Anti-inflammatory effects of annexin-1: stimulation of IL10 release and inhibition of nitric oxide synthesis

www.elsevier.com/locate/intimp

International Immunopharmacology 3 (2003) 1363–1369

Anti-inflammatory effects of annexin-1: stimulation of IL-10

release and inhibition of nitric oxide synthesis

Viviana Ferlazzoa, Pietro D’Agostinob, Salvatore Milanoa, Rosalba Carusoc,Salvatore Feod, Enrico Cillaric, Luca Parentee,*

aDepartment of Bio-Pathology and Bio-Medical Methodologies, University of Palermo, Palermo, ItalybDepartment of Immuno-Haematology and Transfusion, University of Palermo, Palermo, Italy

cDivision of Clinical Pathology, ‘‘V. Cervello’’ Hospital, Palermo, ItalydDepartment of Cellular and Development Biology, University of Palermo, Palermo, Italy

eDepartment of Pharmaceutical Sciences, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy

Received 12 February 2003; received in revised form 15 March 2003; accepted 21 April 2003

Abstract

Annexin-1 (ANX-1) is an anti-inflammatory protein induced by glucocorticoids. Like glucocorticoids, ANX-1 and derived

peptides inhibit eicosanoid synthesis, block leukocyte migration and induce apoptosis of inflammatory cells. Cytokines may

possess either pro-inflammatory, i.e. interleukin(IL)-1h, tumor necrosis factor (TNF)-a, IL-12 or anti-inflammatory properties,

i.e. IL-4, IL-10. The experiments described in the present study have been performed to answer the question whether the anti-

inflammatory action of ANX-1 may be mediated, at least in part, by the release of IL-10. In macrophage (J774) cell line cultures

primed with lipolysaccharide (LPS), recombinant ANX-1 stimulated IL-10 release in a dose- and time-dependent manner. In the

same cells, the protein and its derived N-terminal peptide (amino acids 2–26) dose-dependently inhibited the release of nitric

oxide (NO). Furthermore, both the whole protein and the peptide down-regulated the mRNA expression of the inducible nitric

oxide sythase (iNOS). The peptide was also able to inhibit the expression of IL-12 mRNA. These results suggest that some of

the anti-inflammatory effects of ANX-1 may be mediated by the release of IL-10, which, in turn, inhibits iNOS mRNA

expression and, hence, NO release. In addition, ANX-1-stimulated IL-10 release may also be responsible for the inhibition of

IL-12 mRNA expression and, consequently, IL-12 synthesis.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Annexin-1; IL-10; Nitric oxide; Inflammation

1567-5769/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S1567-5769(03)00133-4

Abbreviations: ANX-1, annexin-1; IL, interleukin; TNF-a,

tumor necrosis factor-a; LPS, lipopolysaccharide; MTT, 3-(4,5-

dimethylthiazol-2y)2,5-diphenyltetrazolium bromide; DTT, dithio-

threitol; DMSO, dimethylsulfoxide; NO, nitric oxide; iNOS,

inducible nitric oxide synthase; RT-PCR, reverse transcriptase

polymerase chain reaction; ERK, extracellular signal-regulated

kinase; S.E.M., standard error of the means.

* Corresponding author. Tel.: +39-89-962654; fax: +39-89-

962828.

E-mail address: [email protected] (L. Parente).

