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10.1146/annurev.physiol.65.092101.142459
Annu. Rev. Physiol. 2003. 65:701–34
doi: 10.1146/annurev.physiol.65.092101.142459
c 2003 by Annual Reviews. All rights reserved
Copyright °
First published online as a Review in Advance on December 2, 2002
AMINOPHOSPHOLIPID ASYMMETRY: A Matter of
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Life and Death
Krishnakumar Balasubramanian and Alan J. Schroit
Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030; e-mail: [email protected]
Key Words phosphatidylserine, phosphatidylethanolamine, apoptosis,
phagocytosis, receptors
You have two sides, one out and one in. Each lipid that’s on the side that’s in
goes out and when its out it comes in and the next lipid goes out until its in.
When they are all out, the side that’s out comes in and the side that’s been
in goes out. Some lipids are out and don’t come in. Sometimes lipids that are
in go out but are not out and come back in. Other times these lipids go out
and are out because they don’t go in. When that happens the cell is dead.
Adapted from Patrick A. Williamson, as modified from “Understanding Cricket.”
www.redstripebeer.com
■ Abstract Maintenance of membrane lipid asymmetry is a dynamic process that
influences many events over the lifespan of the cell. With few exceptions, most cells
restrict the bulk of the aminophospholipids to the inner membrane leaflet by means
of specific transporters. Working in concert with each other, these proteins correct
for sporadic incursions of the aminophospholipids to the outer membrane leaflet as
a result of bilayer imbalances created by various cellular events. A shift in the relative contribution in each of these activities can result in sustained exposure of the
aminophospholipids at the cell surface, which allows capture of the cells by phagocytes before the integrity of the plasma membrane is compromised. The absence of
an efficient recognition and elimination mechanism can result in uncontrolled and
persistent presentation of self-antigens to the immune system, with development of
autoimmune syndromes. To prevent this, phagocytes have developed a diverse array
of distinct and redundant receptor systems that drive the postphagocytic events along
pathways that facilitate cross-talk between the homeostatic and the immune systems.
In this work, we review the basis for the proposed mechanism(s) by which apoptotic
ligands appear on the target cell surface and the phagocyte receptors that recognize
these moieties.
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INTRODUCTION
With numerous species of lipids expressed in eukaryotic plasma membranes, cells
are faced with the formidable task of organizing the lateral and transverse distribution of membrane phospholipids into functional compartments and specific sites.
Cells have developed a number of mechanisms to deal with this issue, one of which
is to create a dynamic equilibrium to their site of function that ensures interaction of specific lipids with appropriate partner moieties. Targeting phospholipids
to specific membrane sites is essential for maintaining critical signal transduction
cascades (1), cell shape (2), hemostasis (3, 4), and homeostasis (5). Whereas the
importance of sphingolipids, phosphoinositides, and lysophospholipids in these
processes cannot be overemphasized (6–9), aminophospholipids have also been
implicated in a diverse array of processes ranging from cell proliferation to cell
death, to catabolism to inflammation (Table 1).
Many studies have established the unifying concept that plasma membrane
phospholipids are dynamically distributed in an asymmetric fashion such that
the majority of the aminophospholipids reside in the cells’ inner membrane leaflet
with the choline-containing phospholipids being restricted to the outer leaflet (10).
Asymmetry is maintained by active ATP-dependent processes (11), suggesting that
it is critical to normal cell function. Indeed, if cells fail to engage mechanisms to
maintain asymmetry, aminophospholipids appear at the cell surface, which leads
to dramatic changes in cell function. One of the important consequences of altered
membrane asymmetry is the recognition and engulfment of phosphatidylserinebearing vesicles and cells by mononuclear phagocytes (12–14). In this article, we
review the mechanisms that regulate membrane lipid asymmetry, the physiologic
significance of an asymmetric phospholipid distribution, and the phagocyte receptors that mediate the recognition and engulfment of senescent and apoptotic
cells.
TRAFFICKING OF AMINOPHOSPHOLIPIDS
TO THE PLASMA MEMBRANE
The transverse distribution of aminophospholipids at the plasma membrane can
be altered by the delivery of lipids from intracellular sites of synthesis to the
inner leaflet and from extracellular sources to the outer leaflet. There are several pathways for the synthesis of aminophospholipids in eukaryotic cells (Figure 1) (15, 16). Phosphatidylserine (PS) is synthesized by a Ca2+-dependent
PS synthase–catalyzed reaction where serine is exchanged with the headgroup
of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) (17). This occurs mainly in the endoplasmatic reticulum (ER) and in specialized ER-derived
mitochondrial-associated membranes (MAM) (18) that bridge the ER to the mitochondria. Once formed, the newly synthesized lipid is transported from the MAM
to the mitochondrial outer membrane by an ATP-dependent mechanism (19). The
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MEMBRANE AMINOPHOSPHOLIPID ASYMMETRY
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TABLE 1 Aminophospholipids in the outer leaflet of cells
Cell type
Lipid
Remarks
References
Normal (nonpathologic cells)
Chick embryo neurons
PE
Outer leaflet PE increases
during development of
interneronal contacts
Differentiation-dependent
decrease in outer leaflet PE
Cells express outer leaflet
PE in cleavage furrow at
late telophase
Large fraction of both lipids
in outer leaflet of chick
embryo cells
Thin sections of tissue from
embryos perfused with
annexin V
Bone marrow and resting
B cells
Transient PS exposure during
IgE stimulation
Cells require PS expression for
engulfment of apoptotic targets
(58)
Required for assembly of
coagulation complexes
Most apoptotic cells express PS
as a very early event
Indirect evidence based on
MC540 staining and
PS-dependent prothrombinase
activity
(72, 256)
Expressed on a subpopulation
of cells
Expressed on a subpopulation
of cells with high variability
between individuals
Shown in patients samples
and in an in vitro experimental
system
(114, 115, 122)
PS-dependent prothrombinase
assay
Annexin V labeling
Shown by specific targeting
using PS antibodies
(137)
PC-12 cells
PE
Chinese hamster ovary cells
PE
Myoblasts, diffentiating
myotubes in vivo
PE, PS
Megakaryocytes
PS
B lymphocytes
PS
Mast cells
PS
Macrophages
PS
Pathologic cells
Platelets
PS
Apoptotic cells
PS
Erythroblasts
PS
Erythrocytes
Sickle
PS
Thalassemia
PS
Diabetes
PS, PE
Tumor cells
Melanoma
Carcinoma
Tumor vasculature
PS
PS
PS
(59)
(60, 61)
(62, 64)
(63)
(66, 67)
(68)
(84, 243)
(80, 83)
(70, 71)
(116, 117, 123)
(118, 119)
(138, 139)
(142)
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lipid then moves to the inner membrane where it serves as a substrate for PS
decarboxylase I to generate PE. The Golgi and vacuoles also synthesize PE by
decarboxylating PS with decarboxylase II. The mechansism by which aminophospholipids are transported from their sites of synthesis to the plasma membrane has
not been completely established but probably involves both vesicular and cytosolic
protein-mediated transfer mechanisms (20). The delivery of lipids from extracellular sources can also alter the transverse distribution of phospholipids. Lipid transport proteins in plasma, such as HDL and LDL, deliver fatty acids, cholesterol,
and phospholipids to the cells outer membrane leaflet where they are utilized by
the cell. Furthermore, normal cell activities such as endo- and exocytosis, which
involve multiple membrane fusion events, likely induce transient episodes of lipid
intermixing between membrane leaflets. To correct for these potential imbalances,
cells have developed several mechanisms that restore and maintain an appropriate
asymmetric aminophospholipid distribution. This asymmetry is maintained by a
combination of the following activities (Figure 2): (a) flip, an energy-dependent
aminophospholipid translocase-mediated movement of aminophospholipids from
the outer to inner leaflet; (b) flop, an energy-dependent movement of phospholipids
from the inner to outer leaflet; and (c) scrambling, a Ca2+-dependent but energyindependent nonspecific randomization of lipids across the bilayer. Although it
is unequivocal that dynamic reorientation of membrane lipids occurs in normal
physiology and in pathologic duress, identity of these proteins and their regulatory
elements remains a mystery.
