- Email: [email protected]
Accumulation of CRTH2-positive leukocytes in human allergic nasal mucosa
Accumulation of CRTH2-positive leukocytes in human allergic nasal mucosa Hideaki Shirasaki, MD, PhD; Megumi Kikuchi, MD, PhD; Etsuko Kanaizumi, MD, PhD; and Tetsuo Himi, MD, PhD
Background: Prostaglandin D2 (PGD2) has been thought to be a potent mediator involved in allergic rhinitis because PGD2 has been recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation and because PGD2 receptor antagonists relieved nasal allergic symptoms in an animal model of allergic rhinitis. The inflammatory effects of PGD2 are exerted through high-affinity interactions with 2 G protein– coupled receptors: D-prostanoid receptor 1 and chemoattractanthomologous receptor expressed on TH2 cells (CRTH2). CRTH2 may mediate the recruitment of leukocytes during a nasal allergic response. Objective: To evaluate the number of CRTH2-expressing cells in allergic and nonallergic human nasal mucosa by means of immunohistochemical analysis. Methods: Human turbinates were obtained after turbinectomy from 14 patients with nasal obstruction refractory to medical therapy. To identify cells expressing the CRTH2 protein, double immunostaining was performed using anti–CRTH2 antibody and monoclonal anti–leukocyte antibodies. Results: The immunohistochemical study revealed that anti–CRTH2 antibody labeled eosinophils, macrophages, mast cells, T lymphocytes, epithelial cells, and submucosal glands in the nasal mucosa. CRTH2 expressions of these leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa. Conclusion: These results suggest that CRTH2 may play an important role in the recruitment of leukocytes into allergic nasal mucosa. Ann Allergy Asthma Immunol. 2009;102:110–115.
INTRODUCTION The allergic response is a complex process involving the interaction of many mediators. Prostaglandin D2 (PGD2) is a potent inflammatory mediator, and its actions are mediated via specific cell surface receptors coupled to G proteins. PGD2 binds mainly 2 G protein– coupled receptors to exert its bioactivity: D-prostanoid receptor 1 (DP1) and chemoattractant-homologous receptor expressed on TH2 cells (CRTH2 [also known as DP2]).1 Activation of DP1 leads to a G␣smediated increase in intracellular cyclic adenosine monophosphate and agonist-induced calcium flux.2 Bronchodilation and vasodilation occur as a consequence of DP1 activation.3,4 In contrast, CRTH2 couples to a G␣i-type G protein, leading to the inhibition of cyclic adenosine monophosphate, and it increases intracellular calcium levels in various types of cells.5 PGD2 was recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation.6 In addition, it has been shown that CRTH2 antagonist attenuated an antigen-induced increase in nasal airway resistance and local eosinophil infiltration in sensitized guinea pigs.7 These studies suggest that CRTH2 may play an important role in the
Affiliations: Department of Otolaryngology, Sapporo Medical University, School of Medicine, Sapporo, Japan. Disclosures: Authors have nothing to disclose. Received for publication July 7, 2008; Received in revised form October 20, 2008; Accepted for publication November 7, 2008.
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pathogenesis of allergic rhinitis. In a previous study8 using rat anti– human CRTH2 antibody, BM16 demonstrated CRTH2 expression on eosinophils and T lymphocytes in nasal polyps. However, there has been no report of CRTH2 expression in human inferior turbinates. In the present study, immunohistochemical analysis of CRTH2 was performed to confirm the expression and distribution of CRTH2 using a commercial anti– human CRTH2 antibody in human allergic and nonallergic inferior turbinates. MATERIALS AND METHODS Tissue Preparation Human inferior turbinates were obtained after turbinectomy from 14 patients with nasal obstruction refractory to medical therapy. Informed consent was obtained from all the patients, and this study was approved by the ethics committee of Sapporo Medical University. All the patients were nonsmokers, and 7 had perennial allergy against mites as defined by questionnaire and CAP testing (Pharmacia, Uppsala, Sweden). Seven patients with negative CAP test results for the major inhaled allergens, including mites, birch, grass, mugwort, and Japanese cedar, formed the group with nonallergic rhinitis. All medications, including antibiotics, were prohibited for at least 3 weeks before the study. The demographic and clinical characteristics of the patients are summarized in Table 1. The nasal mucosal specimens were dissected from the cartilage and were fixed in 10% formalin for immunohistochemical analysis.
ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY
Table 1. Demographic Characteristics of Allergic and Nonallergic Patients Characteristics
Allergic rhinitis (n ⴝ 7)
Nonallergic rhinitis (n ⴝ 7)
Sex, M/F, No. Age, median (range), y Specific IgE to house dust mite (Dermatophagoides pteronyssinus), median (range), kU/L Total IgE, median (range), kU/L Blood eosinophils, median (range), cells/L Current nasal symptoms, No. of patients Nasal obstruction Sneezing Rhinorrhea
3/4 31 (19–58) 2.7 (1.0–13)
4/3 39 (21–55) ⬍0.35
210 (10–452) 370 (70–690)
110 (10–190) 115 (45–240)
7 4 3
7 0 2
Immunohistochemical Analysis Antibodies. For immunohistochemical analysis of CRTH2 receptor, rabbit anti– human CRTH2 receptor polyclonal antibody against a peptide corresponding to the N-terminal domain of human CRTH2 receptor (Cayman Chemical, Ann Arbor, Michigan) was used at 1:100 dilutions. To identify subsets of cells expressing CRTH2, the following monoclonal antibodies were used: anti– eosinophil cationic protein (EG2 clone) (Pharmacia) for eosinophils, anti-CD68 (KP-1 clone) (Dako, Glostrup, Denmark) for macrophages, anti– mast cell tryptase (AA1 clone) (Dako) for mast cells, and anti–CD3 antibody (F7.2.38 clone) (Dako) for T cells. Immunohistochemical analysis. Deparaffinized sections were initially incubated with 3% hydrogen peroxide in methanol for 10 minutes to quench endogenous peroxidase activity. After microwave treatment (10 minutes at 500 W in citrate buffer), the sections were incubated in blocking solution (10% normal goat serum in phosphate-buffered saline [PBS]) for 30 minutes before incubation in primary antibody. Then, the sections were incubated with anti-CRTH2 polyclonal antibody overnight at 4°C, washed, and incubated for 30 minutes with EnVision⫹, Peroxidase (Dako). A further washing in PBS was followed by developing in diaminobenzidine (DAB; Dako) as a chromogen for signal visualization. The slides were counterstained with Mayer hematoxylin and coverslipped using mounting medium. To identify subsets of cells expressing CRTH2, some sections were stained by means of the immunofluorescence technique. For double staining, deparaffinized sections were incubated overnight at 4°C with a combination of rabbit polyclonal anti– human CRTH2 antibody and 1 mouse monoclonal anti– human leukocyte phenotypical marker antibody after microwave treatment (10 minutes at 500 W in citrate buffer). Sections were washed in PBS and were incubated for 30 minutes with Alexa Fluor 594 –labeled goat anti–mouse IgG (diluted 1:50) (Molecular Probes, Eugene, Oregon) and Alexa Fluor 488 –labeled goat anti–rabbit IgG (diluted 1:50) (Molecular Probes). Sections were mounted using Vectashield mounting medium with 4⬘,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc, Burlingame, California) and were examined using a microscope (BX51) and a charge-coupled device camera (DP70) (Olympus Optical Co, Tokyo, Japan). All the images
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were processed using image analysis software (DP Controller and DP Manager; Olympus Optical Co). Using this method, CRTH2-expressing cells were green, cellular phenotypical markers were stained red, and the combined signal was visualized as yellow. To confirm the specificity of immunohistochemical analysis, a commercial CRTH2/DP2 receptor blocking peptide (Cayman Chemical) was used to block protein-antibody complex formation during immunohistochemical analysis according to the manufacturer’s instructions. Negative controls were obtained by replacing primary antibodies with mouse IgG1 and rabbit immunoglobulin fraction (Dako). Quantitation For double immunofluorescence staining, the slides were counted using a microscope equipped with an eyepiece graticule. A total of 6 fields (1 mm2 each) from each slide were counted by placing the upper edge of the grid at the epithelium. The number of CRTH2-positive cells in each phenotype in allergic and nonallergic nasal mucosa was evaluated by 2 masked investigators (M.K. and E.K.). Results are expressed as the number of CRTH2-positive cells per square millimeter and the percentage of CRTH2-positive cells in each phenotype. Statistical Analysis Values are expressed as mean (SD). Differences between groups are compared using the Mann-Whitney test. P ⬍ .05 was considered statistically significant. RESULTS The CRTH2 distribution in nasal mucosa was examined using a polyclonal antibody against a peptide corresponding to N-terminal amino acids 2 to 21 of human CRTH2. Immunoreactivity for CRTH2 was significantly detected in interstitial cells, submucosal glands, and epithelial cells (Fig 1A). The intensity of the CRTH2 receptor staining was decreased by co-incubation with a specific commercial blocking peptide (Fig 1B). Specificity of the staining was also confirmed by the absence of labeling with normal rabbit immunoglobulin (Fig 1C). To clarify the cell types of the interstitial cells expressing the CRTH2 protein, we performed double immunofluores-
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cence staining for CRTH2 and leukocyte phenotype marker. Fig 2 demonstrates examples of CRTH2-positive T lymphocytes (Fig 2B), eosinophils (Fig 2D), macrophages (Fig 2F), and mast cells (Fig 2H). As shown in Figure 3, we observed many CRTH2-expressing eosinophils (43.7 [14.6] cells/mm2) and T cells (42.0 [15.2] cells/mm2). The difference between the number of CRTH2-expressing eosinophils, macrophages, T cells, and mast cells was significantly higher in patients with allergy vs nonallergy (Fig 3). As shown in Figure 4, the ratio of CRTH2-positive cells in allergic nasal mucosa was highest in eosinophils (84.7% [10.9%]), and moderate
Figure 1. Immunohistochemical staining for chemoattractant-homologous receptor expressed on TH2 cells (CRTH2) receptor in human allergic nasal mucosa. Inferior turbinates were stained with anti– human CRTH2 receptor antibody (A and B) or normal rabbit immunoglobulin (C). Staining was observed mainly on airway epithelium (ep), submucosal glands (g), and interstitial cells. The intensity of the staining was decreased by co-incubation with a specific blocking peptide (B). v indicates blood vessels. Scale bar ⫽ 200 m.
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Figure 2. Identification of subsets of cells expressing the chemoattractanthomologous receptor expressed on TH2 cells (CRTH2) receptor among interstitial cells in human nasal mucosa using nuclear stain with 4⬘,6diamidino-2-phenylindole (blue). The CRTH2/DP2 receptor protein (green) shows co-localization with anti–leukocyte antibody (red), and the combined signal is visualized as yellow (B, D, F, H: overlay images). Identification markers are shown for CD3 (pan–T cells) (A and B), eosinophil cationic protein (eosinophils) (C and D), CD68 (macrophages) (E and F), and mast cell tryptase (mast cells) (G and H). Arrows indicate cells expressing CRTH2 and each phenotypical marker. Scale bar ⫽ 50 m.
