Toll-like receptors in human chondrocytes and osteoarthritic cartilage

Abstract

Background and purpose: Degenerating cartilage releases potential danger signals that react with Toll-like receptor (TLR) type danger receptors. We investigated the presence and regulation of TLR1, TLR2, and TLR9 in human chondrocytes.

Methods: We studied TLR1, TLR2, TLR4, and TLR9 mRNA (qRT-PCR) and receptor proteins (by immunostaining) in primary mature healthy chondrocytes, developing chondrocytes, and degenerated chondrocytes in osteoarthritis (OA) tissue sections of different OARSI grades. Effects of a danger signal and of a pro-inflammatory cytokine on TLRs were also studied.

Results: In primary 2D-chondrocytes, TLR1 and TLR2 were strongly expressed. Stimulation of 2D and 3D chondrocytes with a TLR1/2-specific danger signal increased expression of TLR1 mRNA 1.3- to 1.8-fold, TLR2 mRNA 2.6- to 2.8-fold, and TNF-α mRNA 4.5- to 9-fold. On the other hand, TNF-α increased TLR1 mRNA] expression 16-fold, TLR2 mRNA expression 143- to 201-fold, and TNF-α mRNA expression 131- to 265-fold. TLR4 and TLR9 mRNA expression was not upregulated. There was a correlation between worsening of OA and increased TLR immunostaining in the superficial and middle cartilage zones, while chondrocytes assumed a CD166(×) progenitor phenotype. Correspondingly, TLR expression was high soon after differentiation of mesenchymal stem cells to chondrocytes. With maturation, it declined (TLR2, TLR9).

Interpretation: Mature chondrocytes express TLR1 and TLR2 and may react to cartilage matrix/chondrocyte-derived danger signals or degradation products. This leads to synthesis of pro-inflammatory cytokines, which stimulate further TLR and cytokine expression, establishing a vicious circle. This suggests that OA can act as an autoinflammatory disease and links the old mechanical wear-and-tear concept with modern biochemical views of OA. These findings suggest that the chondrocyte itself is the earliest and most important inflammatory cell in OA.

Figure 1.

A. Toll-like receptors TLR1, TLR2, and TLR9 in 4 primary chondrocyte isolates. B–D. 4 mesenchymal stem cell lines (day 0) differentiating via a progenitor stage (days 7 and 14) to chondrocytes (day 21). Quantitative real-time polymerase chain reaction was used to measure the mRNA copy numbers per 10⁶ β-actin copies. In primary chondrocyte isolates, TLR1 and TLR2 mRNA levels were higher than TLR9 mRNA levels (^† p < 0.001). Overall changes in TLR2 and TLR9 mRNA expression were significant during chondrogenesis.

Figure 2.

Chondrocyte pellets at day 21 produced from human bone marrow-derived mesenchymal stem cells. A–D. Immunostaining of TLR1 (panel A), TLR2 (panel B), and TLR9 (panel C) compared to negative staining control (panel D). Magnification 200×. E–G. Collagen type-II immunostaining (Collagen II, panel E) and Safranin O staining of untreated 3D chondrocyte pellets (control, panel F) compared to pellets treated with 5 ng/mL TNF-α (panel G) and 100 ng/mL TNF-α (panel H) (showing dose-dependent depletion of proteoglycans in the presence of TNF-α). Magnification 100×.

Figure 3.

Folds of change in tumor necrosis factor-α (TNF-α) expression and TLR1, TLR2, TLR4, and TLR9 expression upon stimulation with TLR 1/2-specific ligand Pam 3CSK4. Primary 2D chondrocyte cultures (A) and 3D chondrocyte pellet cultures (B) at day 21 (i.e. produced from human mesenchymal stem cells at day 21). ^† p ≤ 0.05 compared to unstimulated cultures.

Figure 4.

Folds of change in tumor necrosis factor-α (TNF-α), TLR1, TLR2, TLR4, and TLR9 upon stimulation of primary 3D chondrocyte pellet cultures. Stimulation with 5 ng/mL TNF-α (A) and with 100 ng/mL TNF-α (B). ^† p ≤ 0.05. All statistical comparisons were against unstimulated cultures (normalized to 1).

Figure 5.

Collagenase-cleaved COL2A-3/4M neoepitope immunostaining of 3D chondrocyte pellets. Untreated 3D-chondrocyte pellets (A) and the negative staining control (B). 3D chondrocyte pellets stimulated with 5 ng/mL TNF-α (C) and 100 ng/mL TNF-α (D). Magnification 100×.

Figure 6.

Safranin O, TLR1, and TNF-α staining of OARSI-graded osteoarthritis samples (grades G1–G5). TLR2 and TLR9 immunostaining was rather similar to that of TLR1 and is shown in Supplementary figure. Surface (tangential, gliding), middle (transient), and deep (radial) zones are marked, and also tide mark (between cartilage and calcified cartilage) and subchondral bone. The safranin O microphotographs are panoramic images, constructed from several microphotographs to provide an overall view of all zones in one image. Magnification 100×.