Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Extracellular 14-3-3: A Novel Mediator of Inflammation Associated with Selective Activation of Intracellular Pathways.
Marotta1, Anthony, Gui1, Yuan, Ghahary2, Aziz, Kilani2, Ruhangiz, Maksymowych3, Walter P.
Rheumatoid arthritis (RA) results from the interaction and convergence of mediators that contribute to pathological processes. The 14-3-3 family members are ubiquitously expressed intracellular chaperones. We previously demonstrated that the h isoform is specifically present extracellularly in the synovial fluid and matched serum of patients with inflammatory arthritis and that its expression significantly correlated with levels of MMP-1 and MMP-3. Recent data also indicates that serum 14-3-3h is highly specific for RA. We aimed to examine the role of extracellular 14-3-3h in the pathogenesis of RA by investigating its effects on 1) the activation of RA-relevant signaling cascades, 2) induction of pro-inflammatory mediators and 3) the effects of selectively targeting 14-3-3h with monoclonal antibodies.
Cells of the monocytic lineage (THP-1) were stimulated with 12.5ng/ml of recombinant human 14-3-3h and activation of relevant signaling targets was assessed by immunoblot analysis using phosphospecific antibodies. Activation of HL-60, PCS-201-010, Jurkat and Daudi cells was assessed by evaluating the phosphorylation status of MAPK/ERK following 15 minutes of stimulation with 12.5ng/ml. mRNA levels of IL-1b, IL-6, IL-8, CCL2/MCP-1, CCL4/MIP-1b, MMP-1, MMP-9, TNFa, RANKL were assessed in THP-1 cells following 18h incubation with a dose range of 0.10 to 100ng/ml of recombinant human 14-3-3h reflecting in vivo concentrations. For antibody targeting studies, recombinant human 14-3-3h was co-incubated with antibody (0.2 to 20mg/ml) for 18h and levels of transcripts were assessed.
Stimulation assays showed that while monocytic, myeloid, and fibroblast cell lines were activated in response to 14-3-3h, T and B cell lines were not. Extracellular 14-3-3h, at concentrations found in RA patient serum (median 1.12ng/ml and range of 0.12 to 20ng/ml) activated key intracellular signalling cascades that regulate cell proliferation (MAPK/ERK), survival (AKT), inflammation (JAK-STAT), and tissue remodelling (SAPK/JNK). These cell stimulatory effects were specific yet distinct from other reported endokines/extracellular factors since neither the activation of p38MAPK nor NFkB was not observed with 14-3-3h stimulation. Furthermore, extracellular 14-3-3h at levels approximating median serum levels behaved as a potent inducer of IL-1b, IL-8, CCL2/MCP-1, CCL4/MIP-1b, MMP-1, MMP-9 and RANKL transcripts. Higher levels of 14-3-3h, though within the range found in vivo, were required for induction of IL-6 and TNFa. Targeting 14-3-3h with monoclonal antibody compounds attenuated these effects.
Extracellular 14-3-3h is a novel mediator that induces expression of several factors associated with the pathogenesis of RA. In contrast to other endokines that activate both p38MAPK and NFkB as well as up-regulate pro-inflammatory factors, 14-3-3h acts through alternate signalling pathways. Targeting 14-3-3h with monoclonal antibody compounds attenuates its inducing effects underscoring its novelty as a potential therapeutic target.
To cite this abstract, please use the following information:
Marotta, Anthony, Gui, Yuan, Ghahary, Aziz, Kilani, Ruhangiz, Maksymowych, Walter P.; Extracellular 14-3-3: A Novel Mediator of Inflammation Associated with Selective Activation of Intracellular Pathways. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :1836