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Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages
Published on: April 20, 2018
An in vitro method to investigate the microbicidal potential of human macrophages for use in psychosomatic research
Ulrike Kuebler1, Ulrike Ehlert, Claudia Zuccarella
1Department of Psychology, Biological and Health Psychology, University of Bern, Alpeneggstrasse 22, 3012 Bern, Switzerland. petra.wirtz@psy.unibe.ch.
Objective:
Psychological states relate to changes in circulating immune cells, but associations with immune cells in peripheral tissues such as macrophages have hardly been investigated. Here, we aimed to implement and validate a method for measuring the microbicidal potential of ex vivo isolated human monocyte-derived macrophages (HMDMs) as an indicator of macrophage activation.
Methods:
The method was implemented and validated for two blood sampling procedures (short-term cannula insertion versus long-term catheter insertion) in 79 participants (34 women, 45 men) aged between 18 and 75 years. The method principle is based on the reduction of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-dis-ulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) by superoxide anions, the first in a series of pathogen-killing reactive oxygen species produced by phorbol myristate acetate-activated HMDM. Cytochrome c reduction and current generation were measured as reference methods for validation purposes. We further evaluated whether depressive symptom severity (Beck Depression Inventory) and chronic stress (Chronic Stress Screening Scale) were associated with macrophage microbicidal potential.
Results:
The assay induced superoxide anion responses by HMDM in all participants. Assay results depended on blood sampling procedure (cannula versus catheter insertion). Interassay variability as a measure for assay reliability was 10.92% or less. WST-1 reduction scores correlated strongly with results obtained by reference methods (cytochrome c: r = 0.57, p = .026; current generation: r values ≥ 0.47, p values <.033) and with psychological factors (depressive symptom severity: r = 0.35 [cannula insertion] versus r = -0.54 [catheter insertion]; chronic stress: r = 0.36 [cannula insertion]; p values ≤ .047).
Conclusions:
Our findings suggest that the implemented in vitro method investigates microbicidal potential of HMDM in a manner that is valid and sensitive to psychological measures.
Insights
This study developed a new method to measure macrophage microbicidal potential, finding it sensitive to psychological states like depression and stress. The assay is a valid indicator of macrophage activation.
Area of Science:
- Immunology
- Psychoneuroimmunology
- Biomedical Engineering
Background:
- Psychological states influence immune cell function.
- Investigating immune cells in peripheral tissues, like macrophages, is crucial.
- Previous research has not sufficiently explored macrophage activation markers related to psychological well-being.
Purpose of the Study:
- To implement and validate a novel in vitro method for assessing the microbicidal potential of human monocyte-derived macrophages (HMDMs).
- To evaluate HMDMs as a measurable indicator of macrophage activation.
- To explore the association between psychological factors and HMDM microbicidal potential.
Main Methods:
- Developed and validated an assay measuring superoxide anion production by PMA-activated HMDMs using WST-1 reduction.
- Compared two blood sampling procedures: short-term cannula insertion and long-term catheter insertion.
- Validated the assay against reference methods (cytochrome c reduction, current generation) and assessed reliability (interassay variability ≤ 10.92%).
- Correlated assay results with depressive symptom severity and chronic stress levels.
Main Results:
- The assay successfully induced superoxide anion responses in HMDMs across all participants.
- Assay outcomes were influenced by the blood sampling method.
- Strong correlations were observed between WST-1 reduction scores and reference methods (cytochrome c: r = 0.57; current generation: r ≥ 0.47).
- Significant associations were found between HMDM microbicidal potential and psychological factors (depressive symptom severity and chronic stress).
Conclusions:
- The implemented in vitro method is a valid and reliable tool for measuring HMDM microbicidal potential.
- This method is sensitive to psychological measures, highlighting the link between mental state and innate immune function.
- The findings open avenues for further research in psychoneuroimmunology.
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