Annexin-1 regulates macrophage IL-6 and TNF via glucocorticoid-induced leucine zipper.
ABSTRACT Annexin-1 (ANXA1) is a mediator of the anti-inflammatory actions of endogenous and exogenous glucocorticoids (GC). The mechanism of ANXA1 effects on cytokine production in macrophages is unknown and is here investigated in vivo and in vitro. In response to LPS administration, ANXA1(-/-) mice exhibited significantly increased serum IL-6 and TNF compared with wild-type (WT) controls. Similarly, LPS-induced IL-6 and TNF were significantly greater in ANXA1(-/-) than in WT peritoneal macrophages in vitro. In addition, deficiency of ANXA1 was associated with impairment of the inhibitory effects of dexamethasone (DEX) on LPS-induced IL-6 and TNF in macrophages. Increased LPS-induced cytokine expression in the absence of ANXA1 was accompanied by significantly increased LPS-induced activation of ERK and JNK MAPK and was abrogated by inhibition of either of these pathways. No differences in GC effects on MAPK or MAPK phosphatase 1 were observed in ANXA1(-/-) cells. In contrast, GC-induced expression of the regulatory protein GILZ was significantly reduced in ANXA1(-/-) cells by silencing of ANXA1 in WT cells and in macrophages of ANXA1(-/-) mice in vivo. GC-induced GILZ expression and GC inhibition of NF-kappaB activation were restored by expression of ANXA1 in ANXA1(-/-) cells, and GILZ overexpression in ANXA1(-/-) macrophages reduced ERK MAPK phosphorylation and restored sensitivity of cytokine expression and NF-kappaB activation to GC. These data confirm ANXA1 as a key inhibitor of macrophage cytokine expression and identify GILZ as a previously unrecognized mechanism of the anti-inflammatory effects of ANXA1.
- SourceAvailable from: endocrinology-journals.org[show abstract] [hide abstract]
ABSTRACT: Synthetic glucocorticoids (GCs) potently inhibit the expression of pro-inflammatory genes and are widely used in the treatment of inflammatory diseases. However, some patients are resistant to the therapeutic effects of GCs, and many suffer deleterious side effects from these drugs. Furthermore, the precise mechanisms by which GCs inhibit pro-inflammatory gene expression remain unclear. A number of recent papers report that GCs induce the sustained expression of MAP kinase (MAPK) phosphatase 1 (MKP-1), a negative regulator of MAPK signal transduction pathways. The potential relevance of MKP-1 to some of the biological effects of GCs is discussed.Journal of Endocrinology 08/2003; 178(1):5-12. · 4.06 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Annexins are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family with members of the family being expressed throughout animal and plant kingdoms. Structurally, annexins are characterized by a highly alpha-helical and tightly packed protein core domain considered to represent a Ca2+-regulated membrane binding module. Many of the annexin cores have been crystallized, and their molecular structures reveal interesting features that include the architecture of the annexin-type Ca2+ binding sites and a central hydrophilic pore proposed to function as a Ca2+ channel. In addition to the conserved core, all annexins contain a second principal domain. This domain, which NH2-terminally precedes the core, is unique for a given member of the family and most likely specifies individual annexin properties in vivo. Cellular and animal knock-out models as well as dominant-negative mutants have recently been established for a number of annexins, and the effects of such manipulations are strikingly different for different members of the family. At least for some annexins, it appears that they participate in the regulation of membrane organization and membrane traffic and the regulation of ion (Ca2+) currents across membranes or Ca2+ concentrations within cells. Although annexins lack signal sequences for secretion, some members of the family have also been identified extracellularly where they can act as receptors for serum proteases on the endothelium as well as inhibitors of neutrophil migration and blood coagulation. Finally, deregulations in annexin expression and activity have been correlated with human diseases, e.g., in acute promyelocytic leukemia and the antiphospholipid antibody syndrome, and the term annexinopathies has been coined.Physiological Reviews 05/2002; 82(2):331-71. · 30.17 Impact Factor
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ABSTRACT: Neutrophils have been implicated in mediating much of the tissue damage associated with chronic inflammatory diseases such as rheumatoid arthritis, where they are involved in destruction of both cartilage and bone. Glucocorticoids are powerful anti-inflammatory agents, often used in the treatment of this autoimmune disease. They exert significant inhibitory effects on neutrophil activation and functions, such as chemotaxis, adhesion, transmigration, apoptosis, oxidative burst, and phagocytosis. The mechanisms by which glucocorticoids exert these effects on neutrophils are unclear. Evidence from studies of inflammation in human subjects and animal models suggests that annexin-I an endogenous, glucocorticoid-induced protein also known as lipocortin-1, has a pivotal role in modulating neutrophil activation, transmigratory, and phagocytic functions. Furthermore, we present evidence for altered neutrophil functions in rheumatoid arthritis that correspond to a significantly reduced capacity of these cells to bind annexin-I. A proposed novel pathway for glucocorticoid actions on neutrophils involving annexin-I could explain the development of chronic neutrophil activation in diseases such as rheumatoid arthritis.Inflammation Research 11/1998; 47 Suppl 3:S158-65. · 1.96 Impact Factor
Annexin-1 Regulates Macrophage IL-6 and TNF via
Glucocorticoid-Induced Leucine Zipper1
Yuan H. Yang,2Daniel Aeberli, April Dacumos, Jin R. Xue, and Eric F. Morand
Annexin-1 (ANXA1) is a mediator of the anti-inflammatory actions of endogenous and exogenous glucocorticoids (GC). The
mechanism of ANXA1 effects on cytokine production in macrophages is unknown and is here investigated in vivo and in vitro. In
response to LPS administration, ANXA1?/?mice exhibited significantly increased serum IL-6 and TNF compared with wild-type
(WT) controls. Similarly, LPS-induced IL-6 and TNF were significantly greater in ANXA1?/?than in WT peritoneal macrophages
in vitro. In addition, deficiency of ANXA1 was associated with impairment of the inhibitory effects of dexamethasone (DEX) on
LPS-induced IL-6 and TNF in macrophages. Increased LPS-induced cytokine expression in the absence of ANXA1 was accom-
panied by significantly increased LPS-induced activation of ERK and JNK MAPK and was abrogated by inhibition of either of
these pathways. No differences in GC effects on MAPK or MAPK phosphatase 1 were observed in ANXA1?/?cells. In contrast,
GC-induced expression of the regulatory protein GILZ was significantly reduced in ANXA1?/?cells by silencing of ANXA1 in WT
cells and in macrophages of ANXA1?/?mice in vivo. GC-induced GILZ expression and GC inhibition of NF-?B activation were
restored by expression of ANXA1 in ANXA1?/?cells, and GILZ overexpression in ANXA1?/?macrophages reduced ERK MAPK
phosphorylation and restored sensitivity of cytokine expression and NF-?B activation to GC. These data confirm ANXA1 as a key
inhibitor of macrophage cytokine expression and identify GILZ as a previously unrecognized mechanism of the anti-inflammatory
effects of ANXA1. The Journal of Immunology, 2009, 183: 1435–1445.
