Table 1.
Summary of characteristics in the study population.
Figure 1.
Immunohistochemical localization of CCL20 in human IVD tissues and comparison of mean density scores of the positive cells between the three groups.
Fifty IVD tissue samples (Group P, n = 20 and Group E, n = 30) from disc degeneration patients and 3 from scoliosis patients were analyzed. Representative results from each group are shown. No distinct CCL20 expression was detected in the control (a). (b) and (c) represent the CCL20 expression analysis of the protrusion and extrusion groups, respectively. Inflammatory reactions on the edges of extruded IVD tissues are shown in (d). The magnifications of the positive cells were shown in the frame of the top right corner of (b), (c) and (d), respectively. (e) and (f) represent the isotype control of (b) and (c), respectively. Magnification, 400x; scale bars, 20 µm. Image-Pro Plus software was used to perform a quantitative analysis of the IHC results. Compared to the normal control, the expression levels of CCL20, shown as the mean densities of the positive cells, were significantly increased in the patient groups (B, *, P<0.01, significantly different from the control, *△, P<0.01, significantly different from Group E).
Figure 2.
Immunohistochemical localization of TNF-α in human IVD tissues and comparison of mean density scores of the positive cells between the three groups.
Fifty IVD tissue samples (Group P, n = 20 and Group E, n = 30) from disc degeneration patients and 3 from scoliosis patients were analyzed. Representative results from each group are shown. No distinct expression of TNF-α was detected in the control (a). (b) and (c) represent the TNF-α expression analysis of the protrusion and extrusion groups, respectively. (d) demonstrates many inflammatory cells on the edges of extruded IVD tissues that express TNF-α. The magnifications of the positive cells were shown in the frame of the top right corner of (b), (c) and (d), respectively. (e) and (f) represent the isotype control of (b) and (c), respectively. Magnification, 400x; scale bars, 20 µm. Image-Pro Plus software was used to perform a quantitative analysis of the IHC results. Compared to the normal control, the expression levels of TNF-α, shown as the mean densities of the positive cells, were significantly increased in the patient groups (B, #, P<0.01, significantly different from the control, #△, P<0.01, significantly different from Group E).
Figure 3.
Presence of IL-17-producing cells in the degenerated IVD tissues.
Fifty IVD tissue samples (Group P, n = 20 and Group E, n = 30) from disc degeneration patients and 3 from scoliosis patients were analyzed. In Group P or the control group, there were few or no positive cells (data not shown). The representative results of group E are shown. (a), IL-17-producing cells were detected by a rabbit anti-IL-17 polyclonal antibody and a mouse anti-CD4 monoclonal Ab, followed by secondary staining with an Alexa 488-conjugated donkey anti-rabbit and an Alexa 568-conjugated donkey anti-mouse IgG. DAPI mounting medium was used for nuclear staining. (b), surface CCR6 expression on the cells was detected with a rabbit anti CD4 monoclonal Ab and a mouse anti-CCR6 monoclonal Ab, followed by secondary staining with an Alexa 488-conjugated donkey anti-rabbit and an Alexa 568-conjugated donkey anti-mouse IgG. DAPI mounting medium was used for nuclear staining. In the top panel, green and red represents the expression of IL-17 and CD4, respectively, and the double-stained cells represent the IL-17-producing cells. In the bottom panel, green and red represents the expression of CD4 and CCR6, respectively, and the double-stained cells demonstrate the surface expression of CCR6 on T lymphocytes.
Figure 4.
IL-17A, TNF-α, alone or in combination, can increase the secretion of CCL20 protein from NP cells in a dose- and time-dependent manner.
Eight IVD tissue samples from disc degeneration patients were used as the primary cultures. NP cells were stimulated with the indicated concentrations of (a) IL-17A or (b) TNF-α for 48 hours. The cells were treated with the optimal concentrations of (c) IL-17A, TNF-α or both for 0, 24, 48 and 72 hours. The cell-free supernatants were harvested and the expression levels of CCL20 protein were quantified by ELISA. The data are representative of at least three separate experiments, performed in duplicate, and the mean protein levels and standard deviations (SD) are shown. Significant differences were detected by a least significance difference analysis (LSD) (P<0.05). There was statistically significant differences between each concentration of IL-17 when compared to the control; however, statistical significance was verified only for TNF-α concentrations higher than 10 ng/ml (IL-17 treatment: *, P<0.01, vs. control; +, P<0.001, vs. 10 ng/ml; #, p<0.05, vs. 1000 ng/ml; TNF-α treatment: *, p<0.001, vs. control; +, p<0.001, vs. 10 ng/ml).
Figure 5.
IL-17A and TNF-α increase CCL20 mRNA expression in NP cells in a dose-dependent manner.
Eight IVD tissue samples from disc degeneration patients were used as the primary cultures. The cells were treated with the indicated concentrations of (a) IL-17A or (b)TNF-α for 48 hours. The expression levels of CCL20 mRNA were detected and the CCL20 transcripts were quantified by real-time RT-PCR analyses in all experiments. The data are representative of at least two separate experiments, performed in duplicate, and the mean increases in the mRNA expression were recorded, after which we evaluated the standard deviations (SD). Significant differences were detected by a least significance difference analysis (LSD) (P<0.05). The results correspond with the findings obtained in the ELISA tests. (IL-17 treatment: *, P<0.001, vs. control; +, P<0.05, vs. 10 ng/ml; #, p<0.01, vs. 1000 ng/ml; TNF-α treatment: *, p<0.001, vs. control; +, p<0.01, vs. 10 ng/ml).
Figure 6.
Expression of CCR6 mRNA in PBMCs from patients with degenerated IVD and and healthy controls.
Fifty PB samples from disc degeneration patients and ten from healthy controls were used for PBMC isolation and detection of CCR6 mRNA expression. There was a significant difference in CCR6 mRNA expression levels between the isolated PBMCs from the patient group and those from the healthy control group. The CCR6 mRNA expression levels were quantified by real-time RT-PCR for all experiments. The data are representative of at least two separate experiments, performed in duplicate, and the average increase in the mRNA expression in the patient group was reported, after which the standard deviations (SD) was calculated. Significant differences were detected with the unpaired student’s t test (*, P<0.05, significantly different from the control).
Figure 7.
Circulating percentages of Th17 cells and CCR6-positive cells in peripheral blood are increased in IVD degenerated patients when compared with controls.
Heparinized peripheral whole blood cells from 20 patients and 15 healthy controls were stimulated with phorbol myristate acetate (PMA), ionomycin, and monensin for 4 h and subsequently stained with fluorochrome-labeled antibodies as described in Materials and Methods. A(a) Lymphocytes were gated by flow cytometry. A(b) CD3+ T subsets were gated by flow cytometry; the plots in the inset box represent the CD3+ T cells. A(c) Representative IL-17 expression levels in the CD3+CD8−T subsets (CD4+ T subsets) from each group are shown. The percentages of positive cells are shown in the upper left panels. A(d) Representative surface CCR6 expression levels on the CD3+CD8−IL-17+ subsets from each group are shown. The percentages of positive cells are shown in the right panel. (B) The percentage of circulating Th17 cells was significantly higher in IVD degenerated patients (2.973±0.689%) than in the control group (1.039±0.156%; *, p<0.001, significantly different from the control). (C) The rate of surface CCR6 expression on circulating Th17 cells was significantly higher in patients with degenerated IVD (59.69±4.48%) than the control group (28.75±2.09%; *, p<0.001, significantly different from the control).