Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2

Abstract

Prostaglandin (PG)D2, which has long been implicated in allergic diseases, is currently considered to elicit its biological actions through the DP receptor (DP). Involvement of DP in the formation of allergic asthma was recently demonstrated with DP-deficient mice. However, proinflammatory functions of PGD2 cannot be explained by DP alone. We show here that a seven-transmembrane receptor, CRTH2, which is preferentially expressed in T helper type 2 (Th2) cells, eosinophils, and basophils in humans, serves as the novel receptor for PGD2. In response to PGD2, CRTH2 induces intracellular Ca2+mobilization and chemotaxis in Th2 cells in a Galphai-dependent manner. In addition, CRTH2, but not DP, mediates PGD2-dependent cell migration of blood eosinophils and basophils. Thus, PGD2 is likely involved in multiple aspects of allergic inflammation through its dual receptor systems, DP and CRTH2.

Figure 1

Binding of PGD₂ to CRTH2. (A) Expression levels of CRTH2 as determined by flow cytometry. Cells were stained with biotinylated BM16 (solid line) or control IgG2a (dotted line) as described (reference 6). (B) CRTH2 mediates Ca²+ mobilization by PGD₂ and purified mast cell supernatants. Thrombin was used as an irrelevant stimulant. Arrows indicate the time of stimulant addition. (C) Scatchard plot analysis for ³H-PGD₂ binding to K562/CRTH2 or K562/DP. (D) CRTH2-mediated binding of ³H-PGD₂ is selectively inhibited by BM7. Cells pretreated with PBS, BM7 (600 μg/ml), or normal rat IgG (600 μg/ml) at room temperature for 20 min were subjected to ³H-PGD₂ binding analysis without washing (mean ± SD, n = 3). (E) DK-PGD₂ and BW245C serve as selective agonists for CRTH2 and DP, respectively, in Ca²+ mobilization assay.

Figure 2

Functional discrimination between CRTH2 and DP. (A) DP but not CRTH2 mediates PGD₂-induced cAMP generation. Intracellular cAMP levels were determined by enzyme-linked immunoassay (mean, n = 2; Amersham Pharmacia Biotech). (B) CTX and PTX differentially affect PGD₂-induced (25 nM) Ca²+ mobilization in K562/CRTH2 and K562/DP. (C) Chemotactic responses to agonists (PGD₂, DK-PGD₂, and BW245C) are opposite in Jurkat/CRTH2 and Jurkat/DP (mean ± SD, n = 3). Numbers of migrated cells in controls were 727 ± 134 (Jurkat/CRTH2), 439 ± 50 (Jurkat/DP), and 661 ± 152 (Jurkat/neo).

Figure 3

Selective responses of Th2, basophils, and eosinophils to PGD₂. (A) CRTH2 expression in representative Th1 and Th2 lines. Levels of CRTH2 expression are presented as described in the legend to Fig. 1 A. (B) PGD₂ and DK-PGD₂ selectively induce Ca²+ mobilization in Th2 cells via CRTH2. Arrows indicate the time of addition of the test samples (black arrows) and 62.5 nM MCP-1 (white arrows). MCP-1 was used as an irrelevant stimulant. BM7 and rat IgG (each 600 μg/ml) were added 20 min before the stimulant addition. (C) Chemotaxis to PGD₂ and DK-PGD₂ is selectively induced in Th2 but not Th1 lines (mean ± SD, n = 3). The numbers of migrated cells in controls were 4,015 ± 627 (Th1) and 1,374 ± 225 (Th2). (D) Chemotaxis to PGD₂ and DK-PGD₂ of Th2 cells is inhibited by BM7 (mean ± SD, n = 3). Th2 cells were treated with PBS, BM7, or normal rat IgG as described in B before being subjected to chemotaxis assay. Numbers of migrated cells in controls were 4,146 ± 433 (PBS), 3,283 ± 349 (BM7), and 3,246 ± 65 (normal rat IgG). (E) mRNA levels for CRTH2, DP, and β-actin (internal control) in peripheral blood leukocytes and Th lines as determined by RT-PCR. (F) Migration of basophils and eosinophils is induced by PGD₂ (mean ± SD, n = 3). (G) Migration of eosinophils and basophils to PGD₂ and DK-PGD₂ is dose dependent (mean ± SD, n = 3). In this experiment, neutrophil (CD16⁺ cell)-depleted leukocyte samples were used. (H) PGD₂-induced migration of basophils and eosinophils is inhibited by BM7 (mean ± SD, n = 3). Neutrophil-depleted leukocytes were treated as described in D. Asterisks indicate statistical significance with a probability of <0.01 in Student's t test.

Figure 4

A phylogenetic tree for human receptors to classical chemoattractants and major prostanoids. The tree was constructed by the N-J method using CLUSTAL X software. The sequence of the human α2B adrenergic receptor was used as an outer group to obtain a root. Chromosomal locations are shown in parentheses. Accession nos. for the receptors are (from top to bottom) GenBank/EMBL/DDBJ AF005900, D38081, L24470, L22647, L27490, L28175, D25418, U19487, Q13258 (GenPept accession no.), AF119711, AF254664, AB008193, AB008193, AB008535, M62505, L14061, L10820, and M84562. Cys LT, cysteinyl LT receptor; FPRL, formyl peptide receptor–like receptor.