Background and Objectives Adipose-derived mesenchymal stem cells (ASCs) are promising target in autoimmune diseases therapy. Adipocytokines are known to influence immune system and their role in rheumatoid arthritis (RA) is intensively studied. Objectives of our work were:
to investigate whether leptin, low (LMW) and high (HMW) molecular weight adiponectin isoforms and TNF affect IL-6, IL-8, VEGF and TGFβ secretion by RA-ASCs.
to determine if conditioned media from RA-ASCs treated with above mentioned adipocytokines, influence function of rheumatoid FLS: IL-6 and MMP-3 secretion, proliferation and apoptosis.
Materials and Methods Articular adipose tissue and synovial membrane were obtained from 18 RA patients during total knee joint replacement surgery. ASCs were isolated and cultured with/without human recombinant leptin, TNF, adiponectin (LMW, HMW) and IFNγ. After 24 h, secretion of cytokines was measured by ELISA. Conditioned media from ASCs cultures were used to stimulate FLS. After stimulation, FLS proliferation and apoptosis was determined (by incorporation of BrdU and flow cytometry, respectively). Cytokine concentration in FLS culture supernatants was determined by ELISA. The Wilcoxon signed-rank test was used for statistical analysis.
Results HMW adiponectin enhanced IL-6, IL-8, VEGF (p < 0.001) and TGFβ (p < 0.01) production. LMW adiponectin increased secretion of IL-6 and VEGF (p < 0.001), but its influence was much weaker than HMW's. TNF was the most potent in stimulating IL-6 and IL-8 production by ASCs (p < 0.001). The influence of adipocytokine treated-ASC-conditioned media on FLS was assesed by comparison with two controls: untreated ASC-conditioned medium and control FLS medium. ASC-conditioned medium treated with HMW caused down-regulation of IL-6 secretion by FLS comparing to both controls (p < 0.05). TNF-treated ASC-conditioned medium induced significant increase in MMP-3 production by FLS comparing to both controls (p < 0.05). Proliferation of FLS was up-regulated after untreated ASC-conditioned medium (p < 0.01) and this was partly reversed by HMW and TNF-treated ASC-conditioned media (p < 0.05). HMW-treated ASC-conditioned medium increased percentage of FLS in late apoptosis (p < 0.05).
Conclusions HMW and TNF seem to be the most potent in altering RA-ASCs properties. Leptin had no effect and LMW exerted only slight effect. HMW was able to up-regulate factors thought to mediate ASCs immunosupression (TGFβ, IL-6). In addition, its effect on FLS shows that this adipokine may exert positive effect on immunosupressive ASCs function. However, because HMW up-regulated also proangiogenic cytokines (IL-8, VEGF), it is difficult to determine its real impact on RA-ASCs. TNF role in modulating RA-ASCs also need to be elucidated.
Funding Grant No. N/NZ5/00932, National Science Center, Poland.