Advertisement

Isolation and Characterization of Mononuclear Phagocytes in the Mouse Lung and Lymph Nodes

  • Sophie L. Gibbings
  • Claudia V. Jakubzick
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1809)

Abstract

There is a diverse population of mononuclear phagocytes (MPs) in the lungs, comprised of macrophages, dendritic cells (DCs), and monocytes. The existence of these various cell types suggests that there is a clear division of labor and delicate balance between the MPs under steady-state and inflammatory conditions. Here we describe how to identify pulmonary MPs using flow cytometry and how to isolate them via cell sorting. In steady-state conditions, murine lungs contain a uniform population of alveolar macrophages (AMs), three distinct interstitial macrophage (IM) populations, three DC subtypes, and a small number of tissue-trafficking monocytes. During an inflammatory response, the monocyte population is more abundant and complex since it acquires either macrophage-like or DC-like features. All in all, studying how these cell types interact with each other, structural cells, and other leukocytes within the environment will be important to understanding their role in maintaining homeostasis and during the development of disease.

Key words

Macrophage Monocyte Dendritic cell Mononuclear phagocyte Lung Flow cytometry Pulmonary Interstitial 

Abbreviations

AM

Alveolar macrophage

BAL

Bronchoalveolar lavage

DC

Dendritic cell

IM

Interstitial macrophage

IN

Intranasal

IT

Intratracheal

LLN

Mediastinal lung-draining lymph node

MP

Mononuclear phagocyte

Notes

Acknowledgment

Grant support: C.V.J. NIH R01 HL115334 and R01 HL135001.

