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. 2007 Nov 26;204(12):3037-47.
doi: 10.1084/jem.20070885. Epub 2007 Nov 19.

The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions

Matthias Nahrendorf  1 , Filip K Swirski, Elena Aikawa, Lars Stangenberg, Thomas Wurdinger, Jose-Luiz Figueiredo, Peter Libby, Ralph Weissleder, Mikael J Pittet
Affiliations

The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions

Matthias Nahrendorf et al. J Exp Med. .

Abstract

Healing of myocardial infarction (MI) requires monocytes/macrophages. These mononuclear phagocytes likely degrade released macromolecules and aid in scavenging of dead cardiomyocytes, while mediating aspects of granulation tissue formation and remodeling. The mechanisms that orchestrate such divergent functions remain unknown. In view of the heightened appreciation of the heterogeneity of circulating monocytes, we investigated whether distinct monocyte subsets contribute in specific ways to myocardial ischemic injury in mouse MI. We identify two distinct phases of monocyte participation after MI and propose a model that reconciles the divergent properties of these cells in healing. Infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit Ly-6C(hi) and -6C(lo) monocytes via CCR2 and CX(3)CR1, respectively. Ly-6C(hi) monocytes dominate early (phase I) and exhibit phagocytic, proteolytic, and inflammatory functions. Ly-6C(lo) monocytes dominate later (phase II), have attenuated inflammatory properties, and express vascular-endothelial growth factor. Consequently, Ly-6C(hi) monocytes digest damaged tissue, whereas Ly-6C(lo) monocytes promote healing via myofibroblast accumulation, angiogenesis, and deposition of collagen. MI in atherosclerotic mice with chronic Ly-6C(hi) monocytosis results in impaired healing, underscoring the need for a balanced and coordinated response. These observations provide novel mechanistic insights into the cellular and molecular events that regulate the response to ischemic injury and identify new therapeutic targets that can influence healing and ventricular remodeling after MI.

