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*
Department of Pathology, General Pathology Unit, University of Verona, Verona, Italy; and
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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response region or the high-affinity synthetic derivative of
the c-sis-inducible element in neutrophils that have been
cultured for at least 3 h with LPS. Similarly, the ability of
IL-10 to up-regulate suppressor of cytokine signaling (SOCS)-3 mRNA was
dramatically enhanced in cultured neutrophils and, as a result,
translated into the SOCS-3 protein. Since neutrophils’ acquisition of
responsiveness to IL-10 required de novo protein synthesis, we assessed
whether expression of IL-10R1 or IL-10R2 was modulated in cultured
neutrophils. We detected constitutive IL-10R1 mRNA and protein
expression in circulating neutrophils, at levels which were much lower
than those observed in autologous monocytes or lymphocytes. In
contrast, IL-10R2 expression was comparable in both cell types.
However, IL-10R1 (but not IL-10R2) mRNA and protein expression was
substantially increased in neutrophils stimulated by LPS. The ability
of IL-10 to activate Stat3 tyrosine phosphorylation and SOCS-3
synthesis and to regulate IL-1 receptor antagonist and
macrophage-inflammatory protein 1
release in LPS-treated neutrophils
correlated with this increased IL-10R1 expression, and was abolished by
neutralizing anti-IL-10R1 and anti-IL-10R2 Abs. Our results
demonstrate that the capacity of neutrophils to respond to IL-10, as
assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression, and
modulation of cytokine production, is very dependent on the level of
expression of IL-10R1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced class II MHC
expression, reactive oxygen intermediates, and NO production, and
suppressing the synthesis of proinflammatory cytokines as well
(1). In the murine system, IL-10 inhibits the development
of Th1 effector cells from naive CD4+ T
lymphocytes, thereby enhancing development of a Th2 dominant population
(1). Furthermore, because of its capacity to inhibit the
production and release of TNF-
and other proinflammatory cytokines
by phagocytes in response to LPS, IL-10 reduces the lethality of
experimental septic shock in mice (2, 3). The intracellular mechanisms by which IL-10 mediates its biological effects remain largely unknown. IL-10 is known to bind to a multicomponent structure composed of at least two subunits: the IL-10R1 (4, 5, 6) and of the recently identified CRFB4/CRF2-4 molecule (7, 8, 9), now called IL-10R2, both members of the class II family of cytokine receptors. The primary ligand-binding component, IL-10R1, binds IL-10 with high affinity and in the presence of IL-10 associates with the accessory subunit, IL-10R2 (8). Both chains are required for signal transduction (8, 9) and interact with members of the Janus kinase (Jak)3 tyrosine kinase family in a ligand-independent manner: IL-10R1 associates with Jak1 (8), while IL-10R2 associates with Tyk2 (8, 10). IL-10 binding leads to receptor heterodimerization which, in turn, leads to the activation of the Jak kinases and phosphorylation of IL-10R1 on cytoplasmic tyrosine residues (11) followed by direct Stat3 recruitment and tyrosine phosphorylation. Stat1 and Stat5 may be also tyrosine phosphorylated in response to IL-10 (6, 12, 13, 14), but the mechanisms of their recruitment to the IL-10R complex and activation remain unclear. Upon phosphorylation, Stat1 and Stat3 (and Stat5) homo/heterodimerize and translocate to the nucleus where they bind to specific promoter sequences to modulate transcription. Moreover, IL-10 has also been shown to inhibit the p56lyn tyrosine kinase activation and other subsequent events in this pathway, including Ras activation (15), and to activate both phosphatidylinositol 3-kinase and p70 S6 kinase as well (16).
Recent studies performed in our laboratory, as well as by other groups,
have shown that IL-10 can also regulate a number of polymorphonuclear
neutrophil (PMN) functional responses, including cytokine production,
reactive oxygen intermediates and prostanoid production,
platelet-activating factor synthesis and release, phagocytosis,
apoptosis, and membrane Ag expression (17). Among these
responses, the modulation of cytokine/chemokine production by IL-10 is
certainly the most widely reported and has been the focus of intensive
investigation (18). In contrast, the ability of IL-10 to
directly trigger or modulate other responses is somewhat controversial
since initial findings suggesting a given action mediated by IL-10
often could not be independently confirmed (17, 18).
Whether this situation simply reflects the different experimental
approaches used by various investigators, or whether it hints to
unknown biological mechanisms is an issue that needs to be clarified.
In this context, our recent observations on the ability of IL-10 to
up-regulate suppressor of cytokine signaling (SOCS) 3 mRNA expression
in the absence of detectable Stat3/Stat1 tyrosine phosphorylation and
Stat-dependent DNA-binding activities in freshly isolated neutrophils
were very intriguing (19, 20). Indeed, while these data
have provided a possible explanation for the inability of IL-10 to
induce CD64/FcRI expression in neutrophils (19), as
opposed to monocytes (12, 19, 21), they have also
suggested that, at least in human neutrophils, the activation of Stat1
and/or Stat3 is not required for the regulatory effects of IL-10 on
cytokine production. The latter notion is however in sharp contrast
with other studies performed with monocytes from Jak1-deficient mouse
embryos (22), and with neutrophils and macrophages derived
from mice engineered to express a genetic Stat3 deficiency
(23), which have highlighted an obligatory role for
Jak1 and Stat3 in mediating the effects of IL-10 on LPS-induced
cytokine release.
