Antihistamines Suppress Upregulation of Histidine Decarboxylase Gene

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Journal of Pharmacological Sciences xxx (2016) 1e7

55 HOSTED BY Contents lists available at ScienceDirect 56 57 Journal of Pharmacological Sciences 58 59 60 journal homepage: www.elsevier.com/locate/jphs 61 62 63 Full paper 64 65 1 Antihistamines suppress upregulation of histidine decarboxylase gene 66 2 fi 67 3 expression with potencies different from their binding af nities for 68 4 histamine H receptor in toluene 2,4-diisocyanate-sensitized rats 69 5 1 70 6 a, *, 3 a, 1, 3 b a, 1 71 7 Q8 Hiroyuki Mizuguchi , Asish K. Das , Kazutaka Maeyama , Shrabanti Dev , 72 a, 2 c c d 8 Masum Shahriar , Yoshiaki Kitamura , Noriaki Takeda , Hiroyuki Fukui 73 9 a Department of Molecular Pharmacology, Institute of Biomedical Sciences, Tokushima University Graduate School, Sho-machi, Tokushima 770-8505, Japan 74 10 b Division of Pharmacology, Department of Integrated Basic Medical Science, Ehime University School of Medicine, Toon 791-0295, Japan 75 11 c Department of Otolaryngology, Institute of Biomedical Sciences, Tokushima University Graduate School, Kuramoto, Tokushima 770-8503, Japan 76 d 12 Department of Molecular Studies for Incurable Diseases, Institute of Biomedical Sciences, Tokushima University Graduate School, Kuramoto, Tokushima 77 770-8503, Japan 13 78 14 79 15 80 article info abstract 16 81 17 82 18 Article history: Antihistamines inhibit histamine signaling by blocking histamine H1 receptor (H1R) or suppressing H1R Received 7 September 2015 83 19 signaling as inverse agonists. The H1R gene is upregulated in patients with pollinosis, and its expression Received in revised form level is correlated with the severity of nasal symptoms. Here, we show that antihistamine suppressed 84 20 1 February 2016 upregulation of histidine decarboxylase (HDC) mRNA expression in patients with pollinosis, and its 85 21 Accepted 4 February 2016 expression level was correlated with that of H1R mRNA. Certain antihistamines, including mepyramine 86 Available online xxx 22 and diphenhydramine, suppress toluene-2,4-diisocyanate (TDI)-induced upregulation of HDC gene 87 23 expression and increase HDC activity in TDI-sensitized rats. However, d-chlorpheniramine did not 88 24 Keywords: Allergy demonstrate any effect. The potencies of antihistamine suppressive effects on HDC mRNA elevation were 89 fi 25 Antihistamines different from their H1R receptor binding af nities. In TDI-sensitized rats, the potencies of antihistamine 90 26 Gene expression inhibitory effects on sneezing in the early phase were related to H1R binding. In contrast, the potencies of 91 27 Histamine signaling their inhibitory effects on sneezing in the late phase were correlated with those of suppressive effects on 92 Histidine decarboxylase HDC mRNA elevation. Data suggest that in addition to the antihistaminic and inverse agonistic activities, 28 93 certain antihistamines possess additional properties unrelated to receptor binding and alleviate nasal 29 94 30 symptoms in the late phase by inhibiting synthesis and release of histamine by suppressing HDC gene transcription. 95 31 © 2016 Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an 96 32 open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 97 33 98 34 99 35 100 36 101 1. Introduction 37 102 38 103 Pollinosis, a seasonal allergic rhinitis, affects approximately 30% 39 104 of the Japanese population (1). Histamine plays important roles in 40 105 the pathogenesis of allergic rhinitis; its action is mainly mediated 41 Abbreviations: DEPC, diethylpyrocarbonate; PCA, perchloric acid; GAPDH, glyc- 106 eraldehyde-3-phosphate dehydrogenase; H1R, histamine H receptor; HDC, histi- through the histamine H1 receptor (H1R). Antihistamines act as 42 1 107 dine decarboxylase; IL, interleukin; PKCd, protein kinase C-d; PMA, phorbol 12- inverse agonists to inhibit histamine signaling by blocking H1R or 43 108 myristate 13-acetate; PUM1, pumilio RNA binding family member 1; Th1/Th2, suppressing H1R signaling (2,3). Therefore, antihistamines are 44 helper T cell type 1/2; TDI, toluene-2,4-diisocyanate. 109 employed as first-line treatment for the nasal symptoms of 45 * Corresponding author. Department of Molecular Pharmacology, Institute of 110 pollinosis. Q2 46 Biomedical Sciences, Tokushima University Graduate School, 1-78-1 Sho-machi, 111 Tokushima 770-8505, Japan. Tel./fax: þ81 88 633 7264. H1R gene expression strongly correlates with the severity of 47 112 E-mail address: [email protected] (H. Mizuguchi). allergic symptoms in toluene-2,4-diisocyanate (TDI)-sensitized rats 48 113 Peer review under responsibility of Japanese Pharmacological Society. and patients with pollinosis (4,5). Further, the suppression of 49 1 Present address: Pharmacy Discipline Khulna University, Khulna, Bangladesh. 