Thesis performed at INSERM U392, Louis Pasteur University , Strasbourg
Rheumatoid arthritis (RA) is the most common inflammatory arthritis (prevalence = 0.4% of the European population and about 150 000 to 250 000 patients in France). RA is characterized by chronic inflammation of the joints, more particularly of the synovium, and by joint destruction. There is growing evidence that both genetic and environmental factors account for RA-pathogenesis by activating innate immune responses (Figure 1, Firestein et Zvaifler, Arthritis Rheum 2002, Firestein, Nature 2003).
During my stay at INSERM U392, we were interested in studying the role of the innate immune responses in the initiation and perpetuation of articular inflammation during RA. Numerous bacterial components or pathogen-associated molecular patterns (PAMPs) have been detected in the joints. They might contribute to inflammation of the synovium by interacting with different pattern recognition receptors (PRRs). The aim of our work was to determine if protein I/II, a PAMP from oral streptococci, might contribute to the aggressive behaviour of fibroblast-like synoviocytes (FLSs), resident cells of the synovium that play a key role in RA pathogenesis.
Figure 1. Multiple pathogenic mechanisms in RA (adapted from Firestein and Zvaifler 2002, Firestein 2003)
Activation of innate and adaptive immunity may occur in parallel (bidirectional arrows).
We showed that interaction of protein I/II with FLSs leads to the synthesis and release of interleukin (IL)-6 and IL-8, two cytokines involved in cellular activation and recruitment. This interaction does not lead to the release of TNF- a and IL-1, which have been described to play a key role in RA inflammation. We also demonstrated that protein I/II induced IL-18 mRNA in FLSs but gene expression was not associated with the synthesis of the corresponding protein in these cells. These data were confirmed by a global gene expression analysis performed by cDNA array. In addition, protein I/II is able to induce the expression of several genes that might contribute to the aggressive behaviour of FLSs, such as matrix metalloprotease (MMP) 3, a key enzyme for cartilage destruction (Figure 2). Taken together, these results show that interaction of FLSs with PAMPs might contribute to both articular inflammation and destruction.
Figure 2. Interaction of protein I/II with FLSs
In blue: signalling pathways triggered by protein I/II and leading to the synthesis of proinflammatory cytokines.
We also studied signalling pathways triggered by the interaction of protein I/II with FLSs. Our results demonstrate that integrin a 5 b 1 is a receptor for protein I/II on FLSs. Moreover, focal adhesion kinase (FAK) plays a key role in signalling pathways leading to the release of IL-6 and IL-8, independently of its autophosphorylation site. This pathway also involves mitogen-activated protein kinases (MAPKs) ERK1/2 and JNKs, as well as binding activity of AP-1 and nuclear translocation of NF- k B. Thus, FAK, which was known to play an important role in cellular adhesion, differentiation and apoptosis, might also be involved in inflammation. Additional experiments showed that MyD88, an adaptor protein of the IL-1/Toll-like receptor (TLR) pathways is also necessary for protein I/II-induced cytokine release whereas TLR2, TLR4 and TLR6 are not involved (Figure 2). How FAK and MyD88 contribute to these pathways remains to be determined.
In this study, we used FLSs isolated from RA-patients as well as from patients suffering from osteroarthritis (OA), a non-inflammatory joint disease, in order to determine if FLSs responses to protein I/II are dependent on their background. The observed differences in cytokine release were rather quantitative then qualitative. However, by analyzing the transcriptional profile of FLSs in response to protein I/II, we showed that gene expression between RA and OA FLSs were different and that only RA FLSs were able to secrete MMP-3 in response to protein I/II. These differences are not due to differences in the signalling pathways we studied.
Taken together, our results show that PAMPs-PRRs interactions might contribute to FLSs activation. Moreover, these interactions might represent a key event in both initiation and perpetuation of joint inflammation in individuals with the appropriate genetic background for RA. This hypothesis does not presuppose a specific antigen for RA but rather a variety of antigens might trigger innate immune responses, lead to the recruitment and activation of T lymphocytes and loss of self-tolerance.
Firestein, G. S. and Zvaifler, N. J.. How important are T cells in chronic rheumatoid synovitis?: II. T cell-independent mechanisms from beginning to end. Arthritis Rheum 2002; 46: 298-308.
Firestein, G. S. Evolving concepts of rheumatoid arthritis. Nature 2003; 423: 356-361