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[Frontiers in Bioscience 7, d1298-1313, May 1, 2002]
1298
PHAGOCYTOSIS OF APOPTOTIC CELLS IN MAMMALS, CAENORHABDITIS ELEGANS AND DROSOPHILA
MELANOGASTER: MOLECULAR MECHANISMS AND PHYSIOLOGICAL CONSEQUENCES
Nathalie C. Franc
MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, Gower street, London WC1E
6BT, United Kingdom
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Molecular mechanisms of phagocytosis of apoptotic cells in mammals: a great complexity
3.1. Professional phagocytes and receptors
3.2. Amateur phagocytes
3.3. Receptors on amateur phagocytes
3.4. Apoptotic cells surface ligands or ‘eat me signals’, opsonins and bridging molecules
4. Phagocytosis of apoptotic cells and animal models: a genetic dissection
4.1. Caenorhabditis elegans and the clearance of apoptotic cells by ‘amateur’ neighboring cells
4.2. Drosophila melanogaster and the clearance of apoptotic cells by ‘professional’ phagocytes, the macrophages
5. Physiological consequences of apoptotic cell clearance
6. Perspectives
7. Acknowledgments
8. References
1. ABSTRACT
Phagocytosis is the necessary corollary of
apoptosis. It leads to the clearance of apoptotic cells by
phagocytes, which can be ‘professional’ or ‘amateur’. I
review the known molecular aspects of phagocytosis of
apoptotic corpses in mammals, Caenorhabditis elegans
and Drosophila melanogaster from the point of view of
the phagocyte and the apoptotic corpse. I highlight recent
advances made in the field and discuss the physiological
outcomes and consequences of this process. Indeed,
phagocytosis of apoptotic cells is important in shaping or
remodeling tissues to maintain their integrity and
specialized functions during development and wound
healing. It also contributes to the development of
inflammation and/or its resolution after an injury or
infection. This perhaps explains why the molecular
mechanisms of phagocytosis of apoptotic cells are
redundant and complex in mammals and suggests why
they appear to have been mostly conserved through
evolution. Caenorhabditis elegans has already proven to
be useful in genetically dissecting the molecular
mechanisms underlying phagocytosis of apoptotic corpses
by ‘amateur’ neighboring cells. Drosophila melanogaster
will become the model of choice in genetically dissecting
the molecular mechanisms underlying phagocytosis of
apoptotic cells by ‘professional’ phagocytes such as
macrophages.
2. INTRODUCTION
The rapid recognition and ingestion of dying
cells, so-called apoptotic corpses or bodies occurs by
phagocytosis. Apoptosis is the process by which cells are
programmed to die in a timely and orderly manner (1-3).
A significant overproduction of cells occurs at all stages of
development in all organisms, and thus apoptosis is critical
in the regulation and maintenance of homeostasis and
tissue integrity (1-3). Apoptosis is also important for the
swift removal of cells with damaged DNA. Studies of the
initiation and execution of the apoptotic program have
attracted considerable interest over the past two decades.
The use of animal models such as Caenorhabditis elegans
(C. elegans) and Drosophila melanogaster, with the
advantage of powerful genetics, has greatly contributed to
advances made in this field (4-6).
The importance of phagocytosis of apoptotic
cells has often been underestimated. Most cells that
undergo apoptosis are ingested in situ by either
‘professional’ or ‘amateur’ (non-professional) phagocytes.
The combined efforts of many laboratories and the use of
model organisms, in particular C. elegans, have led to the
revelation of some of the molecular mechanisms by which
this occurs (7-9). But why are such efforts made?
Phagocytosis of apoptotic corpses has often been
considered as a mere housekeeping function, yet it plays a
critical role in shaping organs during development, as well
as in tissue remodeling during wound healing. There is
now growing evidence that the orderly removal of
apoptotic corpses is more crucial than originally thought.
Apoptotic cells are generally rapidly removed, prior to the
loss of their plasma membrane integrity, thus preventing
Phagocytosis of apoptotic cells: an overview
1299
damage caused by accidental leakage of their potentially
cytotoxic or antigenic content (1, 10). However, several
studies in mammals showed that macrophages
phagocytosing apoptotic cells can also trigger further
programmed cell death of bystander cells (11-13).
Further, recent studies in C. elegans have demonstrated
that the uptake itself may accelerate apoptosis of cells not
yet fully committed to die (14, 15).
Phagocytosis of apoptotic cells is usually
accompanied by anti-inflammatory signals, which are
critical in the resolution of inflammation and the
regulation of immune responses (16). In some
circumstances, however, clearance of apoptotic cells may
also lead to the activation of macrophages and trigger the
production of pro-inflammatory cytokines (17, 18). This
discrepancy is not yet fully understood. Studies suggest
that failure to dispose of apoptotic cells can lead to
neurodegeneration, and that it may also contribute to
autoimmune diseases (19-23). Interestingly, it was also
recently shown that parasites can enter professional
phagocytes by routes similar to those taken by apoptotic
corpses, and thus subvert the ability of the phagocytes to
participate in their killing, as well as that of infected
phagocytes, providing the parasites with a favorable
environment in which to grow (24, 25).
Finally, more recent findings suggest that the
uptake of apoptotic leukemia cells by dendritic cells (DCs)
may have a protective role against leukemia in vivo (26).
Similar findings using DCs that have engulfed
apoptotic/necrotic melanoma cells further the hope of new
strategies in cancer vaccine development (27). In contrast,
the uptake of apoptotic cells expressing an activated
oncogene could lead to its horizontal transfer into the
phagocytes, which may in turn develop a tumorigenic
phenotype (28). The complexity and importance of
phagocytosis of apoptotic cells therefore deserves our
attention.
This review is an attempt to summarize the
current knowledge of the molecular mechanisms of
apoptotic cell clearance while highlighting recent advances
made in the field, and to discuss the many outcomes and
unknowns of this important biological process.
3. MOLECULAR MECHANISMS OF
PHAGOCYTOSIS OF APOPTOTIC CELLS IN
MAMMALS: A GREAT COMPLEXITY
3.1. Professional phagocytes and receptors
Both ‘amateur’ and ‘professional’ phagocytes
mediate phagocytosis of apoptotic cells. ‘Amateur’
phagocyte generally refers to ‘on scene’ cells that are
capable of recognizing and engulfing an apoptotic cell.
However, the term of ‘amateur’ can also be employed for
cells that are poorly phagocytic, whether or not they have
the ability to migrate to the site of apoptosis. In contrast,
professional phagocytes are generally mobile cells that can
infiltrate various tissues and have a high phagocytic
activity.