114 Q1 upregulated H1R gene expression alleviates nasal symptoms (6e9). 50 2 Present address: Department of Pharmacy Jahangirnagar University, Dhaka, 115 Allergic reactions are characterized further by disrupting the T 51 Bangladesh. 116 3 H.M. and A.K.D. contributed equally to this work. helper cell type 1/2 (Th1/Th2) balance generating a pronounced 52 117 53 http://dx.doi.org/10.1016/j.jphs.2016.02.002 118 54 1347-8613/© 2016 Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license 119 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Mizuguchi H, et al., Antihistamines suppress upregulation of histidine decarboxylase gene expression with potencies different from their binding afﬁnities for histamine H1 receptor in toluene 2,4-diisocyanate-sensitized rats, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.02.002 JPHS202_proof ■ 17 February 2016 ■ 2/7

2 H. Mizuguchi et al. / Journal of Pharmacological Sciences xxx (2016) 1e7

1 Th2 profile (10). Th2 cytokine genes, including interleukin (IL)-4 not take antihistamines or any other treatment were designated as 66 2 and IL-5 genes, are upregulated in TDI-sensitized rats and patients the no treatment group. 67 3 with pollinosis; their expression levels were correlated with that of Total RNA isolated using the RNAqueous Micro Kit (Applied 68 4 H1R gene (4,7,11), suggesting crosstalk between the H1R and Th2 Biosystems, Foster City, CA) was reverse transcribed using the 69 5 cytokine signaling pathways. Intranasal application of IL-4 and High Capacity cDNA Reverse Transcription Kit (Applied Bio- 70 6 histamine increases H1R gene and IL-4 gene expression, respec- systems). The sequences of the HDC primers and TaqMan probe 71 7 tively (7). These findings suggest that H1R signaling is important for are listed in Table 1. The pumilio RNA binding family member 1 72 8 pollinosis development and that drugs designed to suppress H1R (PUM1) primer and probe kit (Hs 00206469-m1, Applied Bio- 73 9 signaling will be effective for treating allergies. systems) was used to generate a standard (5).Thedataare 74 10 Because histidine decarboxylase (HDC) is the only enzyme that expressed as the ratio of HDC mRNA to PUM1 mRNA as previ- 75 11 catalyzes histamine synthesis, HDC gene expression is an impor- ously described. The ethics committee of Tokushima University 76 12 tant regulatory step in histamine signaling (12). Histamine is Hospital and Yashima General Hospital approved this study; 77 13 released from subcutaneous mast cells during the anaphylaxis written informed consent was obtained from each patient before 78 14 phase via an IgE-mediated mechanism, whereas in the post- the study commenced. 79 15 anaphylaxis phase, histamine is produced because of an 80 16 increased HDC activity (13). HDC mRNA levels increase in patients 2.2. Animal studies 81 17 with allergic rhinitis and bronchial asthma (14). Application of TDI 82 18 upregulates HDC mRNA expression and HDC enzymatic activity Six-week-old male Brown Norway rats (200e250 g, Japan SLC, 83 19 and histamine content in the nasal mucosa (7). Moreover, sup- Hamamatsu, Japan) with free access to water were kept in a room 84 20 pression of HDC gene expression alleviates the nasal symptoms of maintained at constant temperature (25 ± 2 C) and humidity 85 21 TDI-sensitized rats. (55% ± 10%) with 12-h light/dark cycle. TDI-sensitization was per- 86 22 Here we show that preseasonal prophylactic treatment with formed as previously reported (12). Antihistamines were orally 87 23 antihistamines suppressed the upregulation of HDC mRNA administered once daily for 3 weeks. The number of sneezes was 88 24 expression in patients with pollinosis and that the expression level measured during the 10-min period just after (early phase reaction) 89 25 of HDC mRNA was strongly correlated with that of H1R mRNA. or 9 h (late phase reaction) after TDI provocation. The antihista- 90 fi 26 Because insuf cient data are available on the effect of antihistamines (10 mg/kg each) were orally administered 1 h before TDI 91 27 mines on HDC gene expression, we used TDI-sensitized rats to provocation, whereas the control group received water. All exper- 92 28 investigate the effects of antihistamines on nasal symptoms, his- imental procedures were performed in accordance with the 93 29 tamine levels in nasal discharges, HDC activity, and HDC gene guidelines of the Animal Research Committee of Tokushima 94 30 expression in the nasal mucosa. Our data suggest that during the University. 