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Current pulmonary research underlines the relevance of the alveolar macrophage (AM) integrated in multicellular co‐culture‐systems of the respiratory tract to unravel, for example, the mechanisms of tissue regeneration. AMs demonstrate a specific functionality, as they inhabit a unique microenvironment with high oxygen levels and exposure to external hazards. Healthy AMs display an anti‐inflammatory phenotype, prevent hypersensitivity to normally innocuous contaminants and maintain tissue homeostasis in the alveolus. To mirror the actual physiological function of the AM, we developed three different polarized [classically activated (M1) and alternatively activated (M2 wh , wound‐healing; M2 reg , regulatory)] macrophage models using a mixture of differentiation mediators, as described in the current literature. To test their immunological impact, these distinct macrophage phenotypes were seeded on to the epithelial layer of an established  in vitro  air–blood barrier co‐culture, consisting of alveolar epithelial cells A549 or H441 and microvascular endothelial cells ISO‐HAS‐1 on the opposite side of a Transwell filter‐membrane. IL‐8 and sICAM release were measured as functionality parameters after LPS challenge. The M1 model itself already provoked a severe inflammatory‐like response of the air–blood barrier co‐culture, thus demonstrating its potential as a useful  in vitro  model for inflammatory lung diseases. The two M2 models represent a ‘non‐inflammatory’ phenotype but still showed the ability to trigger inflammation following LPS challenge. Hence, the latter could be used to establish a quiescent, physiological  in vitro  air–blood model. Thus, the more complex differentiation protocol developed in the present study provides a responsive  in vitro  triple‐culture model of the air–blood‐barrier that mimics AM features as they occur  in vivo . © 2015 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

A responsive human triple‐culture model of the air–blood barrier: incorporation of different macrophage phenotypes