Histamine H2 receptor stimulation upregulates T H 2 chemokine CCL17 production in human M2a macrophages
2017; Elsevier BV; Volume: 141; Issue: 2 Linguagem: Inglês
10.1016/j.jaci.2017.06.023
ISSN1097-6825
AutoresSusanne Mommert, Karl Gregor, Kristine Roßbach, Katrin Schaper, Torsten Witte, Ralf Gutzmer, Thomas Werfel,
Tópico(s)Asthma and respiratory diseases
ResumoThe pathogenesis of atopic dermatitis (AD) in acute and chronic inflammatory situations is characterized by a marked infiltration of myeloid-derived dendritic cells, T cells, and eosinophils in the epidermal and dermal compartments as well as by increased amounts of cytokines or chemokines, which are crucial for the cellular crosstalk. In the onset of acute AD, an increased production of the TH2 cytokines IL-4 and IL-13 by early skin-infiltrating T cells, mast cells, or basophils takes place.1Werfel T. Allam J.P. Biedermann T. Eyerich K. Gilles S. Guttman-Yassky E. et al.Cellular and molecular immunologic mechanisms in patients with atopic dermatitis.J Allergy Clin Immunol. 2016; 138: 336-349Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar In consequence to the pathogenic and tissue-derived changes, monocytes differentiate into macrophages and respond to environmental signals with polarization into distinct functional phenotypes. The M2a macrophage phenotype is induced by a TH2-biased immune response in the presence of IL-4 or IL-13 and characterized by secreting a specific panel of chemokines,E1Mantovani A. Sica A. Sozzani S. Allavena P. Vecchi A. Locati M. The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol. 2004; 25: 677-686Abstract Full Text Full Text PDF PubMed Scopus (4490) Google Scholar, E2Kasraie S. Werfel T. Role of macrophages in the pathogenesis of atopic dermatitis.Mediators Inflamm. 2013; 2013: 942375Crossref PubMed Scopus (78) Google Scholar among them CCL17 (formerly known as thymus- and activation-regulated chemokine) and CCL22 (formerly known as macrophage-derived chemokine), which are highly expressed in dermal dendritic cells (DCs) in AD skin.2Gros E. Bussmann C. Bieber T. Forster I. Novak N. Expression of chemokines and chemokine receptors in lesional and nonlesional upper skin of patients with atopic dermatitis.J Allergy Clin Immunol. 2009; 124: 753-760Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar CCL17 was identified as one of the most sensitive biomarkers for AD because its expression in the epidermis as well as CCL17 serum levels of patients with AD correlate with disease severity.E3Vestergaard C. Bang K. Gesser B. Yoneyama H. Matsushima K. Larsen C.G. A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin.J Invest Dermatol. 2000; 115: 640-646Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, E4Morita E. Takahashi H. Niihara H. Dekio I. Sumikawa Y. Murakami Y. et al.Stratum corneum TARC level is a new indicator of lesional skin inflammation in atopic dermatitis.Allergy. 2010; 65: 1166-1172PubMed Google Scholar, E5Jahnz-Rozyk K. Targowski T. Paluchowska E. Owczarek W. Kucharczyk A. Serum thymus and activation-regulated chemokine, macrophage-derived chemokine and eotaxin as markers of severity of atopic dermatitis.Allergy. 2005; 60: 685-688Crossref PubMed Scopus (134) Google Scholar, E6Mansouri Y. Guttman-Yassky E. Immune pathways in atopic dermatitis, and definition of biomarkers through broad and targeted therapeutics.J Clin Med. 2015; 4: 858-873Crossref PubMed Scopus (96) Google Scholar Histamine is found in lesional skin of AD3Stander S. Steinhoff M. Pathophysiology of pruritus in atopic dermatitis: an overview.Exp Dermatol. 2002; 11: 12-24Crossref PubMed Scopus (208) Google Scholar and has both proinflammatory and anti-inflammatory effects, depending on the expression profile of the 4 histamine receptors (HRs) on the cells taking part in the immunologic responses. Targeting the H1R induces various proinflammatory and cell-activation responses that might foster allergic diseases.E7Thurmond R.L. Gelfand E.W. Dunford P.J. The role of histamine H1 and H4 receptors in allergic inflammation: the search for new antihistamines.Nat Rev Drug Discov. 