A method for isolating human lung macrophages and observations of fluorescent phagocytes from the lungs of habitual cigarette smokers.
We report the development of a simple, efficient, expedient, and inexpensive procedure for isolating a large and relatively pure population of macrophages (Mphi) from residual (i.e., non-tumor) lung tissue obtained from lung cancer patients undergoing either a lobectomy or pneumonectomy. The proposed technique was founded on observations by fluorescent microscopy of fresh, non-fixed, and non-stained human lung tissue. Examinations of 74 specimens from different patients revealed that most of the Mphi reside as non-adherent cells within the sponge-like lung stroma. Very few Mphi were observed in the lungs of nonsmokers. In contrast, many Mphi were visible in the lungs of habitual smokers. For most specimens from smokers, a few of the Mphi were present as randomly distributed single cells; the majority of the Mphi, however, were in clusters that ranged from a dozen to several hundred cells. The Mphi could be released readily by different mechanical techniques. In the procedure reported herein, pulmonary leukocytes (> 75% Mphi) were dislodged easily from lung tissue with the use of an inexpensive, hand-operated, tissue grinder. The grinder consisted of a glass mortar and Teflon pestle that provided sufficient clearance between the mortar and pestle so as to avert damaging the displaced leukocytes. The leukocytes were then segregated by centrifugation on a density gradient. Further purification was achieved by harvesting Mphi that had been allowed to adhere to serum-coated polystyrene culture dishes (> 90% Mphi). In most experiments, the Mphi yield (approximately 5 x 10(6) Mphi /gr of lung) and Mphi viability (> 85%) were good. A significant advantage of this technique is that it avoids jeopardizing the cells to the hazards associated with enzymes that have been used in techniques employed previously for isolating Mphi from the lung and other organs. Thus, the proposed method provides numerous lung Mphi for detailed studies of their morphology, phenotype, and function. Moreover, lung Mphi were cultured as non-adherent, single cells in a serum- and cytokine-free tissue culture medium for more than 6 weeks. Lung Mphi from habitual smokers displayed a high level of fluorescence that was readily apparent when viewed with a fluorescence microscope that had been configured with either a fluorescein or rhodamine filter. Serial sections of single, living Mphi obtained with the use of a confocal laser scanning microscope revealed that the fluorescence originated from cytoplasmic inclusions. Relative fluorescence intensity was measured by cytometry.(ABSTRACT TRUNCATED AT 400 WORDS)
Streck
R J
RJ
Department of Molecular Immunology, New York State Department of Health, Buffalo 14263.
Jezewski
H M
HM
Rodriguez
M I
MI
Hurley
E L
EL
Rich
G A
GA
Braun
K M
KM
Pauly
J L
JL
D001992:Bronchoalveolar Lavage Fluid; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D008175:Lung Neoplasms / Q000473:pathology*; D016676:Macrophages, Alveolar / Q000166:cytology*; D012907:Smoking / Q000473:pathology*