The increased immunity of norbin-deficient mice derives from neutrophils. (A) Macrophage depletion. Ncdn^fl/fl and Ncdn^Δmye mice were treated intravenously with 100 μL and intranasally with 50 μL clodronate liposomes, or were mock-treated with control liposomes, at 72 hours before intranasal infection with 2 × 10⁶S pneumoniae; they were culled 6 hours later and BAL performed. (B) Neutrophil depletion. Ncdn^fl/fl and Ncdn^Δmye mice were treated with 1A8 monoclonal Ly6G antibody or isotype control immunoglobulin G at 24 hours and 0 hours before intranasal infection with 2 × 10⁶S pneumoniae (filled symbols), or mock-infected (open symbols), culled 6 hours later, and BAL performed. BAL was analyzed for bacterial burden by quantification of CFU and for numbers of neutrophils (CD11b^hi, CD24^hi in panel A; CD11b^hi, Ly6G^hi in panel B) and macrophages (CD11c⁺, SiglecF⁺ and CD11b⁺, CD24^lo) by flow cytometry. Data in panel A are mean ± standard error of the mean pooled from 2 independent experiments; 2 to 3 mock-infected and 2 to 3 infected mice/genotype/experiment. Data in panel B are mean ± standard error of the mean pooled from 5 independent experiments; 1 mock-infected and 2 infected mice/genotype/experiment. Dots represent individual mice. Statistics comprised two-way analysis of variance with Šidák’s (panel A) or Tukey’s (panel B) multiple comparisons tests. (C) Histology. Ncdn^fl/fl and Ncdn^Δmye mice were infected intranasally with 2 × 10⁶S pneumoniae (filled symbols), or mock-infection (open symbols), culled 6 hours later, lungs fixed in formalin, and hematoxylin and eosin–stained sections analyzed for the presence of neutrophils according to their characteristic nuclear morphology. For each mouse, 60 fields of view of 40 000 μm² from 6 areas distributed throughout the lung were analyzed. Left-hand panels show one representative field of view; right-hand panels are zoom-ins to show labeling of neutrophils within 10 μm of blood vessels or airway epithelium, or within the interstitium using different color dots. The quantification below shows mean ± standard error of the mean of 3 mice per group. Statistics comprised two-way analysis of variance with Šidák’s multiple comparisons test.