Appetizing rancidity of apoptotic cells for macrophages: oxidation, externalization, and recognition of phosphatidylserine
V. E. Kagan,1
G. G. Borisenko,1
B. F. Serinkan,1
Y. Y. Tyurina,1
V. A. Tyurin,1
J. Jiang,1
S. X. Liu,1
A. A. Shvedova,2
J. P. Fabisiak,1
W. Uthaisang,3 and
B. Fadeel3
1Department of Environmental and Occupational
Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260;
2Health Effects Laboratory Division, Pathology and
Physiology Research Branch, National Institute for Occupational Safety and
Health, Morgantown, West Virginia 26505; and 3Division
of Toxicology, Institute of Environmental Medicine, Karolinska Institutet,
17177 Stockholm, Sweden
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ABSTRACT
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Programmed cell death (apoptosis) functions as a mechanism to eliminate
unwanted or irreparably damaged cells ultimately leading to their orderly
phagocytosis in the absence of calamitous inflammatory responses. Recent
studies have demonstrated that the generation of free radical intermediates
and subsequent oxidative stress are implicated as part of the apoptotic
execution process. Oxidative stress may simply be an unavoidable yet trivial
byproduct of the apoptotic machinery; alternatively, intermediates or products
of oxidative stress may act as essential signals for the execution of the
apoptotic program. This review is focused on the specific role of oxidative
stress in apoptotic signaling, which is realized via
phosphatidylserine-dependent pathways leading to recognition of apoptotic
cells and their effective clearance. In particular, the mechanisms involved in
selective phosphatidylserine oxidation in the plasma membrane during apoptosis
and its association with disturbances of phospholipid asymmetry leading to
phosphatidylserine externalization and recognition by macrophage receptors are
at the center of our discussion. The putative importance of this oxidative
phosphatidylserine signaling in lung physiology and disease are also
discussed.
phosphatidylserine oxidation and externalization; apoptosis; phagocytosis; cytochrome c; macrophage receptor
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APOPTOSIS AND PHAGOCYTOSIS OF CELL CORPSES IN THE LUNG: ROLE OF THE
FAS SYSTEM
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Apoptosis arises from the active initiation and propagation of a series of
highly orchestrated specific biochemical events leading to the demise of the
cell (64). Functional
consequences of this process are the elimination of specific cells within a
population when they are damaged or no longer required for function. For
example, apoptosis is an important determinant for tissue morphogenesis during
development (32) and also
plays a role in the elimination of immune effector cells during lymphocyte
selection (20) and the
resolution of inflammation and fibrosis
(132). Failure of efficient
apoptosis can allow the progression of neoplastic disease, permitting the
persistence and multiplication of cells suffering significant genotoxic
damage. On the other hand, extensive cell loss via apoptosis with concomitant
organ dysfunction can arise from a variety of tissue insults including
oxidative stress as documented in brain
(98), liver
(110), and other organs (for
review see Refs. 125,
126).
In the lung, as in other tissues, apoptosis is of paramount importance as a
regulator of cell homeostasis and also serves as a potential mediator of
tissue injury (46). A number
of lung diseases, such as idiopathic pulmonary fibrosis
(85,
151), acute respiratory
distress syndrome (ARDS) (62),
chronic obstructive lung disease
(137), and bacterial
pneumonia (78), are
characterized by extensive apoptosis within the alveolar epithelium. In
addition, multiple pneumotoxins such as silica
(18,
88), bleomycin
(52), asbestos
(1), and paraquat
(13) can induce apoptosis of
various cells. Apoptosis plays a fundamental role in the distal airway
remodeling during the transition from the canalicular to saccular stages
during lung organogenesis
(82). The development of an
effective alveolar-capillary interface requires extensive cell remodeling and
is documented by extensive apoptosis of both epithelial and interstitial cells
in both the gestational (133)
and postnatal (134) periods.
Although much work has focused on the mechanisms for initiation of apoptosis,
little information exists regarding the downstream events involved in
signaling mechanisms that mark apoptotic cells with "eat-me"
signals to govern the fate of these cell corpses or how the lung responds to
them.
The Fas/Fas ligand (FasL) system has been implicated in apoptosis within
the lung. It appears that many cells within the lung, including the alveolar
epithelium, constitutively express Fas
(45,
89). Activation of Fas by
intranasal administration of an agonistic antibody produced marked alveolar
type II epithelial cell apoptosis and pulmonary inflammation in normal but not
in Fas-deficient (lpr) mice
(99). In addition, soluble
FasL was found in a bioactive form in the bronchoalveolar lavage fluid in
patients during the development of acute respiratory distress syndrome (ARDS)
(100). At least one source of
this FasL has been identified as neutrophils
(138), whose accumulation
within the air spaces represents a hallmark of ARDS. Similarly, FasL was found
in bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis
but not controls (86).
Pulmonary fibrosis can also be mimicked in mice by administration of FasL
(53). Importantly,
bleomycin-induced lung injury, a well-established model of fibrosis, was also
attenuated in lpr and gld mice, which are deficient in Fas
and FasL, respectively (84).
Lastly, the periods of lung organogensis characterized by high levels of
apoptosis are also marked by increases in tissue expression of FasL
(23), an observation that
strongly suggests that the Fas/FasL system serves to regulate developmental
lung remodeling as well as tissue injury. Thus apoptosis within the lung
contributes to both acute and chronic lung injury, as well as lung fetal and
neonatal development.
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SELECTIVE PHOSPHOLIPID PEROXIDATION DURING OXIDANT- AND
NONOXIDANT-INDUCED APOPTOSIS
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Oxidative stress is one of the most common triggers of apoptosis. In
addition, apoptosis is frequently accompanied by the generation of reactive
oxygen species (ROS), resulting in part from cytochrome c (cyt
c) departure from mitochondria and concomitant disruption of electron
transport with enhanced generation of one-electron-reduced species of
molecular oxygen within the cell
(10,
11,
15,
34,
36,
67,
75,
112,
121,
130). ROS represent
attractive candidates for final common mediators of apoptosis, yet a specific
role for ROS in the execution or resolution of the apoptotic program has not
been established. Although effects of oxidative stress on the apoptotic
machinery, including the caspases
(28,
56), and mitochondrial
proteins forming the permeability transition pore
(14,
21,
81) have been described,
information on peroxidation of phospholipids and in particular on selective
oxidation of their specific classes is scarce. This is mainly due to the fact
that quantitative assays for oxidation of different classes of phospholipids
are not readily available, which, in turn, is due in large part to a highly
effective system of remodeling and repair of oxidatively modified
phospholipids (106) that
interferes with their accurate measurement.