The results reported in the current study reconcile the above-mentioned conflicting reports insofar as we show herein that human neutrophils can acquire the capacity to respond to IL-10 in terms of Stat3 activation if they are cultured with LPS for a few hours. This occurs because the gene and surface expression of the IL-10R1, which is only present at low levels in circulating or freshly isolated neutrophils, is significantly up-regulated in cultured neutrophils, reaching sufficient levels to confer IL-10 inducibility of responses such as Stat activation, modulation of proinflammatory cytokine production, and enhanced SOCS-3 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A neutralizing mouse anti-hIL-10R1 mAb (3B6, IgG1) was prepared by immunization of BALB/c mice with soluble hIL-10R1-FLAG-His6 as previously described (5). Mouse anti-hIL-10R2 mAbs (all IgG1) were prepared by immunizing BALB/c mice with a recombinant protein containing the extracellular domain of hIL-10R2 fused to the Fc portion of human IgG1. This protein was expressed transiently in COP5 cells (pCDM8 vector; Invitrogen, Carlsbad, CA) and purified by HiTrapA affinity chromatography (Pharmacia Biotech, Uppsala, Sweden). Candidate mAbs were identified first by ELISA, then by their ability to detect recombinant hIL-10R2 expressed in transfected Ba/F3 cells (5). The neutralizing anti-hIL-10R2 mAb 1A8.3 was identified by its ability to block responses to hIL-10 of human TF1 cells expressing recombinant hIL-10R1 (TF1-hIL-10R) and human PBMC (5). Anti-IL-10R2 mAb 4B2.1 is a non-neutralizing mAb and was used for FACS staining.
Cell purification and culture
Highly purified granulocytes (>98.5%) and PBMC were isolated
under endotoxin-free conditions from buffy coats of healthy donors as
previously described (24). The granulocyte populations
contained usually <4% eosinophils (n = 30) as
revealed by May-Grünwald-Giemsa staining. Culture conditions
slightly differed depending on the type of assay performed. For the
immunoblot or EMSA experiments, leukocytes were suspended in standard
culture medium (RPMI 1640 medium; BioWhittaker, Walkersville, MD)
supplemented with 10% low-endotoxin FCS (<0.05 endotoxin units/ml;
Euroclone, Paignton, U.K.), treated with the various stimuli,
distributed in either 6/12-tissue culture well plates, or in
polystyrene flasks (BioWhittaker), and then cultured for 20 min at
37°C in a 5% CO2 atmosphere before cell
disruption (see below). Alternatively, neutrophils or PBMC were
cultured in the absence or presence of 100 ng/ml LPS (from
Escherichia coli serotype 026:B6; Sigma, St. Louis, MO) for
the times indicated in either standard culture medium or in serum-free
medium on poly(2-hydroxyethyl methacrylate (poly-Hema)-covered surfaces
(25, 26) (to achieve nonadherent conditions), before
stimulation for 20 min (unless differently specified) and cell lysis.
Stimuli used were: 200 U/ml IL-10 (from DNAX and Schering-Plough, Palo
Alto, CA, or from PeproTech, Piscataway, NJ) (19, 20), 100 U/ml IFN- (Hoffmann-LaRoche, Basel, Switzerland)
(19), 1000 U/ml IFN- (Roferon; Roche Laboratories,
Nutley, NJ), 1000 U/ml G-CSF (Granulokine; Hoffmann-LaRoche), or 10
ng/ml GM-CSF (PeproTech). In selected experiments, neutrophils were
cultured in standard or serum-free medium for 4 h with or without
LPS in the presence or absence of 20 µg/ml cycloheximide (CHX; Sigma)
before stimulation with IL-10 or G-CSF and cell lysis for protein or
RNA extraction. In other experiments, LPS was preincubated with 10
µg/ml polymyxin B sulfate (PMX; Sigma) before cell addition. For the
experiments aimed to determine cytokine release, neutrophils were
suspended at 5 x 106/ml in standard culture
medium with or without 200 U/ml IL-10, stimulated with 100 ng/ml LPS
15–20 min later, and then cultured for up to 21 h in 24-tissue
culture wells. At the times indicated in the results, cell-free
supernatants were harvested and stored at -20°C. In some of these
latter experiments, 5 µg/ml anti-IL-10R1 or anti-IL-10R2 and
appropriate control Abs were added to the cell cultures, either 30 min
before IL-10 or at various times after IL-10 and LPS. All reagents used
were of the highest available grade and were dissolved in clinical
grade pyrogen-free water (19, 20, 24).
