Endothelial Monocyte-Activating Polypeptide II Mediates Macrophage Migration in the Development of Hyperoxia-Induced Lung Disease of Prematurity

Abstract

Myeloid cells are key factors in the progression of bronchopulmonary dysplasia (BPD) pathogenesis. Endothelial monocyte-activating polypeptide II (EMAP II) mediates myeloid cell trafficking. The origin and physiological mechanism by which EMAP II affects pathogenesis in BPD is unknown. The objective was to determine the functional consequences of elevated EMAP II levels in the pathogenesis of murine BPD and to investigate EMAP II neutralization as a therapeutic strategy. Three neonatal mouse models were used: (1) BPD (hyperoxia), (2) EMAP II delivery, and (3) BPD with neutralizing EMAP II antibody treatments. Chemokinic function of EMAP II and its neutralization were assessed by migration in vitro and in vivo. We determined the location of EMAP II by immunohistochemistry, pulmonary proinflammatory and chemotactic gene expression by quantitative polymerase chain reaction and immunoblotting, lung outcome by pulmonary function testing and histological analysis, and right ventricular hypertrophy by Fulton's Index. In BPD, EMAP II initially is a bronchial club-cell-specific protein-derived factor that later is expressed in galectin-3⁺ macrophages as BPD progresses. Continuous elevated expression corroborates with baboon and human BPD. Prolonged elevation of EMAP II levels recruits galectin-3⁺ macrophages, which is followed by an inflammatory state that resembles a severe BPD phenotype characterized by decreased pulmonary compliance, arrested alveolar development, and signs of pulmonary hypertension. In vivo pharmacological EMAP II inhibition suppressed proinflammatory genes Tnfa, Il6, and Il1b and chemotactic genes Ccl2 and Ccl9 and reversed the severe BPD phenotype. EMAP II is sufficient to induce macrophage recruitment, worsens BPD progression, and represents a targetable mechanism of BPD development.

Keywords: bronchopulmonary dysplasia; endothelial monocyte-activating polypeptide II; inflammation; lung.

Figure 1.

Endothelial monocyte-activating polypeptide II (EMAP II) secreted by airway-conducting epithelial cells of bronchopulmonary dysplasia (BPD) mice recruits macrophages. (A) Experimental schematic of neonatal mouse oxygen exposure to induce BPD. (B) EMAP II protein expression and (C) quantification in whole-lung lysates of normoxia and hyperoxia mice (normalized to β-actin, pooled samples of at least n = 3 for Day 3, n = 2 for Day 30, n = 3–4 for Day 10, n = 6–10 for other days; at least two independent experiments). Main effect of oxygen, **P = 0.0000322, interaction of oxygen:age, P = 0.788. (D) Representative images of immunohistochemical co-staining for EMAP II expression (red) and Clara cell secretory protein (green). Purple indicates co-expression. Scale bar, 20 μm. (E) Representative images of immunohistochemical staining for EMAP II expression (red) and galectin-3 (green). Purple indicates co-expression. Scale bar, 100 μm. Note that compared with lungs exposed to normoxia, those exposed to hyperoxia and harvested on Day 15 were severely dysplastic so that both the bronchial epithelium and the alveoli could not be imaged in the same capture field, although the same magnification as that used in other images was used. (F) EMAP II concentration in tracheal aspirates by immunoblotting and quantification. Main effect of day, P = 0.0187; interaction of oxygen:day, P = 0.711; n = 3 per day. Data are presented as mean ± SEM. ACTB, protein name of β-actin.

Figure 2.

EMAP II protein mediates macrophage chemoattraction in vivo. (A–E) Mice treated with either EMAP II or vehicle (injection) from Days 3 to 15. (A) Schematic of EMAP II treatment in neonatal mice. (B) Representative immunohistochemical images of distal alveoli in lung sections of Day 15 mice showing macrophage (galectin-3, red) and (C) quantification by blinded analysis of galectin-3–positive cells per HPF (n = 4, ****P = 0.00000235). (D and E) Immunoblot probed for IL1β in whole lung lysate of Day 15 mice (normalized to β-actin, **P = 0.01, n = 4). Scale bar, 100 μm. Results are representative of four (B and C) or two (D and E) independent experiments. Data are presented as mean ± SEM. HPF, high-powered field; PFT, pulmonary function tests; rEMAP II, recombinant EMAP II.

