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Acting first on synoviocytes or PBMC did not affect the inhibitory effect of the anti-pdpn

Acting first on synoviocytes or PBMC did not affect the inhibitory effect of the anti-pdpn. PBMC-synoviocyte co-culture compared to PBMC alone (values less than or equal to 0.05 were considered as significant. Results Interaction between RA synoviocytes and PBMC induces IL-6 and IL-1 production PBMC produce pro-inflammatory cytokines, such as IL-6 and IL-1, which are implicated in the Th17 differentiation [16C18]. Resting PBMC alone produced IL-6 at low levels and their activation by PHA had Kira8 (AMG-18) a modest effect (1.4??3.4?ng/ml vs. 13.4??11.8?ng/ml, Kira8 (AMG-18) Fig.?1a). IL-1 production was almost undetectable in control condition (7.2??16.1?pg/ml, Fig. ?Fig.1b),1b), and PHA activation highly increased its secretion (2630.1??2397.3?pg/ml, interleukin, peripheral blood mononuclear cells, phytohemagglutinin, rheumatoid arthritis To investigate the importance of cellCcell contact, a transwell system was used. The insert had a pore size of 0.4?m, which prevents direct cellCcell contact but allows the exchange of soluble factors. In this transwell system, IL-6 and IL-1 production was significantly decreased compared to control (89.1??58.6?ng/ml vs. 289.5??130.9?ng/ml, interleukin, peripheral blood mononuclear cells, phytohemagglutinin, rheumatoid arthritis Actual IL-17 secretion in supernatants was measured by ELISA. Without PHA, IL-17 production was undetectable in resting PBMC (Fig.?2b); but it was present at a very low level in co-culture of PBMC and synoviocytes (1.1??2.2?pg/ml). TCR activation by PHA did not increase significantly IL-17 secretion in PBMC Kira8 (AMG-18) alone (Fig.?2b). In contrast, there was a significant increased production of IL-17 in co-culture with activated PBMC (1.1??2.2?pg/ml vs. 185.5??220.3?pg/ml, adipose-derived stem cells, human umbilical vein endothelial cells. interleukin, peripheral blood mononuclear cells, phytohemagglutinin, rheumatoid arthritis To confirm that pro-inflammatory cytokine production resulting from cell interactions may occur inside the inflamed synovium, co-culture experiments with synoviocytes and PBMC from the same RA patient were tested. As observed in Fig.?3b, co-cultures with autologous cells gave similar results as co-cultures with RA synoviocytes and healthy PBMC. This indicated the absence of contribution of alloreactivity in the effect. Indeed, cell interactions were sufficient to induce IL-6 (Fig.?3b). IL-17 was markedly more produced in co-culture with autologous activated PBMC (Fig.?3b). In parallel, co-cultures between PBMC from patient 1 and synoviocytes from patient 2 and the other way around were tested. Results were similar in both systems (Fig.?3b) indicating the critical role of cell interactions in the pro-inflammatory cytokine production. Monocytes do not contribute to the high IL-17 production Considering the role of IL-6 and IL-1 in the Th17 pathway and the role of cell interactions in maintaining inflammation, the potential contribution of monocytes in this loop was investigated. To study their involvement in our co-culture system, monocytes were removed by adherence. As IL-1 is mainly produced by monocytes in PBMC, the reduction of IL-1 production can be considered as a good marker for the removal of monocytes. As observed in Fig.?4a, Kira8 (AMG-18) the production of IL-1 was indeed significantly inhibited in all conditions without monocytes (interleukin, peripheral blood mononuclear cells, phytohemagglutinin, rheumatoid arthritis To confirm the crucial role of synoviocytes and Th17 cells in the high IL-17 secretion, co-cultures between synoviocytes and Th17 clones (ratio 1:1) were performed. As observed in Fig.?4d, there was no IL-1 production compared to co-cultures with PBMC. This result was expected as the major source of IL-1 was not present. In co-cultures with Th17 clones, IL-6 secretion was induced in control condition as with PBMC, even the level of production was lower than with PBMC (90.2??10.0?pg/ml vs. 712.1??12.5?pg/ml), and with Th17 clones, PHA activation increased IL-6 secretion (635.6??12.5?pg/ml vs. 90.2??10.0?pg/ml, Fig.?4e). As with PBMC, the detection of IL-17 production was possible only with PHA activation (701.7??39.1?pg/ml vs. 15.2??0.2?pg/ml, Fig.?4f) and the interaction with synoviocytes largely increased this secretion (7013.0??458.5?pg/ml vs. 701.7??39.1?pg/ml, Fig.?4f). These results confirmed the crucial role of TCR activation and of cellCcell contact in the high IL-17 production and make synoviocytes and Th17 cells the two major cell types involved in this elevated secretion. Podoplanin plays a major role in high IL-17 secretion during co-culture between activated PBMC and RA synoviocytes The role of direct physical cell interactions in the high IL-17 production Kira8 (AMG-18) is critical. As podoplanin (pdpn) can be expressed by different cell types, including synoviocytes, its potential role was studied with a blocking anti-pdpn antibody. A doseCresponse curve was performed with different concentrations of anti-pdpn antibody (Ab), 0, 1, 5, 10 and 20?g/ml, to determine the optimum concentration of anti-pdpn Ab. The concentration of 5?g/ml of antibody pre-incubated for 4?h gave the higher inhibition of cytokine production (data not shown). In the LASS2 antibody co-culture of synoviocytes and activated PBMC, the presence of anti-pdpn Ab inhibited significantly IL-17 secretion by 64.9??24.0?%. (and compared to control (control?=?1) (b) is represented as well as the percentage of cytokine inhibition compared to control.

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