Dataset Information

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells.

ABSTRACT: Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly, chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

SUBMITTER: Lyons-Cohen MR

PROVIDER: S-EPMC5512285 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells.

Lyons-Cohen Miranda R MR Thomas Seddon Y SY Cook Donald N DN Nakano Hideki H

Journal of visualized experiments : JoVE 20170405 122

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly, chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environme ...[more]

PMID: 28448013

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Project description:BackgroundAsthma exacerbations evoke emergency room visits, progressive loss of lung function and increased mortality. Environmental and industrial toxicants exacerbate asthma, although the underlying mechanisms are unknown. We assessed whether 3 distinct toxicants, salicylic acid (SA), toluene diisocyanate (TDI), and 1-chloro-2,4-dinitrobenzene (DNCB) induced airway hyperresponsiveness (AHR) through modulating excitation-contraction coupling in human airway smooth muscle (HASM) cells. The toxicants include a non-sensitizing irritant (SA), respiratory sensitizer (TDI) and dermal sensitizer (DNCB), respectively. We hypothesized that these toxicants induce AHR by modulating excitation-contraction (EC) coupling in airway smooth muscle (ASM) cells.MethodsCarbachol-induced bronchoconstriction was measured in precision-cut human lung slices (hPCLS) following exposure to SA, TDI, DNCB or vehicle. Culture supernatants of hPCLS were screened for mediator release. In HASM cells treated with the toxicants, surrogate readouts of EC coupling were measured by phosphorylated myosin light chain (pMLC) and agonist-induced Ca2+ mobilization ([Ca2+]i). In addition, Nrf-2-dependent antioxidant response was determined by NAD(P) H quinone oxidoreductase 1 (NQO1) expression in HASM cells.ResultsIn hPCLS, SA, but not TDI or DNCB, potentiated carbachol-induced bronchoconstriction. The toxicants had little effect on release of inflammatory mediators, including IL-6, IL-8 and eotaxin from hPCLS. In HASM cells, TDI amplified carbachol-induced MLC phosphorylation. The toxicants also had little effect on agonist-induced [Ca2+]i. CONCLUSION: SA, a non-sensitizing irritant, amplifies agonist-induced bronchoconstriction in hPCLS via mechanisms independent of inflammation and Ca2+ homeostasis in HASM cells. The sensitizers TDI and DNCB, had little effect on bronchoconstriction or inflammatory mediator release in hPCLS.ImplicationsOur findings suggest that non-sensitizing irritant salicylic acid may evoke AHR and exacerbate symptoms in susceptible individuals or in those with underlying lung disease.

Dataset Information

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells.

Publications

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells.

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