Research paperA novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester
Introduction
Since Ilya Mechnikov first described phagocytosis, this phenomenon has been recognized as an important function of immune cells (Klemparskaya, 1983). Although many cell types are capable of phagocytosis, some cells are specialized to do just that very efficiently. These cells, such as neutrophils, macrophages and dendritic cells, help to remove intruding microbes, inert material, cellular debris and apoptotic cells (Stuart and Ezekowitz, 2008). This permits maintenance of tissue and organ function and overall continuity of the organism. The efficiency of phagocytosis is determined by the phagocytosing cell type as well as the phagocytized object. It is well established that phagocytosis of certain microbes elicits a strong proinflammatory response and activation in the phagocytes leading to accelerated phagocytosis and recruitment of other immune cells (Blander and Medzhitov, 2004, Watts, 2004). Such a response is desirable in a local infection because it helps to clear the microbes and to mount an innate and adaptive immune response against the intruder (Schnitzler et al., 1999). On the other hand, during ontogenesis, many cells undergo apoptosis as tissue remodeling takes place. These dead cells need to be removed by phagocytosis without eliciting an inflammation. Generally, this is established by the immunosuppressive effect of apoptotic cell phagocytosis on phagocytes themselves (Huynh et al., 2002, Asano et al., 2004). Hanayama et al. have shown that deficient phagocytosis of apoptotic B-cells in the spleen leads to the generation of anti-DNA autoantibodies and to a lupus erythematosus-like disease in mice (Hanayama et al., 2004). They have shown that macrophages deficient in milk fat globule EGF-factor 8 (MFGE8), an opsonizing protein secreted by certain phagocytes with a high affinity to phosphatidylserine expressed on apoptotic cells, are able to bind apoptotic cells on their surface but are inefficient in the engulfment of these targets. The absence of MFGE8 has been proposed to have detrimental proinflammatory effects in several disease models including autoimmunity, atherosclerosis and intestinal mucosa regeneration after injury, linking engulfment of apoptotic cells and inflammation (Hanayama et al., 2004, Ait-Oufella et al., 2007, Bu et al., 2007). There are several phagocytosis assay protocols, based on microscopic evaluation or FACS analysis with labeled targets. In most cases, these assays are sufficient, however, they may overestimate actual engulfment of targets due to the detection of some surface-bound bacteria or other material. Thus, in some circumstances the determination of true engulfment of apoptotic cells is desirable. The purpose of this report is to demonstrate a novel and simple approach to a phagocytosis assay that determines the engulfment of apoptotic cells by flow cytometry or fluorescent microscope.
Section snippets
Animals
In our study, we used male Sprague–Dawley rats (6–8 weeks old; 250–300 g BW; Charles River), C57BL6/J wild-type (WT) mice (6–8 weeks old; 20–25 g; Taconic), and C57BL6/J MFG-E8−/− mice (6–8 weeks old; 20–25 g; generous gift by S. Nagata, Osaka University, Japan). All experiments were performed according to national guidelines for the use of animals in research and approved by the Animal Care and Use Committee of the Feinstein Institute for Medical Research.
Reagents
We purchased the following reagents:
Discussion and conclusion
Phagocytosis of microorganisms and “altered self” (e.g., apoptotic cells) is important in tissue remodeling and embryogenesis, host-defense and innate immune response, and serves as a nutrient source in unicellular organisms and is required for antigen presentation in mammals. The act of phagocytosis is a sequence of events. It involves the engagement with the object, activation of intracellular signaling pathways, cytoskeleton rearrangement, and internalization. Phagocytized material
Acknowledgement
We sincerely thank Dr. Asha Varghese for critical reading of the manuscript.
References (15)
- et al.
Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosis in mice
Circulation
(2007) - et al.
Masking of phosphatidylserine inhibits apoptotic cell engulfment and induces autoantibody production in mice
J. Exp. Med.
(2004) - et al.
Regulation of phagosome maturation by signals from toll-like receptors
Science
(2004) - et al.
Milk fat globule-EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium
J. Clin. Invest.
(2007) - et al.
Identification of a factor that links apoptotic cells to phagocytes
Nature
(2002) - et al.
Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice
Science
(2004) - et al.
Bacterial strategies for overcoming host innate and adaptive immune responses
Nat. Immunol.
(2002)
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Supported by NIH grants R01 GM057468, R01 GM053008, and R01 AG028352.