A6.1Interaction between activated lymphocytes and synoviocytes promotes IL-17 secretion, partly mediated by podoplanin

Background and objectivesRheumatoid arthritis (RA) is characterised by an immune cell infiltrate, notably of Th17 cells and a migration of blood derived mononuclear cells that interact with synoviocytes. Here, we study the interactions between synoviocytes and peripheral blood mononuclear cells (PBMC) with focus on the effect of cell contact on the Th17 pathway. The goal is to differentiate intracellular expression from secretion and to identify a mechanism which leads to high IL-17 secretion. In this way, we focus our interest on podoplanin, a transmembrane protein which is increased in RA synovium and known to modulate IL-8 secretion during synoviocyte-platelet interaction.Materials and methodsA co-culture system with RA synoviocytes and PBMC was developed. Synoviocytes were cultured overnight in 96-well plates and healthy PBMC were seeded at a 1:5ratio for 48 h, in the presence or not of TCR activation with PHA. Transwell system was used to study cell-cell contact. An antibody against podoplanin was pre-incubated 4h with PBMC before co-culture. Supernatants were collected at 48 h and IL-6, IL-1 beta and IL-17 production measured by ELISA. Extracellular (CD3, CD4) and intracellular (IL-17) staining of PBMC was analysed by flow cytometry at 48 h.ResultsInteraction between synoviocytes and PBMC was sufficient to induce IL-6 and IL-1 beta production. Flow cytometry analysis showed no difference in the percentage of Th17 cells in activated-PBMC alone or cultured with synoviocytes (3.1 plus or minus 1.7% vs. 3.9 plus or minus 2.7%, respectively, p = 0.4), indicating that Th17 differentiation requires only T cell activation but not cell-cell contact. Conversely IL-17 production was highly increased only in co-cultures with activated-PBMC (346.8 plus or minus 141.6 pg/ml) vs. activated-PBMC alone (37.5 plus or minus 37.1 pg/ml, p = 0.0001). Transwell experiments confirm that contact between synoviocytes and PBMC was critical for IL-17 secretion, as a minor production was observed in transwell system (284.7 plus or minus 106.2 pg/ml in co-culture vs. 5.3 plus or minus 6.1pg/ml in transwell system). Inhibition of podoplanin decreased significantly IL-17 secretion (45.1 plus or minus 19.3%, p = 0.03).ConclusionsThe interaction between resting PBMC and synoviocytes alone induces IL-6 and IL-1 beta production. TCR activation is required for Th17 differentiation but both PBMC activation and cell interaction with synoviocytes are needed to have high IL-17 secretion, p