95 31 late-phase response, mepyramine and diphenhydramine signifi- 96 32 cantly suppressed TDI-induced upregulation of HDC mRNA, HDC 97 2.3. Real-time quantitative reverse transcription polymerase chain 33 activity, and histamine content in the nasal mucosa with potencies 98 3 reaction 34 that differed from their Ki values for [ H]mepyramine binding af- 99 35 finity to H1R. 100 Rat nasal mucosa samples collected using RNAlater (Applied 36 101 Biosystems, Foster City, CA, USA) 4 h after provocation were ho- 37 102 mogenized using a Polytron (Model PT-K; Kinematica AG, Littau/ 38 2. Materials and methods 103 Luzern, Switzerland) in 10 volumes of ice-cold TRIzol; total RNA 39 104 was prepared as previously described (7). RNA samples (8 mg) were 40 2.1. Analysis of HDC mRNA expression in the nasal mucosa of 105 reverse-transcribed using SuperScript II reverse transcriptase 41 patients with pollinosis 106 (Invitrogen); real-time PCR was conducted as previously described 42 107 (7). TaqMan primers and probes were designed using Primer Ex- 43 We determined HDC mRNA levels using the remaining nasal 108 press software (Applied Biosystems). The primers used to detect rat 44 mucosa samples of patients previously analyzed for H1R mRNA 109 HDC mRNA are listed in Table 1. To standardize the starting mate- 45 levels (5). Patient information, preparations for scraping the nasal 110 rial, Rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 46 mucosa, evaluation of nasal symptoms, and other experimental 111 Control Reagents (VICProbe; Applied Biosystems) were used. Data 47 conditions were previously described (5). Among 25 patients with 112 are expressed as the ratio of HDC mRNA to GAPDH mRNA. 48 allergic rhinitis caused by Japanese cedar pollen, 8 patients (3 113 49 males and 5 females; mean age, 47.9 years) who were treated at 114 50 the Department of Otolaryngology, Yashima General Hospital 115 e 51 before the peak pollen period (February 20 March 26, 2009) Table 1 116 52 were administered prophylactic treatment with antihistamines Nucleotide sequences of primers and probes. Q5 117 53 (ebastine, 10 mg/day p.o., n ¼ 5; and fexofenadine, 120 mg/day 118 Primer/probe name Sequence 54 p.o., n ¼ 3). The treatment lasted for 17.9 ± 11.0 days (mean ± SD) 119 55 before the follow-up visit during the peak pollen period when Human HDC mRNA 120 Sense primer 50-CCTGAATGCAGCTCTCAATGTG-30 56 nasal symptoms were evaluated and nasal mucosa scrapings were Anti-sense primer 50-CAGAGTCCCTGAAGTATATCCTCAGAC-30 121 57 obtained from patients by an otolaryngologist as previously Probe FAM-TTGCCCTCTGCAGGCCATGGTTTA-TAMRA 122 58 described (5). 123 Rat HDC mRNA 59 Sneezing, watery rhinorrhea, and nasal obstruction were sepa- Sense primer 50-GCAGCAAGGAAGAACAAAATCC-30 124 60 rately scored on a 0e5 point scale before nasal sampling. Under Anti-sense primer 50-CAACAAGACGAGCGTTCAGAGA-30 125 61 local anesthesia using 4% lidocaine, the surface of each patient's Probe FAM-AAAGCGCATGAGCCCAATGCTGAT-TAMRA 126 62 inferior nasal concha was scratched with a small spatula to obtain a Mouse HDC mRNA 127 63 sample of the nasal mucosa. Of the initial 25 patients, the Sense primer 50-TCTACCTCCGACATGCCAACT-30 128 0 0 64 remaining patients (8 males and 9 females; mean age, 46.3 years) Anti-sense primer 5 -CCACAGCTTAATGGAGCGAAAG-3 129 Probe FAM-CACGGACTTCATGCATTGGCAGATCC-TAMRA 65 who first visited the hospital during the peak pollen period but did 130

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1 2.4. Measurement of HDC activity and histamine content in the expression levels were positively correlated with nasal symptoms 66 2 nasal mucosa (Fig. 1B) and H1R expression levels (Fig. 1C). 67 3 68 4 Nasal mucosa was removed from the nasal septum 9 h after TDI- 3.2. Effect of antihistamines on TDI-induced upregulation of HDC 69 5 provocation and homogenized with 10 volumes of ice-cold HDC gene expression, HDC activity, histamine content in the nasal 70 6 buffer containing 0.1 M potassium phosphate buffer, pH 6.8, mucosa, and histamine levels in nasal lavage fluid of TDI-sensitized 71 7 0.2 mM DTT, 0.01 mM pyridoxal 50-phosphate, 1% polyethylene rats 72 8 glycol (approximately 300 kDa), and 100 mg/ml PMSF. The ho- 73 9 mogenates were centrifuged at 10,000 g for 15 min at 4 C, and Compared with controls, HDC mRNA expression in TDI- 74 10 one-half of the supernatant (supernatant A) was dialyzed thrice sensitized rats was significantly increased after provocation with 75 11 against HDC buffer for 6 h at 4 C (supernatant B). Histamine TDI. Mepyramine and diphenhydramine strongly suppressed TDI- 76 12 content in supernatant A was determined using high-performance induced upregulation of HDC gene expression in TDI-sensitized 77 13 liquid chromatography (12). The HDC activity of supernatant B was rats (Fig. 2A). The other antihistamines, except d-chlorphenir- 78 14 determined by incubation for 4 h at 37 C in 0.25 mM L-histidine. amine, tended to suppress the TDI-induced increase in HDC mRNA 79 15 HDC activities were calculated according to histamine levels (minus levels. We next measured HDC activity in the nasal mucosa of TDI- 80 16 the blank). Protein concentrations were determined using the sensitized rats 9 h after TDI provocation. TDI application for 3 81 17 bicinchoninic acid protein assay reagent (Sigma) and bovine serum weeks induced a significant increase in HDC activity (Fig. 2B). 