Figure 4.
CCL2 and CCL7 mRNA expression by splenic Ly6Chimonocytes following enhanced erythrophagocytosis requires the presence of RPMs. At defined time points after transfusing wild-type recipients with 350 μL of fresh or old RBCs, splenocytes were isolated by FACS, and CCL2 and CCL7 mRNA expression was measured by qPCR in (A) RPMs and (B) Ly6Chi monocytes; these data show that, after old RBC transfusions, expression of these chemokines did not change, or even decreased, in RPMs, whereas they both increased substantially in Ly6Chi monocytes at 5 hours posttransfusion. CCL2-GFP reporter mice were transfused with 200 μL of PBS, fresh GFP+ RBCs, or old GFP+ RBCs, and then sacrificed at 2 hours posttransfusion. (C) Gating strategy for splenic RPMs and Ly6Chi monocytes along with representative dot plots showing induced CCL2 promoter–dependent GFP expression; the percentages of cells expressing GFP are provided. (D) Frequency of GFP expression, demonstrating upregulation by Ly6Chi monocytes, but not by RPMs. (E) Frequency of CCL2 expression by RPMs and Ly6Chi monocytes showing no statistically significant changes posttransfusion. (F) Gating strategy for Ly6Chi monocytes in the blood and bone marrow, along with representative dot plots showing CCL2 promoter–dependent GFP expression; the percentages of cells expressing GFP are provided. To determine the dependence of Ly6Chi monocyte CCL2 and CCL7 mRNA expression on the presence of RPMs, Spic−/− mice were used for the experiments in panels G-K. (G-H) RPMs (VCAM-1hi, F4/80hi) are absent in Spic−/− mice, as compared with Spic+/+ controls. (I) The proportion of RPMs in Spic+/+ recipient mice that ingested old GFP+ RBCs. (J) The proportions of Ly6Chi monocytes in Spic+/+ and Spic−/− mice that ingested old GFP+ RBCs. (K) Fold regulation of CCL2 and CCL7 mRNA expression, as measured by qPCR, in splenic Ly6Chi monocytes from Spic+/+ and Spic−/− mice. *P < .05; **P < .01; ***P < .001; ****P < .0001; ANOVA with Sidak or Tukey multiple comparisons test or unpaired t test. Data are representative of 3 independent experiments, except for Spic+/+ and Spic−/− results, which were pooled for all experiments.

CCL2 and CCL7 mRNA expression by splenic Ly6Chimonocytes following enhanced erythrophagocytosis requires the presence of RPMs. At defined time points after transfusing wild-type recipients with 350 μL of fresh or old RBCs, splenocytes were isolated by FACS, and CCL2 and CCL7 mRNA expression was measured by qPCR in (A) RPMs and (B) Ly6Chi monocytes; these data show that, after old RBC transfusions, expression of these chemokines did not change, or even decreased, in RPMs, whereas they both increased substantially in Ly6Chi monocytes at 5 hours posttransfusion. CCL2-GFP reporter mice were transfused with 200 μL of PBS, fresh GFP+ RBCs, or old GFP+ RBCs, and then sacrificed at 2 hours posttransfusion. (C) Gating strategy for splenic RPMs and Ly6Chi monocytes along with representative dot plots showing induced CCL2 promoter–dependent GFP expression; the percentages of cells expressing GFP are provided. (D) Frequency of GFP expression, demonstrating upregulation by Ly6Chi monocytes, but not by RPMs. (E) Frequency of CCL2 expression by RPMs and Ly6Chi monocytes showing no statistically significant changes posttransfusion. (F) Gating strategy for Ly6Chi monocytes in the blood and bone marrow, along with representative dot plots showing CCL2 promoter–dependent GFP expression; the percentages of cells expressing GFP are provided. To determine the dependence of Ly6Chi monocyte CCL2 and CCL7 mRNA expression on the presence of RPMs, Spic−/− mice were used for the experiments in panels G-K. (G-H) RPMs (VCAM-1hi, F4/80hi) are absent in Spic−/− mice, as compared with Spic+/+ controls. (I) The proportion of RPMs in Spic+/+ recipient mice that ingested old GFP+ RBCs. (J) The proportions of Ly6Chi monocytes in Spic+/+ and Spic−/− mice that ingested old GFP+ RBCs. (K) Fold regulation of CCL2 and CCL7 mRNA expression, as measured by qPCR, in splenic Ly6Chi monocytes from Spic+/+ and Spic−/− mice. *P < .05; **P < .01; ***P < .001; ****P < .0001; ANOVA with Sidak or Tukey multiple comparisons test or unpaired t test. Data are representative of 3 independent experiments, except for Spic+/+ and Spic−/− results, which were pooled for all experiments.

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