The Tissue Fibrinolytic System Contributes to the Induction of Macrophage Function and CCL3 during Bone Repair in Mice

Abstract

Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg-/-), urokinase-type plasminogen activator (uPA-/-) or tissue-type plasminogen activator (tPA-/-) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA-/- mice until day 6, compared with wild-type (uPA+/+) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA-/- mice at the bone injury site on day 4, compared with those in uPA+/+ mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA-/- and Plg-/-, but not in tPA-/- mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-α, IL-1β, IL-6, IL-4 and IFN-γ mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA-/- and Plg-/-, but not in tPA-/- mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA+/+, but not in uPA-/- mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.

Fig 1. Delayed bone repair in Plg ^-/-, uPA ^-/- and tPA ^-/- mice.

(A, C, E) Quantitative computed tomography (qCT) images of the damaged site of femurs in Plg ^+/+ and Plg ^-/- (A), uPA ^+/+ and uPA ^-/- (C) as well as tPA ^+/+ and tPA ^-/- mice (E). The arrowheads indicate the site of bone defect. (B, D, F) Quantification of the bone defect area as assessed by qCT in Plg ^+/+ and Plg ^-/- (B), uPA ^+/+ and uPA ^-/- (D) as well as tPA ^+/+ and tPA ^-/- mice (F). The data represent the mean ± SEM from 8 (B, F) and 6 (D) mice. ^## P < 0.01 and ^# P < 0.05 (Mann-Whitney U test).

Fig 2. Decrease in the number of Osterix-positive cells at the damaged site on day 4 after femoral bone defect in Plg ^-/-, uPA ^-/- and tPA ^-/- mice.

(A) Quantification of Osterix-positive cells per 0.1 mm² of the microscopic fields at the damaged site on day 4 in Plg ^-/-, uPA ^-/-, tPA ^-/- and each wild-type mice. (B) The number of alkaline phosphatase (ALP)-positive cells per 0.1 mm² of the microscopic fields in the damaged site on day 7 in Plg ^-/-, uPA ^-/-, tPA ^-/- and each wild-type mice. (C) The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) per 1 mm of bone surface at the damaged site on day 7 in Plg ^-/-, uPA ^-/-, tPA ^-/- and each wild-type mice. The data represent the mean ± SEM from 4–5 (A) and 5–7 (B, C) mice. ^## P < 0.01 and ^# P < 0.05 (Mann-Whitney U test).

Fig 3. Impaired vessel formation at the damaged site after femoral bone defect in Plg ^-/-, uPA ^-/- and tPA ^-/- mice.

(A) Photographs of CD31-positive blood vessels at the damaged site on day 4 after femoral bone defect. Scale bars indicate 50 μm. (B, C) Quantification of blood vessels at the damaged site on day 4 after femoral bone defect. The number (B) and total luminal area (C) of CD31-positive blood vessels per 0.1 mm² of the microscopic fields in the damaged site. The data represent the mean ± SEM of 5 mice. (D) Western blot analysis of VEGF, TGF-β, HIF-1α and BMP-2 levels in the damaged and contralateral intact femurs on day 4 after a bone defect in uPA ^+/+ and uPA ^-/- mice. The results represent experiments performed on 5 mice in each group. (E) Relative levels of VEGF, TGF-β and BMP-2 mRNA in the damaged and contralateral intact femurs on day 4 after a bone defect in uPA ^+/+ and uPA ^-/- mice. The data are expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA values. The data represent the mean ± SEM of 6 mice. ^## P < 0.01 (Mann-Whitney U test). **P < 0.01 and *P < 0.05 (Tukey’s test).

Fig 4. Decrease in the number of macrophages and ratio of macrophage phagocytosis at the damaged site on day 4 after femoral bone defect in Plg ^-/-and uPA ^-/-, but not in tPA ^-/- mice.

(A) Photographs of F4/80-positive cells at the damaged site on day 4 after femoral bone defect. Scale bars indicate 50 μm. (B) Quantification of F4/80-positive cells at the damaged site on day 4 after femoral bone defect. The data represent the mean ± SEM of 5 mice. (C) The ratio of macrophage phagocytosis at the damaged site on day 4 assessed by transmission electron microscopy. The data represent the mean ± SEM of 4 mice. (D) Transmission electron microscopic photographs of macrophages at the damaged site on day 4 in uPA ^+/+ and uPA ^-/- mice. The results represent experiments performed on 4 mice in each group. Arrowheads indicate erythrocytes in macrophages. Scale bars indicate 2 μm. N: nucleus. ^## P < 0.01 and ^# P < 0.05 (Mann-Whitney U test).

Fig 5. Effects of plasminogen, uPA or tPA deficiency on the levels of cytokines in the damaged and contralateral intact femurs on day 4.

(A–G) Real time-PCR analysis of tumor necrosis factor (TNF)-α (A), interleukin (IL)-1β (B), IL-6 (C), IL-4 (D), IL-13 (E), IL-10 (F) and interferon (IFN)-γ (G) mRNA in the femurs on day 4. The data are expressed relative to GAPDH mRNA values. The data represent the mean ± SEM of 5–6 mice. **P < 0.01 and *P < 0.05 (Tukey’s test). ^† P < 0.05 (Steel-Dwass test).

Fig 6. Effects of uPA, plasminogen or tPA deficiency on the levels of CCL2, CCL3 and CCL4 mRNA in the damaged and contralateral intact femurs on day 4.

(A–C) Real time-PCR analyses of CCL2 (A), CCL3 (B) and CCL4 (C) mRNA in the femurs on day 4. The data are expressed relative to GAPDH mRNA values. The data represent the mean ± SEM of 5–6 mice. **P < 0.01 and *P < 0.05 (Tukey’s test).

Fig 7. Effects of neutralizing anti-CCL3 antibody on the number of macrophages at the damaged site and the decrease in area of bone defect on day 4 in uPA ^+/+ and uPA^-/- mice.

(A) Microphotographs of immunostaining for F4/80 at the damaged site on day 4 after a femoral bone defect in uPA ^+/+ and uPA ^-/- mice treated with normal IgG (Cont) or neutralizing anti-CCL3 antibody (Anti-CCL3). The results represent experiments performed on 5 mice in each group. Scale bars indicate 50 μm. (B) Quantification of the number of F4/80-positive cells per 0.1 mm² in the microscopic fields in the damaged site on day 4 after a femoral bone defect in uPA ^+/+ and uPA ^-/- mice treated with normal IgG or neutralizing anti-CCL3 antibody. The data represent the mean ± SEM of 5 mice. (C) Quantification of the bone defect area as assessed by qCT on days 1 and 4 in uPA ^+/+ and uPA ^-/- mice treated with normal IgG or neutralizing anti-CCL3 antibody. The data represent the mean ± SEM from 5 mice. **P < 0.01 and *P < 0.05 (Tukey’s test).