IDENTIFICATION OF PHOSPHOLIPID TRANSPORTERS
Most of the pivotal studies on membrane phospholipid asymmetry have been performed in human red blood cells (RBC). Although these cells have provided a
wealth of information on the biochemical properties of aminophospholipid transporters, further progress has been impeded by the inability to identify the active
protein components in these cells by molecular biology techniques. Most of the
information on the identity of phospholipid transporters has been made available
from research in yeast and cells from a patient suffering from a congenital bleeding
disorder termed Scott syndrome.
Mg+2-ATPase (Aminophospholipid Translocase)
This activity is associated with an ATP-dependent rapid movement (within seconds to several minutes) of both PS and PE from the outer to inner membrane
leaflet in virtually all eukaryotic cells. Based on its ATP dependence and sensitivity to fluoride and vanadate, Seigneuret & Devaux (21) postulated the activity to
be associated with an aminophospholipid-specific transport protein. Several proteins ranging from blood group antigens to various ATPases have been implicated
in aminophospholipid transport. Observations that Rhnull cells express disproportionate amounts of endogenous PE in their outer leaflet raised the possibility that
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MEMBRANE AMINOPHOSPHOLIPID ASYMMETRY
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Rh antigens might participate in the transbilayer movement of aminophospholipids (22). Although a radiolabeled sulfhydryl inhibitor of PS transport and a
photoaffinity-labeled PS bound to ∼32-kDa red cell proteins that precipitated with
monoclonal Rh antibodies (23), amino acid sequencing revealed no consensus ATP
binding sites (24), and Rhnull cells transported exogenously supplied aminophospholipid analogs normally (23, 25). These results therefore exclude the participation of Rh in lipid transport but do not rule out the participation of a distinct 32-kDa
protein that is coprecipitated with Rh. Another group of proteins with properties
consistent with the transporter are the 80–120 kDa Mg2+-ATPases isolated from
red cell membranes (26–30) and from bovine chromaffin granules (31). Cloning
of the bovine chromaffin granule ATPase indicated that it belongs to a subfamily
of P-type ATPases (32). Disruption of the yeast DRS2 gene, a homolog of the
mammalian ATPase, inhibited the transport of exogenously supplied fluorescent
PS in support of its function as an aminophospholipid transporter (32). Moreover,
a recently identified plant gene involved in cold tolerance homologous to the yeast
DRS2 was able to reconstitute PS transport activity in transport-deficient DRS2
yeast mutants (33). However, similar DRS2 gene deletion experiments carried
out in different laboratories failed to inhibit fluorescent PS transport or alter the
distribution of endogenous lipids across the membrane (34, 35). Thus whereas
the aminophospholipid transporter requires the participation of a Mg2+-ATPase,
the varied and sometimes conflicting results raise the possibility that several distinct
proteins cooperate to form a functional aminophospholipid transport complex.
Multidrug Resistance Proteins
These proteins, members of the ABC transporter family, are involved in the trafficking of biological molecules and drugs across the plasma membrane. They
are broadly classified into P-glycoprotein (MDR), multidrug resistance-associated
protein (MRP), and mitoxantrone resistance protein (MXR) by their size and the
number of transmembrane loops (36, 37). The substrates for MDR and MXR
are largely hydrophobic or amphiphilic, whereas those recognized by MRP are
principally anionic and likely require cotransport of glutathione disulfide (GSSG)
(and/or GSSX) to be operative. Experiments using fluorescent and spin-labled
phospholipid analogs provided evidence for the existence of an ATP- and proteindependent nonspecific flopase that transports lipid from the inner to outer membrane leaflet (38, 39). More recent studies, however, indicate that transport is a
result of MRP1 activity that expels lipids out of the cells because their fluorescent and spin-labeled reporter tags engenderd the lipid with drug-like properties (40, 41). Although these results seem to eliminate a role for the purported
nonspecific flopases in the externalization of authentic phospholipids, several
lipid-specific flopases have been identified that include human MDR3 (42, 43)
and ABC1 (44), which function as bona fide PC- and PS-specific flopases, respectively. Taken together, these results suggest that the steady-state equilibrium
distribution of membrane phospholipids is maintained by joint activities of an
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aminophospholipid-specific flipase and cholinephospholipid-specific flopase
(MDR family) and that acquisition of the PS-expressing apoptotic phenotype requires activation of ABC1 coincident with Ca2+-mediated inhibition of the aminophospholipid translocase (Figure 2) (44).
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Scramblase
Studies with platelet and red cell membranes revealed the existence of a Ca2+dependent scrambling activity that seemed to result in complete intermixing of
lipids between bilayer leaflets irrespective of headgroup specificity (45). Sims
and coworkers reported the isolation and cloning of a putative 37-kDa prolinerich protein (PLSCR1) responsible for this activity (46, 47). Cells from Scott
syndrome patients, who suffer from a bleeding disorder, exhibit an inability to
vesiculate or expose PS when stimulated with Ca2+ (48, 49). Reconstitution studies
using scramblase isolated from Scott erythrocytes, however, indicated that the
protein itself was functional (50). Based on this observation, the authors raised the
possibility either that Scott syndrome is a result of an inability of Ca2+ to bind and
activate the scramblase function or that an inhibitor present in Scott cells is lost
during purification. Interestingly, transfection of Raji cells with the scramblase
gene conferred increased exposure of PS in response to enforced Ca+2 influx (51),
but the same cells failed to express PS in response to several apoptotic stimuli
(52). Moreover, human umbilical vein endothelial cells (HUVECs) induced with
IFN-α to express a 10-fold increase in scramblase showed no change in cell surface
PS upon Ca2+ ionophore treatment (53), and cells from PLSCR1-null mice were
still able to mobilize PS to the cell surface upon activation (54). In light of these
arcane and conflicting observations, it would seem premature to assign the term
scramblase to the 35-kDa protein, especially considering that scramblase-deficient
Raji cells can still express PS in response to FAS ligation when depleted of ATP
(55). Obviously, more experimentation is needed to clarify the role of this protein
in lipid transport (56). The recently discovered canine Scott-like syndrome should
provide a valuable model for delineating the role of these proteins (57).
MEMBRANE LIPID ASYMMETRY IN PHYSIOLOGY
Cell Biogenesis
Cells expend an appreciable amount of energy to ensure that most of the plasma
membrane aminophospholipids are oriented toward the cytoplasm, suggesting
that their appearance at the outer leaflet has important biological consequences.
While most studies stipulate that PS exposure is the kiss of death, hence the
importance of keeping these lipids away from the cell surface, perturbations in
aminophospholipid asymmetry are not always deadly. Several studies have suggested coordination between phospholipid asymmetry, cell cycle, and cell differentiation. Studies using primary cultures of chick embryo neurons showed an
increase in the exposure of PE and a concomitant decrease in the exposure of PC
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MEMBRANE AMINOPHOSPHOLIPID ASYMMETRY
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upon formation of intraneural contacts (58). Other studies using PC-12 neuronal
cells suggested that the distribution of PE at the plasma membrane outer leaflet
decreased during nerve growth factor-induced neuronal differentiation (59). Using
a PE-specific tetracyclic polypeptide (Ro09-0198), Emoto et al. have shown that
Chinese hamster ovary cells express cell surface PE at the cleavage furrow during
late telophase (60). Moreover, immobilization of PE with Ro09-0198 inhibited the
disassembly of the contractile ring components (myosin II and radixin), resulting
in the formation of a long cytoplasmic bridge connecting the daughter cells (61).
