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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3868-3877
IMMUNOBIOLOGY
Engagement of CD11b and CD11c  2 integrin by antibodies or
soluble CD23 induces IL-1 production on primary human monocytes
through mitogen-activated protein kinase-dependent pathways
Roger Rezzonico,
Rachel Chicheportiche,
Veronique Imbert, and
Jean-Michel Dayer
From the Division of Immunology and Allergy, Clinical Immunology
Unit (Hans Wilsdorf Laboratory), Department of Internal Medicine,
University Hospital, Geneva, Switzerland; and INSERM CJF 96-05, Facultè de Mèdecine, Nice, France.
 |
Abstract |
 2 integrins are involved in the recruitment of leukocytes to
inflammatory sites and in cellular activation. We demonstrate that
ligation of CD11b (Mac-1, CR3) or CD11c (p150, CR4) alpha chains of
2 integrins by mAbs or soluble chimeric CD23 (sCD23) on human
freshly isolated monocytes rapidly stimulates high levels of
interleukin-1 production. This induction takes place at the transcriptional level and is regulated by members of the
mitogen-activated protein kinase (MAPK) family. Indeed, stimulation of
monocytes through engagement of CD11b or CD11c results in the
phosphorylation and activation of ERK1, ERK2, and p38/SAPK2 MAP
kinases. U0126, a potent inhibitor of the upstream activator of ERK1/2,
ie, MEK1/2, suppresses IL-1 messenger RNA (mRNA) expression in a
dose-dependent fashion, showing the implication of this pathway in the
transcriptional control of IL-1 production. On the other hand,
inhibition of p38 by SB203580 indicates that this MAPK is involved in
the control of IL-1 production at both transcriptional and
translational levels. Together these data demonstrate that ligation of
CD11b and CD11c 2 integrins by mAbs or sCD23 fusion proteins
triggers the activation of 2 distinct MAPK signaling pathways that
cooperate in controlling IL-1 synthesis at different levels.
(Blood. 2000;95:3868-3877)
© 2000 by The American Society of Hematology.
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Introduction |
The integrin family consists of heterodimeric
membrane-bound glycoproteins that mediate homotypic and heterotypic
cell-cell adhesion, as well as cell-matrix interactions in a broad
range of biologic functions.1 The leukocyte-specific 2
integrin subfamily includes LFA-1, Mac-1 (CR3), and p150,95 (CR4), each consisting of an association of an  chain (CD11a, CD11b, or CD11c) and a common chain (CD18).2,3
Lack of 2 (CD18) integrin expression, as in the leukocyte adhesion
deficiency syndrome, results in an impairment of a variety of immune
functions, of which neutrophil transendothelial migration, macrophage
oxidative burst and phagocytosis, and lymphocyte proliferation are a
few. This results in recurrent bacterial infections,
presumably because of impaired chemotaxis and bacterial
phagocytosis.4
A variety of counterreceptors and soluble ligands for 2 integrins
has already been identified. CD11a/CD18 is mainly expressed on
mononuclear leukocytes and binds to ICAM-1 (CD54),5 ICAM-2 (CD102),6,7 and ICAM-3 (CD50),8,9 members of
the immunoglobulin superfamily. CD11b/CD18 is expressed on mature
neutrophils, monocytes, and natural killer (NK) cells. It also binds to
ICAM-1,10 as well as to several soluble ligands, including
the complement fragment iC3b,11 fibrinogen,12
coagulation factor X,13 and LPS.14 CD11c/CD18
is found on a variety of cells such as monocytes, macrophages, granulocytes, some T and B lymphocytes, and dendritic cells. In contrast with LFA-1 and Mac-1, the ligands and the functional role of
p150,95 have not been well defined, but appear to be similar to those
of Mac-1. Indeed, CD11c/CD18 has been shown to bind iC3b,15 fibrinogen,16 LPS,17 and like the other
leukocyte integrins, it is involved in the adhesion of monocytes to
endothelium.18 Furthermore, it has recently been reported
that CD11b/CD18 and CD11c/CD18 can exhibit an additional adhesive
function because of their CD23-binding
ability.19-21
In addition to the crucial function of integrins in a variety of
cell-adhesion reactions during immune-inflammatory mechanisms, it has
been established that engagement of 2 integrins by natural ligands
(notably, soluble CD23) and certain mAbs also generates outside-in
cellular signaling, leading to cell activation. In monocytes, this
activation includes the induction of procoagulant activity, production
of inflammatory cytokines (tumor necrosis factor [TNF],
IL-1, and IL-6), generation of nitric oxide, and up-regulation of
cell-surface molecule expression.19-24
However, most of the studies investigating signaling pathways and,
particularly, protein kinases induced by triggering of 2 integrins
have been performed on polymorphonuclear neutrophils (PMN).25-27 Conversely, the intracellular events implicated
in 2 integrin-mediated human monocyte activation and,
particularly, those leading to inflammatory cytokine synthesis are less characterized.
Interleukin-1 is mainly produced by monocytes/macrophages and plays
a pivotal role in immuno-inflammatory processes that lead to tissue
destruction in chronic diseases, such as rheumatoid arthritis
(RA).28 Furthermore, soluble CD23, a multifunctional cytokine whose production is increased in RA,29 plays a
role in inflammatory mechanisms and has been shown to induce IL-1 production through activation of macrophages via its interaction with
CD11b and CD11c.19,30-32
The signaling pathways leading to IL-1 production have mainly been
studied on monocytes stimulated by LPS, antigen-antibody complexes,
phorbol esters, or cytokines. In this study, we have investigated the
molecular events involved in the regulation of IL-1 production
induced by triggering of CD11b/CD18 and CD11c/CD18 2 integrins at
the surface of human monocytes using mAbs or human recombinant fusion
proteins for soluble CD23. We have particularly examined the role of
mitogen-activated protein kinases (MAPKs), a family of serine/threonine
kinases activated by various extracellular stimuli, playing an
important role in the regulation of the expression of IL-1 in
monocytes and macrophages.33-36
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Material and methods |
Reagents
RPMI-1640 medium, phosphate-buffered saline (PBS), penicillin,
streptomycin, and L-glutamine were supplied by Life Technologies (Paisley, UK). Low-endotoxin fetal calf serum (FCS) was purchased from
Seromed (Biochrom KG, Berlin, Germany). Ficoll-Paque was from Pharmacia (Uppsala, Sweden). Polymyxin B sulfate, neuraminidase, 5,6-dichlorobenzimidazoleriboside (DRB), and all other chemicals were
purchased from Sigma Chemical (St Louis, MO). [-32P]
dCTP (3000 Ci/mmol) and [-32P] UTP (3000 Ci/mmol) were
from Hartmann Analytic Gmbh (Braunschweig, Germany). U0126 was from
Promega (Madison, WI). SB203580 was purchased from Calbiochem (La
Jolla, CA). Culture media were shown to contain less than 0.15 U/mL
endotoxin as measured by the chromogenic Limulus amebocyte
lysate assay.