1. Introduction

Annexins are structurally related, calcium-depen-

dent, phospholipid-binding proteins that have been

implicated in diverse cellular roles, including control

of inflammatory responses, membrane fusion, cell

differentiation and proliferation, exocytosis, and in-

teraction with cytoskeletal proteins [1]. These proteins

V. Ferlazzo et al. / International Immunopharmacology 3 (2003) 1363–13691364

are defined structurally by a conserved core domain

containing four to eight repeats and by variable N-

terminal regions. The conserved repeats account for

the shared abilities of annexins to bind phospholipids

in a calcium-dependent manner, whereas the specific

N-terminal regions are probably responsible for dif-

ferent effects [2]. Annexin-1 (ANX-1) has been orig-

inally identified in leukocytes as a glucocorticoid-

inducible protein that inhibits phospholipase A2, thus

preventing the formation of pro-inflammatory eicosa-

noids. Human ANX-1 and its N-terminal peptides

(amino acids 1–188 and 2–26) mimic anti-inflamma-

tory actions of glucocorticoids in many experimental

models [3], and anti-annexin antibodies reverse glu-

cocorticoid effects both in vitro and in vivo [4]. Like

glucocorticoids, ANX-1 and derived peptides inhibit

eicosanoid synthesis [5], block leukocyte migration

[6] and induce apoptosis of inflammatory cells [7]. All

these effects contribute to the potent anti-inflammato-

ry action exerted by both glucocorticoids and induced

proteins.

Cytokines are polypeptides produced by activated

cells like macrophages and lymphocytes regulating

both immune and inflammatory responses. Cytokines

mediate several functions on multiple cell types;

however, some cytokines such as interleukin (IL)-

1h, tumor necrosis factor (TNF)-a, IL-6, IL-8, IL-12

have mainly pro-inflammatory effects, while other

cytokines such as IL-4 and IL-10 possess overall

anti-inflammatory properties [8]. Glucocorticoids po-

tently inhibit the synthesis of pro-inflammatory cyto-

kines [9], while ANX-1 may counteract some of the

inflammatory signs caused by cytokines like IL-1 and

TNF-a [10]. However, to our knowledge, there are no

published data linking annexin-1 to anti-inflammatory

cytokines like IL-10.

The experiments described in the present study

have been performed to answer the question whether

the anti-inflammatory action of ANX-1 may be me-

diated, at least in part, by the release of IL-10.

2. Materials and methods

2.1. Macrophage line

The murine macrophage cell line J774 was obtained

from the American Tissue Culture Collection (ATCC,

Rockville, MD). The cells weremaintained in complete

culture medium.

2.2. Reagents

Tissue culture medium consisted of RPMI-1640

(TechGen International, Les Ulis, France) supple-

mented with glutamine (2 mM), antibiotics (penicillin

and streptomycin) and 2% heat-inactivated foetal calf

serum (FCS; Seromed, Biochrom, Berlin, Germany).

Lipopolysaccharide (LPS from E. coli, serotype

026:B6), 3-(4,5-dimethylthiazol-2y)2,5-diphenyltetra-

zolium bromide (MTT), dithiothreitol (DTT) and

dimethylsulfoxide (DMSO) were purchased from

Sigma (Poole, UK). Human recombinant ANX-1

and the N-terminal acetylated synthetic peptide (ami-

no acids 2–26, Ac 2–26) were kindly provided by

Dr. E. Solito (Imperial College School of Medicine,

London, UK) and by Dr. M. Perretti (The William

Harvey Research Institute, London, UK), respective-

ly. Before experimental use, peptides were diluted in

complete RPMI and adjusted to the experimental

concentrations (20 and 50 Ag/ml). Tissue culture

plasticware was purchased from NUNC (Roskilde,

Denmark).

2.3. Induction of nitric oxide and cytokine synthesis

J774 macrophages (1�106) were cultured in 24-

well plates for 2 h at 37 jC in a humidified incubator

containing 5% CO2 in air. Nonadherent cells were

then removed by washings and the adherent ones

cultured with various stimuli as detailed in the legends

to the figures. At various time intervals, culture super-

natants were collected and nitric oxide (NO) and

cytokine measurement carried out.

2.4. Measurement of NO2�

NO2� in the culture supernatant was determined

by Griess reaction as previously described [11].

Briefly, 100 Al/well of the sample was incubated

with an equal volume of Griess solution (1%

sulfanilamide in 5% phosphoric acid + 1% alpha-

naphthyl-amine in distilled water) at room tempera-

ture for 10 min. The absorbance was evaluated with

a Titertek ELISA reader (Flow, Rockville, MD) at

550 nm. The levels of NO2� reflect NO synthesis.

