The interaction between neutrophils, MPO, and DCs in LNs. (Ai-ii) Accumulation of neutrophils (identified as Ly-6G⁺CD11b^hiCD11c⁻MHC-II^neg side-scatter^hi cells; flow cytometry) in draining LNs of normal (n = 6) and subcutaneously OVA/LPS-injected mice (n = 6/time point). (Aiii) Neutrophil infiltration in normal (n = 6) and draining (inguinal) or nondraining (axillary) LNs from mice injected with OVA/LPS (4 hours; n = 6). (B) MPO activity in extracts of draining LNs from normal and OVA/LPS-injected mice (4 hours; n = 6/group). (C) Frozen acetone-fixed sections (4-μm-thick) of draining LNs from WT mice collected 4 hours after OVA/LPS injection were stained for neutrophils (red), MPO (green), and DCs (blue). Confocal images were captured using a NikonC1 inverted confocal laser scanning microscope (×40/0.45 Nikon Plan Apo objective lens), acquired with the use of line sequential scanning (acquisition software: NIS Elements AR 3.0), converted to .TIFF files (Image J software) and processed by Photoshop CS software (Adobe Systems). Neutrophils interact with DCs (magenta). MPO is present inside (yellow) and outside (green) of neutrophils and extracellular MPO contacts DCs (indicated by arrows). Original magnification, ×400. (Di) Surface expression of CD68 (flow cytometry) on LN neutrophils and the proportion (Dii) and total number (Diii) of CD68⁺ neutrophils in draining LNs of normal (n = 6) and OVA/LPS-injected mice (4 hours; n = 6). (Ei-ii) Neutrophil apoptosis (Annexin-V staining; flow cytometry) in draining LNs of normal (n = 6) and OVA/LPS-injected mice (4 hours; n = 6). (F) The effect of DCs on MPO release by neutrophils. BM neutrophils were cultured (in 10% RPMI; 24 hours) with or without BM-derived DCs (1:1 ratio). Control wells contained DCs cultured alone. MPO activity was measured in supernatants. Data are representative of 2-3 independent experiments. *P < .05, **P < .01, ***P < .0001; ND, not detected.