Monoclonal antibodies and recombinant chimeric proteins
Anti-CD11a antibodies were from The Binding Site (Birmingham, UK)
(IgG1, clone BU17) and Pharmingen (San Diego, CA) (IgG2b, clone
G43-25B), anti-CD11b antibodies were obtained from R & D Systems
(Minneapolis, MN) (IgG1, clone 44) and Serotec (Oxford, UK) (IgG1,
clone ICRF44), and anti-CD11c antibodies were from The Binding Site
(IgG1, clone BU15) and R & D Systems (IgG1, clone 3.9). The isotype mAb
controls were purchased from Pharmingen.
Human recombinant fusion proteins for soluble CD23 were the kind gift
of Dr M. Bird (Glaxo Wellcome, Stevenage, UK). hZZ-CD23 is a fusion
protein consisting of the lectin domain of human CD23 linked to the
protein A IgG binding domain (ZZ) that was produced in insect cells as
previously described.37 ZZ-CD23 can form oligomers in
solution. For our studies, we used polymeric ZZ-CD23 material purified
by gel filtration as previously described for mouse
ZZ-CD23.20 ZZ-Pselectin and ZZ-Eselectin fusion proteins were used as negative controls in all experiments. MBP-CD23 chimeric protein consists in maltose binding protein fused to the C-terminal 25-kd form of human CD23. It was expressed in soluble form in Escherichia coli, purified by affinity chromatography on
amylose resin and processed to remove endotoxin by repeated passage
through Pierce Detoxi-gel. MBP-CD23 mainly consists in oligomers in
solution, but conversely to ZZ-CD23, this material was not purified to
remove monomeric MBP-CD23.
Isolation of human monocytes
Monocytes from fresh peripheral blood of normal healthy volunteers
were prepared as previously described.38 Briefly,
peripheral blood mononuclear cells (PBMCs) isolated over a Ficoll
density gradient were incubated at 50 × 106
cells/mL in RPMI 1640 medium containing 10% heat-inactivated FCS for
40 minutes at 4°C under rotation, leading to monocyte aggregation,
followed by 10 minutes of incubation on ice. Pellets of aggregated
monocytes were separated by a gradient of FCS and further depleted of T
and NK cells by rosetting with neuraminidase-treated sheep red blood
cells. Monocyte purity routinely consisted of more than 90%
CD14+ cells, less than 1% CD3+ cells, and less
than 1% CD19+ cells. Cellular viability was shown to be
more than 90% using trypan blue exclusion. Polymyxin B (1 µg/mL) was present throughout the whole isolation procedure and
during the activation experiments to rule out any response due to
contaminations by low-endotoxin levels.39 Furthermore, to
prevent activation on adhesion, monocytes were cultured and stimulated
in polypropylene tubes, unless indicated otherwise.
Monocyte activation and measurement of IL-1
Freshly isolated monocytes were cultured in flat-bottom 96-well
tissue culture trays at 100 to 150 × 103 cells per
well in complete RPMI medium in the presence of polymyxin B. In some
experiments, monocytes were preincubated with specific cell permeable
inhibitors for various times and then cultured in the presence of
anti-2 integrin mAbs or recombinant sCD23 fusion proteins for an
additional 14 to 16 hours. Then, cells were lysed by the addition of
0.1 volume of 10% NP40, and IL-1 production was measured in total
cell lysates by enzyme-linked immunosorbent assay (ELISA) (EIA IL-1
kit; Immunotech, Marseille, France) as described
previously.40 The limit of detection of this assay was 10 pg/mL. In some experiments, IL-1 secretion was determined by
measuring the cytokine concentration in culture supernatants.
RNA extraction and Northern blot analysis
Human monocytes (5-10 × 106 cells) were starved
for 14 hours in medium supplemented with 1% FCS in polypropylene tubes
(Falcon). Cells were harvested, resuspended in 500 µL
RPMI/HEPES containing 1% FCS and incubated in 2-mL tubes (Eppendorf)
with or without effectors at 37°C. Total RNA was isolated by lysing
the cells with TRIzol reagent (Life Technologies) according to the
manufacturer's instructions. RNAs (4 µg) were separated,
transferred, hybridized to 32P-labeled cDNA probes specific
for IL-1 (pAT153-hIL-1 gift from Glaxo) or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH),41 and
autoradiographs quantified as previously described.42
Nuclei isolation and run-on transcription assay
Freshly isolated human monocytes (3 × 107 cells)
were starved and then stimulated in polypropylene tubes as described
for RNA extraction. Preparation of nuclei, transcription assay, and
hybridization were performed as previously described.43
Biosynthetically radiolabeled mRNAs (5 × 106 cpm)
were hybridized onto slot-blotted complementary DNA (cDNA) (10 µg per
slot of linearized pAT153-hIL-1 or pBSGAPDH) for 48 hours at
65°C, then washed and treated with RNAse A. Filters were dried and
exposed to Amersham Hyperfilms MP at  80°C.
