TRPA1 and TRPV1 activation is a novel adjuvant effect mechanism in contact hypersensitivity

Journal of Neuroimmunology 207 (2009) 66–74

Contents lists available at ScienceDirect

Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

TRPA1 and TRPV1 activation is a novel adjuvant effect mechanism in contact hypersensitivity Takahiro Shiba a, Takashi Maruyama a, Kohta Kurohane a, Yusaku Iwasaki b, Tatsuo Watanabe b, Yasuyuki Imai a,⁎ a b

Laboratory of Microbiology and Immunology and the Global COE Program, University of Shizuoka Graduate School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan Laboratory of Food Chemistry and the Global COE Program, University of Shizuoka Graduate School of Nutritional and Environmental Sciences, Shizuoka 422-8526, Japan

a r t i c l e

i n f o

Article history: Received 12 September 2008 Received in revised form 6 December 2008 Accepted 8 December 2008 Keywords: Allergy Skin TRPA1 TRPV1

a b s t r a c t We have revealed that local stimulation of sensory neurons is involved in the adjuvant effect of dibutyl phthalate (DBP) in a fluorescein isothiocyanate-induced mouse contact hypersensitivity model. Transient receptor potential (TRP) A1 and TRPV1 seemed to be candidate DBP targets. Here we directly demonstrated that DBP activates a subset of neurons in mouse dorsal root ganglia responsive to TRPA1 and TRPV1 agonists. TRPA1 and TRPV1 activation was further demonstrated using cultured cells expressing TRP channels. Among structurally different phthalate esters, there is a positive relationship between the activation of TRPA1- or TRPV1-expressing cells and the adjuvant effect. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Various chemicals can induce contact hypersensitivity upon repeated exposure of the skin to them. Fifteen to twenty percent of adult Danish population was reported to have contact hypersensitivity to one or more chemicals present in their environments (Nielsen et al., 2001). Although previous studies have focused on chemicals exhibiting antigenicity, chemicals with adjuvant effects in the environment may also play a role in this increase in hypersensitivity. For example, phthalate esters, which are widely used as plasticizers for plastics and found in the environment, are candidate chemicals with adjuvant effects (Larsen et al., 2002). It has been reported that certain types of phthalate esters in house dust were implicated in allergic symptoms in children (Bornehag et al., 2004). In addition, some phthalate esters

Abbreviations: AITC, allyl isothiocyanate; APC, antigen presenting cell; BSA, bovine serum albumin; CAP, capsaicin; CGRP, calcitonin gene-related peptide; CHO, Chinese hamster ovary; CHS, contact hypersensitivity; DBP, dibutyl phthalate; DC, dendritic cell; DEHP, di(2-ethylhexyl) phthalate; DEP, diethyl phthalate; DHPP, diheptyl phthalate; DHXP, dihexyl phthalate; DINP, diisononyl phthalate; DMP, dimethyl phthalate; DMSO, dimethylsulfoxide; DPNP, dipentyl phthalate; DPP, dipropyl phthalate; DRG, dorsal root ganglia; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HBSS, Hanks' balanced salt solution; HEPES, N-2-hydroxyehtylpiperazine-N′-2-ethanesulfonic acid; HEK, human embryonic kidney; IL, interleukin; MEM, minimum essential medium; SEM, standard error of the mean; TRP, transient receptor potential. ⁎ Corresponding author. Laboratory of Microbiology and Immunology, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan. Tel.: +81 54 264 5716; fax: +81 54 264 5715. E-mail address: [email protected] (Y. Imai). 0165-5728/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.12.001

with short alkyl chains are used for cosmetics and mosquito repellents for topical use (Api, 2001; Vartak et al., 1994). Because of this, it is important to determine whether phthalate esters applied to the skin exert an adjuvant effect on cutaneous allergic responses. In immunological experiments, dibutyl phthalate (DBP) has been empirically included in the solvent system for fluorescein isothiocyanate (FITC), which is widely used as a hapten in mouse contact hypersensitivity (CHS) models. We previously demonstrated that DBP and some other phthalate esters with certain alkyl chain lengths exhibit an adjuvant effect during sensitization to FITC (Imai et al., 2006; Sato et al., 1998). These phthalate esters promote trafficking of FITC-presenting dendritic cells (DC) to draining lymph nodes in the sensitization phase of FITC-induced CHS (Imai et al., 2006; Sato et al., 1998). We have also found that DBP induces the migration of CD301a+ dermal macrophages to draining lymph nodes (Sato et al., 1998). The initiation of this process was reconstituted in an in vitro organ culture of skin explants. If mouse skin was cultured after application of DBP, a decrease in the number of CD301a+ macrophages was observed in explants. On the other hand, if DBP was applied to the excised skin before organ culture, no decrease was observed (Chun et al., 2000). These results suggested that a rapid response involving the whole body, including the nervous system, might be necessary for the effect of DBP. The immune system has some connection to the peripheral nerve system (Beresford et al., 2004; Liu et al., 2006). Anatomically, epidermal Langerhans cells have been shown to be closely associated with calcitonin gene-related peptide (CGRP)-containing nerve fibers (Beresford et al., 2004; Hosoi et al., 1993). Functionally, it has been shown that CHS is abolished through systemic deletion of capsaicin