To characterize phospholipid oxidation during oxidative stress-induced
apoptosis, we have developed a protocol based on metabolic labeling of
cellular phospholipids with a natural oxidation-sensitive and highly
fluorescent fatty acid, cis-parinaric acid (PnA). This reagent has
been extensively used in its free (nonesterified) form for structural
measurements in membranes as well as in assays of oxidative stress in simple
model systems (63,
87). Metabolic labeling yields
cells containing the major phospholipid classes [phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol,
diphosphatidylgycerol, and sphingomyelin (SPH)] fluorescently labeled with PnA
and extremely low intracellular concentrations of free PnA
(124). Because free PnA is
not available for phospholipid repair, resolution of major phospholipid
classes by fluorescence HPLC can be used to quantify their oxidative damage
(as a decreased content of fluorescent PnA residues in respective phospholipid
classes). The level of PnA labeling of endogenous phospholipids
(
13mol%) is low enough to have minimal effects on cell viability
and functions yet sufficient to permit quantitative detection of oxidative
stress (124). Importantly,
the PnA-based assay can identify the selectivity of phospholipid oxidation on
the basis of their polar head groups, and it is obviously independent of the
fatty acid composition of phospholipids
(30,
124).
Using metabolic labeling of phospholipids with PnA, we were able to
establish that different phospholipids undergo nonrandom peroxidation during
oxidant-induced apoptosis in a number of different cell lines
(Table 1). In particular,
preferential oxidation of PS was typical of apoptosis induced by a number of
oxidants, such as organic hydroperoxides, paraquat, and azo-initiators of
peroxyl radicals. Most notably, there was a strong correlation between PS
peroxidation and its externalization during apoptosis (see below). We observed
that in all cases when enhanced PS peroxidation occurred, PS externalization
took place as well and vice versa; the lack or inhibition of preferential PS
peroxidation during apoptosis was accompanied by the lack of PS
externalization. The fundamental association of PS oxidation with apoptosis
was strengthened by experiments in which we used a vitamin E homolog,
2,2,5,7,8-pentamethyl-6-hydroxy-chromane (PMC). Here, we employed the
lipophilic azo-initiator of radicals
2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN) to generate
membrane-confined oxidative stress and induce apoptosis in HL-60 cells
(36). As an effective radical
scavenger, PMC was able to completely protect all phospholipids against
oxidation with the remarkable exception of PS. Furthermore, PMC failed to
protect HL-60 cells against apoptosis following AMVN
(Table 1).
The temporal sequence of PS oxidation and externalization on the cell
surface is compatible with a causal link between these two events. Indeed, if
this is the case, PS oxidation should occur within the plasma membrane where
PS translocation events during apoptosis are known to occur. To address this
issue, we performed subcellular fractionation experiments in PnA-labeled cells
challenged with tert-butyl hydroperoxide (t-BuOOH). We
documented that t-BuOOH induced apoptosis and prominent PS oxidation
in cells and different organelles. Most importantly, the highest rate of PS
oxidation was detected in the plasma membrane compared with other organelles
such as mitochondria, nuclei, lysosomes, and microsomes
(Fig. 1). The causative link
between PS oxidation and apoptosis was further supported by our experiments
with DMSO-differentiated HL-60 cells showing inducible NADPH oxidase activity
(4). Activation of the NADPH
oxidase by phorbol 12-myristate 13-acetate (PMA) or zymosan caused massive
production of superoxide- and hydrogen peroxide
(H2O2)-associated with oxidation of essentially all
major phospholipids such as PC, PE, and PS. Exposure to PMA also induced
apoptosis in these cells as evidenced by PS externalization on the cell
surface, caspase activation, chromatin condensation, and nuclear and DNA
fragmentation. All these effects were suppressed by inhibitors of the NADPH
oxidase, diphenylene iodonium (DPI), or staurosporine. Remarkably, the
pancaspase inhibitor Z-Val-Ala-Asp-fluoromethylketone was able to
significantly protect PS against PMA- (or zymosan-) induced oxidation, whereas
oxidation of other phospholipids was insensitive to the inhibitor.

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Fig. 1. Rate of oxidation of cis-parinaric acid (PnA)-labeled
phosphatidylserine (PS) during apoptosis induced by tert-butyl
hydroperoxide (t-BuOOH) in HL-60 cells. PnA-labeled HL-60 cells (2
x 106) were incubated in the presence of t-BuOOH
(150 µM) for 20 min at 37°C. Lipid oxidation was terminated by addition
of 10 µM butylated hydroxytoluene (BHT), and cells were incubated an
additional 40 min. Lipids were extracted and resolved by HPLC. The rate of
t-BuOOH-induced oxidation of PnA-PS was calculated as the difference
between the specific PnA content of PS in t-BuOOH treated cells and
that in control cells divided by 20 min. PM, plasma membranes; MS, microsomes;
LS, lysosomes; N, nuclei; MT, mitochondria. *P < 0.05
vs. MS, LS, N, or MT, n = 6.
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The above data indicate that PS oxidation may be largely associated with
the execution of apoptotic program. This association, however, is obscured
during oxidant-induced apoptosis due to high background nonspecific oxidation.
Therefore, we have used several models of nonoxidant-induced apoptosis in
which we studied phospholipid peroxidation and revealed that PS was again
selectively oxidized compared with other more abundant phospholipids
(Table 1). In particular,
decreased levels of Bcl-2 protein expression achieved via manipulating the
level of bcl-2 mRNA translation in the squamous nonsmall cell lung
cancer line NCI-H226 by use of a synthetic antisense-bcl-2
oligonucleotide resulted in selective oxidation of PS in the subpopulation of
cells with externalized PS. No significant differences in oxidation of
cis-PnA-labeled PE and PC in cells were found after treatment with
nonsense or antisense-bcl-2 oligonucleotides
(80). Similarly, by exposing
HL-60 cells to staurosporine, a protein kinase inhibitor without direct
prooxidant activity, we were able to induce apoptosis in HL-60 cells without
triggering confounding nonspecific oxidation reactions. PS underwent
preferential oxidation at an early stage of apoptosis, whereas the most
abundant phospholipid, PC, and GSH, the most abundant cytosolic thiol,
remained unoxidized (97).
Finally, Fas triggering of Jurkat T lymphocytes resulted in oxidative stress
with specific PS oxidation and externalization, whereas Raji cells, which are
defective for PS exposure, did not undergo PS oxidation
(74)
(Fig. 2). Expectedly, anti-Fas
triggering of PS oxidation/externalization was accompained by phagocytosis of
apoptotic Jurkat cells (but not Raji cells) by J774A.1 macrophages
(Fig. 3).

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Fig. 2. Cell type-specific externalization of PS. Jurkat cells (A) and
Raji cells (B) were triggered to undergo apoptosis in response to Fas
ligation for 4 h and subsequently costained with Hoechst 33258 (blue) and
annexin V (green) for visualization of nuclear condensation and PS exposure,
respectively.