Western blot analysis and immune complex kinase assay
Nonadherent monocytes were starved as indicated above and stimulated
in 2-mL polypropylene tubes at a concentration of 107
cells/mL. Cells (5-10 × 106) were treated with or
without the effectors for the indicated times at 37°C. After
incubations, monocytes were washed twice with 1 mL ice-cold PBS and
total cell lysates were prepared as previously described.36
For Western analysis, 50 to 100 µg proteins were separated by
SDS-PAGE and transferred to Hybond-ECL membrane (Amersham). The blots
were probed with antiphospho-p44/42 (Santa Cruz Biotech, CA, and New
England Biolabs [NEB], Beverly, MA), antiphospho-p38 (NEB),
anti-ERK2, or anti-p38 (Santa Cruz). Secondary horseradish
peroxidase-conjugated rabbit antimouse or goat antirabbit antibodies
were from DAKO (Copenhagen, Denmark). Antibody-bound proteins were
detected by the Amersham ECL system. For immune complex kinase assay,
active ERK1/2 was immunoprecipitated from total cell lysates
(1-1.5 × 107 cells) with polyclonal rabbit
antiphospho-ERK1/2, followed by incubation with protein A-Sepharose
(Pharmacia). Immune complexes were suspended in a kinase buffer
containing 30 mmol/L HEPES, pH 7.5, 30 mmol/L NaCl, 30 mmol/L
MgAcetate, 10% glycerol, 0.1% NP40, 2 mmol/L DTT, 1 mmol/L
glycerophosphate, 200 µmol/L Na3VO4, and 200 µmol/L ATP to which 1.5 µg Elk1 fusion protein (NEB) was added for
a 30-minute incubation at 30°C. Samples were analyzed by SDS-PAGE
and Western blot, probed with phospho-specific Elk1 antibody (NEB).
| |
Results |
Anti-CD11b and anti-CD11c mAbs as well as sCD23 fusion proteins
trigger IL-1 release on human monocytes
Monocytes are an important source of proinflammatory cytokines. As
previously reported by others,19,31 we show that incubation of enriched human monocytes (more than 90% CD14+ cells)
with soluble anti-CD11b mAbs markedly stimulated IL-1 production to
3585 ± 149 and with anti-CD11c mAbs to 10 599 ± 522 pg/mL,
whereas incubation with anti-CD11a or isotype control antibodies had no
effect (Figure 1). This increase in the
synthesis of IL-1 was accompanied by an important release of the
cytokine in culture supernatants in the amount of 935 ± 113 and
5107 ± 413 pg/mL, respectively. Similar results were obtained
using 2 different mAbs specific for each 2 integrin chain
(CD11a, b, c) (not shown). Interestingly, anti-CD11c appears to be more
efficient in increasing IL-1 production than anti-CD11b. Indeed,
anti-CD11c at the concentration of 5 µg/mL induced a 3-fold higher
production of IL-1 than did anti-CD11b at 20 µg/mL. Furthermore,
we observed that stimulation of monocytes by 2 distinct sCD23 fusion
proteins, ZZ-CD23 and MBP-CD23 (via CD11b and CD11c counterreceptors),
were also potent inducers of IL-1 synthesis (4590 ± 431 and
2872 ± 99 pg/mL, respectively) and secretion (1851 ± 279
and 690 ± 57 pg/mL, respectively). This increase in IL-1
synthesis, measured in total cell lysates, was detected as early as 3 hours after 2 integrin engagement, whereas secretion of IL-1 in
culture supernatants appeared only after 6 hours.
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| Fig 1.
Induction of IL-1 production by engagement of 2
integrins on the surface of freshly isolated human monocytes.
Human monocytes (150 × 103 cells per well) were
incubated for 16 hours with antibodies or recombinant fusion proteins
at the following concentrations: isotype control IgG1 (5 µg/mL);
anti-CD11a (clone BU17, 5 µg/mL); anti-CD11b (clone ICRF44, 20 µg/mL); anti-CD11c (clone BU15, 5 µg/mL); ZZ-CD23 and MBP-CD23 at
0.5 µg/mL; and ZZ-Eselectin and ZZ-Pselectin at 5 µg/mL. Secreted
IL-1 was detected by ELISA in the culture supernatants, total
production of the cytokine being determined in total cell lysates after
addition of 0.1 volume of 10% NP40 to monocytes. Data are mean ± SD from triplicates of 1 experiment representative of 4 others. Similar
results were obtained with anti-CD11a (clone G43-25B), anti-CD11b
(clone 44), and anti-CD11c (clone 3.9) mAbs (not shown).
|
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Thus, we confirm that triggering of monocyte CD11b or CD11c 2
integrins either by mAbs or sCD23 fusion proteins is a potent way of
activating the release of large amounts of IL-1.
Anti-CD11b and anti-CD11c mAbs as well as sCD23 chimeras stimulate
pro-IL-1 gene transcription
To further investigate the molecular mechanisms underlying the
stimulation of IL-1 production, our attention focused on the regulation of pro-IL-1 mRNA expression. To this end, human
monocytes starved for 14 hours in medium, supplemented with 1% FCS,
were cultured in nonadherent conditions in polypropylene tubes to
reduce activation due to adhesion. Cells were then incubated for 1 hour with increasing concentrations of anti-CD11b, anti-CD11c, ZZ-CD23, MBP-CD23, or a high concentration of anti-CD11a, ZZ-Eselectin and
ZZ-Pselectin as negative controls, and expression of pro-IL-1 mRNA
was determined by Northern blot hybridization with specific probes. As
shown in Figure 2, the steady-state level
of pro-IL-1 mRNA was up-regulated in a dose-dependent manner by
incubation with anti-CD11b or anti-CD11c mAbs, reaching a maximal level
with 5 to 10 µg/mL mAbs (panels A and B). At variance with their
effect on the IL-1 protein (Figure 1), anti-CD11b and anti-CD11c
mAbs stimulated pro-IL-1 mRNA to the same level. ZZ-CD23 and
MBP-CD23 increased pro-IL-1 mRNA level in a similar manner, with a
maximal effect at 2 µg/mL (panels C and D). Conversely,
ZZ-selectin fusion proteins and anti-CD11a mAbs did not modulate
IL-1 mRNA level, indicating that up-regulation was not due to
nonspecific activation mediated by the ZZ-domain of chimeras or through
interaction of mAbs with Fc receptors expressed on monocytes.
