Separation of human alveolar macrophages by flow cytometry

Alveolar macrophages (AM), which represent the major resident population of immunocompetent cells in the lower respiratory tract, have been implicated in the pathogenesis of acute lung injury in view of their exceptional capacity to release a large array of inflammatory mediators. The ex vivo analysis of these cells, accessible to bronchoalveolar lavage (BAL) is hampered by the fact that, under conditions of respiratory failure, the AM pool is heavily expanded by polymorphonuclear neutrophils (PMN), which necessitates separation of these cell populations. In the present study, we describe a flow cytometric approach to sort human AM obtained from BAL samples of both healthy volunteers (n = 10) and patients with severe pneumonia demanding mechanical ventilation (n = 10), using forward scatter and high autofluorescence characteristics to discriminate AM from PMN and lymphocytes. This technique yielded highly purified AM populations (>95%) as evidenced by morphological analysis, cytochemistry, and CD71 and CD14 expression of the sorted cells. The flow sorting process, per se, did not induce the expression of the acute-phase cytokine tumor necrosis factor-alpha (TNF-alpha) in control AM as determined by reverse transcriptase-polymerase chain reaction. Unstimulated and lipopolysaccharide-induced TNF-alpha protein secretion were comparable in sorted and unsorted AM as demonstrated by enzyme-linked immunosorbent assay. We suggest flow sorting of viable human AM as an efficient and nonperturbing separation technique to yield highly purified cell populations especially from PMN-rich BAL fluids of critically ill patients.