Fig. 1. Effect of annexin-1 on IL-10 production by LPS-treated J774

macrophages. Cells (1�106/ml) were stimulated with 100 ng/ml

LPS for 24 and 48 h. Open columns: LPS; filled columns:

LPS+ annexin-1 20 Ag/ml (0.54 AM); hatched columns: LPS + an-

nexin-1 50 Ag/ml (1.35 AM). Data are expressed as meansF S.E.M.

of four separate experiments. **P < 0.01: significantly different

from cells treated with LPS alone.

munopharmacology 3 (2003) 1363–1369 1365

2.5. Assay for cytokine determination in the culture

supernatants

The levels of IL-10 in culture supernatants were

determined by enzyme-linked immunosorbent assay

(ELISA) commercial kits (Genzyme, Kocklight, Hat-

field, UK) which employ the multiple antibody sand-

wich principle.

2.6. Cell viability assay

Cell viability under different experimental condi-

tions was determined by a modification of Mosmann

assay that employed mitochondrial-dependent reduc-

tion of MTT to formazan as previously described [12].

Cultures were treated with the peptide derivatives (20

and 50 Ag/ml) in the presence or absence of LPS and

incubated for 24 h. Afterwards, cells were pulsed with

100 Al of 0.2 mg/ml MTT reagent for 2 h at 37 jC,followed by 15-min incubation at 37 jC with 100

Al DMSO. After this period, microtiter plates were

read at 595 nm in a ELISA plate reader. The results are

expressed as absolute optical density (OD) readings.

2.7. Total RNA isolation and RT-PCR

Total RNA was extracted from 1�107 treated and

untreated J774 cells by TRIZOL Reagent (GIBCO

BRL) according to manufacturer’s instructions, was

dissolved in 50 Al of RNase-free water and quantitatedby UV adsorbance. Semiquantitative RT-PCR was

performed using the SuperScript One-Step RT-PCR

System (GIBCO BRL). Each reaction was carried out

according to manufacturer’s instruction with 0.5 Ag of

total RNA and 0.2 AM of one of the pair of primers

previously reported [13]. PCR profile was as follows:

denaturing at 94 jC 1 min, annealing at temperature

previously described [13] for each primer set for 1 min,

extension at 72 jC for 1 min, for 30–35 cycles,

followed by one cycle of 72 jC for 7 min. PCR

products (15 Al) were analysed in a 1.4% agarose gel

in 1� Tris acetate–EDTA buffer and stained with

ethidium bromide. To check linearity of the amplifica-

tion, aliquots of 15 Al were withdrawn from each

reaction at 20, 25 and 30 cycles and analysed as above.

All data reported were normalised for the amount of

GAPD amplified product in each sample. Quantifica-

tion was obtained on elettronically acquired images of

V. Ferlazzo et al. / International Im

the agarose gel with the SigmaGel software from

Jandel Scientific.

2.8. Statistical analysis

All experiments were performed three or four times

and the results are expressed as meansF S.E.M. Some

data are reported as meansF S.E.M. of three or four

individual experiments while others are reported as

meansF S.E.M. of a single representative experiment.

However, within each single experiment, the S.E.M.

was within 10%. Significance was tested by Student’s

t-test by variance analysis (Student–Newmann–

Keuls test).

3. Results

3.1. Effect of recombinant annexin-1 on IL-10 release

Fig. 1 shows the effect of recombinant ANX-1 on

IL-10 production. As clearly indicated, J774 macro-

phages in culture released substantial amounts of IL-

10 when stimulated with LPS (100 ng/ml): 513F 100

pg/ml at 24 h and 711F 60 pg/ml at 48 h. Recombi-

nant ANX-1 added to the cultures was able to stim-

ulate IL-10 release. In the presence of ANX-1 (50 Ag/ml, 1.35 AM) at 24 h, macrophages released 859F 50

Fig. 2. Effect of annexin-1 and peptide Ac 2–26 on NO production

by LPS-treated J774 macrophages. Cells (1�106/ml) were

stimulated with 100 ng/ml LPS for 48 h. Open column: LPS; filled

columns: LPS + peptide Ac 2–26 (peptide) 20 Ag/ml (6.6 AM) or

annexin-1 (protein) 20 Ag/ml (0.54 AM); hatched columns:

LPS+ peptide Ac 2–26 (peptide) 50 Ag/ml (16.4 AM) or annexin-

1 (protein) 50 Ag/ml (1.35 AM). Data are expressed as meansFS.E.M. of three separate experiments. *P < 0.05 and **P< 0.01:

significantly different from cells treated with LPS alone.

