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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2880-2889
Interleukin-10 (IL-10) Selectively Enhances CIS3/SOCS3 mRNA Expression
in Human Neutrophils: Evidence for an IL-10-Induced Pathway That Is
Independent of STAT Protein Activation
By
Marco A. Cassatella,
Sara Gasperini,
Chiara Bovolenta,
Federica Calzetti,
Marieke Vollebregt,
Patrizia Scapini,
Martina Marchi,
Ritsu Suzuki,
Asuka Suzuki, and
Akihiko Yoshimura
From the Department of Pathology, University of Verona, Verona,
Italy; the AIDS Immunopathogenesis Unit, San Raffaele Scientific
Institute, Milano, Italy; and the Institute of Life Science, Kurume
University, Aikawamachi, Kurume, Japan.
 |
ABSTRACT |
We have recently shown that, in human neutrophils, interleukin-10
(IL-10) fails to induce specific DNA-binding activities to the
gamma-interferon response region (GRR), a regulatory element located in
the Fc RI gene promoter, which is required for transcriptional activation by IL-10 and interferon (IFN) in monocytic cells. In
this study, we report that IL-10 is also unable to induce the binding
of STAT1 or STAT3 to the serum-inducible element (hSIE/m67), despite
the fact that both proteins are expressed in neutrophils. Whereas
IFN and granulocyte colony-stimulating factor (G-CSF) are efficient
inducers of STAT1 and STAT3 tyrosine phosphorylation in
polymorphonuclear neutrophils (PMN), IL-10 fails to
trigger STAT1 and STAT3 tyrosine and serine phosphorylation, therefore explaining its inability to induce the FcRI expression in these cells. By contrast, we demonstrate that IL-10 alone represents an
efficient stimulus of CIS3/SOCS3 mRNA expression in neutrophils. CIS3/SOCS3 belongs to the recently cloned cytokine-inducible
SH2-containing protein (CIS) gene family (which also includes CIS1,
CIS2, CIS4, CIS5, and JAB) that is believed to be, at least in part,
under the control of STAT transcription factors and whose products are potential modulators of cytokine signaling. Moreover, IL-10 synergizes with lipopolysaccharide (LPS) in upregulating CIS3/SOCS3 mRNA expression in PMN through a mechanism that involves mRNA stabilization. In contrast to CIS3/SOCS3, mRNA transcripts encoding other family members are unaffected by IL-10 in neutrophils. Finally, transfection of CIS3/SOCS3 in murine M1 myeloid cells suppresses LPS-induced growth
arrest, macrophage-like differentiation, and nitric oxide synthesis,
but not IL-6 mRNA expression. Collectively, our data suggest that, in
neutrophils, the activation of STAT1 and STAT3 phosphorylation is
neither required for CIS3/SOCS3 induction by IL-10 nor involved in the
regulatory effects of IL-10 on cytokine production.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTERLEUKIN-10 (IL-10) is an 18-kD
nonglycosylated protein that is mainly synthesized by helper T cells, B
cells, activated monocytes, macrophages, thymocytes, and keratinocytes
and has the capacity to attenuate a wide range of inflammatory and
immune responses.1 Although IL-10 downregulates specific
effector functions in monocytes, natural killer (NK) cells, B cells,
and Th1 cells,1,2 recent studies performed in our
laboratory, as well as by other groups, have shown that a number of
polymorphonuclear neutrophil (PMN) functional responses are also
regulated by IL-10.3 Among the latter, the modulation of
cytokine and chemokine production by IL-10 has been the focus of
intensive investigation.4-9 However, more recently,
we10 and others11,12 found that IL-10 fails to
induce the surface expression of the high-affinity receptor for IgG
(FcRI/CD64) in PMN. The inability of IL-10 to induce FcRI in
neutrophils was in a sense surprising, in view of earlier studies
performed in human monocytes and murine macrophages, which had
demonstrated that FcRI gene and surface expression are upregulated by IL-10, both in vitro and in vivo.13-15 A likely
explanation for this cell-type-specific action of IL-10 is related to
the fact that the FcRI promoter contains a 39-bp region that is
necessary and sufficient for the induction of gene expression by
interferon (IFN) or IL-10.14,16-21 Consistent with
this interpretation, we10 and others14,20-22
reported that gamma-interferon response region (GRR)-binding complexes
containing STAT1 and STAT3 are effectively induced in monocytes and
peripheral blood mononuclear cells (PBMC) in response to IL-10, whereas
we found that, in neutrophils, no GRR-binding activities are inducible
by IL-10.10
In this study, we further investigated the inability of neutrophils to
activate GRR-binding complexes in response to IL-10. In agreement with
previous studies,20,22,23 we observed that the
IL-10-elicited induction of GRR-binding activities is accompanied by
the tyrosine phosphorylation of STAT1 and STAT3 in PBMC.