T. Shiba et al. / Journal of Neuroimmunology 207 (2009) 66–74

(CAP)-sensitive nerve fibers (Beresford et al., 2004). These studies indicated that peptidergic nerve fibers are required for the CHS response. CAP-sensitive nerve fibers express a CAP receptor, transient receptor potential (TRP) V1 (Caterina et al., 1997). TRPV1 is a calcium permeable cation channel that belongs to the TRP family and mediates multiple noxious stimuli such as CAP, high temperature and an acidic environment (Caterina et al., 1997; Dhaka et al., 2006; Jordt et al., 2000; Tominaga et al., 1998). Some studies suggested that activation of TRPV1 on sensory neurons influences the immune system (Bánvölgyi et al., 2005; Murai et al., 2008; Razavi et al., 2006). Some TRPV1expressing sensory neurons have been shown to express TRPA1, another member of the TRP family of calcium permeable cation channels (Story et al., 2003). TRPA1 reacts with various noxious compounds including allyl isothiocyanate (AITC) and cinnamaldehyde, which constitute the pungent ingredients of mustard and cinnamon, respectively (Bandell et al., 2004; Jordt et al., 2004). TRPA1 is also activated by environmental irritants such as acrolein, which elicits nociceptive and inflammatory reactions against air pollutants and cigarette smoke (Bautista et al., 2006). It is known that the activation of TRPV1 or TRPA1 on sensory nerve endings results in the release of neuropeptides such as CGRP (Bautista et al., 2005; Zygmunt et al., 1999). Several studies have revealed inhibitory or stimulatory effects of neuropeptides on the immune system (Asahina et al., 1995; Niizeki et al., 1999; Peters et al., 2006). We have also found another connection between the nervous system and FITC-induced CHS. Local pretreatment with CAP or AITC at the site of FITC treatment suppressed the phase of sensitization to FITC. Such treatment also inhibited trafficking of FITC-presenting activated DCs to draining lymph nodes (Maruyama et al., 2007a). Moreover, pretreatment with a CGRP antagonist at the site of FITC application inhibited the sensitization (Maruyama et al., 2007a). Pretreatment with CAP or AITC may desensitize TRPV1 or TRPA1, which may be involved in the initiation of DC trafficking upon sensitization to FITC. However, no direct evidence that DBP and other phthalate esters activate sensory neurons through TRP channels has been provided. In this study, we focused on the question of whether phthalate esters activate sensory neurons through TRP channels. First, we examined whether DBP activates neurons obtained from mouse dorsal root ganglia (DRG). We also examined whether neurons responsive to DBP are also responsive to CAP or AITC, or both. Second, we examined whether DBP activates TRPV1 or TRPA1 channels using TRPV1- or TRPA1-expressing Chinese hamster ovary (CHO) cells. Using this in vitro system, we examined whether there is a positive relationship between the adjuvant activity and TRP channel activation using several different types of phthalate esters. 2. Materials and methods 2.1. Compounds AITC, dimethyl phthalate (DMP), diethyl phthalate (DEP), DBP, diheptyl phthalate (DHPP), di(2-ethylhexyl) phthalate (DEHP), and diisononyl phthalate (DINP) were purchased from Wako Pure Chemicals (Osaka, Japan). Dipropyl phthalate (DPP), dipentyl phthalate (DPNP), and dihexyl phthalate (DHXP) were purchased from Kanto Chemicals (Tokyo, Japan). CAP was purchased from Sigma (St. Louis, MO). For in vitro experiments, all compounds were dissolved in dimethylsulfoxide (DMSO) (Nacalai Tesque, Kyoto, Japan) to make stock solutions. The stock solutions were diluted on the day of the experiment. The final concentration of DMSO did not exceed 0.1%. 2.2. Nerve cell isolation from mouse DRG The animal care and experiments were performed in accordance with the guidelines for the care and use of laboratory animals of the

67

University of Shizuoka. Five-week-old specific pathogen-free female CD-1 (ICR) mice (Japan SLC Inc., Shizuoka, Japan) were sacrificed, and their DRG were rapidly dissected out. The ganglia were incubated in complete minimum essential medium (MEM), which consists of Earl's balanced salt solution (Sigma) supplemented with 10% fetal bovine serum (FBS) (Hyclone, South Logan, UT), 1% MEM Vitamin solution (Sigma), 1% Glutamax solution (Invitrogen, Carlsbad, CA), 100 units/ml penicillin G (Wako), and 100 μg/ml streptomycin (Wako) containing 0.25 mg/ml collagenase P (Roche, Basel, Switzerland) for 20 min at 37 °C. After washing by centrifugation, the ganglia were re-suspended in the complete MEM containing 100 ng/ml of nerve growth factor-7 S (Sigma). Cells were dissociated by gentle pipetting using Pasteur pipettes with fine open tips, and then cultured overnight at 37 °C under 5% CO2/95% air on coverslips that had been coated with poly-Llysine (Sigma). 2.3. Stable expression of TRPA1 or TRPV1 on CHO cells To avoid cell damage, CHO cells stably expressing TRPA1 were established with a T-REx system according to the manufacturer's instructions (Invitrogen, CA). Human TRPA1 cDNA was amplified by RT-PCR from mRNA obtained from human fibroblasts, WI-38 cells (RIKEN Bio-Resource Center, Tsukuba, Japan). The primer pair was: 5'-GACGTAAGCTTTGGGGTCAATGAAGTGCAG-3' and 5'-AAGACTCGAGGAAGGTCTGAGGAGCTAAGGC-3'. The cDNA was cloned into pcDNA4/TO, and the DNA sequence was verified. This construct was then transfected by means of Lipofectamine 2000 (Invitrogen) into T-REx CHO cells, which stably express the tetracycline repressor protein transcribed from pcDNA6/TR in the cells. After selection in the presence of zeocin (for pcDNA4/TO) and blasticidin (for pcDNA6/ TR), we obtained CHO cells stably expressing TRPA1. The cells were maintained in Ham's F-12 medium (Sigma) containing 10% FBS (Hyclone), 100 units/ml penicillin G, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B (Invitrogen) at 37 °C under 5% CO2/95% air. One day before the experiments were performed, the culture medium was replaced by Ham's F-12 medium containing 10% FBS and 1 μg/ml tetracycline (Invitrogen) to induce TRPA1 expression. Rat TRPV1 cDNA was obtained and subcloned into pcDNA3 (Invitrogen) as described previously (Morita et al., 2006). The cDNA was then transfected into CHO-K1 cells using Lipofectamine 2000. After culturing in the presence of G418, we obtained CHO cells stably expressing TRPV1. 2.4. Calcium imaging under a confocal microscope For calcium imaging, neurons on coverslips were loaded with a fluorescent calcium indicator, 2.5 μM Fluo 4-AM (Dojindo Laboratories, Kumamoto, Japan), in the complete MEM for 30 min at 37 °C. Cells on the coverslips were perfused with Hanks' balanced salt solution (HBSS) containing 20 mM N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES) (pH 7.4 adjusted with NaOH). Samples were added to the solution during perfusion. The fluorescence intensity of each cell (excitation at 485 nm, emission at 540 nm) was recorded under a confocal microscope (MRC-1024; Bio-Rad Laboratories, Hercules, CA) at room temperature, and analyzed with Laser Sharp version 2.1A software (Bio-Rad). Only neurons that responded to ionomycin (Invitrogen) were included in the results as cells with an intact plasma membrane. 2.5. Calcium imaging with a FLEXstation II TRPA1- or TRPV1-expressing CHO cells were cultured in Ham's F-12 medium in the wells of a 96-well, black-walled, clear-bottomed plate (Corning Inc., Corning, NY) at a density of 4.0 × 104 cells/well for 24 h at 37 °C under an atmosphere of 5% CO2/95% air. The cells were then loaded with 3 μM Fluo 4-AM in HBSS containing 20 mM HEPES,