matory genes, and to up-regulate certain anti-inflammatory genes,
has been widely exploited in the treatment of inflammatory dis-
eases with exogenous GC (1). Annexin-1 (ANXA1) was originally
identified as a GC-regulated protein and was previously termed
lipocortin 1. ANXA1 has long been suggested to function as a
cellular mediator of the anti-inflammatory effects of GC. ANXA1
expression and secretion in several cell types is induced by GC (2).
Exogenous ANXA1, or an N-terminal ANXA1 peptide, mimic
many inhibitory effects of GC, including inhibition of leukocyte
recruitment at inflammatory sites (3–5), inhibition of proinflam-
matory mediators such as phospholipase A2, cyclooxygenase-2
(COX2), and NO, induction of apoptosis in inflammatory cells,
and induction of the anti-inflammatory cytokine IL-10 (reviewed
in Ref. 6). The absence of ANXA1 is associated with increased
lethality in experimental endotoxemia (7) and with exacerbation of
lucocorticoids (GC)3are potent anti-inflammatory and
immunosuppressive agents. The ability of endogenous
GC to suppress the expression of a variety of proinflam-
zymosan-induced acute inflammation (8) and several models of
experimental arthritis (9–11). Because of its induction by GC and
anti-inflammatory effects, a role of ANXA1 in the regulation of
GC sensitivity has been reported in models of acute and chronic
inflammation, including carrageenin-induced paw edema, zymo-
san-induced peritonitis, and Ag-induced arthritis (8, 11, 12).
In several of these models, inhibitory effects of ANXA1 on cy-
tokine expression have been observed. For example, in Ag-in-
duced arthritis, ANXA1 deficiency was associated with increased
synovial expression of TNF, IL-6, and other cytokines. In exper-
imental endotoxemia, the absence of ANXA1 is also associated
with increased serum IL-6 and TNF (7). In both these examples,
the dominant cellular source of cytokines is macrophages, and
macrophage TNF and IL-1 release in vitro have been reported to
be increased in the absence of ANXA1 (7).
Macrophage cytokine expression is induced via activation of
numerous signaling pathways, including MAPK and NF-?B path-
ways (13), but the mechanisms of ANXA1 effects on cytokine
expression, during inflammation and under the influence of GC,
are not known. At least two GC-induced regulatory proteins are
involved in controlling MAPK- and NF-?B-regulated cytokines,
namely MAPK phosphatase 1 (MKP-1; also known as DUSP1)
and GC-induced leucine zipper (GILZ) (14, 15). MKP-1 dephos-
phorylates activated MAPK and acts as a key negative regulator of
TLR-induced inflammation in vivo (16–19). Although a stimula-
tory effect of ANXA1 on ERK activation has been suggested in
stably transfected RAW 264.7 cells (20), we recently reported that
in cultured fibroblasts, endogenous ANXA1 inhibits MAPK acti-
vation via up-regulation of MKP-1(21). GILZ interacts directly
with proinflammatory transcription factors, including NF-?B (22)
and AP-1(23). The potential effect of ANXA1 on GILZ has not
been investigated previously.
The present studies were designed to investigate mechanisms of
inflammatory regulation by ANXA1 in macrophages. We demon-
strate that ANXA1 exerts tonic inhibitory effects on LPS-induced
Centre for Inflammatory Diseases, Department of Medicine, Monash University,
Clayton, Victoria, Australia
Received for publication December 1, 2008. Accepted for publication May 13, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the National Health and Medical Research Council,
Australia. D. A. was supported by grants from Novartis and the Swiss Foundation for
Research in Medicine and Biology.
2Address correspondence and reprint requests to Dr. Yuan H. Yang, Centre for In-
flammatory Diseases, Monash University Department of Medicine, Monash Medical
Centre, Locked Bag 29 Clayton, Victoria 3168, Australia. E-mail address:
3Abbreviations used in this paper: GC, glucocorticoid; ANXA1, annexin-1; COX2,
cyclooxygenase-2; DEX, dexamethasone; GILZ, glucocorticoid-induced leucine zip-
per; MKP-1, MAPK phosphatase 1; siRNA, small interfering RNA; WT, wild type;
FPR, formyl peptide receptor.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
macrophage IL-6 and TNF release and MAPK and NF-?B activa-
tion and participates in GC induction of GILZ. These data indicate
that GILZ is a previously unrecognized target of the anti-
inflammatory effects of ANXA1 in macrophages.
Materials and Methods
ANXA1?/?mice were generated as described previously (8). ANXA1?/?
and wild-type (WT) littermates used in this study are of mixed 129/SvJ ?
C57BL/6 background. Mice were bred and housed under specific patho-
gen-free conditions. Litters from heterozygous ANXA1?/?parents were
genotyped to select homozygous ANXA1?/?and WT mice. Genotyping
was performed using a standard PCR protocol as described previously (8).
All experiments were approved by the Monash University Animal Re-
search Ethics Committee.
Endotoxemia and DEX treatment
Endotoxemia was induced in male 8- to 10-wk-old ANXA1?/?and WT
mice. Animals were injected i.p. with 10 mg/kg LPS from Escherichia coli
O111.B4 (Sigma-Aldrich) dissolved in saline. DEX (Sigma-Aldrich) at a
dose of 0.5 mg/kg was injected i.p. 1 h before LPS (1 or 10 mg/kg). Blood
was collected at indicated times. Ethical considerations precluded the use
of a lethality outcome in these studies.
