Macrophage subsets in the human ileum. Lin− (CD3, CD19, CD20, CD56, TCRαβ), live (PI−) singlet cells were identified in digests of resected healthy ileum (n=8) and CD45+ cells purified by magnetic antibody cell sorting. (a) CD14hi and CD14lo cells were identified and HLA-DR expression assessed. (b) Subsets of CD209/CD163− DR− (blue gate), CD209/CD163− DR+ (red gate), and CD209/CD163+ DR+ (green gate) cells were then identified among the CD14hi (top panels) and CD14lo cells (lower panels), and their expression of CD11c assessed. Plots are representative of eight (A) or three (B) individual samples. (c). Anatomical location of resident mφ in healthy human ileum. CD163+ mφ (green) are found immediately below the epithelial layer (46-diamidino-2-phenyl indole (DAPI)—blue) and are a discrete population from CD103+ DC and lymphocytes (red) (× 100–× 400 final magnification). (d) Representative plots identifying CD14hi and CD14lo CD103− cells among CD45+ Lin− cells from samples of healthy ileum, non-inflamed, or inflamed areas of Crohn's disease ileum (top panels). CD11c and CD163 expression were then assessed on the CD14hi subsets from each sample (lower panels). Results representative of 4–8 individual samples. (e) Ratios of CD14hi:CD14lo CD103− mφ in healthy, non-inflamed, and inflamed Crohn's disease ileal samples. Each symbol represents an individual patient. (f) CD64 vs. CD14 expression by live Lin− MHCII+ cells from normal and inflamed Crohn's disease human ileum. The left panels in each pair show CD64 isotype staining. ***P<0.001.