However, in neutrophils, neither STAT1 nor STAT3 becomes tyrosine or
serine phosphorylated in response to IL-10. To gain further
insight into IL-10 signaling, we also investigated whether the
expression of cytokine-induced Src homology 2-containing (CIS) proteins
might be modulated by IL-10 in neutrophils. Members of this
novel family of cytokine-inducible genes are currently generating much
interest, because they have the potential to negatively
regulate the JAK-STAT signaling pathway.24-31 We now report
that IL-10 directly stimulates the expression of CIS3 mRNA,
both in human neutrophils and PBMC, and that stable
transfection of CIS3 into a murine myeloid cell line
suppresses macro- phage differentiation and nitric oxide synthesis
induced by lipopolysaccharide (LPS). The implications of our findings
and the possible role of CIS3 in the context of IL-10 signaling are discussed.
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MATERIALS AND METHODS |
Cell purification and culture.
Highly purified granulocytes (>99.5%) and PBMC were isolated under
LPS-free conditions from buffy coats of healthy donors by
centrifugation on a Ficoll-Hypaque gradient, as previously described.32 Monocytes were isolated from PBMC after
centrifugation over Percoll gradients, as described
earlier.33 After purification, leukocytes were suspended in
RPMI 1640 supplemented with 10% low endotoxin fetal calf serum (FCS;
<0.006 ng/mL; Hyclone Laboratories Inc, Logan, UT) and treated with
either 100 U/mL IFN or up to 2,000 U/mL IL-10 (kindly provided by Dr
K. Moore, DNAX and Schering-Plough Corp, Palo Alto, CA).10
Optimal biological effects were obtained using either 100 U/mL IL-10
from DNAX4,5 or 20 ng/mL IL-10 purchased from Peprotech Inc
(Rocky Hill, NJ). In selected experiments, PMN were also treated with
1,000 U/mL granulocyte colony-stimulating factor (G-CSF; Granulokine;
Hoffmann-LaRoche, Basel, Switzerland), 100 ng/mL phorbol myristate
acetate (PMA; purchased from Sigma, St Louis, MO), 100 ng/mL LPS (from
Escherichia coli, serotype 026:B6; Sigma), or 10 ng/mL
granulocyte-macrophage colony-stimulating factor (GM-CSF; Genetics
Institute, Boston, MA). Leukocytes thus treated were cultured in
polystyrene flasks or polypropylene tubes (Greiner, Nürtingen,
Germany) at 37°C under a 5% CO2 humidified atmosphere
or (for electrophoretic mobility shift assays [EMSA] studies) at room
temperature.10 Murine M1 cells were cultured in Dulbecco's
modified Eagle's medium (DMEM) containing 10% horse serum, as previously described.26,27,34 All reagents were of the highest available grade and all buffers were prepared using pyrogen-free water for clinical use.
Cellular extracts.