Measurement of cytokines and NO
Concentrations of IL-6 and TNF in serum and culture supernatants were
measured using commercially available ELISA (Quantikine M; R&D Sys-
tems). The sensitivity of these assays was 15.6 pg/ml. Supernatant cyto-
kines were also determined by mouse inflammation kit of cytometric bead
array (BD Biosciences) as described in the instructions of the manufac-
turer. The samples were analyzed by flow cytometry with a Cytomation
Mo-Flo flow cytometer (DakoCytomation).
NO was determined by measuring the amount of nitrite in culture su-
pernatants using Griess reagent as described previously (24). Peritoneal
macrophages were stimulated with LPS (1 ?g/ml) in the presence or ab-
sence of DEX for 48 h. The limit of sensitivity of this assay was 1.56
?mol/L, and the absorbance was read at 540 nm. Nitrite concentration was
determined using sodium nitrite (Ajax Chemicals).
Peritoneal macrophage culture
Primary peritoneal macrophages were prepared from mice by saline flush-
ing or using thioglycollate elicitation, as described previously (25). Cells
were cultured in 5% FCS/DMEM at 37°C for 3 h. After washing off non-
adherent cells, adherent monolayer macrophages were cultured overnight
in complete medium. Cells were stimulated with LPS and/or DEX, and/or
specific MAPK inhibitors (Alexis Biochemicals) of p38 (SB203580),
ERK1/2 (PD98059), and JNK (SP600125), in indicated concentrations for
4 h. Macrophages were treated with Boc2 (100 ?M), a pan antagonist of
formyl peptide receptor (FPR) receptors (Gen Script), in the presence or
absence of DEX for 3 h. Cells were also treated with Ac2-26 (100 ?g/ml)
for 3 h. Supernatants were collected, and cells were then resuspended in
TRIzol reagent (Invitrogen) or RNeasy mini kit (Qiagen) for RNA
Western blot analysis
Western blotting was performed as described previously (26). In brief, total
cell protein was measured by BCA Protein assay kit (Quantum Scientific).
Forty micrograms of protein was separated on 10% SDS-polyacrylamide
actions of LPS and DEX in vivo. WT
and ANXA1?/?littermates were in-
jected with LPS alone over 4 h (A and
B) or LPS for 1.5 h (C and D) in the
presence or absence of DEX. Sera
were analyzed by ELISA. LPS (10
mg/kg)-induced IL-6 (A) and TNF (B)
and the inhibitory effect of DEX on
IL-6 and TNF induced by LPS 10
mg/kg (C) or 1 mg/kg (D). Each bar
represents mean ? SEM. ?, p ? 0.05
ANXA1?/?vs WT mice; †, p ?
0.05; ††, p ? 0.01 LPS vs LPS/DEX.
n ? 7–9 mice each group.
Effect of ANXA1 on
1436ANNEXIN-1, GLUCOCORTICOID, AND GILZ
electrophoresis gels and transferred to Hybond-C extra nitrocellulose mem-
branes (Millipore). Membranes were probed with Abs against phospho-
ERK, phospo-p38, or phospho-JNK (mAbs; Cell Signaling Technology),
MKP-1 (polyclonal; Cell Signaling Technology), GILZ (polyclonal; pro-
vided by Prof. C. Riccardi, University of Perugia, Perugia, Italy), and ?-ac-
tin (mAb; Sigma-Aldrich). Anti-mouse and anti-rabbit Abs conjugated to
Alexa Fluor 700 (Rockland) and IRDye 800 (Rockland), respectively, were
used to probe primary Abs. Protein bands were detected and quantified by
Western blotting with the Odyssey system (Li-Cor). Densitometry ratios
were normalized to ?-actin content and expressed as arbitrary units (AU).
Flow cytometry analysis
Flow cytometry was performed as described previously (21). Briefly, peri-
toneal macrophages obtained from WT and ANXA1?/?mice treated with
vehicle or DEX (0.5 mg/kg) for 3 h, or thioglycollate-elicited macrophages
in RPMI 1640 with 5% FBS were stimulated with DEX for 3 h at 37°C
before fixation with 2% formaldehyde for 10 min. The cells were then
pelleted, resuspended in ice-cold methanol, and incubated for 30 min at
4°C. After washing, cells were stained with control rabbit IgG (Abacus
ALS; Vector Laboratories) or rabbit anti-GILZ Ab (Santa Cruz Biotech-
nology) for 1 h and subsequently incubated with donkey anti-rabbit-FITC
(Biolab) for 30 min at room temperature. Samples were washed and re-
suspended in media for analysis.
Total RNA (0.5 ?g) was reverse transcribed using Superscript III reverse
transcriptase (Invitrogen) and oligo(dT)20. PCR amplification was per-
formed on a Rotor-Gene 3000 (Corbett Research). Murine IL-6, TNF,
MKP-1, and ?-actin primers were used as described previously (27). GILZ
primers have been reported previously (22). The primers used for COX2
were 5?-TTG AAG GTG TCG GGC AGC-3? (forward) and 5?-CAG AAC
CGC ATT GCC TCT G-3? (reverse) and for 18S were 5?-GTAACCCGT
For PCR, each of the standard and sample cDNA dilutions was added to
individual capillary tubes. Amplification (40 cycles) was conducted in a
total volume of 10 ?l containing primer concentrations of SYBR Green I
kit (Sigma-Aldrich). Melting curve analysis was performed at the end of
each PCR. Control reactions for product identification consisted of ana-
lyzing the melting peaks (°C) and determining the length of the PCR prod-
ucts (base pair) by agarose gel electrophoresis. Relative quantification of
target mRNA expression was calculated and normalized to ?-actin or 18S
actions of LPS and DEX in perito-
neal macrophages. Peritoneal mac-
rophages were incubated with LPS
(100 ng/ml) in the presence or ab-
sence of DEX for 4 h. Supernatants
were analyzed for IL-6 and TNF by
ELISA (A–C) and mRNA by real-
time PCR (D). A, Cytokine induc-
tion in resting macrophages; B,
cytokine induction in thioglycol-
phages; C, effect of DEX on LPS-
induced IL-6 and TNF release; and
D, effect of transfection of an
ANXA1 plasmid compared with
control transfection (PCDNA3.1)
ANXA1?/?macrophages. Each bar
represents mean ? SEM of six ex-
periments. ?, p ? 0.05, ??, p ? 0.01
ANXA1?/?cells vs WT; †, p ?
