tively (Fig. 2D). Overall, anti-S100A9 treatment led to an approximately 50% reduction in disease intensity (Fig. 2D). Disease incidences in groups treated with anti-TNFa, anti- S100A9, and the isotype control were 53%, 60%, and 80%, respectively (data not shown). Statistical analysis revealed signif- icant differences between anti-S100A9 or anti-TNFa-treated and isotype control groups. No significant difference in disease activity was observed between anti-S100A9 and anti-TNFa-treated animals. As in RA, the murine LPS-CIA model is characterized by strong infiltration of leukocytes into joint articulations, as well as by marked degradation of bone and collagen. In light of the improvement in clinical arthritic scores following anti-S100A9 therapy, we examined the effect of anti-S100A9 on cellular recruitment and joint integrity. To this end, paws were recovered at the peak of inflammation on day 5. Paw sections from 5 animals per group were stained with hematoxylin and eosin (H&E) to determine cellular infiltration and joint architecture, as well as with safranin/fast green to assess collagen degradation. Mice treated with the isotype control Ab exhibited marked bone destruction with a loss of collagen demonstrated by a decrease in safranin staining (Fig. 3A). In comparison, mice treated with anti- S100A9 showed almost no collagen loss or bone degradation. Blinded analyses of sections confirmed that anti-S100A9 treatment markedly reduced bone destruction (mean scores of 0.25 vs. 1.02 for anti-S100A9 and isotype control treatments, respectively) as well as collagen destruction (mean scores of 0.55 vs. 1.26 for anti- S100A9 and isotype control treatments, respectively, p.0.05, one- way analysis of variance [ANOVA] test). Similar results were obtained with anti-TNFa-treated mice (Fig. 3B, no significant differences between anti-S100A9 and anti-TNFa-treated animals). These results indicate that treatment with anti-S100A9 protects against joint destruction. H&E staining of hind and fore paws revealed a significant decrease in cellular infiltration into the synovium in anti-S100A9- treated animals (mean scores of 0.56 vs. 1.33 for anti-S100A9 and isotype control treatments, respectively; Fig. 3). Western blot analyses of paw homogenates showed a 40% to 80% reduction in the expression of the neutrophil marker 7/4 in anti-S100A9- treated animals, compared to animals treated with the isotype control Ab (Fig. 4A). Similarly, a 60% reduction in the monocyte/ granulocyte marker Gr-1 was observed in anti-S100A9-treated animals (Fig. 4B). Anti-S100A9 Prevents the Release of Pro-inflammatory Cytokines RA is characterized by the presence of excessive concentrations of several pro-inflammatory cytokines like TNFa and IL-6. We therefore investigated the effect of anti-S100A9 on cytokine levels. Serum was collected 4 h after LPS injection and at the peak of inflammation (day 5), and IL-6 and TNFa concentrations were measured using ELISA (Fig. 5A). No cytokines were detected in sera 4 h after LPS injection.(data not shown). Interestingly, the serum concentration of IL-6 at day 5 was approximately 6 times lower in mice treated with anti-S100A9 (11.3 ng/ml compared to 67.4 ng/ml, respectively; Fig. 5A). TNFa was not detected at day 5 in mice sera. In contrast, both IL-6 and TNFa were detected by western blot in frozen paw homogenates on day 5 after LPS injection (Fig. 5B). IL-6 expression was reduced and TNFa was almost completely absent in paws of mice treated with anti-S100A9. Thus, treatment with anti-S100A9 resulted in a sharp reduction of cytokines in serum and at the site of inflammation. Role of S100A9 in Arthritis S100A9 Increases Neutrophil Transmigration by Promoting Cell Adhesion The effect of S100A9 on neutrophil migration was investigated to understand the mechanism of action of anti-S100A9. In addition to its presence at the site of inflammation, S100A9 is also found in blood where it has been hypothesized to actively participate in leukocyte migration [24]. To mimic the presence of S100A9 in the blood, different concentrations of S100A9 were incubated with neutrophils on the luminal side of endothelial cells grown on Transwell filters, while the chemoattractant IL-8 (5 ng/ ml) was added on the basolateral side side to attract the cells. Cell migration was allowed to proceed for 2 h at 37uC. S100A9 significantly increased neutrophil migration, independent of the presence of IL-8 (Fig. 6A). The lowest S100A9 concentration at which this effect was observed was 1 mg/ml, and the maximal effect was observed at 10 mg/ml. These concentrations are in the range observed in sera of patients with inflammatory diseases. These data indicate that the presence of S100A9 in the serum is sufficient to weakly stimulate transendothelial migration even in absence of pro-inflammatory molecules known to attract and promote the extravasation of neutrophils. However, the response to S100A9 is additive in presence of chemotactic agents, and more neutrophils can cross the barrier when they are exposed to these DAMPS. We next investigated the individual effects of S100A9 on endothelial cells and neutrophils to decipher its mechanism of action. S100A9 failed to stimulate the expression of adhesion molecules on endothelial cells or to increase endothelial cell permeability (data not shown). However, S100A9 stimulated prolonged adhesion of neutrophils to the b2 integrin substrate fibrinogen. While IL-8 induced a rapid and transient adhesion to fibrinogen, S100A9 stimulated a delayed, but prolonged adhesion of neutrophils (Fig. 7). Interestingly, this effect of S100A9 was first detected after 30 minutes of stimulation; these kinetics are similar to those of the enhancement of neutrophil transmigration in the presence of S100A9 (Fig. 6B). The role of b2 integrins in the promotion of neutrophil transendothelial migration by S100A9 was confirmed by blocking neutrophil transmigration using various b2 integrin-specific Abs (Fig. 8). Thus, the reduction of granulocyte migration in anti-S100A9-treated animals can be at least partially explained by the inhibition of serum S100A9- promoted leukocyte transmigration. S100A9 Induces Cytokine Secretion by Human Monocytes To examine whether the decrease in cytokine levels could be due to a direct effect of S100A9 on cytokine secretion, human monocytes were incubated with 10 mg/ml of recombinant human S100A9 for 24 h, and supernatants were analyzed using cytokine arrays. S100A9 was found to induce the secretion of several pro- inflammatory cytokines including IL-1b, IL-6, and TNF-a, as well as chemokines such as growth-related oncogene a, IL-8, monocyte chemotactic protein-1, and MIP-1a (Fig. 9A). These results were confirmed using ELISA to detect IL-6, TNFa, and MIP-1a (Fig. 9B). Secretion of both IL-6 and TNFa was dose-dependent, with maximal secretion detected at 1 mg/ml of S100A9, a concentration found in the synovial fluid of arthritis patients (Fig. 9C). Secretion of both cytokines was maximal after 6 to 8 h of stimulation, after which time the concentration of TNFa diminished while the concentration of IL-6 remained constant (Fig 9D). Interestingly, S100A9 failed to stimulate the secretion of cytokines from neutrophils (data not shown). PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45478