Abstract. Annexin 1 (ANXA1) is the first characterized
member of the annexin family of proteins able to bind (i.e. to
annex) to cellular membranes in a calcium-dependent man-
ner. ANXA1 may be induced by glucocorticoids in inflam-
matory cells and shares with these drugs many anti-inflam-
matory effects. Originally described as a phospholipase A2
(PLA2)-inhibitory protein, ANXA1 can affect many compo-
nents of the inflammatory reaction besides the metabolism of
arachidonic acid. Recent data have shown that ANXA1 may
specifically target cytosolic PLA2by both direct enzyme
inhibition and suppression of cytokine-induced activation of
the enzyme. ANXA1 inhibits the expression and/or activity
of other inflammatory enzymes like inducible nitric oxide
synthase (iNOS) in macrophages and inducible cyclooxyge-
nase (COX-2) in activated microglia. The inhibition of iNOS
expression may be caused by the stimulation of IL-10 release
induced by ANXA1 in macrophages. Like glucocorticoids,
ANXA1 exerts profound inhibitory effects on both neu-
trophil and monocyte migration in inflammation. Several
mechanisms may contribute to the protein effect on cell
migration, namely the activation of receptors like the formyl
peptide receptor (FPR) and the lipoxin A4receptor (ALXR),
the shedding of L-selectin, the binding to a4b1integrin and
carboxylated N-glycans. Furthermore, again mimicking the
action of glucocorticoids, ANXA1 promotes inflammatory
cell apoptosis associated with transient rise in intracellular
calcium and caspase-3 activation. Finally, ANXA1 has been
recently identified as one of the ‘eat-me’signals on apoptot-
ic cells to be recognised and ingested by phagocytes. Thus,
ANXA1 may contribute to the anti-inflammatory signalling
that allows safe post-apoptotic clearance of dead cells.
Key words: Annexin 1 – Glucocorticoids – Inflammation –
Cell migration – Apoptosis
Inflamm. res. 53 (2004) 125–132
During 1970s it was becoming clear that a good part of the
anti-inflammatory action of glucocorticoids was due to the
inhibition of eicosanoid release and that this inhibition
occurred at the level of substrate release. In other words glu-
cocorticoids were able to block the activation of phospholi-
pase A2 (PLA2), the enzyme that supplies free arachidonic
acid from cellular phospholipids to cyclooxygenase and
lipoxygenase for the formation of inflammatory eicosanoids.
This implies that: (i) PLA2is the rate limiting step in the syn-
thesis of lipid inflammatory mediators; (ii) the control of
PLA2activity is central to regulating the inflammatory
process. In the early 1980’s papers from different laboratories
showed that glucocorticoid inhibition of arachidonic acid
release was dependent on the synthesis and release of
inhibitory proteins. Glucocorticoid treatment resulted in the
production of a factor that mimicked the inhibitory action of
the steroid itself. The factor was then identified as a protein
of 37 kDa and named ‘lipocortin’. These pioneering experi-
ments triggered an enormous amount of work opening a new
field of biological research (for details of those early experi-
ments see reviews by Flower  and Flower and Rothwell
). Subsequent work has shown that lipocortin was able to
mimic the anti-inflammatory effects of glucocorticoids in
several experimental models of inflammation both in vivo
and in vitro. It also became evident that, like glucocorticoids,
the protein can affect many components of the inflammatory
reaction besides the release of arachidonic acid .
It is now known that lipocortin is the first characterized
member of the annexin superfamily of proteins, so called
since their main property is to bind (i.e. to annex) to cellular
membranes in a Ca2+-dependent manner . Annexins are
ubiquitous proteins described in many organisms from mam-
mals to moulds and even plants and implicated in several
aspects of cell biology (for a comprehensive review see Ray-
nal and Pollard ). A conserved core domain that contains
either four or eight repeating units of approx. 70 amino acids
defines annexins structurally. The conserved repeats account
© Birkhäuser Verlag, Basel, 2004
Annexin 1: more than an anti-phospholipase protein
Luca Parente1and Egle Solito1,2
1Department of Pharmaceutical Sciences, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano (Salerno) Italy, Fax: ++ 39 089 962828,
2Department of Neuroendocrinology, Imperial College London, U.K.