Macrophages or neutrophils are the professional
phagocytes. A large number of surface receptors have
been identified on these cells that participate in
phagocytosis of apoptotic cells. On macrophages, these
include (i) the alpha-v/beta-3 integrin, or vitronectin
receptor (29), (ii) the scavenger receptor of class B, CD36
(30-32), (iii) the scavenger receptor of class A, SR-A (33),
(iv) the glycoprotein macrosialin/CD68 (34), (v) the ATP-
binding cassette transporter 1, ABC1 (35, 36), (vi) the
cluster of differentiation 14 (CD14)(37), (vii) the
phosphatidylserine-receptor (PS-receptor)(38), (viii) the
surfactant protein A (SP-A), a collectin (39), (ix) the MER
receptor tyrosine kinase (22), as well as (x) a recently
identified protein complex that involves calreticulin
(CRT), (also called collagenous tail binding C1q receptor
(cC1qR)), and the endocytic receptor CD91, or low
density lipoprotein (LDL) receptor-related protein (LRP),
(also known as the alpha-2 macroglobulin (alpha-2 m)
receptor)(40). Furthermore, IgG-opsonised apoptotic cells
can be recognized and engulfed via both the complement
receptor CR3 and the Fc-gamma receptor pathways (21,
41-43)(Figure 1).
Most of these receptors were characterized by
inhibition studies using apoptotic ligands or monoclonal
antibodies. Interestingly, several of these proteins bind a
wide variety of ligands. For instance, ABC-transporters
belong to the largest family of transporters, and translocate
a wide range of sugars, amino-acids, metal ions, peptides,
proteins, as well as a large number of hydrophobic
compounds and metabolites across membranes (44).
Mutations in ABC-transporters thus affect multiple cell
functions and 14 out of the 48 known human genes have
been associated with genetic disorders such as cystic
fibrosis, neurological diseases, retinal degeneration,
cholesterol and bile transport defects, anemia, and drug
response (44). ABC1 is unique in that it is found both on
the apoptotic cells as well as on the phagocyte (35, 45).
ABC1 was also recently shown to participate in the
redistribution of phosphatidylserine (PS) on both the
apoptotic cell and the phagocyte, although it is not yet
known how ABC1 promotes this important lipid
redistribution across the membrane (46). Flipping of PS to
the outer leaflet of the plasma membrane is an apoptotic
cell surface change found on most if not all apoptotic cells
throughout the animal kingdom (47). A PS-receptor was
recently characterized that participate in phagocytosis of
apoptotic cells (38). Scavenger receptors (SRs), a large
family of receptors defined by their binding affinity for a
wide range of polyanionic ligands, which participate in
their endocytosis, also bind PS (34, 48-50).
Several receptors for phagocytosis of apoptotic
cells, such as SRs, also play a role in phagocytosis of
bacteria and participate in innate immune responses (50).
CD14, a leucine-rich glycoprotein, is also one such
receptor and was first characterized as a
lipopolysaccharide (LPS) receptor that participates in
bacterial clearance (51). However, lipid-derived moieties
are not the only bacterial and apoptotic ligands. Collectins
are calcium-dependent lectins that target carbohydrate
structures on pathogens, resulting in the agglutination and
Phagocytosis of apoptotic cells: an overview
1300
Figure 1. Schematic representation of the mammalian receptors involved in phagocytosis of apoptotic cells by professional
phagocytes, the macrophages. The vitronectin receptor is composed of the alpha-v/beta-3 integrins, and interacts with the class
B scavenger receptor, CD36 via a bridge of TSP-1, a protein of the extracellular matrix. Other scavenger receptors, such as the
scavenger receptor class A, SR-A and CD68 also participate in the clearance of apoptotic cells. The ABC1 transporter is found
on both the macrophage and the apoptotic cells. CD14, a GPI-anchored receptor, which binds LPS, also recognizes apoptotic
cells via binding of the adhesion molecule ICAM-3, a highly glycosylated Ig-superfamily member. The PS-receptor, a predicted
type II transmembrane protein, binds PS, a phospholipid flipped on the outer surface of apoptotic cell membranes. The collectin
SP-A and the collectin-related complement protein C1q both participate in the recognition of apoptotic cells, possibly via
binding to altered sugars at the surface of apoptotic cells. C1q binds to CRT, a protein of the plasma endoreticulum that is
targeted to the surface membrane of the macrophage via its binding to the LDL receptor-related protein, LRP, itself composed of
an alpha chain and a membrane spanning beta chain. MER is a tyrosine kinase also found on RPE cells involved in the
clearance of ROS photoreceptors, which are shed daily and in a large number in the retina. Both the Fc-gamma receptor and
CR3 can also participate in the clearance of apoptotic cells via their opsonization with Ig-G antibodies or complement proteins.
enhanced clearance of microorganisms (52). These
trimeric proteins may assemble into larger oligomers.
Each polypeptide chain consists of a short amino-terminus,
a collagen like region, an alpha-helical coiled-coil, and a
carbohydrate recognition domain (CRD). The CRD
confers the binding specificity onto lectins, and the CRDs
found on lectins with similar binding affinity are generally
highly conserved in amino-acid sequence. Inhibition
studies have suggested that many lectins with different
binding affinities may participate in clearance of apoptotic
cells (39, 40, 53-55). Although most are uncharacterized,
one collectin, SP-A, was recently shown to participate in
the clearance of apoptotic cells by alveolar macrophages
(39).
A number of phagocyte receptors cooperate in
binding and triggering the uptake of apoptotic cells. CD36
cooperates with the vitronectin receptor alpha-v/beta-3
(31). Other data suggest that CD36 may also cooperate
with the PS-receptor (56). Most recently, yet another
combination of proteins was unraveled that triggers
recognition and uptake of apoptotic cells. An antibody
against cC1qR, one proposed receptor for C1q and
collectins, effectively inhibited binding of C1q-coated
apoptotic cells (40). cC1qR has an almost complete
identical amino acid sequence to CRT (57). Although
originally identified as an endoplasmic reticulum protein,
CRT was recently found on the surface of macrophages
(40). cC1qR/CRT does not have a transmembrane domain
and thus may require a partner to trigger its localization to
the membrane. Interestingly, cC1qR/CRT can bind to
LRP, a cell surface receptor, and an anti-LRP antibody
also blocked phagocytosis of apoptotic Jurkat cells by
macrophages in vitro (40, 58). Both cC1qR and LRP co-
localized on the cell surface of macrophages (40). C1q and
MBL, a collectin, which also inhibited phagocytosis of
apoptotic cells, competed in binding to the same receptor on
macrophages (40). Using a receptor modulation experiment,
Ogden and colleagues showed that C1q, cC1qR/CRT and
LRP cooperated to trigger phagocytosis of apoptotic cells by
macrophages, although a direct physical interaction between
all the partners remains to be formally proven (40).
Interestingly, this uptake occurs via a process reminiscent of
macropinocytosis, which involves the formation of large
macropinocytic vacuoles accompanied with concurrent
ingestion of extracellular fluid (40).
Phagocytosis of apoptotic cells: an overview
1301
Finally, the MER receptor tyrosine kinase was
identified by genetic analysis of a mutation that affected
phagocytosis of apoptotic cells by amateur phagocytes and
will therefore be discussed further below (59). MER was
subsequently found on macrophages where it sustains a
similar function (22).