The importance of PE exposure in cytokinesis was further shown using a mutant
cell line deficient in PE owing to a defect in the MAM pathway. These cells were
arrested in late telophase but could be rescued by restoring cellular PE levels with
the addition of exogenous PE or ethanolamine (61). These data underscore the importance of lipid transporters in regulating specific transbilayer aminophospholipid
distributions compatible with various cellular functions and activities.
Fusion of myoblasts to form multinucleate myotubes that constitute skeletal
muscle is another event that appears to involve altered aminophospholipid exposure. Sessions & Horwitz (62) were the first to show the presence of atypical
amounts of PE and PS in right-side-out myoblast plasma membranes. Surfaceexposed PS has also been detected in differentiating myoblasts in vivo (63). Indeed, the expression of PS seems to be critical for differentiation into myotubes
because annexin V inhibits this process (64). Conceivably, the presence of profusogenic PS and PE drives the fusion events (65) critical for the formation of
multinucleated myotubes. PS is externalized in developing and mature B lymphocytes (66) where it localizes in lipid rafts (67). It has also been found in the
outer leaflet of budding megakaryocytes (63) and becomes reversibly expressed
during IgE-dependent stimulation of mast cell degranulation (68). The controlled
and localized exposure of PS might also play an important role in erythropoiesis,
where differentiation of the erythroblast to the incipient reticulocyte depends on
extrusion of the nucleus to one side of the cell (69). Similar to the fate of apoptotic
cells, the segregated nucleus is recognized and engulfed by macrophages. There
is no direct evidence for segregation of PS to the pole of the cell that contains the
extruded nucleus. However, MC540, a fluorescent dye that stains apoptotic cells
also binds the extruded nucleus (70), suggesting that engulfment occurs by the
same PS-dependent mechanism. Indeed, using a system to model erythropoiesis,
Conner et al. showed a direct correlation between PS expression and the propensity
of the cells to be bound by macrophages (71).
Blood Clotting and Wound Repair
Platelet activation following vascular damage involves adhesion to subendothelial
structures and aggregation of platelets to form a primary hemostatic plug at the
wound site. Anionic lipids expressed on activated platelets are important for the coordinated assembly of coagulation factors that promote coagulation and anticoagulant (protein C) reactions. These lipids are indispensable in promoting membrane
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binding and the catalytic activity of the tenase and prothrombinase complexes that
lead to the formation of thrombin, cleavage of fibrinogen, and, finally, deposition of the clot-forming fibrin matrix (72). The tenase complex is initiated by the
interaction of factor VIIIa with negatively charged lipid to create a high-affinity
binding site for the enzyme factor IXa in the presence of Ca2+. This complex
rapidly activates factor X into Xa. Likewise, in the prothrombinase complex, binding of factor Va to an anionic lipid surface promotes Ca2+-dependent binding of
factor Xa, which converts prothrombin to thrombin. In both complexes, PS is the
most effective anionic phospholipid (73). PS is equally important in promoting the
anticoagulant protein C pathway, which leads to disassembly of the prothrombinase complex (74, 75). Whereas formation of the platelet plug and the expression
of PS on the cell surface is critical for control of bleeding, these cells must be
cleared from the site of injury for wound healing to ensue. Although no definitive
studies have been done, activated PS-expressing platelets are likely recognized
and engulfed by mechanisms similar to those for the clearance of apoptotic cells.
Apoptosis
Programmed cell death, or apoptosis, is a process in which cells undergo an orchestrated sequence of events that culminates in the recognition and phagocytosis of
the dying cells by mononuclear phagocytes. Apoptosis can be initiated by a variety
of stimuli that proceed through distinct pathways (76–78). The extrinsic pathway
is mediated by ligation of the so-called death receptors (Fas, tumor necrosis factor,
TRAIL) resulting in the recruitment of adapter proteins and procaspase molecules
that ultimately activate downstream caspases. The intrinsic pathway, on the other
hand, is mediated by a variety of stress signals that converge at the mitochondria
and stimulate the release of cytochrome c, which in turn initiates the caspase cascade. Apoptosis induced by serum starvation and many chemotherapeutic drugs
operates through the intrinsic pathway. To distinguish apoptotic cells from normal
viable cells, the apoptotic cell membrane must express neoepitopes that bind, either directly or indirectly, to phagocytes. One of the hallmarks of cells undergoing
death, determined by their ability to bind annexin V (79), is the reorientation of
PS from the cells’ inner to outer membrane leaflet (80, 81), an event that precedes
DNA condensation (82, 83). Although PS exposure seems to be a general feature
of senescence and apoptosis, there are several exceptions to this rule. As mentioned above, apoptotic Raji cells do not express PS (52), and some macrophages
(84) and B lymphocytes constitutively express PS (66, 67). In addition, while it
is generally accepted that PS exposure as monitored by annexin V labeling is a
marker for apoptosis, there is no compelling data to suggest exclusivity of PS in
these processes. In fact, apoptotic cells express aldehyde adducts (85) that also
bind annexin V (86).
A large body of evidence indicates that lipid peroxidation is directly responsible for the generation of the apoptotic phenotype (87). Many agents that induce
apoptosis are also stimulators of cellular oxidative metabolism (88–90), and many
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MEMBRANE AMINOPHOSPHOLIPID ASYMMETRY
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inhibitors of apoptosis have antioxidant activities or enhance cellular antioxidant
defenses (91, 92). Accumulating evidence suggests that cells undergoing apoptosis generate oxidatively altered cell surface moieties analogous to those found in
oxidized LDL (OxLDL) (85) and that scavenger receptors known to bind OxLDL
are involved in the binding of apoptotic cells. In addition, antibodies specific to
aldehydes produced as a result of lipid peroxidation (4-hydroxynonenal and malondialdehyde) bind to OxLDL (93, 94), atherosclerotic lesions (95), damaged fibroblasts (96), vascular endothelial cells, and apoptotic cells (85). Finally, OxLDL
competes with apoptotic cells for phagocyte uptake (97, 98). Taken together, these
data provide evidence that when cells undergo apoptosis, oxidation-derived ligands
appear at their surface and function as an epitope that is recognized by phagocytes.
Red Cell Senescence and Disease
Homeostasis is, in part, dependent on the disposal of nonfunctional effete cells.
This is a particularly efficient process in the circulatory system, where RBC are
programmed for removal from the bloodstream after approximately 120 days.
While definitive proof is still unavailable, red cell senescence is likely the result
of a series of progressive events that lead to the cells deterioration and ultimate
catabolism (99, 100). Membrane-related changes include loss of membrane lipids,
decreased surface charge density owing to loss of sialic acid, accumulation of lipid
peroxidation products, and increased adhesiveness to endothelial and reticuloendoethelial cells. Red cells also accumulate increasing amounts of surface-exposed
PE (101) and PS over their lifespan in vivo (102, 103) and under in vitro storage conditions (104). Because aging red cells progressively lose ATP-dependent
enzymatic activity, both the ATP-dependent aminophospholipid translocase and
the Ca2+ pump can be affected. Conceivably, this could lead to an increase in cytosolic Ca+2 levels that stimulate scramblase and suppress the aminophospholipid
translocase. Indeed, out-to-in translocation rates and equilibrium distributions of
spin-labeled and fluorescent phospholipid analogs determined in density-separated
red cell populations indicate that the initial velocity of aminophospholipid translocation is reduced in the denser (older) populations (102, 105). Cumulative oxidative
damage to cellular lipids and proteins also occurs during normal RBC aging (106,
107). This process may also contribute to PS exposure by directly inhibiting lipid
transporters (87, 105), by direct oxidation of its (PS) acyl chain (108), or through
the generation of aldehyde lipid adducts that impersonate PS (86, 109). Although
erythrocytes are not considered to undergo true apoptosis because they lack a
nucleus and mitochondria, recent studies raise the possibility that red cells share
similar regulatory mechanisms with nucleated cells. Other than the well-known
similarities of Ca2+ influx/PS exposure, red cells express the apoptotic regulatory
proteins Bcl-X(L) and Bak (110). Moreover, their death can be prevented with
cysteine protease inhibitors (111, 112).