Figure 3.

Lungs treated with EMAP II present BPD–like phenotype. The experimental design is the same as in Figure 2A. (A) Comparison of distal alveolar structure in inflation-fixed lungs (25 mm Hg) of mice killed on Day 15 by (B) mean linear intercept (****P = 0.00000719), and (C) RAC by blinded observer analysis (n = 8, *P = 0.03337). (D) Biophysical parameters of lung function compliance, resistance, and elastance were assessed (n = 3–6; *P = 0.011, **P = 0.023, *P = 0.008, respectively) and representative pulmonary flow loops presented. (E) Right ventricular hypertrophy quantified by Fulton’s index (n = 6 mice per group, **P = 0.00520) and (F) representative deposition of perivascular elastin (arrows) in distal lung tissue sections stained with Masson’s trichrome. Scale bars, (A) 100 μm and (F) 10 μm. Results are representative of three (A–C and F) or two (D and E) independent experiments. Diamonds are data points past 75%. Data are presented as mean ± SEM. LV, left ventricular; RAC, radial alveolar count; RV, right ventricular.

Figure 4.

Neutralizing EMAP II limits macrophage recruitment both in vitro and in vivo. (A) Quantification of Transwell-migrated macrophages in response to EMAP II vehicle (phosphate-buffered saline), nonspecific IgG, and EMAP II preincubated with various concentrations of anti-EMAP II (n = 2–4 replicates, P = 0.0044, one-way analysis of variance across treatments). (B) Schematic of neonatal hyperoxia exposure protocol used to induce BPD, inj. of anti-EMAP II or IgG. (C) Representative immunohistochemical images of distal alveoli in lung sections showing macrophages (galectin-3, red) and (D) galectin-3–positive cells per HPF, quantified by blinded analysis (n = 4 mice, ***P = 0.000457). Results are representative of samples collected from four (D and E) and two (A) independent experiments. Data are presented as mean ± SEM. Inj., injection.

Figure 5.

Rescued lung structure and function of BPD mice treated with anti-EMAP II. The experimental design is the same as in Figure 4C. (A) Comparison of distal alveolar structure in inflation-fixed lungs (25 mm Hg) of mice killed on Day 15 by (B) mean linear intercept and (C) radial alveolar count by blinded observer analysis (n = 8; ****P = 0.0337, P = 0.08898). (D) Biophysical parameters of lung function compliance, resistance, and elastance were assessed among hyperoxia groups (n = 6–8 mice; ***P = 0.00642, ***P = 0.000209, ***P = 0.00183) and representative pulmonary flow loops presented. (E) RV hypertrophy quantified by Fulton index (ratio of RV weight to LV plus septal weight, n = 3, **P = 0.00537) and (F) representative deposition of perivascular elastin (arrows) in distal lung tissue sections stained by Masson’s trichrome. Scale bars, (A) 100 μm and (F) 10 μm. Results are representative of four (A–F) or two (D–F) independent experiments. Diamonds are data points past 75%. Data are presented as mean ± SEM.

Figure 6.

Neutralizing EMAP II limited macrophage recruitment and caused inflammation induced by high oxygen to subside. (A and B) Representative immunoblot probed for IL1β in whole lung lysate of Day 15 mice and quantified (n = 3, normalized to β-actin, *P = 0.0498). (C) mRNA expression of inflammatory Il1b, Il6, and Tnf and chemokine genes Ccl2 and Ccl9 in lungs determined by quantitative polymerase chain reaction calculated on the basis of Hprt, Eef2, and Rpl13a expression (n = 6–7; *P = 0.0195 [II1b], **P = 0.0489 [Tnfa], **P = 0.00594 [II6], **P = 0.00227 [Ccl2], P = 0.0889 [CcI9]). Samples are from three independent experiments (A–C). Data are presented as mean ± SEM. IB, immunoblot.