A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.
Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8.
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (β2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4/80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11.
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.
1. Lung perfusion and single cell suspension
2. Magnetic isolation and CD11c+ cells enrichment
3. Conventional dendritic cell (cDC) isolation
4. Alternate protocol for obtaining a single cell suspension and CD11c+ cells enrichment
5. Representative Results:
Lung cDC are identified as CD11chi/MHC-IIhi cell population. As shown in Fig. 1, cDC represent a smaller percentage of 1.4% when compared to some other tissues such as spleen (4.8%). However, in contrast to spleen, lung CD11c positive cells express different amounts of MHC-II, including a cell population different than that of cDC. After the single-cell preparation, the cell yield of total lung cells was about 3.0 to 3.8 x 107 cells/lung with a viability of 60-70 % from which a little over 1% represented cDC subset. This percentage was increased after the CD11c magnetic isolation where the total lung cDC was incremented about 10 times (>16%) from the original preparation (Fig. 2A). However, CD11c cells expressing low amounts of MHC-II (CD11chi/MHC-IIlo, macrophages) represented a high contaminating cell population (>70%) which was mixed with the lung cDC subset. As shown in Fig. 2B, those cells were eliminated after the cell sorting step when the cDC purity reached >96%. The usual yield of cDC after the whole procedure was 5 x 104 cells/lung. Microphotographs show morphological characteristics of cDC as rounded cells with the typical dendrites (upper panel). On the other hand, CD11chi/MHC-IIlo represented a morphologically different subset that shows cellular protuberances which are typical of macrophages (MΦ, lower panel) 12.
Figure 1. Differential DC frequency in mouse lung and spleen. Lung and spleen tissue from C57BL/6 mice were collected and treated with collagenase. Following collagenase digestion, cells were stained with anti-mouse CD11c-PE-Cy7 and anti-mouse I-A/I-E-FITC. Representative flow cytometry plots show percentages of cDC (CD11chi/MHC-IIhi) in lung and spleen.
Figure 2. Isolation of cDC from mouse lung. Mouse lung single cell suspension was labeled with anti-CD11c microbeads and passed through an automatic cell separator. A) A representative plot shows the percentages of cDC (CD11chi/MHC-IIhi) and Macrophages (MΦ, CD11chi/MHC-IIlo) populations after CD11c enrichment. Further staining of the enriched fraction followed using anti-mouse CD11c-PE-Cy7 and anti-mouse I-A/I-E-FITC. Double positive cells were sorted and analyzed by flow cytometry. To identify their morphology, cells were cytospun and stained with a modified Wright-Giemsa staining. B) Representative plots show percentages of cDC and MΦ after cell sorting. Microphotographs show a representative image of lung cDC and lung MΦ. Scale bar = 20 μm.
Isolation of pulmonary mouse DC is an important technique for the study of a wide range of respiratory stimuli. The process of obtaining these cells includes critical steps that prevent loss of cells as well as cell viability and purity. Perfusing the lung before collection will help to eliminate any peripheral cells as well as reduce contaminant erythrocytes. The use of the automated dissociation can be advanteogus when a large number of lungs are handled, otherwise you can opt for the alternative protocol, keeping in mind that the dispersion of the tissue after collagenase digestion should be done gently when done manually. The incubation period of the lung tissue while in collagenase solution should not be extended beyond the indicated incubation time. After this step, the remaining process must be done using ice-cold buffers and refrigerated centrifuges to reduce cell death. The lysis of contaminating erythrocytes is a step that should not be repeated more than twice during the whole process. Passing the cell suspension through a nylon mesh, at the indicated steps, is critical to prevent clogging of the cell sorter as well as to reduce cell aggregates which will compromise the cell purity. If further culture in vitro of the isolated cells is involved, all steps must be performed under a biosafety cabinet to prevent any contamination. It is not expected that the cell sorting step will compromise the sterility of the sample.
Dendritic cells are crucial for studies of the innate and adaptive immune interactions. The procedure described has the potential to be applied for the isolation of other DC subsets if appropriate antibodies are used during the magnetic isolation and the cell sorting 8. In addition, pulmonary macrophages (CD11chi/MHC-IIlo) can also be purified from the same cell preparation. Despite the limited number of pulmonary DC that can be isolated per animal by using this procedure, this technique provides the most accurate system to study lung DC. However, prior to DC isolation, mice could be treated with Fms-Like tyrosine kinase 3 (Flt3-) Ligand, an agent that increases DC numbers 13 . Although spleen DC may be easier and faster to isolate, the differences in the maturation state of the DC in lung and spleen must be taken into consideration. In contrast to spleen DC, lung DC express an immature phenotype that is switched to a mature one after e.g. a viral infection8,14.
In summary, the isolation of the specific subsets of lung DC through the described protocol, provides a unique tool that can help to determine the specific role and/or contribution of pulmonary DC in the host immune responses and can be applied to any experimental mouse model that involves the study of the respiratory tract.
The authors have nothing to disclose.
The authors would like to thank to Marilyn Dietrich at the LSU Flow Cytometry Core Facility for her help with the cell sorting and Peter Mottram for his assistance with the microphotographs. This work was funded by the Flight Attendant Medical Research Institute, the LSU-Competitive Research Program Award, and the NIH/NIAID Grants P20 RR020159 and R03AI081171.
Name of the reagent | Company | Catalogue number | Comments (optional) |
ACK lysing buffer | Invitrogen | D6-0005DG | |
Anti-mouse CD11c (HL3) | BD Pharmingen | 5580979 | PE-Cy7 conjugated |
Anti-mouse I-A/I-E (269) | BD-Pharmingen | 553623 | FITC conjugated |
Collangenase Type 1A | Sigma | 9891-500MG | |
Cell strainers | BD Falcon | 352340, 352360 | |
CD11c (N418) Microbeads | Miltenyi | 130-052-001 | |
DNase I | Sigma | D5025-150KU | |
Hank’s Balanced Salt solution | Invitrogen | 14170 | |
Hepes buffer solution | Invitrogen | 15630 | |
Petri dishes 60 mm | BD Falcon | 351016 | |
GentleMACS™ C tubes | Miltenyi | 130-093-237 | |
Gentle MACS dissociator | Miltentyi | 130-093-235 | |
AutoMACS-Pro™ | Miltenyi | 130-092-545 | |
FASCS Aria | BD |