Journal: Frontiers in Immunology
Article Title: Crucial Involvement of IL-6 in Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes
doi: 10.3389/fimmu.2019.03150
Figure Lengend Snippet: 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.
Article Snippet: The following mAbs and polyclonal Abs (pAbs) were used for immunohistochemical and double-color immunofluorescence analyses: rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical Company, Japan), mouse anti-IL-6 mAb (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse IL-6 pAbs, rabbit anti-mouse CCL2 pAbs (Novus Biologicals, Centennial, CO), goat anti-mouse MMP-2 pAbs, goat anti-mouse MMP-9 pAbs (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse PLAU (uPA) pAbs, rabbit anti-mouse tPA pAbs, rabbit anti-mouse PAI-1 pAbs (Santa Cruz Biotechnology, Dallas, TX), mouse anti-mouse Col1A2 mAb (Santa Cruz Biotechnology), rabbit anti-mouse myeloperoxidase (MPO) pAbs (Neomarkers, Fremont, CA), rabbit anti-mouse CD3 mAb (Abcam, Tokyo, Japan), cyanine dye 3-conjugated donkey anti-rat IgG pAbs, cyanine dye 3-conjugated donkey anti-goat IgG, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG pAbs, and FITC-conjugated donkey anti-rabbit IgG pAbs (Jackson ImmunoResearch Laboratories, West Grove, PA).
Techniques: Recombinant, Control, Ligation, Gene Expression, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Saline, Quantitative RT-PCR
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![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)].](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9028/pmc07019028/pmc07019028__fimmu-10-03150-g0001.jpg)
![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 −/− .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9028/pmc07019028/pmc07019028__fimmu-10-03150-g0004.jpg)
