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Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus
Published on: September 19, 2013
A novel method for isolating dendritic cells from human bronchoalveolar lavage fluid
B Ten Berge1, F Muskens, A Kleinjan
1Department of Pulmonary Medicine, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
Insights
Researchers developed a new method to isolate dendritic cells (DCs) from bronchoalveolar lavage fluid. This technique yields functional myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) for pulmonary disease research.
Area of Science:
- Immunology
- Pulmonary Medicine
Background:
- Dendritic cells (DCs) are crucial for immune responses and implicated in lung diseases.
- Current methods for isolating lung DCs are invasive or alter cell function.
- Bronchoalveolar lavage (BAL) offers a minimally invasive source of local DCs.
Purpose of the Study:
- To develop and validate a novel method for isolating functional dendritic cells (DCs) from bronchoalveolar lavage (BAL) fluid.
- To enable detailed studies of DC function in pulmonary diseases.
Main Methods:
- Developed a novel sorting strategy using fluorescence-activated cell sorting (FACS ARIA) on BAL cells.
- Identified myeloid DCs (mDCs) as lineage-negative, CD11c+, HLA-DR+ and plasmacytoid DCs (pDCs) as CD11c-, CD123+.
- Assessed purity, morphology, and function (mixed leukocyte reaction, T cell interaction) of sorted DCs.
Main Results:
- Successfully isolated high-purity mDCs and pDCs from BAL fluid.
- Sorted mDCs demonstrated characteristic morphology and induced T cell proliferation and differentiation.
- DC function was preserved after freezing and thawing.
Conclusions:
- A novel FACS-based sorting strategy enables isolation of functional mDCs from BAL fluid.
- This method provides a valuable tool for studying DCs in the context of pulmonary disease.
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.
Conclusion:
Using a novel sorting strategy, functional mDCs can be isolated from BAL fluid, enabling a detailed study in pulmonary disease.
