American Society of Hematology

Figure 3

$CCR2-positive and CCR2-CX3CR1 double-positive macrophages accumulate in fibrin deposits and mediate endocytic fibrin uptake. (A) Endocytic fibrin uptake by CCR2-positive cells. Percentage CCR2-positive (green bar) and CCR2-negative (gray bar) cells 24 hours after implantation of fibrin into CCR2+/RFP transgenic mice. Crosshatched bars embedded within green and gray bars show fraction of, respectively, CCR2-positive and CCR2-negative cells that endocytose fibrin. Data were generated from serial analysis of confocal z-stacks from 5 mice as described in “Materials and methods.” Error bars indicate standard deviation. (B) Examples of CCR2-positive (red, white in top right panel) cells with endocytosed fibrin 24 hours after fibrin implantation. (C) CCR2-positive cells accumulate in fibrin implantation zones. Total number of CCR2-positive cells in control (left bar), sham-operated (middle bar), or fibrin-implanted (right bar) dermis at 24 hours, as determined by the counting of cells in 4 serial z-stacks from the implantation zone. Data are shown as mean ± standard deviation and were generated by analysis of 4 control, 4 sham-operated, and 5 fibrin-implanted mice. Significance was determined by 1-way ANOVA. (D) Endocytic fibrin uptake by CX3CR1-positive cells. Percentage CX3CR1-positive (green bar) and CX3CR1-negative (gray bar) cells 24 hours after implantation of fibrin into Cx3cr1+/GFP transgenic mice. Crosshatched bars embedded within green and gray bars show fraction of, respectively, CX3CR1-positive and CX3CR1-negative cells that endocytose fibrin. Data were generated from serial analysis of confocal z-stacks from 5 mice as described in “Materials and methods.” Error bars indicate standard deviation. (E) Examples of a CX3CR1-positive cell (green, white in top right panel) with endocytosed fibrin (red, white in bottom right corner) 24 hours after fibrin implantation. (F) CX3CR1-positive cells accumulate in fibrin implantation zones. Total number of CX3CR1-positive cells in control (left bar), sham-operated (middle bar), or fibrin-implanted (right bar) dermis at 24 hours, as determined by the counting of cells in 4 serial z-stacks from the implantation zone. Data are shown as mean ± standard deviation and were generated by analysis of 5 control, 8 sham-operated, and 10 fibrin implanted mice. Significance was determined by 1-way ANOVA. (G) CCR2 single-positive and CCR2, CX3CR1 double-positive cells constitute the majority of fibrin-uptaking cells. Percentage of CCR2 single-positive and CCR2, CX3CR1 double-positive (left bar) and CX3CR1-negative (middle bar), and CCR2, CX3CR1 double-negative cells 24 hours after implantation of fibrin into Cx3cr1+/GFP;CCR2+/RFP bitransgenic mice. Crosshatched bars embedded in bars show fraction of, respectively, CCR2-positive and CCR2-negative cells that endocytose fibrin. A total of 32% of the cells in the left bar are CCR2 single positive and 68% are CCR2, CX3CR1 double positive. Data were generated from serial analysis of confocal z-stacks from 4 mice as described in “Materials and methods.” Error bars indicate standard deviation. (H) Examples of CCR2, CX3CR1 double-positive cells (orange/yellow, white top right and middle panels) with endocytosed fibrin (white) 24 hours after fibrin implantation. (I) CCR2-positive cells do not engage in endocytic collagen degradation. Representative example of injection field from Cx3cr1+/GFP;CCR2+/RFP bitransgenic mice 24 hours after injection with fluorescently labeled fibrillar collagen. Collagen-uptaking (white) CCR2-negative cells and a collagen-negative, CCR2-positive (red, white top right corner) cell are shown.$

CCR2-positive and CCR2-CX3CR1 double-positive macrophages accumulate in fibrin deposits and mediate endocytic fibrin uptake. (A) Endocytic fibrin uptake by CCR2-positive cells. Percentage CCR2-positive (green bar) and CCR2-negative (gray bar) cells 24 hours after implantation of fibrin into CCR2^+/RFP transgenic mice. Crosshatched bars embedded within green and gray bars show fraction of, respectively, CCR2-positive and CCR2-negative cells that endocytose fibrin. Data were generated from serial analysis of confocal z-stacks from 5 mice as described in “Materials and methods.” Error bars indicate standard deviation. (B) Examples of CCR2-positive (red, white in top right panel) cells with endocytosed fibrin 24 hours after fibrin implantation. (C) CCR2-positive cells accumulate in fibrin implantation zones. Total number of CCR2-positive cells in control (left bar), sham-operated (middle bar), or fibrin-implanted (right bar) dermis at 24 hours, as determined by the counting of cells in 4 serial z-stacks from the implantation zone. Data are shown as mean ± standard deviation and were generated by analysis of 4 control, 4 sham-operated, and 5 fibrin-implanted mice. Significance was determined by 1-way ANOVA. (D) Endocytic fibrin uptake by CX3CR1-positive cells. Percentage CX3CR1-positive (green bar) and CX3CR1-negative (gray bar) cells 24 hours after implantation of fibrin into Cx3cr1^+/GFP transgenic mice. Crosshatched bars embedded within green and gray bars show fraction of, respectively, CX3CR1-positive and CX3CR1-negative cells that endocytose fibrin. Data were generated from serial analysis of confocal z-stacks from 5 mice as described in “Materials and methods.” Error bars indicate standard deviation. (E) Examples of a CX3CR1-positive cell (green, white in top right panel) with endocytosed fibrin (red, white in bottom right corner) 24 hours after fibrin implantation. (F) CX3CR1-positive cells accumulate in fibrin implantation zones. Total number of CX3CR1-positive cells in control (left bar), sham-operated (middle bar), or fibrin-implanted (right bar) dermis at 24 hours, as determined by the counting of cells in 4 serial z-stacks from the implantation zone. Data are shown as mean ± standard deviation and were generated by analysis of 5 control, 8 sham-operated, and 10 fibrin implanted mice. Significance was determined by 1-way ANOVA. (G) CCR2 single-positive and CCR2, CX3CR1 double-positive cells constitute the majority of fibrin-uptaking cells. Percentage of CCR2 single-positive and CCR2, CX3CR1 double-positive (left bar) and CX3CR1-negative (middle bar), and CCR2, CX3CR1 double-negative cells 24 hours after implantation of fibrin into Cx3cr1^+/GFP;CCR2^+/RFP bitransgenic mice. Crosshatched bars embedded in bars show fraction of, respectively, CCR2-positive and CCR2-negative cells that endocytose fibrin. A total of 32% of the cells in the left bar are CCR2 single positive and 68% are CCR2, CX3CR1 double positive. Data were generated from serial analysis of confocal z-stacks from 4 mice as described in “Materials and methods.” Error bars indicate standard deviation. (H) Examples of CCR2, CX3CR1 double-positive cells (orange/yellow, white top right and middle panels) with endocytosed fibrin (white) 24 hours after fibrin implantation. (I) CCR2-positive cells do not engage in endocytic collagen degradation. Representative example of injection field from Cx3cr1^+/GFP;CCR2^+/RFP bitransgenic mice 24 hours after injection with fluorescently labeled fibrillar collagen. Collagen-uptaking (white) CCR2-negative cells and a collagen-negative, CCR2-positive (red, white top right corner) cell are shown.

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