3.2. Amateur phagocytes
A large number of ‘amateur’ phagocytes have
been identified in mammals that include some epithelial
cells, fibroblasts and endothelial cells, Sertoli cells,
microglial cells, renal mesangial cells, as well as immature
DCs (1, 10, 60). Many of these cells, in particular
epithelial cells, participate in the shaping or remodeling of
organs or tissues, and contribute to the maintenance of
their integrity and specialized functions. For instance,
cells of the retinal-pigmented epithelium (RPE) clear the
enormous number of apoptotic rod outer segments (ROS)
that are shed daily, and play a key role in maintaining
vision (61). In the Royal College of Surgeons (RCS) rat,
RPE cells fail to recognize and engulf shed ROS (62, 63).
This defect leads to subsequent apoptosis of cone and rod
photoreceptors and results in degeneration of the retina,
which ultimately causes blindness (61). Similarly,
olfactory epithelial cells may play a role in maintaining
olfaction by efficiently clearing apoptotic neurons, which
are extensively renewed in the adult olfactory organ (64).
Finally, a massive loss of cells by apoptosis occurs during
involution of the mammary gland where epithelial cells
engulf their dying neighbors (65). This process may be
critical for the normal remodeling of the gland in
preparation for the next wave of lactation. Macrophages
were also observed in these tissues and may participate in
the removal of apoptotic cells.
However, apoptosis often occurs in tissues
where macrophages do not infiltrate, such as the brain and
testis, where the clearance of apoptotic cells is fully
accounted for by ‘amateur’ yet efficient phagocytes. In
the testis, the somatic Sertoli cells clear dying
spermatogenic cells in the seminiferous tubules (66). The
meaning of this apoptotic clearance is not yet well
understood, but Sertoli cells play multiple roles in
nurturing and supporting germ cells survival (67, 68). In
the brain, microglial cells are the primary immune cells of
the central nervous system (CNS) where they engulf
numerous apoptotic neurons, thereby preventing
neurodegeneration and cerebrovascular strokes.
Phagocytosis of apoptotic inflammatory cells by microglia
may also be an important mechanism for the resolution of
inflammatory attacks of the CNS.
Other ‘amateur’ phagocytes serve as a defense
system for a particular tissue or play a critical protective
role in the regulation of immune responses after an injury
or infection. They can contribute to the development of an
inflammation and/or to its resolution. For instance, renal
mesanglial cells clear apoptotic neutrophils. A failure of
this clearing process results in glomerulonephritis, an
inflammation of the kidney that can be temporary and
reversible or may be progressive, resulting in destruction
of the glomeruli and subsequent chronic or permanent
renal failure (19, 69). In the thymus, epithelial nurse cells
clear apoptotic thymocytes and participate in the selection
of specific lymphocyte populations (70). Fibroblasts
phagocytose apoptotic neutrophils, and can also clear a
large number of red blood cells after a trauma or during
internal bleeding (53, 71, 72). In the liver, hepatocytes,
Kupffer cells and endothelial cells can all engulf apoptotic
lymphocytes and senescent erythrocytes (73, 74).
Sinusoidal cells in particular were shown to be able to
phagocytose apoptotic lymphocytes from the circulation,
which allows them to participate in the development of a
tight and specific response, or the resolution of such a
response (75).
Finally and most interestingly, immature DCs
can also participate in the clearance of apoptotic cells (76-
78). DCs are mobile cells with important immune
functions. They are professional antigen-presenting cells
(APCs) that participate in the activation of both CD4 and
CD8-positive T cells. Although they are competent to
phagocytose apoptotic cells, immature DCs are much less
efficient phagocytes than macrophages or neutrophils and
perform better in their absence. Thus, they are considered
as ‘amateur’ phagocytes. Furthermore, and in contrast to
other phagocytes, DCs do not silently destroy the digested
apoptotic cells but process them for antigen presentation
onto major histocompatibility complexes (MHC) of class I
and II (77, 78). Thus, in a normal state, the presentation of
apoptotic cell-derived antigens by DCs suggests that they
may play a role in cross presentation and tolerance,
thereby causing allograft rejections. In contrast, during an
inflammation, DCs phagocytose microorganisms and
necrotic cells and most likely play key roles in mediating
strong T-cell responses.
It is now well accepted that most if not all cell
types can become phagocytic when given with the
opportunity, and the list of ‘amateur ‘phagocytes is ever
growing. Consistent with this idea is the observation that
phagocytosis of apoptotic cells in the interdigits of the
footplate of a PU.1 knock-out mouse, which is devoid of
macrophages, can still occur and is performed by
neighboring mesenchymal cells (79).
3.3. Receptors on amateur phagocytes
Strikingly, most ‘amateur’ phagocytes use
similar phagocytic receptors to those found on
macrophages, some of which were indeed first
characterized while studying ‘amateur’ phagocytes. LOX-
1, a lectin-like oxidized LDL-receptor, is one such
example and is found on vascular endothelial cells where
it participates in the clearance of apoptotic neutrophils
(54). Interestingly, this receptor is also expressed on
macrophages, but it is not yet known whether it is needed
for clearance of apoptotic bodies by those cells. RPE cells
utilize the alpha-v/beta-5 integrin in binding of the shed
photoreceptors and require CD36 for internalization (80-
83). Cultured RPE cells could also trigger the serum-
stimulated uptake of ROS via a mechanism involving
vitronectin (VN)(84). Interestingly, the mutation of the
RCS rat, whose RPE cells fail to remove apoptotic shed
photoreceptors, was recently identified as a small deletion
Phagocytosis of apoptotic cells: an overview
1302
that disrupts the gene encoding a receptor tyrosine kinase
of the Axl/Mer/Tyro3 family, Mertk (59). Mutations in
the human orthologue of Mertk were also found in patients
with retinitis pigmentosa where a similar defect in
phagocytosis of shed photoreceptors was observed (85,
86). The MER receptor tyrosine kinase receptor was
therefore proposed to play a role in phagocytosis of
apoptotic ROS by RPE cells (59). A subretinal injection
of a MER-expressing virus in the RCS rat allowed for a
partial and transient but significant correction of the retinal
dystrophy phenotype associated with this mutation, further
demonstrating an in vivo role for MER in phagocytosis of
apoptotic photoreceptors by RPE cells (87).
Soon after its characterization, MER was also
found on monocytes and macrophages (22). Macrophages
from a functional knockout mouse (mer kd), carrying a
MER protein with a truncated cytoplasmic tail, failed to
efficiently clear apoptotic thymocytes in vivo (22). These
cells showed a defect in engulfment, rather than in binding
of apoptotic cells, which suggests that the cytoplasmic tail
of MER is required to trigger the uptake of apoptotic cells
but not for their proper recognition by macrophages.
Therefore not all receptors are able to trigger the uptake of
apoptotic cells but rather serve as recognition molecules.
A ‘tethering and tickling’ model was proposed whereby
some receptors anchor the apoptotic cells while others are
subsequently recruited or activated to signal and trigger
the cytoskeleton changes required for the extension of the
membrane around the particle during formation of the
phagosome (88). This model would partly explain the
diversity of receptors found on phagocytes and their ability
to interact or cooperate in the binding and uptake of
apoptotic cells.