0.05 LPS/DEX vs LPS.
Effect of ANXA1 on
1437The Journal of Immunology
expression. The results are presented as the fold induction of mRNA ex-
pression relative to the amount present in control samples.
Small interfering RNA (siRNA) and plasmid transfection
Synthetic murine ANXA1 siRNA oligoribonucleotides with symmetric 3?
TT overhangs were purchased from Santa Cruz Biotechnology. Peritoneal
macrophages were transiently transfected with ANXA1 siRNA or control
siRNA using oligofectamine (Invitrogen) and incubated at 37°C for 48 h.
Cells then were treated with DEX (10?7M) and/or LPS (0.1 ?g/ml) for
indicated times (4 h for mRNA and 24 h for supernatant cytokine beads).
A full-length cDNA encoding ANXA1 (provided by Prof. M. Perretti)
and GILZ (provided by Prof. C. Riccardi, William Harvey Research In-
stitute, London, U.K.) was inserted into pcDNA3.1 (Invitrogen) as de-
scribed previously (28). For peritoneal macrophages, 1 ? 106cells were
transfected with 2 ?g of plasmid DNA using the AMAXA system (Amaxa
Biosystems) with mouse macrophage nucleofector kit. As a control, cells
were also transfected with the empty pcDNA3.1 vector. After electropo-
ration, cells were immediately transferred to 6- and 24-well plates with
complete medium and cultured at 37°C for 24 h. Transfected cells were
treated with LPS or DEX or a combination as indicated times. Transfection
efficiency was identified by quantitative RT-PCR and Western blot
NF-?B activation assay and luciferase assay
Phosphorylation of NF-?B p65 Ser536was measured with the ELISA-based
PathScan Phospho-NF-?B p65 (Ser536) kit and total-NF-?B p65 kit (Cell
Signaling Technology), according to the manufacturer’s instructions.
Briefly, whole-cell lysates were prepared from peritoneal macrophages cul-
tured in the presence or absence of DEX and/or LPS for 1 h. Ninety-six-
well microtiter plates were coated with anti-NF-?B capture Abs, and
Ser536-phosphorylated p65 and total p65 were detected using specific Abs.
Results are expressed as absorbance values. All measures were performed
Peritoneal macrophages were transiently cotransfected with an NF-?B
luciferase plasmid construct (NF-?B-Luc plasmid) (29), an ANXA1 plas-
mid, GILZ plasmid, or PcDNA3.1 using Nucleofector kit and the AMAXA
system. In other experiments, peritoneal macrophages were cotransfected
with GILZ-luc (human GILZ-luc; a gift from Prof. M. Pallardy, Univ-Paris
Sud, Cha ˆtenay-Malabry, France) (30, 31) and ANXA1 plasmid. Trans-
fected cells were treated with LPS (100 ng/ml) in the presence or absence
of DEX (10?7M) for 18 h. Luciferase activity was measured using the
Luciferase Assay System (Promega), according to the manufacturer’s in-
structions. Luminescence measured using a Wallac Victor 2 luminometer
Data were analyzed using the Mann-Whitney two-sample rank test to de-
termine the level of significance between means of groups or the Student’s
t test for comparison of continuous variables. Results are expressed as the
mean ? SEM. A p value ?0.05 was considered statistically significant.
Role of ANXA1 in the regulation of cytokines and GC sensitivity
We first sought to confirm the reported effect of ANXA1 on the
secretion of inflammatory cytokines in vivo. Administration of
LPS (10 mg/kg) induced the release of serum IL-6 and TNF in WT
mice over 4 h (Fig. 1, A and B). Significantly greater increases of
serum IL-6 and TNF were observed in ANXA1?/?mice in com-
parison with WT mice (p ? 0.05). The role of ANXA1 in the
inhibitory effects of GC on LPS responses in vivo has not been
investigated previously. We treated WT and ANXA1?/?mice
with DEX (0.5 mg/kg) 1 h before LPS administration for 1.5 h.
DEX significantly inhibited LPS-induced serum IL-6 in WT mice
at both high (10 mg/kg) and low (1 mg/kg) doses of LPS (Fig. 1,
C and D). However, the inhibitory effect of DEX on IL-6 was
reached statistical significance only in ANXA1?/?mice treated
with low-dose LPS (1 mg/kg). In contrast, the effect of DEX on
LPS-induced serum TNF was comparable in ANXA1?/?and WT
mice (Fig. 1, C and D).
Role of ANXA1 in the regulation of cytokines and GC sensitivity
To explore the mechanism of the effects of ANXA1 on cytokine
release, we first sought to confirm these findings in vitro. We first
investigated LPS responses in resting and thioglycollate-elicited
peritoneal macrophages from WT and ANXA1?/?mice. Com-
pared with WT cells, LPS-induced IL-6 and TNF release was
significantly greater in ANXA1?/?resting (Fig. 2A) and thiogly-
collate-elicited (Fig. 2B) macrophages. We also observed that
LPS-induced NO production (WT undetectable, ANXA1?/?
16.3 ? 7 ?M, p ? 0.05) and COX2 mRNA expression (WT 12 ?
LPS-induced MAPK activation in
peritoneal macrophages. Peritoneal
macrophages were treated with LPS
(100 ng/ml) in the presence or ab-
sence of DEX (10?7M). A, LPS-in-
duced MAPK activation and MKP-1
in macrophages were measured by
Western blot analysis. A representa-
tive experiment (left) and the mean
and SE relative densitometry values
of three separate experiments (right).
