harvested and assayed for cytokines by ELISA analysis. The cultured cells were analyzed for either FoxP3 or CTLA4 expression. The mRNA was also isolated from the cultured cells using the Oligotex mRNA isolation kit according to the manufacturer’s instructions for quantitative PCR analysis. Additionally, in some experiments, the cultures were pulsed with 5bromo-2-deoxyuridine for the final 12 h and assayed for BrdU incorporation according to the manufacturer’s instructions. Flow cytometric analysis FACs-sorted FITChi+ and FITC CD11c+ DC were prepared and stained for flow cytometry immediately after sorting as previously described. Briefly, the cells were incubated for 10 min with Fc block to inhibit non-specific binding. The cells were subsequently incubated on ice and stained with anti-mouse OX40L, anti-mouse MHC class II, anti-mouse GITRL, anti-mouse CD80, anti-mouse CD86, antimouse B7RP-1, anti-mouse PD-L1, anti-mouse PD-L2, anti-mouse RANK and the appropriate isotype-matched controls. The cells were washed in cold FACS buffer and fixed using BD Cytofix fixation buffer until analysis using a LSR II analyzer. 6 CRTH2 blocks OVA-induced skin inflammation Results Inhibition of CRTH2 ameliorates repeated epicutaneous sensitization-induced inflammation and antigen-specific Ig production It has previously been shown that repeated epicutaneous sensitization with the protein antigen chicken egg ovalbumin results in the local expression of Th2 cytokines, infiltration of T lymphocytes and eosinophils, as well as OVAspecific antibodies in the serum. As numerous cell types involved in this inflammatory process express CRTH2, we investigated the role of the CRTH2 receptor by using a specific small molecule antagonist. BALB/ c mice were epicutaneously sensitized by securing a patch of gauze saturated with either a 1% OVA/PBS solution or a PBS to a shaved section of back skin, as previously described. Separate cohorts of OVA-patched mice received either Compound A or vehicle delivered p.o. or intraperitoneally administered dexamethasone, during the second and third sensitizations. Histological examination reveals acute inflammation and mononuclear cell infiltration in sections from OVA-sensitized skin but not in the PBS control mice. Strikingly, the OVA/Compound A and OVA/ dexamethasone-treated groups showed a strongly reduced inflammatory infiltrate in the dermis, as well as a reduction in the epidermal INK-128 thickening compared with the OVA/vehicle group. Analysis of gene expression patterns from the patched skin following the third sensitization period showed an upregulation of broad spectrum of genes involved in inflammation. For instance, pro-inflammatory cytokines and chemokines such as tumor necrosis factor -a, IFN-c, IL-1b, IL-24, MIP-1b, eotaxin, TARC, KC and MIP-2 are up-regulated in the OVA-treated animals and reduced by Compound A treatment. Similarly, CD antigen markers from many of these cell types that respond and/or produce these cytokines and chemokines are PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19825521 reduced, including the CD3 antigen cluster, the DC and Langerhans cell markers CD207 and CD209c and CD14, 
suggesting a reduced number of these pro-inflammatory cells in the patched skin sections. Importantly, message for CRTH2 and the leukotriene receptors LTB4 receptor 1 and 2 is reduced upon Compound A treatment. The mRNA levels of genes linked to inflammatory skin diseases and the atopic phenotype in humans are up-regulated by epicutaneous OVA sensitization and decreased by Compoun