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Figure 3. Mean (SD) number of chemoattractant-homologous receptor expressed on TH2 cells (CRTH2)–positive cells in each phenotype in allergic and nonallergic nasal mucosa. Sections were stained by means of double immunofluorescence for CRTH2 and eosinophils, macrophages, mast cells, and T cells. Error bars represent SD.
Figure 4. Mean (SD) percentage of chemoattractant-homologous receptor expressed on TH2 cells (CRTH2)–positive cells in each phenotype in allergic and nonallergic nasal mucosa. Sections were stained by means of double immunofluorescence for CRTH2 and eosinophils, macrophages, mast cells, and T cells. Error bars represent SD.
CRTH2 expression was associated with macrophages (45.4% [13.8%]), mast cells (43.3% [12.4%]), and T cells (33.4% [11.3%]). CRTH2 expressions of these leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa (Fig 4). DISCUSSION Although the CRTH2/TXA2 prostanoid (TP) receptor antagonist ramatroban has been in clinical use, there have
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been only a few studies indicating the expression of CRTH2 in nasal mucosa. Nantel et al8 reported immunohistochemical localization of CRTH2 using rat anti– human CRTH2 antibody BM16. They detected CRTH2-positive eosinophils and T cells in nasal polyps but not in tissues obtained from healthy individuals.8 Okano et al9 reported that the amount of CRTH2 messenger RNA positively and significantly correlated with the number of infiltrating eosinophils in human allergic inferior turbi-
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nates, suggesting involvement in eosinophil recruitment at inflammatory sites in vivo. The present immunohistochemical analysis showed that CRTH2-expressing cells were eosinophils, mast cells, macrophages, and T lymphocytes in human inferior turbinates. The CRTH2-dependent cell migration is not restricted to TH2 cells, eosinophils, and basophils, as initially reported,10 –12 but also to monocytes.13 These studies suggest that the local production of PGD2 may contribute to migration and activation of these inflammatory leukocytes during a local allergic response. In the present study, approximately 40% of mast cells showed significant immunoreactivity to CRTH2, suggesting the possibility of functional CRTH2 receptor on mast cells. However, there has been no report concerning the existence of CRTH2 on mast cells. Expression of CRTH2 was found not only on infiltrated leukocytes but also on vascular endothelial cells, epithelial cells, and submucosal glands in the nasal mucosa. It has been reported that normal human bronchial epithelial cells and the epithelial cell line NCI-H292 express CRTH2 messenger RNA and protein with an inner cell but without cell surface expression.14 This finding leads us to speculate that intracellular expression of CRTH2 on the epithelial cell has some functional potential and indicates that further research is needed. On the other hand, there seem to be no articles describing the existence of CRTH2 on submucosal glands or the effect of CRTH2 as the mucous secretagogue of airways. In the present study, we found that the percentages of CD3⫹ lymphocytes, macrophages, or mast cells expressing CRTH2 were higher in individuals with perennial allergic rhinitis. As yet, there seem to be no other studies describing up-regulation of CRTH2 expression in allergic airways. Electromobility shift assay demonstrated GATA-3 binding to CRTH2 promoter lesions, suggesting up-regulation of CRTH2 transcription by TH2 cytokines such as interleukin 4 (IL-4) and IL-13.15 Previous studies have demonstrated increased local levels of IL-4 and IL-13 after allergen provocation in allergic rhinitis16 and atopic asthma.17 These observations suggest that CRTH2-expressing cells in allergic airways might be up-regulated by locally produced IL-4/IL13. The clinical information on CRTH2 antagonists is limited to data on ramatroban (BAY u3405; Cayman Chemical), marketed in Japan for allergic rhinitis. Ramatroban has been characterized as a dual antagonist against thromboxane prostanoid receptor and CRTH2.18 Ramatroban significantly blocked allergen-induced local eosinophilia19 and nasal mucosal swelling20 in patients with allergic rhinitis. PGD2 is thought to induce nasal blockage by producing volume changes in the nasal venous sinusoids as a result of vasodilation. The PGD2-induced nasal blockage may be mediated by DP1 receptor rather than by CRTH2 receptor because ramatroban afforded no protection against PGD2-induced nasal blockage in humans.21 A previous immunohistochemical study22 showed TP expressed on blood vessels, not on inflammatory leukocytes, in nasal allergic mucosa. In addition,