After stimulation for the indicated times, neutrophils (1 to 2 × 108/condition) or monocytes/PBMC (0.3 to 1 × 108/condition) were diluted in ice-cold phosphate-buffered
saline (PBS) and centrifuged twice at 500g for 5 minutes at
4°C. The cells were then suspended in relaxation buffer and
disrupted in a nitrogen bomb (Parr Instruments, Mobile, IL), and
extracts were prepared exactly as described.35
Alternatively, cells were pelleted, washed in ice-cold PBS, and
resuspended in lysis buffer (20 mmol/L HEPES-KOH, pH 7.5, 350 mmol/L
KCl, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.1 mmol/L EGTA, 20%
[vol/vol] glycerol, 1% [vol/vol] Nonidet P-40, and 5 mmol/L
dithiothreitol [DTT]) containing protease and phosphatase inhibitors (1 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 10 µg/mL leupeptin, 10 µg/mL pepstatin A, 5 mg/mL
 1-antitrypsin, 1 mmol/L Na3VO4, and 10 mmol/L NaF).36 After 15 minutes of incubation on ice, cell
debris were spun down (12,000g for 20 minutes at 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).
EMSA.
Protein-DNA complexes were detected by EMSA analysis of the various
extracts as previously described,10,35 with the following modifications: 4 to 40 µg of cytoplasmic or 5 to 20 µg of nuclear extracts was usually incubated for 10 minutes at room temperature in a
buffer containing 10 mmol/L Tris, pH 7.5, 100 mmol/L KCl, 5 mmol/L
MgCl2, 1 mmol/L DTT, 100 µg/mL poly
(dI-dC).poly (dI-dC), 50 µg/mL salmon sperm, and 10%
glycerol, followed by the addition of a 32P-labeled
double-stranded oligonucleotide probe corresponding to the GRR element
located within the promoter of the FcRI gene16 (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/m67 (5' gtc gaC ATT TCC CGT AAA TCg 3') for 15 minutes.36 Supershift experiments were performed by
incubating the proteins with 0.5 µg of the various anti-STAT
antibodies for 30 minutes at room temperature, before adding the
labeled probe. Anti-STAT1 (E-23, raised against amino acids 688-710),
anti-STAT3 (C20, raised against amino acids 750-769), and anti-STAT5
(C17, raised against amino acids 711-727 of STAT5b p80 of mouse origin, and specific for STAT5a and STAT5b) antibodies were purchased from
Santa Cruz Biotechnology Inc (Santa Cruz, CA).
Immunoprecipitations and immunoblots.
For the immunoprecipitation experiments, nuclear plus cytoplasmic
fractions from cavitated neutrophils (2 mg) or PBMC (0.85 mg) were
incubated for 2 hours at 4°C on a rotating wheel with 20 µL of a
50% slurry of protein A agarose (Boehringer Mannheim, Mannheim,
Germany) in the presence of a 1:300 dilution of anti-STAT3 antibody
(C-20) or anti-STAT1 antiserum (a generous gift from Dr K. Ozato,
National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, MD).37 The
immunoprecipitates were washed 5 times with a rinsing buffer (40 mmol/L
Tris, pH 8, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L
Na3VO4, and 50 mmol/L NaF) and once with TBS
(20 mmol/L Tris, pH 7.6, 137 mmol/L NaCl) before elecrophoretic
separation on 7.5% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and subsequent transfer to nitrocellulose by
electroblotting. Nitrocellulose membranes were incubated overnight at
4°C in blocking buffer (TBS, 0.2% Tween-20, 7.5% bovine serum albumin [BSA]), before 2 hours of incubation at room temperature with
the primary antibodies at the following dilutions: antiphosphotyrosine antibody 4G10 (Upstate Biotechnology Inc, Lake Placid, NY), 1:1,000; anti-STAT3 (K-15; Santa Cruz), 1:2,000; and anti-STAT1 (C-111; Santa
Cruz), 1:1,500.
