Subcutaneous administration of neutralizing antibodies to endothelial monocyte-activating protein II attenuates cigarette smoke-induced lung injury in mice

Abstract

Proapoptotic and monocyte chemotactic endothelial monocyte-activating protein 2 (EMAPII) is released extracellularly during cigarette smoke (CS) exposure. We have previously demonstrated that, when administered intratracheally during chronic CS exposures, neutralizing rat antibodies to EMAPII inhibited endothelial cell apoptosis and lung inflammation and reduced airspace enlargement in mice (DBA/2J strain). Here we report further preclinical evaluation of EMAPII targeting using rat anti-EMAPII antibodies via either nebulization or subcutaneous injection. Both treatment modalities efficiently ameliorated emphysema-like disease in two different strains of CS-exposed mice, DBA/2J and C57BL/6. Of relevance for clinical applicability, this treatment showed therapeutic and even curative potential when administered either during or following CS-induced emphysema development, respectively. In addition, a fully humanized neutralizing anti-EMAPII antibody administered subcutaneously to mice during CS exposure retained anti-apoptotic and anti-inflammatory effects similar to that of the parent rat antibody. Furthermore, humanized anti-EMAPII antibody treatment attenuated CS-induced autophagy and restored mammalian target of rapamycin signaling in the lungs of mice, despite ongoing CS exposure. Together, our results demonstrate that EMAPII secretion is involved in CS-induced lung inflammation and cell injury, including apoptosis and autophagy, and that a humanized EMAPII neutralizing antibody may have therapeutic potential in emphysema.

INTRODUCTION

Pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD) and a major cause of morbidity and mortality (24) that has no available curative pharmacological interventions. The most recognized cause of COPD is chronic exposure to cigarette smoke (CS) (38). CS exposure injures pulmonary endothelial cells that may culminate in cell death, which, along with inflammation, contributes to destruction of lung parenchyma, a hallmark of emphysema (7, 8, 29). Strategies aimed at reducing endothelial cell death and lung inflammation may therefore be therapeutically useful in emphysema.

We have previously demonstrated that endothelial monocyte activating protein 2 (EMAPII), a proinflammatory cytokine that causes endothelial-cell apoptosis (3, 36), plays a key role in the pathogenesis of emphysema-like airspace enlargement in mice (6). EMAPII is present in the cytosol of cells (30), but stresses such as hypoxia, lipopolysaccharide, viral infections, or CS increase EMAPII levels and promote its extracellular secretion (1, 6, 13, 17, 18, 19). Our previous reports of a key role of EMAPII in emphysema development were in large part based on beneficial effects of a treatment with a rat monoclonal anti-EMAPII antibody administered via inhalation for 3 wk, during the 3rd mo of a 4-mo-long CS exposure (6). To expand preclinical assessments of the usefulness of this approach, we asked in the current study 1) whether a subcutaneous mode of administration of the rat anti-EMAPII antibody is effective against CS-induced emphysema-like disease, 2) whether a beneficial treatment effect is seen in another strain of mice such as C57BL/6, and 3) if a humanized form of anti-EMAPII antibody retains the ability to ameliorate CS-induced lung injury. We also assessed how anti-EMAPII antibody therapy impacts more recently identified mechanisms of CS-induced injury other than apoptosis, such as autophagy. Our studies demonstrate for the first time that subcutaneous treatment with anti-EMAPII antibody ameliorates CS-induced lung inflammation, apoptosis, autophagy, and emphysema-like morphofunctional changes in mice.

MATERIALS AND METHODS

Reagents.

All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.

Animal studies.

Animal studies were approved by the Animal Care and Use Committee of Indiana University, Indianapolis, IN, and National Jewish Health, Denver, CO. Female C57BL/6 mice and DBA/2J were obtained from the Jackson Laboratory (Bar Harbor, ME).

CS exposure and anti-EMAPII antibody administration.

