Background Even though it is well known that smokers have an increased risk to develop rheumatoid arthritis (RA) and other autoimmune diseases, nothing is known about the molecular mechanisms that lead to break of tolerance in smokers. The innate immune receptor NKG2D and its ligands MICA and MICB have previously been described to promote autoimmune reactions. Therefore, we analysed whether smoking leads to increased expression of NKG2D ligands and activates the NKG2D system.
Methods RA and osteoarthritis (OA) synovial fibroblasts (SFs) were cultured from synovial tissues from patients undergoing joint replacement surgery. Real-time PCR was used to measure expression levels of NKG2D ligands in synovial tissues and SFs stimulated with 5% cigarette smoke extract (CSE). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and co-cultured with RASFs and OASFs with and without CSE for 72h. Expression of surface NKG2D was measured by flow cytometry.
Results To see whether smokers have increased levels of NKG2D ligands, we measured the expression of MICA, MICB, RAET1E, RAET1G, and ULBP1-3 in synovial tissues of RA patients (5 smokers, 6 non-smokers). Expression levels of MICA and MICB were significantly higher in synovial tissues of smokers than of non-smokers, while expression levels of the other NKG2D ligands were unchanged. Accordingly, RASFs stimulated with CSE in vitro produced 2.0-fold more MICA (p=0.03, n=6) and 1.5-fold more MICB (p=0.02, n=6) compared to unstimulated cells. To test whether the increased production of MICA and MICB by SFs after CSE stimulation was relevant for the activation of the NKG2D pathway on PBMCs, we performed co-culture experiments and measured internalization of the receptor on PBMCs after ligand binding. In co-cultures of PBMCs and OASFs stimulated with CSE, NKG2D was internalized and significantly less surface NKG2D was measureable on PBMCs after 72h compared to co-cultures without CSE. In co-cultures with PBMCs and RASFs however, no change in surface NKG2D was seen with addition of CSE even though RASFs produced similar amounts of MICA and MICB after CSE stimulation than OASFs.
Conclusions Smoking induces the expression of the NKG2D ligands MICA and MICB in synovial tissues. Internalization and degradation of NKG2D after binding of MIC ligands is an important control mechanism to terminate the activation of NKG2D signaling pathways. Since MIC ligands secreted by RASFs fail to induce internalization of the receptor, continuous activation of NKG2D in joints of smokers might be a trigger for the development of RA in susceptible individuals.
Acknowledgements This work was supported by Masterswich, IAR, IMI-BTCure
Disclosure of Interest None Declared