For the direct detection of tyrosine-phosphorylated STAT1 and STAT3 and
of serine-phosphorylated STAT1 and STAT3, detergent lysates prepared
from PMN and PBMC according to the method described by Vollebregt et
al38 were electrophoresed and electroblotted as described
above. Membranes were first blocked for 1 hour at room temperature in
TBS/T (20 mmol/L Tris-HCl, pH 7.6, 137 mmol/L NaCl, 0.1% Tween 20)
containing 5% BSA and then incubated overnight at 4°C in the
presence of the phospho-specific primary antibodies. The latter were
phospho-specific STAT1 antibody (Tyr701; 9171S; New England Biolabs,
Beverly, MA) diluted 1:500 in blocking buffer, phospho-specific STAT3
antibody (Tyr705; 9131S; New England Biolabs) diluted at 1:1,000,
phospho-specific STAT1 antibody (Ser727; 06-802; Upstate Biotechnology)
diluted 1:1,000 in blocking buffer, and phospho-specific STAT3 antibody
(Ser727; 06-803 [Upstate Biotechnology] or 9134S [New England
Biolabs]) diluted at 1:1,000. Membranes were then probed with
anti-STAT1 (E-23; Santa Cruz) or anti-STAT3 (C-20; Santa Cruz) diluted
1:2,000 in blocking buffer. Antibody binding was detected by using
horseradish peroxidase-conjugated antimouse or antirabbit IgG and shown
using the chemiluminescence system (ECL; Amersham, Arlington Heights,
IL) according to the manufacturer's instructions.
Northern blot analyses.
Total RNA was extracted from PMN, PBMC, or M1 cells by the guanidinium
isothiocyanate method and processed for Northern blot analysis, as
already described.32 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
(Pharmacia, Uppsala, Sweden). The probes used consisted of full-length
cDNA fragments encoding CIS,27 IL-8, tumor necrosis factor
(TNF), IL-1 receptor antagonist,4,5 and
actin (kindly provided by Dr G. Trinchieri, Wistar Institute, Philadelphia, PA). The extent of hybridization was quantitatively analyzed in an InstantImager (Packard Instruments, Meriden, CT) and
plotted after actin normalization. For mRNA half-life experiments, data
were plotted on semilogarithmic graphs as the percentage of remaining
mRNA versus time decays in minutes, and the resulting values were
plotted against time. Half-lives were calculated by regression
analysis. For M1 cells, total RNA was extracted after stimulation with
100 ng/mL LPS, hybridized with digoxigenin (DIG)-labeled riboprobes,
and then visualized using alkaline-phosphatase-labeled anti-DIG
antibodies according to the manufacturer's instructions (Boehringer
Mannheim). cDNA for murine IL-6 and G3PDH have been previously
described.27
LPS-induced differentiation of M1 cells.
M1 stable transfectants were obtained by electroporation with pcDNA
carrying Myc-tagged full-length CIS3 and selected with 0.8 mg/mL G418,
as previously described.27 Two to 4 independent clones were
tested for LPS-induced differentiation and growth arrest. Briefly,
105 parental M1 cells and CIS3-expressing clones were
cultured in medium containing 10% horse serum supplemented with 100 ng/mL LPS for 3 days and then subjected to May-Grunwald-Giemsa staining.
Assay for nitric oxide (NO) synthesis by M1 cells.
Parental M1 cells and CIS3 transfectants were cultured in 24-well
tissue culture plates at a density of 106 cells/well at
37°C. After 1 and 3 days of culture in the presence or absence of
LPS, the NO content of culture supernatants was measured using the
Griess reagents. Nitrite concentrations were calculated from a standard
curve derived from the reaction of NaNO2 in the assay.
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RESULTS |
Lack of induction of STAT-binding complexes by IL-10 in neutrophils.
