Efferocytosing macrophages produce IL-4 in vivo. (A) Peritoneal cell counts on day 0, 3, 4, or 6 following intraperitoneal injection of sodium periodate were enumerated and graphed. Immediately after peritoneal lavage, day 3 PEM with ingested neutrophils (arrows) were identified by cytospin and diaminobenzidine histochemical staining for MPO. (B) Day 0 or day 3 peritoneal cells were first labeled with CD115 and then stained for MPO and IL-4. Percentages of MPO-positive or IL-4–positive macrophages were quantified in (C). (D) PEM from day 3 WT or Il4^−/− mice were labeled with CD115 and stained for intracellular IL-4 using anti-IL-4 or isotype. (E) MPO within CD115+IL-4+, CD115+IL-4, and CD115-IL-4 populations in day 3 peritoneal cells were measured by anti-MPO and compared by histogram (right). (F) Amounts of IL-4 in peritoneal lavages from naive mice or day 3 mice were measured by multiplex. Three to 4 mice were used for each time point. (G) Surface IL-4Rα chain on day 0 and day 3 macrophages were compared by flow cytometry. (H) Day 3 peritoneal cells from a 4get BALB/c mouse were labeled with CD115. GFP+CD115+ cells were sorted by FACS and followed by diaminobenzidine histochemistry for MPO on a microscope slide. (I) The sorted cells were then imaged; brown staining indicated the presence of MPO within the cells. GFP, green fluorescent protein.