82 18 albumin as standard. Pretreatment with mepyramine and diphenhydramine signifi- 83 19 cantly suppressed the TDI-induced increase in HDC activity. Aze- 84 20 2.5. Determination of histamine content in nasal-cavity lavage fluid lastine, epinastine, and olopatadine tended to suppress HDC 85 21 activity. In contrast, d-chlorpheniramine, which did not suppress 86 22 Nasal lavage was performed 9 h after TDI provocation using a TDI-induced HDC upregulation, failed to suppress HDC activity. 87 23 slight modification of a published method (15). Rats were anes- Provocation with TDI induced a significant increase in histamine 88 24 thetized with diethyl ether and then polyethylene tubing con- content in the nasal mucosa of TDI-sensitized rats (Fig. 3). Pre- 89 25 nected to a regulated vacuum device positioned in the left nostril; treatment with mepyramine and diphenhydramine significantly 90 26 slight negative pressure was then applied. Washing with 1.5 ml PBS decreased histamine content in the nasal mucosa. Azelastine, epi- 91 27 (37 C) was performed from the right to left nostrils and repeated in nastine, and olopatadine tended to reduce the histamine level in 92 28 reverse order. Nasal exudates were transferred to a new tube; 10 ml the nasal mucosa. d-Chlorpheniramine had no detectable effect. 93 29 perchloric acid (60% w/v) was added, The suspension was centri- Analysis of nasal lavage samples revealed a notable increase in 94 30 fuged at 10,000 g for 15 min at 4 C, transferred to another tube histamine content 9 h after TDI provocation, which was signifi- 95 31 and stored at 20 C. The histamine content in the supernatant was cantly suppressed by mepyramine and olopatadine. Azelastine 96 32 determined as described above. tended to decrease histamine content, whereas d-chlorphenir- 97 33 amine did not have a detectable effect (Fig. 4). 98 34 2.6. Effect of antihistamines on PMA-induced upregulation of HDC 99 35 gene expression in RAW 264.7 cells 3.3. Effect of antihistamines on TDI-induced nasal symptoms of 100 36 TDI-sensitized rats 101 37 RAW 264.7 cells were cultured in Eagle's minimal essential 102 38 medium containing 10% fetal calf serum and 1% anti- To determine whether histamine induced an allergic reaction, 103 39 bioticeantimycotic (Invitrogen) at 37 C in a humidified atmo- we monitored sneezing just after or 9 h after provocation. Pre- 104 40 sphere containing 5% CO2. RAW 264.7 cells (70% confluence) in 6- treatment with antihistamines reduced the number of TDI-induced 105 41 well dishes were treated with 10 mM antihistamines 1 h before sneezes at both times (Fig. 5). The potencies of inhibitory effects of 106 42 the addition of 100 nM of phorbol 12-myristate 13-acetate (PMA). the antihistamines on the number of sneezes just after provocation 107 43 After 5 h, the cells were harvested using 700 ml RNAiso Plus (Takara) with TDI were similar to their respective affinities of binding to H1R 108 44 to isolate total RNA that was reverse transcribed (5 mg) using a High (Table 2) (16e19). In contrast, 9 h after TDI provocation, these ef- 109 45 Capacity cDNA Reverse Transcription kit (Applied Biosystems). fects were similar to the inhibition of HDC mRNA expression. 110 46 Real-time PCR was performed as described above. 111 47 3.4. Effect of antihistamines on PMA-induced upregulation of HDC 112 48 2.7. Statistical analysis gene expression in RAW 264.7 cells 113 49 114 50 The results are shown as mean ± SEM, and the data acquired PMA treatment upregulates HDC mRNA expression in RAW 115 51 using nasal mucosa of patients with pollinosis are shown as 264.7 cells by activating the PKC/ERK signaling pathway (20). When 116 52 mean ± SD. Statistical analyses were performed using the unpaired RAW 264.7 cells were pretreated with mepyramine and azelastine, 117 53 t test or one-way ANOVA using Dunnett's test with GraphPad Prism the upregulation of HDC mRNA expression induced by PMA was not 118 54 software (GraphPad Software, Inc. San Diego CA). Correlations were suppressed, although it was significantly suppressed by d-chlor- 119 55 analyzed using the Spearman rank correlation method. Values of pheniramine (Fig. 6). 120 56 p < 0.05 were considered statistically significant. 