It is becoming apparent that different phagocytes
can use various combinations of receptors to mediate
binding and uptake of apoptotic bodies. For instance,
Sertoli cells use a CD36-related scavenger receptor, the
scavenger receptor of class B type 1 (SR-B1) (80, 89).
However, a lysosomal ABC-transporter related receptor,
ABCB9, was also recently characterized in human and rat
that is highly expressed on these cells and may also be
involved in phagocytosis of apoptotic spermatogenic cells
(90). In the thymus, nursing epithelial cells also use the
CD36-related scavenger receptor, SR-B1, in clearance of
apoptotic thymocytes (91). DCs were found to use both
CD36 and the alpha-v/beta-5 integrin (76). Mesanglial
cells utilize yet another integrin, the vitronectin receptor
alpha-v/beta-3 (69). Fibroblasts engulf apoptotic
neutrophils via a mechanism that also requires the alpha-
v/beta-3 integrin and a mannose/fucose lectin (53).
Microglial cells-mediated phagocytosis of apoptotic
neurons is inhibited by N-acetylglucosamine or galactose,
suggesting the involvement of asialoglycoprotein-like
lectins; by the RGDS peptide, arguing in favor of a role for
the vitronectin receptor; and finally by PS or O-phospho-L
serine enriched vesicles, implicating a role for a PS-
receptor (55). In the liver, engulfing cells essentially use
lectin-like receptors, which recognize galactose and
mannose residues. Indeed, it has been shown that up-
regulating the cell surface expression of mannose
receptors on liver endothelial cells enhances their ability to
clear apoptotic lymphocytes (55).
Why phagocytes employ various combinations
of receptors is unclear. One explanation is that most of
these cells are part of a specific tissue or organ and thus
participate in specialized functions, needing different
specific molecules on each of these cells. Phagocytes may
use different receptors depending on the nature of the
apoptotic cell or its apoptotic stage, or of their own state of
activation. They may also simply have redundant
recognition and uptake mechanisms, which could become
useful in the event of overwhelming apoptosis or be used
as back-up systems in the event of defective mechanisms
of uptake (92, 93). This diversity of receptors, in particular
of those that are also employed in innate immune defenses,
might also have been driven by pathogen variation.
Phagocytic receptors for pathogens would have then
adapted to be exploited by the host and participate in the
recognition and engulfment of various apoptotic cell types.
3.4. Apoptotic cells surface ligands or ‘eat me signals’,
opsonins and bridging molecules
Despite their characterization, the molecular
mechanisms by which these numerous phagocytes and
their receptors participate in the recognition/binding and
engulfment/uptake of apoptotic cells is still poorly
understood. This is partly due to the incomplete
characterization of the cell surface changes on dying cells
that these receptors recognize.
Cells dying by apoptosis undergo major
morphological changes, such as cell shrinkage and
rounding accompanied by cytoplasm and nuclear
condensation, internucleosomal DNA fragmentation, as
well as membrane blebbing (94-96). The maintenance of
membrane integrity is an important hallmark of apoptosis,
yet profound cell surface changes occur that are important
for the recognition and engulfment of apoptotic cells by
phagocytes (94). These include: (i) alterations in
carbohydrates (53, 73, 97), (ii) the important and almost
universal loss of membrane phospholipid asymmetry
resulting in increased exposure of the anionic
phospholipid, PS (98, 99), as well as (iii) changes in the
adhesion molecule ICAM-3 (also known as CD50)
(100). The nature of these molecular alterations
accounts for the nature of the receptors involved in the
clearance of apoptotic corpses. Thus, lectins
participate in the recognition of carbohydrate pattern
alterations. CD14 binds to ICAM-3 (100, 101). PS-
receptor and scavenger receptors bind to PS (38, 48,
49). The ability of several receptors to bind the same
ligand adds yet another level of complexity and security
in the recognition and uptake of apoptotic cells. On
one hand, this could result in two receptors competing
for the same ligand and could affect the efficiency by
which a phagocyte recognizes its target. On the other
hand, if a large number of ligand molecules are present
at the surface of apoptotic cells, their recognition by
several receptors on the phagocyte may simply
strengthen binding and favor rapid uptake.
Phagocytosis of apoptotic cells: an overview
1303
Not all phagocytic receptors act by directly
binding to a unique and specific ligand at the surface of
apoptotic cells. Molecules bridging two receptors on the
phagocyte or opsonins on the apoptotic cells are
sometimes required. These molecules may also strengthen
the interaction between an apoptotic corpse and a
phagocyte. For instance, thrombospondin-1 (TSP-1), a
multimeric glycoprotein that functions as an adhesion
molecule and a component of the extracellular matrix, was
shown to act as a molecular bridge between the vitronectin
receptor alpha-v/beta-3 and CD36 in phagocytosis of
apoptotic cells by macrophages (31, 102). TSP-1 and
alpha-v/beta-3 are also required for phagocytosis of
apoptotic cells by mesanglial cells, although this
interaction occurs in a CD36-independent manner (69).
TSP-1 binds to the apoptotic corpse, although the surface
molecule to which it binds is not yet known.
Opsonising molecules include the anti-apoptotic
GAS6 and the beta2 glycoprotein I (beta2GPI) (19, 103).
GAS6 is a growth arrest specific gene implicated in the
negative regulation of blood coagulation cascade, and
participates in cell proliferation and survival, as well as
cell adhesion and chemotaxis (104-108). Most recently,
GAS6 was shown to enhance platelet aggregation and
secretion and its blockage or deletion allowed for a
protection against thrombosis (109). Interestingly, GAS6
is a ligand for receptor tyrosine kinases of the Axl, Sky
and Mer family (110-113). GAS6 enhances the uptake of
PS, as well as apoptotic cells by macrophages (114). This
enhancement requires its interaction with the surface of the
macrophage, presumably through a receptor tyrosine
kinase. While GAS6 could bind PS, its physiological
ligand on apoptotic cells remains uncertain. Beta2GPI, a
serum glycoprotein, also binds PS, and macrophage uptake
of apoptotic cells was enhanced in its presence (115, 116).
This uptake was further increased by addition of beta2GPI
antibodies (115, 117). Why this occurs is not well
understood.
