Isolation and Antigenic Identification of Hamster Lung Interstitial Macrophages

Lung interstitial macrophages (IMs) are a large, distinctive population of cells with important proliferative capacities. Characterization of their role in health and disease has been hampered by inadequate methods to separate interstitial from residual alveolar macrophages (AMs) in preparations of individual mononuclear cells from lung tissue. In this study, a specific cell-surface antigen (HAM1) present on more than 90% of hamster AMs, but not expressed by hamster IMs, was used to distinguish these populations. After collagenase digestion of lung tissue slices from exhaustively lavaged and perfused hamster lungs, mononuclear phagocytes were isolated by density gradient centrifugation. The mean yield of lung digest macrophages (3.9 ± 1.9 (SD) × 106) was comparable to the yield of lavaged AMs (4.2 ± 1.9 × 106). The proliferative capacity of lavaged AMs, blood monocytes, and lung digest macrophages was compared using a soft-agar colony-forming unit (CFU) assay. Both lung digest macrophages and blood monocytes had significantly more CFUs (68.7 ± 2.6 and 53.5 ± 8.4 CFU/103 cells [mean ± SEM], respectively) than did AMs (16.5 ± 1.7) (p < 0.01). To further define the composition of the lung digest macrophage population, flow cytometric analysis of fixed cells from six experiments was performed using a mouse monoclonal antibody specific for the HAM1 antigen found only on AMs. The lung digest macrophage population consisted of both antigen-negative IMs (78.2% ± 3.7% [SEM]; n = 6) and antigen-positive, residual AMs (21.8% ± 3.7%). Morphometric counts confirmed that substantial numbers of AMs are left behind after lavage and contribute to macrophages obtained from lung tissue. Using flow cytometric cell sorting, a highly enriched, antigen-negative, viable, esterase-positive population of lung IMs was obtained. These purified lung IMs again formed substantially more colonies than did matched lavaged AMs (42.8 ± 8.5 versus 9.7 ± 1.4 CFU/103 cells; n = 4; p ⩽ 0.05). The results show that although lung tissue macrophages from extensively lavaged animals still contain a substantial proportion (approximately 20%) of contaminating, residual AMs, antigenic differences can be used to identify and isolate lung IMs for in vitro characterization. Using this technique, we found significantly greater in vitro proliferative capacity in purified IMs than in AMs, supporting an important role for IM proliferation in maintenance of lung macrophage populations.