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. 2018 Oct 22:2018:3928915.
doi: 10.1155/2018/3928915. eCollection 2018.

The Role of IL-6RA in UHMWPE Promotes Proliferation in Fibro-Like Synovial Cells

Xianlun Pang  1 , Jiang Yang  2 , Xiaoli Zhen  3 , Hai Nie  4 , Hao Qin  5 , Lingyuan Huang  6 , Lijie Zhang  6
Affiliations

The Role of IL-6RA in UHMWPE Promotes Proliferation in Fibro-Like Synovial Cells

Xianlun Pang et al. Biomed Res Int. .

Abstract

UHMWPE granule could induce macrophages and inflammatory responses in interfacial tissues, which eliminated the wear debris of UHMWPE component and further induced dissolution of the surrounding bone, leading aseptic loosening. However, the mechanism of synovial cells, especially fibroblast-like synovial (FLS) cells response to UHMWPE, remains unknown. Herein we choose FLS cells as research object. Vimentin (+) CD68 (-) was identified by flow cytometry and immunofluorescent staining assay, and the cells were identified as FLS cells, which was consistent with the experimental requirements. The inhibitory evaluation showed that UHMWPE could significantly promote the proliferation and inhibit apoptosis of FLS cells in dose- and time-dependent manners and increase the levels of proinflammatory cytokines, including IL-6, IL-1β, TNF-α, PGE2, MMP2, and LOX. UHMWPE also can induce the expression of mIL-6R protein in FLS cells and further investigate the relationship between apoptosis and inflammation. Interestingly enough, when we added the interleukin-6 receptor antagonist (IL-6RA), the expression levels of proapoptosis-related proteins increased; in other words, UHMWPE-induced antiapoptosis diminished by IL-6RA (50 μg/ml). Taken together, these findings clearly demonstrated that UHMWPE promote growth in FLS cells through upregulating inflammatory factors to produce antiapoptotic effect.

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Figures

Figure 1
Figure 1
Culture and identification of fibroblast-like synovial cells. After primary separation and culture, the 5th generation cells were identified by flow cytometry and immunofluorescence. Morphological observation under light microscope (a). Identification of surface markers by flow cytometry (b). CD68 combined with DAPI staining (c). Vimentin combined with DAPI staining (d). The cells were observed on a fluorescence microscope (Olympus Corporation, Japan) at a magnification of 400x. Cell nuclei were visualized by DAPI staining in blue. The expression of vimentin was visualized by FITC staining in green. n=6.
Figure 2
Figure 2
Detection of UHMWPE promote cell proliferation. FLS were incubated with 0-1.0 g/L UHMWPE for 7 d. Cell viability was determined by MTT assay, n=5 (a). FLS were incubated with 1.0 g/L UHMWPE for 0, 1, and 7 d. The images of Edu staining was observed by fluorescence microscope (400x, final magnification), n=5 (b). Cell nuclei were visualized by DAPI staining in blue and proliferative cells were visualized by Edu staining in red. Data was obtained from three independent experiments; results were shown as means ± standard deviation. ∗p < 0.05, compared with dose of 0 g/L; #p < 0.05, compared with 0 d.
Figure 3
Figure 3
Effects of UHMWPE on apoptosis of FLS cells. FLS were incubated with 0-1.0 g/L UHMWPE for 0, 1, and 7 d or incubated with 1.0 g/L UHMWPE for 0, 1, and 7 d. Apoptotic and necrotic cell populations were analyzed by flow cytometry; quantitative analysis of apoptotic cells after UHMWPE treatment, n=5 (a and b). The expression of apoptosis-related proteins in FLS cells was detected by Western blot assay and the changes of Bcl-2, Bax, caspase-3, and cleaved-caspase-3 were statistically analyzed, n=5 (c). Data was obtained from three independent experiments; results were shown as means ± standard deviation. ∗p < 0.05, compared with dose of 0 g/L; #p < 0.05, compared with 0 d.
Figure 4
Figure 4
UHMWPE increased the expression of inflammatory factors. The inflammatory factors (IL-6, IL-1β, TNF-α, and PGE2) and invasion cytokines (MMP2 and LOX) were detected by ELISA, n=5. ∗p < 0.05, compared with dose of 0 g/L; #p < 0.05, compared with 0 d. Data was obtained from three independent experiments; results were shown as means ± standard deviation.
Figure 5
Figure 5
UHMWPE increased the expression of mIL-6R protein. FLS cells were incubated with UHMWPE (0, 0.01, 0.1, and 1g/L) for 7 d. The expression of IL-6R protein was detected by western blotting assay.
Figure 6
Figure 6
The antagonistic effect of IL-6RA on FLS cells treated with UHMWPE for 7 d. The experiment was divided into 3 groups, including UHMWPE (1 g/L), UHMWPE (1 g/L) + IL-6RA (50 μg/ml), and IL-6RA(50 μg/ml). Apoptotic rate was detected by flow cytometry after treatment IL-6RA, n=5 (a). The expression levels of apoptosis-related proteins including Bax, Bcl2, caspase-3, and cleaved-caspase-3 were analyzed by Western blot after treatment IL-6RA, n=5 (b). Data was obtained from three independent experiments; results were shown as means ± standard deviation. ∗p < 0.05; ∗∗p < 0.01 compared with UHMWPE group.
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