There are numerous reports of increased exposure of PS and PE in the outer
leaflet of sickled (113–115), thalassemic (116, 117), and diabetic (118, 119) red
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cells. Although each of these diseases is distinct, disturbances in membrane phospholipid asymmetry resulting in abnormal levels of PS exposure could be partially
responsible for many of their associated pathologies. These could include anemia
due to cell lysis following the assembly of complement proteins on the cell surface
(120, 121) or accelerated cell clearance through PS-dependent peripheral blood
monocyte-mediated phagocytosis (102, 122). Cell surface PS also makes cells
thrombogenic by providing a catalytic surface for activation of the coagulation
cascade. Thus increased thromboembolism in these diseases could be caused by
their hypercoagulability (116, 123) and/or adhesion to the vascular endothelium
(124, 125).
Although perturbations in aminophospholipid asymmetry can be explained by
opposing activities of various transport proteins (126), studies on diabetic red cells
indicate that oxidative stress plays a critical role in the distribution of membrane
lipids. Lipid peroxidation occurs in red cells incubated in hyperglycemic media
(127, 128) and results in a phenotype that, similar to diabetic RBC, expresses
PS (119) and becomes procoagulant (129). This occurs via oxidative pathways
that result in the production of reactive aldehydes that can be inhibited with
vitamin E and N-acetylcysteine (127, 129). In principle, aldehydes can react with
primary amines on lipids and proteins to generate negatively charged moieties
that mimic PS (86). There have been several reports that advanced glycation end
products (AGE) also generate N-acylated aminophospholipids (130). These lipids
have been shown to trigger lipid peroxidation via free radical mechanisms (131)
that lead to the generation of 4-hydroxynonenal (HNE) and malondialdehyde
(MDA) adducts (132). Such a mechanism could also be involved in apoptosis
(133) and might play a role in phagocyte recognition ligand for phagocytosis
(87).
Tumor Cells and Tumor Vasculature
Tumor cells produce tissue factor (TF), cancer procoagulant, and other factors
that can result in thrombosis and disseminated intravascular coagulation in cancer patients (134, 135). Although increased exposure of aminophospholipids does
not appear to be a general feature of tumor cells, their procoagulant activity has
been shown to result, in part, from the expression of PS. Qualitative differences in
the levels of cell surface PS by the PS-dependent prothrombinase activity assay were found in erythroleukemic cells, melanoma cells, and colon carcinoma
cell lines (136, 137) and directly correlated with their propensity to be bound by
macrophages. Similar observations were obtained using annexin V on ovarian and
gastric carcinoma cells (138, 139).
A large body of evidence indicates that endothelial cells become proadhesive
upon apoptosis (140, 141). In vivo this could lead to adhesion of red cells and
platelets and initiation and propagation of the coagulation cascade by providing
a catalytic surface that expresses both TF and PS. It is particularly interesting
to note that endothelial cells in tumor vasculature, but not normal vasculature,
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express significant amounts of PS that can be exploited for tumor therapy (142).
To accomplish this, vascular cell adhesion molecule-1 (VCAM-1) (which is present
in both normal and tumor blood vessels) was targeted with VCAM-1 antibodies
coupled to a truncated form of TF. Because of the specific expression of PS, TFdependent thrombosis occurred only in the tumor vasculature, which led to tumor
regression (142).
MAINTENANCE OF HOMEOSTASIS: PHAGOCYTOSIS
OF ABERRANT CELLS
Homeostasis in multicellular organisms is maintained by processes that balance
life and death through synchronization, communication, and cooperation between
various cells and tissues. This requires the replacement of effete and senescent
cells with fully functional viable cells and results in the generation of an immense
number of cell corpses. This is a remarkable feat considering that for red cells
alone, this represents ∼30 mL of packed cells (∼1012 cells) that are recognized
and eliminated from the circulation on a daily basis. The collection and disposal
of these cells is executed by specialized phagocytes that have developed multiple receptor systems to identify specific apoptotic target ligands. Many studies
on receptor ligand systems have shown that these recognition partners are not
mutually exclusive (Figure 3, Table 2). One ligand can bind multiple receptors,
and one receptor can bind multiple ligands. This likely facilitates the recognition of a diverse array of cells and targets and potentially increases the overall
efficiency of the process. The removal of these cells is so critical to the organism that its disruption is associated with the pathogenesis of many diseases
including autoimmunity (143–146). The importance of this process is underscored
by observations in Caenorhabditis elegans, where the engulfing phagocytes even
coax cells triggered into apoptosis from escaping back to nonapoptotic phenotypes
(147).
MACROPHAGE RECEPTORS FOR APOPTOTIC LIGANDS
Caenorhabditis elegans Death (CED) Proteins
Because of its well-defined and reproducible molecular events, C. elegans has been
widely used as a model in developmental biology. C. elegans differs from mammals
and insects in that it does not have macrophage-like professional phagocytes (148).
Instead, apoptotic cells are recognized and engulfed by neighboring cells (149,
150). Products of the ced-1, ced-6, and ced-7 genes are phagocyte proteins that play
a role in the recognition of the death-signal on apoptotic cells. CED-7 is an ABC
transporter (151) that together with the CED-1 receptor promotes the recognition
of apoptotic corpses (152, 153). The recognition of a bound target is transmitted to
the cytoskeletal machinery that initiates engulfment through CED-6, a specialized
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TABLE 2 Receptor-ligand partners that recognize dying cells
Receptor
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Integrins
α5β3 (vitronectin)
(CD51/CD61)
α5β3/CD36
α5β5/CD36
α?β1
Scavenger receptors
SRA (Types I, II,
and MARCO)
Linker
protein
Ligand
Proposed
function
References
TSP
PS?
Apoptotic
(165–168)
MFG-E8
TSP
TSP
?
PS?
PS?
PS?
?
Apoptotic
Apoptotic
Immunity
Apoptotic
endothelial cells
(172)
(169, 224)
(170)
(171)
?
Ox-LDL
Immunity
(210, 211)
Apoptotic
Lipid
metabolism
Immunity
Apoptotic
(224, 227)
Apoptotic
(235, 236)
Apoptotic
(238)
Apoptotic
(239, 240)
Apoptotic
(190–192)
SRB (CD36); (see
integrins above)
None
SRC (LOX-1; SRCL)
?
SRD (CD68,
macrosialin)
SRE (SREC)
?
PSOX
?
Complement receptors
CR3 (CD11b/
CD18) and
CR4 (CD11c/CD18)
CD93 (cC1qr;
calreticulin)/CD91
(α2-macroglobulin
receptor)
Unclssified receptors
Mer
?
PSr (PS receptor)
?
Acetyl-LDL
AGE
polyanions
PS
Ox-LDL
polyanion
PS
Ox-LDL
polyanions
PS
Ox-LDL
Ox-LDL
Acetyl-LDL
polyanions
PS
Ox-LDL
Ox-proteins
iC3b
PS?
Collectins
(C1q; MBL;
SP-A, SP-D)
PS?
Gas 6
β2GP1
None
PS
PS
PS
(229, 230)
Immunity
(194–196)
Unknown
Apoptotic
(145, 159–161)
(200, 201)
(241, 242)
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adapter protein (154, 155). Although the apoptotic cell ligands in C. elegans have
not been identified, the similarity of the CED-1 receptor to mammalian scavenger
receptors raises the possibility that it might also bind lipid ligands (152). Moreover,
the similarity of CED-7 to ABC1, a PS flopase in mammalian cells (44, 151),
suggests that it plays a similar role in providing the proverbial “eat-you/eat-me”
signals of phagocyte and prey (84, 156).
