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, 119 (3), 362-8

A Dominant Role for Chemoattractant Receptor-Homologous Molecule Expressed on T Helper Type 2 (Th2) Cells (CRTH2) in Mediating Chemotaxis of CRTH2+ CD4+ Th2 Lymphocytes in Response to Mast Cell Supernatants

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A Dominant Role for Chemoattractant Receptor-Homologous Molecule Expressed on T Helper Type 2 (Th2) Cells (CRTH2) in Mediating Chemotaxis of CRTH2+ CD4+ Th2 Lymphocytes in Response to Mast Cell Supernatants

Shân L Gyles et al. Immunology.

Abstract

Human cultured mast cells, immunologically activated with immunoglobuin E (IgE)/anti-IgE, released a factor(s) that promoted chemotaxis of human CRTH2+ CD4+ T helper type 2 (Th2) lymphocytes. Mast cell supernatants collected at 20 min, 1 hr, 2 hr and 4 hr after activation caused a concentration-dependent increase in the migration of Th2 cells. The effect of submaximal dilutions of mast-cell-conditioned media was inhibited in a dose-dependent manner by ramatroban (IC50 = 96 nm), a dual antagonist of both the thromboxane-like prostanoid (TP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), but not by the selective TP antagonist SQ29548, implicating CRTH2 in mediating the chemotactic response of these Th2 cells. The effect of mast-cell-conditioned media was mimicked by prostaglandin D2 (PGD2) and this eicosanoid was detected in the conditioned media from activated mast cells in concentrations sufficient to account for the activity of the mast cell supernatants. Treatment of the mast cells with the cyclo-oxygenase inhibitor diclofenac (10 microm) inhibited both the production of PGD2 and the CRTH2+ CD4+ Th2-stimulatory activity, while addition of exogenous PGD2 to conditioned media from diclofenac-treated mast cells restored the ability of the supernatants to promote chemotaxis of these Th2 cells. The degree of inhibition caused by diclofenac treatment of the mast cells was concordant with the degree of inhibition of chemotactic responses afforded by CRTH2 blockade. These data suggest that PGD2, or closely related metabolites of arachidonic acid, produced from mast cells may play a central role in the activation of CRTH2+ CD4+ Th2 lymphocytes through a CRTH2-dependent mechanism.

Figures

Figure 1
Figure 1
Effect of human mast cell supernatants on chemotaxis of human CRTH2+ CD4+ Th2 lymphocytes. Supernatants were collected at 20 min, 1 hr, 2 hr and 4 hr after addition of anti-IgE/IgE in the presence or absence of diclofenac (10 μm). Supernatants were collected at the same time-points from unactivated mast cells. All supernatants were diluted 1/10 for assay of chemotactic activity using CRTH2+ CD4+ Th2 cells. Data are presented as the mean ± SEM (n = 9 to n = 12) from three pooled experiments. Responses of CRTH2+ CD4+ Th2 cells activated mast cell supernatants were significantly greater than those responses to supernatants from unactivated mast cells or activated mast cells treated with diclofenac (P < 0·01 by anova). There was no significant difference between responses to supernatants from unactivated mast cells and supernatants from activated mast cells incubated with diclofenac (P > 0·05 by Newman–Keuls test).
Figure 2
Figure 2
Effect of direct treatment of CRTH2+ CD4+ Th2 cells with diclofenac on the chemotactic responses of these cells to mast cell supernatants. CRTH2+ CD4+ Th2 cells were stimulated with activated mast cell supernatants (diluted 1/10) in the presence or absence of diclofenac (10 μm). Data are presented as the mean ± SEM (n = 3). There was a significant difference between responses to supernatants from activated mast cells and supernatants from activated mast cells treated with diclofenac (*P < 0·01 by anova) but no difference between responses of untreated CRTH2+ CD4+ Th2 cells and CRTH2+ CD4+ Th2 cells treated with diclofenac (P > 0·05 by Newman-Keuls test).
Figure 3
Figure 3
Levels of PGD2 in supernatants from human mast cells at various time-points after activation with IgE/anti-IgE in the presence or absence of diclofenac (10 μm). Supernatants were collected from unactivated mast cells at the same time-points for comparison. Data are presented as the mean ± SEM (n = 3–5). There was a significant difference between the levels of PGD2 in supernatants from activated mast cells and those in supernatants from unactivated mast cells and supernatants from activated mast cells treated with diclofenac (P < 0·01 by anova). There was no significant difference between the levels of PGD2 in the supernatants from unactivated mast cells and those from activated mast cells treated with diclofenac (P > 0·05 by Newman-Keuls test).
Figure 4
Figure 4
Addition of exogenous PGD2 to conditioned media from activated mast cells treated with diclofenac restores the ability of supernatants to promote chemotaxis of CRTH2+ CD4+ Th2 cells. PGD2 (1–100 nm) was added to supernatants from diclofenac-treated mast cells activated with IgE/anti-IgE and the ability of the supernatants to promote chemotaxis of these Th2 lymphocytes were compared with those from activated mast cells in the presence or absence of diclofenac. Data are presented as the mean ± SEM (n = 3). P < 0·01 by anova; P > 0·05 by Newman-Keuls test for diclofenac-treated supernatants versus diclofenac-treated supernatants + PGD2 (1 nm) and for activated mast cells compared to diclofenac-treated mast cells + PGD2 (10 nm).
Figure 5
Figure 5
Effect of ramatroban on the ability of mast cell supernatant to cause chemotaxis of CRTH2+ CD4+ Th2 cells. Supernatants were collected from mast cells 1 hr after activation with IgE/anti-IgE. (a) Increasing concentrations of these supernatants were assayed for chemotactic activity in the presence and absence of ramatroban (1 μm). (b) Increasing doses of ramatroban were tested against a 1/10 dilution of the mast cell supernatant. (c) CRTH2+ CD4+ Th2 cells were stimulated with a 1/10 dilution of the supernatant with and without 1 μm ramatroban or 1 μm SQ29548. There was a significant difference between responses of untreated CRTH2+ CD4+ Th2 cells to control media and responses of untreated CRTH2+ CD4+ Th2 cells to supernatants from activated mast cells (P < 0·01 by anova) and a significant difference between responses of untreated CRTH2+ CD4+ Th2 cells and CRTH2+ CD4+ Th2 cells treated with ramatroban to activated mast cell supernatants (P < 0·01 by anova). There was no significant difference between the responses of untreated CRTH2+ CD4+ Th2 cells to control media and responses of ramatroban-treated CRTH2+ CD4+ Th2 cells to supernatants from activated mast cells (P > 0·05 by Newman–Keuls test). There was no significant difference between responses of untreated CRTH2+ CD4+ Th2 cells and responses of CRTH2+ CD4+ Th2 cells treated with SQ29548 to supernatants from activated mast cells. All data are presented as the mean ± SEM (n = 3–5).
Figure 6
Figure 6
Effect of ramatroban (1 μm) on migration of CRTH2+ CD4+ Th2 cells induced by PGD2, SDF-1α, MDC or TARC. Data are presented as the mean ± SEM (n = 3). Ramatroban significantly inhibited responses to PGD2 (P < 0·01 by anova) but not responses to TARC, SDF-1α or MDC (P > 0·05 by Newman–Keuls test).

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