Local interleukin-1-driven joint pathology is dependent on toll-like receptor 4 activation

Abstract

Toll-like receptors (TLRs) may contribute to the pathogenesis of chronic inflammatory destructive diseases through the recognition of endogenous ligands produced on either inflammation or degeneration of the extracellular matrix. The presence of endogenous TLR agonists has been reported in rheumatoid joints. In the present study, we investigated the significance of TLR2 and TLR4 activation by locally- produced endogenous ligands in the severity of joint inflammation and destruction. Local joint pathology independent of systemic immune activation was induced by overexpression of interleukin (IL)-1 and TNF in naive joints using adenoviral gene transfer. Here, we report that at certain doses, IL-1-induced local joint inflammation, cartilage proteoglycan depletion, and bone erosion are dependent on TLR4 activation, whereas TLR2 activation is not significantly involved. In comparison, tumor necrosis factor alpha-driven joint pathology seemed to be less dependent on TLR2 and TLR4. The severity of IL-1-induced bone erosion and irreversible cartilage destruction was markedly reduced in TLR4(-/-) mice, even though the degree of inflammation was similar, suggesting uncoupled processes. Furthermore, the expression of cathepsin K, a marker for osteoclast activity, induced by IL-1beta was dependent on TLR4. Overexpression of IL-1beta in the joint as well as ex vivo IL-1 stimulation of patellae provoked the release of endogenous TLR4 agonists capable of inducing TLR4-mediated cytokine production. These data emphasize the potential relevance of TLR4 activation in rheumatoid arthritis, particularly with respect to IL-1-mediated joint pathology.

Figure 1

Induction of high intraarticular expression of IL-1β and TNFα in wild-type (WT), TLR2^−/−, and TLR4^−/− mice. IL-1β (A) or TNFα (B) overexpressing adenoviruses (3.5 × 10⁵ and 1 × 10⁷ PFU per joint, respectively) were injected into the knee joints of naive mice and concentrations of the respective cytokines were measured in the patella washouts collected 1 day after injection using Bioplex cytokine assays. Values represent the mean ± SEM of six mice per group. Injection of 1 × 10⁷ PFU per joint of the control virus Addel did not induce any detectable levels of cytokines in wild-type or TLR-deficient mice (data not shown).

Figure 2

TLR4 dependency of local IL-1-induced joint pathology. A: Minor histological changes at day 4 of intraarticular injection of the Addel control virus (1 × 10⁷ PFU per joint). B–G show the histological scores of arthritis in wild-type (WT) (black bars), TLR2^−/− (gray bars), and TLR4^−/− (white bars) mice at day 4 of intraarticular overexpression of AdIL-1β (3.5 × 10⁵ PFU per joint; B–D) or AdTNFα (1 × 10⁷ PFU per joint; E–G). Synovial inflammation (B and E) was scored on H&E-stained tissue sections. PG depletion (C and F) and bone erosion (D and G) were scored on Safranin O-stained sections. Each parameter was scored on a 0- to 3-point scale in a blinded manner. Values are the mean ± SEM of six mice per group. n.s. = not significant; n.d. = not detectable. *P < 0.05 compared with wild-type by Mann-Whitney U-test.

Figure 3

Representative images of the knee joints at day 4 of AdIL-1β overexpression. Left panels show synovial inflammation on H&E-stained sections, and right panels show PG depletion (loss of red staining) on Safranin O-stained sections. Both parameters were reduced in TLR4^−/− (lower panels) compared with wild-type (WT) (upper panels) and TLR2^−/− (middle panels) mice. Original magnification (×100) for H&E and (×200) for Safranin O staining. B = bone, C = cartilage, F = femur, JS = joint space, P = patella.

Figure 4

TLR4 dependency of local IL-1-induced cartilage destruction despite similar synovial inflammation. Data show joint pathology at day 4 of high AdIL-1β overexpression (3.5 × 10⁶ PFU per joint). Synovial inflammation (A) was scored on H&E-stained, and cartilage destruction (B) was scored on Safranin O-stained sections on a 0- to 3-point scale. Values are the mean ± SEM of at least five mice per group. n.s. = not significant. ^∗∗P < 0.01 compared with wild-type (WT) by Mann-Whitney U-test. C: Representative images of Safranin O-stained tissue sections showing reduced joint pathology in TLR4^−/− mice compared with wild-type and TLR2^−/− mice. Open and stealth arrows indicate cartilage destruction and bone erosion, respectively. Original magnification, ×100.

Figure 5

TLR4 dependency of local IL-1-induced bone erosion (A) and cathepsin K expression (B–D) despite similar synovial inflammation at day 4 of high AdIL-1β overexpression (3.5 × 10⁶ PFU per joint). Cathepsin K mRNA expression (B) was measured by quantitative real-time PCR. The threshold cycle (Ct) value of cathepsin K was corrected for the Ct of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to obtain the ΔCt, then ΔΔCt was calculated compared with the Addel control. Cathepsin K protein expression (C) was detected by immunohistochemistry and quantified using Leica Qwin software. Values in A (n ≥ 5) and B (n = 4) are the mean ± SEM. The horizontal bars in C represent the mean. n.s. = not significant. ^∗P < 0.05 compared with wild-type (WT) by Mann-Whitney U-test. D: Representative images of immunohistochemical staining of cathepsin K, the osteoclast marker involved in bone resorption. Positive cells at the outer surface of the bone, mineralized cartilage and the intratrabecular space are depicted in the figure. Original magnification, ×200. P = patella; F = femur.

Figure 6

IL-1β, but not TNFα, induces the release of endogenous TLR4 agonists from patellae. A: HEK293-mTLR4/MD2/CD14 cells were stimulated with IL-1β, TNFα, and various TLR ligands. Stimulations were performed at least in triplicate. B: HEK293-mTLR4 cells were stimulated with patella washouts obtained at day 4 or 7 of in vivo Ad5del or AdIL-1β overexpression. A volume ratio of 1:10 of patella washouts:culture medium was used. C: HEK293 and HEK293-TLR4 cells were stimulated with supernatants of patellae ex vivo cultured with IL-1β or TNFα (10 ng/ml each) for 24 hours. A volume ratio of 1:10 of the patella supernatants was used in the presence of the TNFα inhibitor Enbrel (1 μg/ml). D: HEK293-TLR4 cells were stimulated with E. coli LPS (100 ng/ml) or supernatants of ex vivo-cultured patella as in C, either or not following 30 minutes of preincubation with TLR4 antagonist (1 μg/ml). IL-8 was measured in 24-hour culture supernatants using the Bioplex cytokine assay. Values are the mean ± SEM n = 6 patellae per group in B–D; n.s. = not significant, n.d. = not detectable. ^∗P < 0.05 by Mann-Whitney U-test.