Time-course experiments were performed in which nonadherent monocytes
were incubated in the presence of anti-CD11a, anti-CD11b, anti-CD11c,
ZZ-CD23, or MBP-CD23 for different times ranging between 15 minutes and
4 hours (Figure 3). Pro-IL-1 mRNA was
detected after 30 minutes, reached a maximal level at 1 to 2 hours, and was maintained after 4 hours of activation by all
the effectors except anti-CD11a mAb, which had no effect. Small
variations in the kinetics of appearance of pro-IL-1 mRNA were
observed in monocytes from one donor to another. However,
these time-course experiments indicate that pro-IL-1 gene is induced early after 2
integrin engagement.
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| Fig 2.
Dose-dependent stimulatory effect of anti-CD11b/c mAbs
and soluble CD23 chimeric proteins on the steady-state level of
pro-IL-1 mRNA.
Nonadherent human monocytes (7 × 106 cells) were
untreated or incubated for 1 hour with various concentrations of the
following effectors: A, anti-CD11b (clone 44), B, anti-CD11c (BU15), C,
ZZ-CD23, D, MBP-CD23 and E, anti-CD11a (BU17, 5 µg/mL), ZZ-Eselectin
(2 µg/mL), and ZZ-Pselectin (2 µg/mL). Then cells were harvested
and RNA isolated and analyzed by Northern blot hybridization with
pro-IL-1 and GAPDH cDNA probes as described in "Materials and
methods."
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| Fig 3.
Time course of pro-IL-1 mRNA induction by 2
integrin engagement.
Monocytes (6-8 × 106 cells) were untreated or
stimulated for various times with the following effectors: A,
anti-CD11a (BU17, 2 µg/mL), B, anti-CD11b (44, 2 µg/mL), C,
anti-CD11c (BU15, 2 µg/mL), D, ZZ-CD23 (1 µg/mL), and E, MBP-CD23
(1 µg/mL). RNA were isolated and analyzed for pro-IL-1
and GAPDH mRNAs expression as previously described. Results are
representative of 3 distinct experiments.
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We next ascertained that the effect we observed on IL-1
production would not be due to any endotoxin contamination (Figure 4). We found that (1) heated mAbs did not
induce pro-IL-1 mRNA expression (not shown), and (2) polymyxin B
(10 µg/mL) did not reverse the up-regulatory effect of anti-CD11b and
anti-CD11c mAbs or sCD23 fusion proteins on the steady-state level of
pro-IL-1 mRNA, whereas it efficiently blocked (60%-70% of
inhibition) the expression of pro-IL-1 mRNA induced by a high
concentration of LPS (200 ng/mL) (Figure 4). Thus, it is unlikely that
monocyte activation should be due to endotoxin contamination.
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| Fig 4.
Effect of polymyxin B on the induction of pro-IL-1
mRNA level.
Nonadherent human monocytes (8 × 106 cells) were
untreated or stimulated for 1 hour at 37°C with either anti-CD11b
(5 µg/mL), anti-CD11c (5 µg/mL), LPS (200 ng/mL), ZZ-CD23 (1 µg/mL), or MBP-CD23 (1 µg/mL), in the presence or absence of
polymyxin B (10 µg/mL).
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To gain insight into the mechanism by which 2 integrin triggering
elicits the induction of IL-1 in monocytes, we first carried out
run-on experiments to determine whether anti-CD11b and anti-CD11c mAbs
or sCD23 fusion proteins controlled the transcription of the
pro-IL-1 gene. Nascent nuclear RNA chains,
biosynthetically labeled with [-32P] UTP, were
isolated from human monocytes previously stimulated for 1 hour with
anti-2 integrin mAbs or sCD23 chimeras, and hybridized to
nitrocellulose filters previously spotted with plasmids harboring either the IL-1 or the GAPDH coding sequence. The results
demonstrate that anti-CD11b and anti-CD11c mAbs, as well as MBP-CD23
and ZZ-CD23, markedly increased the pro-IL-1
gene transcription rate (Figure 5). Under
the same conditions, anti-CD11a mAb and ZZ-Eselectin had no effect
whatsoever.
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| Fig 5.
Determination of the transcriptional activity of
pro-IL-1 gene in isolated nuclei from human monocytes
stimulated by 2 integrin engagement.
Nonadherent human monocytes (30 × 106 cells) were
starved overnight in RPMI 1640 medium supplemented with 1% FCS and
then stimulated for 1 hour with anti-CD11a (2 µg/mL), anti-CD11b (2 µg/mL), anti-CD11c (2 µg/mL), MBP-CD23 (1 µg/mL),
ZZ-Eselectin (1 µg/mL), or ZZ-CD23 (1 µg/mL). Then cells were
lysed, nuclei isolated, and in vitro transcription performed as
described in "Materials and methods." Results are representative
of 3 distinct experiments.
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These data suggest that the activation of human monocytes via the
engagement of CD11b and CD11c 2 integrins increased
pro-IL-1 gene expression by a transcriptional
mechanism. Furthermore, they indicate that the variations in the
synthesis of IL-1 protein mediated by anti-CD11b and anti-CD11c mAbs
(Figure 1) cannot be explained by differences in the rate of
transcription of the pro-IL-1 gene.
Differential effect of anti-CD11b and anti-CD11c mAbs on
pro-IL-1 mRNA stability
To examine the effect of CD11b or CD11c engagement on the stability
of pro-IL-1 mRNA, monocytes were incubated for 1 hour in the
presence of either anti-CD11b or anti-CD11c mAbs, then the
transcription inhibitor DRB was added and the decay of pro-IL-1 mRNA level was monitored as a function of time by quantitative Northern
blot hybridization analyses (Figure 6).
When monocytes were activated with anti-CD11c mAb (panel B), the
steady-state level of pro-IL-1 mRNA decreased rapidly to reach 50%
of the initial value 30 minutes after DRB addition, then persisted at a
significant level (30% of maximal level) for 2 additional hours, and
finally shut off after 4 to 6 hours. When cells were stimulated with
ZZ-CD23 or MBP-CD23 (panels C and D, respectively), the profile of
pro-IL-1 mRNA decay was quite identical to that observed in the
presence of anti-CD11c mAb. Conversely, on monocytes treated with
anti-CD11b mAb (panel A), the level of pro-IL-1 mRNA after 1 hour
of activation totally disappeared within 30 minutes of blocking
transcription. Interestingly, these data demonstrate that the half-life
of pro-IL-1 mRNA is significantly shorter in monocytes activated by
anti-CD11b mAb than in cells treated with anti-CD11c or sCD23. Thus,
the differences in expression of IL-1 protein under CD11b or CD11c
stimulation (Figure 1) could result from distinct posttranscriptional
mechanisms underlying pro-IL-1 mRNA destabilization.