Insect Phagocyte Receptors
Two distinct phagocyte receptors have been identified in Drosophila melanogaster.
The Class C macrophage-specific scavenger receptor was isolated by expression
cloning (157). This receptor (dSR-CI) is restricted to macrophages and hemocytes
in embryonic development and binds acetylated LDL and other polyanionic ligands. Croquemort (CRQ) is a 68-kDa macrophage receptor structurally related to
mammalian CD36 that participates in the recognition and engulfment of apoptotic
corpses during embryogenesis (158). Disruption of the crq gene does not result in
a lethal phenotype, suggesting that both CRQ and dSR-C1 play complementary
and cooperative roles in apoptotic cell removal.
MAMMALIAN RECEPTORS FOR APOPTOTIC LIGANDS
Mammalian cells have developed a diverse array of distinct but redundant receptor
systems for recognizing apoptotic cells. With the exception of Mer, CD14, PS
receptor, complement, and several integrins, most receptors are members of the
scavenger receptor superfamily. Some receptors bind their targets directly; others
bind through intermediary bridging proteins.
Mer
Gas 6, the growth arrest-specific gene 6 product, binds to several receptor tyrosine
kinases through their cell adhesion molecule-like extracellular domains (159).
Binding experiments showed that Mer-expressing U937 monocytes bound PScoated ELISA plates only in the presence of Gas 6 (160). In addition, Gas 6
enhanced the in vitro uptake of PS vesicles to macrophages (161). Taken together,
these data suggest that Mer/Gas 6 could function as phagocyte recognition partners
for PS-expressing apoptotic cells. Indeed, mice with a cytoplasmic truncation in
Mer (mer kd) exhibited deficient clearance of apoptotic cells in vivo and developed
DNA antibodies reminiscent of systemic lupus erythematosus (SLE). Additional
in vitro studies confirmed that the inability to clear apoptotic cells was due to
a defect in phagocytosis and not in the expression of the apoptotic cell ligand.
Although these studies cannot address whether Gas 6/PS are participating partners
for apoptotic cell recognition in vivo, they do show that Mer plays a critical role
in apoptotic cell recognition and phagocytosis (145).
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Integrins
The integrins, a group of cell surface adhesion proteins, play an important role in
many cellular processes including proliferation, adhesion, motility, migration, invasion, and survival (162). Integrins also participate in the engulfment of apoptotic
cells by phagocytes. The integrin family is composed of 15 α and 8 β subunits
paired in over 20 heterodimeric combinations. The majority of the integrins recognize the tripeptide sequence Arg-Gly-Asp (RGD), although some also bind other
sequences (163, 164). Phagocytosis of apoptotic neutrophils by human monocytederived macrophages is mediated by α 5β 3 (CD51/CD61; vitronectin) (165, 166)
and is inhibited with RGD and antibodies to vitronectin (165). Vitronectinmediated phagocytosis requires thrombospondin (TSP) (167, 168), which acts
as a bridge between the apoptotic cell and the phagocyte. Interestingly, uptake is
also inhibited by CD36 antibodies (169), suggesting that α 5β 3-mediated phagocytosis requires the simultaneous binding of PS (apoptotic cell)/TSP complexes to
both α 5β 3 and CD36 on the phagocyte surface. The physiologic significance of this
apparent dual receptor recognition system has not been studied. The specificity
and cooperativity between integrins and apoptotic cells is also dependent on the
type of phagocyte and the type of target. For example, α 5β 5/CD36 on immature
dendritic cells bind apoptotic cells through TSP. However, unlike macrophage uptake via α 5β 3, uptake through the α 5β 5 pathway plays a role in the presentation of
antigens to cytotoxic T cells (170). The nature of the apoptotic cell also determines
the type of integrin required for its recognition. Apoptotic endothelial cell uptake,
for example, can be mediated by a phagocyte α ?β 1 receptor that does not appear
to involve PS on the endothelial cell surface (171).
Although thrombospondin appears to be the principal protein involved in integrin/CD36-mediated uptake, recent data indicate that other secretory proteins can
also fulfill the bridging function. Milk fat globule-EGF-factor 8 (MFG-E8) is
produced by thioglycollate-elicited macrophages and binds to PE and PS on apoptotic cell surfaces (172). MFG-E8/aminophospholipid complexes are recognized
by α 5β 3 integrins on phagocytes through its RGD motif. Because MFG-E8 is a
constituent of milk fat globules produced in mammary glands (173), it might play
a role in recognizing apoptotic mammary epithelial cells during involution (174)
via a mechanism likely to preclude any inflammatory responses.
CD14
CD14 is a 55-kDa protein found in plasma (sCD14) and bound to cells through
GPI anchors (mCD14). The membrane-bound form, first identified as the bacterial lipopolysaccharide (LPS) receptor (175), is expressed on monocytes and
macrophages (176). Binding of the lipid A portion of LPS to CD14 activates phagocytes and initiates the septic shock syndrome (177). CD14 also binds PS (178) and
mediates the recognition and phagocytosis of apoptotic cells (179), implying that
PS is its apoptotic cell ligand. Indeed, release of the GPI anchor on phagocytes
with phospholipase C significantly inhibited the uptake of PS-expressing targets
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(180). However, it is now known that the binding of CD14 to apoptotic cells occurs
through ICAM-3 (intercellular adhesion molecule 3, CD50) (181), a glycosylated
member of the IgG superfamily that is constitutively expressed in leukocytes (182).
ICAM-3 is composed of five extracellular Ig-like domains and binds the leukocyte
integrins LFA-1 (183, 184) and α dβ 2 (185). Although the functional consequences
of ICAM-3/α dβ 2 are not known, ICAM-3/LFA-1 interactions participate in the initiation of immune responses (186). Upon apoptosis, however, ICAM-3 becomes
modified such that it no longer binds LFA-1. Instead, its specificity switches to
CD14 thereby enabling CD14 expressing phagocytes to bind the apoptotic cell
(181).
Complement Receptors
Human phagocytes express complement receptors that participate in opsonization
of invading microorganisms. Similar to activation by these targets (187), apoptotic
cells also activate complement through deposition of C3b with its concomitant
cleavage to iC3b (188, 189). Because PS binds complement (190, 121), it is reasonable to assume that the apoptotic ligand that binds C3b is PS. The target cell/iC3b
complex is then recognized by complement receptors CR3 (CD11b/CD18) and
CR4 (CD11c/CD18) on the phagocyte (191, 192). Interestingly, while uptake
through complement receptors would be expected to induce inflammatory responses, CR3-mediated uptake of apoptotic cells follows a noninflammatory pathway (193).
Calreticulin (CD93)
The collectins are a family of proteins that play a role in innate immunity (194).
Members of this group include mannose-binding lectin (MBL), surfactant proteins A (SP-A) and D (SP-D), and C1q, a member of the classical complement
pathway. These proteins have been implicated as intermediaries in the recognition
of apoptotic cells by phagocytes through ligation of its receptor, CD93 (cC1qr;
calreticulin), to CD91 (α2-macroglobulin receptor) on the phagocyte (195, 196).
The importance of the C1q-mediated recognition pathway is shown by the fact
that, similar to Mer knockouts (145), dominant-negative C1q knockouts exhibit a
phenotype characterized by an autoimmune-like glomeronephritis together with
the accumulation of multiple apoptotic bodies (146, 197).