121 57 4. Discussion 122 58 3. Results 123 59 In the present study, we demonstrate that certain antihista- 124 60 3.1. Effect of preseasonal prophylactic treatment with antihistamine mines, including mepyramine and diphenhydramine, alleviate 125 61 on HDC mRNA expression in the nasal mucosa of patients with allergic symptoms through their antihistaminic effects and by 126 62 pollinosis suppressing histamine synthesis by inhibiting HDC mRNA expres- 127 63 sion. H1R gene expression was highly correlated with the severity 128 64 Preseasonal prophylactic treatment with antihistamines signif- of allergic symptoms, and compounds that suppress H1R gene 129 65 icantly suppressed HDC mRNA expression (Fig. 1A). HDC mRNA expression alleviate the symptoms of allergy of patients with 130

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1 (A) (B) (C) 66 2 16 10 3.5 67 3 68 9 4 3 69 8 5 12 70 2.5 6 7 71 7 6 2 72 8 8 5 73 1.5 9 * 4 74

10 rhinorrhea score 3 1 75 4 11 2 76 H1R mRNA/PUM1 mRNA HDC mRNA/PUM1 mRNA 12 sum of sneezing and watery 0.5 77 1 r = 0.923, p < 0.01 13 r = 0.618, p < 0.01 78 0 14 0 0 79 prophylactic 0246810121416 0246810121416 15 no treatment 80 treatment HDC mRNA/PUM1 mRNA HDC mRNA/PUM1 mRNA 16 81 17 Fig. 1. Effect of preseasonal prophylactic treatment with antihistamines on the upregulation of HDC mRNA (A) and the relationship between the expression levels of HDC mRNA and 82 18 the nasal symptoms (B) and the expression level of H1R mRNA (C). The scraping of the nasal mucosa from the patients with Japanese cedar pollinosis were collected and total RNA 83 19 was isolated using the RNAqueous Micro Kit. HDC mRNA was determined by quantitative real-time RT-PCR. Data for the sum of sneezing and watery rhinorrhea scores and the 84 expression of H1R mRNA were obtained from Mizuguchi et al. (8). In A, Data are expressed as means ± SD. For statistical analysis, unpaired t-test was used. *P < 0.05 vs. no treatment 20 group. In B and C, ﬁlled circles and open squares represent data obtained from patients with or without preseasonal prophylactic treatment with antihistamines, respectively. 85 21 Correlation was analyzed by Spearman's rank correlation test. 86 22 87 23 88 24 (A) (B) 89 25 90 4 0.2 26 91 27 92 28 93 3 0.15 29 * 94 30 * * * 95 31 * 96 32 2 0.1 97 ** 33 ** 98 34 99 35 1 0.05 100

36 HDC mRNA/GAPDH mRNA 101 HDC activity (pmol/min/mg protein) 37 102 0 0 38 Control TDI Mepy Diphen Prom Azelas Epinas Olop Chlor Control TDI Mepy Diphen Azelas Epinas Olop Chlor 103 39 104 40 105 41 106 42 Fig. 2. Antihistamines block TDI-induced upregulation of HDC mRNA (A) and suppress HDC activity (B). In A, HDC mRNA was determined by quantitative real-time RT-PCR 4 h after 107 provocation. In B, HDC activity in the nasal mucosa was measured by HPLC 9 h after provocation. Data are expressed as means ± SEM. Control, vehicle control; TDI, TDI-provoked 43 rats without administration of antihistamines (TDI-control); Diphen, diphenhydramine; Mepy, mepyramine; Diphen, diphenhydramine; Prom, promethazine; Azlas, azelastine; 108 ** * yy y 44 Epinas; epinastine; Olop, olopatadine; Chlor, d-chlorpheniramine. P < 0.01, and P < 0.05 vs. TDI; P < 0.01 and P < 0.05 vs. d-chlorpheniramine (n ¼ 4). 109 45 110 46 pollinosis (4e9). Th2 cytokines also play important roles in the disease (1). In contrast, rhinitis caused by TDI is a non-IgE medi- 111 47 pathogenesis of allergic inflammation (10). For example, in patients ated disease (23). For example, TDI-specific IgE is not detected in 112 48 with pollinosis, histamine influences the expression and actions of mice exposed to TDI, although total IgE and TDI-specific IgG titers 113 49 Th2 cytokines through crosstalk between the H1R and Th2 cytokine are elevated (24). Therefore, nasal symptoms and pathogenesis 114 50 signaling pathways; and Th2 cytokines, in turn, modulate the observed in TDI-sensitized rats may differ from those of antigen- 115 51 production and release of histamine (21,22). induced IgE-dependent rhinitis. However, intranasal application 116 52 We demonstrated that prophylactic treatment with antihista- of TDI induces nasal symptoms, including sneezing, and watery 117 53 mines suppressed TDI-induced upregulation of H1R and IL-4 mRNA rhinorrhea in TDI-sensitized guinea pigs (25), which are similar to 118 54 expressions in TDI-sensitized rats (4). We also showed that direct those observed in patients with allergic rhinitis. TDI-sensitized rats 119 55 administration of IL-4 into the nasal cavities of normal non-TDI- display many characteristic features of human allergic rhinitis (e.g., 120 56 treated rats upregulated H1R mRNA expression (7). Intranasal nasal symptoms), including infiltration of eosinophils and mast 121 57 histamine application increased IL-4 mRNA levels in normal rats cells (26), increased