Received 27 October 2003; returned for revision 6 November 2003; accepted by R. Pettipher 26 November 2003
Correspondence to: L. Parente
for the shared abilities of the proteins to bind phospholipids,
whereas the specific functions of each annexin are probably
related to their specific N-terminal regions . As the first
member of the family, lipocortin is now referred to as annex-
in 1 (ANXA1). Scope of this review is to discuss the recent
evidence on the mechanisms of the anti-inflammatory effects
of annexin 1.
Annexin 1 and PLA2activity
This is a quite controversial issue. When the first samples of
the recombinant ANXA1 become available, the inhibitory
effect of the protein was tested on the secretory form of phos-
pholipase A2(sPLA2). At that time this isoform of the
enzyme was thought to be pivotal to the inflammatory
process since it causes inflammatory signs and can be found
in high amounts in inflamed joint fluids where it may con-
tribute to tissue damage . Enzyme kinetic experiments
showed that ANXA1 inhibited sPLA2, but this appeared to be
through a mechanism of substrate depletion rather than by
direct enzymatic inhibition, thereby questioning the anti-
inflammatory potential of the protein .
Cytosolic phospholipase A2
Recent experiments have evaluated the role in inflammation
of a different isoform of the enzyme, namely the cytosolic
PLA2(cPLA2). A series of pharmacological data obtained
with specific sPLA2 and cPLA2inhibitors have shown that
cPLA2inhibitors suppressed the surface expression of inte-
grins in activated neutrophils and the release of IL-1b from
lipopolysaccharide (LPS)-stimulated monocytes . These
in vitro effects well explain the therapeutic efficacy of such
compounds in a model of chronic inflammation . The
paper also showed that sPLA2inhibitors had no effect in
inflammatory models either in vitro or in vivo. More recent-
ly Grass et al.  have observed that overexpression of
human group II PLA2 in transgenic mice results in epidermal
hyperplasia in the absence of any inflammatory infiltrate.
This evidence together with the fact that the cytosolic
enzyme is (at variance with sPLA2) very selective for releas-
ing arachidonic acid from the sn-2 position of phospholipids
strongly suggests that cPLA2is the predominant enzyme in
inflammatory signalling responsible for eicosanoid release in
inflammation. In order to investigate the mechanism of inhi-
bition of cPLA2by recombinant ANXA1, Kim et al.  have
measured the degree of inhibition at various substrate and
calcium concentrations. The results have revealed that cPLA2
inhibition by ANXA1 was essentially independent of sub-
strate concentration. The direct interaction between cPLA2
and ANXA1 has been confirmed in a recent study by
immunoprecipitation and mammalian two-hybrid methods
. Using ANXA1 deletion mutants it was also shown that
cPLA2activity was inhibited by both the full-length protein
and the C-terminal region, but not by the N-terminal domain
of the protein . Of note, both annexin 2 (ANXA2) and 3
(ANXA3) had no effect on cPLA2activity, while the inhibi-
tion by annexin 5 (ANXA5) was much smaller than that by
ANXA1 . The relationship between endogenous ANXA1
and cPLA2has been confirmed by experiments where U-937
cells were stably transfected with a sense and antisense
cDNA for ANXA1 . The cells transfected with the anti-
sense clone showed consistently higher cPLA2activity than
the cells transfected with the sense clone, the likely result of
the down-regulation of ANXA1 expression by the antisense
clone. Recently, Hannon et al.  have reported an increase
in cPLA2mRNA and protein in mice lacking the ANX-1
gene (see below). These results strongly suggest that the
expression levels of endogenous ANXA1 regulate expression
and/or activity of cPLA2. Since ANXA1 is active at intracel-
lular calcium concentrations, the protein may have important
pathophysiological roles in controlling cPLA2activity. In this
context, Oh et al.  have recently reported that ANXA1,
by inhibiting cPLA2 activity, suppressed the c-fos serum
response element induced by phorbol myristate acetate in
It is difficult to envisage how exogenously added ANXA1
may directly interact with cPLA2. However experiments by
Croxtall et al. [15, 16] have highlighted a different mecha-
nism of cPLA2inhibition by ANXA1 in the A549 human
lung adenocarcinoma cells that only express the cytosolic
form of the enzyme. It was shown that dexamethasone inhib-
ited cPLA2activation induced by epidermal growth factor
(EGF) in these cells. This inhibition was reversed by a mon-
oclonal anti-annexin antibody, but not by a control mono-
clonal. Moreover, short peptide fragments from the ANXA1
N-terminus mimicked the glucocorticoid effect. It would be
worth investigating whether these effects are mediated by
interaction of exogenous ANXA1 with the membrane recep-
tors reported to bind the protein  (see below).
