Fig. 4.

HA-induced inflammation requires NLRP3, ASC and caspase-1 in the air pouch model of synovitis. The s.c. air pouches were raised on the dorsum of 10- to 12-wk-old mice of indicated genotypes (Materials and Methods). 1 mL suspension of HA crystals (4 mg, unless indicated) in PBS or PBS alone was injected into the air pouches. 8 hours postinjection, mice were killed and the pouch exudates were harvested by a lavage with 3 mL PBS containing 5 mM EDTA. (A–D) The infiltrated neutrophils were identified with hematoxylin and eosin (H&E) staining and counted using a hemocytometer. (C–G) 4 mg HA-4 crystals in suspension were injected into air pouches of age- and sex-matched WT, Casp1^−/−, Nlrp3^−/−, or Asc^−/− mice. (E and F) Supernatants of air pouch exudates were collected by centrifugation. IL-1β and IL-6 production were measured by ELISA. Values are shown as the mean ± SEM of at least seven mice per group. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student's t test or one-way ANOVA and post hoc test. (G) Sagittal sections of the air pouches with surrounding tissues were stained with H&E. (Scale bars, 100 μm.) Arrows, pouch cavities; asterisk, neutrophil infiltration. Images are representative of at least seven mice per group. All results were repeated by at least two separate experiments.