68

T. Shiba et al. / Journal of Neuroimmunology 207 (2009) 66–74

0.1% bovine serum albumin (BSA) (Sigma), and 2.5 mM probenecid (Wako) for 60 min at 37 °C under 5% CO2/95% air. After the Fluo 4-AM solution had been discarded, HBSS containing HEPES, BSA and probenecid was added. The measurements were performed at room temperature with a fluorometric imaging plate reader, FLEXstation II (Molecular Devices, Sunnyvale, CA) (excitation at 485 nm, emission at 525 nm). A sample was added at 20 s and ionomycin was added at 110 s after the start of measurement. Data were analyzed using Prism 4 software (GraphPad, San Diego, CA). 2.6. Calcium imaging with a CAF-110 Experiments were performed as described previously (Morita et al., 2006). TRPV1-expressing human embryonic kidney (HEK) 293 cells were detached from a culture dish, using 10 ml of Ca2+-free phosphate-buffered saline containing 0.5 mM EDTA, and then collected by centrifugation. The cells were washed twice with loading buffer (HBSS containing 20 mM HEPES and 0.1% BSA, pH 7.4, adjusted with NaOH), and re-suspended in the same buffer containing 2.5 μM Fluo 4-AM and 0.15% of chromophore EL (Sigma) for 30 min at 37 °C. After washing twice with the loading buffer, the cells were stocked in the loading buffer for less than 4 h at 0–4 °C. A cuvette containing Fluo 4-loaded cells (3 × 105 cells/ml) was placed in a fluorospectrophotometer (CAF-110; Jasco Inc., Tokyo, Japan). After incubation with stirring at 37 °C for at least 1 min, the test compound was added. The maximal response was determined using 0.1% Triton X-100. The sample solution was added at 20 s and the Triton X-100 solution was added at 80 s after the start of measurement.

3. Results 3.1. DBP activates DRG neurons responsive to TRPA1 and TRPV1 agonists To determine whether DBP directly activates sensory neurons, calcium influx into neurons from the mouse DRG was examined by single cell imaging. Cells that had been loaded with a calcium indicator were sequentially stimulated with DBP (1000 μM), AITC (30 μM), and CAP (1 μM). Finally, the cells were treated with ionomycin (5 μM) to verify the integrity of the plasma membrane. Examples of single cell response patterns are shown in Fig. 1(A–C). Some cells responded to DBP and AITC, but not to CAP (Fig. 1A), while others responded to DBP, AITC and CAP (Fig. 1C). There were some cells that did not respond to DBP or AITC but did respond to CAP (Fig. 1B). A summary of the response patterns of 191 viable (ionomycin responsive) cells is shown in Fig. 1D. DBP stimulated 27 cells, i.e. 14% of the viable cells. Among the DBP-responsive cells, 25 cells (93%) also responded to AITC while 11 cells (41%) also responded to CAP. There was only one cell (4%) that responded to DBP and CAP without a response to AITC. As to CAP, 141 cells (74% of viable cells) responded. Among the CAP-responsive cells, 11 cells (8%) responded to DBP. As to AITC, 34 cells (18% of viable cells) responded. Among the AITCresponsive cells, 25 cells (74%) also responded to DBP. There were 30 cells (16% of viable cells) that did not respond to CAP, AITC, or DBP. There was one cell (1% of viable cells) that responded to DBP alone. These results indicate that DBP stimulates a significant proportion of neurons in the DRG, and most DBP-responsive cells are included in a subset of cells responsive to AITC. 3.2. DBP activates TRPA1-expressing cells

2.7. Sensitization and elicitation of a contact hypersensitivity reaction Experiments were performed as described previously (Imai et al., 2006). BALB/c mice (Japan SLC) of 7-weeks-old were used. On day 0, 160 μl of an FITC solution (0.5%, w/v) diluted with one of the following solvents was epicutaneously applied to the shaved forelimb skin of a mouse under anesthesia. The solvents were acetone, or a 1:1 (v/v) mixture of acetone and a phthalate ester (DBP, DPNP, DPXP or DHPP). On day 7, the same amount of the FITC solution in the respective solvent was applied again. On day 14, the ear thickness baseline (0 h) in each animal was measured using a dial thickness gauge (Mitsutoyo, Kanazawa, Japan). Mice were challenged by applying 20 μl of an FITC solution (0.5% in acetone/DBP) on the right auricle, and then the ear thickness was measured after 24 and 48 h. The left auricle was treated with 20 μl of acetone/DBP alone as a control. Ear swelling at X h is defined as follows: [(ear thickness of the right ear at X h) − (ear thickness of the right ear at 0 h)] − [(ear thickness of the left ear at X h) − (ear thickness of the left ear at 0 h)]. 2.8. Trafficking of FITC-bearing antigen presenting cell (APC) Experiments were performed as described previously with modifications (Imai et al., 2006). ICR mice of 7-weeks-old were used. Mice were epicutaneously treated with 160 μl of an FITC solution (0.5% in one of the solvents given in Fig. 6) on shaved forelimb skin. After 24 h, brachial lymph nodes were obtained and pooled for each condition. Single-cell suspensions of lymph nodes were prepared in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co., Tokyo, Japan) by gentle teasing using needles. After washing in phosphate-buffered saline containing 0.1% BSA and 0.1% NaN3, cells were re-suspended in the same buffer. A total of 5 × 105 cells was examined with a flow cytometer (BD FACS Canto II, BD Biosciences, San Jose, CA) using gates for forward and side scatter to collect signals of cell-associated fluorescence to identify FITC-positive cells. Cells with a fluorescence intensity of 100 or more were arbitrarily designated as being FITC-positive.