B, The effect of MAPK inhibitors di-
rected at ERK (50 ?M PD98059) or
JNK (50 ?M SP600125) on LPS-in-
duced cytokines. Each bar represents
mean ? SEM of separate different ex-
periments (n ? 3 duplicated experi-
ments). ?, p ? 0.05 and ??, p ? 0.01
ANXA1?/?cells vs WT.
Effect of ANXA1 on
1438ANNEXIN-1, GLUCOCORTICOID, AND GILZ
3, ANXA1?/?29 ? 5, p ? 0.05) were significantly increased in
ANXA1?/?macrophages. Taken together with the in vivo find-
ings, these data demonstrate that endogenous ANXA1 exerts a
tonic inhibitory effect on macrophage activation in response
We next sought to analyze the role of ANXA1 in the actions of
GC on macrophages. Resting macrophages were treated with LPS
in the presence or absence of DEX. Treatment with DEX at con-
centrations of 10?8to 10?6M significantly suppressed LPS-in-
duced in WT peritoneal macrophages (Fig. 2C). The inhibitory
effect of DEX on IL-6 and TNF was also detected in ANXA1?/?
cells but was significantly impaired in ANXA1?/?cells in com-
parison to WT (Fig. 2C), suggesting ANXA1 contributes to the
anti-inflammatory effects of GC on macrophages.
To confirm this effect of ANXA1, we restored ANXA1 expres-
sion in ANXA1?/?cells by transient transfection. Overexpression
of ANXA1 in ANXA1?/?cells restored DEX inhibition of LPS-
induced IL-6 and TNF mRNA (Fig. 2D). Taken together, with the
in vivo findings, these results demonstrate a role for ANXA1 in
macrophage sensitivity to GC.
Role of ANXA1 in the regulation of ERK and JNK MAPK
We have previously reported that endogenous ANXA1 inhibits
IL-6 release by fibroblasts by inhibition of MAPK activity (21).
We therefore investigated whether MAPK activity was involved
in the increased cytokine release and decreased GC sensitivity
observed in ANXA1?/?macrophages. MAPK activation in re-
sponse to LPS, as measured by phosphorylation of ERK, p38,
and JNK MAPK, was detected in WT macrophages (Fig. 3A).
LPS-induced phosphorylation of ERK and JNK was modestly
but significantly increased in ANXA1?/?cells in comparison
with WT, but LPS-induced phosphorylation ofp38 MAPK was
comparable in WT and ANXA1?/?cells (Fig. 3A). The in-
volvement of ERK and JNK MAPK in the regulation of LPS-
induced IL-6 and TNF by ANXA1 was further investigated
using MAPK inhibitors. Inhibition of either ERK or JNK
MAPK inhibited LPS-induced IL-6 and TNF release from
ANXA1?/?peritoneal macrophages to levels observed from
LPS-stimulated WT cells in the absence of inhibitors (Fig. 3B).
Taken together, these data suggest ANXA1-mediated inhibition
of ERK and JNK phosphorylation may be important in ANXA1
regulation of macrophage responses to LPS.
We next investigated whether altered MKP-1 expression is in-
volved in ANXA1 regulation of MAPK activation in macrophages.
MKP1 is potently induced by GC and is a mechanism through
which GC inhibits the p38 and JNK MAPK pathways. As shown
in Fig. 3A, LPS-induced MKP1 expression was equivalent in WT
and ANXA1?/?cells. As shown in Fig. 4A, MKP1 expression was
dose-dependently induced by DEX in WT macrophages, and par-
allel effects were observed in ANXA1?/?cells, with no significant
differences observed. These results suggested MKP-1 was not re-
sponsible for the observed effects of ANXA1 deficiency on cyto-
kine expression and MAPK activation. Because ERK MAPK is not
significantly inhibited by MKP-1, we examined the effects of DEX
and ANXA1 on ERK activation directly. Macrophages were
treated with DEX for 3 h before stimulation with LPS for 30 and
60 min, and phospho-ERK activity was detected by Western blot-
ting. As noted above, in the absence of ANXA1, LPS-induced
phospho-ERK was significantly increased (Fig. 4B). No significant
inhibitory effect of DEX on LPS-induced ERK activity was ob-
served in either WT or ANXA1?/?cells, suggesting that although
MAPK-dependent effects of ANXA1 were critical to increased
LPS responses they were not involved in the effects of ANXA1 on
GC sensitivity. We therefore examined an alternate GC-induced
modulator of macrophage activity, GILZ. GILZ expression was
dose-dependently induced by DEX in WT macrophages, but the
effect of DEX on GILZ expression in ANXA1-deficient cells was
markedly and significantly impaired (Fig. 4C).
Requirement for ANXA1 for GC-induced GILZ expression in
We next sought to confirm the requirement for ANXA1 in the
regulation of macrophage GILZ expression.
Western blotting was insufficiently sensitive to detect significant
differences in GILZ protein in ANXA1 macrophages compared
with WT cells (Fig. 5A). However, a trend toward lower DEX-
induced GILZ protein was observed, so GC-induced GILZ protein
expression was therefore further analyzed more quantitatively by
ylation, and GILZ. A, Macrophages were treated with DEX for 4 h, and
MKP-1 mRNA was measured by real-time PCR. Each bar represents mean ?
SEM of three separate experiments. B, Macrophages were treated with LPS in
the presence or absence of DEX, and cell lysates were analyzed for ERK
activation. Top panel: Western blots representative of three experiments. Bot-
Cells were treated with DEX for 4 h, and GILZ mRNA were measured by
real-time PCR. Each bar represents mean ? SEM of three separate experi-
ments performed in duplicate. ?, p ? 0.05 ANXA1?/?vs WT.
Role of ANXA1 in GC regulation of MKP-1, ERK phosphor-
1439The Journal of Immunology
flow cytometry. Using this technique, DEX (3 h) was observed to
significantly increase GILZ protein in WT but not ANXA1?/?
macrophages (Fig. 5B). To confirm the effects of ANXA1 on GILZ
in vivo, mice were treated with i.p. injection of vehicle or DEX for
3 h, and GILZ protein was detected in peritoneal macrophages by
flow cytometry. Treatment with DEX in vivo significantly in-
creased GILZ protein in WT but not ANXA1?/?macrophages
To further confirm the role of ANXA1 in GC regulation of
GILZ, silencing of ANXA1 expression was performed in WT mac-
rophages by siRNA. Silencing efficiency was demonstrated by de-
tecting ANXA1 protein and mRNA expression, which were re-
duced by 75% compared with cells transfected with control siRNA
(Fig. 6, A and B). The biological effect of silencing ANXA1 ex-
pression was confirmed, with significantly increased LPS-induced
TNF measured in ANXA1 siRNA-transfected cells (Fig. 6C).
