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Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro
Published on: January 25, 2017
Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro
Lena Lavie1, Larissa Dyugovskaya2, Andrey Polyakov2
1The Lloyd Rigler Sleep Apnea Research Laboratory, Unit of Anatomy and Cell Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Insitute of Technology; lenal@technion.ac.il.
Abstract:
Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (Gϕ). Unlike neutrophils, Gϕ are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. Gϕ also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing Gϕ in order to further investigate their significance and functions.
Insights
Researchers discovered giant phagocytes (Gϕ), a novel, long-lived neutrophil subpopulation. These cells exhibit enhanced phagocytosis and autophagy, offering new insights into immune cell plasticity and function.
Area of Science:
- Immunology
- Cell Biology
Background:
- Neutrophils (PMN) are crucial for pathogen defense but have a short lifespan and can cause tissue damage.
- Recent findings highlight neutrophil plasticity and immunoregulatory roles beyond traditional functions.
- Neutrophil-derived subpopulations with unique characteristics are emerging areas of research.
Purpose of the Study:
- To describe a novel method for preparing and characterizing a unique neutrophil-derived subpopulation in culture.
- To identify and define the key features of these newly discovered giant phagocytes (Gϕ).
- To lay the groundwork for further investigation into the functional significance of Gϕ.
Main Methods:
- Spontaneous culture of neutrophils without external cytokines/growth factors.
- Identification of Gϕ based on size, morphology, and expression of specific cell markers (CD66b, CD63, CD15, CD11b, MPO, NE).
- Assessment of phagocytic activity, oxidative burst, scavenger receptor expression, oxLDL uptake, and autophagy markers (LC3B).
Main Results:
- A novel, long-lived neutrophil-derived subpopulation, termed giant phagocytes (Gϕ), was identified.
- Gϕ exhibit enhanced phagocytosis of neutrophil remnants, latex, and zymosan, and express scavenger receptors (CD68, CD36).
- Gϕ internalize oxidized-low density lipoprotein (oxLDL) and respond with increased reactive oxygen species (ROS) production and autophagy, dependent on phagocytosis and NADPH oxidase.
Conclusions:
- Giant phagocytes (Gϕ) represent a distinct, long-lived, neutrophil-derived phagocytic cell population.
- Their development is linked to phagocytosis, autophagy, and NADPH oxidase-dependent ROS production.
- This study provides a method for Gϕ preparation, essential for future research on their immune functions.
