Figure 2. LysMcre-Hsp90b1-flox macrophages are hypo-responsive to K. pneumoniae.

Alveolar macrophages (30 000 cells/well) and peritoneal macrophages (50 000 cells/well) from control and LysMcre-Hsp90b1-flox mice were stimulated with Pam₃CSK₄ (1 µg/ml), LPS derived from K. pneumoniae (100 ng/ml) or heat killed K. pneumoniae (2e7 CFU/ml) for 20 h after which levels of TNF-α, CXCL1 and CXCL2 in supernatant were determined (A). To assess phagocytic ability, alveolar macrophages (100 000 cells/well) were incubated with FITC labeled heat killed K. pneumoniae (in a bacterium:cell ratio of 100:1) (B). To determine the role of CD18 during phagocytosis, peritoneal macrophages were pretreated with 10ug/ml control or anti-CD18 antibody 30 min before incubating with FITC labelled heat killed K. pneumoniae (C). After pretreatment, residual CD18 expression was determined by flow cytometry (C, left). Phagocytic index was calculated by multiplying the gMFI of FITC positive cells with the percentage FITC positive cells, and subtracting the phagocytic index of 4°C control samples (B and C, right). Data shown represent means and variation amongst cells from four different mice stimulated separately. Comparison using the Mann-Whitney test. * p< 0.05, ** p< 0.01.