Immunoblots
After stimulation, neutrophils and PBMC (2.5–5 x
106/condition) were diluted in ice-cold PBS and
centrifuged twice at 500 x g for 5 min at 4°C. Cells
were then suspended in lysis buffer (20 mM HEPES (pH 7.9), 420 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P-40, 20% (v/v) glycerol, and
1 mM DTT) containing inhibitors of proteases (5 µg/ml leupeptin, 5
µg/ml pepstatin A, 1 mM PMSF, 1 mg/ml 1-antitrypsin), and
phosphatases (1 mM Na3VO4,
10 mM NaF, 10 µM phenylarsin oxide) (20, 27), and
following a 15-min incubation on ice, cell debris were spun down
(12,000 x g, 20 min, 4°C) and the supernatants were
frozen and stored at -80°C. Small aliquots of the various extracts
were routinely processed for protein content determination, by using a
protein assay kit (Bio-Rad, Hercules, CA). For the direct detection of
tyrosine-phosphorylated Stat3, usually 30 µg of lysates prepared from
PMN and PBMC were electrophoresed on 7.5% SDS-PAGE and subsequently
transferred to nitrocellulose (Hybond; Amersham Pharmacia Biotech,
Piscataway, NJ) by electroblotting. Membranes were first blocked for
1 h at room temperature in TBST (20 mM Tris-HCl (pH 7.6), 137 mM
NaCl, and 0.1% Tween 20) containing 5% BSA and then incubated
overnight at 4°C in the presence of the phospho-specific Stat3 Ab
(Tyr705) (9131S; New England Biolabs, Beverly,
MA) diluted at 1/1000 in blocking buffer. Membranes were then
probed with anti-Stat3 (C20, raised against aa 750–769, purchased
from Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1/2000 in
blocking buffer. For the detection of antigenic SOCS-3 (molecular
mass of 29 kDa), 50 µM MG132 (N-CBZ-Leu-Leu-Leu-Ala, a
proteasome inhibitor; Sigma) was added to the cell cultures 1 h
before stopping incubation to prevent degradation of the protein
(28). Neutrophil lysates (100 µg) were electrophoresed
on 12% SDS-PAGE, were electroblotted to nitrocellulose membranes, and
the latter was incubated with 1.4 µg/ml rabbit anti-SOCS-3
polyclonal Abs (ImmunoBiological Laboratories, Tokyo, Japan; dissolved
in TBST containing 1% BSA) (28). Ab binding was detected
by using HRP-conjugated anti-mouse or anti-rabbit IgG (1/4000
dilution in TBST) and revealed using the chemiluminescence system (ECL;
Amersham Pharmacia Biotech) according to the manufacturer’s
instructions.
EMSAs
Protein-DNA complexes were detected by EMSA analysis of the
various extracts as previously described (19, 20), with
the following modifications: 12 µg of whole-cell extracts were
usually incubated for 10 min at room temperature in a buffer containing
10 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl2, 1 mM
DTT, 100 µg/ml poly(dI-dC)·poly(dI-dC), 50 µg/ml salmon sperm,
and 10% glycerol, followed by addition of a
32P-labeled double-stranded oligonucleotide probe
corresponding to the IFN- response region (GRR) element located
within the promoter of the FcRI gene (29) (5'-CTT TTC
TGG GAA ATA CAT CTC AAA TCC TTG AAA CAT GCT-3') or the high-affinity
synthetic derivative of the c-sis-inducible element (SIE),
hSIE (5'-gtc gaC ATT TCC CGT AAA TCg-3') for 15 min (30).
Supershift experiments were performed by incubating the extracts with
0.5 µg of anti-Stat3 and/or anti-Stat1 Abs (C20 and E23,
respectively, from Santa Cruz Biotechnology) for 30 min at room
temperature before adding the labeled probe.
Northern blot analyses
Total RNA was extracted from PMN using the guanidinium isothiocyanate method and processed for Northern blot analysis as already described (24). Individual mRNA species in human cells were detected by autoradiography after hybridization of nylon filters with cDNA probes labeled with 32P using Ready-to-go kits (Amersham Pharmacia Biotech). The probes used consisted of full-length cDNA fragments encoding IL-10R2 (kindly provided by Dr. G. Uzé, Centre National de la Recherche Scientifique, Montpellier, France) (7), SOCS-3 (kindly provided by Dr. A. Yoshimura, Institute of Life Science, Kurume University, Japan) (31), as well as actin (kindly provided by Dr. G. Trinchieri, Schering-Plough, Dardilly, France). The IL-10R1 cDNA fragment was prepared by RT-PCR of mRNA purified from LPS-stimulated monocytes by using oligonucleotides specific for the IL-10R1 (5'-CCG TCT GTG TGG TTT GAA GCA GAA, 3'-GAT GAT GAC GTT GGT CAC GGT GAA, based on accession no. U00672), kindly provided by Dr. C. Albanesi (Istituto Dermopatico dell’Immacolata, Instituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy).
Extracellular staining of IL-10R
Surface expression of IL-10R1 and IL-10R2 in neutrophils, monocytes, and lymphocytes was performed by flow cytometry analysis as previously described (19). Cell staining was performed using predetermined optimal concentrations of mAbs (5 µg/ml) followed by a biotin-conjugated affinity-purified Ab (goat F(ab')2 anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) preadsorbed with human serum) and streptavidin-PE (BD Biosciences, Mountain View, CA)) as fluorochrome. mAbs used in these assays were: 3B6 (anti-IL-10R1), 4B2.1 (anti-IL-10R2), and OKM1 (anti-CR3, kindly provided by Dr. G. Trinchieri, Schering-Plough). Irrelevant purified mouse IgG1 (Sigma) was used as control. Briefly, 50 µl of peripheral whole blood was incubated with appropriate dilutions of the different mAbs for 30 min at 4°C. Alternatively, freshly isolated populations of leukocytes or neutrophils cultured in the presence or absence of 100 ng/ml LPS, as described above, were preincubated for 30 min at 4°C with PBS containing 5% human serum (to avoid nonspecific staining) and then treated with the various mAbs for another 30 min at 4°C. After cell washings, the second biotin-labeled Ab was added for another 30 min at 4°C, followed by several washings and addition of 10 µl of streptavidin-PE (BD Biosciences) for an additional 30 min. After the final washings, cytofluorographic analyses (using at least 104 cells/sample) were performed on a FACScan (BD Biosciences) using CellQuest software. Thresholds were set on control stains.