V. Ferlazzo et al. / International Immunopharmacology 3 (2003) 1363–13691366

pg/ml of IL-10, while at 48 h, the peak of IL-10

release (1430F 250) was observed with 20 Ag/ml

(0.54 AM) of ANX-1. The recombinant protein did

Fig. 3. Effect of annexin-1 (ANX-1) on iNOS mRNA accumulation.

0.5 Ag of total RNA was extracted from J774 cells stimulated with

LPS (100 ng/ml) in the presence of annexin-1 at different

concentrations, 20 and 50 Ag/ml (0.54 and 1.35 AM) for 12 h and

hybridised with 0.2 AM of relative primers. Results are representa-

tive of three experiments.

not alter cell viability as measured by the MTT

reduction assay (O.D.F S.E.M. n = 3): control cells +

LPS, 2.38F 0.3; ANX-1 20 Ag/ml, 2.61F 0.3; ANX-

1 50 Ag/ml, 2.44F 0.3.

3.2. Effect of recombinant annexin-1 and peptide Ac

2–26 on NO synthesis

LPS (100 ng/ml) stimulated J774 macrophages to

release substantial amounts of NO in 48-h incubation

(120F 19 nmol/ml) (Fig. 2). When added to the

cultures, ANX-1 dose-dependently inhibited NO re-

lease: 75F 10 nmol/ml at 20 Ag/ml (0.54 AM) and

59F 7 nmol at 50 Ag/ml (1.35 AM). The N-terminal

acetylated synthetic peptide (amino acids 2–26) was

also able to reduce NO release (85F 12 nmol/ml), but

only at the highest concentration (50 Ag/ml, 16.4 AM)

(Fig. 2).

3.3. Effect of recombinant annexin-1 and peptide Ac

2–26 on iNOS and IL-12 mRNA accumulation

The analysis of mRNA accumulation showed that

LPS (100 ng/ml) greatly stimulated the expression of

iNOS mRNA in J774 macrophages in 12-h incubation

(14.6-fold compared to cells without LPS) (Fig. 3).

Annexin-1 dose-dependently inhibited iNOS mRNA

Fig. 4. Effect of peptide Ac 2–26 (peptide) on iNOS and IL-12

mRNA accumulation. Total RNA (0.5 Ag) was extracted from J774

cells stimulated with LPS (100 ng/ml) in the presence of peptide Ac

2–26 at different concentrations, 20 and 50 Ag/ml (6.6 and 16.4

AM) for 12 h and hybridised with 0.2 AM of relative primers.

Results are representative of three experiments.

V. Ferlazzo et al. / International Immunopharmacology 3 (2003) 1363–1369 1367

accumulation: 12.9-fold at 20 Ag/ml (0.54 AM) and 3-

fold at 50 Ag/ml (1.35 AM) compared to cells without

LPS (Fig. 3). In similar experimental conditions,

peptide Ac 2–26 was also able to inhibit iNOS

mRNA accumulation: 2.9-fold at 50 Ag/ml (16.4

AM) (Fig. 4). The figure also shows that peptide Ac

2–26 dose-dependently reduced the accumulation of

IL-12 mRNA in J774 macrophages in 12-h incubation.

4. Discussion

The anti-inflammatory effects of annexin-1, a pro-

tein induced by glucocorticoids, are multifaceted and

affect many components of the inflammatory re-

sponse. The protein is able to block the release of

inflammatory mediators by directly inhibiting cyto-

solic phospholipase A2 [14] as well as the expression

of the inducible cyclooxygenase and nitric oxide

synthase [15,16]. Annexin-1 may also inhibit leuko-

cyte migration by impairing neutrophil and monocyte

adhesion to vascular endothelium [6,17]. Recently, it

has been shown that annexin-1 promotes the apoptosis

of monomyelocytic cells [7]. These pro-apoptotic

effects may also contribute to its anti-inflammatory

properties.