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the selective CRTH2 antagonist 30089 equals the dual CRTH2/TP antagonist ramatroban regarding inhibition of eosinophil recruitment in a mouse asthma model.23 These observations suggest that the clinical effects of ramatroban on allergic rhinitis may be the improvement of nasal blockage by inhibition of TP receptor–mediated vascular reaction and that ramatroban may inhibit the migration of CRTH2-expressing leukocytes, such as eosinophils, TH2 lymphocytes, mast cells, and macrophages. In conclusion, using an immunohistochemical technique, we evaluated the number of CRTH2-expressing cells in allergic and nonallergic human nasal mucosa. CRTH2 expressions of the leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa. Precise knowledge of the identity and distribution of CRTH2 should be of considerable interest for understanding the role of CRTH2 in allergic rhinitis. ACKNOWLEDGMENTS We thank Tsuyako Watanabe for her expert technical assistance. REFERENCES 1. Sawyer N, Cauchon E, Chateauneuf A, et al. Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2. Br J Pharmacol. 2002; 137:1163–1172. 2. Boie Y, Sawyer N, Slipetz DM, Metters KM, Abramovitz M. Molecular cloning and characterization of the human prostanoid DP receptor. J Biol Chem. 1995;270:18910 –18916. 3. Norel X, Walch L, Labat C, Gascard JP, Dulmet E, Brink C. Prostanoid receptors involved in the relaxation of human bronchial preparations. Br J Pharmacol. 1999;126:867– 872. 4. Walch L, Labat C, Gascard JP, de Montpreville V, Brink C, Norel X. Prostanoid receptors involved in the relaxation of human pulmonary vessels. Br J Pharmacol. 1999;126:859 – 866. 5. Hirai H, Tanaka K, Yoshie O, et al. Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J Exp Med. 2001;193:255–261. 6. Naclerio RM, Meier HL, Kagey-Sobotka A, et al. Mediator release after nasal airway challenge with allergen. Am Rev Respir Dis. 1983;128: 597– 602. 7. Narita S, Asakura K, Kataura A. Effect of thromboxane A2 receptor antagonist (Bay u 3405) on nasal symptoms after antigen challenge in sensitized guinea pigs. Int Arch Allergy Immunol. 1996;109:161–166. 8. Nantel F, Fong C, Lamontagne S, et al. Expression of prostaglandin D synthase and the prostaglandin D2 receptors DP and CRTH2 in human nasal mucosa. Prostaglandins Other Lipid Mediat. 2004;73:87–101. 9. Okano M, Fujiwara T, Sugata Y, et al. Presence and characterization of prostaglandin D2-related molecules in nasal mucosa of patients with allergic rhinitis. Am J Rhinol. 2006;20:342–348. 10. Nagata K, Hirai H, Tanaka K, et al. CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cell-derived factor(s). FEBS Lett. 1999;459:195–199. 11. Monneret G, Gravel S, Diamond M, Rokach J, Powell WS. Prostaglandin D2 is a potent chemoattractant for human eosinophils that acts via a novel DP receptor. Blood. 2001;98:1942–1948. 12. Gervais FG, Cruz RP, Chateauneuf A, et al. Selective modulation of chemokinesis, degranulation and apoptosis in eosinophils through the PGD2 receptors CRTH2 and DP. J Allergy Clin Immunol. 2001;108: 982–988. 13. Gosset P, Bureau F, Angeli V, et al. Prostaglandin D2 affects the maturation of human monocyte-derived dendritic cells: consequence on the polarization of naïve Th cells. J Immunol. 2003;170:4943– 4952.
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14. Chiba T, Kanda A, Ueki S, et al. Prostagalndin D2 induced IL-8 and GM-CSF by bronchial epithelial cells in a CRTH2-independent pathway. Int Arch Allergy Immunol. 2006;141:300 –307. 15. Quapp R, Madsen N, Cameron L. Characterization of the promoter of human CRTH2, a prostaglandin D2 receptor. Biochem Biophys Res Commun. 2007;363:948 –953. 16. Erin EM, Zacharasiewicz AS, Nicholson GC, et al. Topical corticosteroid inhibits interleukin-4, -5 and -13 in nasal secretions following allergen challenge. Clin Exp Allergy. 2005;35:1608 –1614. 17. Kroegel C, Julius P, Matthys H, Virchow JC, Luttmann W. Endobronchial secretion of interleukin-13 following local allergen challenge in atopic asthma: relationship to interleukin-4 and eosinophil counts. Eur Respir J. 1996;9:899 –904. 18. Sugimoto H, Shichijo M, Iino T, et al. An orally bioavailable small molecule antagonist of CRTH2, ramatroban (BAY u3405), inhibits prostaglandin D2-induced eosinophil migration in vitro. J Pharmacol Exp Ther. 2003;305:347–352. 19. Terada N, Yamakoshi T, Hasegawa M, et al. Effect of a thromboxane A2 receptor antagonist, ramatroban (BAY u3405), on inflammatory cells, chemical mediators and non-specific nasal hyperreactivity after allergen challenge in patients with perennial allergic rhinitis. Allergol Int. 1998; 47:59 – 67. 20. Terada N, Yamakoshi T, Hasegawa M, et al. The effect of ramatroban (BAY u 3405), a thromboxane A2 receptor antagonist, on nasal cavity
volume and minimum cross-sectional area and nasal mucosal hemodynamics after nasal mucosal allergen challenge in patients with perennial allergic rhinitis. Acta Otolaryngol Suppl. 1998;537:32–37. 21. Johnston SL, Smith S, Harrison J, Ritter W, Howarth PH. The effect of BAY u 3405, a thromboxane receptor antagonist, on prostaglandin D2-induced nasal blockage. J Allergy Clin Immunol. 1993;91:903–909. 22. Shirasaki H, Kikuchi M, Seki N, Kanaizumi E, Watanabe K, Himi T. Expression and localization of the thromboxane A2 receptor in human nasal mucosa. Prostaglandins Leukot Essent Fatty Acids. 2007;76: 315–320. 23. Uller L, Mathiesen JM Alenmyr L, et al. Antagonism of the prostaglandin D2 receptor CRTH2 attenuates asthma pathology in mouse eosinophilic airway inflammation. Respir Res. 2007;8:16.
