The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions methods
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The injured myocardium sequentially recruits circulating Ly-6C hi and -6C lo monocytes through modulation of chemokin...
Several mechanisms might explain the selective mobilization of Ly-6C hi monocytes during phase I and Ly-6C lo monocytes during phase II. For instance, the injured myocardium may recruit Ly-6C hi cells that convert into Ly-6C lo cells at the onset of phase II. The myocardium may also recruit circulating Ly-6C hi and -6C lo cells sequentially; a biphasic recruitment of monocytes could result from alterations in the relative number of these cells in peripheral blood and/or from preferential recruitment to the target tissue. To address these issues, we sought to determine how circulating Ly-6C hi and -6C lo monocytes contribute to the pool of monocytes/macrophages in injured hearts, and we measured the following two parameters at the onset of phase I and II: (a) the relative abundance of Ly-6C hi and -6C lo monocytes in the blood, and (b) the intrinsic probability (i.e., on a cell-c...
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Mice were killed 3 h and on days 1-5, 7, and 16 after myocardial infarction ( n = 3-10 mice per time point). Peripheral blood was drawn via cardiac puncture with citrate solution (100 mM Na-citrate, 130 mM glucose, pH 6.5), as anti-coagulant and mononuclear cells were purified by density centrifugation ( ). Total blood leukocyte numbers were determined using acetic acid lysis solution (3% HEMA 3 Solution II, 94% ddH 2 O, and 3% glacial acetic acid). Spleens were removed, triturated in HBSS (Mediatech, Inc.) at 4°C with the end of a 3-ml syringe, and filtered through nylon mesh (BD Biosciences). The cell suspension was centrifuged at 300 g for 10 min at 4°C. Red blood cells were lysed with ACK lysis buffer, and the splenocytes were washed with HBSS and resuspended in HBSS supplemented with 0.2% (wt/vol) BSA and 1% (wt/vol) FCS. Infarct tissue and healthy hearts we...
mAbs and flow cytometry.
The following antibodies were used: anti-CD90-PE, 53-2.1 (BD Biosciences), -B220-PE, RA3-6B2 (BD Biosciences),-CD49b-PE, DX5 (BD Biosciences), -NK1.1-PE, PK136 (BD Biosciences), -Ly-6G-PE, 1A8 (BD Biosciences), -Ly-6G-FITC, 1A8 (BD Biosciences), CD11b-APC, M1/70 (BD Biosciences), -F4/80-biotin, C1:A3-1 (AbD Serotec), -F4/80-FITC, C1:A3-1 (BioLegend), -CD11c-biotin, HL3 (BD Biosciences), -CD11c-FITC, HL3 (BD Biosciences), -I-A b -biotin, AF6-120.1 (BD Biosciences), -I-A b -FITC, AF6-120.1 (BD Biosciences), -Ly-6C-FITC, AL-21 (BD Biosciences), -Ly-6C-biotin, AL-21 (BD Biosciences), TNF-α-FITC, MP6-XT22 (AbD Serotec), anti-CD43-FITC, S7 (BD Biosciences), -CD62L-FITC, MEL-14 (BD Biosciences), -CD68-FITC, FA-11 (AbD Serotec), -CD86-biotin, GL1 (BD Biosciences), -CD115-PE,...
Monocyte subtype tracking.
Ly-6C hi or -6C lo monocytes were sorted from the spleen of C57BL/6 mice on a FACSAria (BD Biosciences) and labeled with 111 In-oxine according to the manufacturer's protocol (GE Healthcare). In brief, cells were washed with HBSS, spun, and resuspended in 111 In-oxine for 15 min at 37°C, pH 6.5-7.5, and washed twice with HBSS. 111 In-oxine is a widely used, FDA-approved agent that is fully biocompatible as it alters neither cell survival nor function at the doses used (,,, ); hence, we considered labeled monocytes to behave similarly to unlabeled monocytes. Approximately 2 × 10 5 Ly-6C hi or -6C lo monocytes incorporating 20 µCi were injected independently i.v. into C57BL/6 on day 0 (phase I) or 4 d (phase II) after MI. The total amount of activity injected into each animal was measured with a radioisotope calibrator (Capintec, Inc.). After 24 h, recipient anim...
