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 2 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-associated macrophages; interstitial macrophages; interstitial lung disease Clinical RelevanceFlow cytometry is an important method that allows for delineation of specific cell components of immune responses and disease states. A flow cytometry panel for myeloid cells in human lung samples (bronchoalveolar lavage and lung tissue) has not been performed previously. Here we develop a single flow cytometry panel that allows for the accurate identification of cellular components in human blood, bronchoalveolar lavage, and lung tissue.

Dendritic Cells and Monocytes with Distinct Inflammatory Responses Reside in Lung Mucosa of Healthy Humans

“…The monocytes referred to in our study could also include monocytederived interstitial macrophages as described by others (27,55,56). Cai et al compared blood monocytes and interstitial macrophages in the lungs of rhesus macaques and showed that they express very similar markers, except that interstitial macrophages, unlike monocytes, did not express CCR2, which is important in the recruitment of monocytes from bone marrow to circulation and then to tissues (55,57).…”

“…However, the limitation of these studies using four-color flow cytometry is the risk of including cell populations with similar phenotypic markers as DCs. In studies applying more advanced flow cytometry panels, noncancerous lung tissue from surgical resections of patients were typically used (14,(26)(27)(28), although it is unclear whether these rare populations are truly representative of DCs present in healthy subjects. Indeed, several studies have investigated DCs in the context of lung diseases such as asthma, chronic obstructive pulmonary disease, bronchiolitis, and sarcoidosis (29)(30)(31)(32)(33)(34)(35).…”

Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists

“…Images were taken at time zero, 10 minutes after exposure to a contractile agent, and 15 minutes after exposure to an airway relaxant with an inverted microscope (Nikon Eclipse TS 100; Nikon, Tokyo, Japan), a 43 objective, and a Nikon DS-Ri2 camera. Alternatively, contraction and relaxation of airways in hPLCSs were measured as previously described (5,15 21 indicator, Oregon green BAPTA-1-AM, as previously described (5,15). Fluorescence images were recorded at 30 images per second with a custom-built two-photon laser scanning microscope and analyzed by Video Savant software (IO Industries, London, ON, Canada) with custom-written scripts.…”

“…This is, to our knowledge, the first comprehensive evaluation of the effect of cryopreservation on a human heterogeneous tissue section with a large variety of cell types. To emphasize the bioassay utility of cryopreserved hPCLSs, we characterize the mechanism of action of bitter-taste receptor agonists, a novel class of bronchodilators, as an inhibition of Ca 21 oscillations in airway smooth muscle. Thus, the application of cryopreserved hPCLSs will profoundly empower the ex vivo immunological, physiological, and pharmacological studies on a variety of lung diseases from the molecular to the integrated tissue level.…”

Macrophage Polarization and Function in Cystic Fibrosis

“…During steady state, AMs don't proliferate (108). AMs are mostly CD206 + cells, though AMs found in the airway linings are CD169 + , whereas, AMs those are found in the airway epithelia are CD169 - (109). SMs are the most immunogenically active macrophages in the body and able to produce enormous amount of pro-inflammatory cytokines and highly cytotoxic (100).…”