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| Fig 6.
Analysis of pro-IL-1 mRNA stability in monocytes
activated through CD11b or CD11c engagement.
Northern blot analysis of the decay of pro-IL-1 mRNA in human
monocytes (7 × 106) stimulated with anti-CD11b (5 µg/mL, panel A), anti-CD11c (5 µg/mL, panel B), ZZ-CD23 (1 µg/mL,
panel C), or MBP-CD23 (1 µg/mL, panel D). Cells were untreated (lane
1) or activated for 1 hour (lane 2) with the above effectors. Then DRB
(60 µmol/L) was added and cells were incubated for a further 0.5, 1, 2, 4, and 6 hours (lanes 3 to 7, respectively). The level of ribosomal
18S RNA visualized by ethidium bromide was used as a control of the
total RNA level. Right-hand side of the figure shows the densitometric
scanning quantification of pro-IL-1 mRNA level.
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Activation of monocytes through CD11b or CD11c engagement results in
activation of ERK1/2
MAPKs are commonly activated after cell adhesion or integrin
cross-linking.44-48 Furthermore, these kinases are involved
in the signaling pathways induced by a variety of stimuli (LPS,
cell-contact, Fc R aggregation, microtubule disruption), leading to
IL-1 production in monocytes.33-36
To gain insight into the molecular events responsible for
IL-1 induction in our system, we focused our attention on the
possible role of ERK1 (p44) and ERK2 (p42) MAPKs. Monocytes were
stimulated for increasing periods with either anti-2 integrin mAbs
or sCD23 fusion proteins. Total cell lysates were analyzed for MAPK
activation by Western blot using antibodies specific for the dually
phosphorylated (Thr202/Tyr204) active forms of both ERK1 and ERK2
(Figure 7, panels A-E). Incubation of
monocytes with anti-CD11a mAb resulted in a slight increase in the
basal phosphorylation status of ERK2 but did not affect ERK1
phosphorylation (panel A). In contrast, in cells activated by
anti-CD11b, anti-CD11c, MBP-CD23, and ZZ-CD23 (panels B, C, D, and E,
respectively), we observed a rapid, significant (4- to 8-fold), and
persistent up-regulation in the level of phosphorylation of both ERK1
and ERK2 starting at 2 minutes and persisting for 4 more hours.
Stripping of the blots and reprobing with an anti-ERK2 antibody showed
that the modulation observed was not due to loading variations.
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| Fig 7.
Effect of 2 integrin engagement on the phosphorylation
status and activation of ERK1/2 kinase activity.
Panels A to E: Time course of stimulation of the phosphorylation status
of ERK1 and ERK2 MAP kinases by 2 integrin engagement. Monocytes
(5 × 106 cells) were stimulated for the indicated
times with anti-CD11a (5 µg/mL), anti-CD11b (5 µg/mL), anti-CD11c
(5 µg/mL), MBP-CD23 (1 µg/mL), and ZZ-CD23 (5 µg/mL),
respectively. Cell lysates were analyzed on SDS-PAGE, followed by
Western blot using a polyclonal antibody raised against the dually
phosphorylated ERK1 (44 kd) and ERK2 (42 kd). Western blots were
stripped and reprobed with anti-ERK2 rabbit polyclonal antibody as a
loading control. Results are the most representative of 4 distinct
experiments. Panel F: Effect of 2 integrin engagement on the
activation of ERK1/2 kinase activity. Nonadherent human monocytes
(15 × 106 cells) were untreated or stimulated with
anti-CD11a (5 µg/mL), anti-CD11b (5 µg/mL), anti-CD11c (5 µg/mL),
ZZ-Eselectin (5 µg/mL), ZZ-CD23 (5 µg/mL), or MBP-CD23 (2 µg/mL) in the presence or absence of U0126 (20 µmol/L). After 15 minutes of incubation, cell lysates were prepared and
immunoprecipitated with antiphospho-ERK1/2 antibody. The pelleted
immunoprecipitates were incubated with Elk1-GST fusion protein as a
substrate and phosphorylation of Elk1 was visualized by Western blot
using an antibody specific for phosphorylated Elk1.
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To ascertain whether this increase in ERK1/2 phosphorylation would
correlate with an increased kinase activity, we performed immune
complex kinase assays (Figure 7F). Monocytes were activated for 15 minutes, cell lysates were immunoprecipitated using
anti-phospho-ERK1/2 antibodies, and ERK activity was measured by means
of the phosphorylation of a recombinant Elk1-GST (glutathione
S-transferase) fusion protein. As demonstrated in Figure 7F, Elk1
phosphorylation was increased by anti-CD11b, anti-CD11c, ZZ-CD23, and
MBP-CD23 but not by anti-CD11a and ZZ-Eselectin. Furthermore,
pretreatment of monocytes with U0126 (20 µmol/L), a specific
inhibitor of MEK-1 and MEK-2, 2 upstream activators of
ERK1/2,49,50 totally abolished the activation of ERK1/2
mediated by mAbs and sCD23 fusion proteins.
