Mechanisms of AllergySelective modulation of chemokinesis, degranulation, and apoptosis in eosinophils through the PGD2 receptors CRTH2 and DP☆
Section snippets
Reagents
PGD2, fluprostenol, and PGE2 were obtained from Biomol Research Laboratories. BW245C, 13,14-dihydro-15-keto-PGD2 (DK-PGD2), and latanoprost (free acid) were from Cayman Chemical. Platelet-activating factor (PAF) was from Sigma. Recombinant human IL-5 was produced at the Merck Frosst Centre for Therapeutic Research by using a baculovirus system and purified by means of fast performance liquid chromatography.10
Eosinophil purification
Circulating eosinophils were isolated from heparinized venous blood from healthy
Human eosinophils express both DP and CRTH2
RT-PCR was performed on total RNA from human eosinophils (>95% purity) to establish which PGD2-binding receptors are expressed by human eosinophils. The identity of the PCR products was confirmed by means of Southern blot detection with DP- and CRTH2-specific probes. CRTH2 and DP mRNA were detected in eosinophils from all 7 donors tested. Results from 3 donors are shown in Fig 1.
Discussion
PGD2 interacts preferentially with 2 GPCRs: DP and CRTH2. We have shown that in eosinophils, shape change, chemokinesis, and degranulation are induced by both PGD2 and DK-PGD2. In contrast, PGD2 and BW245C have antiapoptotic effects on cultured eosinophils. BW245C21 and DK-PGD28 are reported to be highly selective agonists for DP and CRTH2, respectively, and PGD2 has similar affinity for CRTH2 and DP.22 Taken together, these data strongly suggest that CRTH2 mediates shape change, chemokinesis,
Acknowledgements
We thank Betty Stamatiou for her eosinophil isolation protocol; Denis Normandin and Simon Wong for the evaluation of eosinophil purity; Dr Donald W. Nicholson and Mike Scheid for the generation of rIL-5; Dr Bruce Daugherty, Dr John Mudgett, and Marco Soares for help with the chemokinesis assay; Diane Ethier for technical assistance with the FACS; Grace Konrath for technical assistance; and Deborah Slipetz for statistical analysis of the data.
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Reprint requests: Francois G. Gervais, PhD, Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, PO Box 1005, Kirkland, Quebec, Canada, H9R 4P8.