ANXA1 siRNA significantly reduced DEX-induced GILZ mRNA
in WT cells (Fig. 6D). In keeping with this, transient transfection
of ANXA1 in ANXA1?/?macrophages increased GILZ protein to
the level of WT cells (Fig. 6E). Restoring ANXA1 expression also
restored DEX responsiveness with respect to GILZ mRNA induc-
tion (Fig. 6F).
Functional effects of GILZ in ANXA1?/?macrophages
Major anti-inflammatory effects of GILZ are mediated through in-
hibition of the NF-?B pathway. This was therefore examined by
analysis of NF-?B p65 (Ser536) phosphorylation and NF-?B re-
porter gene assays. LPS significantly increased NF-?B p65 Ser536
phosphorylation in both WT and ANXA1?/?cells (Fig. 6G). A
greater increase of p65 phosphorylation in ANXA1?/?cells was
observed basally and in response to LPS. DEX partially but sig-
nificantly inhibited LPS-induced p65 Ser536phosphorylation in
WT cells at 1 h; however, this action of DEX was impaired in
ANXA1?/?cells. LPS activation of an NF-?B luciferase reporter
Protein lysates were assessed for GILZ by Western blotting (n ? 3 each group). B, Cells were treated with DEX for 3 h. Intracellular GILZ protein was
examined by flow cytometry after labeling with an anti-GILZ Ab in control (CT?) and DEX-treated (Dex?) cells, with control IgG (denoted as unlabeled)
serving as controls. Representative histograms are shown from WT (left panel) and ANXA1?/?cells (center panel), and the mean ? SEM of four
experiments (right panel). †, p ? 0.05 control vs DEX treatment. C, WT and ANXA1?/?(KO) mice were treated with DEX (0.5 mg/kg) for 3 h and GILZ
protein expression measured by flow cytometry after labeling with an anti-GILZ Ab (WT?, KO?), with unlabelled cells (WT?, KO?) serving as controls.
Representative histograms shown are from control (left panel) and DEX-treated mice (center panel), and the mean ? SEM of four experiments (right
panel). †, p ? 0.05 control vs DEX treatment.
Effect of ANXA1 on GC-induced GILZ expression. A, Peritoneal macrophages were treated with DEX as indicated concentration for 1 h.
1440 ANNEXIN-1, GLUCOCORTICOID, AND GILZ
gene assay was completely inhibited by DEX in WT cells (Fig.
6H). LPS-induced NF-?B reporter activity was significantly in-
creased in ANXA1?/?cells, and DEX inhibition of NF-?B activ-
ity was significantly abrogated. Overexpression of ANXA1 in
ANXA1?/?cells inhibited LPS-induced NF-?B reporter activity
to WT levels, and no further inhibition was observed with DEX
treatment of ANXA1 overexpressing cells (Fig. 6H).
To confirm the contribution of GILZ to the phenotype of
ANXA1-deficient macrophages, we transiently overexpressed
GILZ in ANXA1?/?macrophages. GILZ overexpression in
ANXA1?/?macrophages inhibited LPS-induced IL-6 to levels
comparable to that in DEX-treated control-transfected cells and
restored the ability of DEX to significantly suppress IL-6 and TNF
release (Fig. 7, A and B). Examination of IL-6 and TNF mRNA by
real-time PCR revealed similar trends. Overexpression of GILZ in
ANXA1?/?macrophages resulted in reduction of LPS-induced
IL-6 and TNF mRNA comparable to the expression observed in
DEX-treated control-transfected cells, and this was not further sup-
pressible by DEX (Fig. 7, C and D). Overexpression of GILZ in
ANXA1?/?macrophages significantly reduced LPS-induced
ERK MAPK phosphorylation (Fig. 7E) but failed to inhibit
LPS-induced NF-?B p65 phosphorylation (Fig. 7F). In contrast,
GILZ overexpression in ANXA1?/?macrophages significantly
reduced NF-?B reporter activity basally and in response to LPS
and DEX (Fig. 7G).
ANXA1 regulation of GILZ promoter
To examine how ANXA1 regulates GILZ expression, we first as-
sessed whether ANXA1 regulates GILZ via effects on the GILZ
siRNA and overexpression on macro-
phage GILZ and activation. A and B,
WT macrophages were transiently
(ASi) or control siRNA (CSi) for 48 h
in the presence or absence of DEX for
24 h (protein) or 4 h (mRNA).
ANXA1 was measured by Western
blotting and real-time PCR (n ? 6
each group). C, siRNA knockdown of
ANXA1 in WT cells were treated
with LPS for 24 h and supernatant
TNF measured by ELISA. ?, p ?
0.05 ANXA1 siRNA compared with
control siRNA. D, siRNA knock-
down of ANXA1 in WT cells were
also treated with DEX for 4 h. GILZ
mRNA was measured by real-time
PCR. ?, p ? 0.05 ANXA1 siRNA
compared with control siRNA. E,
GILZ protein was
with PcDNA3.1 or ANXA1 plasmids
and untransfected WT cells. F, GILZ
mRNA was examined in ANXA1?/?
macrophages transfected with control
(PcDNA3) or ANXA1 plasmids for
48 h in the presence or absence of
DEX for 4 h. ??, p ? 0.01 ANXA1
plasmid compared with control trans-
fection. G, NF-?B p65 phosphoryla-
(Ser536)- and total NF-?B p65 kits in
macrophages lysates after LPS treat-
ment for 1 h; H, NF-?B luciferase re-
porter activity was measured in WT
and ANXA1?/?macrophages trans-
fected with control (PcDNA3) or
mean ? SEM of three duplicate exper-
iments each group (C–H). ?, p ? 0.05.
