Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues

Abstract

Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry-based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206(+)) subsets and other CD206(-) leukocytes. The CD206(-) cells include: (1) three monocyte (CD14(+)) subsets, (2) CD11c(+) dendritic cells (CD14(-), CD11c(+), HLA-DR(+)), (3) plasmacytoid dendritic cells (CD14(-), CD11c(-), HLA-DR(+), CD123(+)), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169(+) and CD169(-) macrophages. In lung tissue, CD169(-) macrophages were a prominent cell type. Using confocal microscopy, CD169(+) macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169(-) macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.

Keywords: alveolar macrophages; interstitial lung disease; interstitial macrophages; interstitial-associated macrophages.

Figure 1.

Flow cytometry panel from human bronchoalveolar lavage (BAL) fluid. (A) Flow cytometry was performed on BAL cells derived from healthy human subjects exposed to filtered air. Using a 13-color antibody panel, subpopulations of mononuclear phagocytic cells (monocytes, macrophages, dendritic cells) and granulocytes (neutrophils, eosinophils, basophils, and mast cells) were identified. The cellular gating depicted is representative of three individual samples (n = 3). (B) Flow-based sorting and morphologic evaluation of human BAL cells. To obtain very rare cell types, including neutrophils, flow-based sorting was performed on BAL cells pooled from three human subjects. Despite pooling of BAL cells, insufficient numbers of eosinophils, basophils, and mast cells were recovered for cytospin analysis. However, alveolar macrophages (AMØs) and neutrophils exhibit typical morphology for these cell types. All images were taken at 60× under oil immersion; scale bar: 10 µm. (C) Graphic representation of relative immune cells distribution in BAL (dendritic cells and plasmacytoid dendritic cells) derived from healthy subjects. Immune cells are quantified as a percentage of CD45⁺ cells (small pie) and percentage of myeloid cells (large pie). This immunophenotyping analysis reveals that AMØs are the predominant cell type in normal human BAL fluid. Data for cell percentages are the average of four independent experiments (n = 4). CD, cluster of differentiation; DC, dendritic cells; FSC-A, forward scatter area; FSC-H, forward scatter height; HLA-DR, human leukocyte antigen DR; NK, natural killer; pDC, plasmacytoid dendritic cells; SSC, side scatter.

Figure 2.

Flow cytometry panel of human whole blood. Flow cytometry was performed on whole blood cells. After obtaining the sample, the red cells underwent lysis. The remaining cells underwent antibody staining per protocol. The cells were separated by FSC-A and FSC-H to obtain singlets (R1). From R1, the cells were analyzed for CD45 expression. CD45⁺ cells (R2) then underwent Live/Dead staining, and live cells (R3) were selected for analysis of CD206 staining. Because lung macrophages do not exist in the whole blood, there were no CD206⁺ cells. CD206⁻ cells (R5) were separated into SSC high (R6) granulocytes and SSC low (R7) nongranulocytes. From the granulocytes (R6), neutrophils (CD16⁺CD24⁻) and eosinophils (CD16⁻CD24⁺) were defined. The nongranulocytes (R7) were separated into CD14⁺ monocytes and CD14⁻ nonmonocytes (R8). The monocytes were then delineated on the basis of CD16 expression. Nonmonocytes (R8) were separated based on CD123 expression. CD123⁺ basophils and pDC were defined based on HLA-DR expression. CD123⁻ cells (R9) were separated into CD11c⁺HLA-DR⁺ DC and CD11c⁻ (R10) populations. This population reflected lymphocytes and B cells. Lymphocyte subsets were separated by CD3 and CD56 staining into NK cells (CD56⁺CD3⁻), natural killer T (NKT) cells (CD56⁺CD3⁺), T cells (CD56⁻CD3⁺), and double-negative cells (R11). From this group (R11), HLA-DR⁺ cells were defined as B Cells. Flow panel is representative of n = 3 samples of whole blood.

Figure 3.

Flow cytometry analyses of human lung tissue. (A) Flow cytometry was performed on human lung single cell suspension derived from subjects declined at the time of organ donation. Using the 13-color antibody panel, similar to that of bronchoalveolar lavage, subpopulations of myeloid cells were identified. The depicted cellular gating is representative of four individual experiments (n = 4). (B) Immune cells were sorted and then underwent cytospin to confirm cellular morphology. The following cells were identified CD169⁻ MØ; AMØs; monocytes; DC; and neutrophils. All images were taken at 60× under oil immersion; scale bar: 10 μm. MØ, macrophage.

Figure 4.

Identifying human pulmonary interstitial-associated macrophages. (A) Flow analyses of BAL and human lung tissues demonstrated the presence of CD169⁻ MØs in the lung tissue but not BAL samples. (B) Differential marker expressions between CD169⁻ MØs, AMØs, and monocytes suggests that CD169⁻ MØs are a unique MØ subpopulation. Cell surface expression of CD71, CD86, and CD80 in CD169⁻ IMØ, CD169⁺ AMØs, CD16⁺ monocytes, CD14⁺CD16⁺ monocytes, and CD14⁺ monocytes were examined. This is a representative sample of n = 6 samples from control lung tissue. The data are presented as a histogram in which the shaded histogram is the isotype control and the open histogram is antibody stained. (C) Confocal microscopy was performed on human lung tissue. Sections were stained for CD206 (red), CD169 (cyan), E cadherin (green), and DAPI (blue). Images at 20× were taken of lung tissue. The upper panel includes single stains for CD206, CD169, and DAPI, which were then merged to demonstrate overall tissue architecture. CD206⁺CD169⁺ cells are located in the airspaces and were consistent with AMØs (merged in white). CD206 + CD169^low/− cells are closely associated with DAPI cells. To confirm the location of cells, higher magnification images (63×) were taken of the merged images with DIC and with E Cadherin. Confocal images reflect a representative sample of control lung tissue. DAPI, 4ʹ,6-diamidino-2-phenylindole; DIC, differential interference contrast; IMØ, interstitial macrophage.

Figure 5.

Flow cytometry panel allows for evaluation of human lung tissues derived from smoking exposure and idiopathic pulmonary fibrosis (IPF). (A) Representative flow cytometry analyses of lung tissue from patients with IPF undergoing lung transplant. (B) Graphic representation of cell populations as a percentage of CD45⁺ cells (small pie) and percentage of myeloid cells (larger pie) from human lung tissue in normal patients, patients with a smoking history, and patients with IPF. (C) Percentages of monocytes and MØs as a percentage of CD45⁺ cells from the different lung tissue samples. Data for cell percentages were obtained from n = 13 samples (normal = 6; smoker = 3, and IPF = 4), which were run and analyzed individually. *P < 0.05, **P < 0.005, and ***P < 0.0005 between the individual sources of monocytes and MØs. dpMØ, double positive macrophages; Mono, monocyte; N.D., none detected.