Fig. 6

The effect of IL-1β and SAA3 on the recruitment of MMP9⁺myeloid cells and the expression of MMP9 in BMDCs. (A-B) The number of MMP9⁺ cells in the pre-metastatic lung of Ctrl and Hepa1-6-bearing mice, WT and Il1b^−/− mice, and lenti-Ctrl and Lenti-Saa3 viruses-infected Il1b^−/− mice. Scale bar, 25 μm. (C) Colocalization of CD11b and MMP9 in the pre-metastatic lung. Scale bar, 10 μm. (D) IL-1β and SAA3 stimulation had no effect on Mmp9 mRNA in BMDCs. Mouse BMDCs were stimulated with IL-1β (10 ng/ml) and SAA3 (3 µg/ml) for 12 h followed by qPCR analysis of Mmp9 mRNA. (E-F) IL-1β and SAA3 stimulation had no effect on the protein level, secretion and activity of MMP9 in BMDCs. Mouse BMDCs were stimulated with IL-1β and SAA3 for 24 h followed by immunoblotting analysis of MMP9 in cell lysate and supernatant (SN) of BMDCs (E). MMP2 and MMP9 activities in the supernatant were detected by gelatin zymography (F). (G) Schematic for experimental design in H-I. (H) IL-1β enhanced the chemotactic activity of MLE-12-conditioned medium (MCM) for BMDCs. (I) IKK 16 blocked the chemotactic activity of IL-1β-stimulated-MCM. (J) Silencing of Rela or SAA3 in MLE-12 restrained the chemotactic activity of IL-1β-stimulated-MCM. For H-J, MLE-12 cells were stimulated with IL-1β for 24 h, and then BMDCs were allowed to migrate for 12 h. (K) Schematic for experimental design in L-M. Saline or SAA3 (1.2 µg in 60 ul saline) were injected intratracheally to the lung of mice. Twelve hours later, lungs were harvested. (L) Intratracheal administration of SAA3 recruited MMP9⁺ cells to the lung. MMP9⁺ cells were detected by IHC (left). The number of MMP9⁺ cells was shown (right). Scale bar, 50 μm. (M) Intratracheal administration of SAA3 recruited MMP9⁺ myeloid cells to the lung. Double IF staining of CD11b (red) and MMP9 (green) in the lung of mice stimulated with SAA3 intratracheally for 12 h. Scale bar, 10 μm. Data are displayed as the mean ± SEM; (B, H, I, L) unpaired Student’s t test; (D and J) one-way ANOVA with Dunnett’s correction. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant