Download Apoptotic cell removal

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R795
Apoptotic cell removal
Peter M. Henson, Donna L. Bratton and Valerie A. Fadok
Ingestion by professional or amateur phagocytes is the
fate of most cells that undergo apoptosis. Studies in
both Caenorhabditis elegans and mammals are now
converging to reveal some of the key mechanisms and
consequences of this removal process. At least seven
corpse removal genes in nematodes have mammalian
equivalents, and represent elements of signaling
pathways involved in uptake. In mammals, a wide
variety of apoptotic cell recognition receptors has been
implicated and appears to be divided into two
categories, involved in tethering the apoptotic cell or
triggering an uptake mechanism related to
macropinocytosis. Apoptotic cell removal is normally
efficient and non-inflammatory. By contrast, the process
may become subverted by parasites to yield a more
favorable growth environment, or in other cases lead to
fibrosis. Removal may also clinch the apoptotic process
itself in cells not yet completely committed to death.
Address: Program in Cell Biology and Department of Pediatrics,
National Jewish Medical and Research Center, 1400 Jackson Street,
Denver, Colorado 80206, USA.
Correspondence: Peter M. Henson
E-mail:[email protected]
Introduction
Cell recognition, engulfment and digestion are the final
stages in the process of apoptosis. The outcome of these
actions is the efficient deletion of unwanted or damaged
cells, often considered a counterpoint to mitosis. That
apoptosis is accompanied by rapid removal of the affected
cells in situ has been recognized since the phenomenon
was first characterized [1,2]. As discussed recently by Jack
Cohen [3], a newer view of biology suggests significant
overproduction, at the level of the organism, sperm, egg
and embryo, of individual cells during development. In
each case, excess material is removed and thus removal is
an important element of overall control. Even in the adult
organism, significant cell turnover seems to be the rule
rather than the exception, including systems such as the
brain that were long thought not to exhibit such plasticity.
Turnover of cells in metazoa means apoptosis and, critically, apoptotic cell removal — often called corpse removal.
However the mechanisms of apoptotic cell recognition and
removal are only now becoming apparent, an understanding that represents a fascinating investigative interplay
between genetic approaches in the nematode, Caenorhabditis elegans, and analyses of uptake mechanisms in mammalian systems (Figure 1).
Current Biology 2001, 11:R795–R805
0960-9822/01/$ – see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Combining studies in mammals and the worm
Seven genes have recently been identified in C. elegans that
appear to play a role in apoptotic corpse removal during
larval development or in corpse digestion after engulfment
[4–8]. One of the many strengths of the C. elegans system
goes back to the original cell mapping studies that led to the
identification of 131 specific somatic cells, as well as some
300 germ cells, that are destined for deletion (see [4,7]).
Until recently, the genes involved in removal each appeared
to correspond to steps in the signal pathways leading to
engulfment, rather than to cell recognition steps per se. In
contrast, studies in mammalian systems generated a host of
potential recognition receptors (see [9–13]) but made little
progress towards identifying uptake signaling molecules.
Recent reports provide a crucial synthesis between these
two approaches and highlight a number of important
points, not the least of which is the broad commonality in
mechanisms employed for apoptotic cell removal. It seems
reasonable to suggest that potential redundancy among
the recognition elements would make their identification
by the genetic screening approaches used for C. elegans less
likely. Such redundancy is certainly implicated in the
studies of mammalian systems in vitro. On the other hand,
the involvement of limited, and presumptively unique,
signaling pathways for uptake could readily explain the
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Figure 1
A comparison of elements implicated in
apoptotic cell uptake in (a) mammals and
(b) C. elegans. The electron micrograph of
uptake in the nematode (b) is taken with
permission from [7].
1. Surface changes on the apoptotic cells
Mammal
PS
Oxidized phospholipid
ABC-1
Altered carbohydrates
ICAM3
Nematode
PS?
ced-7
2/3. Tethering and/or stimulation receptors
(a)
Macrophage
Corpse
PSR
PSR?
SR-A
CD36/SR-B
CD36?
