Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Defective T Cell Tolerance in Rheumatoid Arthritis Due to Increased B-Raf and K-Ras Expression.
Deshpande2, Pratima, Singh1, Karnail, Yu2, Mingcan, Li2, Guangjin, Weyand3, Cornelia M., Goronzy3, Jorg J.
Autoantibodies to common auto- and neo-antigens such as IgG Fc and citrullinated peptides are immunological hallmarks of rheumatoid arthritis (RA). Identified disease risk genes suggest that signaling pathways abnormalities are involved in RA pathogenesis. We have examined the hypothesis that defects in T cell receptor (TCR) signaling lower the activation threshold of RA T cells predisposing for a failure in maintaining tolerance.
TCR-induced signaling was compared in 65 patients with seropositive RA and 54 healthy controls by phosphoepitope flow cytometry. The MAP Kinase Signaling Pathway PCR Array was used to screen for differentially expressed genes. SOS1, RasGRP1, DUSP5, DUSP6, K-Ras and B-Raf transcription was quantified by qPCR. Protein expression of B-Raf, C-Raf, K-Ras and N-Ras were compared by Western blotting and by flow cytometry. siRNA for K-Ras and B-Raf from Qiagen was used in silencing experiments. Co-localization of B-Raf or C-Raf with K-Ras or N-Ras in T cells from control and RA patients was examined by confocal microscopy. CD4 T cells from healthy HLA-DR4+ individuals were transfected with K-Ras or B-Raf cloned into the pIRES2-AcGFP1 vector. Cells were stimulated with 1 mM aa 6577 native or R70Cit vimentin peptides.
Summary of Results:
RA T cells responded to TCR stimulation with increased ERK phosphorylation compared to healthy controls of the same age (p<0.0001). Increased ERK responsiveness was a feature of all functional T cell subsets including naive T cells and did not correlate with disease activity. Increased expression of K-Ras and B-Raf in RA T cells, first identified in gene expression arrays of ERK pathway members, was confirmed by qPCR, Western blotting and flow cytometry (p<0.01). None of the RA-implicated cytokines TNF-a, IL-1, IL-6, IL-15 or IL-21 induced B-Raf or K-Ras gene expression in vitro. Partial silencing of K-Ras and B-Raf significantly lowered activation-induced p-ERK levels (p<0.01). In individual cells, B-Raf and K-Ras baseline levels correlated with activation-induced ERK phosphorylation (p<0.001) confirming that B-Raf and K-Ras are rate-limiting in T cells and concentration differences of these signaling molecules in RA are functionally important. In confocal studies, B-Raf/K-Ras clustering was significantly increased in RA T cells two minutes after TCR stimulation (p<0.001). Increased ERK activity was sustained by activation of a positive feedback loop involving p-ERK dependent RKIP phosphorylation and release of sequestered C-Raf. Overexpression of transfected B-Raf or K-Ras lowered the TCR threshold and enabled responses to citrullinated vimentin in healthy HLA-DR4+ individuals.
Restricting ERK activity, in part by Rap-1 mediated inactivation of C-Raf is an important mechanism to maintain anergy to self-antigens or in the absence of costimulation. Expression of B-Raf that can bypass this pathway is low in T cells from healthy individuals. Naive and memory T cells from RA patients have increased B-Raf and K-Ras expressions that enable T cell responses to low affinity and autoantigens. Our data suggest that B-Raf and K-Ras are promising targets to treat autoimmunity.
To cite this abstract, please use the following information:
Deshpande, Pratima, Singh, Karnail, Yu, Mingcan, Li, Guangjin, Weyand, Cornelia M., Goronzy, Jorg J.; Defective T Cell Tolerance in Rheumatoid Arthritis Due to Increased B-Raf and K-Ras Expression. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :2127