levels of cytokines (27e30), elevation of H1R 122 58 (7), and H1R expression level was strongly correlated with that of mRNA and protein expression (31), increased levels of HDC mRNA, 123 59 IL-5 in patients with pollinosis (11). These findings suggest that HDC activity, and histamine content (12). Accordingly, dis- 124 60 H1R gene expression is correlated with that of Th2-cytokine genes tinguishing between immunological sensitization and irritation by 125 61 and that suppression of H1R gene expression affects the expression TDI is difficult, probably because that the underlying mechanisms 126 62 levels of Th2 cytokines. Therefore, we consider that the suppression driving TDI-induced rhinitis share similarities with allergic rhinitis 127 63 of H1R signaling is important for treating allergies. caused by ubiquitous airborne protein allergens. Therefore, we 128 64 In the present study, we used TDI-sensitized rats as a model of consider that TDI-sensitized rats can serve as a model for human 129 65 allergic rhinitis. Allergic rhinitis is defined as an IgE-mediated allergic rhinitis. 130

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1 10 Sensitization and provocation with TDI elevates HDC mRNA 66 2 levels in the nasal mucosa, followed by increases in HDC activity 67 3 and histamine content (12). Here pretreatment with antihista- 68 4 8 mines, except d-chlorpheniramine, suppressed TDI-induced HDC 69 5 mRNA upregulation at different levels, which indicates the exis- 70 6 tence of novel targets and mechanisms of antihistaminic action as 71 7 6 * well as inverse agonistic activities. The potencies of the inhibitory 72 8 * effects of antihistamines on the increase in HDC mRNA levels were 73 9 different from their binding afﬁnities for the H1R (16e19), sug- 74 10 gesting that this effect is unrelated to H1R binding. Moreover, hu- 75 4 11 man nasal mucosal epithelial and vascular endothelial cells express 76 12 H1R (35). The results are consistent with our unpublished immu- 77 13 nohistochemical analysis of TDI-sensitized rats. To our knowledge, 78 2

14 histamine (pmol/mg protein) there are no reports of HDC expression in epithelial and endothelial 79 15 cells; thus, it is unlikely that these cells produce histamine in an 80 16 autocrine manner. These findings support our hypothesis that the 81 0 17 Control TDI Diphen Mepy Azelas Epinas Olop Chlor suppressive effect of antihistamines on the HDC-induced elevation 82 18 of mRNA expression is not related to H1R-binding. 83 19 Antihistamines have pharmacological properties unrelated to 84 20 receptor binding (36). Azatadine, d-chlorpheniramine, mepyr- 85 21 Fig. 3. Antihistamines suppress histamine synthesis. Histamine content in the nasal amine, and promethazine reduce the release of proinflammatory 86 22 mucosa was measured by HPLC 9 h after provocation. Data are expressed as mediators from mast cells and basophils, chemotaxis and activation 87 ± 23 means SEM. Control, vehicle control; TDI, TDI-control; Diphen, diphenhydramine; of inflammatory cells, particularly eosinophils, and expression of 88 Mepy, mepyramine; Azlas, azelastine; Epinas, epinastine; Olop, olopatadine; Chlor, d- * 24 chlorpheniramine. P < 0.05 vs. TDI (n ¼ 4). adhesion molecules in epithelial cell lines induced by immuno- 89 25 logical or nonimmunological stimuli (37). Desloratadine inhibits 90 26 IgE-mediated IL-4 and IL-13 secretion from human basophils (38), 91 27 and antihistamines suppress Th2 response (3,7e9,12,39). However, 92 28 4 no reports demonstrate that antihistamines suppress HDC ex- 93 29 pressions. To the best of our knowledge, the present study reports 94 30 the first evidence demonstrating that antihistamines suppress the 95 31 upregulation of HDC mRNA expression. 96 32 3 Although several studies demonstrate the upregulation of HDC 97 33 mRNA expression, the mechanism is unknown. Because PMA 98 34 upregulates HDC gene expression (40), PKC signaling may induce 99 35 2 the upregulation of HDC gene transcription. For example, the PKC/ 100 36 ERK/CEBPb pathway mediates PMA-induced upregulation of HDC 101 37 ** mRNA expression in RAW 264.7 cells (20). Our present data 102

