Table 1.
Primers for quantitative RT-PCR.
Figure 1.
Effects of classical and alternative macrophage activation on MMP and TIMP mRNA levels.
Steady state mRNA levels were measured by qPCR in M-CSF differentiated macrophages derived from unselected human CD16+/− monocytes (M0) or classically activated with LPS and IFNγ (M1) or alternatively activated with IL-4 (M2) for 18 hours. Panels A and B show more and less abundant mRNAs, respectively. Values are means ± SEM * p<0.05 vs M0 (n = 7).
Table 2.
Comparison of quantitative RT-PCR results among macrophage phenotypes from adhered mononuclear cells and CD16- monocytes (n = 7).
Figure 2.
Effects of classical activators on MMP and TIMP mRNA levels.
Steady state mRNA levels were measured by qPCR in M-CSF differentiated macrophages derived from unselected human CD16+/− monocytes. Cells were kept un-stimulated or treated with LPS (100 ng/mL), IL-1α (20 ng/mL) TNFα (10 ng/mL), or IFNγ (100 ng/mL) for 18 hours. Steady state mRNA levels were measured and the ratio between treated cells and M-CSF alone was calculated. Values are means ± SEM * p<0.05, n = 7.
Figure 3.
Activation of MAP kinases and effects of inhibitors.
M-CSF differentiated macrophages derived from unselected human CD16+/− monocytes (M0) were pre-treated for 45 min with 10 µM PD98059, SB20380, SP600125 or vehicle (0.1% v/v DMSO) and then some were classically activated with LPS and IFNγ (M1). Phospho and total proteins were measured by western blotting. Levels were normalised to total proteins levels. (A) ERK1/2 with and without PD98059, (B) p38 MAPK with and without SB20380, (C) JNK with and without SP600125, (D) Steady-state mRNA levels of MMPs and other genes were measured after 18 hours of stimulation with LPS and IFNγ. Values are log weighted means and 95% confidence intervals * p<0.05 compared to without inhibitor (n = 7).
Figure 4.
Activation of NF-κB and effects of SC514.
M-CSF differentiated macrophages derived from unselected human CD16+/− monocytes (M0) were pre-treated for 45 min with 40 µM SC514 or vehicle (0.1% v/v DMSO) and then some were classically activated with LPS and IFNγ (M1) for 45 minutes. Total IκB proteins and phospho- p65 RelA were measured by western blotting. Levels were normalised to GAPDH as shown. Values are means ± SD * p<0.05 compared to M0 (n = 5). (A) IκB protein (lower band) and phospho- IκB (upper band), (B) Phosho-p65 RelA, (C) Steady-state mRNA levels of MMPs and other genes were measured after 18 hours of stimulation with LPS and IFNγ. Values are log weighted means and 95% confidence intervals * p<0.05 compared to without inhibitor (n = 7).
Figure 5.
Activation of PI-3K and effects of LY294002.
M-CSF differentiated macrophages derived from unselected human CD16+/− monocytes (M0) were pre-treated for 45 min with 10 µM LY294002 and then some were classically activated with for 45 minutes LPS and IFNγ. (A) PI-3-kinase activity as phospho-AKT with and without LY294002 measured by western blotting. Values normalised to total AKT are means ± SD * p<0.05 compared to without inhibitor. (B) Steady-state mRNA levels of MMPs and other genes were measured after 18 hours of no stimulation (M0 conditions). (C) Steady-state mRNA levels of MMPs and other genes were measured after 18 hours of stimulation with LPS and IFNγ (M1 conditions). Panels B and C values are log weighted means and 95% confidence intervals * p<0.05 compared to without inhibitor (n = 7).
Figure 6.
Effects of classical and alternative macrophage activation on MMP protein levels.
M-CSF differentiated macrophages derived from unselected human CD16+/− monocytes (M0) or classically activated with LPS and IFNγ (M1) or alternatively activated with IL-4 (M2) for 48 hours. Where indicated cells were pre-treated with 10 µM PD98059 (PD), SB20380 (SB), SP600125 (SP), or LY 294002 (Ly) or 40 µM SC514 (SC) or vehicle (0.1% v/v DMSO). (A) After antibody capture MMP-1 activity was measured with a quenched fluorescent substrate. (B) Zymography for MMP-2 and MMP-9 in conditioned medium. (C) COX-2 in conditioned medium by western blotting. (D) MMP-10 in conditioned medium by western blotting. Values are means ± SD * p<0.05 vs M0 n = 5.
Figure 7.
M-CSF differentiated macrophages derived from unselected human CD16+/− monocytes were untreated or classically activated with IFNγ. (A) Phospho and total STAT-1 were measured after 45 minutes of IFNγ with and without JAK-2 inhibitor, 10 or 20 µM ABSPI as indicated, or vehicle (0.1% v/v DMSO). (B) Steady-state mRNA levels of MMPs and other genes were measured after 18 hours of stimulation as in panel A. Values are means ± SEM * p<0.05 compared to without inhibitor (n = 4).
Figure 8.
Co-localisation of MMPs with markers of classical activation in human atherosclerotic plaques.
(A) Serial sections were stained by peroxidase with anti-CD68 (A. CD68) and by dual immuno-fluorescence with anti-MMP-1 or anti-MMP-10 (red) together with anti-COX-2 or p65RelA (green) as shown, and the images were superimposed digitally. Nuclear localised p65RelA (NF-kB*) was detected because of the shift in nuclear counterstain colour from dark blue (DAPI alone) to sky blue (blue plus green) in the superimposed image. (B) Areas rich in macrophages identified from the peroxidase stain were identified in the serial section. Cells in the whole field were counted and a percentage of each staining pattern calculated. Values are means ± SEM, n = 6, * p<0.05.
Figure 9.
A map of regulation of the MMP system in human macrophages.
Pink zone: genes up-regulated by classical activators LPS and IFNγ as shown in the boxes below. Green zone: genes up-regulated during alternative activation (IL-4). White zone: no or minor regulation. Red symbols: up-regulated during classical activation. Blue symbols: up-regulated during alternative activation. Red-blue symbols: similar regulation by classical and alternative activation. Green symbols: scarcely regulated. The boxes above indicate dependence of MMP or TIMP activity on the specified kinases or transcription factors.