The Toll-like receptor adaptor proteins MyD88 and Mal/TIRAP contribute to the inflammatory and destructive processes in a human model of rheumatoid arthritis

Abstract

The widespread distribution of Toll-like receptors (TLRs) and their ligands raises the question whether they contribute to the production of inflammatory and tissue destructive molecules in rheumatoid arthritis (RA). We examined the expression and function of TLR2 and TLR4 and their downstream signaling adaptors MyD88 and Mal/TIRAP in synovial membrane cultures from RA tissue. Both TLR2 and TLR4 were detected by flow cytometry, and stimulation with TLR2 and TLR4 ligands augmented the spontaneous production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8, indicating that TLR2 and TLR4 are functional in these cultures. In addition, overexpression of dominant-negative forms of MyD88 and Mal/TIRAP significantly down-regulated the spontaneous production of cytokines tumor necrosis factor-alpha, IL-6, and vascular endothelial growth factor, and enzymes MMP-1, MMP-2, MMP-3, and MMP-13 in RA synovial membrane cell cultures. Because TLR2 and TLR4 require both MyD88 and Mal/TIRAP for signaling, this study suggests that TLR function may regulate the expression of these factors in the RA synovium. Conditioned media from synovial membrane cell cultures stimulated human macrophages in a MyD88- and Mal-dependent manner, suggesting the release of a TLR ligand(s) from these cells. Thus, TLRs not only protect against infection but may also promote the inflammatory and destructive process in RA.

Figure 1

Expression of TLR2 and TLR4 in the RA synovium. A: mRNA from synovial tissue was extracted, DNase-treated, and then analyzed by RT-PCR for the presence of TLR2 and TLR4 in four separate donors. B and C: RA synovial membrane cells from the same donor were examined by FACS for the expression of cell surface or intracellular TLR2 and TLR4. B: A representative histogram plot and the fold induction ± SEM of five independent donors is shown. C: Permeabilized cells were incubated with isotype control, α-TLR2, or α-TLR4 antibody and co-stained with either α-CD3 or α-CD68.

Figure 2

TLR2 and TLR4 stimulation of RA synovial membrane cultures. RA synovial membrane cells were incubated for 24 hours in the presence of media alone or media containing 20 μg/ml LTA, 10 μg/ml PGN, 10 ng/ml Pam₃Cys-Ser-Lys₄, or 10 ng/ml LPS. Supernatants were collected after 24 hours and assayed for the presence of TNF-α (A), IL-6 (B), and IL-8 (C) by ELISA. Fold induction of cytokine production ± SEM of triplicate cultures is shown and is representative of five independent experiments from unrelated RA patients.

Figure 3

Effect of MyD88dn, Maldn, and IκBα expression in cytokine production in RA synovial membrane cells. RA synovial membrane cells were left uninfected or were infected with Adβ-gal, AdMyD88dn, AdMaldn, or AdIκBα for 2 hours. Cells were subsequently cultured for a further 48 hours. A: Cell extracts were obtained and examined for the presence of endogenous or overexpressed MyD88 and Mal/TIRAP by Western blotting. B–F: Supernatants were collected and assayed for the presence of TNF-α (B), IL-1β (C), IL-6 (D), IL-8 (E), and VEGF (F) by ELISA. Mean cytokine production (±SEM) of triplicate cultures from six to eight unrelated patients is shown. For the statistical analysis of these parametric normally distributed data, a one-tailed Student’s t-test was used to compare uninfected control cells with recombinant adenovirus-infected cells (*P < 0.05, **P < 0.01).

Figure 4

Effect of MyD88dn, Maldn, and IκBα expression in MMP production in RA synovial membrane cells. RA synovial membrane cells were left uninfected or were infected with Adβ-gal, AdMyD88dn, AdMaldn, or AdIκBα for 2 hours. Cells were subsequently cultured for a further 48 hours and supernatants collected and assayed for the presence of MMP-1 (A), MMP-2 (B), MMP-3 (C), and MMP-13 (D) by ELISA. Mean cytokine production (±SEM) of triplicate cultures from six to seven unrelated patients is shown. For the statistical analysis of these parametric normally distributed data, a one-tailed Student’s t-test was used to compare uninfected control cells with recombinant adenovirus-infected cells (*P < 0.05, **P < 0.01).

Figure 5

Stimulation of NF-κB activation in macrophages with RA synovial cell culture supernatants. M-CSF-derived human macrophages were infected with an adenovirus containing a NF-κB luciferase reporter gene and AdGFP, MyD88dn, or Maldn. After 24 hours, the cells were stimulated for 6 hours with filtered supernatants harvested from RA synovial membrane cell cultures, after which luciferase activity was measured. Data are shown as relative luciferase activity to the control as the mean (±SEM) (n = 3). For the statistical analysis, a one-tailed Student’s t-test was used to compare ADGFP control cells with ADMyD88dn and AdMaldn cells (*P < 0.05, **P < 0.01).