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Fig. 3. Typical fluorescence photomicrographs showing phagocytosis of target cells
by J774A.1 macrophages. A, B: phagocytosis of Jurkat cells
(A, control; B, apoptotic cells after stimulation with 250
ng/ml of anti-Fas agonistic antibody, 2 h). C, D: nonapoptotic HL-60
cells [C, control; D, after enrichment with nonoxidated PS
(PS)/oxidated PS (PSox) liposomes]. Target cells were fluorescently labeled
with Cell Tracker Orange (red); macrophages were fluorescently labeled with
Hoechst 33342 (blue).
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Because several recent reports suggest that Fas/FasL are implicated in
apoptosis within the lung (discussed above), we further utilized the human
pulmonary adenocarcinoma A549 cell line as a convenient model for alveolar
epithelial cells to evaluate the effect of Fas-triggered apoptosis on membrane
phospholipid oxidation. We were able to demonstrate that induction of
apoptosis in interferon-
-pretreated A549 cells by anti-Fas monoclonal
antibody caused specific PS oxidation along with its externalization
(Table 1).
In sum, three important features of PS oxidation during apoptosis have been
established during the course of our studies. First, the preferential
oxidation of PS is observed only in intact living cells undergoing apoptosis
and not in cell-free liposome preparations incubated with oxidants. Second, PS
oxidation occurs early during execution of the apoptotic program and precedes
the appearance of such hallmarks of apoptosis as DNA fragmentation and, most
importantly, PS externalization. Finally, PS peroxidation is blocked in cells
overexpressing antiapoptotic gene products such as Bcl-2 and is sensitive to
pancaspase inhibitors (4,
33,
34,
74,
77,
92,
142,
158). Overall, our findings
clearly indicate that execution of the apoptotic program involves initiation
of oxidative stress that targets specific phospholipids in the plasma membrane
including PS. These observations raise two important questions: 1)
What are the catalytic mechanisms involved in PS oxidation during apoptosis?
and 2) What are the consequences of PS oxidation for the execution of
apoptosis?
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MECHANISM OF PS OXIDATION DURING APOPTOSIS: EVIDENCE FOR A ROLE OF
CYT C
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Selective PS oxidation suggests that there may be specific catalytic
mechanism(s) responsible for the predominant oxidation of this particular
class of phospholipids (Fig.
4). One possible mechanism relies on the involvement of cyt
c released from mitochondria into the cytosol during apoptosis. Cyt
c release, an early and common marker of apoptosis, is involved in
the formation of the so-called apoptosome complex and the subsequent
activation of caspases downstream of mitochondria
(70). This proapoptotic role
of cyt c seems to be redox independent and does not require the
presence of the heme moiety
(59,
79). However, a significant
part of cyt c is not released as an apoprotein but rather contains
heme with its redox-active iron
(140,
141). Moreover, as cyt
c is a basic protein (pI 10.3) that is positively charged at neutral
pH (90), it may be predisposed
to interact electrostatically with negatively charged phospholipids such as PS
(103,
149). In fact, it has been
demonstrated that cyt c effectively binds to negatively charged
PS-containing membranes (22).
Electrostatic interaction of cyt c with negatively charged sites of
membranes induces disruption of the Met80-Fe(heme) coordination
bond and partial unfolding of the protein globule, thus facilitating
orientation of the heme moiety along the membrane surface
(113). Disturbance of
Met80-Fe(heme) coordination renders Fe more catalytically redox
active, while unfolding of the protein positions its heme catalytic site
closer to phospholipid targets. Oxidized cyt c is less stable, and
the energy of its unfolding is lower than for the reduced form
(109), suggesting that PS in
the inner leaflet of the plasma membrane may preferentially interact with the
oxidized form of cyt c. Importantly, departure of cyt c from
mitochondria is accompanied by a massive production of
H2O2, which may promote oxidation of cyt c heme
(113). Thus one may speculate
that, once released from mitochondria, cyt c becomes oxidized, binds
to PS-rich lipid rafts on the inner surface of plasma membrane, and unfolds to
expose its redox-active heme-iron moiety to phospholipids, particularly to PS,
the electrostatically most attractive phospholipid species. As a result, PS
may be more susceptible to cyt c-catalyzed oxidation than other
phospholipids during apoptosis (Fig.
5).

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Fig. 4. Pathways involved in selective oxidation of PS during apoptosis: catalytic
role for cytochrome c (cyt c). In cells committed to
programmed death after exposure to proapoptotic signals, mitochondrial
permeability transition and activation of pores take place. Two functionally
important proteins, the caspase coactivator cyt c and the
caspase-independent death effector apoptosis-inducing factor (AIF), are
released from mitochondria into the cytosol. Disruption of mitochondrial
electron transport is accompanied by production of superoxide; the latter
dismutates to H2O2, which can readily diffuse into the
cytosol. Electrostatic interactions of positively charged cyt c with
negatively charged PS may facilitate cyt c-catalyzed generation of
highly reactive oxidants (e.g., oxo-ferryl species) in close vicinity of PS,
thus providing for its selective peroxidation. PSox can contribute to
poisoning of aminophospholipid translocase (APT), thus providing for its own
externalization along with PS. Alternatively, PSox may undergo effective
transmembrane diffusion. Finally, PSox and PS synergistically interact as an
"eat-me" signal for phagocytic recognition of apoptotic cells.
This effect of PSox may be realized through its interaction with one of the
known macrophage receptors, such as PS receptor (PSR), CD36, LOX-1, or with an
as-yet unidentified PSox receptor (PSoxR). Finally, antioxidants by blocking
PS oxidizing pathway(s) can affect PS externalization and subsequent clearance
of apoptotic cells. ROS, reactive oxygen species; MPT, mitochondrial
permeability transition; LA/DHLA, -lipoic/dihydrolipoic acid.
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Fig. 5. Cyt c as a catalyst for selective PS oxidation during apoptosis:
release of cyt c from mitochondria into the cytosol is one of the
early and common events in apoptosis. In the cytosol, cyt c
participates in the activation of the caspase cascade. As a basic protein (pI
10.3), it is positively charged at neutral pH and can electrostatically
interact with negatively charged phospholipids such as PS. As a result, redox
(prooxidant) catalytic activity of heme-containing cyt c may be
directed toward selective PS oxidation. Production of superoxide and
H2O2 by disrupted mitochondrial electron transport
facilitates formation of reactive oxidants such as oxo-ferryl species of
heme-containing cyt c in the immediate vicinity of PS thus providing
for PS oxidation. PSox can be externalized and enhance externalization of PS.
Molecular model of cyt c (1AKK
[PDB]
) was obtained using Cn3D software from
the National Center for Biotechnology and Information.
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There are several experimental findings that are compatible with the model
outlined above. In one series of experiments, we gently sonicated HL-60 cells
in the presence of excess amounts of cyt c (200 µM), resulting in
the integration of cyt c into the cells. We found that incorporation
of cyt c into PnA-labeled HL-60 cells (in the presence of exogenously
added t- BuOOH) resulted in preferential oxidation of PS compared
with other phospholipids (Fig.
