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Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro
Published on: November 26, 2018
Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14
1Department of Internal Medicine, Justus-Liebig-University, Giessen 35392, Germany. ulrich.a.maus@innere.med.uni-giessen.de
Abstract:
The evaluation of monocytes recruited into the alveolar space under both physiological and inflammatory conditions is hampered by difficulties in discriminating these cells from resident alveolar macrophages (rAMs). Using the intravenous injected fluorescent dye PKH26, which accumulated in rAMs without labeling blood leukocytes, we developed a technique that permits the identification, isolation, and functional analysis of monocytes recruited into lung alveoli of mice. Alveolar deposition of murine JE, the homologue of human monocyte chemoattractant protein (MCP)-1 (JE/MCP-1), in mice provoked an alveolar influx of monocytes that were recovered by bronchoalveolar lavage and separated from PKH26-stained rAMs by flow cytometry. Alveolar recruited monocytes showed a blood monocytic phenotype as assessed by cell surface expression of F4/80, CD11a, CD11b, CD18, CD49d, and CD62L. In contrast, CD14 was markedly upregulated on alveolar recruited monocytes together with increased tumor necrosis factor-alpha message, discriminating this monocyte population from peripheral blood monocytes and rAMs. Thus monocytes recruited into the alveolar air space of mice in response to JE/MCP-1 keep phenotypic features of blood monocytes but upregulate CD14 and are "primed" for enhanced responsiveness to endotoxin with increased cytokine expression.
Insights
Researchers developed a new method to identify and analyze monocytes in mouse lungs. This technique distinguishes recruited monocytes from resident alveolar macrophages, aiding in inflammatory studies.
Area of Science:
- Immunology
- Cell Biology
- Pulmonary Medicine
Background:
- Distinguishing between recruited monocytes and resident alveolar macrophages (rAMs) in the lungs is challenging.
- Understanding monocyte behavior in the alveolar space is crucial for studying lung inflammation.
Purpose of the Study:
- To develop a method for identifying, isolating, and functionally analyzing monocytes recruited into the mouse lung alveoli.
- To characterize the phenotype of these recruited monocytes and compare them to rAMs and peripheral blood monocytes.
Main Methods:
- Utilized intravenous injection of the fluorescent dye PKH26, which labels rAMs but not blood leukocytes.
- Induced monocyte influx into alveoli using murine JE (homologue of MCP-1).
- Employed bronchoalveolar lavage and flow cytometry to separate and analyze recruited monocytes from PKH26-labeled rAMs.
Main Results:
- The PKH26 labeling technique successfully differentiated recruited monocytes from rAMs.
- Recruited monocytes exhibited a blood monocytic phenotype (F4/80, CD11a, CD11b, CD18, CD49d, CD62L expression).
- Upregulation of CD14 and increased tumor necrosis factor-alpha message distinguished alveolar monocytes from peripheral monocytes and rAMs, indicating they are primed for enhanced responsiveness.
Conclusions:
- The developed technique enables accurate identification and isolation of alveolar-recruited monocytes.
- These monocytes retain blood monocyte features but upregulate CD14, suggesting a primed state for inflammatory responses in the lungs.
- This finding advances the study of monocyte roles in lung physiology and inflammation.