References

  1. 1.
    Janssen WJ, Bratton DL, Jakubzick CV, Henson PM (2016) Myeloid cell turnover and clearance. Microbiol Spectr 4(6). https://doi.org/10.1128/microbiolspec.MCHD-0005-2015
  2. 2.
    MacLean JA, Xia W, Pinto CE, Zhao L, Liu HW, Kradin RL (1996) Sequestration of inhaled particulate antigens by lung phagocytes. A mechanism for the effective inhibition of pulmonary cell-mediated immunity. Am J Pathol 148(2):657–666PubMedPubMedCentralGoogle Scholar
  3. 3.
    Holt PG (2005) Pulmonary dendritic cells in local immunity to inert and pathogenic antigens in the respiratory tract. Proc Am Thorac Soc 2(2):116–120. https://doi.org/10.1513/pats.200502-017AW CrossRefPubMedGoogle Scholar
  4. 4.
    Sung SS, Fu SM, Rose CE Jr, Gaskin F, Ju ST, Beaty SR (2006) A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins. J Immunol 176(4):2161–2172CrossRefGoogle Scholar
  5. 5.
    Vermaelen K, Pauwels R (2005) Pulmonary dendritic cells. Am J Respir Crit Care Med 172(5):530–551. https://doi.org/10.1164/rccm.200410-1384SO CrossRefPubMedGoogle Scholar
  6. 6.
    Vermaelen KY, Carro-Muino I, Lambrecht BN, Pauwels RA (2001) Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes. J Exp Med 193(1):51–60CrossRefGoogle Scholar
  7. 7.
    Jakubzick C, Tacke F, Llodra J, van Rooijen N, Randolph GJ (2006) Modulation of dendritic cell trafficking to and from the airways. J Immunol 176 (6):3578–3584.CrossRefGoogle Scholar
  8. 8.
    Jakubzick C, Helft J, Kaplan TJ, Randolph GJ (2008) Optimization of methods to study pulmonary dendritic cell migration reveals distinct capacities of DC subsets to acquire soluble versus particulate antigen. J Immunol Methods 337(2):121–131. https://doi.org/10.1016/j.jim.2008.07.005 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Desch AN, Randolph GJ, Murphy K, Gautier EL, Kedl RM, Lahoud MH, Caminschi I, Shortman K, Henson PM, Jakubzick CV (2011) CD103+ pulmonary dendritic cells preferentially acquire and present apoptotic cell-associated antigen. J Exp Med 208(9):1789–1797. https://doi.org/10.1084/jem.20110538 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Jakubzick C, Randolph GJ (2010) Methods to study pulmonary dendritic cell migration. Methods Mol Biol 595:371–382. https://doi.org/10.1007/978-1-60761-421-0_24 CrossRefPubMedGoogle Scholar
  11. 11.
    Guilliams M, Lambrecht BN, Hammad H (2013) Division of labor between lung dendritic cells and macrophages in the defense against pulmonary infections. Mucosal Immunol 6(3):464–473. https://doi.org/10.1038/mi.2013.14 CrossRefPubMedGoogle Scholar
  12. 12.
    Guilliams M, Ginhoux F, Jakubzick C, Naik SH, Onai N, Schraml BU, Segura E, Tussiwand R, Yona S (2014) Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol 14(8):571–578. https://doi.org/10.1038/nri3712 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Desch AN, Henson PM, Jakubzick CV (2013) Pulmonary dendritic cell development and antigen acquisition. Immunol Res 55(1–3):178–186. https://doi.org/10.1007/s12026-012-8359-6 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Desch AN, Gibbings SL, Clambey ET, Janssen WJ, Slansky JE, Kedl RM, Henson PM, Jakubzick C (2014) Dendritic cell subsets require cis-activation for cytotoxic CD8 T-cell induction. Nat Commun 5:4674. https://doi.org/10.1038/ncomms5674 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Atif SM, Nelsen MK, Gibbings SL, Desch AN, Kedl RM, Gill RG, Marrack P, Murphy KM, Grazia TJ, Henson PM, Jakubzick CV (2015) Cutting edge: roles for Batf3-dependent APCs in the rejection of minor histocompatibility antigen-mismatched grafts. J Immunol 195(1):46–50. https://doi.org/10.4049/jimmunol.1500669 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kim TS, Braciale TJ (2009) Respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic CD8+ T cell responses. PLoS One 4(1):e4204. https://doi.org/10.1371/journal.pone.0004204 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Kim TS, Gorski SA, Hahn S, Murphy KM, Braciale TJ (2014) Distinct dendritic cell subsets dictate the fate decision between effector and memory CD8(+) T cell differentiation by a CD24-dependent mechanism. Immunity 40(3):400–413. https://doi.org/10.1016/j.immuni.2014.02.004 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kim TS, Hufford MM, Sun J, Fu YX, Braciale TJ (2010) Antigen persistence and the control of local T cell memory by migrant respiratory dendritic cells after acute virus infection. J Exp Med 207(6):1161–1172. https://doi.org/10.1084/jem.20092017 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wakim LM, Bevan MJ (2011) Cross-dressed dendritic cells drive memory CD8+ T-cell activation after viral infection. Nature 471(7340):629–632. https://doi.org/10.1038/nature09863 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    del Rio ML, Rodriguez-Barbosa JI, Kremmer E, Forster R (2007) CD103− and CD103+ bronchial lymph node dendritic cells are specialized in presenting and cross-presenting innocuous antigen to CD4+ and CD8+ T cells. J Immunol 178 (11):6861–6866Google Scholar