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Figures

Figure 1.
Figure 1.
The ischemic myocardium mobilizes Ly-6Chi and -6Clo monocytes in two distinct phases. Cell suspensions from healthy hearts or infarcts of C57BL/6 mice were stained with anti-CD11b, -CD90, -B220, -CD49b, -NK1.1, -Ly-6G, -Ly-6C, -F4/80, -I-Ab, and -CD11c mAbs. Monocytes/macrophages/dendritic cells were identified as CD11bhi (CD90/B220/CD49b/NK1.1/Ly-6G)lo. (A) Representative dot plots from individual mice within the monocyte/macrophage/dendritic cell gate depict Ly-6Chi monocytes (Ly-6Chi [F4/80/I-Ab/CD11c]lo), Ly-6Clo monocytes (Ly-6Clo [F4/80/I-Ab/CD11c]lo), and macrophages/dendritic cells (Ly-6Clo [F4/80/I-Ab/CD11c]hi) in healthy hearts and within the infarct at specified days after MI. Percentages of cells are shown as the mean ± the SEM. (B) Relative percentage of monocytes and macrophages/dendritic cells. (C) Relative percentage of Ly-6Chi and -6Clo monocytes. (D) Total number of neutrophils per milligram of tissue. (E) Total number of monocytes and macrophages/dendritic cells per milligram of tissue. (F) Total number of Ly-6Chi and -6Clo monocytes per mg tissue. (G) Time course of leukocyte infiltration to infarct. The mean and the SEM are shown. Results are pooled from 6 independent experiments with 3–10 mice per group.
Figure 2.
Figure 2.
The ischemic myocardium sequentially recruits circulating Ly-6Chi and -6Clo monocytes. (A) Number of total leukocytes in peripheral blood of C57BL/6 mice before MI and at the onset of phase I (day 1 after MI) and phase II (day 4). (B) Number of circulating Ly-6Chi and -6Clo monocytes at the same time points. Values in the top right quadrants indicate the ratio between the two subsets. (C) In vivo cardiac accumulation of 111In-oxine-labeled Ly-6Chi and -6Clo monocytes 24 h after adoptive transfer on day 0 (phase I) and day 4 (phase II). Values in the top right quadrants indicate the ratio between the two subsets. (D) Adoptively transferred 111In-oxine–labeled Ly-6Chi monocytes compete with endogenous Ly-6Chi monocytes. Increased numbers of endogenous circulating Ly-6Chi monocytes (x axis) decrease the ratio between transferred and endogenous Ly-6Chi monocytes (label frequency, y axis) that migrated into the peritoneal cavity of mice with peritonitis (Table S1). (E) Pie graph representing the relative proportion of Ly-6Chi and -6Clo monocyte subsets expected to accumulate in infarct tissue, based on the mean abundance of each subset in peripheral blood (B) and the intrinsic capacity of circulating cells from each subset to accumulate at infarct tissue (C). Data represent three independent experiments and are shown as the mean ± the SEM. Table S1 is available at http://www.jem.org/cgi/content/full/jem.20070885/DC1.
Figure 3.
Figure 3.
Sequential recruitment of Ly-6Chi and -6Clo monocytes depends on CCR2 and CX3CR1, respectively. (A) RT-PCR expression profile of MCP-1, MIP-1α, fractalkine, and VCAM-1 in the heart tissue before MI and during phase I (day 1) and II (day 4). (B) Monocyte subset expression profile of CCR2, CCR5, CX3CR1 (for review see reference [29]), and VLA-4 (as assessed by flow cytometry). (C) Number of Ly-6Chi and -6Clo monocytes in infarcts of wild-type, CCR2−/−, and CX3CR1−/− mice in phase I (day 1) and II (day 7). Numbers are normalized to milligrams of tissue. The mean and the SEM are shown. n = 3–5. *, P < 0.05.
Figure 4.
Figure 4.
Ly-6Chi and -6Clo monocytes commit for different functions. Relative phagocytic activity, MMP activity, cathepsin activity, TNF-α production (with or without stimulation with PMA/ionomycin), and VEGF expression in Ly-6Chi monocytes (shaded histogram), Ly-6Clo monocytes (black line), and control CD11b− cells (gray line) retrieved from the ischemic myocardium. The mean and the SEM are shown. n = 3–5. *, P < 0.05; **, P < 0.01.
Figure 5.
Figure 5.
In vivo relevance of the biphasic response to healing. (A) Representative dot plots from individual mice depict Ly-6Chi monocytes (bottom right), Ly-6Clo monocytes (bottom left), and macrophages/dendritic cells (top left) at the infarct after depletion of circulating monocytes with clodronate-loaded liposomes (Clo-Lip). Mice were analyzed on day 4 (Clo-Lip injection on day 0; depletion of phase I) and on day 7 (Clo-Lip injection on day 3; depletion of phase II). Control animals (Ø) did not receive Clo-Lip. Percentages of cells are shown as the mean ± the SEM. (B) Total number of monocytes per milligram of tissue at the infarct before MI, at the end of phase I (day 4) and during phase II (day 7), in the absence (–) or presence (+) of Clo-Lip. The mean and the SEM are shown. n = 3–5. (C) Immunohistochemical analysis 7 d after MI depicts representative infarct sections from undepleted (Ø), phase I–depleted (I), and phase II–depleted (II) C57BL/6 mice. Representative sections stained with anti–Mac-3, anti–NIMP-R14, Masson, α-actin, PSR, and anti-CD31 are shown. The mean and the SEM are shown. n = 7. (D) Immunohistochemistry depicts representative infarct sections from apoE−/− mice 7 d after MI. The mean and the SEM are shown. n = 5. *, P < 0.05; **, P < 0.01. Bars: (Mac-3, NIMP-R14, α-actin, and CD31) 20 μm; (PSR and Masson) 100 μm.
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