38 histamine (pmol/ml) demonstrated that mepyramine and azelastine did not inhibit 103 1 39 PMA-induced upregulation of HDC gene expression in RAW 104 40 264 cells, although they suppressed HDC gene expression in TDI- 105 41 sensitized rats. In contrast, d-chlorpheniramine did not suppress 106 42 0 HDC expression in TDI-sensitized rats, although it inhibited PMA- 107 Control TDI Mepy Azelas Olop Chlor 43 induced HDC upregulation in RAW 264 cells. Therefore, it is un- 108 44 likely that PKC signaling upregulates HDC mRNA expression 109 45 induced by TDI. NF-kB represents another candidate because its 110 46 111 Fig. 4. Antihistamines restrain histamine release in nasal discharge in nasal allergy expression is elevated in patients with allergic rhinitis (41). Dexa- 47 model rats. Nasal discharge from both vestibules was collected by polyethylene tubing methasone significantly reduces the upregulation of HDC mRNA 112 48 connected to a regulated vacuum 9 h after provocation. Histamine content was expression in TDI-sensitized rats (12), and the glucocorticoid 113 49 measured by HPLC. Data are expressed as means ± SEM. Control, vehicle control; TDI, receptoredexamethasone complex binds to AP-1 or NF-kB and 114 TDI-control; Mepy, mepyramine; Azlas, azelastine; Olop, olopatadine; Chlor, d-chlor- 50 ** * y yy suppresses their transcriptional activities. Therefore, the suppres- 115 pheniramine. P < 0.01 and P < 0.05 vs. TDI; P < 0.01 and P < 0.05 vs. d-chlor- 51 pheniramine (n ¼ 4). sive effect of dexamethasone on TDI-induced HDC mRNA upregu- 116 52 lation may be explained by the inhibition of NF-kB signaling. 117 53 However, we have no data to prove this, and there are no reports of 118 54 Because histamine upregulates H1R gene expression (32), the NF-kB-induced HDC gene expression. 119 55 regulation of HDC expression is considered to be important. Pre- It was reported that the expression of HDC gene was transcrip- 120 56 seasonal prophylactic treatment with antihistamine suppressed the tionally regulated by DNA methylation at the region including the GC 121 57 upregulation of HDC mRNA expression in patients with pollinosis box sequence in the promoter (42). Demethylation of this GC box by 122 58 (Fig. 1). Further, HDC mRNA expression level was strongly corre- the treatment with 5-azacytidine induced a high level of HDC mRNA 123 59 lated with that of H1R mRNA. These findings suggest that the expression, suggesting that HDC expression levels were positively 124 60 suppression of HDC expression decreases histamine-induced H1R correlated with hypomethylation of the HDC promoter region (43).It 125 61 mRNA expression level and alleviates nasal symptoms, supporting was also reported that Sp-1 binds to the GC box in the HDC promoter 126 62 our hypothesis that the regulation of HDC expression is important and protects from de novo methylation (42). Since long-term effect 127 63 for treating allergies. The importance of histamine in allergies is was observed in TDI-sensitized rats, DNA methylation status or Sp-1 128 64 also demonstrated by the results of studies using HDC-deficient dependent transcription may be affected by antihistamines such as 129 65 mice (33,34). mepyramine and diphenhydramine. Investigation of the effect of 130

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1 (A) (B) 66 2 67 3 68 4 40 just after provocation 16 9 h after provocation 69 5 70 6 71 7 30 12 72 8 * * 73 9 * 74 20 ** 8 * * 10 ** 75 11 ** 76 number of sneezes number of sneezes 12 10 4 77 13 78 14 79 0 0 15 Control TDI Mepy Diphen Azelas Epinas Olop Chlor Control TDI Mepy Diphen Azelas Epinas Olop Chlor 80 16 81 17 82 18 83 19 Fig. 5. Pretreatment with antihistamines reduced the number of sneezes in nasal allergy model rats. The number of sneezes was counted for 10 min just after provocation (A) and 84 9 h after provocation (B). Data are expressed as means ± SEM. Control, vehicle control; TDI, TDI-control; Diphen, diphenhydramine; Mepy, mepyramine; Azlas, azelastine; Epinas, 20 ** * y 85 epinastine; Olop, olopatadine; Chlor, d-chlorpheniramine. P < 0.01 and P < 0.05 vs. TDI; P < 0.05 vs. d-chlorpheniramine (n ¼ 4). 21 86 22 87 these antihistamines on the DNA methylation of the HDC promoter To assess the clinical significance of these results, we measured 23 88 or Sp-1-dependent HDC gene transcription is under way in our the number of sneezes for 10 min just after and 9 h after provo- 24 89 laboratory. cation. Each antihistamine significantly suppressed sneezing at 25 90 The novel mechanism of the regulation of HDC gene expression both times, and the potency of suppression in the early phase was 26 91 described here is not uniform among antihistamines. For example, similar to receptor-binding affinity. In contrast, in the late phase, 27 92 we showed that mepyramine and diphenhydramine significantly the suppressive potency might be correlated with histamine syn- 28 93 suppressed HDC activity and histamine content in the nasal mucosa thesis, suggesting that certain antihistamines suppressed the nasal 29 94 of TDI-sensitized rats, whereas they were also suppressed by aze- symptom by inhibiting histamine synthesis through the suppres- 30 95 lastine, epinastine, and olopatadine, although the effects of the sion of HDC mRNA expression. 