Cytokine measurements
Antigenic IL-1 receptor antagonist (IL-1ra) and
macrophage-inflammatory protein 1 (MIP-1) were measured in the
cell-free-supernatants by using specific ELISA developed with Abs
purchased from commercial sources: BioSource International (Camarillo,
CA) for IL-1ra and R&D Systems (Minneapolis, MN) for MIP-1. The
detection limits of these ELISA were 50 pg/ml for IL-1ra and 30 pg/ml
for MIP-1 (32).
Statistical analysis
Data are expressed as means ± SEM. Statistical evaluation was performed using Student’s t test and considered to be significant if p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that IL-10 usually fails to induce
detectable tyrosine phosphorylation of Stat3 in human neutrophils (Ref.
20 and Fig. 1
A).
In this regard, no detectable induction of Stat3 tyrosine
phosphorylation by IL-10 was observed in freshly isolated neutrophils
whether they were stimulated in the absence of serum or in
polypropylene tubes instead of tissue culture well plates. Furthermore,
Stat3 was found constitutively phosphorylated on tyrosine residues in
freshly isolated neutrophils of a number of donors (Fig. 1, B and C), but, despite this, IL-10 was either
completely ineffective (Fig. 1B) or only weakly stimulatory
(Fig. 1C). In these latter particular cases, however,
tyrosine-phosphorylated Stat3 was detected only in purified cytoplasmic
fractions and not in nuclear fractions (data not shown). By contrast,
Stat3 became highly tyrosine phosphorylated if neutrophils were
stimulated with IFN- (Fig. 1A) or G-CSF
(33), indicating that the lack of responsiveness of
freshly isolated PMN to IL-10 cannot be attributed to a general
inability of the cells to activate the Jak1/Stat3 signaling pathway. In
addition, Stat3 was highly phosphorylated on tyrosine residues in
IL-10-treated autologous PBMC (Fig. 1A). Unexpectedly, in
all donors, IL-10 proved to be a very potent inducer of Stat3 tyrosine
phosphorylation if neutrophils are first cultured for 4 h with LPS
in standard medium (RPMI 1640 medium supplemented with 10%
low-endotoxin FCS) (Fig. 1). Under the latter conditions, a strong
tyrosine phosphorylation of Stat3 in response to IL-10 (100–500 U/ml)
is already evident by 5 min, reaches a maximum at 15 min, and is still
detectable at 45 min (data not shown). Culture in LPS-free standard
medium also renders the neutrophils responsive to IL-10 in terms of
Stat3 tyrosine phosphorylation, but less markedly so than in
LPS-treated cells (Fig. 1). However, the ability of standard medium to
render neutrophils responsive to IL-10 was not abrogated by PMX (data
not shown), thereby excluding an eventual role of contaminating
endotoxin in the medium or in the FCS. Importantly, the ability of
neutrophils to strongly respond to IL-10 after a 4-h period of culture
was essentially unaffected by the presence of serum in the culture
medium (Fig. 1D). As expected, the effect of LPS in
neutrophils cultured under serum-free conditions was completely
abrogated by PMX (Fig. 1D) and required LPS concentrations
above 10 ng/ml (Fig. 1D), with no significant difference
between 100 and 1000 ng/ml (data not shown). Finally, time course
experiments revealed that neutrophils acquire the capacity to
phosphorylate Stat3 on tyrosine residues upon IL-10 stimulation only if
cultured with LPS for at least 3 h (data not shown), regardless of
the presence of serum in the medium. Taken together, these experiments
indicate that LPS renders neutrophils responsive to IL-10 in terms of
Stat3 tyrosine phosphorylation, but that simple in vitro culture may
also be effective, albeit to a lesser extent.
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We next determined whether IL-10-mediated tyrosine phosphorylation
of Stat proteins in cultured neutrophils was paralleled by their
functional abilities to exert DNA-binding activities. Fig. 2A shows that although no
DNA-binding activities are detectable in whole-cell extracts of freshly
isolated PMN stimulated with IL-10, GRR-binding activities (and
hSIE-binding activities as well, see below) are consistently detected
in preparations from neutrophils stimulated with IL-10 after a 4-h
culture with or without LPS, and contain both Stat3 and Stat1 (Fig. 2B).
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To start elucidating the mechanisms whereby neutrophils
acquire the capacity to respond to IL-10 in terms of Stat3 tyrosine
phosphorylation, we examined the effect of the protein synthesis
inhibitor CHX. Fig. 3A shows
that Stat3 tyrosine phosphorylation in response to IL-10 is abrogated
if neutrophils are cultured in the presence of CHX. However, under
similar experimental conditions, tyrosine phosphorylation of Stat3 in
preparations from G-CSF-stimulated neutrophils is consistently
detectable at almost identical levels (Fig. 3A), thus
excluding any eventual aspecific toxicity of CHX. Identical findings
were obtained in experiments performed under serum-free conditions
(data not shown) or by testing the functional ability of phosphorylated
Stats to bind target sequences (hSIE) in EMSA (Fig. 3B).