ERK1/2 activity, a prerequisite of 2 integrin signaling
of IL-1 synthesis in monocytes
To determine the role of the ERK1/2 pathway in the up-regulation of
IL-1 synthesis induced by CD11b and CD11c triggering, we next
assessed the effects of U0126 on the expression of pro-IL-1 mRNA
(Figure 8, panels A-D) and on IL-1
production (Figure 8E). Nonadherent monocytes were preincubated for 30 minutes with increasing concentrations of U0126 (0.2 to 40 µmol/L),
then stimulated for 1 hour with anti-CD11b, anti-CD11c, MBP-CD23, and
ZZ-CD23 for 1 hour (Figure 8, left part of panels A to D,
respectively). Expression of pro-IL-1 mRNA induced by anti-CD11b
and anti-CD11c mAbs was inhibited by U0126 in a dose-dependent manner
with a complete inhibition at 20 to 40 µmol/L. In contrast, U0126
inhibited only 60% to 80% of the level of IL-1 messenger induced
by MBP-CD23 and ZZ-CD23. Because the expression of pro-IL-1 mRNA
and activation of ERK1/2 are long-lasting events, we analyzed the
effect of U0126 (40 µmol/L) on the level of pro-IL-1 mRNA 1, 3, and 6 hours, respectively, after monocyte activation by anti-CD11b,
anti-CD11c, MBP-CD23, and ZZ-CD23 (right part of panels A to D,
respectively). Pro-IL-1 mRNA level induced by anti-CD11b and
anti-CD11c mAbs was totally abolished by U0126, whatever the length of
activation. This experiment confirmed that 20% to 40% of the signal
remained resistant to U0126 in monocytes stimulated by sCD23 fusion
proteins. In addition to the effect of U0126 on pro-IL-1 mRNA, we
also analyzed its action on IL-1 production (Figure 8E). U0126
inhibited IL-1 production stimulated by anti-CD11b or anti-CD11c
mAbs in a dose-dependent fashion with an IC50 of 0.5 to 1 µmol/L.
Similar results were obtained with PD98059, another MEK1/2-specific
inhibitor51 (not shown). The same experiment was performed
on monocytes activated with MBP-CD23 or ZZ-CD23. Results indicate that
U0126 also inhibited the production of IL-1 with an IC50 of 0.5 µmol/L. However, in accordance with our previous observations at the
IL-1 mRNA level, inhibition was not complete because at 30 µmol/L
of U0126, approximately 30% of the maximal production remained.
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| Fig 8.
Effect of U0126 on the production of IL-1 induced by
2 integrin engagement.
Panels A-D: Effects of U0126 on the level of pro-IL-1 mRNA induced
by ligation of 2 integrins. Left-hand side: Monocytes were
preincubated for 30 minutes with increasing concentrations of U0126 and
then stimulated with anti-CD11b (5 µg/mL, A), anti-CD11c (5 µg/mL,
B), MBP-CD23 (1 µg/mL, C) or ZZ-CD23 (1 µg/mL, D) for 1 hour at
37°C. Right-hand side: Cells were untreated or preincubated for 30 minutes with U0126 (40 µmol/L) and then stimulated for the indicated
times with anti-CD11b (5 µg/mL, A), anti-CD11c (5 µg/mL, B),
MBP-CD23 (1 µg/mL, C) or ZZ-CD23 (1 µg/mL, D). Panel E:
Dose-dependent inhibitory effect of U0126 on the production of IL-1.
Human monocytes (150 × 103 cells per well) were
pretreated for 30 minutes with various concentrations of U0126 and then
incubated for an additional 16 hours with anti-CD11b (20 µg/mL),
anti-CD11c (5 µg/mL), ZZ-CD23 (2 µg/mL), or MBP-CD23 (1 µg/mL).
IL-1 production was determined in total cell lysates by ELISA. Data
are means from triplicates of 1 experiment representative of 2 others
(in each condition the SD was less than 5%).
|
|
Taken together, these results indicate that the activation of ERK1 and
ERK2 seems to be mandatory for mediating the effect of anti-CD11b and
anti-CD11c mAbs on pro-IL-1 mRNA levels and on IL-1 production.
Regarding IL-1 production induced by sCD23 fusion proteins, the
partial inhibitory effect of U0126 suggests that, in this case, IL-1
transcription and synthesis are controlled mainly by an
ERK1/2-dependent pathway and to a lesser extent by another,
ERK1/2-independent, pathway.
Activation and role of p38/SAPK2 in monocytes triggered by
anti-CD11b and anti-CD11c mAbs or sCD23 fusion proteins.
We hypothesize that other congeners of the MAPK family could
participate in the 2 integrin-mediated induction of IL-1
synthesis. To this end, we tested the possible activation of p38/SAPK2,
which was reported to regulate the production of IL-1 induced by LPS stimulation.33,52 p38 activity was assessed by Western blot using an antibody raised against its dually phosphorylated active form.
Results shown in Figure 9A indicate that
the weak basal activation of p38 in untreated monocytes was not
affected when cells were incubated in the presence of anti-CD11a mAb.
In contrast, stimulation of monocytes with anti-CD11b mAb, anti-CD11c
mAb, MBP-CD23, or ZZ-CD23 (Figure 9 panels B to E, respectively) for times ranging between 2 minutes and 2 to 4 hours resulted in a marked
and transient activation of p38 MAPK (8- to 20-fold induction) with a
maximal effect at 15 to 30 minutes. ZZ-Eselectin treatment was without
any effect (not shown).
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| Fig 9.
Time course of stimulation of the phosphorylation of
p38/SAPK2 kinase in human monocytes activated by anti-CD11b mAb,
anti-CD11c mAb, or sCD23 fusion proteins.
Monocytes (5 × 106 cells) were stimulated as
depicted in the legend of Figure 7, ie, anti-CD11a (5 µg/mL),
anti-CD11b (5 µg/mL), anti-CD11c (5 µg/mL), MBP-CD23 (1 µg/mL), and ZZ-CD23 (5 µg/mL) in panels A to E, respectively. Cell
lysates were analyzed by Western blot using a specific antiphospho-p38
antibody. Western blots were stripped and reprobed with anti-p38 rabbit
polyclonal antibody as a loading control. Results are the most
representative of 4 distinct experiments.
|
|
Because the pyridinyl imidazole compound SB203580 is largely reported
to be a specific inhibitor of p38 kinase activity, both in vitro and in
vivo,53-56 we used this inhibitor to determine whether the
activation of p38 MAPK plays a role in the control of pro-IL-1 mRNA
expression and IL-1 production (Figure
10). Monocytes were used untreated or
preincubated for 90 minutes with 10 µmol/L SB203580, a concentration
which proved to abolish p38 kinase activity.53 Cells were
activated for various times ranging from 30 minutes to 4 hours with
anti-CD11b, anti-CD11c mAbs, ZZ-CD23, or MBP-CD23, and pro-IL-1
mRNA level was determined by Northern blot hybridization (Figure 10,
panels A-D, respectively). SB203580 inhibited 70% to 80% of the
pro-IL-1 mRNA level after short periods (0.5-1 hours) of monocyte
activation, but it had no inhibitory effect at later times of cell
activation (2-4 hours). Consistent with recent reports, p38 kinase
activity seems to be involved in the control of the immediate early
induction of pro-IL-1 mRNA transcription.57
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| Fig 10.
