Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis

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Abstract

Purpose

Progress to the clinic may be delayed or prevented when vacuolated or “foamy” alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures.

Methods

Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation.

Results

Cell health, morphology and lipid content were comparable ( p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed.

Conclusions

A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

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Most cited references 37

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Cell size of alveolar macrophages: an interspecies comparison.

F Krombach, S Münzing, A Allmeling … (1997)

Alveolar macrophages (AM) play a critical role in the removal of inhaled particles or fibers from the lung. Species differences in AM size may affect the number and size range of particles/fibers that can be actually phagocytized and cleared by AM. The purpose of this study was to compare the cell size of rat, hamster, monkey, and human AM by selective flow cytometric analysis of cell volume. Resident AM from CD rats, Syrian golden hamsters, cynomolgus monkeys, and nonsmoking, healthy human volunteers were harvested by standard bronchoalveolar lavage procedures. Morphometric analysis of AM was performed using a flow cytometer that generates volume signals based on the Coulter-type measurement of electrical resistance. We found that hamster and rat AM had diameters of 13.6 +/- 0.4 microns (n = 8) and 13.1 +/- 0.2 microns (n = 12), respectively. Comparatively, the AM from monkeys (15.3 +/- 0.5 microns, n = 7) and human volunteers (21.2 +/- 0.3 microns, n = 10) were larger than those from rats and hamsters. The AM from humans were significantly larger (p < 0.05) than those from all other species studied, corresponding to a 4-fold larger cell volume of human AM (4990 +/- 174 microns 3) compared to hamster (1328 +/- 123 microns 3) and rat (1166 +/- 42 microns 3) AM. In summary, we have found marked species differences in the cell size of AM. We suggest that the number and size range of particles/fibers that can be phagocytized and cleared by AM may differ among species due to inherent or acquired species differences in AM cell size.

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An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials

Martin Wiemann, Antje Vennemann, Ursula Sauer … (2016)

Background Most in vitro studies investigating nanomaterial pulmonary toxicity poorly correlate to in vivo inhalation studies. Alveolar macrophages (AMs) play an outstanding role during inhalation exposure since they effectively clear the alveoli from particles. This study addresses the applicability of an in vitro alveolar macrophage assay to distinguish biologically active from passive nanomaterials. Methods Rat NR8383 alveolar macrophages were exposed to 18 inorganic nanomaterials, covering AlOOH, BaSO4, CeO2, Fe2O3, TiO2, ZrO2, and ZnO NMs, amorphous SiO2 and graphite nanoplatelets, and two nanosized organic pigments. ZrO2 and amorphous SiO2 were tested without and with surface functionalization. Non-nanosized quartz DQ12 and corundum were used as positive and negative controls, respectively. The test materials were incubated with the cells in protein-free culture medium. Lactate dehydrogenase, glucuronidase, and tumour necrosis factor alpha were assessed after 16 h. In parallel, H2O2 was assessed after 1.5 h. Using the no-observed-adverse-effect concentrations (NOAECs) from available rat short-term inhalation studies (STIS), the test materials were categorized as active (NOAEC < 10 mg/m3) or passive. Results In vitro data reflected the STIS categorization if a particle surface area-based threshold of <6000 mm2/mL was used to determine the biological relevance of the lowest observed significant in vitro effects. Significant effects that were recorded above this threshold were assessed as resulting from test material-unspecific cellular ‘overload’. Test materials were assessed as active if ≥2 of the 4 in vitro parameters undercut this threshold. They were assessed as passive if 0 or 1 parameter was altered. An overall assay accuracy of 95 % was achieved. Conclusions The in vitro NR8383 alveolar macrophage assay allows distinguishing active from passive nanomaterials. Thereby, it allows determining whether in vivo short-term inhalation testing is necessary for hazard assessment. Results may also be used to group nanomaterials by biological activity. Further work should aim at validating the assay. Electronic supplementary material The online version of this article (doi:10.1186/s12951-016-0164-2) contains supplementary material, which is available to authorized users.

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Amiodarone: review of pulmonary effects and toxicity.