Furthermore, highlighting the specificity of the up-regulatory effect
of LPS on IL-10 responsiveness, G-CSF-induced Stat3 tyrosine
phosphorylation was greatly attenuated in LPS-treated neutrophils (Fig. 3A), in keeping with the reported ability of LPS to
down-regulate the G-CSF receptors in cultured neutrophils (34, 35). Taken together, these data suggest that novel protein
synthesis is required to render cultured neutrophils fully responsive
to IL-10 in terms of Stat3 tyrosine phosphorylation and activation.
Accordingly, previous findings (36) also demonstrated that
new protein synthesis is necessary for the IL-10-mediated cytokine
modulation in human neutrophils. Based on the pattern of Stat3 protein
expression revealed by our Western blot analyses (Fig. 1
), we would
tend to exclude that this newly synthesized protein is Stat3 itself. We
would also exclude Jak1 or Tyk2 since, as shown in Fig. 1A,
the effect of IFN- (which like IL-10 utilizes Tyk2 and Jak1 to
transmit its intracellular signal) is very potent in freshly isolated
neutrophils, but decreases after cell culture, especially with
LPS.
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Previous studies using biotinylated IL-10 have demonstrated that
freshly isolated neutrophils possess detectable IL-10 binding sites,
albeit to a much lesser extent than peripheral blood monocytes or
lymphocytes (19, 37). Because the receptor for IL-10
consists of two components (IL-10R1 and IL-10R2) whose expression and
regulation in neutrophils has never been characterized, we investigated
whether a modulation of the IL-10R mRNA and surface expression might be
involved in LPS-mediated induction of IL-10 responsiveness observed
herein. Initial studies revealed that although both IL-10R1 and IL-10R2
transcripts are constitutively expressed in freshly isolated human
neutrophils, IL-10R1 mRNA expression is consistently lower than in
autologous PBMC (data not shown). However, a 90-min culture of
neutrophils in RPMI 1640 medium (with or without FCS) significantly
increased the gene expression of IL-10R1; this effect was even more
pronounced in the presence of LPS (Fig. 4A). By contrast, IL-10R2 mRNA
levels showed only minimal variation between freshly isolated
neutrophils and cultured cells (Fig. 4), suggesting that also the
IL-10R2 gene has its own set of regulators. The
increase in IL-10R1 mRNA steady-state levels in response to LPS was not
inhibited by CHX (data not shown) and was time dependent, peaking at
3 h and gradually decreasing thereafter (Fig. 4B).
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). Identical results were obtained if
cells were analyzed for IL-10R expression right after isolation (data
not shown). However, surface expression of IL-10R1 was significantly
up-regulated if neutrophils were cultured for 4 h in culture
medium with or without serum or LPS,
whereas that of IL-10R2 was not (Figs. 6 and 7). The up-regulation of neutrophil
IL-10R1 surface expression was always more evident if LPS was present
in standard medium (Figs. 6 and 7), and was completely blocked by CHX
(Fig. 7). By comparison, the surface expression of CR3 (CD11b/CD18) was
not significantly affected by CHX under the same culture conditions
(Fig. 7). Taken together, these data show that IL-10R1 receptor
expression is markedly up-regulated in LPS-cultured neutrophils, and
that this phenomenon relies on de novo protein synthesis.
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Since IL-10 is a potent modulator of cytokine/chemokine release in
neutrophils cultured overnight with LPS (17, 18),
including those of IL-1ra (up-regulatory effect, Fig. 8A) (38) and
MIP-1 (inhibitory effect, Fig. 8C) (36), we
analyzed the functional role of IL-10R1 and IL-10R2 on IL-1ra and
MIP-1 production using specific neutralizing anti-IL-10R Abs. As
shown in B and D of Fig. 8, the modulatory action
of IL-10 on LPS-induced IL-1ra and MIP-1 secretion is mostly
abrogated if neutrophils are cultured in the presence of
anti-IL-10R1- or anti-IL-10R2-neutralizing Abs, indicating that
both IL-10R chains are essential for mediating the effect of IL-10
(1, 39). Importantly, Fig. 8 also makes it clear that a
characteristic feature of the IL-10-mediated modulation of
cytokine/chemokine production by LPS-stimulated neutrophils is that it
starts to occur only after 3–4 h of cell culture, as we and others
have reported (36, 38, 40, 41). Similarly, IL-10 only
starts to modulate the LPS-induced accumulation of cytokine/chemokine
mRNA after 2–3 h in culture (36, 38, 40, 41). Remarkably,
the effect of neutralizing anti-IL-10R1 or anti-IL-10R2 mAbs
(measured after an overnight culture) does not appear to be affected
by the time of their addition to the neutrophil cultures (Fig. 9), i.e., whether it is concurrently
with or up to 3–4 h after LPS and IL-10. The latter findings are
consistent with the requirement of a lag time necessary to induce
the synthesis and expression of IL-10R1 by LPS-treated neutrophils, as
shown above.
