Phospholipases effector phase of arthritis. To test this hypothesis, we examined the effect of TFM C on CAIA induced by injecting a mixture of monoclonal antibodies against type II collagen followed by lipopolysaccharide administration two days later. The major players in CAIA are innate immune cells while adaptive immune cells are not required for disease development. Therefore, CAIA has value as an animal model to study the effector phase of arthritis. In vehicle treated mice, severe arthritis occurred one week after CII antibody injection, and administration of celecoxib inhibited arthritis slightly. In contrast, administration of TFM C significantly suppressed CAIA compared to vehicle or celecoxib treatment. We next analyzed the histological features in the joints of four paws from vehicle, TFMC and celecoxib treated mice 12 days after disease induction. Quantification of the taurine histological severity of arthritis is shown in Figure 3B and typical histological features are presented in Figure 3C. Massive cell infiltration, cartilage erosion, and bone destruction were observed in joints of vehicle treated or celecoxib treated mice but not in those of TFM C treated mice.
These results indicate that TFM C exhibits a strong disease inhibitory effect in CAIA in contrast to vehicle or celecoxib. Everolimus TFM C inhibits the mast cell activation in CAIA Next, we sought to understand the mechanism through which TFM C treatment suppressed arthritis in CAIA. Since mast cells have been demonstrated to be critical for initiation of antibody induced arthritis, we evaluated the effect of TFM C on the activation of mast cells. Because degranulation is the clearest histological hallmark of mast cell activation, joint mast cells were visually assessed for an intact versus degranulating phenotype after staining with berberine toluidine blue. The proportion of degranulated mast cells was significantly lower in TFM C treated mice compared to that in celecoxib or vehicle treated mice. TFM C supresses the activation of macrophages Innate immune cells and inflammatory cytokines, such as IL 1 and TNF a are critical for disease development in CAIA. Thus, we next determined the effect of TFM C on the production of inflammatory cytokines from macrophages.
Splenic macrophages from mice treated with TFM C, celecoxib or control vehicle, were stimulated with LPS ex vivo, and the cytokines in the culture supernatants were measured by ELISA. The production of IL 1, IL 6 and TNF a from macrophages was efficiently suppressed in TFM C treated mice compared to vehicle treated mice. In celecoxibtreated mice, although the production of IL 1b was decreased, the production of other cytokines such as IL 6 and TNF a was not suppressed, and the IL 6 production was even enhanced compared to vehicle treated mice. TFM C suppresses leukocyte influx in thioglycollateinduced peritonitis The other key players in antibody induced arthritis are neutrophils. Neutrophils are recruited to joint tissue and depletion of neutrophils has been shown to supress disease susceptibility and severity in CAIA. An intraperitoneal injection of thioglycollate causes leukocytes influx into the peritoneum from bone marrow and circulation, and neutrophils are the major cell population which first emigrate to the peritoneal cavity.