SREC?
ced-1
CD68
αvβ3/αvβ5 integrins
β1 integrins
CD14
CR3
β2GP receptor
Lectin(s)
Collectin receptors (calreticulin/CD91)
4. Signaling molecules
CrkII
DOCK180
rac-1
ced-6
ABC-1
ELMO
(b)
Sheath cell
Corpse
ced-2
ced-5
ced-10
ced-6
ced-7
ced-12
5. Digestion
Acid DNAase?
nuc-1
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identification of genes within such pathways in the nematodes. These mechanisms for cell removal may also extend
to necrotic cells and cell debris, even if the consequences
of such clearance may sometimes be different [5,14,15].
An example of this synergism is the recent, elegant paper
by Zhou et al. [7]. In this work, the last but one of the
seven C. elegans genes to be characterized, ced-1, does seem
to encode a potential recognition and signaling molecule
that may initiate cell uptake. ced-1 was originally identified
in the genetic screens for persistent cell corpses and encodes
a protein that plays a critical role in engulfment. The protein
is expressed in the membranes of phagocytosing cells and
clusters around corpses during removal. Deletion of potential signaling domains within the intracellular portion of
the molecule leads to defective apoptotic cell clearance,
even in the face of unaltered clustering. These data suggest
that CED-1 both binds to the target apoptotic cell and initiates signaling for uptake.
While the specific ligand that might be recognized by
CED-1 remains unknown, clustering, which is taken to
be equivalent to ligand engagement, appears to be associated with the presence of the product of ced-7. This latter
gene encodes a protein similar to ABC-1 in mammals [16],
a member of the ATP binding cassette transporter family
[17]. Defects in ABC-1 in man are associated with Tangier
disease [18–20] and the molecule appears to be involved
in cholesterol or phospholipid transport. Zhou et al. [7]
suggest the possibility that CED-7 changes phospholipid
distribution on the apoptotic cell (see below) in C. elegans
and that this is what CED-1 recognizes.
It seems reasonable to consider the apoptotic cell removal
process to comprise five steps, namely: surface alterations
and ligand expression on the apoptotic cell; recognition of
these ligands by tethering receptors on the phagocyte; initiation of the appropriate signaling pathways in the phagocyte, which might be mediated by the tethering
interaction but may also involve separate or additional
uptake receptors; the process of engulfment; and finally,
phagosome–endosome interactions leading to digestion. It
is also important to emphasize that uptake is not limited to
professional phagocytes, such as macrophages or immature
dendritic cells in mammals, but may be performed by most
cell types. An example of this is seen in the normal developmental clearance of cells from the interdigital webs in mice
wherein removal occurs even in animals that do not contain
macrophages — PU-1–/– mice — but at a slower rate [21].
Surface changes on apoptotic cells leading to
recognition and removal
Despite all the effort spent characterizing the mechanisms
of apoptosis, comparatively little has been focused on
surface changes on the apoptotic cell. A very clearly defined
change is the exposure of phosphatidylserine on the apoptotic cell surface, shown first on lymphocytes [22] and now
known to occur with essentially all apoptotic cells in
mammals. Phosphatidylserine also appears to be involved
in apoptotic cell removal in amphibians [23] and is seen
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during apoptosis in Drosophila [24]. Whether apoptotic
cells in C. elegans express phosphatidylserine on their surface
has still to be shown definitively.
While the mechanism of phosphatidylserine exposure is
still poorly understood at the molecular level, it appears to
require activation of a phospholipid ‘scramblase’ that
enhances bidirectional phospholipid flip-flop across the
membrane bilayer, and inactivation of an aminophospholipid translocase that would normally return the phosphatidylserine to the inner membrane leaflet. Effects on
both these systems can be seen during normal cell function
but are reversible; the key difference in apoptosis may be
the permanence of the effect. Candidate regulatory genes
for these activities have been identified [25–28] although
their exact role, identity or mode of action is by no means
clear. A search of the C. elegans gene database reveals a
molecule with considerable homology to the mammalian
scramblase. And a putative aminophospholipid translocase
— a member of the P-type ATPase family — is widely distributed, from plants to man [25,29–31]. Although the
validity of this candidate has been challenged, more recent
data in Arabidopsis support a role for these P-type ATPases
as aminophospholipid translocases [29].
Another class of surface changes that has been implicated
in apoptotic cell removal is alteration of membrane carbohydrates. The original observation of apoptotic cell recognition by macrophages suggested a lectin-like activity
[32,33]. Alterations to surface charge on apoptotic cells were
also attributed to carbohydrate moieties [32,34] although
specific sugars on the apoptotic cell surface were not identified in either of these studies. On the other hand there is
some evidence for increased expression of mannose on
apoptotic cells leading to their removal within the liver
[35,36]; this is an area that needs much more detailed
investigation.
A number of mechanisms for such changes to membrane
carbohydrates can be envisioned. New or altered glycosylated molecules may be supplied to the cell-surface from
internal membranous structures, possibly by direct fusion
of elements from the endoplasmic reticulum with the
plasma membrane. In addition, the normal turnover of
membrane glycoproteins is likely to be significantly altered
and decreased in apoptosis leading to gradual depletion of
key molecules or their normal glycosylation. Any enzymatic
alteration of surface oligosaccharides would require either
the presence of enzymes in or on the membrane surface —
so-called ectoenzymes — or in the extracellular medium.
This issue represents a potential problem in all studies of
apoptosis carried out in vitro, as some necrosis and cytolysis
is bound to occur and could contribute intracellular
enzymes to the system that might not be present during
the efficient removal usually seen in vivo.
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A completely different possibility is that appropriate glycoproteins become laterally redistributed or clustered in the
membrane of apoptotic cells. This could be a critical determinant of recognition. For example, members of the family
of defense collagens [37] that includes the C-type lectins
known as Collectins as well as the first component of the
classical complement pathway, C1q, have been shown to
bind to highly localized sites on apoptotic cells [38–40].
Mannose binding lectin is one of these proteins and not
surprisingly its attachment to apoptotic cells is blocked by
high concentrations of free mannose. It is noteworthy that
preliminary evidence shows that phosphatidylserine expression and membrane phospholipid redistribution can often
be patchy (our unpublished observations) and highly localized. The relationship between phospholipid redistribution
and other putative membrane changes to proteins or their
carbohydrate substituents is poorly described.
What is also not clear is the linkage between the two broad
signaling pathways leading to nuclear apoptotic changes —
those that involve mitochondria and those that do not —
and surface changes. How the aminophospholipid scramblase is inactivated is not known. As far as phospholipid
redistribution is concerned, cleavage of PKCδ by caspase 3
induces its unregulated activation, which, we suggest,
leads in turn to a more persistent induction of the phospholipid scramblase [41]. What is not clear is how the
actual phospholipid scrambling effect is mediated, which
is where questions about the involvement of the ABC transporters might come in. These are known to transport phospholipids across cell membranes and it is certainly possible
that ABC1 serves such a function in the apoptotic cell.
However this suggestion, which has been raised by a
number of groups (see [42,43]) is at present merely an
interesting speculation.
If ABC transporters are involved in phospholipid scrambling, the generality of phosphatidylserine expression during
apoptosis dictates the participation of more than one
member of this family as ABC1 is not generally distributed
among different cell types. The phospholipid scramblase
appears to require calcium and phosphorylation [41,44] for
its activity, self aggregates and is known to scramble phospholipids in artificial membrane systems [45], albeit with
low efficiency. It might therefore alter the membrane structure by itself to mediate transbilayer movement. However,
although potentially present, it was not identified in the
genetic screen for abnormal corpse removal in C. elegans.
Another element that must be put into the equation is the
suggestion that, in both mammals and the nematode,
phosphatidylserine is expressed on both the apoptotic cell
and the phagocyte during removal [7,43,46]. At this point,
therefore, the role of ABC1 is difficult to unravel. In the
mouse it seems to play a role in the phagocyte as
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macrophages from knockout mice show defects in apoptotic cell uptake [43]. The problem here is that most cell
types have the capacity to recognize and ingest apoptotic
cells — presumably a very primitive function — once
again in the face of a restricted distribution of ABC1.
would require a receptor on the phagocyte and an important recent paper [57] shows that Mer, a member of the
Axl, Mer, Tyro3 family of receptor tyrosine kinases, may
play such a role. Critically, mer–/– mice show a marked
defect in removal of cells in the thymus.
