Mast Cell Maturation is Driven via a Group III Phospholipase A 2 … · 2013-05-20 · Ikeda 8, Ryo...

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Supplementary Figures and Tables

Mast Cell Maturation is Driven via a Group III Phospholipase A2-Prostaglandin

D2-DP1 Receptor Paracrine Axis

Yoshitaka Taketomi1,2, Noriko Ueno1, Takumi Kojima1, Hiroyasu Sato1,2, Remi Murase1,2, Kei Yamamoto1, Satoshi

Tanaka3, Mariko Sakanaka4, Masanori Nakamura5, Yasumasa Nishito6, Momoko Kawana2, Naotomo Kambe7, Kazutaka

Ikeda8, Ryo Taguchi9, Satoshi Nakamizo10, Kenji Kabashima10, Michael H Gelb11, Makoto Arita12, Takehiko Yokomizo13,

Motonao Nakamura14, Kikuko Watanabe15, Hiroyuki Hirai16, Masataka Nakamura17, Yoshimichi Okayama18, Chisei Ra18,

Kosuke Aritake19, Yoshihiro Urade19, Kazushi Morimoto20, Yukihiko Sugimoto20, Takao Shimizu14, Shuh Narumiya21,

Shuntaro Hara2 & Makoto Murakami1

1Lipid Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; 2School of Pharmacy, Showa University, Tokyo, Japan; 3Department of Immunobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 4School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, Hyogo, Japan; 5School of Dentistry, Showa University, Tokyo, Japan; 6Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; 7Department of Dermatology, Chiba University Graduate School of Medicine, Chiba, Japan; 8Institute for Advanced Biosciences, Keio University, Yamagata, Japan; 9College of Life and Health Sciences, Chubu University, Aichi, Japan; 10Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan; 11Departments of Chemistry and Biochemistry, University of Washington, Washington, USA; 12Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan. 13Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan; 14Faculty of Medicine, The University of Tokyo, Tokyo, Japan; 15Department of Nutrition, Koshien University, Hyogo, Japan; 16Department of Advanced Medicine and Development, Bio Medical Laboratories, Saitama, Japan; 17Human Gene Sciences Center, Tokyo Medical and Dental University, Tokyo, Japan; 18Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Science, Tokyo, Japan; 19Department of Molecular Bihavioral Biology, Osaka Bioscience Institute, Osaka, Japan; 20Department of Pharmaceutical Biochemistry, Graduate School of Medicine and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan; 21Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Correspondence should be addressed to M.M. ([email protected]).

Supplementary information contains Supplementary Figures 1-7 and Tables 1, 2

Nature Immunology doi:10.1038/ni.2586

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Supplementary Figure 1. Expression of sPLA2s in mast cells and their roles in mast cell-dependent anaphylaxis.

(a) Release of lactate dehydrogenease (LDH) from pMCs after treatment for 30 min with or without 5 µg/ml PLA2G3, 5

µg/ml BV-PLA2 or 100 ng/ml antigen (Ag) (n = 4). (b) Flow cytometry of C57BL/6-derived BMMCs (FcεRIα+Kit+),

BMBasos (FcεRIα+CD200R3+), BMDCs (CD11c+MHCIIhi) and BMDMs (CD11b+F480+). Cells shown by squares were used. (c) Real-time PCR of various sPLA2 isoforms in C57BL/6-derived BMMCs before (immature) and 4 days after

(mature) coculture with Swiss 3T3 fibroblasts in the presence of SCF (n = 5). ND, not detected. Pla2g2a is naturally disrupted

in this mouse strain. (d) RBL-2H3 cells were lentivirally transfected with human PLA2G3 (mature form; WT), its

catalytically inactive mutant (HQ), or a control, LacZ. These cells were sensitized for 1 h with IgE and activated for 15 min

with Ag to assess β-HEX release and PGD2 generation (n = 3). (e) Represntative photos of dye extravasation (red arrows) in IgE-Ag-induced PCA in Pla2g3+/+ (+/+) and Pla2g3-/- (–/–) mice or in PLA2G3tg/+ (TG) and wild-type (WT) mice. (f) Quantification of

ear edema in IgE-Ag-dependent PCA in Pla2g2d–/–, Pla2g2e–/–, Pla2g2f–/–, Pla2g5–/–, Pla2g10–/– and their littermate (+/+) controls. Data

(mean ± s.e.m., *P < 0.05; **P < 0.01) are compiled from one (a, b) and two (c-f) experiments.