β 2-Glycoprotein I (β2GP1) Receptor
β2GP1 (apoplipoprotein H) is a 50-kDa plasma glycoprotein that binds negatively
charged phospholipids (198) through its C terminal. Analysis of PS vesicles recovered from mice after intravenous injection showed that the vesicles preferentially
bound β2GP1 and that the clearance rate of PS liposomes was significantly prolonged in β2GP1-deficient mice (199). This suggested that β2GP1 could play a
role in the clearance of PS-expressing apoptotic cells. Indeed, apoptotic cells bound
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β2GP1 and enhanced their recognition and uptake by phagocytes (200, 201). Biochemical analysis revealed that β2GP1/phagocyte interactions are electrostatic
in nature and that protein glycosylation is not critical for phagocyte recognition. Macrophage binding experiments showed that β2GP1 bound to macrophages
only after first binding to its lipid ligand, suggesting that a conformationally altered structure (202, 203) binds to a specific phagocyte receptor that has not been
identified.
Scavenger Receptor Superfamily
The structurally diverse scavenger receptor superfamily constitutes a group of cell
surface proteins that exhibit a diverse array of unique and overlapping specificities. They participate in the binding and uptake of several endogenous macromolecules including anionic ligands [aminophospholipids, oxidized proteins and
lipids, and AGE, lipoproteins (LDL and HDL)] and microorganisms. Several of
these receptors are involved in the binding and uptake of apoptotic cells through
a direct interaction with PS, through bridging proteins bound to PS, or through
other unidentified ligands.
SCAVENGER RECEPTOR CLASS A (SRA) SRA is a homotrimeric group of proteins
in which each subunit has an apparent molecular mass of 90 kDa (204). They are
further grouped into type I, II, and III that differ in mRNA splicing (205). These
proteins are constitutively expressed in many cell types including Kupffer cells,
resident macrophages (206, 207), and endothelial cells (206). SRA-null mice are
less susceptible to atherosclerosis (208), have lowered resistance to infection, and
have an increased vulnerability to septic shock than their wild-type counterparts
(209). In vitro experiments using macrophages from these mice indicated that SRA
participates in the binding of acetylated-LDL, oxidized-LDL, AGE-BSA, bacteria, and apoptotic cells (210, 211). Interestingly, the binding of apoptotic cells to
phagocytes is inhibited by known SRA ligands and by SRA antibodies, but not
by PS vesicles (212). This suggests that the apoptotic cell ligand is likely not PS,
although the participation of specialized multicomponent domains that contain PS
cannot be excluded (98). Although binding to SRA can account for a substantial
fraction of apoptotic cell uptake in vitro, recent experiments in SRA-null mice
showed normal clearance of oxidized red cells (212) and apoptotic thymocytes
(213) in vivo, suggesting that multiple redundant recognition systems exist for the
clearance of apoptotic cells.
A unique member of the class A scavenger receptor family is MARCO (macrophage receptor with collagenous structure) (214, 215). This receptor is restricted to
macrophages in the splenic marginal zones and lymph nodes (216). The strategic
location of these receptors in the spleen suggests that they play a role in bacterial
clearance.
SCAVENGER RECEPTOR CLASS B (SRB) SRB-I and SRB-II are two splice variants
of the CD36-related family of receptors (217, 218) that mediate the selective
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MEMBRANE AMINOPHOSPHOLIPID ASYMMETRY
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uptake of high-density lipoprotein (HDL) cholesteryl esters (217) and the efflux
of free cholesterol (219). The importance of SRB in HDL metabolism was shown
by the observation that overexpression in mice resulted in a dramatic reduction in
plasma HDL-cholesterol levels together with an increased secretion of cholesterol
into the bile (220). Conversely, CD36-null animals exhibited high levels of HDLcholesterol (221). Studies using the human monocytic cell line THP-1 showed that
SR-BI is predominantly associated with caveolae (222), the site of free cholesterol
efflux (223). SRB, together with thrombospondin and vitronectin, also plays a role
in apoptotic cell uptake (169, 224) (see above). This is particularly interesting because both the vitronectin receptor and SRB are associated in caveolae (225, 226).
It should be noted, however, that CD36 also binds PS directly (227), suggesting
that it can also directly mediate the binding of PS-expressing cells independent of
the integrins/TSP pathway.
SCAVENGER RECEPTOR CLASS C A member of the C-type scavenger receptor family was cloned from a human placental cDNA library. This receptor, the scavenger
receptor C-type lectin (SRCL), has a C-type lectin and a carbohydrate recognition
domain (228). SRCL is abundantly expressed in adult human tissues and binds
bacteria, implicating a role in host defense (228). An Ox-LDL receptor, LOX-1,
was cloned from bovine aortic endothelial cells and found to structurally resemble
the C-type lectin family (229). Like other scavenger receptors, LOX-1 mediates
the uptake of senescent and apoptotic cells (230). Uptake through this receptor is
inhibited with PS, polyanions, and OxLDL. Similar to SRCL, LOX-1 also plays a
role in adhesion and host defense (231).
SCAVENGER RECEPTOR CLASS D Human CD68 and its mouse counterpart, macrosialin, are glycosylated transmembrane proteins expressed in macrophages (232).
This receptor is a member of the lysosomal-associated membrane protein (LAMP)
family (233). It is predominantly associated with endosomes (234) but has also
been found on cell surfaces where it binds oxidized LDL and PS, indicating that
it could also play a role in apoptotic cell uptake (235–237). As mentioned for
LOX-1, PS-mediated recognition might involve intermediary bridging proteins.
SCAVENGER RECEPTOR CLASS E Scavenger receptor for endothelial cells (SREC)
was cloned from human umbilical vein endothelial cells (238). It binds Ox-LDL
and structurally resembles CED-1 (152). Cells expressing SREC bind acetylatedLDL with high affinity. Binding is inhibited with typical scavenger receptor ligands including Ox-LDL, malondialdehyde-LDL (MDA-LDL), maleyl-BSA, and
other polyanions. The exact in vivo function of this receptor is not known, although its similarity to CED-1 implicates a role in the recognition of apoptotic
cells.
PSOX Expression cloning using a macrophage cDNA library identified a receptor
that bound both PS and Ox-LDL, suggesting that it may also participate in apoptotic
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cell uptake (239). This protein is expressed in lipid-laden macrophages at the intima
of the atherosclerotic plaques (240), implicating a role in foam cell transformation.
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PS Receptor (PSr)
Unlike the diverse array of linker proteins that tether apoptotic cells to phagocytes
through PS, Fadok et al. (241) reported the cloning of a PS receptor candidate that
directly binds apoptotic cells without soluble linker intermediates. This receptor
is expressed in many cells types including macrophages, fibroblasts, and epithelial cells. Monoclonal IgM (mAb 217) raised against the receptor failed to bind
macrophages in the presence of PS vesicles, suggesting that the target antigen on
the cell surface recognizes PS. Furthermore, apoptotic cell uptake was inhibited by
mAb 217, PS and phosphoserine, which argues for the presence of a PS-specific
receptor. Antibody binding indicates that the PSr is not only present on professional phagocytes but also on many other cell types. The generalized distribution of
PSr suggests that it is a primitive receptor that mainly participates in the localized
recognition and phagocytosis of apoptotic cells by surrounding resident tissue.
Recent studies indicate that engulfment of apoptotic cells requires the engagement of the PS receptor with PS and the engagement of a second distinct receptor
with its specific tethering ligand on the apoptotic cell surface (242). Once a target is tethered, engagement of PSr induces macropinocytosis, which results in the
engulfment of the attached targets. Interestingly, growth factors can circumvent
the requirement for PSr engagement in inducing macropinocytosis. Whereas the
tether can be fulfilled by any of the direct or indirect receptor systems described
above, it seems that PS itself can provide both signals because viable cells devoid
of apoptotic cell-specific ligands are engulfed by phagocytosis after the insertion
of exogenous PS into the target cells plasma membrane (243).
