Role of S100A9 in Arthritis Figure 9. S100A9 induces the secretion of cytokines by monocytes. Cells (1 6106 cells/ml) were incubated with S100A9 (10 mg/ml) for 24 h. Supernatants were harvested and cytokines were measured by (A) cytokine arrays or (B) ELISA. CTRL: unstimulated cells. Values are the mean 6 SEM of 5 different experiments. *p,0.05, t test. (C) Monocytes were stimulated with different concentrations of S100A9 (0.1–40 mg/ml) and IL-6 or TNF-a concentrations were measured in the supernatant. (D) Monocytes were incubated with 10 mg/ml of S100A9 and IL-6 or TNF-a concentrations were assessed as a function of time. Solid line: IL-6 dosage (open circles: unstimulated cells, solid circles: S100A9 stimulated cells), dotted line: TNF-a (white squares: unstimulated cells, solid squares: S100A9 stimulated cells). doi:10.1371/journal.pone.0045478.g009 barrier through the extended activation of b2 integrins. The delayed migration effect of S100A9 suggests that it may induce inside-out signaling, which is required to increase activation of b2 integrins [38], or promote the release of secondary activators such as lipid mediators, which are known to upregulate the ligand- binding activity of b2 integrins [39,40]. These results are consistent with those from Eue et al., which demonstrated that the addition of anti-S100A9 or anti-S100A8/A9 reduced mono- cyte migration across endothelial cells [17]. Based on these observations, it is clear that S100A9 is linked to the migration of leukocytes, particularly neutrophils, to the site of inflammation. Recruitment of innate immune cells into joint articulations leads to local inflammation through the release of proinflammatory mediators that activate resident immune and non-immune cells in the synovium. Cytokines like TNFa and IL-6 are overproduced in RA synovial tissues and play multiple roles in the release of other inflammatory mediators, chemoattractants, and matrix-modifying enzymes. As expected, high levels of TNFa and IL-6 were detected in the paws of arthritic mice. Interestingly, when mice were injected with anti-S100A9, we observed a reduction in TNFa that can be correlated with the decrease in monocyte/granulocyte infiltration. Similar effects were obtained for IL-6, with the difference that low-level expression of IL-6 was still detected in the paws of animals treated with anti-S100A9 Abs. This observation could be partly explained by the fact that synovial fibroblasts produce considerable amounts of this cytokine in the absence of stimuli, and this participates in steady-state bone homeostasis [41,42]. Cartilage degradation and bone erosion are due to a complex interplay between immune cell infiltration, cytokines, matrix- modifying enzymes such as matrix metalloproteinases (MMPs), and osteoclast/osteoblast turnover. It has been recently demon- strated that S100A8 and S100A9 play active roles in these three aspects. Indeed, S100A92/2 mice, which also lack the S100A8 protein, show reduced bone erosion in antigen-induced arthritis through a mechanism partly related to a decrease in osteoclasts [31,43]. Moreover, S100A8- and S100A9-mediated stimulation of human chondrocytes induces the expression of pro-inflammatory cytokines and MMPs that promote cartilage degradation and prevents the formation of new cartilage matrix molecules [44]. In addition, it was recently reported that TNFa- and IL-17-mediated joint erosion depends on the expression of S100A8, MMPs, and a PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e45478