Isolation and Identification of Interstitial Macrophages from the Lungs Using Different Digestion Enzymes and Staining Strategies

Abstract

Interstitial macrophages (IMs) are present in multiple organs. Although there is limited knowledge of the unique functional role IM subtypes play, macrophages, in general, are known for their contribution in homeostatic tissue maintenance and inflammation such as clearing pathogens and debris and secreting inflammatory mediators and growth factors. IM subtypes have been identified in the heart, skin, and gut, and more recently we identified three distinct IMs in the lung. IMs express on their surface high levels of MerTK, CD64, and CD11b, with differences in CD11c, CD206, and MHC II expression, and referred to the three pulmonary IM subtypes as IM1 (CD11c^loCD206⁺MHCII^lo), IM2 (CD11c^loCD206⁺MHCII^hi), and IM3 (CD11c^hiCD206^loMHCII^hi). In this chapter, we highlight how to extract IMs from the lung using three different digestion enzymes: elastase, collagenase D, and Liberase TM. Of these three commonly used enzymes, Liberase TM was the most effective at IM extraction, particularly IM3. Furthermore, alternative staining strategies to identify IMs were examined, which included CD64, MerTK, F4/80, and Tim4. Thus, future studies highlighting the functional role of IM subtypes will help further our understanding of how tissue homeostasis is maintained and inflammatory conditions are induced and resolved.

Keywords: Alveolar macrophages; Dendritic cells; Interstitial macrophages; Lung; Monocytes; Mononuclear phagocytes; Pulmonary.

Fig. 1

Identification of pulmonary interstitial macrophages in steady state

Fig. 2

Alternative gating strategy to identify interstitial macrophages