P090 Pro-inflammatory cytokines and cell interactions between PBMC and synoviocytes induce retraction and formation of pseudopodia

Abstract

Career situation of first and presenting authorStudent for a master or a PhD.IntroductionRheumatoid synovitis is infiltrated by immune cells, which interact with synoviocytes inducing abnormal proliferation and massive production of pro-inflammatory cytokines.ObjectivesThe aim was to evaluate the effect of inflammatory environment and cell interactions on morphological parameters of synoviocytes alone or in co-culture with peripheral blood mononuclear cells (PBMC).MethodsSynoviocytes from different donors: healthy (HE) or rheumatoid arthritis (RA) were exposed or not to inflammatory conditions (IL-17 alone, TNF alone and their combination) during 48 hours and observed with different microscopes (optical, digital holographic and holographic-tomographic). Quantification of morphological parameters included cell confluence, area, motility speed and number of pseudopodia/cell. Co-cultures between normal PBMC and synoviocytes with or without phytohemagglutinin (PHA) or cytokines (IL-17/TNF) were used to mimic the in vivo situation.ResultsInflammatory cytokines induced a change in synoviocyte morphology, inducing a retracted cell with a higher number of pseudopodia. Several parameters decreased in inflammatory conditions: cell confluence (Ctrl:31.7%±2.5%, IL-17:21.8%±2.0%, TNF:19.2%±1.5%, IL-17/TNF:19.8%±1.6%, p<0.01), area (Ctrl:4491±254 µm², IL-17:3537±265 µm², TNF:2862±217 µm², IL-17/TNF:2583±211 µm², p<0.01) and motility speed (Ctrl:446±7 µm/h, IL-17:177±2 µm/h, TNF:161±2 µm/h, IL-17/TNF:159±1 µm/h, p<0.01). The cell membrane exhibited a much larger number of pseudopodia in inflammatory conditions (ctrl: only 18% of cells had more than 4 pseudopodia vs. IL-17/TNF: 82%, p<0.01). The same impact on cell morphology was observed in co-culture of synoviocytes and PBMC, affecting both cell types: synoviocytes were retracted (HE: Ctrl:5092±274 µm², cocult:3037±168 µm², cocult +PHA:3644±184 µm², cocult +IL-17/TNF: 2949±154 µm², p<0.01) and inversely PBMC proliferated in inflammatory and PHA conditions (HE: Ctrl:47.9±1.4 µm², cocult:80.6±3.2 µm², cocult +PHA:94.2±2.9 µm², cocult +IL-17/TNF:114.6±3.4 µm², p<0.01), indicating that cell activation induced a morphological change of cells. With RA but not normal synoviocytes, co-culture was not sufficient to activate both PBMC and synoviocytes. The morphological effect came only from the inflammatory environment and not from cell interactions as if it did not exist and did not occur (RA: Ctrl:4491±254 µm², cocult:4573±275 µm² (ns), cocult +PHA:3220±184 µm², cocult +IL-17/TNF:2313±122 µm², p<0.01).ConclusionsThe inflammatory environment or cell interactions induced massive changes in synoviocytes, with cell retraction and increase of pseudopodia number, leading to better interactions with other cells. Except in the case of RA, the inflammatory environment was absolutely required for such changes.Disclosure of InterestNone declared.