Flow Cytometry Reveals Similarities Between Lung Macrophages in Humans and Mice

Bharat, Ankit; Bhorade, Sangeeta; Morales‐Nebreda, Luisa; McQuattie‐Pimentel, Alexandra C.; Soberanes, Saul; Ridge, Karen M.; DeCamp, Malcolm M.; Mestan, Karen K.; Perlman, Harris; Budinger, G. R. Scott; Misharin, Alexander V.

doi:10.1165/rcmb.2015-0147le

Cited by 158 publications

(176 citation statements)

References 14 publications

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“…Independent of our work, in this issue of the Journal, Bharat and colleagues (pp. 147-149) performed a complementary characterization of macrophages from human lung tissue (35), which validates these observations as reproducible and reliable methods of separating lung macrophage subsets. The flow cytometry profile described here should allow a more detailed examination of human AMØs and IMØs to determine their function in normal homeostasis and disease states.…”

Section: Discussionmentioning

confidence: 68%

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

Hotten

Malakhau

et al. 2016

Am J Respir Cell Mol Biol

212

183

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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.

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Section: Discussionmentioning

confidence: 68%

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

Hotten

Malakhau

et al. 2016

Am J Respir Cell Mol Biol

212

183

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“…Using multiparameter cytometry and geneexpression profiling, the identification of MP populations in human tissues has become possible (26,41). However, the ability to identify tissue resident MPs has been limited by the health and availability of human tissues.…”

Section: Discussionmentioning

confidence: 99%

Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes

Desch

Gibbings

Goyal

et al. 2016

Am J Respir Crit Care Med

153

202

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Rationale: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors.Objectives: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs.Methods: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. Measurements and Main Results:We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood.Conclusions: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics. Correspondence and requests for reprints should be addressed to Claudia V. Jakubzick, Ph.D., Department of Pediatrics and Immunology, National Jewish Health, 1400 Jackson Street, Denver, CO 80206. E-mail: jakubzickc@njhealth.org This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org The human respiratory tract has a branching structure that terminates in millions of alveoli, whose luminal surface covers an approximate area of 50 to 100 m 2 . In comparison to other barrier surfaces, such as the skin (2 m 2 ) and the gut (10 m 2 ), this surface area is massive, and therefore, comprises the body's largest interface with the ambient environment. Because of normal respiratory function, the average human exchanges 7,000 to 9,000 L of gas each day and inhales billions of particles, allergens, and microbes. Accordingly, the human lung constitutes a major site for the innate and adaptive immune responses. In this context, cells in the mononuclear phagocyte system (MPS), which consists of macrophages, monocytes, monocytederived cells, and dendritic cells (DCs), play critical roles. T...

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“…In contrast to recent studies that focused on distinguishing alveolar macrophages, interstitial monocytes, and/or extra-or intravascular monocytes from whole-lung homogenate (11,12,32), our study focused on the assessment of coexpression of a range of markers on airway phagocytes obtainable via BAL. This evaluation revealed multiple subpopulations of alveolar macrophages in both humans and cynomolgus macaques The potential significance of this heterogeneity regarding Mtb infection is suggested by our observations that the frequency of these subpopulations within BAL appears to differ between humans and macaques, and also between the preinfection BAL samples of macaques that ultimately developed LTBI or active TB.…”

Section: Discussionmentioning

confidence: 99%

“…However, strategies for the characterization of these cells are not well standardized. Recent studies in both humans and macaques have categorized lung and airway phagocytes using a limited number of markers and have focused largely on the anatomic localization of these populations (7)(8)(9)(10)(11)(12). In contrast, we hypothesized that Mtb infection could induce the migration of cells from peripheral blood or other lung compartments into the alveolar spaces and could alter the activation and maturation states of these populations.…”

Section: Clinical Relevancementioning

confidence: 99%

Diversity of Human and Macaque Airway Immune Cells at Baseline and during Tuberculosis Infection

Silver

Myers

Jarvela

et al. 2016

Am J Respir Cell Mol Biol

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Immune cells of the distal airways serve as "first responders" of host immunity to the airborne pathogen Mycobacterium tuberculosis (Mtb). Mtb infection of cynomolgus macaques recapitulates the range of human outcomes from clinically silent latent tuberculosis infection (LTBI) to active tuberculosis of various degrees of severity. To further advance the application of this model to human studies, we compared profiles of bronchoalveolar lavage (BAL) cells of humans and cynomolgus macaques before and after Mtb infection. A simple gating strategy effectively defined BAL T-cell and phagocyte populations in both species. BAL from Mtb-naive humans and macaques showed similar differential cell counts. BAL T cells of macaques were composed of fewer CD41 cells but more CD81 and CD4 1 CD8 1 double-positive cells than were BAL T cells of humans. The most common mononuclear phagocyte population in BAL of both species displayed coexpression of HLA-DR, CD206, CD11b, and CD11c; however, multiple phagocyte subsets displaying only some of these markers were observed as well. Macaques with LTBI displayed a marked BAL lymphocytosis that was not observed in humans with LTBI. In macaques, the prevalence of specific mononuclear phagocyte subsets in baseline BAL correlated with ultimate outcomes of Mtb infection (i.e., LTBI versus active disease).Overall, these findings demonstrate the comparability of studies of pulmonary immunity to Mtb in humans and macaques. They also indicate a previously undescribed complexity of airway mononuclear phagocyte populations that suggests further lines of investigation relevant to understanding the mechanisms of both protection from and susceptibility to the development of active tuberculosis within the lung.

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Flow Cytometry Reveals Similarities Between Lung Macrophages in Humans and Mice

Cited by 158 publications

References 14 publications

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

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

Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes

Diversity of Human and Macaque Airway Immune Cells at Baseline and during Tuberculosis Infection

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