Effects of annexin 1 on inducible cyclooxygenase
and nitric oxide synthase
Recent experiments have shown that other enzymes involved
in inflammation, besides phospholipase A2, are likely targets
of ANXA1 action in controlling the inflammatory process.
Wu et al.  have demonstrated that a polyclonal antibody
to ANXA1 prevented the beneficial hemodynamic effects as
well as inhibition of inducible nitric oxide synthase (iNOS)
expression by dexamethasone in rats with septic shock. In
addition, dexamethasone and a N-terminal fragment of
ANXA1 inhibited the induction of iNOS in a macrophage
cell line activated with LPS, an effect abrogated by the spe-
cific polyclonal antibody. Pretreatment of macrophages with
dexamethasone up-regulated the expression of ANXA1 on
the cell surface. Dexamethasone also inhibited the induction
of cyclooxygenase-2 (COX-2). This inhibition was unaffect-
ed by treatment with the polyclonal anti-ANXA1 antibody.
The authors conclude that an enhanced extracellular expres-
sion of ANXA1 contributes to the inhibition by glucocorti-
coids of iNOS, but not of COX-2, expression in vitro and in
The inhibitory effect of ANXA1 on iNOS expression has
been confirmed in rat microglia by Minghetti et al. .
In microglial cells dexamethasone and an ANXA1 N-termi-
nal fragment inhibited the expression of COX-2 and iNOS
as well as the release of PGE2and nitric oxide in a dose-
dependent manner. The inhibition of the expression of both
enzymes was abrogated by a specific polyclonal antibody
126L. Parente and E. Solito
against ANXA1. The suppression of the release of nitric
oxide and PGE2may contribute to the reported neuroprotec-
tive effects of ANXA1  since inflammatory metabolites
may participate in the establishment of neurone and oligo-
dendrocyte damage in ischemic and neurodegenerative dis-
orders [20, 21]. It is of note that in situ hybridization studies
have revealed a marked up-regulation of ANXA1 mRNA in
the microglia following experimental brain injury. The pro-
tein was also secreted by the activated microglia . Inter-
estingly, the inhibition of COX-2 expression appears to be a
unique feature of ANXA1 in microglia inasmuch as it has
not been observed in macrophages , in A549 cells ,
or in synoviocytes .
A very recent paper has suggested that some of the anti-
inflammatory effects of ANXA1 may be mediated by the
release of interleukin (IL)-10 . Results showed that in
macrophages primed with LPS recombinant ANXA1 stimu-
lated IL-10 release in a dose- and time-dependent manner. In
the same cells the protein and a derived N-terminal peptide
dose-dependently inhibited the release of nitric oxide. Fur-
thermore, both the whole protein and the peptide down-reg-
ulated the mRNA expression of iNOS. The peptide was also
able to inhibit the expression of IL-12 mRNA. IL-10 is a
potent anti-inflammatory cytokine able to inhibit expression
of both inducible cyclooxygenase  and nitric oxide syn-
thase . IL-10 is also a potent inhibitor of IL-12 produc-
tion by phagocytic cells . These data suggest that
ANXA1 stimulates the release of IL-10 which, in turn,
inhibits iNOS mRNA expression and, hence, nitric oxide
release. Furthermore, the release of IL-10 by ANXA1 may
also be responsible for the inhibition of IL-12 mRNA expres-
sion and, consequently, IL-12 synthesis. As for the mecha-
nisms involved in the IL-10 stimulation by ANXA1 it is
known that innate immune stimulators such as CpG DNA up-
regulate IL-10 production in macrophages by activating the
extra-cellular signal-regulated kinase (ERK) pathways .