Requests for reprints should be addressed to: Hideaki Shirasaki, MD, PhD Department of Otolaryngology Sapporo Medical University S-1 W-16, Chuo-ku Sapporo, 060-8543, Japan E-mail: [email protected]
Answers to CME examination—Annals of Allergy, Asthma & Immunology, February 2009 Ramanuja S and Kelkar P: Habit cough. Ann Allergy Asthma Immunol. 2009; 102:91–95. 1. b 2. e 3. c 4. b 5. e
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Background: Prostaglandin D2 (PGD2) has been thought to be a potent mediator involved in allergic rhinitis because PGD2 has been recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation and because PGD2 receptor antagonists relieved nasal allergic symptoms in an animal model of allergic rhinitis. The inflammatory effects of PGD2 are exerted through high-affinity interactions with 2 G protein– coupled receptors: D-prostanoid receptor 1 and chemoattractanthomologous receptor expressed on TH2 cells (CRTH2). CRTH2 may mediate the recruitment of leukocytes during a nasal allergic response. Objective: To evaluate the number of CRTH2-expressing cells in allergic and nonallergic human nasal mucosa by means of immunohistochemical analysis. Methods: Human turbinates were obtained after turbinectomy from 14 patients with nasal obstruction refractory to medical therapy. To identify cells expressing the CRTH2 protein, double immunostaining was performed using anti–CRTH2 antibody and monoclonal anti–leukocyte antibodies. Results: The immunohistochemical study revealed that anti–CRTH2 antibody labeled eosinophils, macrophages, mast cells, T lymphocytes, epithelial cells, and submucosal glands in the nasal mucosa. CRTH2 expressions of these leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa. Conclusion: These results suggest that CRTH2 may play an important role in the recruitment of leukocytes into allergic nasal mucosa. Ann Allergy Asthma Immunol. 2009;102:110–115.
INTRODUCTION The allergic response is a complex process involving the interaction of many mediators. Prostaglandin D2 (PGD2) is a potent inflammatory mediator, and its actions are mediated via specific cell surface receptors coupled to G proteins. PGD2 binds mainly 2 G protein– coupled receptors to exert its bioactivity: D-prostanoid receptor 1 (DP1) and chemoattractant-homologous receptor expressed on TH2 cells (CRTH2 [also known as DP2]).1 Activation of DP1 leads to a G␣smediated increase in intracellular cyclic adenosine monophosphate and agonist-induced calcium flux.2 Bronchodilation and vasodilation occur as a consequence of DP1 activation.3,4 In contrast, CRTH2 couples to a G␣i-type G protein, leading to the inhibition of cyclic adenosine monophosphate, and it increases intracellular calcium levels in various types of cells.5 PGD2 was recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation.6 In addition, it has been shown that CRTH2 antagonist attenuated an antigen-induced increase in nasal airway resistance and local eosinophil infiltration in sensitized guinea pigs.7 These studies suggest that CRTH2 may play an important role in the
Affiliations: Department of Otolaryngology, Sapporo Medical University, School of Medicine, Sapporo, Japan. Disclosures: Authors have nothing to disclose. Received for publication July 7, 2008; Received in revised form October 20, 2008; Accepted for publication November 7, 2008.
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pathogenesis of allergic rhinitis. In a previous study8 using rat anti– human CRTH2 antibody, BM16 demonstrated CRTH2 expression on eosinophils and T lymphocytes in nasal polyps. However, there has been no report of CRTH2 expression in human inferior turbinates. In the present study, immunohistochemical analysis of CRTH2 was performed to confirm the expression and distribution of CRTH2 using a commercial anti– human CRTH2 antibody in human allergic and nonallergic inferior turbinates. MATERIALS AND METHODS Tissue Preparation Human inferior turbinates were obtained after turbinectomy from 14 patients with nasal obstruction refractory to medical therapy. Informed consent was obtained from all the patients, and this study was approved by the ethics committee of Sapporo Medical University. All the patients were nonsmokers, and 7 had perennial allergy against mites as defined by questionnaire and CAP testing (Pharmacia, Uppsala, Sweden). Seven patients with negative CAP test results for the major inhaled allergens, including mites, birch, grass, mugwort, and Japanese cedar, formed the group with nonallergic rhinitis. All medications, including antibiotics, were prohibited for at least 3 weeks before the study. The demographic and clinical characteristics of the patients are summarized in Table 1. The nasal mucosal specimens were dissected from the cartilage and were fixed in 10% formalin for immunohistochemical analysis.
ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY
Table 1. Demographic Characteristics of Allergic and Nonallergic Patients Characteristics
Allergic rhinitis (n ⴝ 7)
Nonallergic rhinitis (n ⴝ 7)
Sex, M/F, No. Age, median (range), y Specific IgE to house dust mite (Dermatophagoides pteronyssinus), median (range), kU/L Total IgE, median (range), kU/L Blood eosinophils, median (range), cells/L Current nasal symptoms, No. of patients Nasal obstruction Sneezing Rhinorrhea
3/4 31 (19–58) 2.7 (1.0–13)
4/3 39 (21–55) ⬍0.35
210 (10–452) 370 (70–690)
110 (10–190) 115 (45–240)
7 4 3
7 0 2
Immunohistochemical Analysis Antibodies. For immunohistochemical analysis of CRTH2 receptor, rabbit anti– human CRTH2 receptor polyclonal antibody against a peptide corresponding to the N-terminal domain of human CRTH2 receptor (Cayman Chemical, Ann Arbor, Michigan) was used at 1:100 dilutions. To identify subsets of cells expressing CRTH2, the following monoclonal antibodies were used: anti– eosinophil cationic protein (EG2 clone) (Pharmacia) for eosinophils, anti-CD68 (KP-1 clone) (Dako, Glostrup, Denmark) for macrophages, anti– mast cell tryptase (AA1 clone) (Dako) for mast cells, and anti–CD3 antibody (F7.2.38 clone) (Dako) for T cells. Immunohistochemical analysis. Deparaffinized sections were initially incubated with 3% hydrogen peroxide in methanol for 10 minutes to quench endogenous peroxidase activity. After microwave treatment (10 minutes at 500 W in citrate buffer), the sections were incubated in blocking solution (10% normal goat serum in phosphate-buffered saline [PBS]) for 30 minutes before incubation in primary antibody. Then, the sections were incubated with anti-CRTH2 polyclonal antibody overnight at 4°C, washed, and incubated for 30 minutes with EnVision⫹, Peroxidase (Dako). A further washing in PBS was followed by developing in diaminobenzidine (DAB; Dako) as a chromogen for signal visualization. The slides were counterstained with Mayer hematoxylin and coverslipped using mounting medium. To identify subsets of cells expressing CRTH2, some sections were stained by means of the immunofluorescence technique. For double staining, deparaffinized sections were incubated overnight at 4°C with a combination of rabbit polyclonal anti– human CRTH2 antibody and 1 mouse monoclonal anti– human leukocyte phenotypical marker antibody after microwave treatment (10 minutes at 500 W in citrate buffer). Sections were washed in PBS and were incubated for 30 minutes with Alexa Fluor 594 –labeled goat anti–mouse IgG (diluted 1:50) (Molecular Probes, Eugene, Oregon) and Alexa Fluor 488 –labeled goat anti–rabbit IgG (diluted 1:50) (Molecular Probes). Sections were mounted using Vectashield mounting medium with 4⬘,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc, Burlingame, California) and were examined using a microscope (BX51) and a charge-coupled device camera (DP70) (Olympus Optical Co, Tokyo, Japan). All the images
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were processed using image analysis software (DP Controller and DP Manager; Olympus Optical Co). Using this method, CRTH2-expressing cells were green, cellular phenotypical markers were stained red, and the combined signal was visualized as yellow. To confirm the specificity of immunohistochemical analysis, a commercial CRTH2/DP2 receptor blocking peptide (Cayman Chemical) was used to block protein-antibody complex formation during immunohistochemical analysis according to the manufacturer’s instructions. Negative controls were obtained by replacing primary antibodies with mouse IgG1 and rabbit immunoglobulin fraction (Dako). Quantitation For double immunofluorescence staining, the slides were counted using a microscope equipped with an eyepiece graticule. A total of 6 fields (1 mm2 each) from each slide were counted by placing the upper edge of the grid at the epithelium. The number of CRTH2-positive cells in each phenotype in allergic and nonallergic nasal mucosa was evaluated by 2 masked investigators (M.K. and E.K.). Results are expressed as the number of CRTH2-positive cells per square millimeter and the percentage of CRTH2-positive cells in each phenotype. Statistical Analysis Values are expressed as mean (SD). Differences between groups are compared using the Mann-Whitney test. P ⬍ .05 was considered statistically significant. RESULTS The CRTH2 distribution in nasal mucosa was examined using a polyclonal antibody against a peptide corresponding to N-terminal amino acids 2 to 21 of human CRTH2. Immunoreactivity for CRTH2 was significantly detected in interstitial cells, submucosal glands, and epithelial cells (Fig 1A). The intensity of the CRTH2 receptor staining was decreased by co-incubation with a specific commercial blocking peptide (Fig 1B). Specificity of the staining was also confirmed by the absence of labeling with normal rabbit immunoglobulin (Fig 1C). To clarify the cell types of the interstitial cells expressing the CRTH2 protein, we performed double immunofluores-
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cence staining for CRTH2 and leukocyte phenotype marker. Fig 2 demonstrates examples of CRTH2-positive T lymphocytes (Fig 2B), eosinophils (Fig 2D), macrophages (Fig 2F), and mast cells (Fig 2H). As shown in Figure 3, we observed many CRTH2-expressing eosinophils (43.7 [14.6] cells/mm2) and T cells (42.0 [15.2] cells/mm2). The difference between the number of CRTH2-expressing eosinophils, macrophages, T cells, and mast cells was significantly higher in patients with allergy vs nonallergy (Fig 3). As shown in Figure 4, the ratio of CRTH2-positive cells in allergic nasal mucosa was highest in eosinophils (84.7% [10.9%]), and moderate
Figure 1. Immunohistochemical staining for chemoattractant-homologous receptor expressed on TH2 cells (CRTH2) receptor in human allergic nasal mucosa. Inferior turbinates were stained with anti– human CRTH2 receptor antibody (A and B) or normal rabbit immunoglobulin (C). Staining was observed mainly on airway epithelium (ep), submucosal glands (g), and interstitial cells. The intensity of the staining was decreased by co-incubation with a specific blocking peptide (B). v indicates blood vessels. Scale bar ⫽ 200 m.
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Figure 2. Identification of subsets of cells expressing the chemoattractanthomologous receptor expressed on TH2 cells (CRTH2) receptor among interstitial cells in human nasal mucosa using nuclear stain with 4⬘,6diamidino-2-phenylindole (blue). The CRTH2/DP2 receptor protein (green) shows co-localization with anti–leukocyte antibody (red), and the combined signal is visualized as yellow (B, D, F, H: overlay images). Identification markers are shown for CD3 (pan–T cells) (A and B), eosinophil cationic protein (eosinophils) (C and D), CD68 (macrophages) (E and F), and mast cell tryptase (mast cells) (G and H). Arrows indicate cells expressing CRTH2 and each phenotypical marker. Scale bar ⫽ 50 m.