Histopathological analysis.
Histopathology for assessment of healing was performed in mice killed 7 d after MI for the following groups: wild-type C57BL/6 mice, wild-type mice depleted with clodronate-loaded liposomes immediately after coronary ligation, wild-type mice depleted with clodronate-loaded liposomes 3 d after MI, and apoE -/- C57BL6 mice. Hearts were excised and rinsed in PBS and embedded in OCT (Sakura Finetek). Serial 6-µm thick sections were used for immunohistochemical staining for neutrophils (NIMP-R14, Abcam), monocytes/macrophages (Mac-3, M3/84; BD PharMingen), myofibroblasts (α-actin, RB-9010-PO; Neomarkers), and endothelial cells forming capillaries (CD31, MEC13.3; BD Biosciences). Reaction was visualized as a three-step staining procedure in combination with biotinylated secondary antibodies (BA4001; Vector Laboratories) and AEC Substrate kit (Vector Laboratories). To a...
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Several mechanisms might explain the selective mobilization of Ly-6C hi monocytes during phase I and Ly-6C lo monocytes during phase II. For instance, the injured myocardium may recruit Ly-6C hi cells that convert into Ly-6C lo cells at the onset of phase II. The myocardium may also recruit circulating Ly-6C hi and -6C lo cells sequentially; a biphasic recruitment of monocytes could result from alterations in the relative number of these cells in peripheral blood and/or from preferential recruitment to the target tissue. To address these issues, we sought to determine how circulating Ly-6C hi and -6C lo monocytes contribute to the pool of monocytes/macrophages in injured hearts, and we measured the following two parameters at the onset of phase I and II: (a) the relative abundance of Ly-6C hi and -6C lo monocytes in the blood, and (b) the intrinsic probability (i.e., on a cell-cell basis) of circulating Ly-6C hi and -6C lo monocytes being recruited to injured hearts. To this end, we first analyzed peripheral blood mononuclear cells ex vivo; although the total leukocyte numbers remained relatively stable over time ( ), myocardial infarction triggered acute Ly-6C hi monocyto...
Mice were killed 3 h and on days 1-5, 7, and 16 after myocardial infarction ( n = 3-10 mice per time point). Peripheral blood was drawn via cardiac puncture with citrate solution (100 mM Na-citrate, 130 mM glucose, pH 6.5), as anti-coagulant and mononuclear cells were purified by density centrifugation ( ). Total blood leukocyte numbers were determined using acetic acid lysis solution (3% HEMA 3 Solution II, 94% ddH 2 O, and 3% glacial acetic acid). Spleens were removed, triturated in HBSS (Mediatech, Inc.) at 4°C with the end of a 3-ml syringe, and filtered through nylon mesh (BD Biosciences). The cell suspension was centrifuged at 300 g for 10 min at 4°C. Red blood cells were lysed with ACK lysis buffer, and the splenocytes were washed with HBSS and resuspended in HBSS supplemented with 0.2% (wt/vol) BSA and 1% (wt/vol) FCS. Infarct tissue and healthy hearts were harvested, minced with fine scissors, and placed into a cocktail of collagenase I, collagenase XI, DNase I, and hyaluronidase (Sigma-Aldrich) and shaken at 37°C for 1 h, as previously described ( ). Cells were then triturated through nylon mesh and centrifuged (15 min, 500 g, 4°C). Tot...