To determine whether DBP activates cells through TRPA1mediated signaling, TRPA1 was expressed on CHO cells in a tetracycline inducible system. TRPA1-expressing CHO cells were loaded with a calcium indicator, and then calcium influx in response to samples was measured by means of a microplate assay (Fig. 2). DBP induced elevation of intracellular calcium in a dose-dependent manner (Fig. 2A). Involvement of TRPA1 was verified by the response to AITC (Fig. 2B). The dose response curve (Fig. 2C) revealed that EC50 for DBP was 24.6 μM, which was 2.7 times higher than that for AITC. The maximal response produced by DBP was 82% of that of the positive control (ionomycin), which was a little higher than that by AITC. Elevation of intracellular calcium was not detected in CHO cells without expression of TRPA1 (data not shown). The results indicated that TRPA1 was activated by DBP. 3.3. DBP activates TRPV1-expressing cells Calcium imaging of DRG neurons revealed partial overlapping of the responses to DBP and CAP. We directly examined the effect of DBP using CHO cells expressing TRPV1 constitutively. DBP induced elevation of intracellular calcium in a dose-dependent manner (Fig. 3A). Involvement of TRPV1 was verified by the response to CAP (Fig. 3B). Calcium elevation was slower in the case of DBP compared with that of CAP. The dose response curve (Fig. 3C) revealed that EC50 for DBP was 30.5 μM, which was about 15,000 times higher than that for CAP. The maximal response produced by DBP was 64% of that of the positive control (ionomycin), which was 74% of the maximal response for CAP. Elevation of intracellular calcium was not detected in CHO cells without expression of TRPV1 (data not shown). We confirmed the effect of DBP using another experimental system, in which the calcium response was observed in TRPV1-expressing HEK293 cells (Fig. 4A) in suspension. In this experimental setting, EC50 for DBP was about 150 times higher than that for CAP, and the maximal response produced by DBP was 48% of that produced by CAP. Taken together, the results indicated that DBP is a partial agonist for TRPV1.

T. Shiba et al. / Journal of Neuroimmunology 207 (2009) 66–74

69

CHO cells were stimulated with various types of phthalate esters, and then the calcium responses were compared (Fig. 5). DMP, DEP, DPP, DPNP, and DHXP each induced a significant response of cells expressing TRPA1. Compared with DBP (Fig. 2), similar maximal responses were obtained with DMP, DEP and DPP, but somewhat lower responses with DPNP and DHXP. As for EC50, those for DEP, DPP, DBP, DPNP and DHXP were similar but that for DMP was higher than those for the others. Furthermore, DMP induced calcium influx at 3000 μM in control CHO cells (data not shown). DHPP and DEHP produced a weak response, whereas DINP had no effect at all.

Fig. 1. DBP induces intracellular elevation of calcium in DRG neurons. (A–C) Changes in the intracellular calcium level in individual neurons in response to sequential treatment with DBP (1000 μM), AITC (30 μM), and CAP (1 μM). Neurons were isolated from mouse DRG and then loaded with a calcium indicator, Fluo 4-AM. Intracellular calcium levels were determined under a confocal microscope. Bars indicate the period of sample application. Each trace represents the intracellular calcium level in an individual cell (ordinate) during the time of the experiment (abscissa). Ionomycin (5 μM) was used to make cells permeable to calcium as a positive control. (D) Summary of calcium responses to various stimuli. Each oval represents the number of neurons that responded to the reagent. NR, no response. In total, 191 cells were examined.

3.4. Various phthalate esters activate TRPA1 and TRPV1 We examined whether other phthalate esters are also capable of stimulating TRPA1 or TRPV1 channels. TRPA1- or TRPV1-expressing

Fig. 2. DBP induces a calcium response in TRPA1-expressing CHO cells. (A) TRPA1expressing CHO cells were loaded with Fluo 4-AM, and then treated with various concentrations of DBP. The intracellular calcium level (arbitrary unit; ordinate) was recorded during the time (abscissa) of the experiment in a microplate format. Ionomycin (5 μM) was used to define the maximum level of calcium. The traces corresponding to the different DBP concentrations are as follows: 1000 μM (closed circles), 100 μM (open diamonds), 30 μM (open triangles), 10 μM (open squares), and 1 μM (open circles). The number in each trace indicates the DBP concentration. (B) Calcium responses to various concentrations of AITC. The AITC concentrations were as follows: 100 μM (closed circles), 30 μM (open diamonds), 10 μM (open triangles), 3 μM (open squares), and 1 μM (open circles). (C) Dose response curves in which the concentration (abscissa) of DBP (closed triangles) or AITC (open squares) was plotted against the maximal calcium response (ordinate) upon each treatment. Error bars represent the standard error of the mean (SEM) for replicate determinations (n = 6–9).

70

T. Shiba et al. / Journal of Neuroimmunology 207 (2009) 66–74

As to TRPV1-expressing CHO cells, DPP, DPNP and DHXP induced similar responses to that of DBP. DMP induced some response, but EC50 was higher than for the others. DEP, DHPP and DEHP each induced a weak response, while DINP had no effect at all. When TRPV1-expressing HEK293 cells were used, DPP, DBP, DPNP, and DHXP each produced a significant response (Fig. 4B), which is consistent with the results for TRPV1-expressing CHO cells. In control experiments, elevation of intracellular calcium was not observed in CHO cells without expression of TRPA1 or TRPV1 channels except with 3000 μM DMP (data not shown). Thus, for TRPA1 activation, phthalate esters with alkyl chain carbon numbers C2 to C6 (DEP, DPP, DBP, DPNP and DHXP) were optimal, while for TRPV1 activation, C3 to C6 were optimal.

Fig. 4. DBP and various phthalate esters elicit calcium influx into TRPV1-expressing HEK293 cells. (A) Dose response curves in which the concentration (abscissa) of DBP (closed triangles) or CAP (open squares) was plotted against the calcium response (ordinate). The calcium response is displayed relative to the calcium level obtained in the presence of 10 μM CAP. Error bars represent the SEM for replicate determinations (n = 3–5). (B) Maximal calcium responses caused by various phthalate esters. DMSO was used as a negative control and CAP was used as a positive control. The concentrations were as follows: CAP 10 μM, and phthalate esters 1000 μM. Each bar represents the calcium level upon each treatment relative to the maximal calcium response (Triton X100 treatment). Error bars represent the SEM for replicate determinations (n = 3).

