Compartmental Origin of Pulmonary Macrophages Harvested from Mechanically Disrupted Lung Tissue

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Abstract
Alveolar macrophages (AM) have been extensively studied, but whether these lavageable cells are representative of the lung's interstitial macrophage population is unknown. Previous investigators have compared AM with macrophages obtained from mechanically disrupted whole lung tissue and have found both populations to be morphologically and functionally similar. In such studies, neither the anatomical origin of the macrophages harvested from lung tissue postlavage nor the quantitative removal of AM by bronchoalveolar lavage (BAL) was demonstrated. Accordingly, the extent to which unlavaged AM contributed to the macrophage population obtained from mechanically disrupted lung tissue remains obscure. The present study was undertaken to determine whether macrophages harvested by mechanical disruption of lung tissue following exhaustive BAL are in fact primarily of interstitial origin. We addressed this problem by labeling rat AM with opsonized sheep red blood cells (SRBC-IgG) in situ, exhaustively lavaging the lungs to remove AM, and then mincing the residual lung tissue to liberate unlavaged macrophages for subsequent in vitro analyses. We recovered 24.5 ± 1.3 × 106 AM with 18 lavages, 79.5 ± 2.5% of which had phagocytized SRBC-IgG. Mincing the lavaged tissue resulted in the further release of 2.7 ± 0.4 × l06 viable cells, approximately 85% of which morphologically appeared to be macrophages. The percentage of these cells that were prelabeled with SRBC-IgG and the numerical distribution of the SRBC-IgG among them were virtually identical to those among AM. The percentage of SRBC-IgG-containing macrophages was increased only slightly (11.4 ± 1.9%) by further incubating macrophages harvested from minced lung with the SRBC-IgG in vitro, and most of this increase could be accounted for by unlavaged AM that did not phagocytize the test particles in situ. Moreover, multiparameter flow cytometric analyses of macrophages obtained by BAL and from minced lung tissue demonstrated that these populations were identical in terms of size, light scatter properties, and laser-excited blue and green-yellow autofluorescence characteristics. We conclude that (1) exhaustive lavage fails to retrieve a significant number of AM, and (2) macrophages obtained by mechanically disrupting lung tissue are derived primarily from the alveolar space compartment and not the interstitial compartment, as some investigators contend.