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We have recently reported that SOCS-3 mRNA steady-state levels are
specifically up-regulated by IL-10 in freshly isolated neutrophils
(20). In the light of the findings described above, we
wanted to determine whether this function of IL-10 was modulated in
cultured neutrophils. The experiment shown in Fig. 10A demonstrates that this
is indeed the case, as induction of SOCS-3 mRNA expression by IL-10
resulted dramatically stronger in cultured than in freshly isolated
neutrophils (by an average of 
6- to 7-fold). Similarly to the
acquisition of Stat3 activation (Fig. 3), the enhanced accumulation of
SOCS-3 mRNA in response to IL-10 was almost completely abrogated if
neutrophils were cultured in the presence of CHX (Fig. 10A).
CHX itself superinduced SOCS-3 mRNA expression in either freshly
isolated or in cultured PMN, without however influencing the effect of
IL-10 in freshly isolated PMN (Fig. 10A). Taken together,
these data demonstrate that the increased ability of IL-10 to
up-regulate SOCS-3 mRNA expression in cultured neutrophils relies on de
novo protein synthesis. Once again, the results are consistent with the
notion that the up-regulation of IL-10R1 expression in neutrophils is
required to render them completely responsive to IL-10.
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The availability of specific anti-SOCS-3 Abs (28)
enabled us to demonstrate, for the first time, that neutrophils are
able to synthesize SOCS-3 protein in response to IL-10 (Fig. 10B). However, induction of SOCS-3 Ag was consistently
detected only in lysates prepared from neutrophils stimulated with
IL-10 after culture (with or without LPS), but not right after
isolation (Fig. 10B), correlating with the higher levels of
SOCS-3 mRNA expression induced by IL-10 in cultured cells (Fig. 10A). By contrast, GM-CSF, a cytokine known to potently
modulate expression of SOCS members in neutrophils and other myeloid
cells (20, 31), was found to potently induce SOCS-3
protein in freshly isolated as well as in cultured neutrophils (Fig. 10B), thus excluding a general inability of
freshly isolated PMN to synthesize SOCS-3. Importantly, in addition to
reversing the effects of IL-10 on cytokine production
(Figs. 8 and 9), both anti-IL-10R1- and
anti-IL-10R2-neutralizing Abs completely suppressed the induction
of SOCS-3 protein (Fig. 10C) and Stat3 tyrosine
phosphorylation (Fig. 10D) by IL-10 in cultured neutrophils,
suggesting a causal relationship among these events.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI gene promoter in human neutrophils freshly
isolated from the blood of normal donors (19, 20). These
characteristics are in sharp contrast with the effect of IL-10 on the
same responses in other cell types, such as monocytes and PBMC
(12, 13, 19, 20, 42, 43). Since IL-10 is an established
regulator of cytokine production in LPS-treated neutrophils (17, 18), our data raised the possibility that the modulation of
cytokine production by IL-10 in human neutrophils might occur
independently of Stat3 protein activation. In conflict with this
notion, however, were some studies that, directly or indirectly,
suggested an obligatory role for Stat3 in mediating this action of
IL-10 (22, 23). In particular, neutrophils and macrophages
derived from mice engineered to express a genetic Stat3 deficiency in
the myeloid cell compartment fail to respond to IL-10 and secrete high
levels of TNF- upon stimulation with IL-10 plus LPS
(23). We observed that contrary to its effect on freshly
isolated human neutrophils, IL-10 is a very effective inducer of Stat3
tyrosine phosphorylation in bone marrow-derived murine neutrophils (S.
Gasperini and M. A. Cassatella, unpublished data). Together, these
various observations encouraged us to further study the molecular
mechanisms by which IL-10 signals in human neutrophils, also in
consideration of other peculiar features concerning the action of
IL-10. One of these features is that the molecular mechanism whereby
IL-10 inhibits LPS-inducible gene expression in human neutrophils has
not been defined at all. We know, for instance, that IL-10 does not
inhibit activation of NF-
B in freshly isolated human neutrophils
activated by LPS or TNF- (P. P. McDonald and M. A.
Cassatella, unpublished data), in line with some studies performed in
human monocytic cells (44, 45). Another characteristic
feature is that we (38, 40) and others (36, 41) have often highlighted that, particularly in human
neutrophils, the extracellular release of cytokines/chemokines, as well
as the LPS-induced accumulation of mRNA encoding these
cytokines/chemokines, are significantly modulated by IL-10 only after
4 h of cell culture and not at early time points of incubation.
The latter observations suggest that IL-10 requires some time to be
able to act on cultured neutrophils. In agreement with this necessity,
it is well known that inhibition of cytokine gene expression by IL-10
in neutrophils and monocytes requires new protein synthesis (36, 46). Therefore, we reasoned that in the particular case of human
neutrophils, correlating early events, such as the undetectable Stat3
phosphorylation, with late events, such as the modulation of cytokine
production, although substantially acceptable, might have been perhaps
too speculative.
The findings presented in the current study establish that also in
human neutrophils IL-10 may activate a strong tyrosine phosphorylation
of Stat3- and Stat-binding activities to the GRR and hSIE probes, but
this activation occurs only if IL-10R1 expression is up-regulated. This
latter event is obtained when neutrophils are appropriately stimulated,
for instance with LPS, and as a result synthesize more IL-10R1.
