The long-term objective of this research is to develop highly directed and long-lasting therapy for rheumatoid arthritis (RA).
The specific aims are to, for the first time: 1) Study physical associations between heat shock protein (HSP70) and citrullinated-fibrinogen 2) Demonstrate mechanism of delivery of cit-fibrinogen into immature dendritic cells (DC). The recognition that cit-proteins (especially cit-fibrinogen) are key auto-antigens (Ags) initiating and perpetuating immunopathology in RA may represent a huge step toward achieving more effective therapy. To date, however, many of the events related to the handling of cit-Ags are unknown, including mechanisms of cit-autoAg uptake/processing by antigen presenting cells such as DC. The hypothesis is that in RA, citrullinated Ags such as cit-fibrinogen are transported via HSP into immature DC to activate autoimmune lymphocyte responses. In RA, HSP are highly abundant and likely to facilitate the transport of auto-Ags into immature DC. Because citrullination of proteins results in unfolding and partial denaturing, citproteins are prime candidates for association with HSP, which partner with proteins in non-native states. Emphasis will be placed on studying inducible HSP70 and cit-fibrin, which are specifically elevated in RA synovial fluid (SF) vs non-RA SF and considered key self-cit-Ags in RA, resp.
In Aim 1, HSP70-cit-fibrinogen interactions will be studied using surface plasmon resonance (SPR) and co-immunoprecipitation (IP) techniques. For SPR, purified proteins will be used. HSP70-cit-fibinogen complexes present in RA SF, RA serum, non-RA SF and normal serum will be revealed by IP.
In Aim 2, immature DC will be primed in vitro to undergo an inflammatory/heat stress response under precise conditions present in the RA joint. After the addition of cit-fibrinogen, uptake/intracellular trafficking of cit-fibinogen will be assessed by IP and confocal microscopy. Due to the increased affinity of cit-Ags to HLA-DR4, studies of cit-fibrinogen-HLA-DR interactions and cit-fibrinogen uptake in DC prepared from RA patients expressing the common epitope will be included. These studies will employ synthetic cit-fibrinogen 10mer peptides (containing cit-epitopes) and native fib peptides and purified HLA-DR4 and HLA-DR1 (non RA linked). Understanding how key self-antigens in RA are handled by the immune system at the earliest stages of the autoimmune process may reveal a universal disease mechanism that can be specifically interrupted, before the onset of severe disease activity.
| Martin, Carla A; Kurkowski, Danielle L; Valentino, Alisa M et al. (2009) Increased intracellular, cell surface, and secreted inducible heat shock protein 70 responses are triggered during the monocyte to dendritic cell (DC) transition by cytokines independently of heat stress and infection and may positively regulate DC growth J Immunol 183:388-99 |