Recognition of apoptotic cells
Whether the putative receptor, Mer, acts via Gas-6 and
exposed phosphatidylserine in vivo is not yet known — it
could also act along with some of the other recognition
molecules as a signaling agent. It is important to note in
this section that while many receptors have been suggested to participate in recognition of apoptotic cells
in vitro and even in reconstituted transfected cells, far fewer
candidates have been shown to play this role in vivo. In
part this is likely to reflect the significant redundancy, and
by implication, importance, of the removal processes.
Two possible issues of importance may be gleaned from
the above observations regarding surface changes on apoptotic cells. First, the classes of molecules that are altered,
carbohydrates and phospholipids, are recognized in vivo by
so-called pattern recognition molecules, which have recently
been associated with the innate immune system. Secondly, aggregation of glycoproteins and phospholipids in
the membrane of the apoptotic cell further fits this
concept in that many of these recognition processes are
thought to be low affinity, high avidity systems whose
effectiveness is markedly enhanced by local aggregation.
In mammals, phosphatidylserine expression on the apoptotic cell membrane appears to be a critical element for
recognition or uptake, despite the abundance of potential
recognition receptors [11–13,47,48]. In the absence of
phosphatidylserine expression, uptake is minimal and deliberate replenishment of outer leaflet phosphatidylserine
restores uptake [48]. The selectivity and stereospecificity
of this restoration system strongly implicates the newly
cloned phosphatidylserine receptor [12] in such uptake. A
simple model suggests a variety of tethering ligands with a
critical involvement of the phosphatidylserine receptor for
initiation of the engulfment process.
While a C. elegans homologue for the phosphatidylserine
receptor was identified from the gene database [12], it too
never appeared in the genetic screen for corpse removal.
To this point, its possible role in apoptotic cell removal in
nematodes is unclear (although it has been implicated in
amphibians [23]). Careful examination of the C. elegans
phosphatidylserine receptor candidate gene does reveal a
deletion in a portion of the putative intracellular domain
that might contain a tyrosine phosphorylation site. Whether
this suggests that in the nematode the receptor serves
more a tethering than signaling function remains to be
determined. In mammals, the receptor also appears to play
a key role in suppressing the inflammatory response during
apoptotic cell removal [12,48,49].
The phosphatidylserine receptor in the phagocyte membrane would be expected to interact directly with phosphatidylserine on the apoptotic cell. Other phosphatidylserine-binding membrane proteins include the
scavenger receptors (see below and [50–54]). Gas-6 is a
soluble protein that can recognize and bind phosphatidylserine on apoptotic cells [55,56] and could act as a
bridging molecule. Its ability to participate in uptake
As noted above, the first study of apoptotic cell recognition suggested the involvement of a lectin [33]. More
recently, the complement protein C1q was reported to
bind to blebs on apoptotic keratinocytes [38,39] and to
localized sites on other apoptotic cell types [40]. The same
was seen for mannose binding lectin and surfactant proteins A and D (Vandivier W and Ogden CA, unpublished
observations). Moreover, each of these molecules has been
observed to enhance uptake of apoptotic cells into
macrophages in vitro [40,58] and, for C1q [59] and surfactant protein D, in vivo as well (Vandivier W and Ogden
CA, unpublished observations). These molecules appear
to bind apoptotic cells through their globular head groups
— the pattern recognition regions — and signal for uptake
through their conserved collagen-like tails. Because of the
potential involvement of mannose on the apoptotic cell,
the mannose receptor may be an additional candidate, but
there is as yet no evidence to support its involvement in
mediating apoptotic cell recognition.
On the other hand, scavenger receptors in the A and B
group have been suggested to recognize apoptotic cells,
particularly SR-A [9,60] and CD36 [61–65] or SR-B1
[51–53,66]. In Drosophila, a member of the SR-B family,
Croquemort, has been shown to participate in corpse
removal [67,68]. The ligands involved on the apoptotic
cells are not known. Closely associated with CD36 activity
are the αv integrins: αvβ3 on macrophages [65] and αvβ5 on
dendritic cells [63] and retinal pigment epithelial cells
[69]. Bridging by thrombospondin has been suggested in
some of these CD36-mediated systems although, once
again, the apoptotic cell ligands are unknown. As CD36
can directly bind anionic phospholipids [50,54] the potential interactions in this system are likely to be complex.
