Research paper
A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester

https://doi.org/10.1016/j.jim.2008.11.019Get rights and content

Abstract

Apoptotic cell phagocytosis has recently raised considerable interest, particularly due to its intricate molecular mechanisms and negative immunologic impact of incompetent clearance of apoptotic cells. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. Labeling with pHrodo succinimidyl ester (SE), a pH-sensitive fluorescent dye, makes engulfed apoptotic cells detectable due to the increased post-phagocytic light emission. This is a valuable tool for phagocytosis studies via FACS. We designed an ex vivo assay, using apoptotic pHrodo-labeled lymphocytes as prey and anti-CD11b-labeled tissue macrophages. To demonstrate its validity of detecting internalized apoptotic lymphocytes, we used MFGE8−/− macrophages, known to have impaired phagocytic ability. Uptake of apoptotic lymphocytes was accelerated and enhanced in splenic macrophages after stimulation with recombinant MFGE8, while peritoneal macrophages were able to compensate for the delayed uptake. This novel assay is a quick and reliable method to evaluate the internalization of apoptotic cells.

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)

  • Ait-OufellaH. et al.

    Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosis in mice

    Circulation

    (2007)
  • AsanoK. et al.

    Masking of phosphatidylserine inhibits apoptotic cell engulfment and induces autoantibody production in mice

    J. Exp. Med.

    (2004)
  • BlanderJ.M. et al.

    Regulation of phagosome maturation by signals from toll-like receptors

    Science

    (2004)
  • BuH.F. 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)
  • HanayamaR. et al.

    Identification of a factor that links apoptotic cells to phagocytes

    Nature

    (2002)
  • HanayamaR. et al.

    Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice

    Science

    (2004)
  • HornefM.W. et al.

    Bacterial strategies for overcoming host innate and adaptive immune responses

    Nat. Immunol.

    (2002)
There are more references available in the full text version of this article.

Cited by (237)

  • PARP1 controls the transcription of CD24 by ADP-ribosylating the RNA helicase DDX5 in pancreatic cancer

    2023, International Journal of Biochemistry and Cell Biology
    Citation Excerpt :

    To verify the role of CD24 in regulating the macrophage-mediated antitumor immune response in pancreatic cancer, we generated Capan-1 and Panc-1 cells with stable CD24 knockdown (Figure S2). To measure phagocytic clearance by live-cell microscopy, GFP+ pancreatic cancer cells with or without CD24 knockdown were labeled with a pH-sensitive dye, pHrodo red (Miksa et al., 2009), and were then cocultured with macrophages. During the 12 hour culture period, we found that pancreatic cancer cells with CD24 knockdown were more readily engulfed by low-pH phagolysosomes (Fig. 2 A and Figure S3A).

View all citing articles on Scopus
1

Supported by NIH grants R01 GM057468, R01 GM053008, and R01 AG028352.

View full text