Two additional molecules, the first component
of the complement cascade, C1q, and a member of the
collectin family of molecules, the mannose-binding lectin
(MBL) were also recently shown to act as opsonins,
promoting phagocytosis of apoptotic cells. Humans with
C1q deficiency show an increased risk for bacterial
infections and autoimmune diseases such as systemic
lupus erythrematosus (SLE) and glomerulonephritis, both
of which are accompanied by elevated levels of free
apoptotic cells and auto-antibodies production that could
be the result of a defect in clearance of apoptotic cells (19,
21). Similarly, C1q knockout mice developed
autoantibodies and glomerulonephritis, and showed a
defect in clearance of apoptotic cells in vivo (19, 118). An
MBL deficiency in humans is associated with increased
susceptibility to infection and diseases such as chronic
hepatitis B viral infection, cystic fibrosis, as well as SLE
(119-121). These data suggested a potential role for both
C1q and MBL in phagocytosis of apoptotic cells. C1q was
shown to bind many apoptotic cell types, including
apoptotic vascular endothelial cells and keratinocytes, as
well as both non-apoptotic and apoptotic Jurkat cells in
vitro (40, 122, 123). Interestingly, C1q binding was
observed on the entire surface of non-apoptotic cells while
it was found to concentrate in the blebbing areas of
apoptotic cells, which might be required for its interaction
with the phagocyte receptor (40). A possible explanation
for this re-localization of C1q is that it may be necessary
to trigger recognition of apoptotic cells at a relatively early
stage of apoptosis when cells start blebbing. In contrast,
MBL bound to apoptotic cells exclusively, and may be
used to recognize cells at a later stage of apoptosis when
dying cells have fragmented into apoptotic corpses. As
expected, both mannosidase-treatment and the addition of
mannose inhibited the binding of MBL to apoptotic cells,
and decreased the efficiency of their uptake by human
monocyte-derived macrophages (40). Antibodies against
C1q and MBL at least partially inhibited phagocytosis of
apoptotic Jurkat cells in vitro (40). C1q and MBL thus
seem to act as opsonins to enhance the uptake of apoptotic
Jurkat cells by human monocyte-derived macrophages
(40). These observations led to the identification of
cC1qR and LRP as a new complex of proteins that
cooperate in the binding and uptake of C1q-opsonised
apoptotic cells (40).
4. PHAGOCYTOSIS OF APOPTOTIC CELLS AND
ANIMAL MODELS: A GENETIC DISSECTION
4.1. C. elegans and the clearance of apoptotic cells by
‘amateur’ neighboring cells
The redundancy and complexity in the
mechanisms of recognition and uptake of apoptotic cells
by phagocytes in mammals has made their molecular
analysis difficult and many laboratories have turned to the
power of genetics in organisms such as C. elegans.
Studies using this animal model have already significantly
contributed to advances made in the field, in particular
with regard to the identification of intracellular
components of the phagocytic machinery that signal
cytoskeleton rearrangement during engulfment.
The lineage of all cells in C. elegans has been
thoroughly defined (124, 125). 131 somatic cells and over
300 germ cells undergo apoptosis and are rapidly cleared
by their neighboring cells. Failure of cells to recognize
and engulf their dying neighbors results in persistence of
apoptotic corpses (126). These are easily distinguished
because of their characteristic round shape, accompanied
by cell shrinkage and dilated nuclei, and appear as
refractile disks under Nomarski optics. This has allowed
for an easy screening of mutations that affect the
engulfment of apoptotic cells (126). At least 6 mutations
were first identified that led to the characterization of
genes that are required for the engulfment of apoptotic
cells. All belong to the ced (C. elegans death) family of
genes and have been conserved throughout evolution with
homologues in Drosophila and mammals. Genetic
analysis identified two complementation groups
organizing these mutations in at least two pathways: the
CED-1, -6, and -7 pathway, and the CED-2, -5, and -10
pathway (126). Mutants in the latter pathway also showed
a severe defect in migration of the distal tip cells (DTCs)
in the gonads (127). Most recently, a seventh mutant, ced-
Phagocytosis of apoptotic cells: an overview
1304
Figure 2. Schematic representation of the two pathways
involved in clearance of apoptotic cells by their neighbors
in C.elegans. CED-1 is a scavenger receptor that shares
sequence homologies with the mammalian scavenger
receptor SREC, as well as with the LDL-related receptor
protein, LRP, recently proposed to be the functional
mammalian orthologue of CED-1. CED-1 clusters at the
site of binding to the apoptotic corpse and participates in
its clearance. CED-6 is an adapter protein that acts
downstream of CED-1 and CED-7. Its mammalian
homologue GULP was recently shown to interact with
both CED-1 and LRP. CED-7 is the ABC1 homologue
and acts in the same pathway as CED-1 and CED-6. As
for its mammalian counterpart, CED-7 is found on both
the apoptotic cell and the phagocyte and is believed to
participate in flipping PS at the surface of the corpse.
CED-2, CED-5 and CED-10 are homologous to
mammalian CrkII, DOCK180 and RAC respectively.
CED-2 is an adapter protein that contains both SH2 and
SH3 domains. CED-5 has an SH3 domain and interacts
with CED-2 and CED-12. CED-12 encodes a protein with
a PH domain and SH3 binding motif, and interacts with
the carboxy-terminus of CED-5. CED-12 has a
mammalian homologue ELMO, which also interact with
DOCK180. CED-10 is a small GTPase and acts
downstream of CED-2, 5 and 12. The CED-2, 5, 10 and
12 pathway triggers the necessary cytoskeletal changes
that trigger cell shape and engulfment by the phagocyte.
These proteins are also required for the proper migration
of distal tip cells in C.elegans.
12, was characterized based on a similar migration defect,
and was shown to have a severely reduced engulfment
activity, which argued in favor of CED-12 being part of
the CED-2, -5 and -10 pathway (128-130) (Figure 2).
All seven genes have been fully characterized.
ced-1 encodes a scavenger-receptor that acts as a receptor
for apoptotic cells and shares sequence similarities with
human SREC found on endothelial cells (131). CED-1 is
required on the engulfing cell and clusters at the site of
membrane binding to the apoptotic corpse (131). While
CED-1 is homologous to SREC, the mammalian LRP
receptor also shares homologies with CED-1 and was
recently proposed to be its functional orthologue (132).
Ced-6 encodes an adapter protein with a phosphotyrosine
binding (PTB) site, a leucine-zipper motif for
homodimerization, and a potential proline-rich motif, and
acts downstream of CED-1 and CED-7 (131, 133). Its
human homologue GULP was recently shown to interact
both with CED-1 and LRP and to participate in
phagocytosis of apoptotic cells (132, 134). The ced-7
gene product is similar to the ABC-1 transporter and is
required for CED-1 clustering (129, 131). As for ABC1,
CED-7 is also found on the apoptotic cell and believed to
act by exposing a phospholipid ligand on the cell surface
of the dying cell (131). CED-2 is similar to CRKII, a
signaling molecule containing SH2 and SH3 domains
(127). CED-5 belongs to the CDM family of proteins,
which include CED-5, its mammalian homologue
DOCK180 and the Drosophila protein myoblast city
(MBC), and interacts with CRKII (135-137). CED-5 was
proposed to function in the engulfing cell and to trigger the
extension of its membrane around the dying cell (136).
CED-2 and CED-5 were shown to physically interact in
vitro (127). Ced-10 encodes a small GTPase of the
Rho/Rac/cdc42 family that is most similar to Rac (127).
CRKII, DOCK180 and Rac were all shown to be involved
in triggering cytoskeletal changes that lead to the
engulfment of apoptotic cells in mammalian systems (138,
139).