Interestingly, responsiveness to IL-10 in terms of Stat3 tyrosine
phosphorylation was observed also in neutrophils cultured for 4 h
in the absence of LPS. Although in the latter experimental conditions
IL-10-mediated activation of Stat3 was usually lower than that observed
with LPS-containing medium, the data suggest that factors other than
endotoxin may "prime" neutrophils for IL-10 responsiveness. We also
show that responsiveness to IL-10 by cultured neutrophils is acquired
through a molecular mechanism dependent on new protein synthesis. A
series of evidence indicate that this (or maybe one of these) newly
synthesized protein(s) is the IL-10R1, as opposed to the classical
components of the Jak-Stat signaling pathway that can be activated by
IL-10 (namely, Jak1, Tyk2, Stat1, or Stat3). IL-10R1 is in fact
expressed on the membrane of circulating or freshly isolated
neutrophils at minimal but sufficient levels to bind IL-10 (19, 37), but dramatically augments on the cell surface after
incubation with LPS (Figs. 3, 5, and 6). This increased surface
expression of IL-10R1 correlates well with the capacity of IL-10 to
either activate Stat3 tyrosine phosphorylation and Stat3-dependent
DNA-binding activities to target sequences or to modulate IL-1ra and
MIP-1 release by LPS-stimulated neutrophils. Consistent with the
fact that a fully functional receptor complex is not present in freshly
isolated neutrophils, suppression of the effects of IL-10 (modulation
of cytokine production) was also obtained if neutralizing
anti-IL-10R1 (and anti-IL-10R2 as well) Abs were added to the
neutrophil culture 3.5 h after IL-10.
The other subunit composing the IL-10R complex, IL-10R2, was highly expressed in circulating or freshly isolated neutrophils and did not appear to be significantly influenced by the culture conditions. Furthermore, and in agreement with previous findings obtained by using biotinylated IL-10 (19) or a different anti-IL-10R1 Ab (47), we confirm that whole blood or freshly isolated monocytes and lymphocytes (PBMC) express surface levels of both IL-10R1 and IL-10R2 that are consistently higher than those found in autologous neutrophils. This might explain why IL-10 rapidly activates Stat tyrosine phosphorylation in freshly isolated PBMC, as opposed to neutrophils (20). However, the relevance of IL-10R2 in mediating IL-10 signaling (in association with the IL-10R1 chain) was demonstrated in neutrophils by the capacity of neutralizing anti-IL-10R2 mAbs to suppress the effects of IL-10 as much as the neutralizing anti-IL-10R1. In this regard, LPS was previously shown to selectively induce IL-10R1 expression in murine L929 fibroblasts, which do not constitutively express IL-10R1 (48), but despite this, the cells remained unresponsive to IL-10 with respect to the activation of Stat(s). Similarly, transfection of the murine IL-10R1 chain into L929 fibroblasts conferred to these cells the ability to bind murine IL-10, but again, IL-10 failed to induce Stat activation in these cells (48), suggesting the lack of a component critical for the generation of the IL-10 signaling. The data presented herein demonstrate that in human neutrophils IL-10R2 is already present at sufficient levels whereas IL-10R1 needs to be up-regulated, at least for inducing optimal activation of Stat3. Thus, both chains of the IL-10R complex appear to be critical for Stat signaling (39). By contrast, the Jak/Stat signaling pathway may not be required for the activation of other selective actions induced by IL-10 (16, 20, 49). We note recent findings suggesting that IL-10R2 is a shared receptor chain for IL-10 and the IL-10-related T cell-derived inducible factor (IL-T cell-derived inducible factor/IL-22) (50, 51) and possibly other IL-10 homologues (1, 51). Whether neutrophils express the ligand binding chains for these proteins (e.g., CRF2–9 for IL-T cell-derived inducible factor/IL-22) remains to be determined.
In the present work, we also demonstrate that the ability of IL-10 to up-regulate the expression of SOCS-3 mRNA in neutrophils (20) was strongly enhanced if PMN are stimulated with IL-10 after a 3- to 4-h period of culture. This augmented responsiveness to IL-10 was inhibited by CHX, likely as a result of the inhibitory effect of the drug on the IL-10R1 expression. Very importantly, induction of SOCS-3 mRNA by IL-10 in cultured neutrophils was also followed by the synthesis of antigenic SOCS-3. No intracellular SOCS-3 protein was in fact detected in freshly isolated neutrophils stimulated with IL-10, despite a significant accumulation of SOCS-3 mRNA (20). Furthermore, activation of Stat3 tyrosine phosphorylation and synthesis of SOCS-3 protein were both prevented if cultured neutrophils were stimulated with IL-10 in the presence of neutralizing anti-IL-10R1 and anti-IL-10R2 mAbs. Together with previous observations (20), our results suggest that, in human neutrophils, SOCS-3 protein is synthesized in response to IL-10 only in coincidence of a strong induction of Stat3 tyrosine phosphorylation and only when SOCS-3 mRNA is accumulated at very high levels.
