Crucial Involvement of IL-6 in Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes

Abstract

After the ligation of the inferior vena cava (IVC) of wild-type (WT) mice, venous thrombi formed and grew progressively until 5 days and resolved thereafter. Concomitantly, intrathrombotic gene expression of Il6 was enhanced later than 5 days after IVC ligation. IL-6 protein expression was detected mainly in F4/80-positive macrophages in thrombus. When Il6-deficient (Il6^-/-) mice were treated in the same manner, thrombus mass was significantly larger than in WT mice. Moreover, the recovery of thrombosed IVC blood flow was markedly delayed in Il6^-/- compared with WT mice. F4/80-positive macrophages in thrombus expressed proteolytic enzymes such as matrix metalloproteinase (Mmp) 2, Mmp9, and urokinase-type plasminogen activator (Plau); and their mRNA expression was significantly reduced in Il6^-/- mice. Consistently, the administration of anti-IL-6 antibody delayed the thrombus resolution in WT mice, whereas IL-6 administration accelerated thrombus resolution in WT and Il6^-/- mice. Moreover, IL-6 in vitro enhanced Mmp2, Mmp9, and Plau mRNA expression in WT-derived peritoneal macrophages in a dose-dependent manner; and the enhancement was abrogated by a specific Stat3 inhibitor, Stattic. Thus, IL-6/Stat3 signaling pathway can promote thrombus resolution by enhancing Mmp2, Mmp9, and Plau expression in macrophages.

Keywords: IL-6; macrophages; matrix metalloproteinases; proteolytic enzymes; thrombosis.

Figure 1

Intrathrombotic expression of IL-6 in wild-type (WT) mice after inferior vena cava (IVC) ligation. (A) Il6 gene expression was examined by real-time reverse transcription (RT)–PCR as described in section Materials and Methods. All values represent mean ± SEM (n = 6). (B) Intrathrombotic IL-6 protein levels were determined by ELISA. All values represent mean ± SEM (n = 6). (C) Immunohistochemical analysis of intrathrombotic IL-6 expression (original magnification, ×100, upper panel; ×400, lower panel). (D) A double-color immunofluorescence analysis of IL-6-expressing cells in the thrombus. The samples were immunostained with the combination of anti-F4/80 mAb and anti-IL-6 pAbs as described in section Materials and Methods. The fluorescent images were digitally merged in the right panel. Representative results from six independent experiments are shown here [original magnification, ×400; blue, nuclear staining by 4′,6-diamidino-2-phenylindole (DAPI)].

Figure 2

Inferior vena cava (IVC) ligation-induced deep vein thrombus formation in wild-type (WT) and Il6^−/− mice. (A) Macroscopic appearance of venous thrombi in WT and Il6^−/− mice at 5 and 10 days after IVC ligation. Representative results from six independent animals are shown here. (B) Thrombus mass of WT and Il6^−/− mice at the indicated time intervals after IVC ligation. All values represent the mean ± SEM (n = 6). **p < 0.01, WT vs. Il6^−/−. (C) Histopathological analyses of venous thrombi obtained from WT and Il6^−/− mice at 5, 10, and 14 days after IVC ligation. Venous thrombi were stained with hematoxylin and eosin (H&E) or Masson trichrome solution (Masson). Representative results from six independent experiments are shown here (original magnification, ×100). (D) Immunohistochemical detection of Col1A2 proteins in the thrombi obtained from WT and Il6^−/− mice at 5, 10, and 14 days after IVC ligation. (E) Intrathrombotic Col1 gene expression in WT and Il6^−/− mice at the indicated time intervals after IVC ligation. All values represent the mean ± SEM (n = 6). *p < 0.05, WT vs. Il6^−/−. (F) Laser Doppler analysis of thrombosed blood flow. All values represent the values mean ± SEM (n = 6 animals). **p < 0.01, WT vs. Il6^−/−.