3.5. Adjuvant effect and TRPA1/TRPV1 activation are correlated for various types of phthalate esters

Fig. 3. DBP induces a calcium response in TRPV1-expressing CHO cells. (A) TRPV1expressing CHO cells were loaded with Fluo 4-AM, and then treated with various concentrations of DBP. Experiments were performed as described in the legend to Fig. 2. The DBP concentrations were the same as in Fig. 2A. The number on each trace indicates the DBP concentration. (B) Calcium responses to various concentrations of CAP. The CAP concentrations were as follows: 100 nM (closed circles), 30 nM (open diamonds), 10 nM (open triangles), 1 nM (open squares), and 0.1 nM (open circles). (C) Dose response curves in which the concentration (abscissa) of DBP (closed triangles) or CAP (open squares) was plotted against the maximal calcium response (ordinate) upon each treatment. Error bars represent the SEM for replicate determinations (n = 5–8).

We previously demonstrated that the adjuvant effect of phthalate esters on FITC-induced CHS differed depending on the type (length of alkyl chain) of phthalate ester. Phthalate esters with alkyl chain carbon numbers C2 to C4 were shown to exhibit an adjuvant effect (Imai et al., 2006). Because phthalate esters with alkyl chain carbon numbers C5 (DPNP) and C6 (DHXP) were shown to activate TRPA1 and TRPV1, we reexamined the adjuvant effect as well as the effect on DC trafficking using some phthalate esters that had not been tested previously (C5 to C7). The phthalate esters with alkyl chain carbon numbers C4 to C7 (DHPP) exhibited a significant adjuvant effect in the FITC-induced CHS model (Fig. 6) and enhancement of FITC-presenting DC trafficking (Fig. 7). Together with the results of our previous studies, the relationship between in vitro TRP channel activation by various phthalate esters, and the in vivo adjuvant effect and effect on DC trafficking are summarized in Table 1. 4. Discussion Using neurons isolated from mouse DRG, we found that the subset of DBP-responsive neurons virtually completely overlapped with that of AITC-responsive ones, and DBP activates part of CAP-responsive ones. Other groups previously showed that AITC-responsive neurons

T. Shiba et al. / Journal of Neuroimmunology 207 (2009) 66–74

71

Fig. 5. Elevation of intracellular calcium in TRPA1- or TRPV1-expressing CHO cells induced by various phthalate esters. Dose response curves in which the concentrations (abscissa) of various phthalate esters were plotted against the maximal calcium response (ordinate) in TRPA1-expressing (closed squares) or TRPV1-expressing (open circles) CHO cells upon each treatment. Error bars represent the SEM for replicate determinations (n = 4–8).

were included in the subset of CAP-responsive neurons (Story et al., 2003). Our results also indicated that there is some overlap between AITC-responsive and CAP-responsive neurons. However, some AITCresponsive neurons did not respond to CAP. DBP was able to activate neurons that respond to AITC but not to CAP. DBP evoked calcium influx into TRPA1-expressing CHO cells (Fig. 2). The dose response curve revealed that EC50 of DBP is of the same order as that of AITC, and the maximal responses were similar for DBP and AITC. DBP also evoked calcium influx into TRPV1-expressing CHO cells (Fig. 3) and TRPV1-expressing HEK293 cells (Fig. 4A). EC50 of CAP for the activation of TRPV1-expressing cells was much lower than that of DBP. However, the EC50 values of DBP for the activation of TRPV1- and TRPA1-expressing CHO cells were similar. Thus, DBP may be able to activate TRPA1 and TRPV1 with similar potency. In contrast, the maximal calcium response to DBP was lower in the case of TRPV1-expressing cells than TRPA1-expressing ones. This result may suggest that DBP preferentially activates TRPA1 rather than TRPV1.

A recent study involving TRPV1-deficient mice revealed an elevated response in oxazolone-induced CHS (Bánvölgyi et al., 2005). The apparent contradiction with our results may be caused by difference in the weight of involvement and in the roles during sensitization process between TRPV1 and TRPA1. Alternatively, activation of TRPV1 may produce different effects depending on the type of haptens. Studies on FITC-induced CHS using TRPV1 and TRPA1 receptor knockout mice would be useful to directly demonstrate the effects of phthalate esters on TRPV1 and TRPA1 in vivo. We have revealed that the adjuvant effects of phthalate esters depend on the length of the alkyl chains, and most of such differences could be explained by the enhancement of DC trafficking (Imai et al., 2006; Maruyama et al., 2007b) (Figs. 6 and 7). We have tested phthalate esters with alkyl chain carbon numbers C1 to C9 (Table 1). Taken together with those of our previous studies, the results indicated that phthalate esters with C2 to C7 have an adjuvant effect on FITC-induced CHS, and those with C3 to C7 significantly enhance

72

T. Shiba et al. / Journal of Neuroimmunology 207 (2009) 66–74 Table 1 Effects of various phthalate esters on in vitro TRP channel activation and in vivo skin sensitization using an FITC-induced mouse CHS model TRPV1 Enhanced DC Enhancement in Phthalate Side chain TRPA1 ear swelling testa ester carbon number activationa activationa traffickinga DMP DEP DPP DBP DPNP DHXP DHPP DEHP DINP

1 2 3 4 5 6 7 8 9

NDb + + + + + − − −

NDb − + + + + − − −

− − + + + + + − −

− + + + + + + − −

a

Activation or enhancement: +, positive effect; −, no effect; ND, not determined. Not definitive because a high concentration of DMP evoked a nonspecific response in CHO cells without expression of TRP channels. b

Fig. 6. Various phthalate esters promote ear swelling in the FITC-induced contact hypersensitivity model. After sensitization with an FITC solution in acetone, or in a 1:1 mixture of acetone and a phthalate ester, ear swelling at 24 h (A) and 48 h (B) was determined after challenge with FITC in acetone/DBP. The solvent conditions are shown on the abscissa and the ordinate indicates ear swelling (in μm). The values for individual mice are plotted, and the means for each group are shown by bars. Statistical significance (compared with the acetone control) was analyzed by means of one-way ANOVA, followed by Dunnett's test.

trafficking of DC presenting FITC. As for the activation of TRPA1expressing CHO cells, phthalate esters with C2 to C6 were optimal (Figs. 2 and 5). Thus, there is a positive relationship between the adjuvant activity and TRPA1 channel activation among different types of phthalate esters.