Figure 1A shows that no GRR-binding
activities are detectable in cytoplasmic (or nuclear) extracts of
IL-10-treated PMN. In sharp contrast, GRR-binding activities are
consistently detectable in preparations from IL-10-stimulated PBMC or
from IFN-stimulated cells (Fig 1A). Identical results were obtained
when increasing amounts of neutrophil and monocyte extracts were
analyzed by EMSA using a serum-inducible element (hSIE) probe (Fig 1B)
that binds STAT protein complexes with a higher affinity than the
GRR.39,40 Figure 1C shows that the hSIE-binding complexes
induced in monocytes by IL-10 contain both STAT1 and STAT3, but not
STAT5 (data not shown), in keeping with previous observations made
using a GRR probe.10 By comparison, the hSIE/m67-binding
activities induced by IFN in PMN contain only STAT1 (not shown).
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| Fig 1.
Lack of induction of DNA-binding complexes by IL-10 in
neutrophils. (A) PMN and autologous PBMC were incubated for 20 minutes
in the presence or absence of 100 U/mL IL-10 or IFN. Cytoplasmic
extracts were then prepared by nitrogen cavitation and analyzed in
EMSA, using a 32P-labeled GRR oligonucleotide. For PMN
extracts, 40 µg of protein was used in the binding reactions, whereas
10 µg of protein was used for PBMC extracts. This experiment is
representative of at least 10. (B) PMN and autologous monocytes were
incubated for 15 minutes with 100 U/mL IL-10 or IFN, and the
resulting whole-cell extracts were analyzed in EMSA, using a
32P-labeled hSIE/m67 oligonucleotide. Amounts of extract
used are indicated. This experiment is representative of 2. (C)
Characterization of the hSIE/m67-binding complexes induced in
IL-10-treated monocytes. Purified monocytes were treated for 20 minutes with 100 U/mL IL-10, and whole-cell extracts were analyzed in
EMSA. Binding reactions were performed in the presence or absence of
specific anti-STAT antibodies as indicated, before the addition of the
hSIE/m67 probe. This experiment is representative of 4.
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STAT1 and STAT3 are not phosphorylated after IL-10 stimulation in
neutrophils.
We previously showed that homodimers and heterodimers of these proteins
can be induced to bind STAT-binding probes (GRR and hSIE/m67) in
response to G-CSF stimulation.41 Thus, the lack of
responsiveness of PMN to IL-10 in terms of GRR- or hSIE/m67-binding activities cannot be attributed to a general inability of these cells
to activate STAT1 and STAT3. Because tyrosine phosphorylation of STAT
proteins is required for DNA binding,42,43 we initially investigated whether STAT1 and STAT3 become tyrosine phosphorylated after IL-10 stimulation of PMN, as previously shown for monocytes and
lymphocytes.20,22,23 For this purpose, neutrophils and autologous PBMC were stimulated for 15 minutes with either IL-10 (up to
1,000 U/mL) or IFN, before disruption of the cells by nitrogen
cavitation. The resulting cytoplasmic preparations were immunoprecipitated with anti-STAT3 or anti-STAT1 antibodies and analyzed by immunoblot using an anti-phosphotyrosine (4G10) antibody. As shown in Fig 2, IFN and (to a lesser
extent) IL-10 both induced the tyrosine phosphorylation of STAT1 in
PBMC, whereas, in neutrophils, STAT1 became tyrosine phosphorylated
only in response to IFN. Similarly, IL-10 promoted the
phosphorylation of STAT3 on tyrosine residues in PBMC, but failed to do
so in PMN (Fig 2). Stripping and reprobing the nitrocellulose membranes
with anti-STAT1 or anti-STAT3 antibodies ascertained that
comparable amounts of proteins had been immunoprecipitated in these
experiments. Identical results were obtained using neutrophil or PBMC
whole-cell extracts (data not shown).
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| Fig 2.
Defective activation of STAT1 and STAT3 tyrosine
phosphorylation in IL-10-treated PMN. PMN and autologous PBMC were
incubated in the presence or absence of 100 U/mL IL-10 or IFN for 15 minutes before lysis. STAT1 and STAT3 were immunoprecipitated and the
membranes were blotted with antiphosphotyrosine antibodies (4G10).
Similar amounts of immunoprecipitated material were loaded in each
lane, as judged from subsequent reblotting with appropriate antibodies.
