Supernatants collected 1 hr after activation with IgE/anti-IgE were used to assess the effect of ramatroban. 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 Protirelin 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. and setting we found that PGD2 is usually a dominant mast cell-derived mediator promoting chemotaxis of Th2 lymphocytes and that this response is usually mediated by CRTH2. Materials and methods ReagentsPGD2 and PGD2-MOX enzyme immunoassay kits were purchased from Cayman Chemical (Ann Arbor, MI). Ramatroban (BAY u3405) was synthesized by Evotec OAI (Abingdon, Oxon, UK) and is also available commercially from Cayman Chemical. Mono-poly-resolving medium was purchased from Dainippon Pharmaceuticals (Osaka, Japan). Magnetic antibody cell sorting CD4+ isolation and anti-CRTH2 microbead kits were purchased from Miltenyi Biotec (Bisley, Surrey, UK). Human recombinant stem cell factor and human recombinant IL-6 were purchased from R & D Systems (Abingdon, Oxon, UK). CD34+ progenitors from human cord blood, Iscoves altered Dulbeccos medium and X-VIVO 15 medium were purchased from Cambrex BioScience (Wokingham, Berkshire, UK). Antibodies against human tryptase and chymase were purchased CSF2RA from Chemicon International (Chandlers Ford, Hampshire, UK). Human myeloma IgE was purchased from Biodesign International (Saco, ME). Human recombinant IL-2, human recombinant IL-4, goat anti-human IgE and diclofenac were purchased from Sigma (Poole, Dorset, UK). FicollCHypaque was purchased from Amersham Biosciences (Amersham, Buckinghamshire, UK). The 96-well ChemoTx plates were purchased from Neuroprobe (Gaithersburg, Protirelin MD). Human mast cell culture and activationHuman mast cells were cultured from CD34+ progenitor cells as described by Nakahata and Toru.16 CD34+ progenitor cells from human cord blood were cultured at a density of 1 1 105 cells/ml with Iscoves modified Dulbeccos medium containing 10% human serum, 0.55 m 2-mercaptoethanol, penicillin/streptomycin, human recombinant stem cell factor (100 ng/ml) and human recombinant IL-6 (50 ng/ml) in 5% CO2 at 37 C for 8C10 weeks. Half of the culture medium was replaced twice a week with fresh medium made up of the same concentration of cytokines. The expression of tryptase and chymase of the cells was tested with immunostaining using the method described by Craig for 2 min to collect any cells on the underside of the filters. The upper membrane was carefully removed and cell migration was quantified by fluorescence-activated cell sorting. Background cell migration was determined by measuring the response to media alone. Data analysesAll data involving multiple comparisons were analysed using one-way anova followed by the NewmanCKeuls test. Probability values of 0.05 were considered statistically significant. Results Release of CRTH2+ CD4+ Th2 cell stimulatory activity from IgE-activated human mast cells At all the time-points tested supernatants collected from IgE/anti-IgE-treated mast cells contained activity that stimulated significantly greater chemotactic responses of CRTH2+ CD4+ Th2 cells than supernatants from Protirelin unactivated mast cells (Fig. 1). The chemotactic activity was detectable as early as 20 min after treatment, increased with time, reached a maximum response at about 1C2 hr and was sustained for at least 4 hr. Supernatants from unactivated mast cells had a similar effect to the X-VIVO 15 medium unfavorable control, which caused a background migration of 16.64 0.01% maximum response (= 6). Open in a separate window Physique 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.
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