Increased Autophagy Enhances the Resistance to Tumor Necrosis Factor-Alpha Treatment in Rheumatoid Arthritis Human Fibroblast-Like Synovial Cell

Abstract

Tumor Necrosis Factor-alpha (TNF-α) was reported to increase autophagy in rheumatoid arthritis human fibroblast-like synovial cell (RA-HFLS). We investigated different levels of TNF-α exposed to RA-HFLS by focusing on the relationship of autophagy and apoptosis. RA-HFLS and normal human fibroblast-like synovial cell (HFLS) were stimulated by TNF-α in the presence or the absence of 3-methyladenine (3-MA) or chloroquine (CQ). Cell apoptosis was detected by flow cytometry. Autophagy was determined through the expression levels of LC3, Beclin1, and P62 measured by Western Blot analysis as well as Confocal Laser Scanning Microscopy. The basal autophagy level was significantly higher in RA-HFLS than in HFLS. Autophagy was enhanced both in RA-HFLS and HFLS when they were treated with TNF-α. With the treatment of TNF-α, a slightly higher autophagy level was found in RA-HFLS than in HFLS, without a dose dependent effect. When autophagy was inhibited by 3-MA or CQ, apoptosis increased in both groups. With the stimulation of different doses TNF-α, apoptosis was much higher in HFLS group than in RA-HFLS. Autophagy is a protection mechanism when treated by TNF-α in RA-HFLS.

Figure 1

LC3 has two subtypes, LC3-I and LC3-II. When autophagy was induced, the conversion from LC3-I to LC3-II was correspondingly increased. LC3-II was commonly used as a maker for autophagosomes. RA-HFLS (a) and HFLS (b) were treated with 30 ng/ml TNF-α in presence or absence of 5 mM 3-methyladenine (3-MA) for 48 h. LC3 was detected by western blot, and GAPDH was used as a loading control in all experiments. Values are fold change, divided by the average value of relative optical density of LC3-II in the HFLS without any treatment (c).

Figure 2

Cells were treated with 30ng/ml TNF-α in presence or absence of 5 mM 3-MA for 48 h. Cells were incubated in cultured medium with the mRFP-GFP-LC3 adenoviruses at a MOI of 100 for 36h at 37°C before the treatment with TNF-α and 3-MA. We counted yellow spots and red spots under the confocal microscope (a, c). The red spot represents autolysosome and yellow spot represents autophagosome. The percentage of yellow staining cells and red staining cells were shown in the bar graphs to evaluate autophagosome formation in each group (b, d). Values are mean± standard deviation of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 3

Cells were treated with 30ng/ml TNF-α in presence or absence of 30 uM CQ for 48 h. Cells were incubated in cultured medium with the mRFP-GFP-LC3 adenoviruses at a MOI of 100 for 36h at 37°C before the treatment with TNF-α and CQ. We counted yellow spots and red spots under the confocal microscope. The red spot represents autolysosome and yellow spot represents autophagosome. The percentage of yellow staining cells and red staining cells were shown in the bar graphs to evaluate autophagosome formation in each group. Values are mean± standard deviation of three independent experiments.

Figure 4

Cells were treated with TNF-α at concentrations 0, 10, 40, and 100 ng/ml for 48 h. LC3-II, Beclin1, and P62 were detected by western blot. Values are fold change, divided by the average value of relative optical density of these protein in the HFLS without any treatment. The data were analyzed and presented by histogram.

Figure 5

Cells were treated with 0, 10, 40, and 100ng/ml TNF-α in presence or absence of 5 mM 3-MA for 48 h. Apoptosis was determined by flow cytometry following annexin V/propidium iodide (PI) staining (a). The data were analyzed and presented by histogram (b). Values were mean± standard deviation. The two factors analysis of variance (two-way ANOVA) was chosen for analysis of data from cell apoptosis results. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 6

Cells were treated in the presence or absence of d 5 mM 3-MA for 48 h. Autophagy was detected by western blot (LC3-II) and cell apoptosis was determined by flow cytometry. The correlation between autophagy and apoptosis was analyzed with Pearson correlation in RA-HFLS (a) and in HFLS (b).