To investigate the mechanisms underlying itching in atopic dermatitis, we examined whether thromboxane (TX) A2, an arachidonic acid metabolite, is involved in spontaneous scratching, an itch-related response, in NC mice with atopic dermatitis-like skin lesions. The TXA2 receptor (TP) antagonist ONO-3708 inhibited the spontaneous scratching. The mRNA expression of TX synthase (TXSyn) distributed mainly in epidermis and the concentration of TXB2, a metabolite of TXA2, were increased in lesional skin. Scratching caused by the PAR2 agonist SLIGRL-NH2 was suppressed by ONO-3708. SLIGRL-NH2-induced scratching decreased approximately 75% in TP-deficient mice, compared to wild-type mice. In primary cultures of mouse keratinocytes, SLIGRL-NH2 induced the production of TXA2, as evidenced by the increased TXB2, which was inhibited by the TXSyn inhibitor sodium ozagrel and a PAR2-neutralizing antibody. Taken together, these results suggest that epidermal TXA2, which may be produced via PAR2 activation, is involved in itching in atopic dermatitis. Key words: itch; thromboxane A2; TP receptor; thromboxane synthase; atopic dermatitis; proteinase-activated receptor 2.

Accepted Apr 6, 2016; Epub ahead of print Apr 12, 2016

Acta Derm Venereol 2016; XX: XX–XX

Tsugunobu Andoh, Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-1094, Japan. E-mail: andoht@pha.u-toyama.ac.jp

Atopic dermatitis (AD) is a severe, chronic inflammatory skin disease and itching constitutes its major diagnostic criterion (1–3). In AD patients, scratching induced by the itch sensation aggravates dermatitis and increases the frequency of itching (4). Therefore, itch control is very important for improving quality of life and treating AD. Although H1 histamine receptor antagonists often serve as the drug of first choice for treating itching, AD-derived itching responds poorly to these drugs (5), suggesting that histamine is not essential for AD itching. Therefore, a comprehensive understanding of the mechanisms and mediators underlying AD itching is limited.

Glucocorticoids attenuate itching in chronic skin diseases including AD (6–8). The mechanisms underlying the pharmacological action of glucocorticoids including the inhibition of phospholipase A2 (9) and the regulation of the expression of cyclooxygenase (COX)-2, but not COX-1 (10), have been elucidated. These findings suggest that arachidonic acid metabolites, which are regulated by glucocorticoids, are involved in the itch sensation.

Thromboxane (TX) A2 is an arachidonic acid metabolite produced by catalysis with COX and TX synthase (TXSyn) and is altered spontaneously to form inactive TXB2. Additionally, TXA2 exerts its pharmacological activity (e.g., platelet aggregation) via the thromboxane TXA2 receptor (TP), a G-protein coupled receptor (11) also involved in allergic skin inflammation (12). In patients with pruritic diseases, the serum concentration of TXB2, a metabolite (and therefore an indicator) of TXA2, is increased (13–16). Moreover, intradermal injection of TXA2 elicits scratching, an itch-associated response, in mice (17). These results suggest that TXA2 plays an important role in the itching sensation that accompanies several pruritic skin diseases. However, it remains unclear whether TXA2 is involved in AD-associated itching.

NC mice that are maintained for a long time in a conventional environment develop chronic dermatitis, hyperplasia, dry skin, increased serum IgE levels, mast cells in the skin and spontaneous scratching, an itch-related behavior (18, 19); these features are similar to those of AD patients (1–3). In this study, we investigated whether TXA2 is involved in spontaneous itching in NC mice with AD-like chronic dermatitis.

Materials and methods

Animals

Male NC/Nga mice (14–21 weeks old), purchased from Japan SLC (Hamamatsu, Japan), were used in this study. The mice were bred in a specific pathogen-free environment at the Division of Animal Resource and Development, Life Science Research Center, University of Toyama, and in a conventional environment in the experimental animal room in the Department of Applied Pharmacology, University of Toyama. All mice, except for the healthy controls, were kept together with mite-infected mice with chronic dermatitis for 2 weeks at 4–5 weeks of age in a conventional environment in the experimental animal room (19). These mice exhibited severe dermatitis after one and two months. In one series of experiments, TP receptor-deficient mice (TP-R–/–) mice with a C57BL/6 genetic background and wild-type mice were used at 11 weeks of age (20). The mice were housed in a room with controlled temperature (21–23°C), humidity (45–65%), and light cycle (lights on from 08:00 to 20:00 h). Food and water were freely available. All animal experiment procedures were approved by the Committee for Animal Experiments at the University of Toyama and Asahikawa Medical University and were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, 2006).