Fig. 7. Various phthalate esters promote trafficking of FITC-positive cells from the skin to draining lymph nodes. Appearance of FITC-positive cells in draining lymph nodes after epicutaneous application of FITC in acetone, or in a 1:1 mixture of acetone and a phthalate ester (abscissa). Some mice were left untreated (un-sens). The ordinate represents the percentage of FITC-positive cells (arbitrarily defined as cells with a fluorescence intensity of 100 or more in the histogram obtained on flow cytometry) relative to the total lymph node cell number for each solvent condition. Each bar represents the mean ± SEM for three independent experiments. Statistical significance (compared with the acetone control) was analyzed by means of one-way ANOVA, followed by Dunnett's test.

As for activation of TRPV1-expressing CHO cells, phthalate esters with C3 to C6 strongly stimulated the calcium response (Figs. 3 and 5). Compared with TRPA1 activation, DEP (C2) did not stimulate TRPV1expressing CHO cells and the maximal level of calcium response to DPP (C3) was relatively low. In the case of TRPV1-expressing HEK293 cells, phthalate esters with C4 to C6 were optimal while that with C3 also exhibited a significant but weak response (Fig. 4B). In the latter system, the difference in EC50 between DBP and CAP was rather small as compared with in TRPV1-expressing CHO cells, while the maximal calcium response to DBP was much lower (Fig. 4A). These differences may be due to the experimental systems, that is, they depend on whether adherent CHO cells or HEK293 cells in suspension were used. Thus, there is also a positive relationship between the adjuvant activity and TRPV1 channel activation among different types of phthalate esters. Activation of TRPA1 and TRPV1 occurred in parallel in most cases with different types of phthalate esters. An exceptional case was DEP (C2), which activated TRPA1 but not TRPV1. Trafficking of FITCpresenting DCs was not facilitated by DEP whereas interleukin (IL)-4 production by local lymph node cells was enhanced (Imai et al., 2006; Maruyama et al., 2007b). In the FITC-induced CHS system, IL-4 production seems to be closely associated with the response (Dearman and Kimber, 2000; Takeshita et al., 2004; Tang et al., 1996). Consistent with these results, desensitization of TRPA1 by AITC pretreatment was shown to suppress IL-4 production upon sensitization with FITC in the presence of DBP (Maruyama et al., 2007a). In contrast, desensitization of TRPV1 by CAP pretreatment did not affect IL-4 production. TRPA1-mediated signaling by DEP or DBP may make the lymph node environment suitable for IL-4 production. However, the increase in the trafficking of FITC-presenting DCs may be facilitated only in combination with TRPV1 activation. The difference could be due to the difference in tissue distribution between TRPA1 and TRPV1. TRPV1 has been reported to be expressed on immune cells such as DCs and mast cells (Basu and Srivastava, 2005; Stander et al., 2004; Stokes et al., 2004). Thus, TRPV1 activation on non-neural cells may also regulate the immune response. The levels of stimulation of TRPA1 as well as those of TRPV1 were similar for DHPP (C7) and DEHP (C8) (Fig. 5). Despite this, DHPP, but not DEHP, stimulated DC trafficking and exhibited an adjuvant effect (Imai et al., 2006) (Figs. 6 and 7). There is a possibility that some other mechanisms could be involved in the case of DHPP, but we have not identified them so far. We provided the fact that phthalate esters with different types of alkyl chains have different efficacy in the TRP channel activation, but how phthalate esters activate TRPA1 or TRPV1 is unclear at present. Covalent binding of electrophilic agents to cytoplasmic cysteine residues has been shown as one of molecular modes of TRPA1 activation (Macpherson et al., 2007). Covalent modification by phthalate esters is not likely because phthalate esters are not highly

T. Shiba et al. / Journal of Neuroimmunology 207 (2009) 66–74

electrophilic. The fact that hydrophobic compounds such as CAP activate TRPV1 and the involvement of cytoplasmic residues of TRPA1 suggest that appropriate hydrophobicity may be important. Such a physicochemical requirement for receptor activation may be supported by the results that the activation of TRPA1 and TRPV1 occurred in parallel in most cases with different types of phthalate esters. Activation of TRPA1 or TRPV1 on neurons by phthalate esters will lead to the release of neuropeptides such as CGRP from peripheral nerve endings following the elevation of intracellular calcium (Bautista et al., 2005; Zygmunt et al., 1999). CGRP is reported to have various effects on immune cells such as Langerhans cells, mast cells, T cells and B cells (Asahina et al., 1995; Hosoi et al., 1993; Niizeki et al., 1997; Peters et al., 2006; Schlomer et al., 2007; Wang et al., 1992). A previous report indicated that CGRP induces tumor necrosis factor-α release from mast cells (Niizeki et al., 1997). Another study revealed that mast cell-associated tumor necrosis factor promotes DC migration (Suto et al., 2006). Our previous study revealed that sensitization to FITC in the presence of DBP is suppressed by treatment with a CGRP antagonist at the site of skin sensitization (Maruyama et al., 2007a). Others also showed that CGRP was able to boost sensitization in oxazolone-induced CHS (Gutwald et al., 1991). Besides CGRP, a substance P agonist has been reported to promote dinitrofluorobenzene-induced skin sensitization (Niizeki et al., 1999). These studies suggest that neuropeptide release from sensory neurons may provide a key as to the connection with the immune system. Upon stimulation of TRPV1 receptor, not only pro-inflammatory neuropeptides (such as CGRP and substance P) but also antiinflammatory ones (such as somatostatin) are released (Murai et al., 2008; Pintér et al., 2006). Inhibitory effects of somatostatin on immune system have been documented (Pintér et al., 2006). In contrast, an earlier study demonstrated that somatostatin had no effect upon skin sensitization to oxazolone (Gutwald et al., 1991). It would be important to pay attention to the possible modulatory roles of anti-inflammatory neuropeptides as downstream regulators after activation of sensory neurons. Considering the above findings, we hypothesized how stimulation of sensory neurons could enhance skin sensitization to FITC, as follows. (i) Phthalate esters activate TRPA1 and TRPV1 expressed on neurons and increase the intracellular calcium concentration. (ii) Elevation of intracellular calcium triggers the release of neuropeptides such as CGRP from nerve endings. (iii) Neuropeptides promote trafficking of APCs such as Langerhans cells and dermal DCs to draining lymph nodes. (iv) APCs that have migrated to draining lymph nodes induce the differentiation of antigen-specific T cells into effector and memory T cells. (v) Antigen-specific effector and memory T cells leave the lymph nodes and enter the blood circulation to repopulate skin sites. To determine the antigenicity of chemical compounds, the murine local lymph node assay is available as a test involving relatively small numbers of animals (Kimber et al., 2002). However, there is no good test for adjuvants involving a reduced number of experimental animals. The positive relationship among various phthalate esters between the adjuvant activity and TRPA1 and TRPV1 channel activation suggests that in vitro measurement of TRP channel activation may be useful for the screening of candidate compounds to predict adjuvant activity. Despite that we focused on sensory neurons, various types of cells, such as keratinocytes, mast cells and dermal macrophages, also regulate the immune response in skin. These cells could be potential targets of chemical compounds with adjuvant activity through yet unknown mechanisms. On the other hand, the present assay could be adapted for chemicals other than phthalate esters to reveal adjuvant activity. Further investigations involving different kinds of chemicals will show the relationship between TRP channel activation and adjuvant activity. In conclusion, our study has suggested that stimulation of sensory neurons via TRPA1 and TRPV1 is involved in the adjuvant effect during