It is known that host response to injury is finely

regulated by the formation of both pro-inflammatory

and anti-inflammatory cytokines. Proteins like IL-1h,TNF-a and IL-12 stimulate body’s defence mecha-

nisms by promoting inflammatory reactions in order

to fight inflammatory stimuli and restore homeostasis

[8]. On the other hand, cytokines such as IL-4 and IL-

10, by lowering inflammatory responses, prevent

defence mechanisms from causing damage by over-

shooting [18]. In particular, IL-10 has potent anti-

inflammatory and immunosuppressive activities in

systemic inflammatory diseases like rheumatoid ar-

thritis [19] as well as in a variety of experimental

models of local inflammation [20]. Among its anti-

inflammatory effects IL-10 is able to inhibit expres-

sion of both inducible cyclooxygenase [21] and nitric

oxide synthase [22]. IL-10 is also a potent inhibitor of

IL-12 production by phagocytic cells [23].

In this context, we decided to investigate the effect

of annexin-1 on IL-10 release. The results shown in

Fig. 1 clearly demonstrate that annexin-1 stimulated

IL-10 release from a macrophage cell line (J774)

primed with LPS. Next, we investigated in the same

cells the effect of annexin-1 and a related peptide, Ac

2–26, derived from its N-terminus on nitric oxide

release. This peptide mimics many of the anti-inflam-

matory actions of the whole protein [24]. The data in

Fig. 2 show that the whole protein inhibited NO

release from J774 cells in a dose-dependent manner,

while the peptide caused a significant reduction of NO

release only at the highest dose (50 Ag/ml). These

results were confirmed by experiments analysing

iNOS mRNA accumulation. Annexin-1 dose-depen-

dently down-regulated the expression of iNOS mRNA

(Fig. 3), whereas an inhibitory effect of the peptide

was observed only at 50 Ag/ml (Fig. 4). The whole

protein was approximately 20 times more potent than

the peptide on a molar basis. Fig. 4 also shows that the

peptide strongly inhibited the expression of IL-12

mRNA. The effect of the whole protein on IL-12

mRNA expression was not investigated because of its

limited supply; however, it is generally accepted that

the biological properties of the amino terminus pep-

tide are indeed very similar, if not identical, to those of

the whole protein [24].

On the basis of the above results, we would suggest

that some of the anti-inflammatory actions of annexin-

1 may be mediated by the release of IL-10. In other

words, annexin-1 stimulates the release of IL-10,

which, in turn, inhibits iNOS mRNA expression

and, hence, NO release. Furthermore, the release of

IL-10 by annexin-1 may also be responsible for the

inhibition of IL-12 mRNA expression and, conse-

quently, IL-12 synthesis. Indeed, the inhibitory effect

of IL-10 on IL-12 pathways has already been de-

scribed [23,25]. The present evidence does not leave

out other possible mechanisms of action by annexin-1

like the interference with release and/or effects of

specific transcription factors, as previously suggested

[16].

It is of interest that previous reports have shown

that annexin-1 and IL-10 share selective anti-inflam-

matory and immunosuppressive effects. Both proteins

reduce leukocyte migration elicited in vivo by IL-1h[26,27], inhibit antigen-driven Th1- and Th2-like

responses [28,29], and promote apoptosis of inflam-

matory cells [7,30]. Future experiments will address

the issue of the signalling pathways involved in the

stimulation of IL-10 release by annexin-1. It has been

recently demonstrated that innate immune stimulators

V. Ferlazzo et al. / International Immunopharmacology 3 (2003) 1363–13691368

such as CpG DNA up-regulate IL-10 production in

macrophages by activating the extracellular signal-

regulated kinase (ERK) pathways [31]. Since endog-

enous annexin-1 promotes constitutive activation of

ERK [32], it is conceivable that ERK signalling

pathway is involved in the effect of annexin-1.

To our knowledge, this is the first report showing

the stimulation of IL-10 release by annexin-1. These

results shed new light on the mechanisms of the anti-

inflammatory action of annexin-1 and widen the fields

of possible therapeutic applications of the protein and

derived peptides.

Acknowledgements

L.P. is supported by grants from the University of

Salerno (60% 2002, 2003).