6) (77).
Furthermore, in cell-free model systems, PS proved to be selectively oxidized
by a cyt c/ascorbate/H2O2-catalytic system
compared with PC (73). In
another series of experiments, we used a different approach to disrupt
mitochondria and release cyt c into cytosol, utilizing the DP-1
peptide. DP-1 is composed of two functional domains: a protein transduction
domain, PTD-5 (a 12-mer peptide sequence RRQRRTSKLMKR from M13 phage library),
and an antibiotic peptide, KLA [(KLAKLAK)2]. PTD-5 is used to guide
KLA to target cells and allow for its internalization. PTD-5 is positively
charged due to a high content of arginine and lysine residues (
70%) and
causes efficient and rapid internalization of conjugated proteins into cells
in vitro and in vivo (102).
KLA, an antimicrobial peptide, is designed to target and disrupt bacterial
cells, as well as mitochondria in eukaryotic cells
(31,
69). Therefore, the PTD-5/KLA
conjugate (DP-1) is able to preferentially disrupt mitochondria and induce
release of cyt c but spare damage to the plasma membrane and other
organelles in cells. Incubation of Jurkat cells with DP-1 peptide caused a
rapid (within 5 min) release of cyt c from mitochondria into cytosol.
Importantly, cyt c released from mitochondria could be completely
recovered in the cytosolic fraction. Furthermore, we found that DP-1-induced
cyt c release was accompanied by a more than fourfold increase in
H2O2 production by Jurkat cells. Next, using the
PnA-based assay, we showed that PS was the only phospholipid that was
significantly oxidized after incubation of Jurkat cells with 10 µM DP-1 (J.
Jiang, B. F. Serinkan, Z. Mi, P. D. Robbins, and V. E. Kagan, unpublished
observations). DP-1-induced PS oxidation was accompanied by significant
externalization of PS in Jurkat cells and enhanced recognition and
phagocytosis of these cells by J774A.1 macrophages. Together, these data
suggest that release of cyt c may be involved in PS oxidation. The
possibility that this site-specific oxidative stress and selective PS
oxidation can play a signaling role in the resolution of apoptosis, through
PS-dependent pathways such as PS externalization, recognition of apoptotic
cells by specialized macrophage receptors, and subsequent phagocytosis, is
discussed below.

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Fig. 6. Effect of cyt c on tert-butyl hydroperoxide
(t-BuOOH)-induced phospholipid oxidation in HL-60 cells. PnA-labeled
HL-60 cells loaded with cyt c (cyt c was incorporated into
PnA-labeled cells by mild sonication) were incubated in the presence of
t-BuOOH (150 µM) for 20 min. Lipid oxidation was terminated by
addition of 10 µM BHT, and cells were incubated an additional 40 min.
Lipids were extracted and resolved by HPLC. Data are means ± SE;
*P < 0.05 vs. phosphatidylinositol (PI),
phosphatidylcholine (PC), or phosphatidylethanolamine (PE).
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PS ASYMMETRY AND EXTERNALIZATION (RAFTING) DURING APOPTOSIS:
THRESHOLD PHENOMENA AND ROLE IN MACROPHAGE CLEARANCE
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The plasma membrane, the barrier between intra- and extracellular milieus,
plays a pivotal role in the communication of cells with their environment. The
majority of aminophospholipids (PE and PS) are predominantly confined to the
inner leaflet of the plasma membrane, whereas choline-containing phospholipids
(PC and SPH) are localized mainly in its outer leaflet
(6,
25,
93,
120,
167). This phospholipid
asymmetry is maintained by an ATP-dependent aminophospholipid translocase that
specifically transports PS and PE from the outer to the inner leaflet (for
review see Ref. 6).
Aminophospholipid translocation is Ca2+ inhibitable and
sensitive to the sulfhydryl-reactive agent N-ethylmaleimide (NEM), as
well as to vanadate, an inhibitor of P-type ATPases. The molecular identity of
this "flippase" is not firmly established, although a candidate
P-type ATPase termed ATPase II that possesses phospholipid transporting
properties has been identified
(29,
54,
105,
108,
143,
146).
Loss of membrane phospholipid asymmetry with subsequent externalization of
PS is known to be an important signaling mechanism, e.g., to stimulate the
coagulation cascade during platelet activation and to mediate cell recognition
by macrophages (131). This
process is believed to be mediated by a Ca2+-activated
phospholipid scramblase that facilitates the bidirectional movement of all
classes of phospholipids across the the lipid bilayer
(6). Zhou and coworkers
(161,
163) identified a 37-kDa
protein in platelets with scramblase activity and have demonstrated a
correlation between the expression of this scramblase and the
Ca2+ ionophore-induced externalization of PS. However,
this protein is normal in Scott syndrome patients whose blood cells are
defective for scramblase activity and PS exposure
(6,
167), suggesting that
additional molecules are required for scramblase activation. Moreover, red
blood cells from scramblase null mice normally mobilize PS to the surface upon
Ca2+ ionophore stimulation
(164). PS externalization is
considered a general phenomenon in cells undergoing apoptosis
(43,
95), although a few instances
of apoptosis in the absence of PS exposure have also been described
(38,
47). PS exposure is dependent
on extracellular Ca2+
(7,
57,
96) and occurs downstream of
the activation of caspases
(37,
94,
155). In addition, previous
studies have indicated that PS exposure during apoptosis is dependent on the
concomitant inhibition of the aminophospholipid translocase and activation of
the phospholipid scramblase (7,
157). However, recent data
suggest that scramblase expression is not a critical determinant of apoptotic
PS exposure (43,
162). Moreover, the inhibitor
of the aminophospholipid translocase, NEM, can induce PS exposure in the
absence of other indices of apoptosis
(43,
154), and the NEM-induced
redistribution of PS in Raji cells is sufficient to trigger macrophage removal
of these cells (74). Together,
these findings serve to underscore the role of aminophospholipid translocase
inhibition for the outward movement of PS during apoptosis. Importantly,
although disturbances of phospholipid asymmetry during apoptosis are caspase
dependent, neither aminophospholipid translocase nor scramblase has been
reported as a direct target of caspases, the major proteolytic executors of
apoptosis (41,
166). Therefore, alternative
mechanisms affecting these enzymatic activities must be responsible for
disturbances of phospholipid asymmetry during apoptosis. We have recently
shown that intracellular ATP can modulate aminophospholipid translocation and
PS exposure during Fas-mediated apoptosis, irrespective of the scramblase
status of the cell (49), and
we have observed that Bcl-2 overexpression maintains intracellular ATP levels
and abrogates PS exposure in Fas-triggered SKW6.4 cells (W. Uthaisang, S.