Despoina Markoulaki, Likurgos Kolilekas, E Manali … (2010)

Amiodarone, a bi-iodinated benzofuran derivative, is, because of its high effectiveness, one of the most widely used antiarrhythmic agents. However, adverse effects, especially potentially fatal and non-reversible acute and chronic pulmonary toxicity, continue to be observed. This review provides an update of the epidemiology, pathophysiology, clinical presentation, treatment and outcome of amiodarone pulmonary effects and toxicity. Lung adverse effects occur in approximately 5% of treated patients. The development of lung complications appears to be associated with older age, duration of treatment and cumulative dosage, high levels of its desethyl metabolite, history of cardiothoracic surgery and/or use of high oxygen mixtures, use of iodinated contrast media, and probably pre-existing lung disease as well as co-existing respiratory infections. Amiodarone-related adverse pulmonary effects may develop as early as from the first few days of treatment to several years later. The onset of pulmonary toxicity may be either insidious or rapidly progressive. Cough, new chest infiltrates in imaging studies and reduced lung diffusing capacity in the appropriate clinical setting of amiodarone use, after the meticulous exclusion of infection, malignancy and pulmonary oedema, are the cardinal clinical and laboratory elements for diagnosis. Pulmonary involvement falls into two categories of different grades of clinical significance: (i) the ubiquitous 'lipoid pneumonia', the so-called 'amiodarone effect', which is usually asymptomatic; and (ii) the more appropriately named 'amiodarone toxicity', which includes several distinct clinical entities related to the differing patterns of lung inflammatory reaction, such as eosinophilic pneumonia, chronic organizing pneumonia, acute fibrinous organizing pneumonia, nodules or mass-like lesions, nonspecific interstitial pneumonia-like and idiopathic pulmonary fibrosis-like interstitial pneumonia, desquamative interstitial pneumonia, acute lung injury/acute respiratory distress syndrome (ARDS) and diffuse alveolar haemorrhage. Pleural/pericardial involvement may be observed. Three different and intertwined mechanisms of lung toxicity have been suggested: (i) a direct toxic effect; (ii) an immune-mediated mechanism; and (iii) the angiotensin enzyme system activation. Mortality ranges from 9% for those who develop chronic pneumonia to 50% for those who develop ARDS. Discontinuation of the drug, control of risk factors and, in the more severe cases, corticosteroids may be of therapeutic value. Supportive measures for supervening ARDS in the intensive care setting may become necessary.

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Author and article information

Contributors

Victoria Hutter:

ORCID: http://orcid.org/0000-0003-1346-3680

+44 (0)1707 285018 , v.hutter@herts.ac.uk

Journal

Journal ID (nlm-ta): Pharm Res

Journal ID (iso-abbrev): Pharm. Res

Title: Pharmaceutical Research

Publisher: Springer US (New York )

ISSN (Print): 0724-8741

ISSN (Electronic): 1573-904X

Publication date (Electronic): 24 May 2017

Publication date PMC-release: 24 May 2017

Publication date (Print): 2017

Volume: 34

Issue: 12

Pages: 2466-2476

Affiliations

[1 ]ISNI 0000 0001 2161 9644, GRID grid.5846.f, Centre for Topical Drug Delivery and Toxicology School of Life and Medical Sciences, , University of Hertfordshire, ; Hatfield, Hertfordshire, AL10 9AB UK

[2 ]ISNI 0000 0001 2165 3025, GRID grid.8267.b, Department of Pharmaceutical Biochemistry and Molecular Diagnostics Pharmacy Faculty, , Medical University of Lodz, ; 1 Muszynskiego Street, 90-151 Lodz, Poland

[3 ]ISNI 0000 0001 2322 6764, GRID grid.13097.3c, Institute of Pharmaceutical Science, , King’s College London, ; Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH UK

[4 ]ISNI 0000 0001 2322 6764, GRID grid.13097.3c, Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, Faculty of Life Sciences and Medicine, , King’s College London, ; 150 Stamford Street, Waterloo Campus, London, SE1 9NH UK

[5 ]ISNI 0000 0001 2162 0389, GRID grid.418236.a, Medicines Research Centre, , Glaxo-Smith-Kline, ; Gunnels Wood Road Stevenage, Hertfordshire, SG1 2NY UK

[6 ]GE Healthcare Life Sciences, Maynard Centre, Forest Farm, Whitchurch, Cardiff, CF14 7YT UK

[7 ]ISNI 0000 0001 0679 2801, GRID grid.9018.0, Institute of Pharmaceutical Technology and Biopharmacy, , Martin Luther University Halle-Wittenberg, ; Wolfgang-Langenbeck-Str. 4, 06108 Halle (Saale), Germany

Author information

Victoria Hutter http://orcid.org/0000-0003-1346-3680

Article

Publisher ID: 2176

DOI: 10.1007/s11095-017-2176-5

PMC ID: 5736774

PubMed ID: 28540501

SO-VID: a81932aa-b1f5-4408-a8bd-43d666cf74a6

License:

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.