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Supplementary Figure 2. Unusual features of tissue-resident mast cells in Pla2g3-/- mice. (a, b) Transmission electron microscopy of dermal (a) and peritoneal (b) mast cells in Pla2g3+/+ (+/+) and Pla2g3–/– (-/-) mice

before and 2 min after antigen (Ag) challenge. Scale bars, 2 µm. (c, d) Flow cytometory of FcεRIα and c-Kit expression on skin (c) and peritoneal (d) mast cells in Pla2g3+/+ and Pla2g3–/– mice. In the analysis of skin mast cells, CD45+ hematopoietic

cells gated from the collagenase-dispersed skin were sorted for FcεRIα and c-Kit (c). Mean fluorescent intensity (MFI) values

of surface FcεRIα expression are shown. (e) Histamine release (quantity and percentage) from pMCs after stimulation for 30

min with 1 µM A23187. (f) Real-time PCR of mucosal mast cell markers (Mcpt1 and Mcpt2) in the small intestine of Pla2g3+/+ and Pla2g3–/– mice (n = 5). (g) Real-time PCR of inflammatory cell markers in the ear of Pla2g3+/+ and Pla2g3–/–

mice (n = 7). (h) Flow cytometry of immune cell populations in the spleen of Pla2g3+/+ and Pla2g3–/– mice (n = 3). Data

(mean ± s.e.m., *P < 0.05; **P < 0.01) are complied from one (e-h), two (a-c) or three (d) experiments.


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Supplementary Figure 3. Adoptive transfer of BMMCs lacking or overexpressing PLA2G3 into KitW-sh/W-sh mice. (a-d) Pla2g3+/+ (+/+) and Pla2g3–/– (-/-) BMMCs were intradermally (a, b) and intravenously (c, d) transfered into KitW-sh/W-sh

(Wsh) mice. After 6 and 12 weeks of reconstitution, respectively, touidine blue+ mast cells (blue arrows) in skin sections were

counted (a, c). Scale bars, 25 µm. Magnified views of toluidine blue+ mast cells are shown in boxes (a). Quantification of ear edema in IgE-Ag-dependent PCA in Pla2g3+/+ or Pla2g3–/– BMMC-reconstituited and non-reconstituted KitW-sh/W-sh mice (d).

(e) After intradermal transfer of PLA2G3tg/+ (TG) and littermate control (WT) BMMCs for 6 wk, the reconstituited and

non-reconstituted KitW-sh/W-sh mice were subjected to IgE-Ag-dependent PCA. Representative photos of dye leakage (red

arrows) are shown (b, d, e). Data (mean ± s.e.m., *P < 0.05; **P < 0.01) are complied from one (e) or two (a-d) experiments.


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Supplementary Figure 4. Properties of IL-3-driven BMMCs lacking or overexpressing PLA2G3. (a) Proliferation of Pla2g3+/+ (+/+) and Pla2g3–/– (-/-) BMMCs in IL-3-supplemented medium (n = 9). (b) Flow cytometry of

surface c-Kit and FcεRIα expression on Pla2g3+/+ and Pla2g3–/– BMMCs. MFI values of FcεRIα expression are shown (n = 5). (c, d) sPLA2 activity release from Pla2g3+/+ (+/+) and Pla2g3–/– (-/-) BMMCs (c) or from PLA2G3tg/+ (TG) and wild-type

(WT) BMMCs (d) with or without IgE-Ag stimulation for 30 min (n = 4). (e-j) Release of histamine (e, h), PGD2 (f, i) and

LTC4 (g, j) by BMMCs from Pla2g3+/+ and Pla2g3–/– mice (e-g) or from Pla2g3tg/+ and WT mice (h-j) after stimulation for

