Journal Article

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

Hotten, Danielle F., Hollingsworth, John W., Malakhau, Yuryi, Gunn, Michael D., Tighe, Robert M., Yu, Yen-Rei A., Ghio, Andrew J., Noble, Paul W., Kraft, Monica, Volker, Ellen

2016

Overview

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.

• Publisher: Oxford University Press,American Thoracic Society
• Subject: Adolescent / Adult / Alveoli / Biomarkers - blood / Bronchoalveolar Lavage Fluid - cytology / Bronchoalveolar Lavage Fluid - immunology / Bronchus / CD11c antigen / CD123 antigen / CD14 antigen / Cell culture / Confocal microscopy / Dendritic cells / Disease / Epithelium / Female / Flow Cytometry / Gene expression / Granulocytes / Histocompatibility antigen HLA / Humans / Immune system / Immunophenotyping - methods / Infections / Lavage / Leukocytes (basophilic) / Leukocytes (eosinophilic) / Leukocytes (granulocytic) / Leukocytes (neutrophilic) / Lung - cytology / Lung - immunology / Lungs / Lymphocytes / Macrophages / Macrophages, Alveolar - immunology / Male / Mast cells / Methods / Microscopy, Confocal / Monocytes / Myeloid cells / Myeloid Cells - immunology / Neutrophils / Original Research / Pulmonary fibrosis / Respiratory tract / Sialic Acid Binding Ig-like Lectin 1 - blood / Studies / Young Adult