Local apoptosis mediates clearance of macrophages from resolving inflammation in mice

Abstract

Chronic inflammatory diseases such as atherosclerosis are characterized by an accumulation of macrophages. To design therapies that would reduce macrophage burden during disease, understanding the cellular and molecular mechanisms that regulate macrophage removal from sites of resolving inflammation is critical. Although past studies have considered the local death of macrophages or the possibility that they emigrate out of inflammatory foci, methods to quantify death or emigration have never been employed. Here, we applied quantitative competition approaches and other methods to study resolution of thioglycollate-induced peritonitis, the model in which earlier work indicated that emigration to lymph nodes accounted for macrophage removal. We show that migration to lymph nodes occurred in a CC chemokine receptor 7-independent manner but, overall, had a quantitatively minor role in the removal of macrophages. Blocking migration did not significantly delay resolution. However, when macrophages resistant to death were competed against control macrophages, contraction of the macrophage pool was delayed in the apoptosis-resistant cells. These data refute the concept that macrophages are dominantly cleared through emigration and indicate that local death controls macrophage removal. This finding alters the emphasis on which cellular processes merit targeting in chronic diseases associated with accumulation of macrophages.

Figure 1

Phenotype and dynamics of resident and inflammatory macrophages during acute peritonitis. Quantification of total leukocytes (A) or granulocytes (neutrophils and eosinophils) (B) in the peritoneal cavity at the steady state and during a 14-day period after i.p. administration of thioglycollate. (C) Fluorescence-activated cell sorter (FACS) plots illustrating the gating strategy used for identification of macrophages at the steady state and during a 14-day period after i.p. administration of thioglycollate. Macrophages were found in CD115^hi gates (first row) at all times; CD115 expression on these cells was reduced at day 1. Three macrophage populations were discerned on the basis of F4/80 intensity, and these populations are depicted in the second through the fourth rows of the dot plots. F4/80^low macrophages (solid-line gates) and F4/80^hi macrophages (finely dotted gates) were resident macrophages, and inflammatory peritoneal macrophages (wide-dashed gates) appeared only in response to thioglycollate. (D) FACS plots illustrating the gating of inflammatory macrophages and corresponding cell counts in the peritoneum of CCR2-deficient mice and controls 1 and 5 days after initiation of peritonitis. (E) Comparison of F4/80, CD36, CD11c, and MHC-II cell surface expression levels between resident (F4/80^low and F4/80^hi) and inflammatory (F4/80^int) macrophages 5 days after initiation of peritonitis. (F) Quantification of resident macrophages and (G) inflammatory macrophages in the peritoneal cavity during the steady state and a 14-day period after i.p. administration of thioglycollate. Data are derived from at least 2 experiments performed with 5 replicates per experimental condition.

Figure 2

Gene expression analysis of inflammatory macrophage populations. (A) Hierarchical clustering of steady-state and inflammatory macrophage populations (3 replicates for each populations) based on the 15% of genes with the greatest variability. (B) Volcano plot (P value vs fold change, with each dot representing 1 probe set), highlighting the 76 genes upregulated in MHC-II⁻ inflammatory macrophages (green) and the 93 genes upregulated in MHC-II⁺ inflammatory macrophages (purple). Some differentially expressed probe sets were tagged with the gene name they correspond to. (C) Heat map depicting genes differentially expressed (P < .05; fold > 2) between MHC-II⁻ and MHC-II⁺ inflammatory macrophages. (D) Pathways associated with differentially expressed genes between MHC-II⁻ and MHC-II⁺ inflammatory macrophages.

