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. 2020 Nov 16:13:925-932.
doi: 10.2147/JIR.S280419. eCollection 2020.

Human Lung Macrophages Challenged to Oxidants ex vivo: Lysosomal Membrane Sensitization is Associated with Inflammation and Chronic Airflow Limitation

Hans Lennart Persson  1   2 , Apostolos Sioutas  1   2 , Petra Jacobson  1   2 , Linda K Vainikka  3   4
Affiliations

Human Lung Macrophages Challenged to Oxidants ex vivo: Lysosomal Membrane Sensitization is Associated with Inflammation and Chronic Airflow Limitation

Hans Lennart Persson et al. J Inflamm Res. .

Abstract

Background: The lung macrophage (LM) is involved in most inflammatory processes of the human lung by clearance of dying cells and by wound repair. Upon cellular stress by oxidant challenge in vivo lysosomes may rupture in LMs and leakage of cellular content and cell debris may trigger airway inflammation and fibrosis, which may lead to chronic airflow limitation (CAL).

Objective: The aim of this study was to determine whether lysosomal membrane permeabilization (LMP) in LMs challenged to oxidants ex vivo is associated with airway inflammation and CAL, the latter assessed as the reduced forced expiratory volume in one second (FEV1) expressed as % of predicted.

Materials and methods: Twenty-eight subjects were investigated; 13 lung-healthy subjects and 15 subjects with a variety of inflammatory disorders, demonstrating CAL on dynamic spirometry (defined as an FEV1/FVC ratio < 0.70). LMs were harvested by broncho-alveolar lavage (BAL) and challenged ex vivo by oxidants. LMP in oxidant-exposed LMs was assessed as the emitted acridine orange (AO) green fluorescence from oxidant-exposed LMs (using macrophage-like murine J774 cells as positive controls). Inflammatory cells in BAL were counted and lung volumes were recorded.

Results: Oxidant-induced LMP in LMs was significantly greater among subjects with CAL and particularly among those with ongoing inflammation. Previous tobacco history did not influence LMP. Among subjects with CAL, oxidant-induced LMP correlated negatively with FEV1% of predicted.

Conclusion: Lysosomes of LMs harvested from patients with CAL demonstrate an increased sensitivity to oxidants, which may trigger mechanisms behind CAL, eg, chronic airway inflammation and fibrotic re-modelling. The study suggests a mechanistic role for LMP in LMs on airway inflammation, suggesting an anti-inflammatory effect by drugs that prevent increased LMP.

Keywords: BAL; COPD; LMP; acridine orange; lung macrophages; pulmonary fibrosis.

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Conflict of interest statement

The authors have no financial or non-financial conflicts to disclose in relation to the present study.

Figures

Figure 1
Figure 1
In parallel experiments murine macrophage-like J774 cells and LMs, harvested from a patient with CAL or not, were subjected to the same oxidant challenge ex vivo. Increase of cytosolic and nuclear AO-induced green fluorescence, reflecting increased LMP (leakage of AO from lysosomes to the cytosol), was assessed every 2 min and expressed as AU. AO-induced green peak fluorescence of J774 cells and LMs are indicated in the figure image. An index was calculated by dividing the green peak fluorescence value of the LMs with the green peak fluorescence value of the J774 cells at 60 min of GO-exposure. The index value was used to quantify LMP in LMs exposed ex vivo to oxidants. The positive control, oxidant-exposed J774 cells, varied < 10% over time. For further details, see Material and Method section.
Figure 2
Figure 2
AO green peak fluorescence index, as measure of increased LMP, in oxidatively stressed LMs harvested from control subjects (Con; n = 13) and subjects with CAL (CAL; n = 15). In the group with CAL subjects X-smokers (X-S) did not differ significantly, when compared with never-smokers (N-S). Means ± 1 S.D. are indicated. NS = no significance.
Figure 3
Figure 3
Correlation between LMP, expressed as the AO-green peak fluorescence index and CAL (n = 15), expressed as FEV1% of predicted.
See this image and copyright information in PMC

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