Leukocyte counts and macrophage phenotypes in induced sputum and bronchoalveolar lavage fluid from normal subjects

Abstract

Bronchoalveolar lavage (BAL) is a well-standardized method for retrieving cells and soluble material from the lung. An alternative, less invasive method to recover such material is induced sputum (IS). The method provides an opportunity to perform frequent observations, in order to follow a dynamic course of pulmonary inflammatory disease and effects of treatment. Recent research on IS has focused on markers of inflammation in asthma and the method has, in that context, proven to be reliable, for instance in the evaluation of airway eosinophilia [1][2][3][4][5][6][7][8][9].There is still, however, a need to determine the degree of correlation between IS and BAL material and to establish baseline values for IS variables in healthy individuals. The main difference between the processing of IS and BAL is the incubation of sputum material with a disulphide reducing agent, dithiothreitol (DTT), which is carried out to obtain cell dispersion [10]. Incubation of samples with DTT allows a high repeatability in differential cell counts, but has been shown to interfere with immunological detection of some cellular antigens, for example, the intracellular expression of eosinophil cationic protein, EG2 [10].The most predominant cell in the alveolar and airway lining fluid is the macrophage. Studies of material recruited from the airways and alveoli separately [11] have implied the presence of morphologically different subsets of macrophages deriving from different levels of the bronchoalveolar tree.The present study was conducted to determine intraindividual differences in cell-count data and macrophage phenotypes in IS and BAL collected from healthy smokers and nonsmokers and to investigate any measurable DTTmediated effect on macrophage immunocytochemical staining results. Materials and methods SubjectsIS and BAL fluid samples were collected from 16 subjects; nine smokers, six females and three males, mean age 27.9 yrs (range 21-38) and seven nonsmokers, five females and two males, mean age 26.6 yrs (range 19-38). The smokers mean cigarette consumption was 10.4±7.2 pack-yrs (mean±SD), and present consumption exceeded 10 cigarettes·day -1 for the last 5 yrs.All subjects had normal pulmonary radiographs and showed no clinical signs of respiratory disease. IS samples BAL and IS samples were processed and cell viability and cell counts were assessed. The macrophages were characterized by seven monoclonal antibodies (RFD1, RFD7, CD11b, CD54, CD68, CD71 and HLA-DR) using an indirect immunoalkaline phosphatase method.Intraindividual comparison of IS and BAL showed that IS samples from smokers and nonsmokers contained a lower total cell count (p<0.0l smokers, p<0.05 nonsmokers), a lower percentage of macrophages (both p<0.05) and a higher percentage of neutrophils (both p<0.05) than BAL samples. In addition, nonsmokers sputum samples contained a lower proportion of lymphocytes (p<0.05) than BAL. The macrophage expression of RFD7 and CD71 was higher in smokers sputum samples (both p<0.05) than in BAL, while nonsmokers sputum macro...

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