Since endogenous ANXA1 promotes constitutive activation
of ERK  it is conceivable that ERK signalling pathway is
involved in the effect of the protein.
Effects of annexin 1 on leukocyte migration in
It is well established that one of the major anti-inflammato-
ry properties of glucocorticoids is the ability to inhibit
leukocyte migration. A series of papers from the laboratory
of Rod Flower and Mauro Perretti has revealed that ANXA1
may have profound effects on migration of neutrophils and
monocyte/macrophages thereby mediating the inhibitory
action of glucocorticoids. In a murine model of zymosan
peritonitis subcutaneous administration of dexamethasone
inhibited monocyte and neutrophil accumulation . The
steroid was no longer active in mice passively immunized
against full length recombinant ANXA1. An N-terminus
fragment of the protein was also able to inhibit leukocyte
migration. The role of ANXA1 as a mediator of anti-migra-
tory effects of glucocorticoids was reinforced by data by
Mancuso et al.  who showed, using an intravital micro-
scopy technique, that the in vivo administration of an antiin-
flammatory dose of dexamethasone increased ANXA1 lev-
els in circulating leukocytes. Most of the adherent leuko-
cytes subsequently detached and returned to the blood
stream, whereas those that had entered into the diapedesis
process exhibited a 3- to 4-fold longer latency than control
cells before transmigration. As expected these actions were
mimicked by an ANXA1 N-terminus peptide and inhibited
by polyclonal antibodies against the protein.
Effects on neutrophils
The above results were confirmed in a following paper 
where the intravenous administration of the recombinant pro-
tein promoted detachment of neutrophils adherent to the
endothelium of murine postcapillary venules. It was also
shown that the structurally related annexin 5 (ANXA5) had
no effect. A chimeric molecule constructed from the N-ter-
minus of ANXA1 attached to the core of region of ANXA5
produced cell detachment with kinetics similar to ANXA1
again suggesting that the N-terminal aminoacids of ANXA1
have a crucial role for the protein action. Subsequent data
 obtained through fluorescence-activated cell-sorting
(FACS) analysis and confocal microscopy demonstrated that
endogenous ANXA1 is mobilized and externalised following
neutrophil adhesion to endothelial monolayers in vitro or to
venule endothelium in vivo. The externalised protein acts to
promote the detachment of adherent neutrophils from
endothelium thus down-regulating neutrophil diapedesis.
This has been recently confirmed in a model of acute inflam-
mation by immunofluorescence and immunocytochemistry
Effects on monocytes
The adhesion of monocytic cells to endothelium is also
strongly inhibited by exogenous and endogenous ANXA1
as recently shown by Solito et al. . Exogenous pro-
tein inhibited the adhesion of U-937 cells to TNF-a-stimu-
lated endothelial monolayers. Again the N-terminal domain
appeared to be crucial in the protein-induced inhibition. The
role of endogenous ANXA1 was suggested by the fact that
a specific antibody against the protein reversed the inhi-
bition of adhesion induced by dexamethasone. Moreover
U-937 cells stably transfected with antisense clone for
ANXA1 (see above) displayed a reduced degree of dexam-
ethasone-induced inhibition of the adhesion to the endothe-
lium than control cells transfected with the vector alone. The
ability of ANXA1 to suppress the migration of monocytic
cells has been recently confirmed by using transfected U-
937 cells in vitro and in vivo . Cells stably transfected
with the sense clone overexpressing the active fragment of
ANXA1 showed a reduced transendothelial migration in vit-
ro in response to stromal cell-derived factor-1a (SDF-1a,
CXC chemokine ligand 12). For in vivo studies a validated
model of cell migration into human rheumatoid synovium
grafted onto SCID mice was used. The overexpression of
the bioactive fragment of ANXA1 inhibited U-937 migra-
tion to the synovial transplants in response to intragraft
injection of SDF-1a.
