Autor:innen:
M. Arnold (Bad Nauheim, DE)
J. Fuchs (Bad Nauheim, DE)
I. Aykara (Bad Nauheim, DE)
K. Frommer (Bad Nauheim, DE)
T. Laibe (Bad Nauheim, DE)
S. Rehart (Frankfurt, DE)
U. Müller-Ladner (Bad Nauheim, DE)
E. Neumann (Bad Nauheim, DE)
Background:
In rheumatoid arthritis (RA), osteoclasts are one of the most important mediators of bone erosion. In addition, RA synovial fibroblasts (RASF) have a major influence on joint erosion in RA. They affect osteoclast differentiation e.g. by increased RANKL production or secretion of other pro-osteoclastogenic factors such as IL-6. In addition to the pro-osteoclastogenic effect of soluble factors such as RANKL and IL-1, visfatin inhibits osteoclast differentiation. Furthermore, visfatin increases the secretion of pro-inflammatory factors by RASF, such as IL-6 or matrix degrading enzymes. In this study, the effect of RASF with/without activation by visfatin and IL-1 on osteoclastogenesis was evaluated.
Methods:
Blood from healthy donors and RA patients was used for PBMC isolation. RANKL, TGF-β and hM-CSF were added to induce osteoclast differentiation. RASF-conditioned media (CM) were prepared from confluent RASF cultured for 48h. Differentiating PBMCs in monoculture were compared to PBMC cultured with CM from RASF (CM: 10%, 20%, 30%) as well as in direct co-culture with RASF with/without stimulation with IL-1 (0.05ng/ml), visfatin (25ng/ml). After two weeks in culture, cells were stained using TRAP staining. 3-5 images per well were used for quantification dependent on the variability of the wells. IL-6 was measured by ELISA in supernatants collected at day 14.
Results:
IL-6 production increased by IL-1 (e.g. co-culture: 2,8-fold) and visfatin (CM-visfatin: 10%=4,3-fold, 20%=5,4-fold, 30%=4,2-fold; co-culture: 9,5-fold) compared to unstimulated control in all settings. In addition, IL-6 was increased with the addition of CM compared to unstimulated controls (healthy donors CM 30%: unstimulated p=0.0342, IL-1 p=0.0133, visfatin p=0,0133; RA: unstimulated p=0.0133, IL-1 p=0.0342, visfatin p=0.0133, n=3 each). Of note, baseline IL-6 concentrations were higher in PBMC from RA patients compared to healthy donors. Co-culture showed an additional increase in IL-6 levels in all settings (e.g. monoculture: IL-1 4.71±5.75pg/ml, visfatin 141.09±182.79pg/ml; co-culture: IL-1 7241±10398pg/ml, visfatin 24535±16994pg/ml;). During osteoclast differentiation, addition of CM showed similar osteoclastogenesis with similar proportion of osteoclasts with 2 and 3-5 vs. higher numbers of nuclei per cell compared to control. In coculture with RASF osteoclasts showed a stronger TRAP signal compared to monoculture especially for unstimulated and IL-1 stimulated co-cultures.
Conclusions:
Both, in monoculture with CM and in coculture, IL-6 levels were increased compared to control, whereas in RA patients the IL-6 levels were higher compared to healthy donors. The CM containing secreted factors of RASF did not have a prominent influence on osteoclastogenesis. However, the presence of RASF increased the TRAP signal showing an increased activity of differentiated osteoclasts especially in unstimulated and IL-1 stimulated co-cultures but not with addition of visfatin.