Ced-12, the newly identified gene involved in
engulfment of apoptotic cells, encodes a protein with a
pleckstrin homology (PH) domain and SH3 binding motif
(128-130). PH domains are found in many proteins that
act in signal transduction pathways or in cytoskeleton
reorganization. A ced-12 mutation strongly exacerbates
the engulfment defect seen in loss-of function mutation of
ced-1, 6 and 7, but does not affect that of a ced-5 mutant
(128-130). Moreover, CED-10 overexpression could
bypass the requirement for CED-12, arguing that CED-10
acts downstream of CED-12 in a same pathway (128-130).
CED-12 functions in engulfing cells, and was further
shown to physically interact with the carboxy-terminal
sequence of CED-5 (128). ELMO1, the mammalian
homologue of CED-12 also interacted with CED-5, as did
CED-12 with DOCK180, the mammalian homologue of
CED-5 (129, 130). Consistently, ELMO1 and DOCK180
also physically interacted (130). Using a yeast three-
hybrid system, Wu and colleagues further demonstrated
that CED-5 acts as a bridge to link CED-2 and CED-12
(129). Whether this complex forms in vivo or what might
trigger these proteins to interact with each other is not
known. Several lines of evidence imply the involvement
of Rac and its homologue CED-10 in the signaling
pathway that regulates the cytoskeleton dynamics during
engulfment in mammals and C. elegans, respectively (127,
138, 139). Surprisingly, Zhou and colleagues showed that
overexpression of CED-12 in Swiss 3T3 cells could also
regulate cytoskeletal rearrangements via yet another small
GTPase, Rho (128). While activation of Rac induces
polymerization of actin leading to ruffles and filopodia
formation, Rho induces actin-assembly into bundles and
stress fibers (140). Thus CED-12 might act through
different small GTPases to reorganize the cytoskeleton in
various functions. What would trigger CED-12 to interact
with a particular small GTPase in a specific situation is not
understood.
Phagocytosis of apoptotic cells: an overview
1305
4.2. Drosophila melanogaster and the clearance of
apoptotic cells by ‘professional’ phagocytes, the
macrophages
Drosophila melanogaster is also often used to
genetically dissect the molecular mechanisms of biological
processes of interest. While in C.elegans, apoptotic
corpses are cleared by amateur phagocytes, the
neighboring cells, several cell types, both of the amateur
and professional classes, have been reported to participate
in the clearance of apoptotic cells in Drosophila (141)
(142-144). Thus, in this respect, Drosophila is more
similar to mammals and recently became a model
organism of choice to study the molecular mechanisms of
apoptotic cell clearance by professional phagocytes (145,
146).
In Drosophila, epithelial and glial cells are the
amateur phagocytes, and macrophages, also called
plasmatocytes, are the professional phagocytes (141-144).
Drosophila macrophages have similar functions to their
mammalian counterparts: they participate in phagocytosis
of apoptotic cells during development, as well as of
microorganisms in innate immune responses, and have
scavenger activities (141, 146-149). They are also the
major producers of extracellular matrix proteins and are
believed to play a role in maintaining tissue integrity in the
fly embryo by depositing a lattice of extracellular matrix
around it (149). This may also account for the ability of
macrophages to migrate freely throughout the embryo,
which lack a sophisticated circulatory system as found in
Vertebrates. Moreover, Drosophila macrophages have a
scavenger activity and can endocytose a wide variety of
polyanionic molecules (150, 151). A Drosophila
scavenger receptor, dSR-C1, which is expressed on
embryonic macrophages, was recently shown to
participate in phagocytosis of bacteria (150, 152). In the
absence of dSR-C1, Drosophila SL2 Schneider cultured
cells showed a 25% reduction in their ability to engulf
bacteria, arguing that the molecular mechanisms of
recognition and engulfment of bacteria may be redundant
in the fly (152).
Drosophila macrophages arise from the
mesoderm in the head of the embryo and in the gnathal
segments at about stage 10 of development (149). At this
stage, approximately 40 precursors on either side of the
head of the embryo undergo 4 mitotic divisions and give
rise to approximately 700 hemocytes, or blood cells, which
all belong to the plasmatocyte lineage (149). They then
migrate throughout the embryo following distinct
stereotyped paths (149). By late stage 11, programmed
cell death has initiated and plasmatocytes that encounter
apoptotic cells become phagocytic (141, 148, 149). Their
ability to recognize and engulf apoptotic cells prompted
Tepass and colleagues to name these cells macrophages.
We previously reported the characterization of a
CD36-related receptor, Croquemort (CRQ or CROQ),
which is specifically expressed on macrophages during
Drosophila embryogenesis (145). In fact, this gene
product appears at late stage 11 when programmed cell
death is initiated (145). This observation and the sequence
homology with CD36, a mammalian receptor previously
shown to participate in phagocytosis of apoptotic cells
prompted us to further study its potential role in clearing
apoptotic cells. Using a combination of transfection in a
heterologous system and inhibition studies, we revealed
that the crq gene could confer the ability to recognize and
engulf apoptotic murine thymocytes on cultured Cos-7
kidney cells (145). We next analyzed several deficiencies
that remove the crq locus and showed that they had a
defect in phagocytosis of apoptotic corpses by
macrophages in the embryo (146). This defect could be
rescued by a crq transgene, demonstrating that crq is
essential for efficient phagocytosis of apoptotic cells in
vivo (146). The severity of the defect observed, a
reduction of approximately 90% of the phagocytic index,
argues that the CRQ-pathway is the main event required
for phagocytosis of apoptotic cells and that there will not
be much redundancy in the mechanisms of clearance of
apoptotic cells in the fly embryo. Interestingly,
macrophages in the crq-deficient embryos had no defect in
phagocytosis of bacteria, revealing distinct pathways for
the phagocytosis of apoptotic corpses and bacteria by
Drosophila macrophages (146). These results also
demonstrated conservation in the mechanisms of
phagocytosis of apoptotic cells, as various CD36-family
members also participate in this process in mammals (see
above). Finally, these results promoted Drosophila as
being a suitable model system to genetically dissect the
molecular mechanisms underlying phagocytosis.
5. PHYSIOLOGICAL CONSEQUENCES OF
APOPTOTIC CELL CLEARANCE
Although present, the clearance of apoptotic
cells is not an essential function in C. elegans; mutants
that fail to clear apoptotic cells live to the adult stage
(126). Interestingly, two recent studies in C. elegans have
demonstrated that genes involved in uptake mechanisms of
apoptotic cells may in turn accelerate apoptosis of cells not
yet fully committed to die (14, 15). In double mutants
with a partial loss-of-function of ced-3, a gene encoding
the C. elegans homologue of caspase-3 required for
programmed cell death, and loss-of-function of the cell
death engulfment genes, cells were observed that appeared
to be in the process of dying but yet reverted to a fully
viable cell as they failed to be engulfed (14, 15). Cells in
animals with partial loss-of-function of ced-3 alone
eventually proceeded through programmed cell death and
were engulfed by their neighbors, thus arguing for a role
of the engulfment genes in triggering full-programmed cell
death in cells on the verge of death (14, 15). Several
previous studies in mammals also showed that
phagocytosis of apoptotic cells could trigger the induction
of further programmed cell death by macrophages (11-13).
