Background Radiological observations of joint destruction in RA are preceded by inflammatory process observed in bone-marrow (BM) together with detection of autoantibodies in peripheral blood. Previous studies have supported the concept that bone marrow can actively participate in the pathogenesis of RA by TLR triggered B cell activity (1) and the presence of activated T cell subpopulations (2). However, the suppressive properties of bone-marrow-resident cells remain poorly understood. In the present work investigating suppressive properties of natural CD4+Foxp3+ regulatory T cells (Tregs), we show increased production of main pro-inflammatory cytokine TNF by otherwise suppressive Tregs isolated from bone marrow of RA patients.
Objectives To compare the functional activity and cytokines profile of Tregs isolated from RA and osteoarthritis (OA) bone marrow samples.
Methods Bone marrow samples were obtained as a standard procedure during hip bone replacement surgery from RA and OA patients. CD4+CD25+++ Tregs and CD4+CD25- responder T cells (Tresp) were isolated from bone marrow mononuclear cells (BMMC) by cells sorter (FACS Aria BD) for Tregs functional activity measurement. As a control, Treg and Tresp populations were isolated from peripheral blood of healthy volunteers. Isolated cells were cultured alone or together in 1:1 ratio and were additionally stimulated in some samples by anti-CD3/CD28 antibodies. Pro-inflammatory cytokines (TNF and IFN-gamma) and anti-inflammatory cytokines (IL-10, TGF-beta, IL-35) were estimated by specific ELISAs in the supernatants removed before addition of [3H] thymidine to proliferation assays. Data are shown as mean ± SEM. The study was approved by local Ethical Committee.
Results Concentration of TNF produced by Tregs isolated from RA BM was comparable with TNF produced by responder T cells (14.2±2.7 pg/ml in Tregs vs 12.7±1.8 pg/ml in Tresp) and higher then TNF produced by Tregs isolated from OA BM (8,5±3.1 pg/ml) or healthy volunteers blood (7.7±1.9 pg/ml). However, despite high TNF production, Tregs from RA BM remain functionally active and suppress proliferation and production of TNF and IFN-gamma in coculture with Tresp (63.0±6.0%, p=0.012; 34.4±10.4%; p=0.03; 33.7±21.1%; p=0.046 respectively). Low production of IFN-gamma by Tregs was further lowered by additional TCR stimulation in all investigated groups of patients. Production of IL-10 by Treg or Tresp was negligible under investigated conditions in all investigated groups of patients (below the first standard of 4.4 pg/ml in ELISA). Similarly, no IL-35 was detected in any of investigated probes. However, we observed significant amounts of TGF-beta produced by Treg and Tresp populations. Treg from RA BM produced more TGF-beta than Tresp (116.5±32.6 pg/ml vs 77.6±49.9 pg/ml).
Conclusions Natural Tregs from bone marrow of RA patients despite TNF production remain suppressive. Thus, CD4+Foxp3+ Treg present in RA BM share suppressive capacities with pro-inflammatory feature, that may contribute to RA pathogenesis.
W. Rudnicka et al.; Eur J Immunol 2009; 1211-20
E. Kuca-Warnawin et al.; Ann Rheum Dis 2011; 227-33
Disclosure of Interest None declared