Abstract
Accessibility and ease of leukocyte extraction led to the peritoneal cavity becoming one of the most commonly used sites to obtain primary macrophages for in vitro analyses and to model inflammation. However, the advent of multiparameter flow cytometry has highlighted the complexity of the mononuclear phagocyte compartment of the serous cavities, which contains multiple populations of macrophages, dendritic cells, and monocytes that coexist with other leukocytes. Given that serous cavity macrophages are known to contribute to both the maintenance of tissue homeostasis and the generation and resolution of inflammation, a thorough understanding of the cells that comprise the peritoneal macrophage compartment, how to identify them from related mononuclear phagocytes, and the processes required to isolate them for ex vivo and in vitro analysis is important if we are to fully understand their function in different tissue contexts. Here, we detail commonly used methods to isolate leukocytes from the peritoneal and pleural cavities and describe reliable strategies to identify the discrete populations of mononuclear phagocytes in these sites.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Charalampidis C, Youroukou A, Lazaridis G et al (2015) Pleura space anatomy. J Thorac Dis 7:S27–S32
Ghosn EEB, Cassado AA, Govoni GR et al (2010) Two physically, functionally, and developmentally distinct peritoneal macrophage subsets. Proc Natl Acad Sci U S A 107:2568–2573
Nguyen HH, Tran BT, Muller W et al (2012) IL-10 acts as a developmental switch guiding monocyte differentiation to macrophages during a murine peritoneal infection. J Immunol (Baltimore, Md.: 1950) 189:3112–3120
Davies LC, Rosas M, Smith PJ et al (2011) A quantifiable proliferative burst of tissue macrophages restores homeostatic macrophage populations after acute inflammation. Eur J Immunol 41:2155–2164
Cain DW, O’Koren EG, Kan MJ et al (2013) Identification of a tissue-specific, C/EBPβ-dependent pathway of differentiation for murine peritoneal macrophages. J Immunol (Baltimore, Md.: 1950) 191:4665–4675
Accarias S, Genthon C, Rengel D et al (2016) Single-cell analysis reveals new subset markers of murine peritoneal macrophages and highlights macrophage dynamics upon Staphylococcus aureus peritonitis. Innate Immun 22:382–392
Okabe Y, Medzhitov R (2014) Tissue-specific signals control reversible program of localization and functional polarization of macrophages. Cell 157:832–844
Bain CC, Hawley CA, Garner H et al (2016) Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities. Nat Commun 7:ncomms11852
Kim KW, Williams JW, Wang YT et al (2016) MHC II+ resident peritoneal and pleural macrophages rely on IRF4 for development from circulating monocytes. J Exp Med 213:1951–1959
Liao CT, Rosas M, Davies LC et al (2016) IL-10 differentially controls the infiltration of inflammatory macrophages and antigen-presenting cells during inflammation. Eur J Immunol 46:2222–2232
Tamoutounour S, Henri S, Lelouard H et al (2012) CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis. Eur J Immunol 42:3150–3166
Bain CC, Scott CL, Uronen-Hansson H et al (2013) Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunol 6:498–510
Tamoutounour S, Guilliams M, Montanana Sanchis F et al (2013) Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin. Immunity 39:925–938
Jakubzick C, Gautier EL, Gibbings SL et al (2013) Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes. Immunity 39:599–610
Gautier EL, Shay T, Miller J et al (2012) Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat Immunol 13:1118–1128
Schlitzer A, McGovern N, Teo P et al (2013) IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity 38:970–983
Scott CL, Bain CC, Wright PB et al (2015) CCR2(+)CD103(−) intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells. Mucosal Immunol 8:327–339
Louis C, Cook AD, Lacey D et al (2015) Specific contributions of CSF-1 and GM-CSF to the dynamics of the mononuclear phagocyte system. J Immunol (Baltimore, Md.: 1950) 195:134–144
van de Laar L, Saelens W, De Prijck S et al (2016) Yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages. Immunity 44:755–768
Lavin Y, Winter D, Blecher-Gonen R et al (2014) Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159:1312–1326
Cailhier JF, Partolina M, Vuthoori S et al (2005) Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation. J Immunol (Baltimore, Md. : 1950) 174:2336–2342
Cailhier JF, Sawatzky DA, Kipari T et al (2006) Resident pleural macrophages are key orchestrators of neutrophil recruitment in pleural inflammation. Am J Respir Crit Care Med 173:540–547
