Dual role of interleukin-17 in pannus growth and osteoclastogenesis in rheumatoid arthritis

Abstract

Introduction: In a murine model, interleukin (IL)-17 plays a critical role in the pathogenesis of arthritis. There are controversies, however, regarding whether IL-17 is a proinflammatory mediator in rheumatoid arthritis (RA). We previously established an ex vivo cellular model using synovial tissue (ST)-derived inflammatory cells, which reproduced pannus-like tissue growth and osteoclastic activity in vitro. Using this model, we investigated the effects of IL-17 on pannus growth and osteoclastogenesis in RA.

Methods: Inflammatory cells that infiltrated synovial tissue from patients with RA were collected without enzyme digestion and designated as ST-derived inflammatory cells. ST-derived inflammatory cells were cultured in the presence or absence of IL-17 or indomethacin, and the morphologic changes were observed for 4 weeks. Cytokines produced in the culture supernatants were measured by using enzyme-linked immunosorbent assay kits. Osteoclastic activity was assessed by the development of resorption pits in calcium phosphate-coated slides.

Results: Exogenous addition of IL-17 dramatically enhanced the spontaneous production of IL-6 and prostaglandin E₂ (PGE₂) by the ST-derived inflammatory cells, while it had no effect on the production of tumor necrosis factor (TNF)-α and macrophage colony-stimulating factor (M-CSF). Furthermore, IL-17 did not affect the spontaneous development of pannus-like tissue growth and osteoclastic activity by the ST-derived inflammatory cells. On the other hand, IL-17 enhanced pannus-like tissue growth, the production of TNF-α and M-CSF and the development of osteoclastic activity in the presence of indomethacin, an inhibitor of endogenous prostanoid production, while exogenous addition of PGE₁ suppressed their activities.

Conclusions: The present study suggests that IL-17 induces negative feedback regulation through the induction of PGE₂, while it stimulates proinflammatory pathways such as inflammatory cytokine production, pannus growth and osteoclastogenesis in RA.

Figure 1

Effect of interleukin (IL)-17 on the production of (A) IL-6 (A) and (B) prostaglandin E₂ (PGE₂) by the synovial tissue (ST)-derived inflammatory cells. Cells were incubated in the absence or presence of increasing concentrations of IL-17 (0 to 100 ng/ml) for 3 weeks. IL-6 (n = 7) and PGE₂ (n = 3) in the culture supernatants were measured by enzyme-linked immunosorbent assay as described in Materials and methods.

Figure 2

Effect of interleukin (IL)-17 and prostaglandin E₁ (PGE₁) on pannus-like tissue growth in vitro. Synovial tissue (ST)-derived inflammatory cells were incubated in the absence or presence of increasing concentrations of IL-17 (0 to 100 ng/ml) (n = 17) or PGE₁ (100 nM) (n = 9). Morphologic changes were observed under an inverted phase contrast microscope twice weekly for 4 weeks and were scored semiquantitatively on a scale of 0 to 4 according to the degree of tissue development as described in Materials and methods. Box and whisker plots indicate 25th/75th percentile and minimum/maximum, respectively. Bar indicates the median value. *P < 0.001 (by Mann-Whitney U test).

Figure 3

Effect of interleukin (IL)-17 on pannus-like tissue growth in the presence of indomethacin. Synovial tissue (ST)-derived inflammatory cells (n = 7) were incubated with an incremental dose of IL-17 in the absence or presence of indomethacin (100 to 1000 nM). Morphologic changes were observed under an inverted phase contrast microscope twice weekly for 4 weeks and were scored semiquantitatively on a scale of 0 to 4 according to the degree of tissue development as described in Materials and methods. Box and whisker plots indicate 25th/75th percentile and minimum/maximum, respectively. Bar indicates the median value. *P < 0.05 (by Wilcoxon signed-rank test).

Figure 4

Effect of interleukin (IL)-17 on the production of macrophage colony-stimulating factor (M-CSF), tumor necrosis factor α (TNF-α) and IL-6. Synovial tissue (ST)-derived inflammatory cells were incubated with incremental doses of IL-17 in the absence or presence of indomethacin (100 to 1,000 nM) for 1 week. Enzyme-linked immunosorbent assay kits were used to measure the concentration of (A) TNF-α, (B) M-CSF and IL-6 (C) in the culture supernatants derived from seven donors. There were no significant differences in the production of IL-6 between the presence and absence of indomethacin. Box and whisker plots indicate 25th/75th percentile and minimum/maximum, respectively. Bar indicates the median value. *P < 0.05 (by Wilcoxon signed-rank test).

Figure 5

Effect of interleukin (IL)-17 on the osteoclastogenesis. Osteoclastic activity was assessed by the development of resorption pits in calcium phosphate-coated slides as described in Materials and methods. Synovial tissue (ST)-derived inflammatory cells (n = 6) were cultured with incremental doses of IL-17 in the absence or presence of indomethacin on calcium phosphate-coated slides for 2 weeks and examined for the development of resorption pits. The ratio to the number of resorption pits in medium alone was plotted. Bar indicates the median value. *P < 0.05 vs. medium alone (by Wilcoxon signed-rank test).