In Drosophila, it is not known whether mutants
that specifically abolish clearance of apoptotic cells are
viable. Deficiencies that remove the crq gene are
embryonic lethal. However, this lethality is already being
accounted for by other genes in the crq vicinity, which are
also deleted. A single mutation of the crq gene might give
us an answer as to whether phagocytosis of apoptotic cells
Phagocytosis of apoptotic cells: an overview
1306
is essential for the life of the fly. However, crq may also
be involved in other functions and may not allow us to
address this particular question. The life cycle and
development of a fly is very different than that of a worm
as it undergoes profound morphological changes, in
particular during metamorphosis, while developing from
the pupal to the adult stages. Indeed, all tissues with the
exception of the imaginal discs, which will give rise to all
adult appendages, are hydrolyzed at the pupal stage.
Extensive programmed cell death is observed during
pupariation and CRQ has been proposed to participate in
the clearance of dying cells at this stage of development as
well (153). Thus, failure to clear apoptotic cells during
metamorphosis may have more dramatic consequences
than during embryogenesis. It is known that apoptosis
does not affect macrophage differentiation in the fly
embryo (149). However, whether macrophages may be
able to induce apoptosis has not yet been carefully looked
at in Drosophila. While embryos that are devoid of
hemocytes carry on with an apparent normal onset of
apoptosis, macrophages may be competent to kill and
account for at least some apoptosis. A strict identification
and quantification of all apoptotic cells at all stages of
development of such embryos would certainly provide an
answer to that question.
In mammals, the great complexity and the
redundancy in the mechanisms of clearance of apoptotic
cells almost certainly reflect its many outcomes and
implications, as well as its physiological importance. The
use of a particular combination of receptors and ligands
might indeed determine the nature of the responses
triggered by the engulfment of apoptotic corpses. The
dogma is that apoptotic cells are taken up before they
release any cytotoxic and antigenic content (1, 10).
Studies showed that failure to dispose of apoptotic cells
could lead to neurodegeneration or contribute to
autoimmune diseases (19, 21-23). In most cases, the
uptake of apoptotic cells is accompanied by anti-
inflammatory signals, such as the production of
transforming growth factor-beta (TGF-beta), prostaglandin
E2 (PGE2), platelet-activating factor (PAF), and
interleukin-10 (IL-10) among others (16, 154-156). The
synthesis and secretion of various chemokines such as the
macrophage inflammatory protein-2 (MIP-2), KC and
Mip-1alpha by macrophages that had engulfed apoptotic
cells were inhibited, while others such as the macrophage
chemotaxis protein, MCP-1 were not affected (156).
These tight regulations of cytokines and chemokines most
likely account for the resolution of inflammation and the
regulation of immune responses.
However, an in vitro assay recently set up to
study phagocytosis of apoptotic cells by rat microglial
cells has led to rather different observations (157).
Microglial cells could distinguish and engulf apoptotic rat
thymocytes, as well as apoptotic encephalitogenic myelin-
basic protein (MBP) T-cells. The latter are commonly
used in experimental autoimmune encephalomyelitis
studies that model the demyelinating disease multiple
sclerosis (158). Using this assay, Magnus and colleagues
showed that microglial cells that encountered apoptotic
cells secreted lower amounts of pro-inflammatory
cytokines, such as tumor necrosis factor-alpha (TNF-
alpha) and interleukin-12 (IL-12), with no changes in anti-
inflammatory cytokine production, such as TGF-beta and
IL-10 (159). MER, the tyrosine kinase receptor involved
in the recognition of apoptotic ROS by RPE cells also
inhibited tumor necrosis TNF-alpha cytokine production
(160). Furthermore, while pretreatment with the tumor
necrosis factor-alpha (TNF-alpha), a cytokine produced by
Th1cells, or the Th2-type cytokine, TGF-beta, did not
significantly affect phagocytosis of apoptotic thymocytes
by microglial cells, the uptake of apoptotic cells by
interleukin-4 (IL-4)-treated microglial cells was decreased
by about 50% (157). Together, these results suggest a
tight regulation of various cytokines that may control the
inflammation, restrict auto-inflammation and minimize
damage in inflamed tissues of the CNS. However, in some
circumstances, clearance of apoptotic cells may also lead
to macrophage activation and trigger the production of
pro-inflammatory cytokines, which may become important
at a wound site where more phagocytes would be needed
to remove apoptotic and necrotic cells, as well as
pathogens (17, 18).
Strikingly, only a small number of apoptotic
corpses are seen in microglial cells at any given time,
while they are expected to have an extensive phagocytic
role in vivo, due to high levels of programmed cell death in
the CNS. Interestingly, pre-incubation with interferon-
gamma (IFN-gamma) allowed microglial cells to ingest
more apoptotic thymocytes and faster, suggesting a role
for IFN-gamma in their recruitment, as well as in the
enhancement of their phagocytic activity (157). Little is
currently known about how macrophages are recruited to
the site of apoptosis, and whether dying cells might
stimulate the recruitment of macrophages themselves. The
macrophage colony-stimulatory factor, M-CSF, was
recently shown to play an important role in the recruitment
and differentiation of macrophages that scavenge effete
cells in the uterus (161). Indeed, osteopetrotic (op/op)
mice that lack M-CSF had fewer macrophages recruited in
the uterus, where the endometrial epithelial cells, which
secrete M-CSF, normally undergo programmed cell death
and are cleared by macrophages (161). These mice also
showed a defect in clearance of apoptotic epithelial cells
(161).
PS ligation induces the release of TGF-beta, and
the PS-receptor has been proposed to play a crucial role in
switching on or off the synthesis of pro- or anti-
inflammatory cytokines, depending on the particle cleared
by macrophages (155). Interestingly, amastigotes of the
parasite Leishmania spp, which are responsible for disease
propagation, were shown to inhibit macrophage activity by
exposing PS at their surface, a mechanism that allows the
parasite to evade its killing by macrophages (24). The
secretion of TGF-beta and IL-10 was also observed
following Leishmania infection, which was dependent on
PS-receptor engagement with PS, and sustained
Leishmania growth (24). PS exposure also accounted for
the uptake of the parasite (24). Thus, the parasite enters
macrophages by routes that are similar to that taken by
Phagocytosis of apoptotic cells: an overview
1307
apoptotic corpses, and subverts the ability of macrophages
to participate in its killing, favoring its growth and
propagation. Similarly, the pathogenic trypanosome,
Trypanosoma cruzi (T. cruzi) infects macrophages and was
also shown to induce the production of TGF-beta, as well
as prostaglandins and polyamines. This down-modulates
the ability of macrophages to trigger an appropriate
inflammatory response that would lead to T. cruzi killing
and promotes its growth in a favorable and nutritious
environment (25). Many receptors that participate in the
clearance of apoptotic corpses have also been shown to
participate in phagocytosis of microorganisms, in
particular bacteria (see above), which may thus also evade
the immune system. Understanding the molecular
mechanisms of apoptotic cell clearance will further our
knowledge of phagocytosis of bacteria, which could also
become critical in developing novel approaches to fight
infectious diseases.
