopropyl alcohol as a shared component. As a commonly used alternative plasticizer, dibutyl sebacate has been well documented.24,19) However, our previous studies on phthalate esters demonstrated that not only DBP but also phthalate esters with short chain alcohol exhibited adjuvant effect.6,7) As to sebacate esters, there is a report of CHS due to hand cream containing diethyl sebacate.20) It is interesting to carry out systematic studies on aliphatic type alternative plasticizers with short chain alcohols such as dibutyl sebacate and diethyl sebacate.

Acknowledgment

This work was supported by a Grant-in-Aid for Challenging Exploratory Research (Grant number 15K14990) from the Japan Society for the Promotion of Science (JSPS) to YI. We thank Mr. Nicholas J. Halewood for the language editing service.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
© 2018 The Pharmaceutical Society of Japan
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