IMPLICATIONS OF RECEPTOR REDUNDANCY
It is apparent that combinations of multiple receptors and ligands can independently trigger mechanisms for the removal of apoptotic cells by phagocytes. Although this multiplicity and redundancy can be shown in experimental in vitro
systems, it is likely that selective receptor/ligand pairs operate in specific tissues.
For example, recognition via the C1q pathway appears to be operative largely in
the kidneys (146), whereas uptake through Gas 6/Mer is operative in the thymus
(145). Receptor expression is also restricted to the type of engulfing phagocyte.
MARCO (SRA) is restricted to macrophages in the splenic marginal zones and
lymph nodes implying a role specific to bacterial clearance (214, 216). Dendritic
cells and macrophages recognize apoptotic cells through α 5β 5 and α 5β 3, respectively. This suggests that α 5β 5-mediated uptake plays a role in antigen presentation
of apoptotic cell moieties and has a role in maintaining and reinforcing tolerance
to autologous antigens (170, 244).
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Whatever the specific receptor-ligand partner responsible for the uptake of a particular apoptotic cell, the most critical ligand seems to be PS because its blockade
with annexin V abrogates recognition (245–247). Whereas PS serves as a unique
and ubiquitous target on apoptotic cells, the specificity of binding for phagocytes
is most likely determined by the ability of one or more bridging proteins to bind its
specific receptor. Thus bridging protein-deficient C1q-null mice (146) cannot bind
PS to the CD93 phagocyte receptor, and receptor-deficient Mer-null mice (145)
cannot engage the PS/Gas 6 recognition pathway. The importance of CD93 (C1q)
and Mer (Gas 6) pathways in organ-specific apoptotic cell clearance can be seen
from data showing that null mice accumulate apoptotic bodies in the kidneys and
thymus (in corticosteroid-treated animals), respectively. However, even though
deficient clearance of apoptotic cells in these specific organs results in delayed
phagocytosis and likely persistent self-antigen exposure that lead to an SLE-like
autoimmune disease (144), deletion of either gene is not lethal. This suggests that
the burden to clear apoptotic cells, for the most part, is taken over by one or more
alternate ligand/receptor pathways.
Irrespective of the participatory ligand/receptor pair, one of the most important
features of apoptotic cell phagocytosis is that their clearance does not induce an
inflammatory response. In fact, phagocytes that engulf apoptotic cells become
actively antiinflammatory by releasing several suppressive mediators, including
TGF-β and prostaglandin E2 (PGE2) (248, 249), by a process that is apparently
triggered by ligation of the PSr (250). Indeed, even cells undergoing apoptosis in
areas of inflammation release antiinflammatory mediators that promote healing
(251).
CONCLUDING REMARKS
The appearance of aminophospholipids on the outer leaflet is clearly a pivotal event
in many cell functions. This is controlled by an intricate network of transporters that
selectively mobilize individual lipid species between membrane leaflets. Although
the functionality of the proteins responsible for generating a specific equilibrium
distribution of aminophospholipids between leaflets is known, their identity remains elusive. Contrary to the general supposition that the presence of PS in the
cells outer leaflet is a death signal, there are many examples where cells express
both PE and PS, yet they are not recognized by phagocytes. For example, PE
is transiently exposed during cell division at the cleavage furrow, and PS is expressed in viable lymphocytes, mast cells, and macrophages. These observations
suggest that other moieties, possibly in concert with PS, engender the cell with
specific phagocyte targets. Conceivably, the target could be provided by the appearance of a specific eat-me signal or the disappearance of a specific don’t-eat-me
signal (Figure 4). The eat-me signal could be provided by any combination of
oxidized surface moieties or possibly by a specific spatial reorganization of existing membrane components including PS. The latter possibility is particularly
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interesting because it provides the cell with a switching mechanism to transcend
from one physiologic state (nonapoptotic) to another (apoptotic). Indeed, in viable
cells, PS seems to be predominantly localized in lipid rafts (252) where, in spite of
its presence, it is likely not recognized by phagocytes. Induction of apoptosis could
trigger the release of the sequestered lipid from the rafts leading to its dispersal
throughout the membrane for recognition by the phagocyte. Such a hypothesis is
compatible with data showing that insertion of exogenously supplied PS into viable
cells renders them edible (243). Alternatively, viable cells could display specific
don’t-eat-me moieties that keep phagocytes at bay. In red cells, this signal could be
provided by specific antigens such as CD47, which are lost over its lifespan (253).
In leukocytes, on the other hand, homophilic binding of CD31 induces signaling
pathways that prevent engagement of the phagocyte to its target. These pathways
are disrupted upon apoptosis, negating the inhibitory effects of CD31 (254). The
signal could be provided by pro-apoptotic Bad. Similar to the distribution of PS,
Bad is localized in lipid rafts but relocates to other sites upon apoptosis (255). Thus,
apoptosis-dependent redistribution of Bad could operate as a don’t-eat-me signal
when clustered in lipid rafts, as an eat-me signal when dispersed to other sites, or
both.The nature of the ligand-receptor complexes engaged on apoptotic and senescent cells most likely determines the biological consequences of the phagocytic
event. Thus whereas certain pathways can potentiate immune tolerance, others
generate antiinflammatory responses important in the resolution of inflammation
and tissue repair. Understanding the relative contribution and interactions between
these different receptor-ligand pairs will be important in unraveling the molecular
events critical to these processes.
ACKNOWLEDGMENTS
This work was supported by the Elsa U. Pardee Foundation, the John Q. Gaines
Foundation, and by National Institutes of Health grant GM-64610.
The Annual Review of Physiology is online at http://physiol.annualreviews.org
LITERATURE CITED
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Expression of SR-PSOX, a novel cellsurface scavenger receptor for phosphatidylserine and oxidized LDL in human atherosclerotic lesions. Arterioscler.
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Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RB, Henson PM.
2000. A receptor for phosphatidylserinespecific clearance of apoptotic cells. Nature 405:85–90
Hoffmann PR, de Cathelineau AM, Ogden CA, Leverrier Y, Bratton DL, et al.
2001. Phosphatidylserine (PS) induces PS
receptor-mediated macropinocytosis and
promotes clearance of apoptotic cells. J.
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Fadok VA, de Cathelineau A, Daleke
DL, Henson PM, Bratton DL. 2001.
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Krahling S, Callahan MK, Williamson P,
Schlehel RA. 1999. Exposure of phosphatidylserine is a general feature in the
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2000. Phosphatidylserine-dependent phagocytosis of apoptotic glioma cells by
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Figure 1 Biosynthesis and trafficking of aminophospholipids. PS is synthesized in
the endoplasmic reticulum (ER) by base exchange with PC. PS is transported from the
ER to the mitochondrial-associated membrane (MAM), which then delivers it to the
outer membrane of the mitochondria (M) by an ATP-dependent process. The lipid is
then transported to the mitochondrial inner membrane where it is converted to PE by
PS decarboxylase I. PS is also transported from the ER to the Golgi (G) by an unknown
mechanism, where it is converted to PE by PS decarboxylase II. The mechanism by
which PE and PS is transported to the plasma membrane (PM) is not clear, but could
involve vesicular or protein-mediated mechanisms. Whereas intracellular lipid transfer
proteins (LTP) move aminophospholipids from the organelles to the PM inner leaflet
without perturbing asymmetry, the introduction of aminophospholipid to the outer
leaflet by extracellular LTP (HDL and LDL) distorts the cells equilibrium distribution.
Transport via a vesicular mechanism can create a disturbance in aminophospholipid
asymmetry by two pathways. Because aminophospholipid synthesis occurs in the lumen of the organelles, fusion of the vesicle with the plasma membrane results in a
reorientation of the vesicles inner leaflet to the plasma membranes outer leaflet. Moreover, fusion of the vesicle with the PM involves transient hexagonal phases at the site
of fusion that itself permits interleaflet mixing.