References

[1] Raynal P, Pollard HB. Annexins: the problem of assessing the

biological role for a gene family of multifunctional calcium-

and phospholipid-binding proteins. Biochim Biophys Acta

1994;1197:63–93.

[2] Rosengarth A, Gerke V, Luecke H. X-ray structure of full-

length annexin 1 and implications for membrane aggregation.

J Mol Biol 2001;306:489–98.

[3] Yang Y, Leech M, Hutchinson P, Holdsworth SR, Morand EF.

Antiinflammatory effect of lipocortin 1 in experimental arthri-

tis. Inflammation 1997;21:583–96.

[4] Perretti M, Ahluwalia A, Harris JG, Harris HJ, Wheller SK,

Flower RJ. Acute inflammatory response in the mouse: exac-

erbation by immunoneutralization of lipocortin 1. Br J Phar-

macol 1996;117:1145–54.

[5] Kim S-W, Rhee HJ, Ko J, Kim YJ, Kim HG, et al. Inhibition

of cytosolic phospholipase A2 by annexin I. Specific interac-

tion model and mapping of the interaction site. J Biol Chem

2001;276:15712–9.

[6] Perretti M, Croxtall JD, Wheller SK, Goulding NJ, Hannon R,

Flower RJ. Mobilizing lipocortin 1 in adherent human leuko-

cytes downregulates their transmigration. Nat Med 1996;2:

1259–62.

[7] Solito E, de Coupade C, Canaider S, Goulding NJ, Perretti M.

Transfection of annexin 1 in monocytic cells produces a high

degree of spontaneous and stimulated apoptosis associated

with caspase-3 activation. Br J Pharmacol 2001;133:217–28.

[8] Sundy JS, Patel DD, Haynes BF. Cytokines in normal and

pathogenic inflammatory responses. In: Gallin JI, Snyderman

R, editors. Inflammation: basic principles and clinical cor-

relates. Philadelphia: Lippincott Williams & Wilkins; 1999.

p. 433–41.

[9] Parente L. The development of synthetic glucocorticoids. In:

Goulding NJ, Flower RJ, editors. Glucocorticoids. Basel: Bir-

khauser Verlag; 2001. p. 35–51.

[10] Flower RJ, Rothwell NJ. Lipocortin-1: cellular mechanisms

and clinical relevance. Trends Pharmacol Sci 1994;15:71–6.

[11] Milano S, Arcoleo F, D’Agostino P, Cillari E. Intraperitoneal

injection of tetracyclines protect mice from lethal endotoxemia

down-regulating inducible nitric oxide synthase in various

organs and cytokines and nitrate secretion in the blood. Anti-

microb Agents Chemother 1997;41:117–21.

[12] D’Agostino P, Ferlazzo V, Milano S, La Rosa M, Di Bella G,

et al. Chemically modified tetracyclines induce cytotoxic ef-

fects against J774 Tumour cell line by activating the apoptotic

pathway. Int Immunopharmacol 2003;3:63–73.

[13] D’Agostino P, Ferlazzo V, Milano S, La Rosa M, Di Bella G,

et al. Anti-inflammatory effects of chemically modified tetra-

cyclines by the inhibition of nitric oxide and interleukin-12

synthesis in J774 cell line. Int Immunopharmacol 2001;1:

1765–76.

[14] Kim S-W, Rhee HJ, Ko J, Kim YJ, Kim HG, et al. Inhibition

of cytosolic phospholipase A2 by annexin I. Specific interac-

tion model and mapping of the interaction site. J Biol Chem

2001;276:15712–9.

[15] Minghetti L, Nicolini A, Polazzi E, Greco A, Perretti M, et al.

Down-regulation of microglial cyclo-oxygenase-2 and induci-

ble nitric oxide synthase expression by lipocortin 1. Br J

Pharmacol 1999;126:1307–14.

[16] Wu C-C, Croxtall JD, Perretti M, Bryant CE, Thiemermann C,

et al. Lipocortin 1 mediates the inhibition by dexamethasone

of the induction by endotoxin of nitric oxide synthase in the

rat. Proc Natl Acad Sci U S A 1995;92:3473–7.

[17] Solito E, Romero IA, Marullo S, Russo-Marie F, Weksler BB.

