Abstract
Phagocytosis is the process by which phagocytes, including macrophages, neutrophils and monocytes, engulf and kill invading pathogens, remove foreign particles, and clear cell debris. Phagocytes and their ability to phagocytose are an important part of the innate immune system and are critical for homeostasis of the host. Impairment in phagocytosis has been associated with numerous diseases and disorders. Different cytokines have been shown to affect the phagocytic process. Cytokines including TNFα, IL-1β, GM-CSF, and TGF-β1 were found to promote phagocytosis, whereas high mobility group box-1 (HMGB1) inhibited the phagocytic function of macrophages. Here, we describe two commonly used methods to assess the phagocytic function of cultured macrophages, which can easily be applied to other phagocytes. Each method is based on the extent of engulfment of FITC-labeled latex minibeads by macrophages under different conditions. Phagocytic activity can be assessed either by counting individual cells using a fluorescence microscope or measuring fluorescence intensity using a flow cytometer.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Metchnikoff I (1884) A disease of Daphnia caused by a yeast. A contribution to the theory of phagocytes as agents for attack on disease-causing organisms. Archiv Pathol Anat Physiol Klin Med 96:177–195
Flannagan RS, Jaumouillé V, Grinstein S (2012) The cell biology of phagocytosis. Annu Rev Pathol 7:61–98
Underhill DM, Goodridge HS (2012) Information processing during phagocytosis. Nat Rev Immunol 12:492–502
Swanson JA (2008) Shaping cups into phagosomes and macropinosomes. Nat Rev Mol Cell Biol 9:639–649
Garin J, Diez R, Kieffer S et al (2001) The phagosome proteome insight into phagosome functions. J Cell Biol 152:165–180
Greenberg S, Grinstein S (2002) Phagocytosis and innate immunity. Curr Opin Immunol 14:136–145
Rabinovitch M (1995) Professional and non-professional phagocytes: an introduction. Trends Cell Biol 5:85–87
Martin TR, Frevert CW (2005) Innate immunity in the lungs. Proc Am Thorac Soc 2:403
Zhang P, Summer WR, Bagby GJ et al (2000) Innate immunity and pulmonary host defense. Immunol Rev 173:39–51
Savill J, Dransfield I, Gregory C et al (2002) A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2:965–975
Hodge S, Hodge G, Scicchitano R et al (2003) Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells. Immunol Cell Biol 81:289–296
Berenson CS, Garlipp MA, Grove LJ et al (2006) Impaired phagocytosis of nontypeable Haemophilus influenzae by human alveolar macrophages in chronic obstructive pulmonary disease. J Infect Dis 194:1375–1384
McClure CD, Schiller NL (1996) Inhibition of macrophage phagocytosis by Pseudomonas aeruginosa rhamnolipids in vitro and in vivo. Curr Microbiol 33:109–117
Smith ME (2001) Phagocytic properties of microglia in vitro: implications for a role in multiple sclerosis and EAE. Microsc Res Tech 54:81–94
Andrews T, Sullivan KE (2003) Infections in patients with inherited defects in phagocytic function. Clin Microbiol Rev 16:597–621
Taylor A, Finney-Hayward T, Quint J et al (2010) Defective macrophage phagocytosis of bacteria in COPD. Eur Respir J 35:1039–1047
Donnelly LE, Barnes PJ (2012) Defective phagocytosis in airways disease. Chest J 141:1055–1062
O’Reilly PJ, Hickman-Davis JM, Davis IC et al (2003) Hyperoxia impairs antibacterial function of macrophages through effects on actin. Am J Respir Cell Mol Biol 28:443–450
Morrow DMP, Entezari-Zaher T, Romashko J III et al (2007) Antioxidants preserve macrophage phagocytosis of Pseudomonas aeruginosa during hyperoxia. Free Radic Biol Med 42:1338–1349
Suttorp N, Simon LM (1983) Decreased bactericidal function and impaired respiratory burst in lung macrophages after sustained in vitro hyperoxia. Am Rev Respir Dis 128:486–490
Patel VS, Sitapara RA, Gore A et al (2013) High Mobility Group Box-1 mediates hyperoxia-induced impairment of Pseudomonas aeruginosa clearance and inflammatory lung injury in mice. Am J Respir Cell Mol Biol 48:280–287
Phipps JC, Aronoff DM, Curtis JL et al (2010) Cigarette smoke exposure impairs pulmonary bacterial clearance and alveolar macrophage complement-mediated phagocytosis of Streptococcus pneumoniae. Infect Immun 78:1214–1220
Davis KA (2006) Ventilator-associated pneumonia: a review. J Intensive Care Med 21:211–226
Hartl D, Latzin P, Hordijk P et al (2007) Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease. Nat Med 13:1423–1430
Ren Y, Savill J (1995) Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis. J Immunol 154:2366–2374
Lotze MT, Tracey KJ (2005) High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 5:331–342
Entezari M, Weiss DJ, Sitapara R et al (2012) Inhibition of HMGB1 enhances bacterial clearance and protects against P. aeruginosa pneumonia in cystic fibrosis. Mol Med 18:477–485
van Zoelen MA, Ishizaka A, Wolthuis EK et al (2008) Pulmonary levels of high-mobility group box 1 during mechanical ventilation and ventilator-associated pneumonia. Shock 29:441–445
Liu G, Wang J, Park YJ et al (2008) High mobility group protein-1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine. J Immunol 181:4240
Kennedy TP, Nelson S (2013) Hyperoxia, HMGB1, and ventilator-associated pneumonia: reducing risk by practicing what we teach. Am J Respir Cell Mol Biol 48:269–270
Barile FA (2013) Cell culture methodology. In: Principles of toxicology testing, vol 2, 2nd edn, CRC press, Boca Raton, pp 163–186
Drevets DA, Campbell PA (1991) Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J Immunol Methods 142:31–38
Dunn P, Tyrer H (1981) Quantitation of neutrophil phagocytosis, using fluorescent latex beads. Correlation of microscopy and flow cytometry. J Lab Clin Med 98:374–381
Lehmann AK, Sørnes S, Halstensen A (2000) Phagocytosis: measurement by flow cytometry. J Immunol Methods 243:229–242
Acknowledgments
This work was supported by grants (LLM) from National Heart and Blood Institute (HL093708) and St. John’s University.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Sharma, L. et al. (2014). Assessment of Phagocytic Activity of Cultured Macrophages Using Fluorescence Microscopy and Flow Cytometry. In: Vancurova, I. (eds) Cytokine Bioassays. Methods in Molecular Biology, vol 1172. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0928-5_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0928-5_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0927-8
Online ISBN: 978-1-4939-0928-5
eBook Packages: Springer Protocols