2008; 7: 41-53Crossref PubMed Scopus (473) Google Scholar, E8Jutel M. Akdis M. Akdis C.A. Histamine, histamine receptors and their role in immune pathology.Clin Exp Allergy. 2009; 39: 1786-1800Crossref PubMed Scopus (228) Google Scholar The H2R has a similar expression pattern on immune cells compared with the H1R but opposed to the H1R, stimulation of the H2R attenuates various functions on allergen-specific T cells,4Jutel M. Watanabe T. Klunker S. Akdis M. Thomet O.A. Malolepszy J. et al.Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors.Nature. 2001; 413: 420-425Crossref PubMed Scopus (506) Google Scholar DCs,5Gutzmer R. Diestel C. Mommert S. Kother B. Stark H. Wittmann M. et al.Histamine H4 receptor stimulation suppresses IL-12p70 production and mediates chemotaxis in human monocyte-derived dendritic cells.J Immunol. 2005; 174: 5224-5232Crossref PubMed Scopus (211) Google Scholar and basophils.6Novak N. Mete N. Bussmann C. Maintz L. Bieber T. Akdis M. et al.Early suppression of basophil activation during allergen-specific immunotherapy by histamine receptor 2.J Allergy Clin Immunol. 2012; 130: 1153-1158Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Regarding the H4R, various immunomodulatory effects mediated on DCs,7Gschwandtner M. Mommert S. Kother B. Werfel T. Gutzmer R. The histamine H4 receptor is highly expressed on plasmacytoid dendritic cells in psoriasis and histamine regulates their cytokine production and migration.J Invest Dermatol. 2011; 131: 1668-1676Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar T cells,8Mommert S. Gschwandtner M. Koether B. Gutzmer R. Werfel T. Human memory Th17 cells express a functional histamine H4 receptor.Am J Pathol. 2012; 180: 177-185Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar natural killer cells,E10Ehling S. Rossbach K. Dunston S.M. Stark H. Baumer W. Allergic inflammation is augmented via histamine H4 receptor activation: the role of natural killer cells in vitro and in vivo.J Dermatol Sci. 2016; 83: 106-115Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, E9Mommert S. Dittrich-Breiholz O. Stark H. Gutzmer R. Werfel T. The histamine H4 receptor regulates chemokine production in human natural killer cells.Int Arch Allergy Immunol. 2015; 166: 225-230Crossref PubMed Scopus (12) Google Scholar and keratinocytesE11Glatzer F. Gschwandtner M. Ehling S. Rossbach K. Janik K. Klos A. et al.Histamine induces proliferation in keratinocytes from patients with atopic dermatitis through the histamine 4 receptor.J Allergy Clin Immunol. 2013; 132: 1358-1367Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar have been described. The aim of this study was to evaluate the role of histamine in chemokine production, in particular of CCL17 and CCL22, in human macrophages and in monocytes. The investigation of the role of the HRs in inflammatory diseases was approved by the local ethics committee of the Hannover Medical School and was conducted according to the Declaration of Helsinki principles. Fully differentiated human M2 macrophages were stimulated with IL-4 or IL-13 for different time periods (see additional information in the Methods section in this article's Online Repository at www.jacionline.org). The upregulation of CCL17 mRNA expression and protein production by IL-4 or IL-13 was time-dependent and showed the highest expression levels after 48 hours (data not shown). We assessed effects of histamine and agonists specific for H1R, H2R, and H4R on IL-4– or IL-13–activated M2a macrophages. Interestingly, we observed a significant upregulation of the IL-4– or IL-13–induced expression of CCL17 at mRNA- and protein level by histamine, by amthamine (specific H2R agonist), or by 4-methylhistamine (4-MH) (H2R and H4R agonist), indicating a critical role of the H2R for this effect. In the presence of the selective H2R antagonist ranitidine, the effects of histamine, of amthamine, and of 4-MH on CCL17 expression were reversed, confirming a functional role of the H2R in this reaction (Fig 1, A, B, C, E and F, and Fig 2, A-F). In contrast, preincubation with the selective H4R antagonist JNJ7777120 did not reverse the histamine or the 4-MH–induced upregulation of CCL17 (Fig 1, D and G). The H2R-mediated upregulation of CCL17 was observed to a lesser extent in IL-4– or IL-13–activated human monocytes (see Fig E4, A and B, in this article's Online Repository at www.jacionline.org) but not in (1) resting M2 macrophages, (2) in human IL-10–activated M2c, or (3) in classically activated M1 macrophages (data not shown). H1R or H4R agonists did not influence the CCL17 expression (Fig 1, A and B, and Fig 2, G and H). The upregulation of CCL17 production via H2R in IL-4–activated human M2a macrophages occurred in a time- and dose-dependent manner (see Fig E3, A-C, in this article's Online Repository at www.jacionline.org).Fig 2Stimulation via H2R significantly upregulates CCL17 mRNA expression and protein production in IL-13–activated human M2a macrophages. IL-13 (15 ng/mL)-activated M2a macrophages were stimulated with histamine, 2-pyridylethylamine (H1R agonist), amthamine (H2R agonist), 4-MH (H2R/H4R agonist), and ST-1006 (H4R agonist). The upregulation of CCL17 mRNA expression and protein production in response to histamine, amthamine, and 4-MH was significantly blocked by preincubation with the selective H2R antagonist ranitidine. The concentration of 10 μM was used for all ligands. A-C, CCL17 mRNA expression was detected by quantitative PCR. D-F, CCL17 protein concentrations were analyzed by ELISA. The mRNA- and protein expression were normalized to the IL-13–stimulated samples (IL-13–stimulated M2a macrophages produced in median 216 pg/mL CCL17, ranging from 5.5 pg/mL to 549 pg/mL). G and H, Stimulation of the H1R or H4R shows no effect. 2-Pyrid, 2-pyridylethylamine; Amth, amthamine; Hist, histamine; JNJ, JNJ7777120; NS, nonstimulated; Ran, ranitidine. Significant differences, as determined by the Wilcoxon signed rank test, are indicated as follows: *P < .05; **P < .01; medians are shown in the graphs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) On comparing basal and IL-4– or IL-13–induced production of CCL17 in M2 macrophages obtained from healthy donors versus cells obtained from patients with AD, we observed almost similar CCL17 expression levels in both groups induced by IL-4 but reduced expression of CCL17 induced by IL-13 in patients with AD. The H2R-mediated upregulation of CCL17 was observable in cells from patients with AD to a lesser extent (see Fig E1, A-D, in this article's Online Repository at www.jacionline.org). Patients with AD have a background of a general systemic TH2 polarization. Therefore, we speculate that cells from patients with AD are adapted to an inflammatory microenvironment, leading to unresponsiveness to further stimulation. Analyzing the characteristics of the different HRs, H2R is usually considered rather as a receptor mediating immune-suppressive and protolerogenic functions. H2R was shown to reduce TLR-induced expression of cytokines/chemokines in monocytes and DCs5Gutzmer R. Diestel C. Mommert S. Kother B. Stark H. Wittmann M. et al.Histamine H4 receptor stimulation suppresses IL-12p70 production and mediates chemotaxis in human monocyte-derived dendritic cells.J Immunol. 