Vol. 53, 2004Anti-inflammatory effects of annexin 1 127
Mechanisms of anti-migratory action
Needless to say, the quest for the molecular mechanisms of
the inhibitory effects of ANXA1 on leukocyte adhesion and
transmigration is wide open. Although the picture is far from
complete, some recent data may help unravel the puzzle.
Very interesting data have been recently published by
Walther et al. . The paper suggests that ANXA1 and
related N-terminus peptides bind to and activate the formyl
peptide receptor (FPR) on neutrophils to inhibit their
transendothelial passage. The hypothesis is based on several
observations. Firstly, the compounds Boc 1 and Boc 2,
antagonists of the peptide formyl-Met-Leu-Phe (FMLP), the
classical FPR ligand, abolished the inhibitory effects of
ANXA1 peptides on neutrophil transmigration. Secondly,
ANXA1 peptides, like FMLP, caused internalization of
FPR-GFP in HeLa cells. Thirdly, N-terminus peptides and
FMLP stimulated transient [Ca2+]irises in cells stably trans-
fected with FPR. The same pattern of stimulation of FPR by
both ANXA1 peptides and FMLP is in apparent discrepan-
cy with the fact that ANXA1 and related peptides have anti-
inflammatory effects, whereas FMLP acts as a pro-inflam-
matory mediator. This issue was addressed in the same paper
 by comparing effects of low and high doses of the pep-
tides. It was found that high concentrations (100–200 mM)
of the ANXA1 peptides elicited, like FMLP, the production
of superoxide anions from neutrophils. Lower concentra-
tions of 10–20 mM, though effectively inhibiting transmi-
gration, did not trigger a respiratory burst. Moreover, low
concentrations of the peptides were able to induce receptor
desensitization to FMLP effects, thereby partially antago-
nising neutrophil activation elicited by FMLP. Low concen-
trations of ANXA1 peptides also stimulated transient [Ca2+]i
rises, as above reported, without activating mitogen-activat-
ed protein (MAP) kinase pathway. Finally, both an ANXA1
N-terminus peptide and FMLP at low concentrations
induced L-selectin shedding from neutrophil surface (see
below). These results suggest that ANXA1 peptides at low
doses block neutrophil transmigration, while activating the
cells at higher concentrations. In this context other authors
 have reported that ANXA1 inhibited L-selectin expres-
sion in both neutrophils and monocytes, but not in lympho-
cytes. The protein also induced L-selectin shedding from
neutrophils. Interestingly, the FPR antagonist Boc abolished
L-selectin shedding induced by FMLP, but not that induced
by ANXA1, suggesting that the ANXA1-induced shedding
was not mediated by the FPR. Since in this paper  the
full-length protein was used, it is possible that the N-termi-
nus peptides of ANXA1 and the full-length protein may act
through different mechanisms implying the involvement of
receptor(s) different from FPR.
The possible existence of other mechanisms and/or
receptors contributing to ANXA1 anti-migratory effects has
also been proposed by Perretti et al. . These authors have
investigated the effect of ANXA1 and related N-terminus
peptides on leukocyte migration in a model of acute inflam-
mation, the zymosan-induced peritonitis in wild-type and
FPR knock-out (KO) mice. The inhibition of neutrophil
migration caused by the intravenous administration of two
ANXA1 N-terminus peptides was abolished in wild-type
mice by the concomitant treatment with the FPR antagonists
Boc1 and Boc2. Moreover, the inhibitory effect of peptides
was no longer observed in FPR KO mice. This suggests that
N-terminus peptides act essentially through FPR supporting
previous data in vitro . On the other hand, the inhibition
of neutrophil migration induced by the full-length ANXA1
was only partially reversed in both wild-type Boc-treated and
FPR KO mice implying the involvement of other receptor(s)
A different receptor with high homology to FPR that
may be targeted by ANXA1 to inhibit neutrophil migration
is the lipoxin A4receptor (ALXR), also known as FPR-like 1
(FPRL1) receptor. The stimulation of this receptor by lipox-
in A4and aspirin-triggered lipoxins results in the inhibition
of neutrophil trafficking and responses in inflammation .