The data shown are representative of 4 independent experiments.
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Time course experiments on whole-cell extracts prepared by detergent
lysis and analyzed with antibodies that specifically recognize the
phosphotyrosine forms of STAT1 and STAT3 confirmed that STAT1 and STAT3
are phosphorylated on tyrosine residues after treatment of neutrophils
with IFN and G-CSF, respectively, but not with IL-10
(Fig 3A). In PBMC, a strong tyrosine
phosphorylation of STAT3 in response to IL-10 was already evident by 5 minutes, reached a maximum at 15 minutes, and was still detectable at
45 minutes; STAT1 tyrosine phosphorylation followed an analogous time
course, albeit with a less intense signal (Fig 3A).
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| Fig 3.
Time course of STAT1 and STAT3 phosphorylation in
IL-10-treated PMN and PBMC. Cells were treated with 100 U/mL IL-10,
100 U/mL IFN, and 1,000 U/mL G-CSF (A) or 20 ng/mL IL-10
(Peprotech), 100 U/mL IFN, and 100 ng/mL PMA (B) before lysis under
denaturating conditions as described in Materials and Methods. One
hundred fifty micrograms of PMN lysate and 80 µg of PBMC lysate were
loaded on the gels; and immunoblots were performed using antibodies
specific for tyrosine or serine phosphorylated forms of STAT1 and
STAT3. Subsequent reblotting with the indicated anti-STAT1 or
anti-STAT3 antibodies was performed to ensure that similar amounts of
material were deposited in each lane. The data for each panel are
representative of 4 independent experiments.
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Under the same conditions, we also assessed the ability of IL-10 to
induce serine phosphorylation of STAT1 and STAT3. Serine phosphorylation appears to increase the magnitude of gene transcription induced by tyrosine phosphorylation, even though it is not sufficient per se to activate STAT proteins.44 Western blot analyses
showed that, whereas STAT3 was unphosphorylated on ser-727 in resting neutrophils and PBMC, IL-10 treament only led to a time-dependent serine phosphorylation of STAT3 in PBMC (Fig 3B). Despite this, G-CSF
(not shown) and PMA were effective inducers of STAT3 serine phosphorylation in neutrophils (Fig 3B). With respect to STAT1, low
constitutive levels of serine phosphorylated protein were present
in PBMC, which remained unchanged after IL-10 treatment. In
contrast, PMA (and to lesser extent) IFN both augmented STAT1 serine
phosphorylation in PBMC (Fig 3B). No STAT1 serine phosphorylation was
detected in neutrophils after any treatment (Fig 3B).
Expression of CIS family members in neutrophils and their induction
by IL-10.
A new family of STAT-regulated genes, the CIS family, has recently been
the focus of much attention, because its members are potential negative
regulators of cytokine signaling.24-31 In view of their
potential involvement in regulating IL-10 signaling, we examined
whether the gene expression of CIS proteins might be influenced, in
turn, by IL-10 in human neutrophils. For this purpose, we performed
Northern blot analyses on total RNA isolated from PMN incubated with
IL-10 (100 U/mL) and/or LPS (100 ng/mL) for 2 and 6 hours. Because of
the well-established capacity of both GM-CSF and IFN to upregulate
CIS mRNA expression in myeloid cells,27,45 total RNA was
also extracted from neutrophils incubated for 2 hours with either agent
for comparison purposes. Figure 4 shows
that CIS3 mRNA (but not CIS1 or CIS2 mRNA) is constitutively expressed
in unstimulated neutrophils. Whereas the gene expression of CIS1, CIS3,
and JAB (not shown) was upregulated by both GM-CSF and IFN, IL-10
and LPS selectively enhanced the expression of CIS3 mRNA (Fig 4).