Agents (see Appendix S11)

Primary cultures of mouse keratinocytes (see Appendix S11)

Behavioral experiments (see Appendix S11)

Real-time PCR (see Appendix S11)

Reverse transcription and PCR (see Appendix S11)

Enzyme immunoassay for TXB2

Animals were anesthetized with sodium pentobarbital (80 mg/kg) and transcardially perfused with phosphate-buffered saline (PBS). The skin was isolated using an 8-mm-diameter punch, immediately shredded with scissors, and added to 1 ml of ice-chilled ethanol containing 10 μM indomethacin and 10 μM zileuton to inhibit cyclooxygenase and 5-lipoxygenase, respectively. Following homogenization, each sample was centrifuged at 600 × g at 4°C for 10 min. The supernatant (0.8 ml) was mixed with 5 ml of distilled water (pH 3.0) and applied to a C18 Sep-Pak cartridge (Waters, Milford, MA, USA) equilibrated with methanol. The cartridges were washed with hexane followed by distilled water and the lipids were then eluted with methyl formate. The eluted fraction was then evaporated and the resulting residue was suspended in an enzyme immunoassay buffer (Cayman Chemical, Ann Arbor, MI, USA) for the assay of TXB2. The amount of TXB2 was determined using an enzyme immunoassay kit (Cayman Chemical). In the case of cultured keratinocytes, the culture medium was changed to mouse keratinocyte growth medium (CnT-Prime basal medium; CELLnTEC Advanced Cell Systems AG), as the assay medium. Samples for the assay of TBX2 were obtained from the supernatant 5 min post SLIGRL-NH2 administration. Following collection of culture medium, the cultured keratinocytes were treated with 1% Triton X-100 to solubilize the cell proteins. Total protein concentration was determined using a Bio-Rad Protein Assay Kit (Hercules, CA, USA) and TXB2 concentration was measured using an enzyme immunoassay kit (Cayman Chemical). The concentration of TXB2 in keratinocytes was normalized to a skin section (8-mm diameter) and total protein concentration, respectively.

Immunostaining

Under anesthesia with sodium pentobarbital (80 mg/kg, intraperitoneal), the animals were transcardially perfused with PBS following by with 4% paraformaldehyde (PFA). The skin of the rostral back was isolated, post-fixed with 4% PFA and then immersed in 30% sucrose solution for 2 days. The tissue was then embedded in Tissue-Tek1 O.C.T. Compound (Sakura Fineteck Co., Ltd., Tokyo, Japan) and the frozen samples were sectioned at 16 μm with a cryostat (Leica, Wetzlar, Germany). The sections were washed with PBS and then treated with Protein Block® (DAKO Co., Hamburg, Germany) followed by 0.3% Triton X-100 in PBS. The sections were treated with anti-TXSyn antibody (Cayman Chemical) at 4°C overnight. For some experiments, sections were treated with the antibody (Cayman Chemical) pre-incubated with its specific antigen prior to staining. Subsequent to washing with PBS, the preparations were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG (Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. Fluorescence signals were observed using a fluorescence microscope (BX-61/DP70, Olympus, Tokyo, Japan). Fluorescent intensity was analyzed with NIH Image software (National Institute of Health, Bethesda, MD, USA).

Statistical analysis

Data are presented as mean ± S.E.M. The statistical significance of differences between group means was analyzed using the Student’s t-test (two groups) or one-way analysis of variance (ANOVA) followed by a post-hoc Holm–Šidák test (3 or more groups). A p-value < 0.05 was considered statistically significant.

Results

Spontaneous scratching in NC mice with chronic dermatitis

Fig. 1A shows the number of spontaneous bouts of hind paw scratching towards any region of the body in individual healthy and chronic dermatitis NC mice. Although the healthy mice did not exhibit spontaneous scratching, most age-matched chronic dermatitis mice exhibited apparent spontaneous scratching. In the following experiments, we used healthy NC mice that were given intradermal injections of SLIGRL-NH2. In other experiments, healthy and chronic dermatitis NC mice were compared.