It has been shown that ANXA1 and lipoxin A4directly inter-
act with human ALXR/FPRL1 to synergize in inhibiting neu-
trophil activities in inflammation . The interaction of
ANXA1 with different receptors has been recently reviewed
by Mauro Perretti . More experimental work is warrant-
ed to precisely define the intracellular signalling pathways
mediated by these receptors.
Effect on adhesion molecules
A possible mechanism in regulating cell migration is the inter-
ference of ANXA1 with adhesion molecules that mediate
leukocyte-endothelium interactions. In experiments utilizing
confocal microscopy technique Solito et al.  have observed
that ANXA1 colocalized with the a4b1 integrin on the surface
of monocytic cells. It is known that a4b1 integrin interacts with
vascular cell adhesion molecule (VCAM)-1 expressed by
endothelial cells to mediate several neutrophil functions such
as tethering, rolling and firm arrest . In this light the rela-
tionship between VCAM-1 and ANXA1 was investigated. It
was shown that the extracellular domain of VCAM-1 reversed
the ANXA1-induced inhibition of U-937 cells adhesion to
endothelium. Co-immunoprecipitation experiments finally
demonstrated that ANXA1 and VCAM-1 competed for bind-
ing to the a4b1 integrin in U-937 cells .
A class of novel carboxylated N-glycans constitutively
expressed on endothelial cells has been recently described.
The functional block of these glycans by monoclonal anti-
bodies inhibited the extravasation of neutrophils and mono-
cytes in a murine model of peritoneal inflammation . A
subsequent paper by the same laboratory  has reported
that two proteins from activated neutrophils were able to bind
to the glycans, namely S100A8 and ANXA1. The binding of
ANXA1 to the glycans required an intact N-terminus.
Among the effects of ANXA1 on adhesion molecules,
the above reported stimulation of L-selectin shedding 
must be included. This has been very recently confirmed
by de Coupade et al.  who have proposed that endoge-
nous ANXA1 externalized on cell surface by glucocorti-
coids could facilitate L-selectin shedding through a calci-
um-dependent interaction with the selectin. The effects of
ANXA1 on adhesion molecules may well contribute to its
inhibitory effects on leukocyte-endothelium adhesion and
may promote the search for other molecules involved.
128 L. Parente and E. Solito
Annexin 1 null mice
The capability of ANXA1 to exert multiple effects in inflam-
mation has received an exciting confirmation by very recent
data obtained in mice lacking the ANXA1 gene . These
mice exhibited a complex phenotype that includes up-regula-
tion of the expression of COX-2 and cPLA2in some organs
like lungs and thymus. In these and other tissues (spleen,
stomach, kidney) an up-regulation of other annexins like
ANXA2, 4 and 6 was also observed whereas these annexins
were downregulated in the ovary and the liver. In car-
rageenin- and zymosan-induced inflammation, ANXA1 null
mice exhibited an exaggerated response characterized by an
increase in leukocyte migration and IL-1b generation. The
null mice were also partially resistant to the anti-inflamma-
tory effects of dexamethasone. Finally, leukocytes from the
null mice exhibited anomalies in migratory behaviour,
phagocytosis, and expression of adhesion molecules .
Apoptosis, glucocorticoids, annexin 1
A detailed discussion on apoptosis is beyond the scope of this
article. Excellent, updated reviews are available [47–51].
Here the connections of apoptosis to the inflammatory
process and the recent described effects of ANXA1 on apop-
tosis will be discussed in connection to the apoptotic effects
Apoptosis is a physiological cell death process in which
cells are removed from the body without eliciting an inflam-
matory response. During apoptosis a number of specific mor-
phological alterations take place, including cell shrinkage,
chromatin condensation, DNA fragmentation with the forma-
tion of apoptotic bodies. Apoptotic cells and bodies are rapid-
ly recognised and engulfed by neighbouring cells and phago-
cytes, thereby preventing complications that would result
from a release of intracellular contents (see below). Apoptosis
is a finely regulated process in which many specific proteins
play pro- and anti-apoptotic roles. Bcl-2 and related cytoplas-
mic proteins are key regulators of apoptosis (reviewed in
). Some members of this superfamily of proteins promote
cell survival by inhibiting the activation of caspases, whereas
other members promote apoptosis by blocking the effects of
the pro-survival proteins. Caspases are a family of cysteine
proteases considered as the central executioners of apoptotic
death (reviewed in [49, 51]). These enzymes can contribute
directly to cell disassembly by destroying nuclear lamina 
or indirectly cleaving several proteins involved in cytoskeletal
regulation . Caspases can also inactivate proteins that pro-
tect living cells from apoptosis like the inhibitor of caspase-
activated deoxyribonuclease (ICAD), the enzyme responsible
for DNA fragmentation .