Neither IL-10 nor LPS had any effect on the gene expression of either
JAB or CIS5 (data not shown). In the same experiments (Fig 4), the
LPS-induced accumulation of IL-8 mRNA was inhibited by IL-10 (by
~50% at 6 hours), whereas that of IL-1ra mRNA was greatly enhanced,
in agreement with previous studies.4-7 As shown in Fig 4,
and better explored in the experiment depicted in
Fig 5, both the IL-10-mediated and
LPS-mediated increase in CIS3 mRNA steady-state levels was
time-dependent, peaking at 2 to 3 hours in the case of IL-10
stimulation (Figs 4 and 5) and gradually decreasing thereafter.
Remarkably, coincubation of PMN with IL-10 and LPS yielded a
synergistic augmentation of CIS3 gene expression; this synergism was
already evident at 3 hours and persisted even when, in IL-10-treated
neutrophils, CIS3 mRNA levels had returned to baseline levels (Fig 5).
Further investigation showed that the IL-10-induced accumulation of
CIS3 mRNA occurred in a dose-dependent manner and that the pattern of
CIS3 and IL-1ra mRNA inducibility markedly differed between neutrophils
and autologous PBMC (Fig 6). Importantly,
the latter observation also provides convincing evidence that the
findings that we report here for CIS family members in neutrophils are
unlikely to result from the small proportion of contaminating PBMC that
are present in our neutrophil preparations.
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| Fig 4.
Effect of IL-10 and LPS on the gene expression of various
CIS family members, IL-8, and IL-1ra in neutrophils. PMN were
preincubated with or without 100 U/mL IL-10 for 15 minutes before the
addition of LPS, IFN, or GM-CSF for the times indicated. Total RNA
was extracted and analyzed by Northern blotting. This experiment is
representative of 4.
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| Fig 5.
Time course of CIS3 mRNA expression in PMN treated with
IL-10 and/or LPS. PMN were incubated for the indicated times with 500 U/mL IL-10 in the presence or absence of LPS, before total RNA
extraction and Northern blot analysis for CIS3 mRNA expression. The
extent of hybridization was quantitatively analyzed in an InstantImager
(Packard Instruments) and plotted after actin normalization. This
experiment is representative of 3.
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| Fig 6.
Dose-dependent effect of IL-10 on CIS3 mRNA expression in
neutrophils. PMN were incubated in the presence or absence of
increasing concentrations of IL-10 for 2 hours before total RNA
extraction. For comparative purposes, RNA was also extracted from PMN
stimulated with GM-CSF and from autologous PBMC. CIS3 and IL-1ra gene
expression was analyzed by Northern blot. This experiment is
representative of 2.
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Effect of IL-10 on CIS3 mRNA stability.
In an attempt to elucidate the mechanisms whereby IL-10 modulates the
accumulation of CIS3 transcripts in LPS-treated PMN, we examined the
influence of IL-10 on CIS3 mRNA stability. PMN were stimulated with LPS
for 3.5 hours in the presence or absence of IL-10 and then treated with
actinomycin D to block the formation of additional transcripts. At
increasing intervals thereafter, the cultures were processed for
Northern blot analysis, and changes in the amount of cytokine mRNA were
quantitated by InstantImager scanning. Whereas our preliminary
experiments established that neither IL-10 nor LPS stimulation
significantly alters CIS3 mRNA stability (not shown),
Fig 7 shows that IL-10 treatment markedly prolonged CIS3 mRNA half-life in LPS-stimulated cells (65 v 175 minutes, respectively). Under the latter conditions, IL-10 did not
significantly affect the stability of TNF and actin mRNA isolated
from LPS-treated PMN (Fig 7). A similar effect of IL-10 on the turnover
rate of CIS3 transcripts was exerted in cells stimulated with LPS for 5 hours, a time point at which the differences in CIS3 mRNA levels were
still evident (not shown).
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| Fig 7.
Effect of IL-10 on the turnover rate of CIS3 mRNA in
LPS-stimulated PMN. PMN were cultured with 1 µg/mL LPS in the
presence or absence of 100 U/mL IL-10. After 3.5 hours, actinomycin D
(5 µg/mL) was added and the cells were further cultured for the
indicated times. Total RNA was prepared and analyzed by Northern
blotting with CIS3, TNF , and actin cDNA probes. This experiment is
representative of 3.
