How to dispose of dead or dying cells?

Where does a cell go after programmed cell death? It’s not so much an existential question as it is a practical matter. Cells that undergo apoptosis or one of the many newly described, nonapoptotic forms of programmed cell death (e.g., necroptosis, ferroptosis, pyroptosis) can’t linger. They must be removed efficiently to maintain tissue homeostasis and to avoid initiation of necrotic events that lead to a local immune response. Removal involves the immunologically silent uptake of whole apoptotic cells or cell fragments into specialized phagocytes that have been named efferocytes. Efferocytosis—the specific process by which these dead cells are engulfed and digested—was coined from the Latin word efferre, which translates “to take to the grave.” This term is used to differentiate the disposal of cells that met a programmed cell death from other common phagocytic processes such as the engulfment of pathogens and other foreign particles.

How do cells that carry out efferocytosis recognize their target?

Dead cells can be ingested by both professional phagocytes (e.g., macrophages, monocytes, dendritic cells) and by cells from near-neighbor tissues (e.g., epithelial cells, endothelial cells, fibroblasts). Apoptotic cells release a number of chemotactic molecules as “find-me” signals and display various ligands on their surface as “eat-me” signals (Figure 1). “Find-me” signals include triphosphate nucleotides (e.g., ATP, UTP), the chemokine CX3CL1, and the signaling lipids, lysophosphatidylcholine, and sphingosine-1-phosphate. The most well-known “eat-me” signal is externalized negatively charged phosphatidylserine, but modified carbohydrates and externalized proteins such as calreticulin can also provoke efferocytosis. Cells capable of carrying out efferocytosis are equipped with specialized receptors to detect these “find-me” and “eat-me” signals.

Figure 1. The process of efferocytosis involves the recognition, engulfment, and clearance of apoptotic cells.

It has been suggested that healthy cells avoid being efferocytosed by displaying “don’t eat-me” signals that prevent their removal. The transmembrane protein CD47 is thought to deliver anti-efferocytic signals via an inhibitory receptor on phagocytes called signal regulatory protein α. Loss of CD47 during cell death would remove this inhibition and enable uptake and removal of the dead cell. Exploitation of this process is of interest to novel cancer treatments, since many cancers express high levels of CD47.

How is efferocytosis involved in immune responses?

The overall process of efferocytosis is so efficient that even when significant cell removal is ongoing (>100 billion cells are removed daily from a normal adult mammal and during embryogenesis), only minimal evidence of actual dying cells is apparent. Efferocytes are actively anti-immunogenic due to their production of anti-inflammatory cytokines, and their ability to suppress the inflammatory signaling of the ingested cell. The anti-inflammatory protein, annexin A1, and specialized pro-resolving mediators, such as resolvin E1 and protectin D1, which function in promoting the resolution of inflammation, have all been shown to be upregulated during the process of efferocytosis. Without proper efferocytosis, apoptotic cells can undergo a secondary necrosis that may lead to the dangerous release of damage-associated molecular patterns (DAMPs), which can simulate the immune system and induce an inappropriate inflammatory response. In the extreme, loss of the efferocytic process can result in the presentation of self-antigens in an inflammatory context, potentially resulting in autoimmunity. On the other hand, when tumor cells or cells infected with viruses are ingested, efferocytic dendritic cells are known to cross-present antigens from the engulfed material to cytotoxic T cells, resulting in an appropriate immune response to these insults. Investigation of this process is of great interest for the development of cancer immunotherapies and vaccines.

How can I detect efferocytosis?

Cayman scientists have developed the Efferocytosis Assay Kit as a tool to study modulators of efferocytosis, programmed cell death, specialized pro-resolving mediators, and cross-presentation. The kit conveniently combines the fluorescent probes needed to visualize phagocytic (effector) cells (CytoTell™ Blue) and target cells (CFSE) with a compound that induces apoptosis (staurosporine). To perform the assay, viable cells are stained with a cell permeable version of CFSE, then exposed to staurosporine to induce apoptosis. These cells are then fed to phagocytic cells stained with CytoTell™ Blue. Both sets of labeled cells are cultured together at a predetermined ratio and analyzed by flow cytometry or fluorescence microscopy. Because CytoTell™ Blue has a peak excitation of 405 nm and a peak emission of 450 nm, it can be multiplexed with green CFSE fluorescence (491/518 nm ex./em. max) for multicolor cell analysis. Phagocytes that have efferocytosed apoptotic cells will appear both blue and green (Figure 2).

Figure 2. Workflow of Cayman's Efferocytosis Assay.

How can I adapt this kit to my experiment?

During cell culture, apoptotic cells can be treated with modulators that change the ability of phagocytes to detect them. Conversely, effector cells can be treated with modulators that alter their ability to recognize apoptotic cells. This kit can also be used to follow the fate of efferocytosed apoptotic cells in relation to antigen presentation/cross-presentation, inflammation, effector cell death, and many additional biological processes.

Learn more about the features of this kit.

Download our Apoptosis Assay Guide (PDF) to learn about additional cell-based assays from Cayman.

Suggested Reading

Elliott, M.R., Koster, K.M., Murphy, P.S. Efferocytosis signaling in the regulation of macrophage inflammatory responses. J. Immunol. 198(4), 1387-1394 (2017).

Henson, P.M. Cell removal: Efferocytosis. Annu. Rev. Cell. Dev. Biol. 6(33), 127-144, (2017).

Karaji, N. and Sattentau, Q.J. Efferocytosis of pathogen-infected cells. Front. Immunol. 8, 1863 (2017).

Kumar, S. and Birge, R.B. Efferocytosis. Curr. Biol. 26(13), R558–R559 (2016).

Tzelepis, F., Verway, M., Daoud, J., et al. Annexin1 regulates DC efferocytosis and cross-presentation during Mycobacterium tuberculosis infection. J. Clin. Invest. 125(2), 752–768 (2015).

Schwab, J.M., Chiang, N., Arita, M., et al. Resolvin E1 and protectin D1 activate inflammation-resolution programmes. Nature. 447(7146), 869-874 (2007).