CS exposure of mice was performed for 5 h/day, 5 days/wk, using a total body exposure with the TE-10E smoking apparatus (Teague Enterprises, Davis, CA) (4). C57BL/6 or DBA/2J mice (2 mo old, female, n = 5–15/group) were exposed to ambient air control (AC) or to CS. CS exposure consisted of 11% mainstream and 89% side stream and was delivered at a concentration of 100 mg/m³ total particulate matter, which models the exposure of first-hand smokers. Control mice underwent the same sleep cycle disruption and stimulation via handling. The rat monoclonal neutralizing antibody against mouse EMAPII was produced and purified as previously described (6, 31). We have previously demonstrated that the anti-EMAPII antibody detects both pro- and mature-EMAPII (31). Furthermore, this anti-EMAPII antibody had neutralizing properties against EMAPII function, such as migration of human peripheral blood monocytes and human endothelial cell apoptosis (31). Rat IgG control antibody was obtained from Abcam (ab37361; Cambridge, MA). The humanized anti-EMAPII antibody was provided by Lakepharma (Belmont, CA), and the human isotype IgG control was obtained from Evitria (Schlieren, Switzerland). We performed several independent experiments as follows. Rat anti-EMAPII antibody or control IgG was administered to C57BL/6 mice via subcutaneous injection (0.125 mg/mouse, 3 times/wk, for 3 wk) during the 4th mo of a 6-mo CS or AC exposure (Fig. 1A) or immediately after a 6-mo CS or AC exposure (Fig. 3A). Rat anti-EMAPII antibody or control IgG was administered to DBA/2J mice via nebulization (0.05 mg/mouse, 3 times/wk, for 4 wk) immediately following a 4-mo CS or AC exposure (Fig. 3D). Humanized anti-EMAPII antibody or human isotype IgG control was administered to DBA/2J mice via subcutaneous injection (0.075 mg/mouse, 3 times/wk, for 3 wk) during a single month CS or AC exposure (Fig. 4A).

Fig. 1.

Subcutaneous administration of a monoclonal rat anti-endothelial monocyte-activating protein 2 (EMAPII) antibody ameliorates cigarette smoke (CS)-induced emphysema-like airspace enlargement in C57BL/6 mice. A: schematic of experimental protocol for CS or ambient air control (AC) exposure and nonhumanized anti-EMAPII antibody treatment or IgG control (n = 5–15/group). C57BL/6 mice were injected subcutaneously with anti-EMAPII antibody or isotype IgG 3 times/wk for 3 wk during 6 mo of CS or AC exposure. Each arrow represents an injection. m, Months old. B: lung static compliance (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test; n = 5–15). ns, Not significant; EMAP Ab, anti-EMAPII antibody. C: mean linear intercept (MLI) of lung parenchyma (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test; n = 4–5). D: surface area-to-volume (SA/V) ratio of lung (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test; n = 4–5). E: representative hematoxylin- and eosin-stained lung sections of lung parenchyma, showing airspace enlargement and alveolar wall destruction in CS-exposed mice treated with IgG, and preserved alveolar architecture in mice treated with rat anti-EMAPII antibody.

Fig. 3.

Administration of rat anti-endothelial monocyte-activating protein 2 (EMAPII) antibody following chronic cigarette smoke (CS) exposure mitigated CS-induced increase of static lung compliance and airspace size in mice. A: schematic of experimental protocol for CS or ambient air control (AC) exposure and treatment with nonhumanized rat anti-EMAPII antibody or IgG control in C57BL/6 mice (n = 10/group). Rat anti-EMAPII antibody or control IgG was administered to C57BL/6 mice via subcutaneous injection (3 times/wk, 3 wk) after 6 mo of CS or AC exposure. Each arrow represents an injection. m, Months old. B: lung static compliance of C57BL/6 mice (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test; n = 10). ns, Not significant. C: mean chord length (MCL) of lung parenchyma of C57BL/6 mice (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test; n = 10). D: schematic of experimental protocol for CS or AC exposure and nonhumanized rat anti-EMAPII antibody treatment or IgG control in DBA/2J mice (n = 5/group). Rat anti-EMAPII antibody or control IgG was administered to DBA/2J mice via nebulization (3 times/wk, 4 wk) following 4 mo of CS or AC exposure. Each arrow represents an inhalation. m, Months old. E: lung static compliance of DBA/2J mice (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test; n = 5). F: MCL of lung parenchyma of DBA/2J mice (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test; n = 5).

Fig. 4.

