A novel method for isolating dendritic cells from human bronchoalveolar lavage fluid.
BACKGROUND
Dendritic cells (DCs) play a pivotal role in linking the innate and adaptive immune response and have been implicated in a variety of pulmonary diseases. Currently, studies on the role of DCs are limited by difficulties in isolating DCs from the lung. Surgical lung specimens are not readily available and purification of DCs from digested lung tissue is likely to induce phenotypical and functional changes. DCs obtained from the alveolar spaces are thought to represent the local microenvironment and can be obtained using minimally invasive techniques. We developed a novel method of isolating DCs from bronchoalveolar lavage (BAL) fluid.


METHODS
After removal of macrophages, the remaining BAL cells were stained with a lineage mix (CD3-, CD14-, CD16-, CD19-, CD56-FITC), CD11c and HLA-DR and sorted with a FACS ARIA. DAPI was used as a dead-live marker. mDCs were low autofluorescent, lineage mix negative, CD11c+ and HLA-DR+ cells. pDCs were CD11c(-) but CD123+. Morphological assessment of sorted mDCs and pDCs was performed. Sorted mDCs were tested in a mixed leukocyte reaction (MLR) with naive CD4+ T cells and evaluated for T cell differentiation and cytokine production. With confocal microscopy DC-T cell interaction was assessed.


RESULTS
Using our sorting strategy, mDCs and pDCs, with a high purity upon FACS analysis of the sorted fraction, were obtained. These cells showed the morphological characteristics of DCs. Most importantly, mDCs were able to induce T cell proliferation and differentiation in a MLR, and interact with T cells as assessed by confocal microscopy. These results indicate the presence of functional DCs. Freezing and thawing of the BAL cells did not affect phenotype or T cell stimulatory capacity of the isolated DCs.


CONCLUSIONS
Using a novel sorting strategy, functional mDCs can be isolated from BAL fluid, enabling a detailed study in pulmonary disease.
Ten Berge
B
B
Department of Pulmonary Medicine, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
Muskens
F
F
Kleinjan
A
A
Hammad
H
H
Hoogsteden
H C
HC
Lambrecht
B N
BN
Van den Blink
B
B
D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology* / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D018613:Microscopy, Confocal
D015703:Antigens, CD; D016207:Cytokines; D006684:HLA-DR Antigens