Effects of SB203580 on the production of IL-1 induced
by 2 integrin engagement.
Panels A-D: Northern blot analysis of the effects of SB203580 on the
level of pro-IL-1 mRNA. Monocytes were untreated or preincubated
for 90 minutes with SB203580 (10 µmol/L) and then stimulated with
anti-CD11b (5 µg/mL, A), anti-CD11c (5 µg/mL, B), ZZ-CD23 (1 µg/mL, C), or MBP-CD23 (1 µg/mL, D) for the indicated times. Panel
E: Dose-dependent inhibitory effect of SB203580 on the production of
IL-1. Human monocytes (150 × 103 cells per well)
were pretreated for 90 minutes with SB203580 at various concentrations
and then incubated for an additional 16 hours with anti-CD11b (20 µg/mL), anti-CD11c (5 µg/mL), ZZ-CD23 (2 µg/mL), or
MBP-CD23 (1 µg/mL). IL-1 production was determined in total cell
lysates by ELISA. Data are means from triplicates of 1 experiment
representative of 2 others (in each condition the SD was less than
10%).
|
|
Because the commonly described mechanism of SB203580-mediated
inhibition of cytokine synthesis takes place at the translational level,54 we also tested the effect of this inhibitor on the production of IL-1 by monocytes (Figure 10E). SB203580 inhibited in
a dose-dependent manner the production of IL-1 induced by engagement
of anti-CD11b, anti-CD11c mAbs, or sCD23 fusion proteins with IC50 of
20, 100, and 30 nmol/L, respectively. However, the inhibition of
p38/SAPK2 was not sufficient to abrogate IL-1 synthesis, because
20% to 30% of the maximal cytokine production persisted at 1 µmol/L
of SB203580. Similar results have been obtained with SB202190,54 another highly specific blocker of p38 kinase
activity (not shown).
Altogether, these data suggest that, in our system, a p38
MAPK-dependent pathway is involved in the control of IL-1 production both at the transcriptional and translational levels.
| |
Discussion |
We and others have previously reported that 2 integrins (CD11a,
CD11b, and CD11c) play an important role in the production of IL-1
on human monocytes stimulated by direct contact with activated T
lymphocytes.58-60 However, because these molecules are
expressed on the surface of both monocytes and T lymphocytes, it was
difficult to clearly define the role that 2 integrins play on each
cell type in this process.
In this study, we investigated the molecular events involved in the
control of IL-1 production on freshly isolated human monocytes
activated through 2 integrin engagement. In accordance with previous
data,19 we confirm that engagement of CD11b and CD11c by
mAbs provides a potent signal of activation, resulting in a marked
production of IL-1. Interestingly, anti-CD11c mAbs were more
effective in inducing IL-1 synthesis than were anti-CD11b mAbs.
However, these results contradict other reports that indicate that
ligation of 2 integrins was inefficient in mediating signal transduction, leading to early gene expression and particularly to
IL-1 expression.61,62 Taken together, these observations indicate that some, but not all, anti-CD11b and anti-CD11c mAbs are
capable of stimulating both IL-1 production and secretion in human
monocytes. Similarly, certain anti-CD11b mAbs induced the respiratory
burst, whereas other mAbs did not.63 In contrast, the
anti-CD11a mAbs we used (BU17 and G43-25B) were inefficient in inducing
IL-1 production. This is in agreement with a recent study in which 2 other anti-CD11a IgG (25.3 and B-B15 from Immunotech) did not stimulate
cytokine synthesis by human monocytes.19 Altogether, these
results rule out the possibility that the stimulatory effects of
anti-CD11b and anti-CD11c mAbs were mediated via their binding to Fc receptors.
Interestingly, 2 distinct recombinant fusion proteins of soluble CD23,
a natural ligand of CD11b and CD11c, produced effects similar to those
triggered by anti-CD11b and anti-CD11c mAbs, suggesting that the
stimulation of IL-1 production through 2 integrin ligation was
physiologically relevant. The specificity of action of sCD23 by binding
to CD11b and CD11c has been clearly demonstrated in that Fab fragments
of anti-CD23, anti-CD11b, and anti-CD11c mAbs inhibited IL-1
production induced by sCD23 in monocytes.19 Furthermore,
production of other proinflammatory mediators such as nitric oxide,
oxidative burst, and other proinflammatory cytokines (TNF, IL-6),
induced by sCD23 and anti-CD11b or anti-CD11c IgG were also
specifically blocked by Fab fragments of anti-CD23, anti-CD11b, and
anti-CD11c mAbs.19-21 These blocking effects of anti-CD11b
and anti-CD11c Fab fragments and the ability of soluble CD23 to
oligomerize,64 suggest that cross-linking of CD11b or CD11c
could be necessary to mediate monocyte activation.
Production of IL-1 by monocytic cells is a complex and finely
regulated process.28 Our study brings new insights in the molecular mechanisms controlling IL-1 synthesis induced by ligation of 2 integrins. Engagement of CD11b or CD11c on monocytes by mAbs or
sCD23 fusion proteins markedly induced both steady-state level of
proIL-1 mRNA and transcription rate of the
pro-IL-1 gene. However, some differences
exist between the mechanisms of each effector. Indeed, we notably
observed that pro-IL-1 transcripts have a much shorter half-life
in monocytes incubated with anti-CD11b mAbs than in cells activated by
anti-CD11c mAbs or sCD23 fusion proteins. Furthermore, the half-life of
pro-IL-1 mRNA in all our conditions appears much lower than that
observed in response to LPS.65 Therefore, the presence of
such unstable pro-IL-1 transcripts suggests that the transcription
rate must be sufficiently sustained in monocytes activated through 2
integrins to allow translation, whereas this is not necessary on LPS
activation. Moreover, the 3' untranslated region of pro-IL-1
mRNA contains an AU-rich sequence that has been implicated in rapid
message turnover.66-68 Thus, the distinct
posttranscriptional regulation of pro-IL-1 mRNA degradation induced
by ligation of CD11b or CD11c could account for differences in the
efficiency of translation and consequently in the production of
IL-1.