Orrenius, and B. Fadeel, unpublished observation). In addition, the
aminophospolipid translocase is sensitive to oxidative and nitrosative
modification of its SH groups
(35,
65), and it is therefore
tempting to speculate that oxidative stress may also play a role in
translocase inhibition and the subsequent loss of phospholipid asymmetry.
Importance of lipid rafting for the externalization of PS and its
recognition. Apoptosis-associated exposure of PS on the cell surface is
only one feature typical of the global and complex biochemical (flipping of
SPH, redistribution of cholesterol) and biophysical (changes of fluidity,
segregation of lipids, and formation of microdomains) rearrangements of the
plasma membrane during apoptosis culminating in its dramatic blebbing and
vesiculation (107,
148). Lipid rafts are
specialized membrane subdomains that have a high cholesterol and sphingolipid
(
50 and 20%, respectively) content and are organized in a tightly packed,
liquid-ordered manner (2,
51). Various proteins (e.g.,
the multifunctional scavenger receptor CD36) selectively partition into
detergent-resistant lipid rafts, suggesting that clustering of proteins within
rafts is a process regulated by specific lipid-protein interactions
(51). Aggregation of rafts
following receptor ligation may be a general mechanism for promoting immune
cell signaling. Recent studies have provided evidence that raft integrity is
essential for the plasma membrane redistribution of PS in
Ca2+ ionophore-stimulated tumor cells
(83). It has been speculated
that the formation of rafts also participates in PS exposure during apoptosis
(40). Such aggregation of PS
molecules may facilitate their subsequent recognition by one or more PS
receptor(s) (PSR) on the macrophage surface (see below). Interestingly,
clustering of ced-1, a putative PSR in Caenorhabditis elegans, is
seen in response to cell corpse recognition in the nematode system
(165). Although it is not
known whether PS oxidation is involved in PS aggregation and formation of
rafts in the plasma membrane during apoptosis, it is noteworthy that
aggregation of phospholipid hydroperoxides resulting in the formation of their
clusters has been documented
(72). Dillon et al.
(27) have shown that annexin V
colocalizes with markers of lipid rafts in the outer membrane of activated,
nonapoptotic B cells, suggesting that PS exposure can occur in specific
membrane microdomains in the absence of cell death. Moreover, mature B cells
expose PS on their surface, where it colocalizes with antigen receptors and
forms caps that are required for receptor-mediated signaling events that
trigger Ig production (27).
Similarily, transient, nonapoptotic exposure of PS is seen during development
of skeletal and heart muscle and has been suggested to be essential for
myotube formation in mice
(153). Finally, normal
macrophages themselves were reported to display PS on their surface, and this
was suggested to be required for phagocytosis of PS-expressing target cells
(12). These observations beg
the question of how cells that express the common PS-dependent eat-me signal
can escape macrophage recognition. One may speculate that the functional
outcome of PS externalization may ultimately depend on the density of PS on
the cell surface. A low or intermediate level of PS or transient exposure of
PS may not suffice to trigger clearance, whereas more extensive
externalization of PS during apoptosis may reach a threshold necessary for
phagcoytosis to occur (see below). Alternatively, viable nonapoptotic
PS-positive cells may fail to express additional necessary cofactors, such as
chemotactic mediators or accessory surface ligands (such as oxidized PS),
required for the stimulation of macrophages.
Evaluations of amounts of externalized PS in nonapoptotic and apoptotic
cells. Because apoptosis is accompanied by PS oxidation in the plasma
membrane, two different populations of PS, nonoxidized PS (PS) and oxidized PS
(PSox), are likely to be present in the outer membrane leaflet of apoptotic
cells. These two PS species may behave differently with regard to their
topography in the membrane as well as their effects on enzymatic and
nonenzymatic pathways involved in the maintenance of PS asymmetry across the
plasma membrane. PSox may act as a poison or "suicide substrate"
for the aminophospholipid translocase, thus inhibiting translocation of both
PSox and PS. This would result in the appearance of both PS and PSox on the
surface of the plasma membrane. Alternatively, PSox could be preferentially
externalized during apoptosis as a result of poor substrate recognition by the
translocase and/or a high rate of spontaneous (nonenzymatic) transbilayer
flip-flop. Methods for the quantitative assessment of PS content on the
surface of apoptotic cells are clearly needed to determine the levels of
native PS, PSox, and potentially other molecular species of PS and the
mechanism of their redistribution within the plasma membrane during apoptosis.
Although annexin V-based assays for PS externalization have been extensively
used in numerous studies, the results are usually interpreted in terms of
distribution of cell populations with an arbitrary (above threshold) amount of
externalized PS rather than the absolute amount (concentrations) of PS
expressed on the cell surface. Unfortunately, quantification of PS on
apoptotic cells has not received significant attention. Indeed, since the
pioneering work by Fadok and colleagues
(43), who discovered PS
externalization in apoptotic murine thymocytes using chemical derivatization
with fluorescamine, a nonpermeable reagent for primary amines, there are only
few examples of PS quantitative measurements (e.g., in the human leukemia T
cell line Jurkat and the human leukemia HL-60 cell line)
(Table 2). However, neither the
fluorescamine-based assay nor the annexin V-based flow cytometric assay
permits the quantitative estimation of amounts of PS on the surface of
nonapoptotic cells. Using a modification of the annexin V method, the annexin
V iron-beads/electron paramagnetic resonance assay, we were recently able to
quantify PS on the surface of Jurkat cells and HL-60 cells
(Fig. 7,
Table 2). We found that
apoptotic HL-60 cells and Jurkat cells externalized up to 25280-fold
more PS than nonapoptotic controls. This suggests that PS is a prominent
signal for clearance of apoptotic cells by macrophages (G. G. Borisenko, T.
Matsura, S.-X. Liu, V. A. Tyurin, J. Jiang, B. F. Serinkan, and V. E. Kagan,
unpublished observations).

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Fig. 7. Estimation of PS externalization using EPR-based annexin V-iron beads
assay. Shown are the EPR spectra of iron-beads recorded from Jurkat cells (2
x 106 cells) labeled with annexin V iron beads (A,
B) or with basic (without annexin V) iron beads (C, D). Signals
from annexin V iron beads and basic iron beads report PS-specific and
nonspecific binding, respectively. A: incubation of cells with PC/PS
liposomes (0.3 mM phospholipids) resulted in enrichment of the external
leaflet of plasma membrane with exogenous PS as evidenced by annexin V-iron
bead binding (thin line, signal after treatment with liposomes; bold line,
without liposomes). B: increased annexin V-iron bead EPR signal from
apoptotic cells (after treatment with camptothecin, 50 µM for 3 h) revealed
externalization of endogenous PS on the cell surface. C, D:
nonspecific binding of the basic iron beads was not affected by either
incubation with liposomes (C) or by treatment with camptothecine
(D). EPR spectra of iron beads were recorded at room temperature
under the following conditions: 10 mW, microwave power; 9.445 GHz, microwave
frequency; 300 mT, center field; 300 mT, sweep width; 2 mT, field modulation;
0.3 s, time constant; 1 min, time scan.
|
|
The importance of PS on the cell surface for recognition and phagocytosis
by macrophages was first demonstrated by Schroit and colleagues
(136,
145) in the early 1980s in
experiments using red blood cells with artificially manipulated levels of
exogenous PS. These investigators hypothesized that recognition of PS-exposing
cells by macrophages involves specific ligand-receptor interactions
(136). Further studies
demonstrated that activated monocytes are able to bind different tumor cell
lines with elevated levels of PS, but not normal human keratinocytes cells
with relatively low PS content
(152). These results imply
that a threshold of PS expression may exist for macrophage recognition of
PS-externalizing cells (152).
