Tim-3 mediates phagocytosis of apoptotic cells by peritoneal exudate macrophages. (A) Peritoneal resident Mac1⁺ cells (PRMs) and peritoneal exudate Mac1⁺ cells (PEMs) were stained with biotinylated RMT1-17, RMT2-14, RMT3-23, or RMT4-54, followed by FITC-anti-CD11b mAb and PE-avidin (thick histograms); then CD11b⁺ cells were analyzed by flow cytometry. Thin histograms indicate background staining with biotinylated control rat IgG2a. (B) Macrophages were pretreated with control rat IgG2a (rIgG), RMT3-23, or RMT4-54, and then cultured with CFSE-labeled apoptotic cells for 30 minutes at 37°C. Cells were stained with PE-Mac1, and percentage of recognition of CFSE-apoptotic cells by Mac1⁺ cells was quantified by flow cytometry. Columns represent mean ± SD of triplicates (*P < .05; **P < .01 compared with rIgG). (C) Peritonitis was elicited by intraperitoneal injection of thioglycolate. Three days later, the mice were intraperitoneally injected with rIgG, RMT3-23, or RMT4-54 (200 μg/head), and then with CFSE-labeled apoptotic cells. Two hours later, peritoneal cells were harvested, and recognition of CFSE-labeled apoptotic cells by Mac1⁺ cells was quantified by flow cytometry. The experiments (n = 4-5 per group) were performed 3 times independently with a similar result. Columns represent mean ± SD of 4 mice in a representative experiment (*P < .05 compared with rIgG). Similar results were obtained in 3 (A,C) or 2 (B) independent experiments.