2005; 174: 5224-5232Crossref PubMed Scopus (211) Google Scholar, E12Gschwandtner M. Bunk H. Kother B. Thurmond R.L. Kietzmann M. Werfel T. et al.Histamine down-regulates IL-27 production in antigen-presenting cells.J Leukoc Biol. 2012; 92: 21-29Crossref PubMed Scopus (29) Google Scholar, E13Glatzer F. Mommert S. Kother B. Gschwandtner M. Stark H. Werfel T. et al.Histamine downregulates the Th1-associated chemokine IP-10 in monocytes and myeloid dendritic cells.Int Arch Allergy Immunol. 2014; 163: 11-19Crossref PubMed Scopus (18) Google Scholar as well as to induce tolerance by suppressing proliferation of allergen-stimulated T cells and increase of IL-10 productionE14Meiler F. Zumkehr J. Klunker S. Ruckert B. Akdis C.A. Akdis M. In vivo switch to IL-10-secreting T regulatory cells in high dose allergen exposure.J Exp Med. 2008; 205: 2887-2898Crossref PubMed Scopus (387) Google Scholar or by suppressing basophil activation and mediator release.6Novak N. Mete N. Bussmann C. Maintz L. Bieber T. Akdis M. et al.Early suppression of basophil activation during allergen-specific immunotherapy by histamine receptor 2.J Allergy Clin Immunol. 2012; 130: 1153-1158Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Our data challenge this role of H2R, because the proinflammatory TH2-related chemokine CCL17 was upregulated via H2R in M2a macrophages and in monocytes, pointing toward a dual role of this receptor. In contrast to the studies of McIlroy et al9McIlroy A. Caron G. Blanchard S. Fremaux I. Duluc D. Delneste Y. et al.Histamine and prostaglandin E up-regulate the production of Th2-attracting chemokines (CCL17 and CCL22) and down-regulate IFN-gamma-induced CXCL10 production by immature human dendritic cells.Immunology. 2006; 117: 507-516Crossref PubMed Scopus (80) Google Scholar in immature monocyte-derived DCs, we did not observe that the elevated expression of CCL17 in response to histamine is accompanied by an upregulation of CCL22 in M2a macrophages (see Fig E2, A and B, in this article's Online Repository at www.jacionline.org) or monocytes (Fig E4, C and D). This supports the hypothesis that the physiology between CCL17 and CCL22 is at least partly nonredundant.E15Hashimoto S. Nakamura K. Oyama N. Kaneko F. Tsunemi Y. Saeki H. et al.Macrophage-derived chemokine (MDC)/CCL22 produced by monocyte derived dendritic cells reflects the disease activity in patients with atopic dermatitis.J Dermatol Sci. 2006; 44: 93-99Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar In accordance with the observation in human M2a macrophages, we also detected increased expression of CCL17 upon stimulation with IL-4 in bone marrow–derived macrophages obtained from BALB/c mice. H2R stimulation also increased the IL-4–induced CCL17 production in these murine cells (data not shown). In conclusion, we describe a novel function for H2R in M2a macrophages and in monocytes, promoting a TH2-polarized response by upregulating the production of CCL17 as key chemokine in AD that may lead to enhanced recruitment of CCR4-expressing TH2 cells, regulatory T cells, and CLA+ T cells to the site of inflammation. These results may be helpful to identify novel therapeutic target structures in allergic and inflammatory diseases driven at least in part by histamine. We thank Brigitta Koether, Kira Herwig, and Judith Schaper for excellent technical assistance and Holger Stark for providing the H4R agonist ST-1006. Residual blood samples from platelet apheresis disposables used for routine platelet collection and of regular anonymus healthy donors served as source material for the isolation of human PBMCs. Peripheral blood samples were taken from patients with extrinsic AD. PBMCs were separated by density gradient centrifugation on lymphoprep (Fresenius Kabi Norge AS, Oslo, Norway). With a seeding density of 1 × 106 cells per well, PBMCs were plated in a 24-well plate in Iscoves Medium supplemented with AB serum (2.5% v/v). To attach the monocytes, cells were incubated for 1.5 hours at 5% CO2 and 37°C. Nonadherent cells were removed by vigorously washing the adherent cells 