SEPARATION OF HUMAN ALVEOLAR MACROPHAGES BY FLOW-CYTOMETRY

Alveolar macrophages (AM), which represent the major resident populati on of immunocompetent cells in the lower respiratory tract, have been implicated in the pathogenesis of acute lung injury in view of their e xceptional capacity to release a large array of inflammatory mediators . The ex vivo analysis of these cells, accessible to bronchoalveolar l avage (BAL) is hampered by the fact that, under conditions of respirat ory failure, the AM pool is heavily expanded by polymorphonuclear neut rophils (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 characterist ics to discriminate AM from PMN and lymphocytes. This technique yielde d highly purified Abl populations (>95%) as evidenced by morphological analysis, cytochemistry, and CD71 and CD14 expression of the sorted c ells. 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 re action. 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 yie ld highly purified cell populations especially from PMN-rich BAL fluid s of critically ill patients.