73

skin sensitization. This concept may reflect the connection between skin irritation and skin allergies such as CHS. TRPA1 and TRPV1 may play a central role by transmitting noxious stimuli to the brain and immune cells such as APCs by sensing noxious compounds. These findings will contribute not only to our knowledge of the immunological roles of TRP channels on sensory neurons, but also to the development of risk assessment methods for chemicals with an adjuvant effect. Acknowledgments This work was supported partly by a grant-in-aid (18659033, 20390041) and by research funding for the Global COE Program from the Japan Society for the Promotion of Science. References Api, A.M., 2001. Toxicological profile of diethyl phthalate: a vehicle for fragrance and cosmetic ingredients. Food. Chem. Toxicol 39, 97–108. Asahina, A., Hosoi, J., Beissert, S., Stratigos, A., Granstein, R.D., 1995. Inhibition of the induction of delayed-type and contact hypersensitivity by calcitonin gene-related peptide. J. Immunol. 154, 3056–3061. Bandell, M., Story, G.M., Hwang, S.W., Viswanath, V., Eid, S.R., Petrus, M.J., Earley, T.J., Patapoutian, A., 2004. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41, 849–857. Bánvölgyi, A., Pálinkás, L., Berki, T., Clark, N., Grant, A.D., Helyes, Z., Pozsgai, G., Szolcsányi, J., Brain, S.D., Pintér, E., 2005. Evidence for a novel protective role of the vanilloid TRPV1 receptor in a cutaneous contact allergic dermatitis model. J. Neuroimmunol. 169, 86–96. Basu, S., Srivastava, P., 2005. Immunological role of neuronal receptor vanilloid receptor 1 expressed on dendritic cells. Proc. Natl. Acad. Sci. USA 102, 5120–5125. Bautista, D.M., Movahed, P., Hinman, A., Axelsson, H.E., Sterner, O., Hogestatt, E.D., Julius, D., Jordt, S.E., Zygmunt, P.M., 2005. Pungent products from garlic activate the sensory ion channel TRPA1. Proc. Natl. Acad. Sci. USA 102, 12248–12252. Bautista, D.M., Jordt, S.E., Nikai, T., Tsuruda, P.R., Read, A.J., Poblete, J., Yamoah, E.N., Basbaum, A.I., Julius, D., 2006. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269–1282. Beresford, L., Orange, O., Bell, E.B., Miyan, J.A., 2004. Nerve fibres are required to evoke a contact sensitivity response in mice. Immunology 111, 118–125. Bornehag, C.G., Sundell, J., Weschler, C.J., Sigsgaard, T., Lundgren, B., Hasselgren, M., Hagerhed-Engman, L., 2004. The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case-control study. Environ. Health Perspect. 112, 1393–1397. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824. Chun, K.H., Imai, Y., Higashi, N., Irimura, T., 2000. Migration of dermal cells expressing a macrophage C-type lectin during the sensitization phase of delayed-type hypersensitivity. J. Leukoc. Biol. 68, 471–478. Dearman, R.J., Kimber, I., 2000. Role of CD4(+) T helper 2-type cells in cutaneous inflammatory responses induced by fluorescein isothiocyanate. Immunology 101, 442–451. Dhaka, A., Viswanath, V., Patapoutian, A., 2006. Trp ion channels and temperature sensation. Annu. Rev. Neurosci. 29, 135–161. Gutwald, J., Goebeler, M., Sorg, C., 1991. Neuropeptides enhance irritant and allergic contact dermatitis. J. Invest. Dermatol. 96, 695–698. Hosoi, J., Murphy, G.F., Egan, C.L., Lerner, E.A., Grabbe, S., Asahina, A., Granstein, R.D., 1993. Regulation of Langerhans cell function by nerves containing calcitonin generelated peptide. Nature 363, 159–163. Imai, Y., Kondo, A., Iizuka, H., Maruyama, T., Kurohane, K., 2006. Effects of phthalate esters on the sensitization phase of contact hypersensitivity induced by fluorescein isothiocyanate. Clin. Exp. Allergy 36, 1462–1468. Jordt, S.E., Tominaga, M., Julius, D., 2000. Acid potentiation of the capsaicin receptor determined by a key extracellular site. Proc. Natl. Acad. Sci. USA 97, 8134–8139. Jordt, S.E., Bautista, D.M., Chuang, H.H., McKemy, D.D., Zygmunt, P.M., Hogestatt, E.D., Meng, I.D., Julius, D., 2004. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427, 260–265. Kimber, I., Dearman, R.J., Basketter, D.A., Ryan, C.A., Gerberick, G.F., 2002. The local lymph node assay: past, present and future. Contact Dermatitis 47, 315–328. Larsen, S.T., Lund, R.M., Nielsen, G.D., Thygesen, P., Poulsen, O.M., 2002. Adjuvant effect of di-n-butyl-, di-n-octyl-, di-iso-nonyl- and di-iso-decyl phthalate in a subcutaneous injection model using BALB/c mice. Pharmacol. Toxicol. 91, 264–272. Liu, Y., Teige, I., Birnir, B., Issazadeh-Navikas, S., 2006. Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE. Nat. Med. 12, 518–525. Macpherson, L.J., Dubin, A.E., Evans, M.J., Marr, F., Schultz, P.G., Cravatt, B.F., Patapoutian, A., 2007. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 445, 541–545. Maruyama, T., Iizuka, H., Tobisawa, Y., Shiba, T., Matsuda, T., Kurohane, K., Imai, Y., 2007a. Influence of local treatments with capsaicin or allyl isothiocyanate in the sensitization phase of a fluorescein-isothiocyanate-induced contact sensitivity model. Int. Arch. Allergy Immunol. 143, 144–154.

