Neutrophil-DC hybrids kill bacteria rapidly by a CRAMP-mediated mechanism. (A-C) Ly6G⁺/CD11c⁻/MHC II⁻ neutrophils, Ly6G⁺/CD11c⁺/MHC II⁺ hybrids, and/or Ly6G⁻/CD11c⁺/MHC II⁺ traditional DCs purified from GM-CSF–supplemented BM culture (d 6) were compared for CRAMP mRNA expression by quantitative polymerase chain reaction (means ± SD from triplicate samples) (A) and for CRAMP protein production by western blotting (B) and immunofluorescence staining (bar represents 20 μm) (C). (D) The above hybrid and traditional DC populations were incubated for 60 min with live E. coli. After killing extracellular bacteria by exposure to kanamycin (time 0), the samples were incubated for an additional 0.5-3.5 h to test intracellular bacterial killing (means ± SD from triplicate samples). (E) Neutrophil-DC hybrids (left panel) and traditional DCs (right panel) purified from wild-type mice or CRAMP-deficient mice were examined for intracellular bacterial killing activities (means ± SD from triplicate samples). (F) E. coli were cultured for 4 h with a synthetic CRAMP peptide or whole protein extracts from neutrophil-DC hybrids or traditional DCs in the presence or absence of neutralizing anti-CRAMP antibodies or control antibodies. The starting bacterial number before culturing is shown in the top bar (means ± SD from triplicate bacterial cultures). *P < .05, **P < .01, ***P < .001 between the indicated samples (F) or compared with traditional DCs (E) or to the starting bacterial numbers at time 0 (E). Data are representative of at least 2 independent experiments.