Splenic RPMs exhibit local self-maintenance following old RBC transfusion-induced cell death. (A) Bone marrow from UBC-GFP mice was harvested and enriched for Ly6C^hi monocytes by negative selection. GFP⁺, Ly6C^hi, CD115⁺, CD11b⁺ monocytes were then adoptively transferred to wild-type C57BL/6 recipients prior to transfusion of the latter with 350 μL of old RBCs. At 2 hours posttransfusion, GFP⁺ Ly6C^hi monocytes were observed in the spleen. By 5 and 8 days posttransfusion, GFP⁺ RPMs (F4/80^hi, VCAM-1^hi) were observed in the spleen. (B) Wild-type C57BL/6 mice were transfused with 350 μL of fresh or old RBCs and sacrificed at defined time points. Splenic RPMs were stained with Ki67, demonstrating increased proliferation at 1 to 2 days after old RBC transfusions, as compared with RPMs from mice that received fresh RBCs. Histogram (left) of Ki67 staining at 1 day posttransfusion. (C) Ly6C^hi monocyte cell numbers were quantified in the blood of CCR2^−/− mice, demonstrating their absence. (D) Ly6C^hi monocyte cell numbers were quantified in the spleens of CCR2^−/− mice, demonstrating their absence. (E) The splenic RPM population in CCR2^−/− mice is reduced by 5 hours after transfusions of old RBCs, but restored by 2 days posttransfusion. (F) By Ki67 staining, RPMs in CCR2^−/− mice proliferate following transfusion of old RBCs, relative to RPMs in mice that received fresh RBCs. Histogram (left) of Ki67 at 2 days posttransfusion. **P < .01; ***P < .001; ANOVA with the Sidak posttest or unpaired Student t test. Data are representative of 3 independent experiments.