Figure 7. S100A9 increases neutrophil adhesion to fibrinogen. Neutrophils were incubated with S100A9 (40 mg/ml) or IL-8 (5 ng/ml) alone or in combination and allowed to adhere to fibrinogen for different incubation periods at 37uC. The number of adhered neutrophils was determined as described in Materials and Methods. Data shown represent the mean 6 SEM of at least 3 experiments using neutrophils from different donors. doi:10.1371/journal.pone.0045478.g007 Role of S100A9 in Arthritis S100A9, they can swiftly release S100A9 and calprotectin in the absence of gene transcription [34,35]. Once secreted, S100A9 and calprotectin can stimulate neighbour innate immune or non- immune cells to produce inflammatory mediators like TNFa, IL-6, and IL-1b, which are known to exacerbate and sustain inflam- mation. Taken together, our results confirm that S100A9 stimulates the production of pro-inflammatory cytokines and chemokines such as TNFa, IL-6, and MIP-1a by monocytes. These mediators participate in the induction of the acute arthritis phase as well as in disease progression, resulting in infiltration of immune cells into the joints, localized secretion of cytokines and tissue-degrading enzymes, and ultimately cartilage and bone destruction. Treatment with anti-S100A9 was associated with a decrease in cartilage and bone erosion, both of which are closely related to immune cell infiltration. Indeed, treatment with anti-S100A9 strongly decreased the infiltration of neutrophils and monocytes into the paws of arthritic mice. These results highlight the participation of S100A9 and calprotectin in leukocyte migration toward inflammatory sites. Indeed, both are deposited on endothelium by neutrophils at inflammatory sites [36,37] and are presumed to play a role in neutrophil adhesion to endothe- lium. Here we further demonstrate that S100A9 increased neutrophil transmigration, probably by prolonging the time window for the movement of neutrophils across the endothelial Figure 8. The effect of S100A9 on neutrophil migration is abolished by anti-CD11b and anti-CD18 antibodies. S100A9 (40 mg/ml) +/2 antibodies against CD11a, CD11b, and CD18, or the isotype control were added to the upper wells with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower wells and neutrophils were allowed to migrate for 2 h at 37uC. The number of transmigrated cells was determined as described in Materials and Methods. Data shown represent the mean 6 SEM of at least 3 experiments using neutrophils from different donors. *p,0.05, **p,0.01, one-way ANOVA, Dunnett’s multiple comparison test. doi:10.1371/journal.pone.0045478.g008 PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45478