Finally, the role of DCs in phagocytosis of
apoptotic cells, which leads to cross presentation of self-
antigens, has initiated a new line of investigation with
therapeutic potential in cancer vaccination. Tumor cells
can evade the immune system by various means, including
the induction of expression of immunosuppressive
cytokines, the down-regulation or constitutive expression
of pro-apoptotic genes such as Fas and its ligand, and/or
the reduced expression of MHC complexes (1, 162). In
two reports, DCs were fed with apoptotic or necrotic
tumor cells and presented self-antigens derived from these
apoptotic cells that stimulated in vitro or in vivo cellular-
mediated cytotoxic immune responses, thus conferring
protection against further tumor cell development in mice
(26, 27). In contrast, the horizontal transfer of an activated
oncogene into phagocytes that had taken up apoptotic cells
expressing this oncogene was also observed, and
phagocytes that received this oncogene developed a
tumorigenic phenotype characterized by in vitro loss of
contact inhibition and in vivo proliferative advantage (28).
This result suggests that phagocytosis of apoptotic cells
may occasionally be at the origin of cancer development.
6. PERSPECTIVES
Numerous receptors on phagocytes and a few
apoptotic cell-ligand that participate in phagocytosis have
been characterized, yet we still have a limited
understanding of their relationship to each other.
Professional and amateur phagocytes often employ similar
receptors in the recognition and uptake of apoptotic cells,
yet their efficiency of engulfment is strikingly different,
and what makes a professional phagocyte such an efficient
housekeeper as opposed to an amateur phagocyte remains
elusive. A possible explanation for this difference could
be the nature of their membrane or its fluidity, or simply
the ability of professional phagocytes to migrate and travel
much greater distances than an amateur phagocyte, most
of which are resident cells. To date, we know very little
about what triggers migration of professional phagocyte to
the site of programmed cell death, and whether apoptotic
cells signal to the phagocytes at a distance or whether a
fortuitous proximity of the two cells is needed for the
phagocyte to sense the ‘eat me signals’. Such studies
would provide further clues as to what the nature of
apoptotic signaling molecules might be.
Most, if not all apoptotic cells share a common
feature, the exposure of PS at their surface. This could
simply reflect a common and quick need for all apoptotic
cells to signal to phagocytes that they must be removed
while ensuring an efficient and ‘silent’ removal of the
apoptotic bodies, i.e. by triggering anti-inflammatory
signals. However, PS and its receptor alone may not
account for all clearance of apoptotic cells, and the
outcome of phagocytosis of apoptotic cells might be
directed by the presence of other phagocyte receptors and
the recognition of their ligands at the surface of apoptotic
corpses, thus informing the phagocyte about the nature of
the cell which has died.
The physiological and pathological
consequences of phagocytosis of apoptotic cells or its
failure are numerous. With a renewed interest for the
study of phagocytosis of apoptotic cells, we are entering a
new era of investigation of the molecular mechanisms that
govern a number of these important processes, such as
homeostasis, tissue remodeling or shaping and the
maintenance of organ functions during development,
wound healing and innate immunity, autoimmunity and
possibly cancer.
The identification of seven C. elegans genes and
that of their counterparts in mammals illustrate an
evolutionary conservation in the molecular mechanisms by
which apoptotic cell clearance occurs. Studies of the crq
gene in Drosophila show that the molecular mechanisms
of apoptotic cell clearance were also conserved in insects.
It is likely that studies in C.elegans and Drosophila, and
the use of their powerful genetics will continue to provide
valuable information on these mechanisms that will lead to
a better understanding of phagocytosis of apoptotic cells in
mammals.
7. ACKNOWLEDGMENTS
I wish to thank Dr. John Nambu for giving me
the opportunity to contribute to this issue of Frontiers in
Bioscience. I am also extremely thankful to my colleagues
Dr. Mark Marsh, Dr. Dan Cutler, and Dr. Alan Hall for the
critical reading of this manuscript and their continuous
help and support. NC Franc is a member of the Cell
Biology Unit of the MRC Laboratory for Molecular Cell
Biology at the University College London and is funded
by the Medical Research Council.
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Abbreviations: C. elegans: Caenorhabditis elegans, DC:
dendritic cell, SR-A: scavenger receptor of class A, ABC1:
ATP binding cassette transporter 1, CD14: cluster of
differentiation 14, PS-receptor: phosphatydilserine
receptor, SP-A: surfactant protein A, CRT: calreticulin,
cC1qR: collagenous tail binding C1q receptor, LDL: low
density lipoprotein, LRP: LDL receptor-related protein,
alpha2-m: alpha2-macroglobulin, CR3: complement
receptor 3, PS: phosphatidylserine, SRs: scavenger
receptors, LPS: lipopolysaccharide, CRD: carbohydrate
recognition domain, RPE: retinal pigmented epithelium,
ROS: rod outer segments, RCS rat: Royal College of
Surgeons rat, CNS: central nervous system, APC: antigen-
presenting cell, MHC: major histocompatibility complex,
LOX-1: lectin-like oxidized LDL receptor, VN:
vitronectin, SR_B1: scavenger receptor class B type 1,
TSP-1: thrombospondin-1, beta2GPI: beta2 glycopreotein
I, GAS6: growth arrest specific gene 6, MBL: mannose-
binding lectin, SLE: systemic lupus erythrematosus, CED:
C. elegans death gene, DTC: distal tip cell, SREC:
scavenger recepor found on endothelial cells, PTB:
phosphotyrosine binding, GULP: engulfment LRP-related
protein., MBC: myoblast city, PH domain: pleckstrin
homology domain, dSR-C1: Drosophila scavenger
receptor class C type 1, CRQ or CROQ: Croquemort,
TGF-beta: transforming growth factor-beta, PGE2:
prostaglandin E2, PAF: platelet-activating factor, IL:
interleukin, MIP-2: macrophage inflammatory protein-2,
MCP: macrophage chemotaxis protein, MBP: myelin-
basic protein, TNF-alpha: tumor necrosis factor-alpha,
IFN-gamma: interferon-gamma, M-CSF macrophage
colony-stimulatory factor, op: osteopetrotic, T.cruzi:
Trypanosoma cruzi
Key Words: Phagocytosis, Apoptotic cells, Drosophila,
C. elegans, Mammals, Professional Phagocyte, Amateur
Phagocyte, Review
Send correspondence to: Dr Nathalie C. Franc, MRC
Laboratory for Molecular Cell Biology and Cell Biology
Unit, University College London, Gower street, London
WC1E 6BT, United Kingdom, Tel: (+44)(0)20-7679-7255,
Fax: (+44)(0)20-7679-7805, E-mail: n.franc@ucl.ac.uk
