Chapter 3: Mouse Model of Influenza Infection and Characterisation of Lung
3.7 Surface markers of cells isolated from the Lung Parenchyma
Next, we investigated the APC populations within the lung parenchyma. To isolate cells from the lung tissue, the lungs were finely cut into small pieces and subjected to enzymatic digestion using collagenase to release the cells. However, as the lung parenchyma has an extremely fibrous structure, collagenase digestion was insufficient to completely disrupt the tissue into single cell suspension and necessitated mechanical disruption by repeated forceful pipetting and physically pushing the tissue pieces against the surface of a cell strainer to break it up. As the frequency of DCs in the lung parenchyma is significantly lower compared to lymphoid organs such as the spleen, the use of a technique to enrich for DCs would greatly facilitate analysis and further downstream purification. To do this, we adapted a procedure previously employed by our laboratory for isolating splenic DCs (Wong et al. 2008; Wong et al.
2009) which utilises OPTIPREP density centrifugation. Using OPTIPREP at a density of g=1.064g/ml, CD11c+ APCs from lung tissue were enriched approximately 4-fold from 6% to 23% (Figure 3.7.1). This also had an added advantage of removing large cellular debris as well as dead cells that resulted from enzymatic digestion and physical disruption of the lung tissue which typically formed about 30-40% of all cells obtained.
After enrichment with OPTIPREP, we were also able to further purify CD11c+ cells to ~90% by using MACS beads. Using the same combination of CD11c, CD11b and MHC II, we could identify three major groups of APCs in lung tissue. They were (i) CD11c+ CD11b+ MHCII+ dendritic cells, (ii) CD11c+ CD11b-ve MHCIIhi dendritic
84 cells and (iii) CD11c+ CD11b-ve MHCII-ve/low interstitial macrophages (Fig 3.7.2).
Alveolar macrophages surface markers were identical to that of interstitial macrophages and therefore we could not distinguish between these two macrophages aside from their anatomical location (Fig 3.6). However, it is needful to bear in mind that lavage may not be able to wash out all airspaces and hence lung interstitial macrophages may also comprise alveolar macrophages that were washed out.
Consistent with observations by other groups (del Rio et al. 2007), lung parenchymal DCs did not express such markers such as CD8 and CD4 which are commonly used to differentiate splenic DCs (Figure 3.7.3). Lung DCs could also be distinguished from macrophages by their side scatter (SS) and autofluorescence on the FL1 channel (Figure 3.7.4). Hence, for all subsequent analysis of lung DC populations, we gated on SSlow events or low autofluorescent cells on FL1 (when the channel was not in use) so as minimize spillover of macrophages into the DC populations during analysis.
Figure 3.7.1 Enrichment of lung APCs from whole lung digest using OPTIPREP Representative FACS plot showing the percentage of CD11c+ lung APCs from whole lung digest from C57BL6 mice with or without enrichment using OPTIPREP density centrifugation. FACS plots are gated on live cells. n = 6 mice.
CD11c
MH C I I
6.3% 23.1%
No OPTIPREP OPTIPREP
85
Figure 3.7.2 Surface markers of lung antigen presenting cells from the lung parenchyma
Typical FACS profile of lung APCs after isolation with CD11c magnetic beads. BAL was performed on C57BL6 mice before lungs were excised and digested in colleganase. Cells were enriched for APCs by density centrifugation using OPTIPREP following which CD11c+ cells were purified by positive selection using MACS beads. Using CD11c, CD11b and MHC II markers, 3 populations can be identified: (i) CD11c+MHCII+CD11bhi DCs, (ii) CD11chiMHCII+CD11blo/neg DCs and (iii) CD11chiCD11bnegMHCIIlo/neg interstitial macrophages.
CD11c
CD11b
MH C I I MH C I I
(i) (ii)
(iii)
86 Figure 3.7.3 Lung DCs do not express CD8 and CD4
Lung DCs of C57BL6 mice were enriched from the lung parenchyma using anti- CD11c MACS beads. Cells were split into 3 groups and stained for CD11c, MHC II and either CD8, CD4 or isotype antibody conjugated to the same fluorochrome.
Data representative of 2 experiments.
Isotype
CD8
CD4 CD11c
MH C II
87 Figure 3.7.4 Lung DCs and macrophages can be additionally distinguished by side scatter and autofluorescence
Representative FACS plot of lung cells after enrichment for APCs using OPTIPREP.
DCs were defined as CD11c+MHCIIhi cells whereas macophages were defined as CD11c+MHCIIlo/neg cells. FACS plot shows a backgating analysis of the side scatter and FL1 autofluorescence profile of DCs and macrophages. Data representative of 3 experiments.
DC gate
FS
SS
Ungated Macrophage gate
CD11c
MHC II SS
FS
DC Macrophage
FL1 Empty
DC Macrophage
Backgatinganalysis