Abstract
BackgroundConcomitant use of methotrexate (MTX) improves the clinical efficacy of anti-TNF agents in the treatment of rheumatoid arthritis (RA). We aimed to clarify the cytotoxic effect of MTX on transmembrane TNF (tmTNF)-expressing cells treated with anti-TNF agents.MethodsJurkat T cells stably expressing tmTNF were used for the following experiments. Cytotoxicity induced by an anti-TNF agent (infliximab, adalimumab, or certolizumab pegol) with concomitant MTX were compared with that by MTX alone or by an anti-TNF agent alone using flow cytometry. Apoptosis-induction mediated by reverse signal through tmTNF, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) were evaluated. Folic acid and Y-27632, a Rho kinase inhibitor, were used as inhibitors to study intracellular signaling pathway in apoptosis induced by MTX and anti-TNF agents.ResultsApoptosis of tmTNF-expressing cells was significantly increased by the concomitant administration of MTX and an anti-TNF agent, compared with MTX alone or an anti-TNF agent alone. The apoptosis induction by concomitant MTX was most pronounced in infliximab-treatment. Reverse signal transduction, but not CDC or ADCC/ADCP, was responsible for the coordinate effect of MTX and an anti-TNF agent on tmTNF-expressing cells. Folic acid inhibited MTX-mediated apoptosis, while Y-27632 suppressed JNK activation and infliximab-induced apoptosis via revere signal through tmTNF.ConclusionThe apoptotic effect was enhanced by combination of MTX and an anti-TNF agent in tmTNF-expressing cells. The intracellular pathways induced by MTX and anti-TNF agents seem to be independent. These findings might explain at least in part improved the clinical response upon co-therapy of MTX and an anti-TNF agent in RA.
Highlights
Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that exerts pleiotropic effects on various type of cells and plays a major role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis (RA) [1,2,3]
The intracellular signaling pathway mediated by MTX and an anti-TNF agent were independent. These results suggest that simultaneous administration of MTX and an anti-TNF agent enhances apoptosis in tmTNFexpressing cells compared with the monotherapy of MTX or an anti-TNF agent
In order to clarify the mechanisms of improved clinical efficacy induced by concomitant treatment of MTX and an anti-TNF agent other than inhibiting generation of anti-drug antibodies (ADAb), we took note of the apoptotic effect mediated by reverse signal through Transmembrane TNF (tmTNF) in tmTNF-expressing cells
Summary
Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that exerts pleiotropic effects on various type of cells and plays a major role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis (RA) [1,2,3]. Transmembrane TNF (tmTNF), a precursor from of soluble TNF, mediates its biological functions in a cell-to-cell contact manner. TmTNF binds to TNF receptor type 1 or 2 on cell surface of target cells as a ligand. Anti-TNF agents bind to both of soluble TNF and tmTNF, resulting in neutralization of their biological activities [5]. Anti-TNF agents induce cell death to tmTNF-expressing cells via the binding to tmTNF [6,7,8]. Destruction of tmTNF-expressing cells via anti-TNF agents is mediated by antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and apoptosis via reverse signals through tmTNF [4, 7, 9]. We aimed to clarify the cytotoxic effect of MTX on transmembrane TNF (tmTNF)-expressing cells treated with anti-TNF agents
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