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Updated: Jun 16, 2026
Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
Two physically, functionally, and developmentally distinct peritoneal macrophage subsets
Eliver Eid Bou Ghosn1, Alexandra A Cassado, Gregory R Govoni
1Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA. eliverg@stanford.edu
Abstract:
The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.
Insights
Two distinct macrophage subsets reside in the peritoneal cavity: large peritoneal macrophages (LPM) and small peritoneal macrophages (SPM). SPM are derived from blood monocytes and are crucial for immune responses following stimulation.
Area of Science:
- Immunology
- Cell Biology
- Macrophage Biology
Background:
- The peritoneal cavity (PerC) harbors diverse immune cells, with macrophages (MØ) frequently studied.
- Existing knowledge on PerC MØ heterogeneity and development is incomplete.
- Understanding PerC MØ subsets is crucial for interpreting ex vivo functional studies.
Purpose of the Study:
- To define and characterize distinct macrophage subsets within the adult mouse peritoneal cavity.
- To investigate the origin, development, and functional responses of these macrophage subsets.
- To elucidate the impact of stimulation on PerC macrophage populations.
Main Methods:
- Isolation and characterization of peritoneal macrophages (MØ) from adult mice.
- Flow cytometry analysis using surface markers (CD11b, F4/80, MHC-II).
- In vivo and in vitro stimulation with lipopolysaccharide (LPS) and thioglycolate.
- Assessment of nitric oxide (NO) production and phagocytic activity.
Main Results:
- Two MØ subsets identified: large peritoneal MØ (LPM) and small peritoneal MØ (SPM).
- LPM (90% in unstimulated PerC) express high CD11b/F4/80, disappear upon stimulation.
- SPM express low CD11b/F4/80, high MHC-II, originate from blood monocytes post-stimulation.
- Both subsets are phagocytic and produce NO; LPS differentially affects their NO production in vitro and in vivo.
Conclusions:
- The peritoneal cavity contains at least two distinct macrophage subsets with differing origins and responses.
- SPM are derived from blood monocytes and are critical responders to inflammatory stimuli.
- Findings reveal novel insights into PerC MØ heterogeneity, development, and function, impacting data interpretation.
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