31 96 latter were not statistically significant. In contrast, d-chlorphenir- In conclusion, we demonstrate here that the long-term effects of 32 97 amine had no detectable effect. The potencies of the inhibitory certain antihistamines are mediated through inhibition of HDC 33 98 effects of antihistamines were different from their binding affinities gene expression and down-regulation of the H1R signaling 34 99 for the H1R. Because mepyramine, diphenhydramine, epinastine, pathway. These findings provide a new perspective on the long- 35 100 and d-chlorpheniramine are inverse agonists and olopatadine is a term effects of antihistamines on allergies. Specifically, mepyr- 36 101 neutral antagonist (3), it is unlikely that the ability to suppress HDC amine and diphenhydramine inhibited allergic responses during 37 102 mRNA expression was correlated with inverse agonistic activity. the late phase. Therefore, detailed knowledge on the mechanism 38 103 The diversity among antihistamines in the suppression of HDC that regulates HDC gene expression may be helpful for the devel- 39 104 mRNA expression is of interest because uncovering their structur- opment of new drugs for treating allergies. 40 105 eeactivity relationships may facilitate the discovery of new drugs 41 106 for treating allergies. When we investigated the levels of histamine 42 107 in the nasal discharges, similar results were obtained. For example, 43 108 mepyramine and olopatadine significantly suppressed the TDI- 44 5 109 induced release of histamine. The ability of olopatadine to inhibit 45 110 histamine release was not related to the suppression of HDC mRNA 46 111 expression but related to its function as a membrane stabilizer (43). 47 4 112 Here the inhibitory effect of azelastine was not statistically signif- 48 113 icant, and d-chlorpheniramine did not affect histamine release. * 49 114 50 3 115 51 116 Table 2 3 52 Ki values of antihistamines used in this study determined by [ H]mepyramine 117 53 Q6 displacement assay. 2 118 54 119 Antihistamines Ki (nM) 55 120 Mepyramine 1.7a, 4.5b 56 a b 1 121 d-Chlorpheniramine 7.5 , 8.0 HDC mRNA/GAPDH mRNA 57 Diphenhydramine 17b 122 58 Promethazine 2.9b 123 59 Azelastine 7.0c 0 124 c 60 Epinastine 3.8 Control PMA Mepy Azelas Chlor 125 Olopatadine 41d 61 126 62 Data were taken from: Fig. 6. Effect of antihistamines on PMA-induced upregulation of HDC gene expression 127 a m 63 Ref. (16). in RAW 264.7 cells. RAW 264.7 cells were treated with 10 M of antihistamine 1 h 128 b Ref. (17). before stimulation with 100 nM PMA. After a 5-h treatment with PMA, total RNA was 64 c Ref. (18). isolated, and the HDC mRNA levels were determined by real-time quantitative RT-PCR. 129 * 65 d Ref. (19). Data are presented as the means ± SEM. (n ¼ 4). P < 0.05 vs. PMA. 130

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1 Conflicts of interest (18) Fukuda S, Midoro K, Yamasaki M, Gyoten M, Kawano Y, Fukui H, et al. Char- 66 2 acterization of the antihistamine effect of TAK-427, a novel imidazopyridazine 67 derivative. Inflam Res. 2003;52:206e214. 3 The authors declare no financial conflicts of interest. (19) Sharif NA, Xu SX, Yanni JM. Olopatadine (AL-4943-A): ligand binding and 68 4 functional studies on a novel, long acting H1-selective histamine antagonist 69 and anti-allergic agent for use in allergic conjunctivitis. J Ocul Pharmacol Ther. 5 Q7 Uncited reference 70 1996;12:401e407. 6 (20) Shiraish M, Hirasawa N, Kobayashi Y, Oikawa S, Murakami A, Ohuchi K. 71 7 (44). Participation of mitogen-activated protein kinase in thapsigargin-and TPA- 72 8 induced histamine production in murine macrophage RAW 264.7 cells. Br J 73 Pharmacol. 2000;129:515e524. 9 Acknowledgments (21) Igaz P, Novak I, Lazar E, Horvath B, Heninger E, Faauls A. Bidirectional 74 10 communication between histamine and cytokines. Inflamm Res. 2001;50: 75 11 This work was financially supported in part by grants-in-aid for 123e128. 76 12 (22) Marone G, Granata F, Spadaro G, Genovese A, Triggiani M. The histamine- 77 Research on Allergic disease and Immunology from the Ministry of cytokine network in allergic inflammation. J Allergy Clin Immunol. 13 Health Labor and Welfare of Japan (10103185) to HF, grants-in-aid 2003;112:S83eS88. 78 14 for Scientific Research (B) (26305004 to HF) and Scientific (23) Tanaka K, Okamoto Y, Nagaya Y, Nishimura F, Takeoka A, Hanada S, et al. 79 15 Research (C) (15K07933 to HM) from the Japan Society for the A nasal allergy model developed in the guinea pig by intranasal application of 80 2,4-toluene diisocyanate. Int Arch Allergy Appl Immunol. 1998;85:392e397. 16 Promotion of Science, and a grant from the Osaka Medical Research (24) Johnson VJ, Yucesoy B, Reynoids JS, Fluharty K, Wang W, Richardson D, et al. 81 17 Q3,4 Foundation for Incurable Diseases. 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