ced-1, like the other six genes involved in C. elegans apoptotic cell removal, is also similar to a mammalian molecule,
in this case, the scavenger receptor from endothelial cells,
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SREC [70]. SREC binds and internalizes acetylated lowdensity lipoproteins. However, it is not widely distributed
among mammalian cell types and so cannot be solely
responsible for apoptotic cell recognition. Nor does it
exhibit the specificity for phosphatidylserine recognition,
in contrast to the phosphatidylserine receptor, that characterizes this phospholipid’s involvement in apoptotic cell
clearance. SREC has yet to be shown capable of mediating
apoptotic cell uptake, even in endothelial cells. Intriguingly, although their sequences appear unrelated, SREC
and the phosphatidylserine receptor are both found on the
tip of human chromosome 17; the biological significance of
this association is unknown.
CD14 represents an interesting candidate for apoptotic
cell recognition. This soluble or glycophosphoinositidelinked protein is involved in the appropriate interaction of
lipopolysaccharides with Toll-like receptor 4. It has been
implicated in recognition of apoptotic cells, particularly
apoptotic lymphocytes. Most of the recognition processes
do not seem to show much specificity for apoptotic cell
type so this selectivity may either be a special case or may
merely represent lack of detailed investigation in other
systems. The counter-ligand on the apoptotic cell has
been suggested to be ICAM3 [71] which is upregulated or
exposed on apoptotic lymphocytes through an unknown
mechanism. This again brings in something new, namely
a protein ligand. While it seems likely that CD14 represents a tethering rather than signaling ligand as it does not
exhibit a transmembrane domain, it may prove important
in immune system development and function. These
observations also raise the possibility that there will be
other cell specific protein–protein interactions involved in
apoptotic cell removal.
Finally, one cannot ignore the possibility that apoptotic
cells in mammals might be rendered more susceptible to
phagocytosis in vivo by more conventional antibody–antigen
interactions, the process of opsonization. For example, a
number of studies have suggested that apoptotic cells initiate attachment of complement C3bi followed by deposition of IgG, and subsequent uptake through complement
receptor 3, CR3 [72–74]. Once again, however, the structures on the apoptotic cells that might initiate this
opsonization are not fully defined.
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engulfment. CED-12 has recently been shown to participate in the CED-2, CED-5, CED-10 pathway in C. elegans.
Two mammalian homologues of CED-12, ELMO1 and 2,
seem to form a trimolecular complex with CrkII and
DOCK180 that results in activation of Rac-1 (Ravichandran K, personal communication). However, mutation of
any one of these four genes in C. elegans diminishes, but
does not eliminate, corpse removal.
CED-1, CED-6 and CED-7 seem to participate in an
alternative path for uptake: double mutants of any of these
three genes with one of the former group results in much
more profound abnormalities in apoptotic cell clearance
than the single mutation. CED-1 has already been discussed above and may represent a C. elegans recognition or
signaling receptor. CED-7 is an ABC transporter that may
be involved in both apoptotic cell and phagocyte. As discussed above, its activity, while very poorly understood, is
most likely to be involved in membrane alterations. CED6 resembles an adaptor protein with a phosphotyrosine
binding domain, and its mammalian homolog is implicated
in phagocytes during apoptotic cell engulfment [77,78].
How CED-6 interacts with putative surface receptors or
downstream effector molecules is not yet clear.
In contrast, evidence for two separate uptake pathways in
mammals is lacking. Possibly one or more of the different
recognition molecules could initiate uptake by using different signal pathways but, at least to us, this seems
unlikely. It has been suggested that uptake into professional phagocytes such as macrophages could be different
from ingestion by tissue cells [81]. However, the effect is
largely one of ingestion rate and efficiency and in our
hands does not show differences in receptor usage or
uptake mechanism. Even in C. elegans it is not clear how
the proposed pathways are related.
Uptake signaling pathways
As actin dependent processes are presumably involved,
Rho family GTPases are expected in both pathways. Rac-1
is the key candidate in the CED-2, 5, 10 cell uptake path
and studies in murine macrophages suggest the participation of Rac-1 and Cdc42 [80]. A key problem with many of
these investigations is the complexity of trying to sort out
signaling processes with multiple, or even unknown, recognition molecules and receptors, and initial signal transduction molecules. The ability to address the processes initiated
by an individual receptor would be of great value.