74

T. Shiba et al. / Journal of Neuroimmunology 207 (2009) 66–74

Maruyama, T., Shiba, T., Iizuka, H., Matsuda, T., Kurohane, K., Imai, Y., 2007b. Effects of phthalate esters on dendritic cell subsets and interleukin-4 production in fluorescein isothiocyanate-induced contact hypersensitivity. Microbiol. Immunol. 51, 321–326. Morita, A., Iwasaki, Y., Kobata, K., Iida, T., Higashi, T., Oda, K., Suzuki, A., Narukawa, M., Sasakuma, S., Yokogoshi, H., Yazawa, S., Tominaga, M., Watanabe, T., 2006. Lipophilicity of capsaicinoids and capsinoids influences the multiple activation process of rat TRPV1. Life Sci. 79, 2303–2310. Murai, M., Tsuji, F., Nose, M., Seki, I., Oki, K., Setoguchi, C., Suhara, H., Sasano, M., Aono, H., 2008. SA13353 (1-[2-(1-Adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea) inhibits TNF-α production through the activation of capsaicin-sensitive afferent neurons mediated via transient receptor potential vanilloid 1 in vivo. Eur. J. Pharmacol. 588, 309–315. Nielsen, N.H., Linneberg, A., Menne, T., Madsen, F., Frolund, L., Dirksen, A., Jorgensen, T., 2001. Allergic contact sensitization in an adult Danish population: two crosssectional surveys eight years apart (the Copenhagen Allergy Study). Acta Derm. Venereol. 81, 31–34. Niizeki, H., Alard, P., Streilein, J.W., 1997. Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity. J. Immunol. 159, 5183–5186. Niizeki, H., Kurimoto, I., Streilein, J.W., 1999. A substance P agonist acts as an adjuvant to promote hapten-specific skin immunity. J. Invest. Dermatol. 112, 437–442. Peters, E.M., Ericson, M.E., Hosoi, J., Seiffert, K., Hordinsky, M.K., Ansel, J.C., Paus, R., Scholzen, T.E., 2006. Neuropeptide control mechanisms in cutaneous biology: physiological and clinical significance. J. Invest. Dermatol. 126, 1937–1947. Pintér, E., Helyes, Z., Szolcsányi, J., 2006. Inhibitory effect of somatostatin on inflammation and nociception. Pharmacol. Ther. 112, 440–456. Razavi, R., Chan, Y., Afifiyan, F.N., Liu, X.J., Wan, X., Yantha, J., Tsui, H., Tang, L., Tsai, S., Santamaria, P., Driver, J.P., Serreze, D., Salter, M.W., Dosch, H.M., 2006. TRPV1+ sensory neurons control β cell stress and islet inflammation in autoimmune diabetes. Cell 127, 1123–1135. Sato, K., Imai, Y., Irimura, T., 1998. Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity. J. Immunol. 161, 6835–6844. Schlomer, J.J., Storey, B.B., Ciornei, R.T., McGillis, J.P., 2007. Calcitonin gene-related peptide inhibits early B cell development in vivo. J. Leukoc. Biol. 81, 802–808.

Stander, S., Moormann, C., Schumacher, M., Buddenkotte, J., Artuc, M., Shpacovitch, V., Brzoska, T., Lippert, U., Henz, B.M., Luger, T.A., Metze, D., Steinhoff, M., 2004. Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures. Exp. Dermatol. 13, 129–139. Stokes, A.J., Shimoda, L.M., Koblan-Huberson, M., Adra, C.N., Turner, H., 2004. A TRPV2PKA signaling module for transduction of physical stimuli in mast cells. J. Exp. Med. 200, 137–147. Story, G.M., Peier, A.M., Reeve, A.J., Eid, S.R., Mosbacher, J., Hricik, T.R., Earley, T.J., Hergarden, A.C., Andersson, D.A., Hwang, S.W., McIntyre, P., Jegla, T., Bevan, S., Patapoutian, A., 2003. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819–829. Suto, H., Nakae, S., Kakurai, M., Sedgwick, J.D., Tsai, M., Galli, S.J., 2006. Mast cellassociated TNF promotes dendritic cell migration. J. Immunol. 176, 4102–4112. Takeshita, K., Yamasaki, T., Akira, S., Gantner, F., Bacon, K.B., 2004. Essential role of MHC II-independent CD4+ T cells, IL-4 and STAT6 in contsact hypersensitivity induced by fluorescein isothiocyanate in the mouse. Int. Immunol. 16, 685–695. Tang, A., Judge, T.A., Nickoloff, B.J., Turka, L.A., 1996. Suppression of murine allergic contact dermatitis by CTLA4Ig. Tolerance induction of Th2 responses requires additional blockade of CD40-ligand. J. Immunol. 157, 117–125. Tominaga, M., Caterina, M.J., Malmberg, A.B., Rosen, T.A., Gilbert, H., Skinner, K., Raumann, B.E., Basbaum, A.I., Julius, D., 1998. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21, 531–543. Vartak, P.H., Tungikar, V.B., Sharma, R.N., 1994. Comparative repellent properties of certain chemicals against mosquitoes, house flies and cockroaches using modified techniques. J. Commun. Dis. 26, 156–160. Wang, F., Millet, I., Bottomly, K., Vignery, A., 1992. Calcitonin gene-related peptide inhibits interleukin 2 production by murine T lymphocytes. J. Biol. Chem. 267, 21052–21057. Zygmunt, P.M., Petersson, J., Andersson, D.A., Chuang, H., Sorgard, M., Di Marzo, V., Julius, D., Hogestatt, E.D., 1999. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400, 452–457.