Impact of Hypoxia on Neutrophil Degranulation and Inflammatory Response in Alpha-1 Antitrypsin Deficiency Patients

Abstract

Background: Alpha-1 antitrypsin deficiency (AATD) is an inflammatory disorder where neutrophils play a key role. Excessive neutrophil activation leads to local hypoxia and tissue damage. Most research on neutrophil function has been conducted under atmospheric conditions (21% O₂), which may not represent physiological or pathological conditions. This study aimed to determine the effects of hypoxia on neutrophil degranulation and cytokine production in AATD patients.

Methods: Neutrophils isolated from 54 AATD patients (31 MZ; 8 SZ; 15 ZZ) and 7 controls (MM) were exposed to hypoxia (1% O₂) for 4 h. Neutrophil degranulation was assessed by measuring elastase (NE), myeloperoxidase (MPO), lactoferrin, and matrix metalloproteinase-9 (MMP-9) levels using immunoassay-based methods. Pro-inflammatory (IL-8, IL-1 beta, IL-6, and TNF-alpha) and anti-inflammatory (IL-4 and IL-10) cytokine levels were assessed by a Luminex-based method.

Results: Our results indicate a significantly increased release of NE (p = 0.015), MPO (p = 0.042), lactoferrin (p = 0.015), and MMP-9 (p = 0.001) compared to controls. Pro-inflammatory cytokines show a significant rise in IL-8 (p = 0.019), a trend towards increased IL-1 beta (p = 0.3196), no change in IL-6 (p = 0.7329), and reduced TNF-alpha (p = 0.006). Anti-inflammatory cytokines show increased IL-4 (p = 0.057) and decreased IL-10 (p = 0.05703).

Conclusions: Increased neutrophil degranulation and inflammatory phenotype are observed in AATD neutrophils under physiological hypoxia.

Keywords: COPD; alpha-1 antitrypsin deficiency; hypoxia; liver; neutrophil; rare respiratory diseases.

Figure 1

Neutrophils from ZZ AATD asymptomatic children show increased degranulation. Neutrophils were incubated under hypoxia for 4 h, primed with TNF-α (20 ng/mL for 30 min), and activated with fMLP (100 nM for 10 min). The presence of granule proteins was measured in the supernatants. (A) Elastase activity in the supernatants of AATD patients and healthy volunteers; (B) myeloperoxidase levels in the supernatants of AATD patients and healthy volunteers; (C) lactoferrin levels in the supernatants of AATD patients and healthy volunteers; (D) matrix metalloproteinase-9 (MMP-9) levels in the supernatants of AATD patients and healthy volunteers. * p < 0.05; ** p < 0.01.

Figure 2

Neutrophil pro-inflammatory cytokine profile of control individuals and AATD patients. Neutrophils were incubated under hypoxia for 4 h, primed with TNF-α (20 ng/mL for 30 min), and activated with fMLP (100 nM for 10 min). Cytokine concentration was measured in the tissue culture supernatants. (A) Interleukin-8 (IL-8) concentration in the supernatants of AATD patients and healthy volunteers; (B) interleukin-6 (IL-6) concentration in the supernatants of AATD patients and healthy volunteers; (C) interleukin-1 beta (IL-1 beta) concentration in the supernatants of AATD patients and healthy volunteers; (D) tumor necrosis factor alpha (TNF-alpha) concentration in the supernatants of AATD patients and healthy volunteers. * p < 0.05.

Figure 3

Neutrophil anti-inflammatory cytokine profile of control individuals and AATD patients. Neutrophils were incubated under hypoxia for 4 h, primed with TNF-α (20 ng/mL for 30 min), and activated with fMLP (100 nM for 10 min). Cytokine concentration was measured in the tissue culture supernatants. (A) Interleukin-4 (IL-4) concentration in the supernatants of AATD patients and healthy volunteers; (B) interleukin-10 (IL-10) concentration in the supernatants of AATD patients and healthy volunteers. * p < 0.05.