Our results suggest that simultaneous administration of MTX and IFX enhances apoptosis of activated immune cells expressing tmTNF in synovial cells in RA individuals compared with MTX or IFX monotherapy. 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 (S)-JQ-35 providers. Results Apoptosis of tmTNF-expressing cells was significantly increased from the concomitant administration of MTX and an anti-TNF agent, compared with MTX only or an anti-TNF agent only. 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 transmission through tmTNF. Summary The 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 providers seem to be self-employed. These findings might clarify at least in part improved the medical response upon co-therapy of MTX and an anti-TNF agent in RA. test/college student = 5/group). (D) TmTNF-expressing Jurkat cells were incubated with 0.1 M MTX for 24 h, 0.01 M ETN for 12 h, or combination of MTX and ETN for 12 h after MTX for 12 h. The proportion of Annexin V-positive cells was indicated. Ideals are mean (S)-JQ-35 (SEM) of each group (= 6/group). (E) TmTNF-expressing Jurkat cells were incubated with (S)-JQ-35 0.1 M MTX for 24 h, 0.01 M CZP for 12 h, or combination of MTX and CZP for 12 h after MTX for 12 h. The proportion of Annexin V-positive cells was indicated. Ideals are mean (SEM) of each group (= 6/group). * 0.05, ** 0.01, Mann-Whitney checks. At first, we analyzed for apoptosis induced by MTX only, IFX only and MTX plus IFX in tmTNF-expressing cells to see their combined cytotoxic effects. IFX is definitely a chimeric anti-TNF full IgG1. Treatment with MTX only for 20 h induced apoptosis in 7.2% of tmTNF-expressing cells, on the other hand untreated control showed apoptosis in 2.4% of these cells ( 0.01). Activation with IFX only for 6 h also significantly improved apoptotic cells to 21.3% compared to control ( 0.01). Of notice, apoptotic cells dramatically improved up to 34.2% under co-administration of MTX and IFX (20 h of MTX, and IFX was present for the last 6 h) (Figures 1B,C). Consequently, synergistic apoptotic effect was observed in co-administration of MTX and IFX. Subsequently, to investigate whether this apoptosis-inducing effect differs among anti-TNF providers, similar experiments were carried out using additional anti-TNF providers; etanercept (ETN), a fusion protein of extracellular website of TNF receptor 2 and IgG1-Fc, and certolizumab pegol (CZP), a PEGylated Fab fragment of the anti-TNF antibody. As these two anti-TNF providers are much weaker in inducing apoptosis of tmTNF-expressing cells than IFX, the incubation time in the presence of these providers was long term from 6 to 12 h. After 24 h of incubation with MTX only, Annexin V-positive apoptotic cells were 8.4% of (S)-JQ-35 the tmTNF-expressing cells and apoptotic cells in control were 2.8%. Treatment of ETN only for 12 h induced apoptosis in 5.3% of the tmTNF-expressing cells. In the situation of co-stimulation, the percentage of apoptotic cells induced by co-stimulation of MTX and ETN was 14.0%, approximately equal to the sum of percentages of apoptotic cells by MTX alone and ETN alone (Number 1D). Furthermore, related additive effect with MTX Pde2a was observed in CZP treatment. Proportions of apoptotic cells in control, MTX only, CZP alone, and MTX plus CZP were 3.3, 8.3, 7.6, and 15.8%, respectively (Number 1E). Taken collectively, MTX showed an additive apoptotic effect when co-stimulated with ETN or CZP in tmTNF-expressing T cells. The Additive Effect via Combination of MTX and an Anti-TNF Agent Were Observed in Neither Complement-Dependent Cytotoxicity, Antibody-Dependent Cell-Mediated Cytotoxicity, nor Antibody-Dependent Cellular Phagocytosis To examine whether synergistic or additive effects between MTX and an anti-TNF agent are observed in cytotoxic assays other than apoptosis by reverse signal through tmTNF, we performed CDC and ADCC/ADCP assay. Transmembrane TNF-expressing cells were cultured with MTX for 25 h, IFX for 1 h or MTX plus IFX for 1 h after undergone 24 h of MTX treatment in the medium containing refreshing or heat-inactivated serum to evaluate CDC activities (Number 2). IFX-induced prominent cell death in human refreshing serum, but not in heat-inactivated serum, indicating that IFX offers.