1441 The Journal of Immunology
promoter. Transfection of ANXA1 in ANXA1?/?cells signifi-
cantly increased GILZ reporter luciferase activity in comparison
with control plasmid (Fig. 8A), confirming a transcriptional effect
of ANXA1 on GILZ expression. Whether usage of ANXA1 re-
ceptors is required for ANXA1 regulation of GILZ was next in-
vestigated. In the mouse, the FPR family is complex, with multiple
FPR-related receptors (32). Blocking murine FPR using the pan
antagonist Boc2 (33) did not significantly reduce DEX-induced
GILZ mRNA in WT or ANXA1?/?cells. The biologically active
ANXA1-derived N-terminal peptide Ac2-26 interacts with FPR
plasmid. Transfected cells were treated with LPS and DEX for 24 h. Supernatant IL-6 (A) and TNF (B) concentrations were measured by flow cytometric
bead kits. Transfected cells were treated with LPS (0.1 ?g/ml) for 4 h in the presence or absence of DEX (10?7M). IL-6 (C) and TNF mRNA (D) were
measured using real-time PCR. GILZ expression cells were treated with LPS over 1 h for ERK (E) and NF-?B p65 activity (F). ANXA1?/?cells were
cotransfected with NF?B-luc and GILZ plasmid or PcDNA 3.1, and then NF-?B luciferase reporter activity was measured (G). Bars represent mean ? SEM
of four to seven experiments. ?, p ? 0.05 and ??, p ? 0.01 ANXA1?/?vs WT.
Effect of GILZ overexpression in ANXA1?/?macrophages. ANXA1?/?macrophages were transfected with PcDNA3.1 or murine GILZ
pcDNA3.1 using AMAXA system. GILZ luciferase activity was measured after 48 h. B, WT and ANXA1?/?macrophages were pretreated with Boc2 (100
?M) in the presence or absence of DEX (0.1 ?M) for 3 h. GILZ mRNA was analyzed by quantitative PCR. C, GILZ mRNA was also examined in
ANXA1?/?cells treated with ANXA1 peptide Ac2-26 (100 ?g/ml) for 3 h. Bars represent mean ? SEM of three duplicate experiments. ?, p ? 0.05.
Regulation of GILZ promoter by ANXA1. A, ANXA1?/?cells were cotransfected with GILZ promoter-luc and ANXA1 plasmid or
1442ANNEXIN-1, GLUCOCORTICOID, AND GILZ
(34) and FPRL-1/ALXR (35, 36). Treatment of ANXA1?/?mac-
rophages with ANXA1 N-terminal Ac2-26 did not increase GILZ
mRNA in ANXA1?/?cells. These results suggest that ANXA1
regulation of GILZ expression does not require ANXA1 binding to
FPR family members.
Activation of cytokine expression by macrophages is critical for
early host defense against microbial infections and for subsequent
development of adaptive immunity, as well as for the expression of
many inflammatory diseases. Endogenous GC exert an essential
inhibitory effect upon immune responses, tempering the potentially
life-threatening effects of the inflammatory response through a
variety of mechanisms. The current studies examine the mecha-
nisms through which a key GC-induced protein, ANXA1, regu-
lates cytokine expression by macrophages.
Our results confirm the recent report (7) that deficiency of
ANXA1 is associated with increased sensitivity to LPS in vivo,
further reflected by increases in vitro macrophage cytokine release
in response to LPS. Damazo et al. (7) reported similar increases in
IL-6 and TNF to those we have demonstrated, in a study which
also demonstrated increased leukocyte recruitment responses in
the absence of ANXA1. We have extended these observations by
examining the contribution of ANXA1 to GC inhibition of LPS-
induced IL-6 and TNF, demonstrating that ANXA1 is required for
the full inhibitory effect of GC on macrophages in vitro and pro-
viding for the first time a mechanism for these observations.
The mechanism(s) through which ANXA1 regulates cytokine
expression, either in the setting of LPS stimulation or GC inhibi-
tion, has not been previously established. We recently reported that
cultured fibroblasts from ANXA1-deficient mice demonstrated
markedly increased spontaneous IL-6 release, secondary to in-
creased MAPK activation in the setting of reduced expression of
the MAPK inhibitory phosphatase MKP-1 (21). We here demon-
strate the involvement of ERK and JNK MAPK in the amplified
IL-6 and TNF release by ANXA1-deficient macrophages stimu-
lated with LPS. Increased LPS-induced phosphorylation of ERK,
and to a lesser extent JNK, MAPK was observed in ANXA1?/?
macrophages, and the contribution of increased ERK and JNK
phosphorylation was confirmed by abrogation of increased IL6 and
TNF release in ANXA1?/?cells treated with MAPK inhibitors.
Contrasting effects of ANXA1 on MAPK activation have been
reported previously (20). ANXA1 was initially reported to up-
regulate ERK MAPK activation, based on experiments in which
overexpression of ANXA1 resulted in constitutive activation of
ERK. Early ERK activation was recently demonstrated in vitro in
response to exogenous ANXA1 protein and its N-terminal peptide
Ac2-26 (37). Our previous studies of the effects of endogenous
ANXA1 in fibroblasts were, in contrast, consistent with an inhib-
itory effect of ANXA1 on MAPK phosphorylation (21). In this
study, we demonstrate an inhibitory effect of endogenous ANXA1
on macrophage MAPK activation in response to LPS. The present
results suggest that inhibition of ERK and JNK phosphorylation is
involved in endogenous ANXA1 inhibition of LPS-induced mac-
rophage IL-6 and TNF expression. Disparities between the re-
ported effects of ANXA1 based on the use of exogenous ANXA1
on the one hand and deletion of endogenous ANXA1 on the other
are difficult to reconcile. However, given the confirmation here of
increased cytokine expression in vivo and in vitro in the absence of
ANXA1, and our previous report of increased MAPK activation in
ANXA1-deficient fibroblasts, we believe the current data demon-
strate that the physiological effect of endogenous ANXA1 on
MAPK activation is inhibitory.
