Figure 1.
Immunohistochemical staining of DP2 in human nasal polyp mast cells.
(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.
Figure 2.
mRNA expression of PGD2 receptors in human mast cells.
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.
Figure 3.
Flow cytometry analysis of DP2 and FcεRI expression on human mast cells.
Expression of DP2 and FcεRI on hPBDMC and LAD2 were examined by flow cytometry. A representative result of dot plot (left) and MFI (Mean Fluorescent Intensity, right) from five to eight independent experiments calculated using WinMDI ver.2.9 software (mean±SEM) are shown. (A) Total expression of DP2 in hPBDMC (n = 8). (B) Total expression of DP2 in LAD2 (n = 5). (C) Surface expression of DP2 in hPBDMC (n = 8). (D) Surface expression of DP2 on LAD2 (n = 5). (E) Surface expression of FcεRI on hPBDMC (n = 15–21). (F) Surface expression of FcεRI (n = 5) on LAD2. (G) Surface expression of DP2 on DP2 transfectant, K562/B19 (n = 3) was examined as a control. In dot plot, the percentage of positive cells was shown inside, and gray, black and red dots represent unstained, stained with isotype control and specific antibody, respectively. †p<0.05, ††p<0.01, †††p<0.001 compared with unstained; **p<0.01, ***p<0.001 compared with isotype control by repeated measures ANOVA followed by the Tukey post-test.
Figure 4.
DP2 agonist-induced Ca2+ flux in human mast cells.
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.
Figure 5.
Pertussis toxin abolished DP2 agonist-induced Ca2+ flux in human mast cells.
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 vs agonist, PTX vs PTX/agonist), *p<0.05; **p<0.01 compared with each agonist treatment (agonist vs PTX/agonist) by repeated measures ANOVA followed by the Tukey post-test.
Figure 6.
DP2 antagonists did not abolish DP2 agonist-induced intracellular Ca2+ flux.
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.
Figure 7.
No effects of DP2 agonist on human mast cell degranulation induced by IgE-crosslinking.
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.
Figure 8.
Single cell analysis of DP2 expression on human mast cells with ImageStream.
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.
Figure 9.
Effect of intrinsic PGD2 on DP2 expression in human mast cells.
LAD2 were incubated for indicated time periods in the presence or absence of 10 µg/mL aspirin and then any change of surface and total DP2 expression was examined by flow cytometry. ΔRelative MFI [Relative mean fluorescent intensity (MFI) from stained cells with DP2 Ab - Relative MFI from stained cells with isotype Ab], where relative MFI is [(MFI from stained cells with Ab - MFI from unstained cells)/MFI from unstained cells], from five independent experiments was calculated using WinMDI ver.2.9 software (mean ± SEM). No statistical significance difference was found between sham and aspirin treatment groups by Two-way ANOVA followed by the Bonferroni post-test.