Figure 3

Kinetics of inflammatory macrophage migration to LNs. (A) FACS plots depicting the appearance of inflammatory macrophages (F4/80⁺CD36⁺ or F4/80⁺ MHC-II⁺) in the mediastinal LN over the course of 8 days after i.p. administration of thioglycollate. (B) Total LN cells (Left) or total cells with F4/80⁺ CD36⁺ cells gated out (Right) based on cell surface expression of CD11c and MHC-II. Bottom plot overlays F4/80⁺ CD36⁺ gated cells on total LN cells, plotted to show CD11c vs MHC-II. (C) Labeling of peritoneal inflammatory macrophages 24 hours after i.p. injection of the phagocytic tracer dye PKH26 in mice inflamed 2 days earlier by thioglycollate injection. (D) PKH26⁺ cells were identified in the mediastinal LN 3 days after injection of PKH26 in the peritoneal cavity (5 days after inflammation was induced by thioglycollate) and analyzed for F4/80 and CD36 cell surface expression. Far-right plot shows PKH26 levels after gating on all F4/80⁺ CD36⁺ LN cells. (E) Inflammatory macrophages were retrieved from CD45.1 mice that had been injected 1 day earlier with thioglycollate. These macrophages were adoptively transferred into CD45.2 mice at the same stage of inflammation. CD45.1⁺ inflammatory macrophages were gated in the mediastinal LN cell suspension (left FACS plot) 4 days later and analyzed for CD36 expression. (F-H) Inflammatory macrophages in the peritoneum, draining LN and omentum at different times after induction of peritonitis by thioglycollate (n = 4-6 mice per group per time point). Data are representative of at least 2 independent experiments.

Figure 4

Blocking inflammatory macrophages migration marginally impaired their clearance during resolution. (A) Heat map showing the expression of chemotactic receptors in MHC-II⁻ and MHC-II⁺ inflammatory macrophages 5 days after intraperitoneal administration of thioglycollate (3 replicates were generated for each population). (B) Kinetics of MHC-II⁻ and MHC-II⁺ inflammatory macrophages during the course of thioglycollate-induced peritonitis in CCR7-deficient mice and controls (n = 5 mice per group per time). (C) Accumulation of inflammatory macrophage in the mediastinal LN of CCR7-deficient mice and controls 5 days after intraperitoneal administration of thioglycollate (n = 5 mice per group). (D) Mobilization to the mediastinal LN of adoptively transferred F4/80⁺ CD36⁺ CD45.1⁺ inflammatory macrophages in CD45.2 mice injected with thioglycollate in the presence of PTX or iPTX (n = 5-7 per group). To maximize the recovery, cells and PTX or iPTX (single injection) were injected in the peritoneum at day 2, and LNs were analyzed at day 5. (E) Baseline number of inflammatory macrophage in the peritoneum at day 5 and numbers of macrophages recovered at day 8 after treatment with either active (PTX) or inactive (iPTX) pertussis toxin at day 5. Each symbol represents one mouse. (F) The number of F4/80⁺CD36⁺ macrophages recovered in the mediastinal LN at day 8 from the experiment in panel E, illustrating that active pertussis toxin prevented emigration to the LN.

Figure 5

Inflammatory macrophages contraction during resolution is controlled by apoptotic cell death. (A) FACS plot illustrating the gating strategy used for neutrophils (ii, CD115⁻ Gr-1⁺) and Ly-6C^hi monocytes (i, CD115⁺ Gr-1⁺) in the inflamed peritoneal cavity. Quantification of neutrophils (B) as well as Ly-6C^hi monocytes (C) up to day 5 in mice injected with thioglycollate (n = 5 per time). (D) Quantification of annexin V staining in inflammatory macrophages from day 3 to 8 after intraperitoneal administration of thioglycollate (n = 5 per time). (E-F) Ratios of F4/80^int inflammatory macrophages competed after injection into thioglycollate-inflamed peritoneum at day 5 (injected) and recovered at day 8 (recovered). Two competitions are shown: between macrophages derived from WT and CD68-Bcl2 (Mφ-hBcl2^Tg) transgenic mice and between macrophages derived from WT and Bim^−/− mice. Each symbol represents data from 1 mouse. (G) Quantification of F4/80^int inflammatory macrophages in the peritoneal of irradiated recipient mice transplanted with bone marrow from CD68-Bcl2 (Mφ-hBcl2^Tg) or WT mice during a 12-day period after i.p. administration of thioglycollate. (H) Heat maps depict gene expression patterns of mRNA transcripts that mediate or are induced in response to efferocytosis. Three replicates are shown for different macrophage populations.