Prostaglandin D2 receptors in human mast cells

Abstract

To the Editor, Mast cells are important effector cells playing a critical role in regulation of immune responses in allergic diseases. Mast cells are one of the main sources of pro-inflammatory eicosanoids, such as prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs), which can affect allergic type-2 responses, activating cells such as Th2, ILC2, eosinophils, and basophils and also acting in autocrine or paracrine fashion on mast cells themselves.1 It has been established that cysLTs can potently regulate mast cells functions through cysteinyl leukotriene type 1 receptor (CysLT1) but it is still unknown whether PGD2 can induce activation of human mast cells. 2,3 Conflicting reports have been published showing positive or negative staining for PGD2 receptors in human tissue mast cells using immunochemistry.4,5 As a number of novel antagonists for PGD2 receptors have entered clinical trials, information regarding whether human mast cells could be a target for this new class of drugs seems to be of particular interest.6,7 In order to analyze whether human mast cells may be activated by PGD2, we first examined the expression of PGD2 and cysLTs receptors at the mRNA level in two human mast cell lines, LAD2 and LUVA (Figure 1A). Interestingly, LUVA cells expressed high levels of PTGDR2 (gene encoding PGD2 receptor type 2, DP2, also known as CRTH2 and CD294) in comparison with much lower levels detected in LAD2 cells. PTGDR (gene encoding PGD2 receptor type 1, DP1) transcripts have been detected at background level or undetected in some samples suggesting that DP1 receptor is not expressed in those cells. As previously reported2,8 both cell lines express CYSLTR1 and lower levels of CYSLTR2 (genes encoding cysLT receptor type 1 and 2, respectively). To confirm these observations at the protein level, DP2 surface expression was measured in both cell lines (Figure 1B) by flow cytometry. LUVA cells consistently presented high DP2 expression, with more than 90% cells showing positive staining. There was no DP2 surface expression detected in LAD2 cells. Functional DP2 receptors couple in most cells to G protein αi and signal through intracellular calcium mobilization. Next, we determined whether DP2 expression in LUVA cells can lead to calcium mobilization upon PGD2 stimulation (Figure 1C). Both cell lines responded in a concentration-dependent manner to LTD4 but there was no positive response to any of the PGD2 concentrations tested. DP2 is a high affinity receptor for PGD2 and can be fully activated by low nanomolar concentrations of PGD2, with 100 nmol/L showing often maximum activity.9 In order to detect any low affinity responses to PGD2 in mast cells, concentrations of up to 1 μmol/L were tested in both cell lines with similar negative responses observed (not shown). As LUVA cells express high levels of DP2, we also considered an additive effect of PGD2 on LTD4 induced calcium mobilization. Calcium mobilization was therefore measured when LUVA cells were simultaneously stimulated with a range of LTD4 concentrations together with 100 nmol/L PGD2 and calcium mobilization was measured (Figure 1C). There was no difference in calcium responses between LTD4/PGD2 and LTD4 only stimulated cells suggesting a lack of response to PGD2 through calcium mobilization in human mast cells. We next evaluated whether other signaling pathways may be activated by PGD2 in LUVA cells, namely changes in intracellular cyclic adenosine monophosphate (cAMP) concentrations. To our surprise, PGD2 did not significantly induce or inhibit intracellular cAMP generation induced by forskolin (Figure 1D) suggesting that DP2 receptor in LUVA cells may be nonfunctional as none of tested canonical G protein coupled receptor signaling pathways was affected. PGD2 acting through DP2 has been shown to potently induce gene transcription, chemotaxis, or cell degranulation in many cells so even in the absence of detectable signaling additional functional responses were also tested. As shown in Figure 1E, LTD4 but not PGD2 increased mRNA expression of CSF2 in LUVA cells, that was fully inhibited by CysLT1 antagonist montelukast, but not by DP2 antagonist (CAY10471) or CysLT2 antagonist (HAMI3379). No additive effect of PGD2 on LTD4 induced gene expression was observed. Similarly, when chemotaxis responses were examined (Figure 1F), LTD4 induced significant chemotaxis of LUVA cells but no significant chemotaxis or chemotactic additive effect was seen for PGD2 stimulated cells. Neither LTD4 nor PGD2 induced significant cell degranulation measured by β-hexosaminidase assay (Figure S1). As a positive control for PGD2 activity, DP2 expression has been measured in human basophils by flow cytometry, showing downregulation of DP2 expression upon PGD2 exposure, an effect inhibited by pretreatment with CAY10471. There was no downregulation of DP2 expression in LUVA cells (Figure S2). Altogether, neither LAD2 nor LUVA human mast cell lines were activated by PGD2 despite high expression of DP2 detected in LUVA cells. We next sought to verify whether our observations from human mast cell lines could be confirmed in primary human mast cells. Mononuclear cells were isolated from nasal polyps and nasal turbinates as described in Supporting information, and mast cells were defined as viable singlet cells that were positive for CD45+, c-kit+, and FcεRI+ (Figure 2A,B, Figures S3 and S4). Peripheral blood basophils were used for comparison, defined as viable singlet cells that were CD45+, FcεRI+, and CD123+. Mast cells and basophils were flow sorted, RNA extracted and PGD2 receptor expression measured by RT-PCR (Figure 2E). Background or nondetectable levels of PTGDR were observed in mast cells and in basophils. In contrast, very high levels of PTGDR2 mRNA were