Hypoxia Increases the Potential for Neutrophil-mediated Endothelial Damage in Chronic Obstructive Pulmonary Disease

Abstract

Rationale: Patients with chronic obstructive pulmonary disease (COPD) experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterized by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage was assessed. Histotoxic protein concentrations were measured in normoxic versus hypoxic neutrophil supernatants and plasma from patients experiencing COPD exacerbation and healthy control subjects. Measurements and Main Results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from patients with COPD. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage, and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia, and hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of patients with COPD had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive "hypersecretory" neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in plasma of patients with COPD. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD.

Keywords: cardiovascular disease; cell degranulation; neutrophil elastase; vascular endothelium.

Figure 1.

Patients experiencing chronic obstructive pulmonary disease (COPD) exacerbation have a circulating signature indicating increased protease activity. Plasma from patients experiencing COPD exacerbation or age- and sex-matched healthy control subjects was assessed for content of (A) neutrophil elastase-specific fibrinogen cleavage product AαVal³⁶⁰ or (B) PR3-specific fibrinogen cleavage product AαVal⁵⁴¹ by immunoassay (n = 12 COPD, n = 14 healthy; cohort 1). Results represent mean ± SEM, (A) unpaired t test and (B) Mann-Whitney test. *P < 0.05 and **P < 0.01.

Figure 2.

Hypoxia increases elastase release from platelet-activating factor (PAF)-primed neutrophils in a PI3Kγ-dependent manner. (A–C) Neutrophils from healthy human donors were incubated under normoxia or hypoxia in the presence or absence of PI3Kγ-selective inhibitor (AS605240, 3 μM) or PI3Kδ-selective inhibitor (CAL-101, 100 nM) as indicated. After 4 hours, cells were treated with PAF (1 μM, 5 min) and/or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (100 nM, 10 min) or vehicle control as indicated. For reoxygenation, unstimulated hypoxic cells were moved to normoxia with the addition of twice-volume normoxic media for 30 minutes before treatment with PAF and fMLP. Supernatant neutrophil elastase (NE) activity was measured and is expressed as fold change relative to hypoxic activated neutrophils (A: n = 5, B: n = 4, C: n = 4–6). (D–F) Femoral bone marrow neutrophils were isolated from PI3Kγ-null (PI3Kγ^−/−), PI3Kδ-hyperactive (E1020K), PI3Kδ-kinase dead (D910A), or wild-type mice from the relevant genetic background. After 4 hours, cells were treated with cytochalasin B (Cyt B; 5 μg/ml, 5 min) and fMLP (10 μM, 10 min) or vehicle control. Supernatant NE activity was measured and is expressed as fold change relative to wild-type hypoxic activated neutrophils (D: 3–4 mice per genotype per experiment, n = 5 independent experiments; E and F: 3–4 mice per genotype per experiment, n = 3 independent experiments). Results represent mean ± SEM, two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 3.

Supernatants from hypoxic activated neutrophils cause increased endothelial cell damage in a partially protease-dependent manner. Neutrophils from healthy donors were incubated under normoxia or hypoxia for 4 hours and then treated with platelet-activating factor (PAF) (1 μM, 5 min) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) (100 nM, 10 min) or vehicle control. Supernatants from normoxic versus hypoxic, PAF/fMLP versus vehicle control-treated neutrophils were incubated with confluent human pulmonary artery endothelial cells (hPAECs) for (A and B) 24 hours, (C) 6 hours, or (D) 48 hours in the presence or absence of alpha-1-antitrypsin (α1AT, 46 μg/ml) as indicated. (A and B) hPAECs were fixed and stained with rhodamine-phalloidin and DAPI. Supernatants were from normoxic (NP) or hypoxic (HP) PAF/fMLP-treated neutrophils. Cell detachment was quantified using ImageJ, expressed as percentage detachment of whole field of view. (A) Representative confocal images from (B) five independent experiments; scale bars, approximately 20 μm. (C) hPAECs were stained with FITC-AnV and propidium iodide (PI) for flow cytometric assessment of apoptosis with apoptotic (AnV⁺PI⁻) cells expressed as percentage of total population (n = 4). (D) Survival of hPAECs was measured by MTT assay (n = 6–12). Results represent mean ± SEM, two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. AnV = annexin V; FITC = fluorescein isothiocyanate.

Figure 4.