Based on the current findings, we cannot exclude that other unidentified polypeptides are also synthesized de novo and contribute to render neutrophils fully responsive to IL-10. Certainly, the capacity of IL-10 to induce a strong Stat3 tyrosine phosphorylation and to modulate cytokine gene expression and release in LPS-activated neutrophils seem to correlate, supporting previous studies demonstrating a critical role of the Jak1/Stat3 pathway in the IL-10-mediated deactivation of phagocytes (22, 23). Our findings also provide some indications helping to clarify the reasons explaining the controversial data existing in the literature on the ability of IL-10 to directly trigger or modulate specific effector functions in human neutrophils (17). Based on the present results, it can be reasonably speculated that if the conditions or the materials and reagents used for neutrophil isolation and culture are not stringently controlled (for instance by a careful monitoring of endotoxin content/contamination of solutions, buffers, and culture media), the cells can easily become "primed" during the isolation procedure and, as a consequence, be rendered more responsive to IL-10 through up-regulation of IL-10R1. Finally, our findings suggest that LPS not only activates neutrophils to produce and release a host of proinflammatory mediators, but also prepares these cells to be subsequently able to respond immediately to anti-inflammatory signals such as IL-10, presumably to help limit the extent of inflammatory reactions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marco A. Cassatella, Department Pathology, Strada Le Grazie 4, I-37134 Verona, Italy. E-mail address: marco.cassatella{at}univr.it
3 Abbreviations used in this paper: Jak, Janus kinase; PMN, polymorphonuclear neutrophil; SOCS, suppressor of cytokine signaling; h, human; CHX, cycloheximide; PMX, polymyxin B sulfate; GRR, IFN- response region; SIE, c-sis-inducible element; IL-1ra, IL-1 receptor antagonist; MIP-1, macrophage-inflammatory protein 1.
Received for publication April 17, 2001. Accepted for publication June 28, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Biol. Chem. 271:17174. and STAT3 complexes in human T cells and monocytes. J. Immunol. 155:1079.[Abstract]
RI/CD64) gene and STAT protein binding to the IFN- response region (GRR) are regulated differently in human neutrophils and monocytes by IL-10. J. Immunol. 160:911.R surface expression and cytotoxic activity: distinct regulation of antibody dependent cellular cytotoxicity by IFN-, IL-4, and IL-10. J. Immunol. 149:4048.[Abstract]
and lipopolysaccharide enhancement of phagocyte respiratory burst capability: studies on the gene expression of several NADPH oxidase components. J. Biol. Chem. 265:20241.-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator. J. Leukocyte Biol. 68:277.B in human neutrophils during phagocytosis of bacteria independently of oxidant generation. FEBS Lett. 432:40.[Medline]
is mediated through common DNA response elements. Proc. Natl. Acad. Sci. USA 88:11305., IL-1, IL-8, MIP-1, MIP-1, and IP-10 by human neutrophils stimulated with group B meningococcal outer membrane vesicles. Infect. Immun. 68:6917. response region within the promoter of the FcRI/CD64 gene in human neutrophils. FEBS Lett. 386:239.[Medline]
and IL-1 in mediating the production of IL-8 triggered by lipopolysaccharide. J. Exp. Med. 178:2207. responsive promoter elements. J. Immunol. 153:165.[Abstract]
B) which are involved in IL-6 regulation. Leukemia 10:1308.[Medline]
) is a shared component of both IL-10 and IL-TIF (IL-22) receptor complexes. J. Biol. Chem. 276:2725.This article has been cited by other articles:
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|
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 |
|
 N. Tamassia, V. Le Moigne, M. Rossato, M. Donini, S. McCartney, F. Calzetti, M. Colonna, F. Bazzoni, and M. A. Cassatella Activation of an Immunoregulatory and Antiviral Gene Expression Program in Poly(I:C)-Transfected Human Neutrophils J. Immunol., November 1, 2008; 181(9): 6563 - 6573. [Abstract] [Full Text] [PDF]
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|
 N. Tamassia, F. Calzetti, N. Menestrina, M. Rossato, F. Bazzoni, L. Gottin, and M. A. Cassatella Circulating neutrophils of septic patients constitutively express IL-10R1 and are promptly responsive to IL-10 Int. Immunol., April 1, 2008; 20(4): 535 - 541. [Abstract] [Full Text] [PDF]
|
|
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 |
|
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|
|
|
|
 |
|
 C. Ratthe, M. Pelletier, S. Chiasson, and D. Girard Molecular mechanisms involved in interleukin-4-induced human neutrophils: expression and regulation of suppressor of cytokine signaling J. Leukoc. Biol., May 1, 2007; 81(5): 1287 - 1296. [Abstract] [Full Text] [PDF]
|
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|
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|
|
|
|
 |
|
 P. M.-C. Dang, C. Elbim, J.-C. Marie, M. Chiandotto, M.-A. Gougerot-Pocidalo, and J. El-Benna Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity FASEB J, July 1, 2006; 20(9): 1504 - 1506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Srisodsai, R. Kurotani, Y. Chiba, F. Sheikh, H. A. Young, R. P. Donnelly, and S. Kimura Interleukin-10 Induces Uteroglobin-related Protein (UGRP) 1 Gene Expression in Lung Epithelial Cells through Homeodomain Transcription Factor T/EBP/NKX2.1 J. Biol. Chem., December 24, 2004; 279(52): 54358 - 54368. [Abstract] [Full Text] [PDF]
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|
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|
 |
|
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