Gene complementation studies in C. elegans suggest two
related pathways for internalization of the apoptotic cells
by phagocytes [7,75,76]. CED-2, CED-5 and CED-10 represent homologs of mammalian CrkII, DOCK180 and Rac1 and constitute one such pathway. Studies in mammalian
systems also show these molecules are involved in apoptotic cell removal (see[43,77–80]). They are presumed to
act at the point of cytoskeletal reorganization during
It is clear that a critical issue in understanding apoptotic
cell removal is whether the uptake processes and signaling
pathways are unique. They are clearly different from Fc
receptor driven phagocytosis. Stimulated macropinocytosis
driven by growth factors such as epidermal growth factor
(EGF), platelet-derived growth factor (PDGF) and macrophage colony-stimulating factor (M-CSF) signaling through
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Figure 2
Phagocytosis of cells by the zipper
mechanism or by stimulated
macropinocytosis. It is suggested that most
forms of apoptotic cell ingestion result from
the macropinocytotic process.
Zipper mechansim
Stimulated
macropinocytosis
Membrane
ruffles
Current Biology
their receptor tyrosine kinases may be a closer model.
However, very preliminary evidence obtained in collaboration with Dr Kodimangalam Ravichandran suggests to us
that upstream signaling elements resulting from growth
factor stimulation may be different from those seen in
apoptotic cell uptake. On the other hand, downstream molecules such as Rac-1 are common. The nematode studies
seem to support this implication.
From the point that Rho family GTPases become involved,
the signaling for cytoskeletal reorganisation, ruffling, membrane extension and eventual fusion, along with other elements of internalization, may not be specific for apoptotic
cells or their recognition receptors. However, key issues are
the relationship between Cdc42 and Rac; how and when
Rac-1 becomes localized to the site of uptake; whether the
interaction of an apoptotic cell initiates activation and
membrane alterations at only the local site of contact and
stimulation; and any potentially unique processes involved
in subsequent phagosome disposition, pH changes, lysosome and vesicle fusion. Because of the overall importance
of the apoptotic cell removal process and its consequences,
these are critical questions for future investigation.
Mechanisms of uptake
Ingestion of particles into cells occurs by a number of different mechanisms reflecting different receptors, signaling
pathways and physical uptake processes. Immunoglobulin
Fc receptor stimulation induces phagocytosis by a process
that involves the sequential ligation of the receptors by Ig
molecules on the particle as in a zipper [82–85]; this results
in a phagosome with membranes in close contact with
the ingested particle. Uptake through CR3–αmβ2 integrin
involves the particle appearing to sink into the phagocytic
cell, without the development of an obvious phagocytic
cup. We have recently suggested that uptake of apoptotic
cells through the types of receptors discussed above occurs
by a process more akin to macropinocytosis (Figure 2).
In stimulated macropinocytosis, induced, localized membrane ruffling is associated with enclosure of extracellular
fluid followed by internalization within a spacious phagosome. Particles attached to the surface are taken in at the
same time. In this system the receptors for macropinocytosis initiation could be on either the particle or the apoptotic cell, or could interact independently if the apoptotic
cell were already tethered to the phagocyte membrane.
We have called this a ‘tether and tickle’ mechanism ([86],
Hoffmann P and de Cathelineau A, unpublished observations) and thereby imply a two-step uptake process with a
requirement for apoptotic cell attachment and signaling
that, in many cases, may be mediated by different receptors (Figure 3).
The tether and tickle mechanism resembles that accompanying uptake of pathogenic Salmonella into epithelial cells.
Here the virulence factor SopE [87,88] is injected into the
cell by the bacterial Type III secretion system where it
activates members of the Rho family of GTPases and
causes membrane ruffling and macropinocytosis [89,90].
Salmonella lacking the virulence factor may still attach to
the cell surface but are not ingested unless ruffling and
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macropinocytosis are induced by alternative stimuli, such
as growth factors [91].