Hypoxia selectively increases granule and cytoplasmic histotoxic protein secretion from activated neutrophils. Neutrophils from healthy donors were incubated under normoxia or hypoxia in the (A and B) presence or (C–E) absence of ethylenediaminetetraacetic acid (1 mM) and sivelestat (10 μM) for 4 hours and then treated with platelet-activating factor (PAF) (1 μM, 5 min) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) (100 nM, 10 min). (A and B) Trypsin-digested supernatants were individually labeled with isobaric tags and subjected to tandem mass spectrometry. (A) Principal component analysis showed separation of normoxic versus hypoxic supernatant samples by PC1 (dashed line) with samples from individual donors indicated by dashed lines connecting hypoxic and normoxic samples (n = 5). (B) Volcano plot representation of differential protein expression between paired normoxic and hypoxic supernatants where the vertical dashed lines represent log₂ fold change (FC) of protein abundance = ±1, and the horizontal dashed line represents adjusted P value = 0.05 (n = 5). (C–E) Neutrophil supernatant content of resistin (C: n = 17), NGAL (neutrophil gelatinase-associated lipocalin) (D: n = 7), and cyclophilin A (E: n = 7) was measured from independent samples by ELISA. Results represent mean ± SEM, (B) paired t test with P value adjusted by Benjamini-Hochberg procedure and (C–E) paired t test. *P < 0.05 and **P < 0.01. MPO = myeloperoxidase; NE = neutrophil elastase; PC = principal component.

Figure 5.

Hypoxia further augments histotoxic protein release from chronic obstructive pulmonary disease (COPD) versus healthy neutrophils. (A) Neutrophils from healthy donors or patients experiencing COPD exacerbation were incubated under normoxia and treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) (100 nM, 30 min) or vehicle control. Shape change was assessed by flow cytometric analysis of forward scatter, expressed as percentage shape-changed cells of total population (n = 5–12). (B–F) Neutrophils from healthy donors or patients experiencing COPD exacerbation were incubated under normoxia or hypoxia for 4 hours and then treated with platelet-activating factor (PAF) (1 μM, 5 min) and fMLP (100 nM, 10 min) or vehicle control. Supernatant content of elastase (B: n = 7–14), NGAL (neutrophil gelatinase-associated lipocalin) (C: n = 7–12), cyclophilin A (D: n = 3–7), resistin (E: n = 7–12), and MPO (myeloperoxidase) (F: n = 3–6) was measured by ELISA or activity assay. Supernatant neutrophil elastase activity is expressed as fold change relative to healthy hypoxic activated neutrophils. All samples were obtained from cohort 1. Results represent mean ± SEM, two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 6.

Hypoxia accentuates endothelial–neutrophil interaction induced by chronic obstructive pulmonary disease (COPD) versus healthy neutrophil supernatants. Neutrophils from healthy donors or patients experiencing COPD exacerbation were incubated under normoxia or hypoxia for 4 hours and then treated with platelet-activating factor (PAF) (1 μM, 5 min) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) (100 nM, 10 min). Supernatants from normoxic (NP) or hypoxic (HP) PAF- or fMLP-treated neutrophils were incubated with confluent human pulmonary microvascular endothelial cells (hPMECs) for 4 hours in the presence of serum (2%). Washed hPMECs were perfused with healthy neutrophils. (A) Representative images (original magnification, ×100) showing arrested/rolling neutrophils (N) as bright phase and transmigrated neutrophils (TN) as dark phase. (B) Endothelial–neutrophil interactions (total number of rolling, adhered, and transmigrated neutrophils after bolus neutrophil injection) were captured with time-lapse imaging (n = 3). (C) Quantification of neutrophil rolling and adherence was performed using ImagePro software (n = 3). All neutrophil supernatant samples were obtained from cohort 1. Results represent mean ± SEM, two-way ANOVA. *P < 0.05.

Figure 7.

Plasma from patients with chronic obstructive pulmonary disease (COPD) has increased content of hypoxia-upregulated histotoxic granule proteins and vascular injury biomarkers. Plasma from healthy donors or patients experiencing COPD exacerbation was assessed for content of (A) neutrophil elastase (NE), (B) MPO (myeloperoxidase), (C) NGAL (neutrophil gelatinase-associated lipocalin), (D) resistin, (E) cyclophilin A, (F) ICAM-1 (intercellular adhesion molecule-1), and (G) VCAM-1 (vascular cell adhesion molecule-1) by ELISA (NE, NGAL, and cyclophilin A) or chemiluminescence immunoassay (MPO, resistin, ICAM-1, and VCAM-1) (n = 36 healthy, n = 36 COPD; 4 samples from cohort 1 and 32 samples from cohort 3). Results represent mean ± SEM, Mann-Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.