The following antibodies were used: anti-CD90-PE, 53-2.1 (BD Biosciences), -B220-PE, RA3-6B2 (BD Biosciences),-CD49b-PE, DX5 (BD Biosciences), -NK1.1-PE, PK136 (BD Biosciences), -Ly-6G-PE, 1A8 (BD Biosciences), -Ly-6G-FITC, 1A8 (BD Biosciences), CD11b-APC, M1/70 (BD Biosciences), -F4/80-biotin, C1:A3-1 (AbD Serotec), -F4/80-FITC, C1:A3-1 (BioLegend), -CD11c-biotin, HL3 (BD Biosciences), -CD11c-FITC, HL3 (BD Biosciences), -I-A b -biotin, AF6-120.1 (BD Biosciences), -I-A b -FITC, AF6-120.1 (BD Biosciences), -Ly-6C-FITC, AL-21 (BD Biosciences), -Ly-6C-biotin, AL-21 (BD Biosciences), TNF-α-FITC, MP6-XT22 (AbD Serotec), anti-CD43-FITC, S7 (BD Biosciences), -CD62L-FITC, MEL-14 (BD Biosciences), -CD68-FITC, FA-11 (AbD Serotec), -CD86-biotin, GL1 (BD Biosciences), -CD115-PE, 604B5-2E11 (AbD Serotec), -Mac-3-FITC, M3/84 (BD Biosciences), -Gr-1-PeCy7, RB6-8C5 (BD Biosciences). Strep-PerCP (BD Biosciences) was used to label biotinylated antibodies. Monocytes were identified as CD11b hi (CD90/B220/CD49b/NK1.1/Ly-6G) lo (F4/80/I-A b /CD11c) lo Ly-6C hi/lo. Macro...
Ly-6C hi or -6C lo monocytes were sorted from the spleen of C57BL/6 mice on a FACSAria (BD Biosciences) and labeled with 111 In-oxine according to the manufacturer's protocol (GE Healthcare). In brief, cells were washed with HBSS, spun, and resuspended in 111 In-oxine for 15 min at 37°C, pH 6.5-7.5, and washed twice with HBSS. 111 In-oxine is a widely used, FDA-approved agent that is fully biocompatible as it alters neither cell survival nor function at the doses used (,,, ); hence, we considered labeled monocytes to behave similarly to unlabeled monocytes. Approximately 2 × 10 5 Ly-6C hi or -6C lo monocytes incorporating 20 µCi were injected independently i.v. into C57BL/6 on day 0 (phase I) or 4 d (phase II) after MI. The total amount of activity injected into each animal was measured with a radioisotope calibrator (Capintec, Inc.). After 24 h, recipient animals were killed with CO 2 and hearts were collected. cpm for each heart were measured using a 1480 Wizard Wallac Gamma Counter (PerkinElmer Life and Analytical Sciences, Inc.). Percent-injected dose per gram tissue (%ID/g) was calculated after correcting for decay, excretion, and tail radioactivity...
Histopathology for assessment of healing was performed in mice killed 7 d after MI for the following groups: wild-type C57BL/6 mice, wild-type mice depleted with clodronate-loaded liposomes immediately after coronary ligation, wild-type mice depleted with clodronate-loaded liposomes 3 d after MI, and apoE -/- C57BL6 mice. Hearts were excised and rinsed in PBS and embedded in OCT (Sakura Finetek). Serial 6-µm thick sections were used for immunohistochemical staining for neutrophils (NIMP-R14, Abcam), monocytes/macrophages (Mac-3, M3/84; BD PharMingen), myofibroblasts (α-actin, RB-9010-PO; Neomarkers), and endothelial cells forming capillaries (CD31, MEC13.3; BD Biosciences). Reaction was visualized as a three-step staining procedure in combination with biotinylated secondary antibodies (BA4001; Vector Laboratories) and AEC Substrate kit (Vector Laboratories). To analyze the collagen content of the scar, sections were stained with PSR. Collagen content was visualized with polarized light ( ). Necrotic debris was analyzed on Masson trichrome-stained sections. Neutrophils, macrophages, and myofibroblasts (magnification 400 × ) and collagen content and...
Machine-readable layer
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