Ansel KM, Harris RBS, Cyster JG (2002) CXCL13 is required for B1 cell homing, natural antibody production, and body cavity immunity. Immunity 16:67–76
Rosas M, Davies LC, Giles PJ et al (2014) The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal. Science 344:645–648
Gautier EL, Ivanov S, Williams JW et al (2014) Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival. J Exp Med 211:1525–1531
Daems WT, de Bakker JM (1982) Do resident macrophages proliferate? Immunobiology 161:204–211
Ratajczak MZ, Jaskulski D, Pojda Z et al (1987) Omental lymphoid organ as a source of macrophage colony stimulating activity in peritoneal cavity. Clin Exp Immunol 69:198–203
Wijffels JF, Hendrickx RJ, Steenbergen JJ et al (1992) Milky spots in the mouse omentum may play an important role in the origin of peritoneal macrophages. Res Immunol 143:401–409
Ginhoux F, Guilliams M (2016) Tissue-resident macrophage ontogeny and homeostasis. Immunity 44:439–449
van Furth R, Cohn ZA, Hirsch JG et al (1972) The mononuclear phagocyte system: a new classification of macrophages, monocytes, and their precursor cells. Bull World Health Organ 46:845–852
Schulz C, Gomez Perdiguero E, Chorro L et al (2012) A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336:86–90
Hoeffel G, Wang Y, Greter M et al (2012) Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. J Exp Med 209:1167–1181
Gomez Perdiguero E, Klapproth K, Schulz C et al (2015) Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature 518:547–551
Hoeffel G, Chen J, Lavin Y et al (2015) C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages. Immunity 42:665–678
Sheng J, Ruedl C, Karjalainen K (2015) Most tissue-resident macrophages except microglia are derived from fetal hematopoietic stem cells. Immunity 43:382–393
Bain CC, Bravo-Blas A, Scott CL et al (2014) Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat Immunol 15:929–937
Epelman S, Lavine KJ, Beaudin AE et al (2014) Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation. Immunity 40:91–104
Molawi K, Wolf Y, Kandalla PK et al (2014) Progressive replacement of embryo-derived cardiac macrophages with age. J Exp Med 211:2151–2158
Hashimoto D, Chow A, Noizat C et al (2013) Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 38:792–804
Yona S, Kim KW, Wolf Y et al (2013) Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38:79–91
Guilliams M, Ginhoux F, Jakubzick C et al (2014) Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol 14:571–578
Wu X, Briseño CG, Durai V et al (2016) Mafb lineage tracing to distinguish macrophages from other immune lineages reveals dual identity of Langerhans cells. J Exp Med 213:2553–2565
Gautier EL, Ivanov S, Lesnik P et al (2013) Local apoptosis mediates clearance of macrophages from resolving inflammation in mice. Blood 122:2714–2722
Davies LC, Rosas M, Jenkins SJ et al (2013) Distinct bone marrow-derived and tissue-resident macrophage lineages proliferate at key stages during inflammation. Nat Commun 4:1886
Newson J, Stables M, Karra E et al (2014) Resolution of acute inflammation bridges the gap between innate and adaptive immunity. Blood 124:1748–1764
Gundra UM, Girgis NM, Gonzalez MA et al (2017) Vitamin A mediates conversion of monocyte-derived macrophages into tissue-resident macrophages during alternative activation. Nat Immunol 18:642–653
Mowat AM, Bain CC (2017) Alternative monocytes settle in for the long term. Nat Immunol 18:599–600
Rosas M, Thomas B, Stacey M et al (2010) The myeloid 7/4-antigen defines recently generated inflammatory macrophages and is synonymous with Ly-6B. J Leukoc Biol 88:169–180
Caton ML, Smith-Raska MR, Reizis B (2007) Notch-RBP-J signaling controls the homeostasis of CD8- dendritic cells in the spleen. J Exp Med 204:1653–1664
Guilliams M, Dutertre CA, Scott CL et al (2016) Unsupervised high-dimensional analysis aligns dendritic cells across tissues and species. Immunity 45:669–684
Miller JC, Brown BD, Shay T et al (2013) Deciphering the transcriptional network of the dendritic cell lineage. Nat Immunol 13:888–899
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Bain, C.C., Jenkins, S.J. (2018). Isolation and Identification of Murine Serous Cavity Macrophages. In: Rousselet, G. (eds) Macrophages. Methods in Molecular Biology, vol 1784. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7837-3_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7837-3_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7836-6
Online ISBN: 978-1-4939-7837-3
eBook Packages: Springer Protocols