  21. 21.
    Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama M, Calderon B, Schraml BU, Unanue ER, Diamond MS, Schreiber RD, Murphy TL, Murphy KM (2008) Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science 322(5904):1097–1100. https://doi.org/10.1126/science.1164206 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Tussiwand R, Everts B, Grajales-Reyes GE, Kretzer NM, Iwata A, Bagaitkar J, Wu X, Wong R, Anderson DA, Murphy TL, Pearce EJ, Murphy KM (2015) Klf4 expression in conventional dendritic cells is required for T helper 2 cell responses. Immunity 42(5):916–928. https://doi.org/10.1016/j.immuni.2015.04.017 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Gibbings SL, Thomas SM, Atif SM, McCubbrey AL, Desch AN, Danhorn T, Leach SM, Bratton DL, Henson PM, Janssen WJ, Jakubzick CV (2017) Three unique interstitial macrophages in the murine lung at steady state. Am J Respir Cell Mol Biol 57:66–76. https://doi.org/10.1165/rcmb.2016-0361OC CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Janssen WJ, Barthel L, Muldrow A, Oberley-Deegan RE, Kearns MT, Jakubzick C, Henson PM (2011) Fas determines differential fates of resident and recruited macrophages during resolution of acute lung injury. Am J Respir Crit Care Med 184:547–560. https://doi.org/10.1164/rccm.201011-1891OC CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mould KJ, Barthel L, Mohning MP, Thomas SM, McCubbrey AL, Danhorn T, Leach SM, Fingerlin TE, O'Connor BP, Reisz JA, D'Alessandro A, Bratton DL, Jakubzick CV, Janssen WJ (2017) Cell origin dictates programming of resident versus recruited macrophages during acute lung injury. Am J Respir Cell Mol Biol 57:294–306. https://doi.org/10.1165/rcmb.2017-0061OC CrossRefGoogle Scholar
  26. 26.
    McCubbrey AL, Barthel L, Mohning MP, Redente EF, Mould KJ, Thomas SM, Leach SM, Danhorn T, Gibbings SL, Jakubzick CV, Henson PM, Janssen WJ (2018) Deletion of c-FLIP from CD11bhi macrophages prevents development of Bleomycin-induced lung fibrosis. Am J Respir Cell Mol Biol 58:66–78. https://doi.org/10.1165/rcmb.2017-0154OC CrossRefPubMedGoogle Scholar
  27. 27.
    Misharin AV, Morales-Nebreda L, Reyfman PA, Cuda CM, Walter JM, McQuattie-Pimentel AC, Chen CI, Anekalla KR, Joshi N, Williams KJN, Abdala-Valencia H, Yacoub TJ, Chi M, Chiu S, Gonzalez-Gonzalez FJ, Gates K, Lam AP, Nicholson TT, Homan PJ, Soberanes S, Dominguez S, Morgan VK, Saber R, Shaffer A, Hinchcliff M, Marshall SA, Bharat A, Berdnikovs S, Bhorade SM, Bartom ET, Morimoto RI, Balch WE, Sznajder JI, Chandel NS, Mutlu GM, Jain M, Gottardi CJ, Singer BD, Ridge KM, Bagheri N, Shilatifard A, Budinger GRS, Perlman H (2017) Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. J Exp Med 214(8):2387–2404. https://doi.org/10.1084/jem.20162152 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Jakubzick CV, Randolph GJ, Henson PM (2017) Monocyte differentiation and antigen-presenting functions. Nat Rev Immunol 17(6):349–362. https://doi.org/10.1038/nri.2017.28 CrossRefPubMedGoogle Scholar
  29. 29.
    Larson SR, Atif SM, Gibbings SL, Thomas SM, Prabagar MG, Danhorn T, Leach SM, Henson PM, Jakubzick CV (2016) Ly6C(+) monocyte efferocytosis and cross-presentation of cell-associated antigens. Cell Death Differ 23(6):997–1003. https://doi.org/10.1038/cdd.2016.24 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Gibbings SL, Goyal R, Desch AN, Leach SM, Prabagar M, Atif SM, Bratton DL, Janssen W, Jakubzick CV (2015) Transcriptome analysis highlights the conserved difference between embryonic and postnatal-derived alveolar macrophages. Blood 126(11):1357–1366. https://doi.org/10.1182/blood-2015-01-624809 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Jakubzick C, Gautier EL, Gibbings SL, Sojka DK, Schlitzer A, Johnson TE, Ivanov S, Duan Q, Bala S, Condon T, van Rooijen N, Grainger JR, Belkaid Y, Ma'ayan A, Riches DW, Yokoyama WM, Ginhoux F, Henson PM, Randolph GJ (2013) Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes. Immunity 39(3):599–610. https://doi.org/10.1016/j.immuni.2013.08.007 CrossRefPubMedGoogle Scholar
  32. 32.
    Jakubzick C, Bogunovic M, Bonito AJ, Kuan EL, Merad M, Randolph GJ (2008) Lymph-migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph nodes. J Exp Med 205(12):2839–2850. https://doi.org/10.1084/jem.20081430 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Yona S, Kim KW, Wolf Y, Mildner A, Varol D, Breker M, Strauss-Ayali D, Viukov S, Guilliams M, Misharin A, Hume DA, Perlman H, Malissen B, Zelzer E, Jung S (2013) Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38(1):79–91. https://doi.org/10.1016/j.immuni.2012.12.001 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of PediatricsNational Jewish HealthDenverUSA
  2. 2.Department of Microbiology and ImmunologyUniversity of ColoradoDenverUSA

Personalised recommendations