 

Fig. 1. (A) Comparison of spontaneous scratching between healthy and dermatitis mice. Spontaneous hind paw scratching towards any region of the body was counted for 1 h at 21 weeks of age. Healthy mice were kept under specific pathogen-free (SPF) conditions and dermatitis mice were transferred to a conventional environment (CNV) at 4–5 weeks of age. Open circles represent the values for individual animals; the horizontal bars represent the means (n = 10 each). *p < 0.05 (Student’s t-test). (B) Effects of ONO-3708 on spontaneous scratching in NC mice with chronic dermatitis. Either ONO-3708 or the vehicle (VH) was administered orally 1 h prior to observation. The broken line represents the mean number of scratch bouts in healthy mice. Values represent the mean and S.E.M. (n = 16). *p < 0.05, as compared with VH-treated mice (Holm–Šidák test).

Effects of TP antagonist on spontaneous scratching in NC mice with chronic dermatitis

The TP receptor antagonist ONO-3708 (30 or 100 mg/kg) significantly inhibited spontaneous scratching in NC mice with chronic dermatitis, compared with the vehicle-treated group (Fig. 1B).

Effects of the TP receptor antagonist and TP receptor-deficiency on SLIGRL-NH2-induced scratching

PAR2 is involved in spontaneous scratching in NC mice with chronic dermatitis (24). Intradermal injection of SLIGRL-NH2 (100 nmol/site), a PAR2 agonist peptide, elicited scratching in healthy NC mice (Fig. 2). ONO-3708 (30 and 100 mg/kg) significantly inhibited scratching induced by SLIGRL-NH2 compared with the vehicle-treated group (Fig. 2 A). While SLIGRL-NH2 (100 nmol/site) induced scratching in wild-type mice, the scratching was decreased by approximately 75% in TP-deficient mice, compared with that in wild-type mice (Fig. 2B). The mRNA expressions of TP receptor, TXSyn and PAR2 in the skin and DRG in wild-type and TP-deficient mice are shown in Fig. S11.

Fig. 2. Effects of the thromboxane (TX) A2 receptor (TP) antagonist ONO-3708 on SLIGRL-NH2-induced scratching. (A) SLIGRL-NH2 (100 nmol/site) was injected intradermally into the rostral back of healthy NC mice. ONO-3708 or the vehicle (VH) was administered orally 1 h prior to SLIGRL-NH2 injection. The broken line represents the mean number of scratch bouts in the saline-injected group. Values represent the mean and S.E.M. (n = 8). *p < 0.05, as compared with VH-treated group (Holm–Šidák test). (B) SLIGRL-NH2 (100 nmol/site) or saline was injected intradermally into the rostral back of TP-deficient (TP-R–/–) or the wild-type (WT) mice.

Values represent the mean and S.E.M. (n = 6–8). *p < 0.05, as compared with saline-injected group (WT), #p < 0.05, as compared with SLIGRL-injected group (WT) (Holm–Šidák test).

Expression of TXSyn mRNA and TXB2 concentration in NC mouse skin

The expression of TXSyn mRNA (Fig. 3A) and TXB2, a stable degradation product of TXA2 (Fig. 3B), were significantly higher in the lesional skin of NC mice with chronic dermatitis than in the skin of healthy NC mice.

Fig. 3. (A) Expression level of TXSyn mRNA in the skin of healthy (lesional) or dermatitis mice (NC). The mRNA levels were determined in 8-mm diameter punch skin of the rostral back by real-time PCR. The mRNA levels were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase in each sample and then to the mean level observed in the healthy control mice. Values represent the means and S.E.M. (n = 9). *p < 0.05 (Student’s t-test). (B) TXB2 concentration in the skin of healthy or dermatitis (lesional) mice. The TXB2 concentration in the rostral back skin of healthy or dermatitis mice was measured by an enzyme immunoassay. Values represent the mean and S.E.M. (n = 10). *p < 0.05 (Student’s t-test).

Distribution of TXSyn in the skin of NC mice

In contrast to healthy skin, the lesional skin of NC mice with chronic dermatitis exhibited epidermal hyperplasia (Fig. 4). Immunohistochemical staining and fluorescent intensity analysis in the epidermis and dermis revealed that TXSyn is predominantly expressed in epidermal keratinocytes (Fig. 4). Staining was eliminated when pre-absorbed antibody was used (Fig. 4 bottom).