MAPK family members are known to play a key role in the control of
pro-IL-1 expression induced by a variety of
stimuli.33-36 According to a recent report, cross-linking
of CD11a and CD11b by mAbs results in the activation of ERK1/2 and the
induction of procoagulant activity in the THP-1 monocytic cell
line.69 The work presented herein was designed to evaluate
the role of MAPKs in the control of IL-1 production in response to
ligation of 2 integrins on primary human monocytes. We demonstrated
for the first time that engagement of CD11b or CD11c by mAbs on
nonadherent monocytes activated both the p42/44 MAPK and p38/SAPK2
pathways, whereas anti-CD11a mAbs had no effect on these
kinases. Our results indicate that the stimulation of these MAPK
pathways by anti-CD11b and anti-CD11c mAbs is probably physiologically
relevant because it can also be activated by sCD23. To our knowledge,
this is the first report showing that stimulation of human monocytes by
sCD23 leads to the activation of ERK1/2 and p38 MAPKs.
Whereas LPS induces a rapid and transient activation of
ERK1/2,50,70 the ligation of CD11b or CD11c by mAbs or
sCD23 fusion proteins results in a long-lasting activation of these MAP
kinases, which suggests that this pathway plays a major role in
monocyte activation. In keeping with this notion, we demonstrated that the inhibition of the ERK1/2 pathway by the specific U0126 compound abolished IL-1 mRNA expression and IL-1 production induced by anti-CD11b and anti-CD11c mAbs. These results clearly indicate that
activation of ERK1/2 is a mandatory condition for the transcriptional induction of IL-1 expression mediated by 2 integrin triggering. This observation is consistent with the fact that transactivation of
the nuclear factor NF-IL6 essential for
pro-IL-1 gene induction, is dramatically
up-regulated by MAPK.71-73 Moreover, the lack of effect of
anti-CD11a mAbs on IL-1 production could be explained by the fact
that these antibodies do not stimulate MAPK congeners. It is noticeable
that, in monocytes activated by sCD23 fusion proteins, the inhibition
of IL-1 production by U0126 was not total (70%). This partial
inhibition could be explained by a recent report suggesting that sCD23
could mediate part of its effects on monocytes and notably the
induction of IL-1 synthesis through interaction with the vitronectin
receptor and its associated CD47 molecule.74 Thus, U0126
could block the ERK1/2-dependent production of IL-1 induced by the
interaction of sCD23 with CD11b/CD18 and CD11c/CD18, whereas signaling
mediated by ligation of sCD23 on CD47 could be ERK1/2 independent.
Moreover, it has clearly been established that 2 integrins, and
notably CD11b/CD18 and CD11c/CD18, can form molecular complexes with
other leukocyte receptors such as FcRI, FcRII, FcRIIIB, uPAR
(CD87), and CD14.75 Such molecular associations have been
implicated in the mediation of the respiratory burst induced by some
anti-CD11b mAbs.63 Consequently, such molecular complexes
could also participate in the control of IL-1 expression induced by
anti-CD11b and anti-CD11c mAbs.
The p38 MAPK is a key component in stress-induced signal transduction
pathways, leading notably to inflammatory cytokines production.76,77 Experiments using SB20358056
demonstrate that p38 also actively participates in the signaling
pathways responsible for IL-1 production induced by 2 integrin
ligation. Indeed, inhibition of p38 activity results in a marked
decrease (70%-80%) in pro-IL-1 mRNA level at early times (0.5-1 hours) of monocyte activation by anti-CD11b, anti-CD11c, or sCD23
fusion proteins. This is consistent with the transient activation of p38 by 2 integrin ligation and is in total accordance with a recent
report demonstrating that p38 MAPK can control the transcriptional activation of the IL-1 gene induced by LPS
through interaction with the C/EBP (NF-IL6) and C/EBP
transcription factors.57 However, SB203580 failed to
inhibit the expression of the pro-IL-1 transcript at later times of
monocyte activation by 2 integrins. Thus, the early inhibition of
pro-IL-1 mRNA expression cannot reflect by itself the inhibitory
effect of SB203580 observed on IL-1 protein production. This implies
that SB203580 also mediates its effects via a more classical
translational mechanism.54,78
In conclusion, the above results clearly demonstrate that ERK1/2 and
p38/SAPK2 pathways are essential for the stimulation of IL-1
production induced by the direct engagement of the 2 integrin alpha
chains CD11b and CD11c either with mAbs or their functional ligand
sCD23 in monocytes. This activation of monocytes through 2 integrins
may be relevant to physiologic and pathologic conditions involving
cell-cell interactions such as adherence of activated monocytes to
endothelial cells allowing transmigration, macrophage/T lymphocyte
interaction at the inflammatory site, or other pathologic situations in
which sCD23 level is increased, such as in rheumatoid
arthritis.29,32 Thus, further understanding of the
molecular mechanisms controlling the production of proinflammatory cytokines would be helpful in the design of new anti-inflammatory therapies.
| |
Acknowledgments |
We are indebted to Dr M. Bird for kindly providing ZZ-CD23, MBP-CD23,
and ZZ-selectin fusion proteins. We are also grateful to M. T. Kauffmann for technical assistance and to Dr G. Ponzio, Dr B. Rossi, Dr
N. Rochet, and Mrs R. Rehm for critical reading of the manuscript.
| |
Footnotes |
Submitted October 29, 1999; accepted February 17, 2000.
Supported by the Swiss National Science Foundation grant no.
31.50930.97 and by the Hans Wilsdorf Foundation.
Reprints: Roger Rezzonico, INSERM U364-Facultè de
Mèdecine, Avenue de Valombrose, 06107 Nice cedex 02-France;
e-mail: rezzonic{at}unice.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction