Supernatants were harvested, centrifuged to pellet cells and insoluble debris and assessed for cytokine levels by ELISA or cytokine array. For differentiation experiments, monocytes grown in OptiMEM were treated with dibutyryl-cAMP (db-cAMP, 100 μm), macrophage colony-stimulating factor (M-CSF; 5 ng/ml) or granulocyte–macrophage colony-stimulating factor (GM-CSF; 2 ng/ml) for 4 days before analysis by flow cytometry or assay of cytokine release. For flow cytometric analysis, 100-μl aliquots of cells (5 × 106/ml) were stained with the mAb for individual integrins for 30–60 min on ice before washing in PBS; if required, a BEZ235 order fluorophore-conjugated secondary

reagent was added and a further 30–60 minutes of incubation was conducted before washing and analysis. Appropriate isotype controls were included. Data were collected from a minimum of 104 cells using a FACScan instrument (BD Biosciences) and analysed using CellQuest software (BD Biosciences). Human monocytes release cytokines following stimulation by a range of stimuli. Other groups have demonstrated that exposure of human PBMC to sCD23 promoted TNF-α release, via ligation of the αVβ3 integrin,18 and other cytokines via ligation of β2 integrins.17,35Figure 1(a) illustrates that normal PBMC released TNF-α following stimulation with

lipopolysaccharide (LPS) or sCD23 but not when treated with the extracellular matrix proteins vitronectin (Vn) or fibronectin (Fn), which are additional ligands for αVβ3 and αVβ5. However, these

Serine Protease inhibitor cells expressed high levels of three of the four integrins that are known to bind sCD23; namely αVβ3, αVβ5 and αXβ2 (Fig. 1b). Therefore, it is not clear which of the four possible sCD23-binding integrins would be responsible for acute regulation of release of one or more discrete cytokines or groups of cytokines (Fig. 1c), or whether these integrins generate synergistic or mutually inhibitory signals. To test the broad hypothesis that individual sCD23-binding integrins differentially regulate acute cytokine release Rho from monocytic cells, an antibody array approach was employed to determine the qualitative patterns of cytokine release from THP-1 cells following stimulation with antibodies directed against individual sCD23-binding integrin isoforms (Fig. 1c). The general principle of the assay is shown in Supplementary material, Fig. S1A and the patterns of pairs of anti-cytokine antibodies printed on the array are shown in Supplementary material, Fig. S1B. The pattern of release of cytokines driven by sCD23 in monocytic cells is complex and may reflect the fact that up to four distinct sCD23 binding integrins can be ligated on the same cell, with each potentially giving rise to a distinct effect on cytokine synthesis and release.