To further explore this hypothesis, we enriched the external leaflet of the
plasma membrane of nonapoptotic Jurkat cells and HL-60 cells with known
amounts of exogenous PS and determined the sensitivity of macrophages for PS
on the surface of the target cells (G. G. Borisenko, T. Matsura, S.-X. Liu, V.
A. Tyurin, J. Jiang, B. F. Serinkan, and V. E. Kagan, unpublished
observations). The dependence of the phagocytic capacity of macrophages on the
amounts of PS integrated into the outer leaflet of plasma membrane in Jurkat
cells or HL-60 cells unequivocally demonstrated a nonlinear response with a
sensitivity threshold required to initiate macrophage recognition of
externalized PS. Hence, at least a
510-fold increase of the PS
content in the outer leaflet of plasma membrane was required to trigger
macrophage recognition and uptake. During apoptosis, e.g., induced by the
chemotherapeutic agent camptothecin, both cell lines expressed externalized PS
in amounts far in excess of the recognition threshold and were thus
effectively phagocytosed.
 |
MACROPHAGE RECOGNITION OF PS VS. PSOX: OXIDIZED
PHOSPHOLIPIDS ENHANCE PHAGOCYTOSIS OF APOPTOTIC CELLS
|
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Many receptors have been implicated in the removal of dying, apoptotic
cells by macrophages. Not only do different populations of phagocytes use
discrete receptors, but a single phagocyte may express a number of receptors
that cooperate in the ingestion of their prey
(119). These different
receptors include the class A scavenger receptor
(114,
116),
V
3-integrin (vitronectin receptor)
(42,
44), CD68
(16), CD14
(26), lectin-like oxidized
low-density lipoprotein receptor, and CD36
(44,
123). An interesting
alternative point of view is that CD31 acts as a cell surface molecule that
normally prevents phagocyte ingestion of viable cells by transmitting
"detachment" signals
(9). CD31-mediated detachment
is disabled in apoptotic cells by an unknown mechanism, resulting in the
promotion of tethering of cell corpses to phagocytes. In addition, serum
proteins such as
2-glycoprotein I
(5,
19) and C1q, the first
component of complement
(101), bind to apoptotic
cells and enhance their uptake. Many of the above receptors can bind PS, but
not all of them are specific for this phospholipid
(26,
115). Furthermore, activated
macrophages were recently reported to secrete a glycoprotein, milk fat globule
epidermal growth factor 8, that specifically binds to PS on apoptotic cells
and to
V
3-integrin on the macrophage
surface, thus serving as a "molecular bridge" between the two
cells (60). Regardless of the
receptors engaged or disengaged in phagocytosis, ingestion does not occur in
the absence of PS exposure
(39,
74). Specifically, recognition
of surface PS was reported to be dependent on the presence of the so-called
PSR, a recently cloned receptor that is expressed by phagocytes and mediates
pinocytosis and initiates uptake of apoptotic cells
(40,
68,
156). Indeed, the requirement
for PS expression and the ensuing ligation of PSR on phagocytes by PS on the
apoptotic cell surface may be essential to signal uptake of cells that are
tethered to phagocytes via other receptors
(144). However, additional
signals, such as oxidative changes, may also be required for the engulfment of
target cells (129). In fact,
many of the receptors implicated in phagocytosis of apoptotic cells can
strongly bind oxidized phospholipids
(48,
129), which arise during
apoptosis and provide additional ligands for recognition receptors
(74,
150). Oxidized epitopes on
the surface of apoptotic cells may thus act as important signals for the
recognition of target cells by macrophages
(16,
128). In particular,
C-reactive protein, a component of the innate immune response, was shown to
bind to oxidized PC species on the surface of "late" apoptotic
(propidium iodide-positive) cells
(17). Moreover, Podrez et al.
(117,
118) recently described a
novel family of oxidized PC homologs that were able to act as a ligands for
the scavenger receptor CD36 and promote macrophage foam cell formation.
Because plasma membrane PS may be a specific target for oxidation, it is
tempting to speculate that a combination of PS and PSox may be essential for
recognition and uptake of apoptotic cells. In support of this notion,
inhibition of PS oxidation in cells during apoptosis has been demonstrated to
block phagocytosis of Jurkat cells and HL-60 cells by macrophages
(4,
74). Moreover, we found that
integration of PSox along with PS into plasma membrane of nonapoptotic cells
significantly stimulated their phagocytosis compared with the incorporation of
similar amounts of native PS alone (Fig.
3) (74). In
addition, liposomes containing PSox acted as potent inhibitors of phagocytosis
of apoptotic cells (anti-Fas-triggered Jurkat cells and
t-BuOOH-treated HL-60 cells)
(74). Together, these findings
indicate that PSox, indeed, may act as an important signal on the cell surface
that can act alone or in combination with native PS to facilitate recognition
of apoptotic cells. Nevertheless, many questions remain regarding the role of
PSox in the signaling for engulfment. For example, what is the fraction of
PSox vs. PS on the cell surface during apoptosis? Can other oxidized
phospholipids such as PCox, PEox, etc. synergistically interact with PS to
facilitate recognition of apoptotic cells? What are the actual concentrations
of PCox, PEox, and other oxidized phosopholipids compared with PSox on the
cell surface during apoptosis? Interestingly, our preliminary experiments
indicate that PSox is capable of markedly reducing the threshold for
recognition of PS-containing cells by macrophages. Another important question
relates to the type of receptors involved in recognition of PS and PSox. It
seems likely that recognition of PS and PSox involves different receptor(s).
Indeed, we found that anti-PSR antibody, but not anti-CD-36 antibody, was able
to inhibit phagocytosis of Jurkat cells with PS integrated into their plasma
membrane. In contrast, both anti-PSR antibody and anti-CD36 antibody were
effective in suppressing phagocytosis of Jurkat cells enriched with both PS
and PSox (Fig. 8); anti-CD36
antibody was also efficient in suppressing the engulfment of Fas-triggered
Jurkat cells, which display both PSox and its nonoxidized counterpart on the
cell surface (C. Elenström-Magnusson and B. Fadeel, unpublished
observations). These data imply that CD36 and PSR might cooperate to recognize
oxidized PS. In conclusion, selective oxidation and externalization of PS in
the plasma membrane of apoptotic cells likely creates conditions whereby
oxidized PS on the external surface of the cell could act as a preferred
ligand (or eat-me signal) for certain macrophage receptors, including
scavenger receptors such as CD36, resulting in the recognition and disposal of
cell corpses.