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Figure 3. Mean (SD) number of chemoattractant-homologous receptor expressed on TH2 cells (CRTH2)–positive cells in each phenotype in allergic and nonallergic nasal mucosa. Sections were stained by means of double immunofluorescence for CRTH2 and eosinophils, macrophages, mast cells, and T cells. Error bars represent SD.
Figure 4. Mean (SD) percentage of chemoattractant-homologous receptor expressed on TH2 cells (CRTH2)–positive cells in each phenotype in allergic and nonallergic nasal mucosa. Sections were stained by means of double immunofluorescence for CRTH2 and eosinophils, macrophages, mast cells, and T cells. Error bars represent SD.
CRTH2 expression was associated with macrophages (45.4% [13.8%]), mast cells (43.3% [12.4%]), and T cells (33.4% [11.3%]). CRTH2 expressions of these leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa (Fig 4). DISCUSSION Although the CRTH2/TXA2 prostanoid (TP) receptor antagonist ramatroban has been in clinical use, there have
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been only a few studies indicating the expression of CRTH2 in nasal mucosa. Nantel et al8 reported immunohistochemical localization of CRTH2 using rat anti– human CRTH2 antibody BM16. They detected CRTH2-positive eosinophils and T cells in nasal polyps but not in tissues obtained from healthy individuals.8 Okano et al9 reported that the amount of CRTH2 messenger RNA positively and significantly correlated with the number of infiltrating eosinophils in human allergic inferior turbi-
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nates, suggesting involvement in eosinophil recruitment at inflammatory sites in vivo. The present immunohistochemical analysis showed that CRTH2-expressing cells were eosinophils, mast cells, macrophages, and T lymphocytes in human inferior turbinates. The CRTH2-dependent cell migration is not restricted to TH2 cells, eosinophils, and basophils, as initially reported,10 –12 but also to monocytes.13 These studies suggest that the local production of PGD2 may contribute to migration and activation of these inflammatory leukocytes during a local allergic response. In the present study, approximately 40% of mast cells showed significant immunoreactivity to CRTH2, suggesting the possibility of functional CRTH2 receptor on mast cells. However, there has been no report concerning the existence of CRTH2 on mast cells. Expression of CRTH2 was found not only on infiltrated leukocytes but also on vascular endothelial cells, epithelial cells, and submucosal glands in the nasal mucosa. It has been reported that normal human bronchial epithelial cells and the epithelial cell line NCI-H292 express CRTH2 messenger RNA and protein with an inner cell but without cell surface expression.14 This finding leads us to speculate that intracellular expression of CRTH2 on the epithelial cell has some functional potential and indicates that further research is needed. On the other hand, there seem to be no articles describing the existence of CRTH2 on submucosal glands or the effect of CRTH2 as the mucous secretagogue of airways. In the present study, we found that the percentages of CD3⫹ lymphocytes, macrophages, or mast cells expressing CRTH2 were higher in individuals with perennial allergic rhinitis. As yet, there seem to be no other studies describing up-regulation of CRTH2 expression in allergic airways. Electromobility shift assay demonstrated GATA-3 binding to CRTH2 promoter lesions, suggesting up-regulation of CRTH2 transcription by TH2 cytokines such as interleukin 4 (IL-4) and IL-13.15 Previous studies have demonstrated increased local levels of IL-4 and IL-13 after allergen provocation in allergic rhinitis16 and atopic asthma.17 These observations suggest that CRTH2-expressing cells in allergic airways might be up-regulated by locally produced IL-4/IL13. The clinical information on CRTH2 antagonists is limited to data on ramatroban (BAY u3405; Cayman Chemical), marketed in Japan for allergic rhinitis. Ramatroban has been characterized as a dual antagonist against thromboxane prostanoid receptor and CRTH2.18 Ramatroban significantly blocked allergen-induced local eosinophilia19 and nasal mucosal swelling20 in patients with allergic rhinitis. PGD2 is thought to induce nasal blockage by producing volume changes in the nasal venous sinusoids as a result of vasodilation. The PGD2-induced nasal blockage may be mediated by DP1 receptor rather than by CRTH2 receptor because ramatroban afforded no protection against PGD2-induced nasal blockage in humans.21 A previous immunohistochemical study22 showed TP expressed on blood vessels, not on inflammatory leukocytes, in nasal allergic mucosa. In addition,