10 min with IgE-Ag (n = 8). (k) IgE-Ag-stimulated Ca2+ influx in Pla2g3+/+ and Pla2g3–/– BMMCs (n = 6). (l) Kinetic

induction of cytokines in IgE-Ag-activated Pla2g3+/+ and Pla2g3–/– BMMCs (n = 7). (m, n) Immunoblotting of total and

phosphorylated PLCγ2, Akt, MAPKs (m) and cPLA2α (n) in IgE-Ag-stimulated Pla2g3+/+ and Pla2g3–/– BMMCs. The ratios

of phosphorylated to total cPLA2α were quantified by densitometry (n = 5) (n). (o) ESI-MS of AA (20:4)-containing phosphatidylcholine in Pla2g3+/+ and Pla2g3–/– BMMCs before and 2 min after IgE-Ag stimulation (n = 3). Data (mean ±

s.e.m., *P < 0.05; **P < 0.01) are compiled from one (c, d), two (b, k, m-o) or three (a, e–j, l) experiments.


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Supplementary Figure 5. Properties of Pla2g3-/-, PLA2G3tg/+ and Pla2g4a-/- BMMCs with or without coculture. (a) Proliferation of Pla2g3+/+ and Pla2g3–/– BMMCs after coculture (Cocx) for 4 days in SCF-supplemented medium (n = 6).

(b) Transmission electron microscopy of Pla2g3+/+ (+/+) and Pla2g3–/– (-/-) BMMCs before and on day 4 of coculture. Scale bars,

2 µm. (c) Hdc expression in PLA2G3tg/+ (TG) and wild-type (WT) BMMCs before and on day 4 of coculture (n = 6). (d)

Flow cytometry of c-Kit and FcεRIα expression on Pla2g3+/+ and Pla2g3–/– BMMCs after coculture for 4 days. MFI values

of FcεRIα expression are indicated (n = 6). (e) Expression of various genes in Pla2g3+/+ and Pla2g3–/– BMMCs before and on day 4 of coculture (n = 8). (f) Ear edema in IgE-Ag-dependent PCA in Pla2g4a+/+ and Pla2g4a–/– mice. (g, h) Mast-cell

counts (n = 6) (g) and histamine levels (n = 5) (h) in Pla2g4a+/+ and Pla2g4a–/– ears. (i, j) IgE-Ag-induced histamine release

for 10 min (i) and histamine contents (j) in Pla2g4a+/+ and Pla2g4a–/– BMMCs (n = 4). (k) Hdc expression in Pla2g4a+/+ and

Pla2g4a–/– BMMCs before and on day 4 of coculture (n = 4). (l, m) PGD2 (l) and LTC4 (m) generation by Pla2g4a+/+ and

Pla2g4a–/– BMMCs after stimulation with indicated doses of Ag for 10 min (n = 8). Data (mean ± s.e.m., *P < 0.05, **P <

0.01) are compiled from one (g, h), two (b-d, f, i-m) or three (a, e) experiments.


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Supplementary Figure 6. IgE-Ag-dependent PCA in various knockout mouse lines that lack eicosanoid receptors or biosynthetic enzymes.

(a) Quantification of ear edema in IgE-Ag-dependent PCA in Ptgdr2–/–, Ptger1–/–, Ptger2–/–, Ptger3–/–, Ptger4–/–, Ptgfr–/–, Ptgir–/–, Tbxa2r–/–, Ltb4r1–/–, Ltb4r2–/–, Ptges–/–, Ptges2–/–, and Alox15–/– mice compared to their wild-type littermates. Positions

of individual molecules examined so far are indicated by red boxes in the lipid mediator-biosynthetic and receptor pathway as

illustrated. Results in mice deficient in DP1 and two PGDSs are shown in Figures 5 and 6, respectively. (b) Ptgdr2 expression

in Pla2g3+/+ and Pla2g3–/– BMMCs before and after coculture (Cocx) for 4 days (n = 8). (c) Hdc expression in Ptgdr2+/+ and

Ptgdr2–/– BMMCs before and on day 2 of coculture (n = 4). (d) Ptgdr2 expression in the ear of Pla2g3+/+ and Pla2g3–/– mice.