Subcutaneous administration of a monoclonal humanized anti-endothelial monocyte-activating protein 2 (EMAPII) antibody ameliorates cigarette smoke (CS)-induced inflammation in the lungs of DBA/2J mice. A: schematic of experimental protocol for CS or ambient air control (AC) exposure and humanized anti-EMAPII antibody treatment or isotype IgG control. Mice were treated with humanized anti-EMAPII antibody or IgG control for 3 wk during 1 mo of CS or AC exposure. Each arrow represents an injection. m, Months old (n = 5–7/group). B–E: no. of Ly6G⁺ neutrophils (B), CD45⁺ leukocytes (C), CD11c⁺CD45⁺ resident (Mφ, D), and CD11b⁺CD45⁺ recruited Mφ (E) determined by flow cytometry in bronchoalveolar lavage fluid (BALF) of mice (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test, n = 5). ns, Not significant.

Morphological evaluation of the lungs and lung compliance measurements.

Following euthanasia, lungs were processed as described previously (28). Morphometric analysis was performed on coded slides as described, using a macro developed by R. M. Tuder for MetaMorph (39). Lung compliance measurements were performed with a computer-controlled small animal ventilator (Flexivent; SCIREQ, Montreal, PQ, Canada) (6).

Bronchoalveolar lavage fluid collection and flow cytometry.

An 18-gauge angiocatheter (4075, JELCO-W; Smiths-Medical, Minneapolis, MN) was inserted in trachea to obtain five serial instillations (1 × 1 and 4 × 0.9 ml) of PBS containing 2 mM EDTA, which consistently provided a return of 4 ml of total bronchoalveolar lavage fluid (BALF). To obtain a total CD45⁺ leukocyte count, aliquots of the five lavages were pooled, blocked with CD16/CD32 (clone 93; eBioscience, ThermoFisher), stained with CD45 (30-F11; BD, Franklin Lakes, NJ), and added to 123count eBeads (eBioscience). To avoid variability introduced by pelleting and aspirating, cells used for total cell counts were stained and fixed without any spinning. The concentration of CD45⁺ cells was calculated using the absolute concentration of the counting beads added and the ratio of total CD45⁺ events to total bead events, and then multiplied by 4 ml to obtain total CD45⁺ counts. To determine the differential cell counts, the five lavages were pooled and spun, blocked with antibodies against CD16/CD32 (eBioscience), and stained with antibodies against CD45 (30-F11; BD), Ly6G (1A8; Biolegend), CD64 (X54–5/7.1; BD), F4/80 (BM8; eBioscience), CD11c (N418; eBioscience), and CD11b (M1/70; eBioscience). Cells were washed and resuspended using wash buffer consisting of PBS with 9% FBS and 0.5 mM EDTA. A minimum of 20,000 CD45⁺ leukocyte events for each sample was collected using an LSR II cytometer (BD) and analyzed using FlowJo (Ashland, OR). Gating for total leukocytes involved excluding debris and doublets followed by CD45⁺ staining. Neutrophils were gated using Ly6G⁺CD64⁻ staining. Macrophages were gated using CD64⁺F4/80⁺ staining and classified as recruited (CD11b⁺CD11c^low) or resident (CD11b^lowCD11c⁺SiglecF⁺) (26).

Determination of chemokine (C-X-C motif) ligand 2/macrophage inflammatory protein 2 and tumor necrosis factor-α in BALF.

BALF of C57BL/6 mice that received treatment with anti-EMAPII antibody or control IgG during 6 mo of CS or AC exposure were concentrated using an Amicon ultra-0.5 centrifugal filter (Millipore, Billerica, MA). Chemokine (C-X-C motif) ligand 2 (CXCL2)/macrophage inflammatory protein 2 (MIP-2), and tumor necrosis factor-α (TNF-α) in concentrated BALF were determined using a commercially available ELISA kit (Quantikine Mouse CXCL2/MIP-2 kit and TNFα kit; R&D Systems, Minneapolis, MN), following the manufacturer’s instructions.

Western blotting.