3 times with PBS. An appropriate amount of RPMI 1640, supplemented with 2 mM l-glutamine, 100 mg/mL penicillin/streptomycin, 12 mM HEPES, and 5% v/v FCS (PAN-Biotech, Aidenbach, Germany; all other media components from Biochrom, Berlin, Germany), and 10 ng/mL macrophage colony-stimulating factor (R&D, San Diego, Calif) was added. Cells were incubated for 5 days at 37°C and 5% CO2 without medium change. On day 5, another 50% by volume of fresh medium was added. On day 8, the medium was completely changed and on day 10 the fully differentiated M2 macrophages were activated with IL-4 (20 ng/mL) (R&D Systems) or IL-13 (15 ng/mL) (R&D Systems). Monocytes were generated from PBMCs obtained from the same source material used for isolation and differentiation of macrophages. The cells were allowed to attach at the culture dishes. After 2 hours, nonadherent cells were removed and the adherent monocytes were cultured overnight without GM-CSF or M-CSF. Then, the cells were stimulated comparable to the M2 macrophages. The following histamine receptor ligands were used in this study: Histamine (Alk-Scherax, Wedel, Germany) as agonist for all HRs; 2-pyridylethylamine (Tocris Bioscience, Bristol, UK) as selective H1R agonist; amthamine (Tocris Bioscience) as selective H2R agonist; 4-MH (4-methylhistamine) as H2R/H4R agonist (Tocris Bioscience); ST-1006 (Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Germany) as H4R agonistE16Sander K. Kottke T. Tanrikulu Y. Proschak E. Weizel L. Schneider E.H. et al.2,4-diaminopyrimidines as histamine H4 receptor ligands–scaffold optimization and pharmacological characterization.Bioorg Med Chem. 2009; 17: 7186-7196Crossref PubMed Scopus (66) Google Scholar; ranitidine (Tocris Bioscience) as selective H2R antagonist; and JNJ7777120 (Sigma Aldrich, Deisenhofen, Germany) as selective H4R antagonist. All histamine receptor ligands were used at a concentration of 10 μM. For assessment of chemokine production, fully differentiated M2 macrophages were stimulated with IL-4 (20 ng/mL) (R&D Systems) or IL-13 (15 ng/mL) (R&D Systems) for 24 hours. IL-4– and IL-13–activated M2a macrophages were treated with histamine or histamine receptor specific ligands (10 μM) for an additional 24 hours. To show the dose dependency, M2a macrophages were stimulated in the range 0.1 μM to 100 μM with agonists as indicated. Total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The cDNA was synthesized by reverse transcription (QuantiTect reverse transcription kit, Qiagen, Germany). Real-time quantitative PCR was performed with Quantitect primer assays for CCL17 (QT00096866), CCL22 (QT00089817), and RPS 20 (ribosomal protein S20) (QT00079247) using SYBR Green according to the manufacturer's instructions (Qiagen, Hilden, Germany) using the LightCycler 480. The amount of the target mRNA relative to the amount of the reference gene, ribosomal protein S20 (rps 20), mRNA in the same sample, was calculated using the comparative Ct method also known as the [delta] [delta] Ct method provided by the Software LC 480 (Roche Molecular Biochemicals, Mannheim, Germany). The Ct values of both the calibrator and the samples of interest are normalized to the appropriate endogenous reference gene rps 20. A total of 1 × 106 fully differentiated monocytes, M2 macrophages, M2c macrophages, or M1 macrophages per well were activated with IL-4, IL-13, IL-10, or IFN-γ + LPS, respectively, for 24 hours and either nonstimulated or stimulated with histamine or the specific H1R, H2R, and H4R agonists for 24 hours (10 μM). For blocking experiments, cells were treated with the H2R antagonist ranitidine or the H4R antagonist JNJ7777120 30 minutes before stimulation with receptor agonists. RNA was stabilized in lysis buffer and cell-free supernatants were taken after 24 hours from this time point. The chemokine concentrations of CCL17 and CCL22 were