DP2 agonist-induced Ca²⁺ flux in human mast cells.

<p>After measuring baseline fluorescence of Fluo-4 AM loaded MC (1.25×10⁵ cells in 50 µL/well), (A, C) DK-PGD₂ or (B, D) 15R-15-methyl PGD₂ was given to the MC and intracellular Ca²⁺ flux was assessed by measuring fluorescence change. (A, B) Cytosolic free Ca²⁺ changes induced by DP2 agonists are presented as ΔFluorescence ratio (fluorescence ratio of agonist treatment – fluorescence ratio of sham treatment), where fluorescence ratio is fluorescence unit at each time point/baseline fluorescence unit. Arrow indicates the time when agonist was given. (C, D) Cytosolic free Ca²⁺ changes induced by DP2 agonist treatment are presented as ΔIntegral for 3 min from ΔFluorescent ratio curves shown in A and B. Results are expressed as mean ± SEM for three separate experiments. *p<0.05, **p<0.01 compared with 100 nM agonist treatment by repeated measures ANOVA followed by the Bonferroni post-test.</p

Single cell analysis of DP2 expression on human mast cells with ImageStream.

<p>Expression of DP2 on LAD2 and hPBDMC were examined with ImageStream after staining live cells for surface expression (A, C) or with fixed and permeabilized cells for total expression (B, D). (A, C) After surface staining, DP2 signals were detected from inside MC (open triangle) rather than on the surface. (B, D) Intracellular punctate staining for DP2 (arrow) was observed in fixed and permeabilized MC before staining. (E, F) K562/B19 (DP2 transfectant) was used as a control for surface expression of DP2 (closed triangle). Representative images of cells stained with isotype matched control Ab (left) and DP2 positive cells (right) from three independent experiments are shown.</p

DP2 antagonists did not abolish DP2 agonist-induced intracellular Ca²⁺ flux.

<p>After measuring baseline fluorescence of Fluo-4 AM loaded LAD2, a DP2 selective antagonist (1 µM CAY10471 or 100 nM CAY10595) or DP2/TP dual antagonist (1 µM ramatroban) was added. After 5 min, 1 µM 15R-15-methyl PGD₂ was added and intracellular Ca²⁺ flux was assessed by measuring fluorescence change. Relative Ca²⁺ flux was calculated from ΔIntegral for 3 min after addition of 15R-15-methyl PGD₂, where sham treatment instead of antagonist considered as 100%. Results are expressed as mean ± SEM for three (ramatroban and CAY10471) and five (CAY10595) separate experiments. There was no statistical difference between sham and antagonist treatment. Note: Higher concentrations of each antagonist could not be used as they caused Ca²⁺ flux by themselves.</p

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

<div><p>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.</p></div

Immunohistochemical staining of DP2 in human nasal polyp mast cells.

<p>(A) Tissue sections from nasal polyps (n = 15) were double stained with rabbit anti-human DP2 and mouse anti-human MC tryptase antibodies or isotype matched control antibodies. DP2 staining is shown in dark red and MC tryptase is shown in blue. Insert shows the cellular staining with examples of single- (white triangle for DP2 single⁺, black triangle for tryptase single⁺) and double-positive cells (open arrow). (B) Percentage of DP2⁺ MC and non MC from total nucleated non epithelial cells (C) Percentage of DP2 positive MC among tryptase⁺ MC. The percentage of DP2 positive MC among MC was calculated by [number of double positive cells/(number of double positive cells + number of tryptase single positive cells)]×100.</p

Pertussis toxin abolished DP2 agonist-induced Ca²⁺ flux in human mast cells.

<p>LAD2 were pretreated with 10 nM pertussis toxin (PTX) for 2 h then Fluo-4 AM was loaded. After measuring baseline fluorescence of Fluo-4 AM loaded MC (1.25×10⁵ cells in 50 µL/well), (A) 1 µM PGD₂, (B) 1 µM DK-PGD₂ or (C) 1 µM 15R-15-methyl PGD₂ was added and intracellular Ca²⁺ flux was assessed by measuring fluorescence change. (A–C) Cytosolic free Ca²⁺ changes by DP2 agonists were presented as Fluorescence ratio (fluorescence unit at each time point/baseline fluorescence unit). Arrow indicates the time when agonist was given. (D) Cytosolic free Ca²⁺ changes in A–C are presented as integral for 3 min. Results are expressed as mean ± SEM for three separate experiments. ^†p<0.05; ^††p<0.01; ^†††p<0.001 compared with each sham treatment (sham <i>vs</i> agonist, PTX <i>vs</i> PTX/agonist), *p<0.05; **p<0.01 compared with each agonist treatment (agonist <i>vs</i> PTX/agonist) by repeated measures ANOVA followed by the Tukey post-test.</p

mRNA expression of PGD₂ receptors in human mast cells.

<p>Expression of DP2 and DP1 mRNA in human MC lines (HMC-1 and LAD2) and primary cultured MC [peripheral blood-derived MC (hPBDMC) and cord blood-derived MC (hCBDMC)]. Three different cultures were shown for hPBDMC and hCBDMC. Human DP2⁺/CD4⁺ T cells cultured in Th2-polarizing conditions were used for a positive control of DP2 and dH₂O instead of cDNA was used as a negative control.</p

No effects of DP2 agonist on human mast cell degranulation induced by IgE-crosslinking.

<p>LAD2 or hPBDMC were sensitized with 100 ng/mL biotinylated human IgE overnight. Cells were washed and resuspended (2×10⁵ cells/200 µL) in HEPES-Tyrode's buffer (HTB), and stimulated with 100 ng/mL streptavidin in the presence or absence of indicated dose of 15R-15-methyl PGD₂ for 30 min. The cells were centrifuged, and the percent release of β-HEX into the supernatant was calculated. β-HEX release (%) are expressed as mean ± SEM for 8–9 separate experiments of LAD2 (A), and 8–10 separate experiments of hPBDMC (B) with five different hPBDMC cultures. **p<0.01, ***p<0.001 compared with sham (0 nM 15R-15-methyl PGD₂ without IgE cross-linking), and no statistical significant difference was found between 15R-15-methyl PGD₂ treatment group by one-way ANOVA followed by the Tukey post-test.</p