Fig. 3.
Hla-mediated production of IL-1β by myeloid lineage cells is impaired in Adam10−/− mice. Tissue IL-1β levels in control and Adam10−/− mice were assessed by ELISA in skin homogenates (a) and BAL fluid (b) from mice infected with S. aureus. To standardize against slight variations in the degree of tissue homogenization, tissue IL-1β was standardized to total protein in each sample. Results are 1 representative experiment of 4 from skin and 5 from BAL fluid. *** p < 0.001, Student's t test. c Recruitment of neutrophils and macrophages to S. aureus-infected skin tissue, examined by immunofluorescence microscopy performed on frozen skin sections. Control and Adam10−/− mice were examined 48 h after infection, demonstrating similar neutrophil recruitment by Ly-6G staining and macrophage infiltration by MoMa-2 staining. The imaged area immediately adjacent to and encompassing the abscess is highlighted with an orange box, while lesions from the overlying epidermis are highlighted in blue boxes. Images are representative of results obtained from analysis of 9 independent mice from 3 experiments. d Flow cytometric analysis of neutrophil and macrophage populations in lung homogenates prepared 8 h following intranasal infection with S. aureus. Cell populations were examined in 9 independent mice from 3 experiments as a function of all CD45+ cells, where the data are a representative experiment. e Ex vivo chemotaxis assay performed on primary neutrophils harvested from control and Adam10−/− mice where chemotaxis in response to media alone or 1 × 106 CFU S. aureus was scored by counting neutrophils that migrated across a Transwell filter toward the chemotactic stimulus. Migration was recorded as a percentage of total neutrophil input into the assay. ** p < 0.01, two-way ANOVA. NS = Not significant. f, g IL-1β production in response to 6-hour culture of primary neutrophils (f) or macrophages (g) from control or Adam10−/− mice with purified, endotoxin-free Hla (25 μg/ml), HKSA (equivalent to 2 × 107 staphylococci), or Hla + HKSA. ** p < 0.01, *** p < 0.001, Student's t test.