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Fig. 8. PS- and PSox-induced phagocytosis elicits different sensitivity toward
anti-PSR and anti-CD35 antibodies. J774A.1 macrophages were pretreated for 30
min at 37°C with MAb against PSR (11 µg/ml) or MAb against CD36
receptor (100 µg/ml) before addition of target cells.
*P < 0.05 vs. control.
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|
 |
ANTIOXIDANTS AS REGULATORS OF APOPTOSIS AND PHAGOCYTOSIS OF APOPTOTIC
CELLS
|
---|
The potentially important signaling function of PS oxidation in the plasma
membrane and the subsequent externalization of PSox on the cell surface
suggest that antioxidants theoretically could play an unusual role in the
regulation of apoptosis and phagocytosis of apoptotic cells. Thus if PS
oxidation is essential for its externalization and, furthermore, if PSox acts
as an additional stimulatory signal facilitating recognition and engulfment of
apoptotic cells, then inhibition of PS oxidation by antioxidant enzymes and/or
water- and lipid-soluble antioxidants might interfere with the execution of
these critical functions and, hence, with the resolution of the apoptotic
process. Obviously, total nonspecific prevention of oxidative stress during
oxidant-induced apoptosis by antioxidants via blocking the initiation of
apoptotic program is a trivial and highly predictable effect that has been
documented in a number of reports
(50,
111). Therefore, models of
nonoxidant apoptosis, where oxidative stress functions only as a specific
component of the execution of apoptotic program, are preferable for studies of
antioxidant effects. In particular, observations related to PS-specific
effects of antioxidants may be of considerable interest. We found that
antioxidant enzymes can indeed modulate PS oxidation-dependent signaling
pathways in apoptosis. This was demonstrated in experiments with Fas-triggered
apoptosis in Jurkat cells
(74). Anti-Fas antibody
triggered selective oxidation of PS accompanied by PS externalization, caspase
activation, and recognition and phagocytosis of apoptotic cells by several
classes of macrophages. Remarkably, high doses of the antioxidant enzymes
superoxide dismutase (SOD)/catalase (50 U/ml of each) were able to inhibit
oxidative stress (e.g., production of superoxide and
H2O2) and block PS oxidation and
recognition/phagocytosis of apoptotic target cell by J774A.1 macrophages
without affecting other biomarkers of apoptosis, such as PS externalization,
caspase-3 activation, and nuclear condensation
(74). Hildeman et al.
(66) showed that an SOD
mimetic, Mn(III) tetrakis (5,10,15,20-benzoic acid) porphyrin, protected T
cells after activation through Fas/TNF-
(i.e., death
receptor)-independent pathways. This protection, however, was due to decreased
mitochondrial damage and subsequent caspase-dependent DNA fragmentation. It
seems, therefore, that ROS may be differentially involved in signaling and
execution pathways during apoptosis depending on the initial triggering
mechanisms.
Not only antioxidant enzymes but also low-molecular-weight chain-breaking
antioxidants can affect apoptotic pathways by inhibiting PS oxidation. For
example, an effective lipid antioxidant, etoposide (VP-16), at
pharmacologically relevant concentrations (50 µM) was able to completely
block PS oxidation in HL-60 cells during H2O2-induced
apoptosis (Y. Y. Tyurina, B. F. Serinkan, V. A. Tyurin, V. Kini, J. C.
Yalowich, B. Fadeel, and V. E. Kagan, unpublished observations). Under these
conditions, etoposide inhibited PS externalization in HL-60 cells as well as
their phagocytosis by J774A.1 macrophages. It is important that effects of
antioxidants on PS oxidation and subsequent PS-dependent pathways are studied
and determined specifically during apoptosis rather than when both apoptotic
and necrotic mechanisms are realized concomitantly. In this regard, Shacter
and colleagues (3,
139) reported that high
concentrations of H2O2 inhibited phagocytosis of
apoptotic cells upon etoposide treatment, largely due to the shifting of cell
death from apoptosis to necrosis. Vitamin E (
-tocopherol) is one of the
major natural lipid-soluble chain-breaking antioxidants of membranes. We
experimentally determined the effects of vitamin E on Fas-triggered oxidation
of PS, apoptosis in Jurkat cells, and their phagocytosis by J774A.1
macrophages. We found that substantial inhibition of Fas-induced phospholipid
oxidation could be achieved only at relatively high pharmacological
concentrations of vitamin E. Complete inhibition of Fas-induced PS oxidation
was observed only when vitamin E levels in cells were >20-fold in excess of
those in nonsupplemented cells. At these high doses, however, vitamin E did
not affect the outcome of apoptosis as shown by PS externalization and nuclear
morphological alterations (B. F. Serinkan, Y. Y. Tyurina, M. Djukic, H. Babu,
A. Schroit, and V. E. Kagan, unpublished observations). No changes in the
effectiveness of phagocytosis of apoptotic cells occurred in the presence of
vitamin E. Notably, physiologically relevant levels of vitamin E were not able
to completely block PS oxidation. It should be mentioned, however, that the
effects of vitamin E on phagocytosis may be realized through its
antioxidant-independent mechanisms of macrophage activation, such as induction
of the expression of cell-cell adhesion molecules
(24,
127).
Thus physiological levels of antioxidants are not likely to be sufficient
to completely block PS oxidation during apoptosis and hence to interfere with
PS-dependent pathways of phagocytosis. High pharmacological doses of
antioxidants, however, are able to cause inhibition of PS oxidation sufficient
to affect PS externalization (etoposide) or phagocytosis (SOD/catalase).
Antioxidants are commonly believed to be effective anti-inflammatory agents
(122). However, it is
important that their use at high doses be considered carefully, as their
specific mechanism of action predicts a potential effect as inhibitors of
PS-dependent pathways in apoptosis and phagocytosis of apoptotic cells, thus
precluding the noninflammatory removal of dying cells (discussed below).
 |
ROLE OF APOPTOTIC CELL CLEARANCE IN LIMITING INFLAMMATORY RESPONSES
IN THE LUNG
|
---|
Neutrophils are an important line of host defense against invading
microorganisms, and the production of ROS in these cells is an essential step
in the killing of ingested bacteria. The apoptotic death of neutrophils and
their subsequent engulfment by macrophages is believed to be a critical
component in the resolution of inflammation, as this serves to remove these
cells from the inflammatory site with minimal damage to surrounding tissues
(132,
159). Conversely, a mismatch
between the rate of apoptosis and the rate of phagocytic clearance of
apoptotic cells may underlie detrimental inflammatory responses, as shown in,
e.g., mice receiving agonistic anti-Fas antibodies
(104). These animals die from
hepatic failure as a result of massive apoptosis of hepatocytes and fulminant
inflammation in the liver, due most likely to "secondary" necrosis
of unengulfed cells. The harnessing of apoptotic mechanisms involved in the
resolution of inflammation and restitution of tissue homeostasis may thus
yield novel therapeutic strategies in conditions of excessive
inflammation.
