Effects of interleukin-1β and tumor necrosis factor-α on macrophage inflammatory protein-3α production in synovial fibroblast-like cells from human temporomandibular joints

Abstract

Background: Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast-like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL-1β or TNF-α to determine which genes were altered.

Methods: Ribonucleic acid was isolated from SFCs after IL-1β or TNF-α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein-3α (MIP-3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL-1β or TNF-α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL-1β or TNF-α after treatment with inhibitors. The MIP-3α levels were measured using an ELISA.

Results: Macrophage inflammatory protein-3α was the gene most upregulated by IL-1β- or TNF-α stimulation. The mRNA and protein levels of MIP-3α increased in response to IL-1β in a time-dependent manner. In contrast, during TNF-α stimulation, the MIP-3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL-1β- and TNF-α-stimulated MIP-3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion: Interleukin-1β and TNF-α increased the MIP-3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP-3α from stimulation with IL-1β or TNF-α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.

Keywords: interleukin-1β; macrophage inflammatory protein-3α; synovial fibroblast-like cell; temporomandibular joint; tumor necrosis factor-α.

Figure 1

Agarose gel depicting relative MIP-3α mRNA levels in synovial fibroblast-like cells treated with IL-1β or TNF-α by endpoint PCR. Cells were either left untreated or treated with 0.1 ng/ml IL-1β or 10 ng/ml TNF-α for 2, 4, or 8 h. GAPDH was analyzed as an internal control.

Figure 2

Real-time PCR analysis of the MIP-3α mRNA levels in synovial fibroblast-like cells treated with IL-1β or TNF-α. The cells were treated with 0.1 ng/ml IL-1β or 10 ng/ml TNF-α for 2, 4, or 8 h. Fold changes were calculated using the ΔΔCT method. Mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.005 compared with the untreated control cells.

Figure 3

Effects of IL-1β and TNF-α on the MIP-3α protein levels in conditioned medium from human TMJ synovial fibroblast-like cells. The cells were stimulated with the indicated concentrations of (A) IL-1β or (B) TNF-α for 24 h, and the MIP-3α protein levels in the conditioned medium were then assayed using an ELISA. Mean ± SD (n = 4 replicates). * P < 0.05, **P < 0.005, compared with the untreated control cells. Significant differences (P < 0.05) in IL-1β and TNF-α were determined using one-way ANOVA.

Figure 4

Enzyme-linked immunosorbent assay of MIP-3α protein levels in conditioned medium from human TMJ synovial fibroblast-like cells over time with cytokine treatment. Cells were treated with 0.1 ng/ml IL-1β or 10 ng/ml TNF-α for 4, 8, 24, or 48 h. Mean ± SD (n = 4). *P < 0.05, **P < 0.005 compared with the untreated control cells.

Figure 5

Network of IL-1β- and/or TNF-α-induced molecules by IPA. Data were analyzed using Ingenuity Pathway Analysis (Ingenuity®System, www.ingenuity.com). The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. Nodes are indicated by various shapes that represent the functional class of the gene product. The lines are displayed with various labels that describe the nature of the relationship between the nodes.