Equal protein amount (20–50 μg), as determined by a bicinchoninic assay (Pierce Biotechnology, Rockford, IL), was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Burlington, MA), followed by routine immunoblotting (37). Briefly, samples were diluted in 4× Laemmli buffer and resolved in Criterion 18-well 4–20% TGX gels (Bio-Rad, Hercules, CA). A semidry transfer apparatus (Bio-Rad) was used. Membranes were probed with the following antibodies: anti-vinculin (ab18058; Abcam), anti-β-actin (A5441; Sigma), anti-LC3B (L7543; Sigma), anti-sequestosome-1 (SQSTM1)/p62 (H00008878-M01; Abnova, Taipei, Taiwan), anti-phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) [9215; Cell Signaling Technology (CST), Danvers, MA], anti-p38 MAPK (9212; CST), anti-phosho-S6 ribosomal protein (S6) (Ser^235/236) (2211; CST), anti-S6 ribosomal protein (2217; CST), anti-Bcl-2 (2876; CST), anti-poly(ADP-ribose) polymerase 1 (PARP1) (cleaved p25) (ab32064; Abcam), anti-Bax (2772; CST), and anti-cleaved caspase-3 antibody (ab13847; Abcam). Appropriate secondary antibodies (goat anti-rabbit/mouse, horseradish peroxidase conjugate) (45001175/45001187; GE Healthcare Life Sciences, Logan, UT) were used in conjunction with Luminata Forte (WBLUF0500; EMD Millipore) for chemiluminescent reaction. Images were taken with a ChemiDoc XRS system with Image Laboratory software (Bio-Rad). Density quantification of protein bands was performed with ImageJ 1.49 (National Institutes of Health, Washington, DC).

Statistical analysis.

All data were expressed as means ± SE or SD. Statistical analyses were performed with SPSS version 17.0 software (SPSS, Chicago, IL) or Prism Graphpad version 6.0 software (GraphPad Software, La Jolla, CA) using one-way ANOVA with Tukey’s multiple-comparisons test or unpaired t-test. P < 0.05 was considered statistically significant.

RESULTS

Subcutaneous treatment with rat anti-EMAPII antibody ameliorates CS-induced emphysema in C57BL/6 mice.

C57BL/6 mice (2 mo old, female) were exposed to CS or AC for 6 mo. Treatment with rat anti-EMAPII antibody or rat control IgG was administered via subcutaneous injection (3 times/wk) for 3 wk during the 4th mo of CS (Fig. 1A). As expected, chronic CS exposure significantly increased lung compliance (Cst; by 12%) compared with age-matched air controls (Fig. 1B). Furthermore, CS significantly increased airspace size, measured by mean linear intercept (MLI) (by 17%; Fig. 1C), and decreased surface area/lung volume (by 12%; Fig. 1D). These results show that 6 mo of CS exposure caused pulmonary emphysema-like disease in C57BL/6 mice treated with IgG. As expected, administration of anti-EMAPII antibody did not affect airspace size in AC groups (data not shown). However, lungs of CS-exposed mice treated with anti-EMAPII antibody had significantly lower Cst and MLI and higher surface area/lung volume following CS exposure, similar to those of AC mice (Fig. 1, B–D). The MLI reflected representative histological findings of lungs from C57BL/6 mice (Fig. 1E) in which chronic CS exposure with control IgG treatment caused airspace enlargement and alveolar wall destruction, whereas treatment with anti-EMAPII antibody was associated with preserved alveolar architecture. These findings indicate that administration of nonhumanized rat anti-EMAPII antibody via subcutaneous injection attenuates CS-induced emphysema-like morphofunctional changes in mice.

Treatment with rat anti-EMAPII antibody attenuates CS-induced lung inflammation and apoptosis in C57BL/6 mice.