Figure 3

The effects of IL-6 deficiency on macrophage infiltration and CCL2 expression in thrombus tissues. (A) Immunohistochemical analysis was performed using anti-F4/80 mAb at day 5 in venous thrombus samples from wild-type (WT) and Il6^−/− mice (original magnification, ×400). Representative results from six independent experiments are shown here. (B) F4/80-positive macrophage numbers were determined as described in section Materials and Methods. All values represent the mean ± SEM (n = 6 animals). *p < 0.05, WT vs. Il6^−/− (C) Intrathrombotic expression of Ccl2 mRNA after inferior vena cava (IVC) ligation was determined by real-time reverse transcription (RT)–PCR as described in section Materials and Methods. All values represent the mean ± SEM (n = 6 animals). **p < 0.01, WT vs. Il6^−/−. (D) Immunohistochemical analyses of intrathrombotic CCL2. Representative results from six independent experiments are shown here. (E) Intrathrombotic CCL2-positive cell numbers were determined. All values represent the mean ± SEM. *p < 0.05, WT vs. Il6^−/−.

Figure 4

Intrathrombotic expression of proteolytic enzymes in wild-type (WT) and Il6^−/− mice. (A–C) A double-color immunofluorescence analysis of MMP-2-, MMP-9-, or PLAU-expressing cells in the thrombus. The fluorescent images were digitally merged in the right panel. Representative results from six independent experiments are shown here [original magnification, ×400; blue, nuclear staining by 4′,6-diamidino-2-phenylindole (DAPI)]. (D–F) Intrathrombotic gene expression of Mmp2 (D), Mmp9 (E), and Plau (F) after inferior vena cava (IVC) ligation. Each gene expression was determined by real-time reverse transcription (RT)–PCR as described in section Materials and Methods. All values represent the mean ± SEM (n = 6 animals). *p < 0.05, **p < 0.01, WT vs. Il6^−/−.

Figure 5

The effects of anti-IL-6 antibody (Ab) and recombinant murine IL-6 (rIL-6) in wild-type (WT) mice on thrombus resolution. (A–F) WT mice were intraperitoneally administered with anti-IL-6 as described in section Materials and Methods. (A) Macroscopic appearance of venous thrombi obtained from WT mice treated with anti-IL-6 Ab or control IgG at 10 days after inferior vena cava (IVC) ligation. Representative results from six independent animals are shown here. Thrombus weights (B) and thrombosed blood flow (C) were measured at 10 days after IVC ligation. All values represent the mean ± SEM (n = 6 animals). *p < 0.05 vs. control IgG. (D–F) Intrathrombotic gene expression of Mmp2 (D), Mmp9 (E), and Plau (F) after IVC ligation. Each gene expression was determined by real-time reverse transcription (RT)–PCR as described in section Materials and Methods. All values represent the mean ± SEM (n = 6 animals). *p < 0.05, **p < 0.01, control Ig vs. anti-IL-6. (G–L) WT mice were intraperitoneally administered with rIL-6 as described in section Materials and Methods. (G) Macroscopic appearance of venous thrombi obtained from WT mice treated with rIL-6 or phosphate-buffered saline (PBS) at 10 days after IVC ligation. Representative results from six independent animals are shown here. Thrombus weights (H) and thrombosed blood flow (I) were measured at 10 days after IVC ligation. All values represent the mean ± SEM (n = 6 animals). *p < 0.05, **p < 0.01, PBS vs. rIL-6. (J–L) Intrathrombotic gene expression of Mmp2 (J), Mmp9 (K), and Plau (L) after IVC ligation. Each gene expression was determined by real-time RT-PCR as described in section Materials and Methods. All values represent the mean ± SEM (n = 6 animals). *p < 0.05, **p < 0.01, control PBS vs. rIL-6.

Figure 6

The effects of recombinant murine IL-6 (rIL-6) on the gene expression of Mmp2, Mmp9, and Plau and on Stat3 signaling in peritoneal macrophages. Peritoneal macrophages were obtained from wild-type (WT) mice and were stimulated as described in section Materials and Methods. The gene expression of Mmp2 (A), Mmp9 (B), and Plau (C) was analyzed by real-time reverse transcription (RT)–PCR. All values represent the mean ± SEM (n = 6 independent experiments). *p < 0.05, vs. no stimulation. (D) Western blotting analysis using anti-GAPDH pAbs confirmed that an equal amount of protein was loaded onto each lane. Representative results from six independent experiments are shown here. (E) The ratios of p-Stat3/Stat3 were densitometrically determined and are shown. All values represent means ± SEM (n = 4 independent experiments). (F–H) The effects of anti-IL-6 or Stattic on IL-6-induced gene expression of Mmp2 (F), Mmp9 (G), and Plau (H). Each gene expression was analyzed by real-time RT-PCR. All values represent the mean ± SEM (n = 4 independent experiments). *p < 0.05; **p < 0.01, vs. no stimulation.