Findings in murine models implicate subpopulations of alveolar macrophages in the pathogenesis of lung injury and fibrosis; however, the relevance of these findings for humans with chronic lung disease is unknown in part because of a lack of proper tools to identify macrophage heterogeneity in the human lung. Here we report a flow cytometry protocol that allows unambiguous identification of alveolar macrophages, interstitial macrophages, and monocytes in the human lung and in bronchoalveolar lavage fluid. We validated this panel using normal lung tissue and tissue from patients with chronic obstructive pulmonary disease and lung fibrosis. We found evidence of heterogeneity within human alveolar macrophage populations, which suggest parallels between murine and human macrophage development and differentiation.

Lung macrophages are essential for maintaining homeostasis in the healthy lung and play an important role in pulmonary diseases (1). For decades, alveolar macrophages, which are abundant in the alveolar space of the normal lung, were considered a homogenous population of cells derived from and continuously replenished by circulating monocytes originating from the bone marrow. In careful studies of murine lung development, several groups of investigators have overturned this paradigm. They discovered that alveolar macrophages originate from fetal