Expression of DP2 (CRTh2), a Prostaglandin D2 Receptor, in Human Mast Cells

in Human Mast Cells. PLoS ONE 9(9): e108595. doi:10.1371/journal.pone.0108595 Expression of DP2 (CRTh2), a Prostaglandin D Receptor, 2 in Human Mast Cells Tae Chul Moon 0 1 2 3 Eduardo Campos-Alberto 0 1 2 3 Tsuyoshi Yoshimura 0 1 2 3 Graeme Bredo 0 1 2 3 Aja M. Rieger 0 1 2 3 Lakshmi Puttagunta 0 1 2 3 Daniel R. Barreda 0 1 2 3 A. Dean Befus 0 1 2 3 Lisa Cameron 0 1 2 3 Roland Seifert, Medical School of Hannover, Germany 0 and Nutritional Science, University of Alberta , Edmonton, AB , Canada , 5 Department of Pathology and Laboratory Medicine, Schulich School of Medicine & Dentistry 1 Edmonton, AB, Canada, 3 Department of Laboratory Medicine and Pathology, University of Alberta Hospitals , Edmonton, AB , Canada , 4 Department of Agricultural , Food 2 1 Pulmonary Research Group, Department of Medicine, University of Alberta , Edmonton, AB , Canada , 2 Department of Biological Sciences, University of Alberta 3 Western University , London, ON , Canada PGD2 has long been implicated in allergic diseases. Recent cloning of a second PGD2 receptor, DP2 (also known as CRTh2), led to a greater understanding of the physiological and pathophysiological implications of PGD2. PGD2 signaling through DP1 and DP2 mediates different and often opposite effects in many cell types of the immune system. Although mast cells (MC) are the largest source of PGD2 in the body, there is little information about their potential expression of DP2 and its functional significance. In this study, we show that tissue MC in human nasal polyps express DP2 protein, and that human MC lines and primary cultured human MC express mRNA as well as protein of DP2. By immunohistochemistry, we detected that 34% of MC in human nasal polyps expressed DP2. In addition, flow cytometry showed that 87% of the LAD2 human MC line and 98% of primary cultured human MC contained intracellular DP2. However, we could not detect surface expression of DP2 on human MC by single cell analysis using imaging flow cytometry. Blocking of endogenous PGD2 production with aspirin did not induce surface expression of DP2 in human MC. Two DP2 selective agonists, DK-PGD2 and 15R-15-methyl PGD2 induced dose-dependent intracellular calcium mobilization that was abrogated by pertussis toxin, but not by three DP2 selective antagonists. MC mediator release including degranulation was not affected by DP2 selective agonists. Thus, human MC express DP2 intracellularly rather than on their surface, and the function of DP2 in human MC is different than in other immune cells such as Th2 cells, eosinophils and basophils where it is expressed on the cell surface and induces Th2 cytokine and/or granule associated mediator release. Further studies to elucidate the role of intracellular DP2 in human MC may expand our understanding of this molecule and provide novel therapeutic opportunities. - Funding: This study was supported by the Canadian Institutes of Health Research (CIHR, 7034, 111267). TCM received a CIHR-Rx&D/Canadian Lung Association/ GlaxoSmithKline (GSK) research program fellowship (XCL-81974) and Alberta Heritage Foundation for Medical Research (now Alberta Innovates - Health Solutions, AIHS) fellowship (200501397). TY received a fellowship from Tokyo Jikei Medical University. EC received an AllerGen/Canadian Allergy, Asthma and Immunology Foundation (CAAIF)/Merck International Clinician-Scientist and Post-Doctoral Research Fellowship. LC received a salary award from AIHS and held a GSK-CIHR Chair in Airway Inflammation, and these were competitive peer reviewed grant competitions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The funding from GlaxoSmithKline as a CIHR-Rx&D/Canadian Lung Association/GSK research program fellowship (XCL-81974) to TCM is a post-doctoral fellowship administered by the Canadian Institutes of Health Research (CIHR) in partnership with the Canadian Lung Association, GlaxoSmithKline and CIHR Rx&D Collaborative Research Program. There is no competing interest with GlaxoSmithKline with this fellowship award. The funding from Merck as an AllerGen/Canadian Allergy, Asthma and Immunology Foundation (CAAIF)/Merck International Clinician-Scientist and Post-Doctoral Research Fellowship to EC is administered by AllerGen, a government funded National Centre of Excellence. There is no competing interest with Merck with this salary award. The funding from GlaxoSmithKline as a GSK-CIHR Chair in Airway Inflammation and from AIHS to LC are salary awards scholarships. Therefore there is no competing interest with GlaxoSmithKline. AIHS is not a commercial source but is a publicly funded, board-governed corporation that operates under an Act of the Alberta provincial legislature. Cover Letter The AstraZeneca Canada Inc. in ADBs title is to acknowledge his chair position as a courtesy, but ADB has no commercial relationship with them. In 1991, Astra Canada endowed a Chair in Asthma Research to the University of Alberta; Astra merged with Zeneca much later and the name of the endowment/chair was altered. This endowment is under the full control of the University and there is no formal affiliation with AstraZeneca. Therefore there is no competing interest with AstraZeneca Canada Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Mast cells (MC) are tissue-resident cells derived from bone marrow progenitors. They are widely distributed throughout the multiple tasks in different locations and functional settings. MC are primary effector cells of allergic inflammation following IgE cross-linking and they also have diverse roles in angiogenesis, wound healing, tissue remodeling, regulation of inflammation, host defense, and innate and adaptive immune responses [14]. Along with mediators such as histamine and proteases in the granules, and de novo synthesized cytokines and chemokines, activated produce an abundance of prostaglandin (PG) D2 and leukotriene (LT) C4 [5,6]. These lipid mediators have bronchoconstricting and vasoactive properties, but also participate in host defense, inflammation, and allergic diseases through diverse activities such as effector cell trafficking, antigen presentation, immune cell activation and fibrosis [68]. PGD2 is a key mediator produced by activated MC [5,9] and antigen presenting cells [10] following allergen exposure in patients with asthma, atopic dermatitis or allergic rhinitis [11 13]. PGD2 contributes directly to smooth muscle contraction [14,15], vascular leak and vasodilation [16] that typically occur in type I hypersensitivity, and also potentiates cellular responses to other physiologically relevant mediators (eg., histamine) released during these allergic reactions [17]. It modulates dendritic cell migration and maturation [18] and induces migration and activation of human Th2 cells [19,20], eosinophils [21,22], basophils [20,23], and macrophages [2 (...truncated)