Several mechanisms contribute to the resolution of
inflammatory reactions. Among these, apoptosis of inflam-
matory cells and their removal from the inflammatory site
through engulfment by professional phagocytes represent
key phenomena as the ingestion of apoptotic cells and bodies
does not trigger the release of pro-inflammatory mediators
from phagocytes (reviewed in [55, 56]). Both glucocorticoids
and ANXA1 may have important roles in these biological
processes aimed to restore homeostasis.
Glucocorticoids promote apoptosis in many cell types
including lymphocytes , eosinophils , monocytes
, but prevent apoptosis of neutrophils [58, 60]. The com-
plex molecular and biochemical pathways of glucocorticoid-
induced apoptosis have been recently reviewed by Distel-
horst . Although many questions still await to be
answered, some points may be fixed.
(i)Both transrepression and transactivation activities of the
glucocorticoid receptor are required for induction of
apoptosis, while target genes remain uncertain. Inhibi-
tion of expression and activity of transcription factors
like AP-1  and NF-kB  by glucocorticoids are
very likely to contribute to their lethal effects.
(ii) Overexpression of Bcl-2 inhibits apoptosis by glucocor-
ticoids by modulating calcium fluxes across the endo-
plasmic reticulum (ER) membrane . It is known that
glucocorticoids up-regulate expression of the inositol
triphosphate receptor (IP3R) in lymphocytes undergo-
ing apoptosis . This suggest that calcium release
from the ER, via the IP3R, leads to enhanced cytosolic
calcium concentration triggering effector pathways of
(iii) Bcl-2 may also work by inhibiting proteasome activa-
tion . In glucocorticoid-induced apoptosis, the mul-
ticatalytic proteasome complex may target and degrade
several pro-survival proteins including transcription
factors like AP-1 and NF-kB , apoptosis inhibitory
proteins like the c-inhibitor of apoptosis (IAP)-1 and the
X-linked inhibitor of apoptosis (XIAP) , and the
cyclin dependent kinase inhibitor, p27Kip1. Interest-
ingly, the involvement of the proteasome in apoptosis is
a peculiar characteristic of the glucocorticoid death
pathway as the proteasome appears dispensable in Fas-
induced apoptosis .
(iv) The involvement of caspases in glucocorticoid-induced
apoptosis has been differently reported. Some reports
have suggested that activation of caspase-3 is necessary
for glucocorticoid-induced apoptosis showing that cas-
pase-3 is activated in dexamethasone-treated lympho-
cytes , while others have indicated that caspase-6
rather than caspase-3 is activated in glucocorticoid-treat-
ed lymphoid cells . Recently, Komoriya et al. 
using flow cytometry and confocal microscopy have
investigated the order of the sequence of events in the
caspase cascade in vivo. In dexamethasone-treated thy-
mocytes the pattern of sequential caspase activation
implied APAF-1-mediated activation of caspase-9 fol-
lowed by caspase-1, caspase-6, caspase-8, and caspases-
3 and -7. The overall picture indicates that there may be
more than one pathway to glucocorticoid-induced apop-
tosis in different cell subtypes, one caspase-9-dependent
pathway and one caspase-3-dependent pathway .