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Figure 2 Aminophospholipid transport activities in mammalian cells. Membrane lipid asymmetry is maintained by the concerted action
of different transporters that, depending on the physiological state of the cell, remain either active (green arrows) or dormant (red arrows).
The drug resistance proteins MDR and Mrp1 are responsible for an ATP-dependent transport of PC from the inner to the outer leaflet. In
contrast, the aminophospholipids PS and PE are transported to the inner leaflet by a Mg2+-ATPase. Cells also contain an ABC1 transporter
(CED-7 in C. elegans) that actively shuttles PS to the outer leaflet. Although not known, this activity is likely dormant in resting/viable cells.
In apoptotic cells, however, it becomes activated together with the scramblase, a non-specific transporter, that facilitates free intermixing
between bilayers. Inset shows the typical equilibrium distribution in normal red blood cells [adapted from (10)].
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Figure 3 Apoptotic cell surface ligands and phagocyte receptors.
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Figure 4 Hypothetical models for the recognition of apoptotic cells by phagocytes. (A) Viable cells utilize different mechanisms to render
themselves inedible to phagocytes. This is accomplished by either restricting specific eat-me signals to sites inaccessible to the phagocytes
or by the expression of don’t touch (eat) me signals at the cell surface that keep the phagocytes at bay. Upon apoptosis, they become edible
by displaying novel ligands, removing inhibitory ligands, or by reorganizing the lateral and/or transverse distribution of PS and/or other
ligands. (B) PS relocates from the inner to outer leaflet and becomes a recognition ligand (sequestered lipid rafts do not play a role in this
model). (C ) A new ligand (specific carbohydrate residues or proteolytically cleaved membrane proteins) appear at the cell surface. (D, E )
Upon apoptosis, PS or other ligands are mobilized out of the lipid rafts to sites accessible by phagocytes. (F ) An inhibitory don’t touch (eat)
me signal is lost allowing phagocytes access to various ligands.
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Annual Reviews
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Annual Review of Physiology,
Volume 65, 2003
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CONTENTS
Frontispiece—Jean D. Wilson
xiv
PERSPECTIVES, Joseph F. Hoffman, Editor
A Double Life: Academic Physician and Androgen Physiologist,
Jean D. Wilson
1
CARDIOVASCULAR PHYSIOLOGY, Jeffrey Robbins, Section Editor
Lipid Receptors in Cardiovascular Development, Nick Osborne
and Didier Y.R. Stainier
Cardiac Hypertrophy: The Good, the Bad, and the Ugly, N. Frey
and E.N. Olson
Stress-Activated Cytokines and the Heart: From Adaptation to
Maladaptation, Douglas L. Mann
23
45
81
CELL PHYSIOLOGY, Paul De Weer, Section Editor
Cell Biology of Acid Secretion by the Parietal Cell, Xuebiao Yao
and John G. Forte
Permeation and Selectivity in Calcium Channels, William A. Sather
and Edwin W. McCleskey
Processive and Nonprocessive Models of Kinesin Movement,
Sharyn A. Endow and Douglas S. Barker
103
133
161
COMPARATIVE PHYSIOLOGY, George N. Somero, Section Editor
Origin and Consequences of Mitochondrial Variation in Vertebrate
Muscle, Christopher D. Moyes and David A. Hood
Functional Genomics and the Comparative Physiology of Hypoxia,
Frank L. Powell
Application of Microarray Technology in Environmental
and Comparative Physiology, Andrew Y. Gracey and
Andrew R. Cossins
177
203
231
ENDOCRINOLOGY, Bert W. O’Malley, Section Editor
Nuclear Receptors and the Control of Metabolism,
Gordon A. Francis, Elisabeth Fayard, Frédéric Picard, and
Johan Auwerx
261
vii
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viii
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Insulin Receptor Knockout Mice, Tadahiro Kitamura, C. Ronald Kahn,
and Domenico Accili
The Physiology of Cellular Liporegulation, Roger H. Unger
313
333
GASTROINTESTINAL PHYSIOLOGY, John Williams, Section Editor
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The Gastric Biology of Helicobacter pylori, George Sachs,
David L. Weeks, Klaus Melchers, and David R. Scott
Physiology of Gastric Enterochromaffin-Like Cells, Christian Prinz,
Robert Zanner, and Manfred Gratzl
Insights into the Regulation of Gastric Acid Secretion Through Analysis of
Genetically Engineered Mice, Linda C. Samuelson and Karen L. Hinkle
349
371
383
NEUROPHYSIOLOGY, Richard Aldrich, Section Editor
In Vivo NMR Studies of the Glutamate Neurotransmitter Flux
and Neuroenergetics: Implications for Brain Function,
Douglas L. Rothman, Kevin L. Behar, Fahmeed Hyder,
and Robert G. Shulman
401
Transducing Touch in Caenorhabditis elegans, Miriam B. Goodman
and Erich M. Schwarz
429
Hyperpolarization-Activated Cation Currents: From Molecules
to Physiological Function, Richard B. Robinson and
Steven A. Siegelbaum
453
RENAL AND ELECTROLYTE PHYSIOLOGY, Steven C. Hebert, Section Editor
Macula Densa Cell Signaling, P. Darwin Bell, Jean Yves Lapointe,
and János Peti-Peterdi
Paracrine Factors in Tubuloglomerular Feedback: Adenosine, ATP,
and Nitric Oxide, Jürgen Schnermann and David Z. Levine
Regulation of Na/Pi Transporter in the Proximal Tubule,
Heini Murer, Nati Hernando, Ian Forster, and Jürg Biber
Mammalian Urea Transporters, Jeff M. Sands
Terminal Differentiation of Intercalated Cells: The Role of Hensin,
Qais Al-Awqati
481
501
531
543
567
RESPIRATORY PHYSIOLOGY, Carole R. Mendelson, Section Editor
Current Status of Gene Therapy for Inherited Lung Diseases,
Ryan R. Driskell and John F. Engelhardt
The Role of Exogenous Surfactant in the Treatment of Acute Lung
Injury, James F. Lewis and Ruud Veldhuizen
Second Messenger Pathways in Pulmonary Host Defense,
Martha M. Monick and Gary W. Hunninghake
585
613
643
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Annual Reviews
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CONTENTS
Alveolar Type I Cells: Molecular Phenotype and Development,
Mary C. Williams
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SPECIAL TOPIC: LIPID RECEPTOR PROCESSES, Donald W. Hilgemann,
Special Topic Editor
Getting Ready for the Decade of the Lipids, Donald W. Hilgemann
Aminophospholipid Asymmetry: A Matter of Life and Death,
Krishnakumar Balasubramanian and Alan J. Schroit
Regulation of TRP Channels Via Lipid Second Messengers,
Roger C. Hardie
Phosphoinositide Regulation of the Actin Cytoskeleton,
Helen L. Yin and Paul A. Janmey
Dynamics of Phosphoinositides in Membrane Retrieval and
Insertion, Michael P. Czech
SPECIAL TOPIC: MEMBRANE PROTEIN STRUCTURE, H. Ronald Kaback,
Special Topic Editor
Structure and Mechanism of Na,K-ATPase: Functional Sites
and Their Interactions, Peter L. Jorgensen, Kjell O. Håkansson,
and Steven J. Karlish
G Protein-Coupled Receptor Rhodopsin: A Prospectus,
Slawomir Filipek, Ronald E. Stenkamp, David C. Teller, and
Krzysztof Palczewski
ix
669
697
701
735
761
791
817
851
INDEXES
Subject Index
Cumulative Index of Contributing Authors, Volumes 61–65
Cumulative Index of Chapter Titles, Volumes 61–65
ERRATA
An online log of corrections to Annual Review of Physiology chapters
may be found at http://physiol.annualreviews.org/errata.shtml
881
921
925