Ligation of some apoptotic cell recognition receptors such
as CD36 or αv integrins induces tethering of cells to macrophages but without uptake. Inclusion of phosphatidylserine on the target cell surface in this circumstance initiates
uptake. By contrast, phosphatidylserine on the target cell
membrane without the tethering ligand is ineffective, we
would suggest, because the affinity/avidity is too low to
initiate the process. Independent stimulation of the phosphatidylserine receptor in the presence of previously tethered cells also initiates their uptake — a bystander
mechanism akin to that outlined above for Salmonella.
This dual process may provide significant checks and balances in the removal process, minimizing for example,
uptake of cells that may transiently express phosphatidylserine during activation or of cells that may
become transiently attached to potential phagocytes in the
absence of the signaling receptor ligation, the tickle part of
the process.
Uptake of apoptotic cells by the tether and tickle mechanism would initially involve spacious phagosomes and the
concurrent ingestion of extracellular fluid. This was found
to be the case ([40], Hoffmann et al. unpublished observations), and was in marked contrast with uptake of cells,
even apoptotic cells, via the Fc receptor after opsonization;
here there is no evidence for fluid uptake into the phagosome. It should be noted that while macropinocytosis initially exhibits a spacious phagosome, this may soon be
followed by condensation of the phagosome to yield more
close apposition of the membranes to the ingested particles [89]. Although not studied to any degree, one might
expect differences in subsequent disposition of the phagosomes, in digestion of the apoptotic cells and final processing of the contents. The significant body of literature
showing macropinocytosis to be a key uptake mechanism
in antigen processing and presentation supports this, as
does the genetic evidence for specific genes involved in
corpse digestion in C. elegans [75,92].
Points of regulation
One aspect of regulation in the removal of apoptotic
corpses seen in mammalian systems is the stimulation of
macrophages to exhibit enhanced uptake. It is intriguing
to note that in most investigators’ hands, only a proportion
of macrophages, primary isolates or even cell lines, actually
engulf apoptotic cells in vitro. Whether this reflects subtypes of cells, varying abilities to undergo the putative
macropinocytotic process, cell–cell contact issues or heterogeneity in receptor expression, remains to be determined. Resident macrophages from lung or peritoneum
are markedly inefficient at apoptotic cell clearance as are
circulating monocytes. However, macrophages that have
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Figure 3
Apoptotic cell
Signaling ligands
Signaling receptors
Phagocyte
Tethering ligands
Tethering receptors
Signaling for membrane
ruffling and
macropinocytosis
Current Biology
A proposed two component uptake mechanism for apoptotic cells
involving a combination of tethering and signaling receptors — a ‘tether
and tickle’ process.
emigrated to, and matured within inflammatory sites are
very effective [93,94].
As monocytes mature into macrophages in vitro they too
develop an increased ability to recognize and ingest apoptotic cells. Part of this maturation may reflect stimulated
increase in expression of the phosphatidylserine receptor
[95,96]. It may also suggest levels of regulation that are
only beginning to be explored. For example, stimulation
of macrophage CD44 [97] and exposure to corticosteroids
[98] or to lipoxins [99] have each been shown to enhance
the ability of macrophages to recognize and engulf apoptotic cells. While the mechanisms for enhancement have
not been clarified, these processes do seem to be selective
for apoptotic cells.
Maturation of monocytes into immature dendritic cells
also increases their ability to ingest apoptotic cells. Immature dendritic cells show increased constitutive macropinocytosis so it may be that uptake in these cells is
regulated more by the nature of the tethering interaction
than specific stimulation of macropinocytotic signaling.
Regulation of uptake efficiency is expected in resident
tissue cells as well but has not yet been investigated to any
significant extent. It would also seem likely that other
combinations of stimuli and environmental factors yield
circumstances that block or delay apoptotic cell recognition and uptake. For example, defective clearance of apoptotic inflammatory cells in cystic fibrosis has recently been
attributed in part to cleavage of the recognition receptors
by elastase (Vandivier et al., unpublished observations).
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Another area of increased interest is the likelihood that
cells, including apoptotic cells, may under certain circumstances express ‘don’t eat me’ signals. The demonstration
that CD47 expression by erythrocytes blocks their phagocytosis, through an interaction with the signal regulatory
protein SIRPα [100,101], lends credence to such a concept.
Whether this system, or others, operates in the context of
apoptotic cell uptake remains to be firmly established.