(e) Production of 15-HETE and PGI2 (measured as the stable endoproduct 6-keto-PGF1α) before and on day 2 of coculture of Pla2g3+/+ and Pla2g3–/– BMMCs with Swiss 3T3 fibroblasts (n = 4). Data (mean ± s.e.m.) are compiled from one (e), two (a, c, d) or three (b) experiments. **, P < 0.01. NS, not significant.


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Supplementary Figure 7. Regulatory expression and function of fibroblastic L-PGDS in mast cell maturation.

(a) L-PGDS immunohistochemistry followed by toluidine blue counterstaining in Ptgds+/+ (+/+) or Ptgds–/– (-/-) skins. Scale

bars, 25 µm. (b) IgE-Ag-dependent PCA in KitW-sh/W-sh (Wsh) mice 6 weeks after intradermal reconstitution with Ptgds+/+ (+/+) or Ptgds–/– (-/-) BMMCs. (c) Hdc expression in Ptgds+/+ or Ptgds–/– BMMCs before and after coculture (Cocx) for 2 days (n =

4). (d) Expression of Hdc relative to Kit in Pla2g3+/+ or Pla2g3–/– BMMCs before and on day 1 of coculture with or without

AT-56 (n = 6). (e) Kinetic PGD2 generation in Ptgds2+/+ and Ptgds2–/– BMMC cocultures (n = 4). (f, g) Ptgds2 expression in

Pla2g3+/+ or Pla2g3–/– BMMCs (n = 8) and fibroblasts (n = 3) (f) or Ptgds expression in Pla2g3+/+ or Pla2g3–/– BMMCs (n =

8) and fibroblasts (n = 6) (g) before and on day 2 of coculture. (h) Ptgds expression in primary mouse skin-derived fibroblasts

before and on day 2 of coculture with wild-type BMMCs. (i) Ptgds expression in fibroblasts before and on day 2 of coculture

with PLA2G3tg/+ (TG) and wild-type (WT) BMMCs. (j) Fibroblastic Ptgds expression after culture for 2 days with or without

5 µg/ml PLA2G3 or BV-PLA2 (n = 3). Data (mean ± s.e.m., P < 0.05; **P < 0.01) are compiled from one (i, j), two (a-e, h) or three (f, g) experiments. (k) The schematic diagram of the PLA2G3-L-PGDS-DP1 circuit for mast cell maturation.


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Supplementary Table 1. Genes that were altered by PLA2G3 deficiency in BMMCs cocultured with fibroblasts.

Signal intensities and fold changes [knockout (KO) relative to wild type (WT)] under each culture condition were assessed by

microarray. Genes with a > 40% reduction are shown in blue and those with a > 50% increase are shown in red. Most genes

listed here were markedly increased after maturation of WT BMMCs to CTMC-like cells but were up-regulated only partially

upon maturation of KO BMMCs. Accordingly, the KO/WT ratios of most genes were reduced in CTMC-like cells (i.e., after

coculture) but not in BMMCs (i.e., before coculture). Exceptions were Ptger3, Ltb4r1, and Ltc4s, the expression levels of

which were reduced after coculture, and Ltc4s, which was expressed at a higher level in KO than in WT CTMC-like cells.

The changes in Ltc4s are consistent with the fact that the LTC4-synthetic capacity drops after maturation into CTMC-like cells.

A representative result from four independent experiments is shown. ND, not detected.