 

Fig. 4. Thromboxane synthase (TXSyn). Representative images of TXSyn (green) immunostaining in the skin of healthy (Top) and dermatitis (Middle) NC mice. Skin samples of dermatitis NC mice were also immunostained with the pre-absorbed TXSyn antibody (Bottom). Dotted lines indicate the boundary between the dermis (D) and epidermis (ED). The values in parenthesis indicate the fluorescence intensity ratio in the ED and D (ED/D); scale bar = 100 μm.

The production of TXA2 induced by SLIGRL-NH2 in primary cultures of mouse keratinocytes

Both TXSyn and PAR2 mRNA were expressed in primary cultures of mouse keratinocytes (Fig. 5A). SLIGRL-NH2 (20–200 μM) induced TXA2 production, as determined by an increase in TXB2 concentration in primary cultures of mouse keratinocytes (Fig. 5B). SLIGRL-NH2-induced TXA2 production was significantly inhibited by the TXSyn inhibitor sodium ozagrel (1–10 μM) (Fig. 5B). In addition, the PAR2-neutralized antibody (Fig. S21) also attenuated the TXA2 production inhibited by SLIGRL-NH2 (Fig. 5C).

 

Fig. 5. The production of thromboxane (TX) A2 induced by SLIGRL-NH2 in primary cultures of mouse keratinocytes. (A) The expression levels of TX synthase (TXSyn) and proteinase-activated receptor 2 (PAR2) mRNA were determined using reverse transcription (RT)-PCR. Typical examples of the bands of RT-PCR products for TXSyn, PAR2 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA are seen in the gel. (B) Effects of the TXSyn inhibitor sodium ozagrel on SLIGRL-NH2-induced TXA2 production. Sodium ozagrel was applied 5 min prior to the application of SLIGRL-NH2. Since TXA2 is altered spontaneously to form inactive TXB2, the TXB2 content in the assay medium was measured using an enzyme immunoassay. Values represent the mean and S.E.M. (n = 6). *p < 0.05, as compared with assay medium alone, #p < 0.05, as compared with SLIGRL-NH2 (200 μM) alone (Holm–Šidák test). (C) Effect of PAR2-neutralized antibody on SLIGRL-NH2-induced TXA2 production. All cultures were exposed to Non-specific IgG (nIgG) (1:500) or PAR2-neutralized antibody (PAR2ab, 1:500) 10 min prior to the application of SLIGRL-NH2 (200 μM). Values represent the means and S.E.M. (n = 6). *p < 0.05, as compared with nIgG alone, #p < 0.05, as compared with nIgG+SLIGRL-NH2 (Holm–Šidák test).

Discussion

Responses to pruritogens differ among mouse species (25). An intradermal injection of histamine (10–1,000 nmol/site) did not induce scratching in healthy NC mice (Fig. S31), suggesting that NC mice respond minimally to histamine. Thus, pruritogens other than histamine are involved in the observed spontaneous scratching in NC mice with chronic dermatitis. The results of the present study show that the TP receptor antagonist ONO-3708 inhibits spontaneous scratching in NC mice with chronic dermatitis. In healthy NC mice, an intradermal injection of the TXA2 analogue U-46619 (1–100 nmol/site) elicited dose-dependent scratching (Fig. S31). Additionally, U-46619-induced scratching was inhibited by ONO-3708 (Fig. S41). ONO-3708 inhibits receptor binding of U-46619 but not TXB2, leukotriene D4, or prostaglandin D2 and E1 (21, 22), suggesting that the action of U-46619 is mediated via the TP receptor. Both TXSyn mRNA expression and TXA2 production, as gauged by TXB2 concentration, were increased in the skin of NC mice with chronic dermatitis. These results suggest that TXA2 and its receptor are involved in spontaneous itching in NC mice with chronic dermatitis.

TXSyn is a key enzyme involved in the synthesis of TXA2 from prostaglandin H2, a metabolite of arachidonic acid (26). TXSyn was predominately expressed in epidermal keratinocytes in the skin of both healthy and dermatitis NC mice. Previously, we have also shown that keratinocytes express TXSyn in ICR mice, which are untreated healthy mice (17). Mast cells in the skin also express TXSyn and produce TXA2 via immunological stimulation (27). However, the TXA2 content is much lower than the contents of other arachidonic acids metabolites, such as prostaglandin D2 and leukotriene C4 (27). In addition, no immunoreactivity against TXSyn was observed in the dermis, where the mast cells are distributed, of either healthy or dermatitis NC mice. Therefore, these results do not support the involvement of mast cell TXA2 in itching in NC mice with chronic dermatitis. However, it is still possible that mast cell-derived TXA2 is involved in itching associated with dermatitis. Taken together, these findings indicate that epidermal keratinocytes constitute the main source of TXA2 production in lesional skin.

