Isolation and Partial Characterization of Subpopulations of Alveolar Macrophages, Granulocytes, and Highly Enriched Interstitial Macrophages from Rat Lung

Abstract

The contribution of interstitial macrophages (IM) to lung defense, homeostasis, and pathophysiology is for the most part unknown. Studies on this cell type are difficult because they are not readily accessible in large numbers or in high purity. In the present work, various nonenzymatic and enzymatic methods were compared with the aim of isolating and characterizing pure populations of lung IM. The results of our studies demonstrate that most procedures currently used to isolate IM yield subpopulations of alveolar macrophages (AM) or IM highly contaminated by AM and granulocytes. We found that lavage of the lung yielded only one half of the total AM present in the tissue. The remainder of the AM could only be obtained by extensive washing of cut and disaggregated lung tissue, which is considered by some investigators to be an effective procedure for IM isolation. According to our results, cells recovered by lavage and washing of cut and disaggregated lung tissue were morphologically and histochemically identical, were strongly positive for nonspecific esterase, highly phagocytic, and appeared to represent subpopulations of AM with apparently varying degrees of adherence to the alveolar walls. We also found that IM could be obtained in high purity by sequential digestion of the remaining lung tissue with 60 and 175 IU/ml of collagenase followed by selective adherence. Digestion of the tissue with 60 IU/ml of collagenase resulted in a highly enriched population of granulocytes and also reduced their contamination in the IM population. The resulting IM were distinct from AM by morphology and histochemistry. Like AM, these cells displayed Fc receptor-mediated phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model.

“…Lungs were excised, minced, and digested enzymatically with 15 ml/lung of digestion solution (RPMI 1640, 5% FCS, penicillin and streptomycin, 1 mg/ml collagenase [Boehringer Mannheim Biochemicals, Indianapolis, IN], and 30 g/ ml DNase [Sigma Chemical Co., St. Louis, MO]) for 30 min at 37 Њ C. The undigested fragments in the cell suspension were dispersed further by repeated aspiration through a 10-ml syringe. This digestion procedure results in minimal contamination with epithelial cells (25). A 100-l digest was removed to assay for lung CFU.…”

Long-Lasting Production of TGF-β1 by Alveolar Macrophages Exposed to Low Doses of Asbestos without Apoptosis

“…Male Wistar rats (Oriental Yeast Co., Ltd., Tokyo, Japan) were instilled with 4 mg of CH or saline via the trachea (16). One or 5 days after instillation, the lungs and the heart were removed from the rats, and 20 ml of BALF was collected by washing the lungs 4 times with 5 ml each-time of calcium-and magnesium-free Hank's balanced salt solution (HBSS) pre-warmed at 37°C (17). The first 5-ml volume of BALF collected by washing carefully was temporarily separated from the subsequent collection to avoid the dilution of cytokines by washing repeatedly.…”

The Fibrinogen Binding Protein of Staphylococcus epidermidis Is a Target for Opsonic Antibodies

“…Macrophages were obtained from rats (average weight, 180 g; Wistar) by bronchoalveolar lavage as described elsewhere (2,6). The macrophages were adjusted to a concentration of 10 6 cells/ml and added to a 24-well cell culture plate (Nunc) at 10 6 cells/well.…”