Isolation of myeloid and plasmacytoid dendritic cells from human bronchoalveolar lavage fluid

Abstract

Studies of bronchoalveolar lavage fluid (BALF) dendritic cells (DC) have been hampered by the scarcity of DC and the lack of DC-specific surface markers. Four surface Ag have been recently described as specific markers for distinct subsets of DC and have been used for the isolation and characterization of fresh noncultured DC from lung resection specimens: BDCA-1 (CD1c) and BDCA-3 for myeloid DC type 1 and type 2, respectively, and BDCA-2 and BDCA-4 for plasmacytoid DC. The aim of this study was to develop a new method for the isolation of BALF DC, using immunomagnetic separation of BDCA+ cells. Mononuclear cells were obtained from BALF after Ficoll-Paque density gradient centrifugation. Monocytes, T cells and B cells were magnetically labelled and depleted. The unlabelled cell fraction was incubated with BDCA-1, BDCA-3 and BDCA-4 beads and the total BDCA+ DC were retained. The ability of isolated DC to induce T-cell responses was evaluated by coculturing the isolated DC with immunomagnetically sorted naive T cells. The above procedure resulted in a population of viable DC that showed a strong capacity in induce T-cell responses. Functionally intact human BALF myeloid DC types 1 and 2 as well as plasmacytoid DC can be easily obtained by immunomagnetic isolation. Considering that bronchoalveolar lavage is a minimally invasive procedure, these cells are optimal candidates with which to elucidate the properties and capabilities of pulmonary DC.