Unexpectedly, we were unable to demonstrate an effect of
ANXA1 on MKP-1 expression in macrophages and found no ev-
idence of altered GC regulation of MAPK activation in the absence
of ANXA1. MKP-1 is a critical GC-induced negative regulator of
MAPK activity and innate immune responses, with increased LPS-
induced MAPK activation and cytokine production observed in
MKP-1-deficient mice (16–19). MKP-1 has chiefly been reported
to dephosphorylate activated p38 and JNK MAPK, with lesser
effects on ERK, and through this mechanism, MKP1 is thought to
be a dominant mediator of the effects of GC on MAPK pathways.
We observed no effect of ANXA1 deficiency on macrophage
MKP-1 expression basally, under LPS stimulation, or in response
to GC and, in parallel with this, observed no evidence for differ-
ences in GC effects on ERK MAPK in ANXA1?/?cells. These
findings are in contrast to previous findings in fibroblasts and sug-
gest that ANXA1 does not regulate MKP-1 expression in macro-
phages. It is possible that other members of the DUSP family may
be regulated by ANXA1, but this has not been investigated here.
However, these findings suggest that notwithstanding the involve-
ment of increased MAPK phosphorylation in response to LPS in
explaining increased LPS-induced cytokine release in ANXA1?/?
cells, a separate pathway must be involved in ANXA1 mediation
of GC effects on macrophage cytokine release.
We observed that in addition to increased ERK MAPK phos-
phorylation, ANXA1?/?macrophages exhibited increased NF-?B
activity in response to LPS. GILZ is a recently described GC-
induced anti-inflammatory protein, the reported actions of which
include effects on ERK MAPK and NF-?B pathways, suggesting it
as a candidate to explain these effects of ANXA1. GILZ binds
directly to the p65 subunit of NF-?B (22), impacts via protein-
protein interactions with c-fos and c-Jun to affect AP-1 transcrip-
tional activation (23), and inhibits ERK1/2 phosphorylation via
interaction with the upstream MAPK pathway (38, 39). The cur-
rent study provides evidence that GILZ is a previously unrecog-
nized target of ANXA1. We observed reduced basal GILZ expres-
sion in ANXA1?/?macrophages, consistent with the increased
ERK activation and ERK-dependent cytokine expression ob-
served. ANXA1 regulation of GILZ expression was confirmed us-
ing ANXA1 silencing by siRNA in WT macrophages, and confir-
mation of reduced GC-induced GILZ expression in the absence of
ANXA1 was obtained in vivo. Restoring ANXA1 in ANXA1?/?
macrophages reconstituted GC inducibility of GILZ, in parallel
with restoration of the inhibitory effects of GC on IL-6 and TNF.
The involvement of GILZ in increased ERK MAPK activa-
tion in the absence of ANXA1 was supported by inhibition of
ERK activation in response to GILZ overexpression in
ANXA1?/?cells. Reduced GC-induced GILZ expression in
ANXA1?/?macrophages also paralleled reduced GC sensitiv-
ity in these cells, and overexpression of GILZ restored GC inhib-
itory effects on LPS-induced IL-6 and TNF in ANXA1?/?mac-
rophages. Finally, increased NF-?B reporter activation observed in
ANXA1?/?macrophages was inhibited by overexpression of ei-
ther ANXA1 or GILZ. Ser536phosphorylation of NF-?B p65 is
required for NF-?B nuclear translocation and is shown here to be
modestly inhibited by endogenous ANXA1. No evidence for in-
volvement of GILZ in this phenomenon was adduced, suggesting
it may be secondary to increased cytokine expression in the ab-
sence of ANXA1 rather than a GILZ-dependent effect. This is not
inconsistent with the observations that GILZ binds directly to
NF-?B p65 (22, 39), although an odd study suggest that GILZ
influences NF-?B p65 phosphorylation state per se in T cells (40).
The mechanism of ANXA1 regulation of GILZ expression re-
mains unknown. The current data indicate that the effect on GILZ
expression is transcriptional, but no effect of the ANXA1 receptor
1443The Journal of Immunology
ligand Ac2-26 or the ANXA1 receptor antagonist Boc2 was ob-
served, suggesting that ANXA1 effects on GILZ transcription do
not require ANXA1 binding with FPR family members.
The findings of this study context can at this stage only be ap-
plied to the in vivo with caution. In vivo modulation of GILZ
expression by DEX was impaired in ANXA1?/?mice, consistent
with this mechanism applying in vivo, and DEX inhibition of LPS-
induced serum IL-6 was impaired in ANXA1?/?mice. However,
no difference in DEX inhibition of LPS-induced TNF was ob-
served in ANXA1?/?mice in vivo. Multiple factors may contrib-
ute to this, including the fact that in vivo cytokine measurements
were made on samples obtained at a single time point, whereas
assays of in vitro culture supernatants reflect cumulative effects of
a given factor. The effects of ANXA1 on DEX regulation of TNF
observed in vitro may not apply at the 1.5-h time point chosen for
the in vivo studies reported here. In addition, the effects of GILZ
on LPS and GC regulation of cytokines in vivo has not been re-
ported. It is also possible that as yet unknown ANXA1-indepen-
dent mechanisms are operative in vivo that do not apply in vitro.
If a GILZ-deficient mouse becomes available, it will be of interest
to observe the effects of ANXA1 on LPS and DEX regulation of
cytokines in GILZ-deficient mice.
In conclusion, these data demonstrate that ANXA1 is a critical
endogenous negative regulator of the expression of IL-6 and TNF
by macrophages, via effects on MAPK phosphorylation, involving
regulation of the expression of GILZ (Fig. 9). GILZ is a previously
unrecognized target of ANXA1, and the functions of GILZ have
the potential to explain many aspects of the proinflammatory phe-
notype of ANXA1-deficient mice and cells. Modulation of GILZ
expression by manipulation of ANXA1 may afford a mechanism of
inhibiting inflammation without GC, which would be an attractive
modality for treatment of acute and chronic inflammatory diseases.
We thank M. Perretti and F. D’Acquisto for generously providing us with
an annexin-1 expression plasmid; C. Riccardi for GILZ plasmid and anti-
GILZ serum; and M. Pallardy and A. Biola-Vidamment for the GILZ lu-
ciferase reporter construct.
The authors have no financial conflict of interest.
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