analyzed using commercially available ELISAs. The respective ELISAs were performed according to the manufacturer's instructions (R&D Systems). For statistical analyses, the software GraphPad Prism Version 5.0 (San Diego, Calif) was used. Wilcoxon matched pairs test was performed and the median is shown in the graph. A P value of less than .05 was regarded as statistically significant (P < .05 was labeled with *, P < .01 was labeled with **, P < .001 was labeled with ***).Fig E2Stimulation with histamine or with selective histamine receptor agonists has no effect on protein production of CCL22 in IL-4– or IL-13–activated human M2a macrophages. Fully differentiated M2 macrophages were activated with IL-4 (20 ng/mL) or IL-13 (15 ng/mL) for 24 hours. M2a macrophages were stimulated with histamine, amthamine (H2R agonist), 4-MH (H2R/H4R agonist), and ST-1006 (H4R agonist) for 24 hours. The concentration of 10 μM was used for all agonists. A, CCL22 protein concentrations in IL-4– activated M2a macrophages. B, CCL22 protein concentrations in IL-13–activated M2a macrophages. Protein concentrations were analyzed by ELISA. The protein expression was normalized to the IL-4– or IL-13–stimulated samples (IL-4–stimulated M2a macrophages produced in median 167117 pg/mL CCL22, ranging from 89,268 pg/mL to 294,926 pg/mL). (IL-13–stimulated M2a macrophages produced in median 115,200 pg/mL CCL22, ranging from 16,960 pg/mL to 1,312,000 pg/mL). ∗P < .05; medians are indicated in the graphs. Amth, Amthamine; Hist, histamine; NS, nonstimulated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Stimulation via H2R significantly upregulates CCL17 mRNA expression and protein production in IL-4–activated human M2a macrophages in a time- and dose-dependent manner. The highest expression of CCL17 was found when the cells were stimulated for 24 hours with a concentration of 10 μM of the HR ligands. A and B, M2a macrophages were stimulated for different time periods with histamine, amthamine, and 4-MH as indicated. C, M2a macrophages were stimulated with different concentrations of histamine, amthamine, and 4-MH as indicated for 24 hours. A and C, CCL17 mRNA expression was detected by quantitative PCR. B, CCL17 protein concentrations were analyzed by ELISA. The mRNA- and protein expression were normalized to the IL-4–stimulated samples (IL-4–stimulated M2a macrophages produced in median 470 pg/mL CCL17, ranging from 97 pg/mL to 770 pg/mL). Amth, Amthamine; Hist, histamine; NS, nonstimulated. Significant differences, as determined by the Wilcoxon signed rank test, are indicated as follows: *P < .05; **P < .01; medians are shown in the graphs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Stimulation via H2R significantly upregulates CCL17 but not CCL22 protein production in IL-4– or IL-13–activated human monocytes. IL-4 (20 ng/mL)- or IL-13 (15 ng/mL)-activated monocytes were stimulated with histamine, amthamine (H2R agonist), 4-MH (H2R/H4R agonist), and ST-1006 (H4R agonist) for 24 hours. The protein expression was normalized to the IL-4 or IL-13 stimulated samples. A and B, CCL17 production was assessed by ELISA (IL-4– or IL-13–stimulated M2a macrophages produced in median 303 pg/mL or 120 pg/mL CCL17, ranging from 147 pg/mL to 655 pg/mL or from 34 pg/mL to 771 pg/mL). C and D, CCL22 production was assessed by ELISA (IL-4– or IL-13–stimulated M2a macrophages produced in median 29,790 pg/mL or 23,310 pg/mL CCL22, ranging from 6,576 pg/mL to 116,900 pg/mL or from 5,237 pg/mL to 104,700 pg/mL). The concentration of 10 μM was used for all ligands. Amth, Amthamine; Hist, histamine; NS, nonstimulated. Significant differences, as determined by the Wilcoxon signed rank test, are indicated as follows: *P < .05; medians are shown in the graphs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)
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