Next, we determined whether subcutaneous treatment with anti-EMAPII antibody attenuates chronic CS-induced inflammation and apoptosis in the lungs of C57BL/6 mice. We measured CXCL2/MIP-2, a functional rodent homolog of the human IL-8, and TNF-α levels in BALF of C57BL/6 mice treated with rat anti-EMAPII antibody or IgG during 6 mo of CS or AC exposure. Treatment with anti-EMAPII antibody inhibited (by 60%) the increase of CXCL2/MIP-2 in BALF during CS exposure (Fig. 2A). Similarly, TNF-α was decreased (by 40%) in BALF of C57BL/6 mice treated with anti-EMAPII antibody during CS exposure (Fig. 2B). Because the proinflammatory and proapoptotic effects of EMAPII were associated with activation of p38 mitogen-activated protein kinase signaling (p38 MAPK) in pulmonary endothelial cells (9, 10), we assessed whether treatment with anti-EMAPII antibody attenuated CS-induced upregulation of p38 MAPK in mice lungs. We measured the activation of p38 MAPK in whole lungs of C57BL/6 mice by Western blotting and noted that, as expected, chronic CS exposure increased by approximately twofold the phosphorylation of p38 MAPK compared with AC-exposed mice treated with IgG (Fig. 2C). Treatment with anti-EMAPII antibody during chronic CS exposure attenuated CS-induced phosphorylation of p38 MAPK to the same level as that of the AC- and IgG-treated group (Fig. 2C). Using Western blotting, we then determined the effect of anti-EMAPII antibody treatment on the proapoptotic protein Bax in the lungs of C57BL/6 mice. Bax is a member of the Bcl-2 family of proteins that triggers apoptosis by forming pores in the mitochondria membrane (14) and has been shown to be upregulated in lungs of humans with COPD (22) and mouse emphysema models (5). Chronic CS exposure tended to increase proapoptotic Bax protein levels in the lungs in IgG-treated mice, whereas rat anti-EMAPII antibody decreased Bax in CS-exposed lungs by 42% (Fig. 2D). Because the pore-forming properties of Bax can be inhibited by appropriate increases in anti-apoptotic Bcl-2 protein (14), we measured Bcl-2 in lungs of mice. Unlike its effect on bax, anti-EMAPII antibody treatment did not change Bcl-2 protein levels in the lungs of mice exposed to either AC or CS when compared with IgG-treated mice (data not shown).

Fig. 2.

Subcutaneous administration of a monoclonal rat anti-endothelial monocyte-activating protein 2 (EMAPII) antibody attenuates cigarette smoke (CS)-induced inflammation and apoptosis in the lungs of C57BL/6 mice. A and B: chemokine (C-X-C motif) ligand 2 (CXCL-2)/macrophage inflammatory protein 2 (MIP-2) (A) and tumor necrosis factor-α (TNF-α) (B) measured in concentrated bronchoalveolar lavage fluid (BALF) of mice treated with anti-EMAPII antibody or isotype IgG control during 6 mo of CS or ambient air control (AC) exposure as described in Fig. 1A (mean ± SD; 2 pooled BALF samples from 5 mice/group). C: Western blots and densitometric analysis of phospho (p) p38 MAPK/p38 MAPK in mice lungs (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test; n = 4). ns, Not significant. D: Western blots and densitometric analysis of proapoptotic Bax protein normalized to vinculin as loading control (means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test, n = 4).

Subcutaneous treatment with humanized anti-EMAPII antibody ameliorates CS-induced inflammation in the lungs of DBA/2J mice.

We reported that inhalation of rat anti-EMAPII antibody attenuated chronic CS exposure-induced lung injury in DBA/2J mice (6). Because the study was conducted by administering antibody during CS exposure, we first investigated if this approach was also effective when administered following chronic CS exposure. The administration of the rat anti-EMAPII antibody by either subcutaneous injection or inhalation, following chronic CS exposure in either C57BL/6 or DBA/2J mice, ameliorated CS-induced increase of static lung compliance (Fig. 3, B and E) and increase of airspace size, measured by mean chord length (Fig. 3, C and F). We next investigated whether a humanized form of the anti-EMAPII antibody mitigated CS-induced lung injury in DBA/2J mice. Mice (2-mo-old females) were treated with humanized anti-EMAPII antibody or a human isotype control IgG via subcutaneous injection (3 times/wk, 3 wk) during 4 wk of CS or AC exposure (Fig. 4A). To evaluate lung inflammation, we measured leukocytes in BALF by flow cytometry. Cells in BALF consisted predominantly of alveolar macrophages (>90%). Although in this experiment there was no significant difference in neutrophils among the three groups (Fig. 4B), 1 mo of CS exposure significantly increased the number of total leukocytes in BALF (CD45⁺ cells; by 50%) compared with unexposed age-matched controls (Fig. 4C). Subcutaneous treatment with humanized anti-EMAPII antibody decreased the number of total leukocytes in BALF in CS-exposed mice (Fig. 4C). Similarly, DBA/2J mice treated with humanized anti-EMAPII antibody tended to have a lower number of both resident and recruited macrophages in the BALF during CS exposure compared with control IgG-treated mice (Fig. 4, D and E).