Accession No. Gene Name BMMCs Fold

(KO/WT)

CTMC-like cells Fold

(KO/WT) +/+ –/– +/+ –/–

Granule-associated

NM_008230 Hdc Histidine decarboxylase (HDC) 445 243 0.55 11054 3676 0.33

NM_010779 Mcpt4 Mus musculus mast cell protease 4 (MMCP-4) 120 107 0.89 16046 1506 0.09

NM_010781 Mcpt6 Mus musculus mast cell protease 6 (MMCP-6) 4321 3207 0.74 22397 11397 0.51

NM_008681 Ndrg1 N-myc downstream regulated gene 1 (NDRG1) 2482 2276 0.92 27977 10223 0.37

Lipid mediator biosynthetic enzymes and receptors

NM_008869 Pla2g4a Phospholipase A2, group IVA (cPLA2α) 120 176 1.47 545 274 0.50

NM_011198 Ptgs2 Prostaglandin-endoperoxide synthase 2 (COX-2) ND ND – 553 114 0.21

NM_019455 Ptgds2 Hematopoietic prostaglandin D2 synthase (H-PGDS) 58 71 1.22 936 542 0.58

NM_011196 Ptger3 Prostaglandin E receptor 3 (EP3) 1109 1187 1.07 423 236 0.56

NM_008967 Ptgir Prostaglandin I receptor (IP) 58 41 0.71 591 221 0.37

NM_008519 Ltb4r1 Leukotriene B4 receptor 1 (BLT1) 1490 1417 0.95 115 57 0.44

NM_008521 Ltc4s Leukotriene C4 synthase (LTC4S) 302 275 0.91 49 126 2.57

Cytokines, chemokines, and their receptors

NM_008361 Il1b Interleukin 1β (IL-1β) 23 18 0.78 3704 1556 0.42

NM_021283 Il4 Interleukin 4 (IL-4) 1163 1105 0.95 716 407 0.57

NM_031168 Il6 Interleukin 6 (IL-6) 2757 2708 0.98 18685 8022 0.43

NM_013693 Tnf Tumor necrosis factor (TNFα) 26 23 0.88 2057 1367 0.66

NM_019418 Tnfsf14 Tumor necrosis factor superfamily, member 14 (LIGHT) 483 612 1.27 7854 4846 0.62

NM_008362 Il1r1 Interleukin 1 receptor, type I (IL-1R) ND ND – 623 279 0.45

NM_011329 Ccl1 Chemokine (C-C motif) ligand 1 (CCL1) ND ND – 1547 468 0.30

NM_013652 Ccl4 Chemokine (C-C motif) ligand 4 (CCL4) 3955 3093 0.78 19997 9165 0.46



NM_008176 Cxcl1 Chemokine (C-X-C motif) ligand 1 (CXCL1) ND ND – 3250 988 0.30




NM_009142 Cx3cl1 Chemokine (C-X3-C motif) ligand 1 (CX3CL1) ND ND – 1504 447 0.30


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Supplementary Table 2. A list of probe/primer sets for real-time PCR.

Name Assay No.

Pla2g1b Mm00478249_m1

Pla2g2d Mm00478250_m1

Pla2g2e Mm00478870_m1

Pla2g2f Mm00478872_m1

Pla2g5 Mm00448162_m1

Pla2g10 Mm00449532_m1

Pla2g3 Mm01191142_m1

PLA2G3 Hs00210447_m1

Pla2g4a Mm00447040_m1

Ptgds Mm01330613_m1

Ptgds2 Mm00479846_m1

Ptgdr1 Mm00436050_m1

Ptgdr2 Mm00438315_s1

Hdc Mm00456104_m1

HDC Hs00157914_m1

Mcpt1 Mm00656886_g1

Mcpt2 Mm00484932_m1

Mcpt4 Mm00487636_g1

Mcpt6 Mm00487645_m1

Cpa3 Mm00483940_m1

Ndrg1 Mm00440447_m1

Kit Mm00445212_m1

KIT Hs00174029_m1

Mitf Mm00434954_m1

Srgn Mm01169070_m1

Itgae Mm00434443_m1

Itgb7 Mm01296188_m1

Mrgpx1 Mm02525847_g1

Mrgpx2 Mm04204397_m1

Il4 Mm00445259_m1

Il6 Mm00446190_m1

Tnf Mm00443260_g1

Itgax Mm00498698_m1

Cd207 Mm00523545_m1

Emr1 Mm00802529_m1

Cd3e Mm01179194_m

Foxp3 Mm00475162_m1


Mast Cell Maturation is Driven via a Group III Phospholipase A 2 … · 2013-05-20 · Ikeda 8, Ryo...

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