Consequences of recognition and removal
Apoptotic cell clearance is efficient and non-inflammatory
and may often be mediated in vivo by neighboring cells.
Great efforts are currently being expended on understanding the sequelae of apoptotic cell deletion, largely because
of its active anti-inflammatory and suggested anti-immunogenic effects. Such effects have been discussed in some
detail (see [9–11,79,102–104]). Following initial observations that uptake does not induce the release of inflammatory mediators, it soon became apparent that inflammation
is actively suppressed, both in vitro and in vivo, through
the production of inhibitory factors such as transforming
growth factor (TGF) β, prostaglandin E2 (PGE2) and interleukin-10 (IL-10).
We, and others, have implicated the phosphatidylserine
receptor and phosphatidylserine expression on the apoptotic cells as a major player in this suppression and have
recently suggested that its effect may be extended to
explain the inhibition of dendritic cell maturation and
antigen presentation in the adaptive immune response
[79,105]. This is in keeping with the suggested distinction
between the interaction with dendritic cells of apoptotic
cells — which inhibits maturation and antigen presentation — and necrotic cells [14,15,102,106–108]. It is worth
noting in this regard that induced apoptosis and cell deletion in normal tissues often leads to replenishment and
repair, in contrast to the removal of excess cells seen
during development. It seems likely to us that growth and
differentiation factors other than TGFβ may be produced
in response to apoptotic cell recognition that could contribute to this return to normal structure and function.
The obverse of this overall beneficial and essentially
normal process are the circumstances where suppression of
inflammation or immunity is essentially harmful. A clear
example of this was shown in tissue responses to Trypanosoma cruzi, the cause of Chagas disease. Here, apoptotic cells enhance the survival of the parasites in macrophages through their induction of TGFβ and PGE2 [109].
Similarly there is increasing evidence that apoptosis in
tumor cells might also have an overall negative effect on
the host as a consequence of local suppression of inflammation and immunity. TGFβ itself has long been associated with the development of fibrotic reactions in tissues
— a process that may lead to an important restoration of
structural integrity in a skin wound, but long-term impairment in function in the lung or liver. These types of
responses emphasize the need for balance in the response
to apoptotic cell removal in normal cell turnover.
In situations where cells are produced to excess, for
example myofibroblasts in fibrotic wound healing, even
here the processes of apoptosis and cell clearance appear
critical to their resolution. A number of studies have
addressed the ability of macrophages not only to engulf
apoptotic cells but also to induce apoptosis in appropriate
targets, especially in the context of tissue reorganization
during development or after inflammatory or fibrotic
changes [110–112]. Although not explicitly investigated in
most of these experimental systems, a coordinated induction of apoptosis and cell removal seems likely to occur.
Potential mechanisms include the production of oxidants,
nitric oxide (NO), tumor necrosis factor (TNF) α or Fas
ligand (FasL), and attachment of the macrophage to the
target cell could readily enhance both apoptosis and subsequent removal.
The genetic approach already discussed in C. elegans has
recently made an intriguing contribution to our understanding of the combined apoptosis and engulfment process
[113,114]. Apoptosis in worms is blocked in the absence of
CED-3 or CED-4. However, in partial ced-3 mutants,
showing only reduced numbers of apoptotic cells, the combined mutation of engulfment genes blocks apoptosis. The
implication is that engulfment can contribute to the induction of cell death initiated by less than optimal activation of
caspases. Once again the mechanisms are not yet defined
although Green and Beere [115] have suggested a possible
early discharge of lysosomal hydrolases akin to processes
described many years ago in mammalian phagocytes
[116–118] and colloquially called ‘frustrated phagocytosis’
or ‘regurgitation while feeding’. The two C. elegans studies
also suggest that the terminal, caspase-dependent ‘execution’ step in apoptosis may not be as irreversible as is often
implied — a concept that is perhaps more readily understood in the apparently more complex regulation processes
seen during apoptosis in mammalian cells [119].
The take-home message for our understanding of apoptotic cell clearance is that the complexities of apoptotic
cell engulfment are beginning to succumb to investigation,
are likely to be unique and primitive, and appear to
exhibit remarkable similarities across disparate members
of the animal kingdom.
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