Subcutaneous treatment with humanized anti-EMAPII antibody attenuates CS-induced apoptosis, autophagy, and repression of mammalian target of rapamycin signaling in the lungs of DBA/2J mice.

To determine whether humanized anti-EMAPII antibody treatment attenuates CS-induced lung apoptosis, we exposed DBA/2J mice to CS for 4 wk, a duration of exposure previously shown to increase markers of apoptosis in this mouse strain (2, 27). To measure apoptosis, we used Western blotting analysis of cleaved PARP1 and cleaved caspase-3 in mouse lung lysates. CS exposure increased cleaved PARP1 by 1.4-fold and cleaved caspase-3 by 2.8-fold compared with the AC IgG group (Fig. 5, A and B). Subcutaneous treatment with humanized anti-EMAPII antibody lowered both CS-induced cleaved PARP1 and cleaved caspase-3 to the same levels as those of the AC IgG group. Our results indicate that humanized anti-EMAPII antibody treatment ameliorates CS-induced inflammation and apoptosis in the lungs of mice.

Fig. 5.

Subcutaneous administration of a monoclonal humanized anti-endothelial monocyte-activating protein 2 (EMAPII) antibody attenuates cigarette smoke (CS)-induced apoptosis, autophagy, and repression of mammalian target of rapamycin (mTOR) signaling in the lungs of DBA/2J mice. A: Western blots and densitometric analysis of cleaved poly(ADP-ribose) polymerase 1 (PARP1) protein expression normalized to vinculin in mice lungs after 1 mo of CS or ambient air control (AC) exposure and treatment with anti-EMAPII antibody or IgG control as described in Fig. 4A (n = 5). B: representative Western blots and densitometric analysis of cleaved caspase-3 protein expression normalized to vinculin in mice lungs (n = 5). C: representative Western blots and densitometric analysis of phosphorylated (p) S6 ribosomal protein (S6)/S6 protein in mice lungs (n = 5–7). D: Western blots and densitometric analysis of sequestosome-1 (SQSTM1) (p62) normalized to β-actin and LC3B-II-to-LC3B-I ratio in mice lungs (n = 5). All graphs show means ± SE; *P < 0.05, 1-way ANOVA with Tukey’s multiple-comparisons test. ns, Not significant.

The mechanism by which EMAPII triggers apoptosis is not well defined. Because CS-induced apoptosis may be triggered by unremitting cell stress responses that include autophagy, we determined the effect of anti-EMAPII antibody on autophagy, including the mammalian target of rapamycin (mTOR) pathway, which regulates lysosomal autophagy (12, 25). We assessed mTOR signaling by measuring the phosphorylation of S6, a downstream target of mTOR signaling. CS exposure for 4 wk decreased the levels of S6 phosphorylation by 34% in IgG-treated mice compared with AC-exposed mice, whereas CS-exposed mice treated with humanized anti-EMAPII antibody exhibited similar levels of S6 phosphorylation as the AC IgG group (Fig. 5C). To measure autophagy, we assessed the expression of autophagosome markers LC3B and p62 (SQSTM1) in the lungs of mice. Increases in LC3B-II relative to LC3B-I indicate either high levels of autophagosome production or decreased autophagosome degradation in the lysosome, known as blocked autophagy flux (21, 41). Increased autophagosome degradation is also measured by a loss of SQSTM1, whereas increased SQSTM1 levels indicate a decreased autophagosome lysosomal degradation (21, 41). CS exposure increased the LC3B-II-to-LC3B-I ratio by 27% and decreased SQSTM1 by 31% (Fig. 5D) in IgG-treated mice, suggesting that CS exposure activated autophagy in lung cells. Treatment with humanized neutralizing EMAPII antibody attenuated the CS-induced increase of the LC3B-II-to-LC3B-I ratio in the lungs of DBA/2J mice but had little effect on SQSTM1 levels. These results suggest that treatment with anti-EMAPII antibody may inhibit the initiation of autophagy at a level upstream of mTOR signaling.

DISCUSSION

Our results demonstrate that subcutaneous administration of either monoclonal nonhumanized or humanized anti-EMAPII antibody attenuates several markers of CS-induced lung injury in mice, including inflammation, apoptosis, autophagy, alveolar architecture, and lung function. We have also demonstrated therapeutic effectiveness of anti-EMAP II antibody even when administered following chronic lung damage induced by CS exposure.