Cystic fibrosis (CF) is an inflammatory disease of the lung characterized
by a sustained influx of inflammatory cells into the airways and release of
proteases from these cells (8).
The fact that inflammation is persistent and that necrotic (or postapoptotic)
cells accumulate in the airways of CF patients suggests that the normal
mechanism for removal of effete cells is impaired. Indeed, Vandivier et al.
(156) recently demonstrated
an abundance of unengulfed apoptotic cells in the airways of CF as well as
non-CF bronchiectasis patients. These investigators also provided evidence
that neutrophil elastase, an intracellular protease that is released by
inflammatory cells into the airways during inflammation, cleaves the PSR on
the surface of phagocytes, thus contributing to the disruption of apoptotic
cell clearance. Consequently, therapies that augment macrophage engulfment of
apoptotic cells, for instance by targeting the PS-dependent pathway of cell
clearance, may be envisaged for conditions of pulmonary decline due to
excessive inflammation.
Another example of chronic inflammation is the rare hereditary disease
known as chronic granulomatous disease (CGD), which is characterized by severe
and sometimes fatal infections, including fungal or bacterial pneumonia
(71). The basic defect arises
from an inability of neutrophils to generate superoxide and
H2O2 due to mutations in the membrane-bound NADPH
oxidase. We have previously shown that neutrophils possess both
caspase-dependent and oxidant-dependent modes of PS exposure, which are
employed during constitutive apoptosis and activation-induced (nonapoptotic)
death, respectively (37,
55). We have yet to determine
whether PS is also oxidized in neutrophils undergoing apoptotic vs.
nonapoptotic cell death. Nevertheless, our studies of CGD neutrophils
demonstrated that although these cells exhibit a normal apoptotic response,
with caspase activation and plasma membrane exposure of PS, they fail to
externalize PS when incubated with the potent neutrophil activator PMA
(37). In addition, our recent
studies using DMSO-differentiated, neutrophil-like HL-60 cells as well as
neutrophils from healthy donors confirm that both PMA and opsonized zymosan
beads, a more physiological stimulator of the NADPH oxidase, are capable of
triggering ROS-dependent, DPI-inhibitable PS externalization
(4). Concomitant oxidation of
PS was also observed in the HL-60 model. These data thus provide evidence for
the involvement of NADPH-derived ROS in the oxidation and externalization of
PS. Our findings were corroborated in a recent report, in which
H2O2-dependent PS externalization and macrophage uptake
of neutrophils ingesting Staphylococcus aureus were documented
(58). It appears reasonable to
speculate that the failure to activate this mode of PS exposure in vivo could
result in defective clearance of cells by macrophages and hence contribute to
the formation of the characteristic granulomatous lesions and subsequent
tissue destruction evidenced in CGD patients. We surmise that the augmentation
of PS- and/or PSox-dependent cell clearance may serve as a therapeutic
approach in CGD and related granulomatous disorders.
Molecules other than PS and PSox may also contribute to the removal of
effete cells during inflammation. For instance, ligation of the cell surface
adhesion molecule CD44 is known to promote the uptake of apoptotic neutrophils
but not of apoptotic lymphocytes
(61). Teder et al.
(147) recently reported that
mice deficient for CD44 exhibit a >10-fold increase in unengulfed apoptotic
cells in the lung tissue after bleomycin treatment compared with wild-type
animals. Importantly, lack of CD44 resulted in increased mortality from lung
injury due to unremitting inflammation, thus serving to underscore the
importance of clearance of apoptotic neutrophils in the resolution of lung
inflammation.
In view of the above observations concerning the importance of PS oxidation
in the plasma membrane during apoptosis, it seems prudent to reconsider the
clinical utilization of antioxidants, believed to act as promising
therapeutics in the regulation of inflammatory responses. Thus it will be
important to establish regimens and conditions for antioxidant treatments that
do not affect important PS oxidation signaling events in apoptotic cells and
hence interfere with macrophage recognition of such cells. Conversely, one can
envision that directed and targeted delivery of PSox to the surface of damaged
cells can be used to enhance their safe clearance and may be useful for
limiting the inflammatory response in the lung and in other tissues.
 |
CONCLUDING REMARKS
|
---|
In summary, generation of ROS is an integral component of the apoptotic
program and results in the preferential oxidation of PS in the plasma membrane
of the dying cell. Oxidized PS, in turn, serves as an important signal through
which macrophages recognize and eliminate apoptotic cells. Oxidative stress
within the apoptotic cell may promote this clearance process either via
enhanced externalization of oxidized PS on the surface of apoptotic cells
and/or through more effective recognition of apoptotic cells exhibiting PSox
(along with its nonoxidized counterpart) on their surface. Furthermore, we
speculate that the mitochondrial release of cyt c during apoptosis is
critically involved in the selective oxidation of PS and its subsequent
externalization. Regardless of the specific mechanism(s) involved, the final
outcome of macrophage recognition of PS and Psox is the nonphlogistic disposal
of potentially harmful cells, e.g., at sites of inflammation. In this context,
the possibility that antioxidants capable of inhibiting PS oxidation might
interfere with PS externalization and/or its recognition by macrophages needs
to be carefully considered. Nevertheless, as outlined in the present review,
apoptosis-dependent, mitochondrially derived oxidative stress is
mechanistically linked with the oxidation of PS and the stimulation of
PS-dependent signaling pathways culminating in the disposal of cells by
macrophages.
 |
ACKNOWLEDGMENTS
|
---|
B. Fadeel acknowledges the continuous support of his mentor, Sten Orrenius,
Karolinska Institutet.
This work was supported by National Heart, Lung, and Blood Institute Grants
1RO1 HL-7075501 and 1RO1 HL-6414501A1. G. G. Borisenko was
supported by the International Neurological Science Fellowship Program (1 FO5
NS4392201) administered by National Institutes of Health/National
Institute of Neurological Disorders and Stroke in collaboration with the World
Health Organization. W. Uthaisang was supported by a scholarship from the
Ministry Staff Development Project, Ministry of University Affairs, Thailand.
B. Fadeel was supported by the Swedish Society for Medical Research.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: V. E. Kagan, Dept. of
Environmental and Occupational Health, Univ. of Pittsburgh, Pittsburgh, PA
15260 (E-mail:
kagan{at}pitt.edu).
 |
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