Separation of human alveolar macrophages by flow cytometry.
Maus, Ulrich, Simone Rosseau, Werner Seeger, and J[umlaut]urgen
Lohmeyer.
Department of Internal Medicine, Justus-Liebig-University, Giessen,
Germany
APStracts 3:0214L, 1996.
Alveolar macrophages (AM) which represent the major resident
population of immunocompe tent 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 alveolar macrophage
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 (PN) 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 morphologic analysis, cytochemistry and CD 71 and CD 14
expression of the sorted cells. The flow-sorting process per se did
not induce the expression of the acute phase cytokine TNF-[alpha] in
control AM as determined by RT-PCR. Unstimulated and
lipopolysaccharide (LPS)-induced TNF-[alpha] protein secretion were
comparable in sorted and unsorted AM as demonstrated by enzyme-linked
immunosorbent assay (ELISA). We suggest flow sorting of viable human
AM as an efficient and non-perturbing separation technique, to yield
highly purified cell populations especially from PMN-rich BAL fluids
of critically ill patients.
Received 26 June 1996; accepted in final form 30 October 1996.
APS Manuscript Number L191-6.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996