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the selective CRTH2 antagonist 30089 equals the dual CRTH2/TP antagonist ramatroban regarding inhibition of eosinophil recruitment in a mouse asthma model.23 These observations suggest that the clinical effects of ramatroban on allergic rhinitis may be the improvement of nasal blockage by inhibition of TP receptor–mediated vascular reaction and that ramatroban may inhibit the migration of CRTH2-expressing leukocytes, such as eosinophils, TH2 lymphocytes, mast cells, and macrophages. In conclusion, using an immunohistochemical technique, we evaluated the number of CRTH2-expressing cells in allergic and nonallergic human nasal mucosa. CRTH2 expressions of the leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa. Precise knowledge of the identity and distribution of CRTH2 should be of considerable interest for understanding the role of CRTH2 in allergic rhinitis. ACKNOWLEDGMENTS We thank Tsuyako Watanabe for her expert technical assistance. REFERENCES 1. Sawyer N, Cauchon E, Chateauneuf A, et al. Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2. Br J Pharmacol. 2002; 137:1163–1172. 2. Boie Y, Sawyer N, Slipetz DM, Metters KM, Abramovitz M. Molecular cloning and characterization of the human prostanoid DP receptor. J Biol Chem. 1995;270:18910 –18916. 3. Norel X, Walch L, Labat C, Gascard JP, Dulmet E, Brink C. Prostanoid receptors involved in the relaxation of human bronchial preparations. Br J Pharmacol. 1999;126:867– 872. 4. Walch L, Labat C, Gascard JP, de Montpreville V, Brink C, Norel X. Prostanoid receptors involved in the relaxation of human pulmonary vessels. Br J Pharmacol. 1999;126:859 – 866. 5. Hirai H, Tanaka K, Yoshie O, et al. Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J Exp Med. 2001;193:255–261. 6. Naclerio RM, Meier HL, Kagey-Sobotka A, et al. Mediator release after nasal airway challenge with allergen. Am Rev Respir Dis. 1983;128: 597– 602. 7. Narita S, Asakura K, Kataura A. Effect of thromboxane A2 receptor antagonist (Bay u 3405) on nasal symptoms after antigen challenge in sensitized guinea pigs. Int Arch Allergy Immunol. 1996;109:161–166. 8. Nantel F, Fong C, Lamontagne S, et al. Expression of prostaglandin D synthase and the prostaglandin D2 receptors DP and CRTH2 in human nasal mucosa. Prostaglandins Other Lipid Mediat. 2004;73:87–101. 9. Okano M, Fujiwara T, Sugata Y, et al. Presence and characterization of prostaglandin D2-related molecules in nasal mucosa of patients with allergic rhinitis. Am J Rhinol. 2006;20:342–348. 10. Nagata K, Hirai H, Tanaka K, et al. CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cell-derived factor(s). FEBS Lett. 1999;459:195–199. 11. Monneret G, Gravel S, Diamond M, Rokach J, Powell WS. Prostaglandin D2 is a potent chemoattractant for human eosinophils that acts via a novel DP receptor. Blood. 2001;98:1942–1948. 12. Gervais FG, Cruz RP, Chateauneuf A, et al. Selective modulation of chemokinesis, degranulation and apoptosis in eosinophils through the PGD2 receptors CRTH2 and DP. J Allergy Clin Immunol. 2001;108: 982–988. 13. Gosset P, Bureau F, Angeli V, et al. Prostaglandin D2 affects the maturation of human monocyte-derived dendritic cells: consequence on the polarization of naïve Th cells. J Immunol. 2003;170:4943– 4952.
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14. Chiba T, Kanda A, Ueki S, et al. Prostagalndin D2 induced IL-8 and GM-CSF by bronchial epithelial cells in a CRTH2-independent pathway. Int Arch Allergy Immunol. 2006;141:300 –307. 15. Quapp R, Madsen N, Cameron L. Characterization of the promoter of human CRTH2, a prostaglandin D2 receptor. Biochem Biophys Res Commun. 2007;363:948 –953. 16. Erin EM, Zacharasiewicz AS, Nicholson GC, et al. Topical corticosteroid inhibits interleukin-4, -5 and -13 in nasal secretions following allergen challenge. Clin Exp Allergy. 2005;35:1608 –1614. 17. Kroegel C, Julius P, Matthys H, Virchow JC, Luttmann W. Endobronchial secretion of interleukin-13 following local allergen challenge in atopic asthma: relationship to interleukin-4 and eosinophil counts. Eur Respir J. 1996;9:899 –904. 18. Sugimoto H, Shichijo M, Iino T, et al. An orally bioavailable small molecule antagonist of CRTH2, ramatroban (BAY u3405), inhibits prostaglandin D2-induced eosinophil migration in vitro. J Pharmacol Exp Ther. 2003;305:347–352. 19. Terada N, Yamakoshi T, Hasegawa M, et al. Effect of a thromboxane A2 receptor antagonist, ramatroban (BAY u3405), on inflammatory cells, chemical mediators and non-specific nasal hyperreactivity after allergen challenge in patients with perennial allergic rhinitis. Allergol Int. 1998; 47:59 – 67. 20. Terada N, Yamakoshi T, Hasegawa M, et al. The effect of ramatroban (BAY u 3405), a thromboxane A2 receptor antagonist, on nasal cavity
volume and minimum cross-sectional area and nasal mucosal hemodynamics after nasal mucosal allergen challenge in patients with perennial allergic rhinitis. Acta Otolaryngol Suppl. 1998;537:32–37. 21. Johnston SL, Smith S, Harrison J, Ritter W, Howarth PH. The effect of BAY u 3405, a thromboxane receptor antagonist, on prostaglandin D2-induced nasal blockage. J Allergy Clin Immunol. 1993;91:903–909. 22. Shirasaki H, Kikuchi M, Seki N, Kanaizumi E, Watanabe K, Himi T. Expression and localization of the thromboxane A2 receptor in human nasal mucosa. Prostaglandins Leukot Essent Fatty Acids. 2007;76: 315–320. 23. Uller L, Mathiesen JM Alenmyr L, et al. Antagonism of the prostaglandin D2 receptor CRTH2 attenuates asthma pathology in mouse eosinophilic airway inflammation. Respir Res. 2007;8:16.
Requests for reprints should be addressed to: Hideaki Shirasaki, MD, PhD Department of Otolaryngology Sapporo Medical University S-1 W-16, Chuo-ku Sapporo, 060-8543, Japan E-mail: [email protected]
Answers to CME examination—Annals of Allergy, Asthma & Immunology, February 2009 Ramanuja S and Kelkar P: Habit cough. Ann Allergy Asthma Immunol. 2009; 102:91–95. 1. b 2. e 3. c 4. b 5. e
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