We have previously reported that treatment with nonhumanized anti-EMAPII antibody via inhalation attenuates CS-induced pulmonary emphysema-like disease in DBA/2J mice exposed to 4 mo of CS (6). However, it is unclear whether this administration route will be effective in humans given the spatial differences between these two species. In this study, we observed that anti-EMAPII antibody treatment initiated either during or following smoking exposure was advantageous to improving lung compliance and airspace size in not only DBA/2J mice but also in C57BL/6 mice. Because most clinical applications of monoclonal antibodies involve systemic administration, in this preclinical study we tested the effect of treatment with monoclonal anti-EMAPII antibody via subcutaneous injection. Consistent with our previous results (6), treatment with monoclonal rat anti-EMAPII antibody via subcutaneous injection attenuated CS-induced pulmonary emphysema-like manifestations in C57BL/6 mice exposed to 6 mo of CS. These results suggest that both inhalation and subcutaneous routes of administration of anti-EMAPII antibody are potentially useful for the treatment of CS-induced lung diseases.

For clinical therapeutic use, rodent-derived monoclonal antibodies require full humanization, in which the constant regions of the antibodies are completely replaced with their human domain equivalent, while the variable regions are filled up with human-like motifs as much as neutralizing activity permits. Our study of the humanized version of the anti-EMAPII monoclonal antibody identified that it was effective to attenuate CS exposure-induced apoptosis and inflammation in the lungs of mice that were exposed to 1 mo of CS, indicating that, in principle, such an approach may be effective at ameliorating emphysema-like end points following chronic CS exposure.

Interestingly, we observed for the first time that the mechanism of action of EMAPII in the lungs during CS exposure involves inhibition of mTOR signaling, a central regulator of cell growth and autophagy. We found that lungs of mice treated with anti-EMAPII antibody have improved mTOR signaling despite ongoing CS exposure. Autophagy, a ubiquitous eukaryotic cytoplasmic quality and quantity control pathway, is increasingly recognized as an important process in the pathogenesis of COPD (5, 20, 23, 33). Autophagy functions in the clearance of misfolded proteins and damaged organelles, as well as recycling of cytosolic components (16, 32), and is regulated by mTOR-dependent and mTOR-independent pathways (25, 35). Although autophagy and mTOR signaling have been studied in the context of an impressive number of biological processes and disease states, including COPD, the exact roles of autophagy and mTOR signaling in the pathogenesis of COPD remain complex and at times controversial (11, 15, 20, 33, 34, 40), related in part to cell-type specificity and magnitude of CS exposure. Because humanized anti-EMAPII antibody treatment attenuated CS-induced increase of LC3B-II abundance and repression of mTOR signaling in the lungs of mice, it indicates a role of EMAPII in the initiation of autophagy. Our study suggests that future investigations into the role of EMAPII in this important mechanism of cell injury and repair are warranted.

In conclusion, we provide preclinical evidence that subcutaneous administration of rat or humanized neutralizing antibodies to EMAPII attenuates CS-induced lung injury in mice. The potent effect of a relatively short treatment intervention with anti-EMAPII antibody in mice suggests that EMAPII could be a therapeutic target in COPD.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants R34-HL-127873 (to M. Clauss and I. Petrache) and 1F30-HL-136169-01A1 from NRSA (to K. Ni), Harrington Discovery Institute Contract Grant CAR17584 (to I. Petrache), a FORCES grant from IUPUI (to I. Petrache), and an MSD Life Science Foundation International Fellowship (to K. Koike).

DISCLOSURES

MAC and IP are coscientific founders of Allinaire Therapeutics, Inc.

AUTHOR CONTRIBUTIONS

K.S.S., M.A.C., and I.P. conceived and designed research; K.K., E.L.B., M.J.J., A.M.M., and K.N. performed experiments; K.K., E.L.B., K.S.S., K.N., and I.P. analyzed data; K.K., E.L.B., K.S.S., A.M.M., K.N., M.A.C., and I.P. interpreted results of experiments; K.K. and I.P. prepared figures; K.K. and I.P. drafted manuscript; K.K., E.L.B., K.S.S., M.J.J., A.M.M., K.N., M.A.C., and I.P. edited and revised manuscript; K.K. and I.P. approved final version of manuscript.