Macrophage activation switching: an asset for the resolution of inflammation

Abstract

Macrophages play a central role in inflammation and host defence against microorganisms, but they also participate actively in the resolution of inflammation after alternative activation. However, it is not known whether the resolution of inflammation requires alternative activation of new resting monocytes/macrophages or if proinflammatory activated macrophages have the capacity to switch their activation towards anti-inflammation. In order to answer this question, we first characterized differential human macrophage activation phenotypes. We found that CD163 and CD206 exhibited mutually exclusive induction patterns after stimulation by a panel of anti-inflammatory molecules, whereas CCL18 showed a third, overlapping, pattern. Hence, alternative activation is not a single process, but provides a variety of different cell populations. The capacity of macrophages to switch from one activation state to another was then assessed by determining the reversibility of CD163 and CD206 expression and of CCL18 and CCL3 production. We found that every activation state was rapidly and fully reversible, suggesting that a given cell may participate sequentially in both the induction and the resolution of inflammation. These findings may provide new insight into the inflammatory process as well as new fields of investigation for immunotherapy in the fields of chronic inflammatory diseases and cancer.

Fig. 1

Morphology of differentially stimulated macrophages. Eight-day differentiated monocyte-derived macrophages (MDM) were cultured for 5 days in the presence of medium alone (NT) or the specified stimulation. Cells were then stained with May–Grünwald–Giemsa stain. Original magnification: × 200.

Fig. 2

(a) Effect of pro- and anti-inflammatory stimuli on CD163 and CD206 expression. Mature macrophages were treated from day 8 to day 12 with a panel of pro- and anti-inflammatory molecules. Levels of CD163 and CD206 expression at the membrane were then assessed by quantitative flow cytometry. Results are expressed as a percentage of untreated cells mean equivalent fluorochrome (MEF) ± s.e.m. for five independent donors. *P < 0·05, Student's unpaired t-test. (b) Effect of pro- and anti-inflammatory stimuli on the production of CCL3 and CCL18. Mature macrophages were treated from day 8 to day 12 with a panel of pro- and anti-inflammatory molecules, and CCL3 and CCL18 production was assessed by enzyme-linked immunosorbent assay (ELISA). Concentration values were normalized on the basis of tetrazolium salt (MTT) assay results, to account for variations in cell viability. Results are expressed as a percentage of untreated cells cytokine production ± s.e.m. for five independent donors. *P < 0·05, Student's unpaired t-test. (c) Summary of the effect of pro- and anti-inflammatory stimuli on tested markers showing the data displayed in (a) and (b). The effect of the different stimuli on the four markers tested are indicated as follows: 0: no effect; + and ++ : slight and strong increase, respectively; – and –: slight and strong decrease, respectively. Only significant effects are considered. The chosen cut-off for a strong versus slight effect are twofold modulations for flow cytometry and threefold for cytokine expression.

Fig. 3

(a) Effect of pro- and anti-inflammatory stimuli on human leucocyte antigen (HLA)-DR expression. Mature macrophages were treated from day 8 to day 12 with a panel of pro- and anti-inflammatory molecules. Level of HLA-DR expression at the membrane was then assessed by quantitative flow cytometry. Results are expressed as a percentage of untreated cells mean equivalent fluorochrome (MEF) ± s.e.m. for five independent donors. *P < 0·05, Student's unpaired t-test. (b) Effect of pro- and anti-inflammatory stimuli on phagocytotic activity. Mature macrophages were treated from day 8 to day 12 with a panel of pro- and anti-inflammatory molecules. Phagocytotic activity was assessed by the capacity of macrophage to engulf fluorescein-labelled Escherichia coli. Results are expressed as a percentage of untreated cells fluorescence ± s.e.m. for three independent donors. *P < 0·05, Student's unpaired t-test.

Fig. 4

Plasticity of macrophage morphology. Mature macrophages were treated for 5 days by different pro- or anti-inflammatory stimuli (first round, left-hand panels). Cells from one well per condition were then stained with May–Grünwald–Giemsa stain. The remaining wells were then washed and cultured for another 5 days in medium alone (central panels, NT) or treated with molecules different from that used during the first round (right-hand panels). Cells were then stained with May–Grünwald–Giemsa stain.

Fig. 5

Reversibility of macrophage activation phenotype. Macrophages were treated with a cytokine or dexamethasone from day 8 until day 12 or from day 8 to day 11. They were then washed and cultured for another 3 or 4 days in medium alone (→) or were counterstimulated with another cytokine. Levels of CD163 and CD206 expression at the membrane were assessed by quantitative flow cytometry on days 12 and 16. CCL3 and CCL18 concentrations in the supernatant were quantified by enzyme-linked immunosorbent assay (ELISA) on days 11 and 14. Data are expressed as mean equivalent fluorochrome bound per cell (membrane expression, left-hand panel), or as pg/ml normalized according to the tetrazolium salt (MTT) test (chemokine secretion, right-hand panel). Data are representative of three independent experiments.