Annexin 1 binds to U937 monocytic cells and inhibits their

adhesion to microvascular endothelium: involvement of the

a4h1 integrin. J Immunol 2000;165:1573–81.

[18] de Waal Malefyt R. Role of interleukin-10, interleukin-4, and

interleukin-13 in resolving inflammatory responses. In: Gallin

JI, Snyderman R, editors. Inflammation: basic principles and

clinical correlates. Philadelphia: Lippincott Williams & Wil-

kins; 1999. p. 837–49.

[19] Katsikis PD, Chu C-Q, Brennan FM, Maini RN, Feldmann M.

Immunoregulatory role of interleukin 10 in rheumatoid arthri-

tis. J Exp Med 1994;179:1517–27.

[20] Cuzzocrea S, Mazzon E, Dugo L, Britti D, De Maio M, Caputi

AP. Absence of endogenous interleukin-10 enhances the evo-

lution of murine type-II collagen-induced arthritis. Eur Cyto-

kine Netw 2001;12:568–80.

[21] Mertz PM, DeWitt DL, Stetler-Stevenson WG, Wahl LM.

Interleukin 10 suppression of monocyte prostaglandin H syn-

thase-2. Mechanism of inhibition of prostaglandin-dependent

matrix metalloproteinase production. J Biol Chem 1994;269:

21322–9.

[22] Cunha FQ, Moncada S, Liew FY. Interleukin-10 (IL-10) in-

hibits the induction of nitric oxide synthase by interferon-

gamma in murine macrophages. Biochem Biophys Res Com-

mun 2003;182:1155–9.

[23] D’Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin

V. Ferlazzo et al. / International Immunopharmacology 3 (2003) 1363–1369 1369

M, Trinchieri G. Interleukin-10 (IL-10) inhibits human lym-

phocyte interferon gamma-production by suppressing natural

killer cell stimulatory factor/IL-12 synthesis in accessory cells.

J Exp Med 2003;178:1041–8.

[24] Perretti M, Wheller SK, Choudhury Q, Croxtall JD, Flower

RJ. Selective inhibition of neutrophil function by a peptide

derived from lipocortin 1 N-terminus. Biochem Pharmacol

1995;50:1037–42.

[25] Moore W, de Waal Malefyt R, Coffmann RL, O’Garra A.

Interleukin-10 and the interleukin-10 receptor. Annu Rev Im-

munol 2001;19:683–765.

[26] Perretti M, Flower RJ. Modulation of IL-1-induced neutrophil

migration by dexamethasone and lipocortin 1. J Immunol

1993;150:992–9.

[27] Perretti M, Szabo C, Thiemermann C. Effect of interleukin-4

and interleukin-10 on leucocyte migration and nitric oxide

production in the mouse. Br J Pharmacol 1995;116:2251–7.

[28] Kamal AM, Smith SF, De Silva Wijayasinghe M, Solito E,

Corrigan CJ. An annexin 1 (ANXA1)-derived peptide inhibits

prototype antigen-driven human T cell Th1 and Th2 responses

in vitro. Clin Exp Allergy 2001;31:1116–25.

[29] Grunig G, Corry DB, Leach MW, Seymour BWP, Kurup VP,

Rennick DM. Interleukin-10 is a natural suppressor of cyto-

kine production and inflammation in a murine model of aller-

gic bronchopulmonary aspergillosis. J Exp Med 2003;185:

1089–100.

[30] Cox G. IL-10 enhances resolution of pulmonary inflammation

in vivo by promoting apoptosis of neutrophils. Am J Physiol

1996;271:L566–71.

[31] Yi A-K, Yoon J-G, Yeo S-J, Hong S-C, English BK, Krieg

AM. Role of mitogen-activated protein kinases in CpG DNA-

mediated IL-10 and IL-12 production: central role of extrac-

ellular signal-regulated kinase in the negative feedback loop of

the CpG DNA-mediated Th1 response. J Immunol 2002;168:

4711–20.

[32] Alldridge LC, Harris HJ, Plevin R, Hannon R, Bryant CE. The

annexin protein lipocortin 1 regulates the MAPK/ERK path-

way. J Biol Chem 1999;274:37620–8.