In the skin of NC mice with chronic dermatitis, expression of TXSyn mRNA was increased. The AP-1 family of transcription factors plays an important role in the promotor activity for TXSyn (28). Increased immunoreactivity to AP-1-related transcription factors (e.g., Jun D, c-Fos, Fos B, Fra-1, and Fra-2) has been observed in the epidermis (29) of AD patients compared to that of healthy subjects. Jun D and c-Fos, in particular, exhibit strong cytoplasmic positivity in the epidermis of AD patients (29). Thus, the increase in AP-1 expression may be related to the elevated TXSyn expression in atopic dermatitis-like skin lesions.

The mechanisms underlying TXA2 production in lesional skin are not completely understood. Previously, we have demonstrated that serine proteinase and PAR2 are involved in itch-related responses in NC mice with chronic dermatitis (24). In this study, the PAR2 agonist peptide SLIGRL-NH2-induced itch-related responses were inhibited by a TP receptor antagonist in healthy NC mice and were decreased in TP receptor-deficient mice, suggesting that TXA2 is involved in PAR2-related responses. PAR2 is predominately expressed in epidermal keratinocytes (24). In primary cultures of NC mouse keratinocytes, the application of a PAR2 agonist peptide induced the production of TXA2, and this response was inhibited by the TX inhibitor and the PAR2-neutralizing antibody. Thus, PAR2 activation may be involved in the production of TXA2 in keratinocytes. PAR2 is activated by trypsin-like serine proteases (e.g., tryptase and kallikrein 5) and a synthetic PAR2-tethered ligand known as PAR2 agonist peptide (30–32). In NC mice with chronic dermatitis, tryptase from mast cells (24) and kallikrein 5 from keratinocytes (33) are involved in itching behavior, suggesting that these serine-proteases play an important role in the production of TXA2. There are at least two possible pathways by which keratinocytes produce TXA2 following PAR2 stimulation. PAR2 is a G-protein-coupled receptor (30) and its activation increases the level of free intracellular Ca2+ ions (30, 34, 35). Thus, the activation of phospholipase A2 via increased free intracellular Ca2+ ions (36) may promote TXA2 production. Production may also occur via Rho kinase-mediated phospholipase A2 activation (37); the activation of PAR2 stimulates Rho kinase activity (38). In addition to the increase in TXSyn expression, these two pathways may contribute to the production of TXA2 in the lesional skin of NC mice with chronic dermatitis.

In the skin, TP receptors are predominantly expressed in keratinocytes and primary afferent neurons (17) (Fig. S31). In addition, TP receptors are expressed in small-size neurons in the dorsal root ganglion (17); these have chiefly unmyelinated C-fibers (39, 40), which play an important role in itch signaling (41, 42). The application of the TXA2 analogue U-46619 increases the concentration of free intracellular Ca2+ ions, which is inhibited by the TP receptor antagonist, in primary cultures of dorsal root ganglion neurons (17). The action of U-46619 is also inhibited by L-type calcium channel blockers, suggesting that the activation of TP receptors elicits membrane depolarization (43). These observations suggest that TXA2 acts on primary afferent neurons or through both autocrine and paracrine mechanisms in keratinocytes. Additionally, intra-epidermal neurites increase in the skin of NC mice with chronic dermatitis (44) and patients with atopic dermatitis (45, 46). These findings also support the hypothesis that TXA2 released from keratinocytes acts directly on primary afferent neurons.

In conclusion, we demonstrated that TXA2 produced by epidermal keratinocytes is involved in spontaneous itch-related behavior in NC mice with AD-like skin lesions. Moreover, we found that the serine protease-PAR2 system partially contributes to the production of TXA2. PAR2, TXSyn and TP receptor may constitute potential new targets for the development of antipruritic drugs for AD.

Acknowledgments

We are very grateful to Ono Pharmaceutical Co. Ltd. for providing the gift of ONO-3708. This study was supported in part by Japan Agency for Medical Research and Development.

The authors declare no conflicts of interest.

1http://www.medicaljournals.se/acta/content/?doi=10.2340/00015555-2437

References