Macrophages isolated from the lung are not representative of their abundance in vivo and are activated during the isolation process. (A) Maximum-intensity projections of whole-mount confocal images of an intact adult mouse lung from Csf1r-FusionRed mice² (left) and from Siglec1-cre x Rosa26-ZSGreen mice (right). Note the stellate morphology of abundant IMs surrounding terminal alveoli. The Csf1r transgene detects IMs, whereas the brighter CD169 transgene detects both IMs and round alveolar macrophages (arrows) with similar relative abundance. (B) The cellular annotations of human lung leukocyte populations from the integrated human lung cell atlas.⁵⁴ Note the minor subpopulation annotated as interstitial Mph perivascular (IM). A separate annotation of cDC2 is based upon the expression of CD1C, CLEC10A, and FCER1A, all of which are inducible in monocytes.⁵⁶ IMs were distinguished from monocyte-derived macrophages based upon the expression of F13A1 and FOLR2.⁵⁴ (C) screen shots of data of the individual genes indicated from the complete human lung atlas viewed in CellXGene. Blue dots show cells with high expression of the indicated transcripts. Cellular annotations from the atlas are indicated in red. There is no defined IM population in this projection. CSF1R and MARCO appear mutually exclusive, with the latter being associated with alveolar macrophages and CSF1R detected weakly in an overlapping set of clusters annotated as monocytes, elicited macrophages, and lung macrophages. FOS (and other immediate early genes, including FOSB, JUN, and EGR1, not shown) are detected throughout the hematopoietic, endothelial, mesenchymal, and epithelial compartments. Bottom panels show representative inducible cytokines and chemokines apparently expressed by subpopulations of AMs and other macrophages (IL1B) as well as T cells (CCL3). Arrows highlight the clusters of expressing cells.