PMID- 19804781
OWN - NLM
STAT- MEDLINE
DCOM- 20091229
LR  - 20181201
IS  - 1872-7905 (Electronic)
IS  - 0022-1759 (Linking)
VI  - 351
IP  - 1-2
DP  - 2009 Dec 31
TI  - A novel method for isolating dendritic cells from human bronchoalveolar lavage 
      fluid.
PG  - 13-23
LID - 10.1016/j.jim.2009.09.009 [doi]
AB  - 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.
FAU - Ten Berge, B
AU  - Ten Berge B
AD  - Department of Pulmonary Medicine, Erasmus MC, Dr. Molewaterplein 50, 3015 GE 
      Rotterdam, The Netherlands.
FAU - Muskens, F
AU  - Muskens F
FAU - Kleinjan, A
AU  - Kleinjan A
FAU - Hammad, H
AU  - Hammad H
FAU - Hoogsteden, H C
AU  - Hoogsteden HC
FAU - Lambrecht, B N
AU  - Lambrecht BN
FAU - Van den Blink, B
AU  - Van den Blink B
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20091003
PL  - Netherlands
TA  - J Immunol Methods
JT  - Journal of immunological methods
JID - 1305440
RN  - 0 (Antigens, CD)
RN  - 0 (Cytokines)
RN  - 0 (HLA-DR Antigens)
SB  - IM
MH  - Antigens, CD/immunology/metabolism
MH  - Bronchoalveolar Lavage Fluid/*cytology/immunology
MH  - CD4-Positive T-Lymphocytes/immunology/*metabolism/pathology
MH  - Cell Differentiation
MH  - Cell Separation/*methods
MH  - Cytokines/metabolism
MH  - Dendritic Cells/*cytology/immunology/*metabolism
MH  - Flow Cytometry
MH  - HLA-DR Antigens/immunology/metabolism
MH  - Humans
MH  - Immunohistochemistry
MH  - Lymphocyte Activation
MH  - Lymphocyte Culture Test, Mixed
MH  - Microscopy, Confocal
EDAT- 2009/10/07 06:00
MHDA- 2009/12/30 06:00
CRDT- 2009/10/07 06:00
PHST- 2009/04/23 00:00 [received]
PHST- 2009/09/04 00:00 [revised]
PHST- 2009/09/25 00:00 [accepted]
PHST- 2009/10/07 06:00 [entrez]
PHST- 2009/10/07 06:00 [pubmed]
PHST- 2009/12/30 06:00 [medline]
AID - S0022-1759(09)00289-0 [pii]
AID - 10.1016/j.jim.2009.09.009 [doi]
PST - ppublish
SO  - J Immunol Methods. 2009 Dec 31;351(1-2):13-23. doi: 10.1016/j.jim.2009.09.009. 
      Epub 2009 Oct 3.