The first indication of the involvement of ANXA1 in apop-
tosis came from a paper by McKanna  showing that
ANXA1 expression dramatically increased in alveolar cells
of mammary ducts undergoing apoptosis in post-lactational
regression. One year later Sakamoto et al.  reported that
Vol. 53, 2004Anti-inflammatory effects of annexin 1 129
exogenous ANXA1 facilitated H2O2-induced apoptosis of rat
thymocytes. More recent studies have shown that overex-
pression of ANXA1 in U-937 cells  and in broncho-alve-
olar epithelial cells  promoted apoptosis associated with
caspase-3 activation. Finally, exogenous ANXA1 stimulated
transient rise in intracellular calcium concentrations accom-
panied by dephosphorylation of the pro-apoptotic protein
Bad and apoptosis of neutrophils . It has been suggested
that following an increase in cytosolic calcium, the calcium-
activated protein phosphatase calcineurin dephosphorylates
Bad, which allows Bad to associate with mitochondria, het-
erodimerize with Bcl-xLand promote apoptosis .
The experimental evidence above discussed suggests that
ANXA1 may mediate the pro-apoptotic effects of glucocor-
ticoids in some cells by activating caspase-3 and/or acting on
calcium fluxes. Other possible mechanisms of apoptosis
shared by glucocorticoids and ANXA1 my be linked to the
inhibition of eicosanoid and nitric oxide synthesis. It has
been shown that both PGE2 and nitric oxide  can pre-
vent apoptosis in lymphocytes. On the other hand, there is an
apparent conflict with regard to the effects on neutrophils,
with glucocorticoids protecting these cells from apoptosis
,  and ANXA1 promoting neutrophil death . Fur-
ther studies are necessary to clarify this contrasting point.
However it is important to keep in mind that in lymphoid
cells glucocorticoids do promote apoptosis, but can also pro-
tect from death induced by other stimuli like antigen-T cell
receptor interaction (reviewed in ). It is possible that also
in neutrophils glucocorticoid may have either positive or neg-
ative effects on apoptosis since NF-kB activation protects
human neutrophil from apoptosis  and inhibition of NF-
kB activation represents one of the pro-apoptotic mecha-
nisms of glucocorticoids , as above discussed.
The clearance of apoptotic cells and bodies
In recent years the mechanisms underlying the recognition of
apoptotic cells by phagocytes and the safe removal of apop-
totic cells and bodies have been widely investigated [56, 83].
Specific ‘eat-me’signals on apoptotic cells serve as markers
for phagocytes to recognize the apoptotic cells and to ingest
them. The best studied of these signals is the exposure of
phosphatidylserine (PS) on the outer leaflet of the plasma
membrane (reviewed in ). It has been shown that PS-
dependent ingestion of apoptotic cells by macrophages
inhibit the release of pro-inflammatory cytokines like IL-
1b,TNF-a, IL-8 , but stimulates the release of anti-
inflammatory cytokines like TGF-b1 [85, 86] and IL-10 .
This may well explain why the ingestion of apoptotic cells
and bodies by phagocytes does not trigger an inflammatory
response. It is also known that glucocorticoids are able to
promote non-inflammatory phagocytosis of apoptotic neu-
trophils by macrophages . Very recently Arur et al. 
have suggested that ANXA1 may be an endogenous ligand
mediating engulfment of apoptotic cells. They have demon-
strated that ANXA1 is recruited to the PS-rich domains of
apoptotic cell surface and that this recruitment requires cas-
pase activity and the release of intracellular calcium. Impor-
tantly, silencing ANXA1 protein by siRNA resulted in defec-
tive tethering and engulfment of apoptotic cells. Given the
potent anti-inflammatory effects of ANXA1 and its ability to
stimulate IL-10 release , it is tempting to suggest that
ANXA1 is part of the anti-inflammatory signalling elicited
by recognition and engulfment of apoptotic cells by phago-
cytes, dampening inflammatory responses and allowing safe
post-apoptotic clearance of dead cells.
Since its discovery as an anti-phospholipase protein, ANXA1
has come a long way to encompass a wide range of anti-
inflammatory properties (see Table 1). However, further
studies are warranted to precisely define the signal transduc-
tion mechanisms of its effects on leukocyte migration and
apoptosis. Hopefully, these studies will tell whether in the
future glucocorticoids could be replaced by ANXA1-derived
compounds in some therapeutic applications against
Acknowledgements. This work is supported by grants from the Univer-
sity of Salerno (60% 2003), from the Italian Ministry of Research
(COFIN 2002), and from the Wellcome Trust (grant no.
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