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Negative effect of CIS3 on LPS signaling.
To address the potential function of CIS3, we stably transfected CIS3
in the murine M1 myeloid cell line and examined the effects thereof on
LPS-induced differentiation, NO synthesis, and cytokine mRNA
expression. This leukemic cell line does not constitutively express
CIS3 and has been widely used as a model system not only for the study
of LPS-induced macrophage-like differentiation,26,46-47 but
also for the JAK/STAT signaling pathway.34,48 As shown in
Fig 8A, stable transfectants expressing
CIS3 were almost completely resistant to LPS-induced differentiation,
whereas LPS-treated parental M1 cells exhibited typical morphological
changes such as vacuolation and chromatin condensation. Further
incubation of M1 cells in LPS resulted in apoptotic cell death, whereas
CIS3 transfectants grew normally (data not shown). In addition, LPS induced a large accumulation of nitrites into the culture medium of
parental M1 cells, whereas no LPS-dependent NO synthesis was seen in
CIS3 transfected cells (Fig 8B). In contrast, LPS-induced expression of
IL-6 mRNA, or of TNF production as well (A. Yoshimura, personal
communication, April 1999), were not affected in
CIS3-transfected cells (Fig 8C).
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| Fig 8.
Negative effects of stable CIS3 transfection on
LPS-signaling in murine M1 cells. (A) Suppression of LPS-induced
differentiation in M1 cells. Parental M1 cells (M1-P) and stable
transfectants expressing CIS3 (M1-CIS3) were incubated without or with
100 ng/mL LPS. After 72 hours of culture, cells were spun down on slide
glass using a cytospin and examined after May-Grunwald Giemsa staining.
(B) Inhibition of NO synthesis of M1 cells by CIS3. Parental M1 cells
(5 × 105; parent) and stable transfectants expressing
CIS3 were cultured with or without 100 ng/mL LPS for the days
indicated. No synthesis was determined by the Griess method. Similar
results were obtained with at least 2 independent clones of each
transfectants. (C) Expression of IL-6 mRNA in M1 cells and
transformants. Parental M1 cells (parent) and stable transfectants
expressing CIS3 were stimulated with 100 ng/mL LPS for the days
indicated. Total RNA was extracted and expression of IL-6 and G3PDH was
analyzed by Northern blotting.
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| |
DISCUSSION |
Several studies have established that, in monocytic cells, IL-10 has
the ability to induce the gene and surface expression of the
high-affinity IgG receptor, FcRI.10,13-15 In marked
contrast, we and others have reported that, in neutrophils, IL-10 fails to exert a similar effect.10-12 In this regard, we recently
demonstrated that, whereas IL-10 induces the binding of multimeric
complexes containing both STAT1 and STAT3 to the GRR sequence of the
FcRI gene promoter, no GRR-binding complexes were induced by IL-10 in autologous neutrophils.10 In the current report, we
confirmed these observations by showing that, in neutrophils, IL-10
fails to elicit any detectable DNA-binding activities to an
oligonucleotide probe, hSIE/m67, which binds STAT protein complexes
with a higher affinity than the GRR.39,40 We also extended
our previous findings by showing that neither STAT1 nor STAT3 is
phosphorylated on serine or tyrosine residues in response to IL-10
stimulation of neutrophils, in keeping with the fact that the
DNA-binding activity of STAT proteins is critically dependent on these
phosphorylations.42,43 It is noteworthy that both STAT1 and
STAT3 can nevertheless undergo tyrosine phosphorylation in neutrophils
in response to stimuli other than IL-10, as shown here in the case of
IFN and G-CSF or in other works performed with GM-CSF-stimulated
neutrophils.49,50 By comparison, we found that IL-10
rapidly triggers the tyrosine phosphorylation of both STAT1 and STAT3
in autologous PBMC, as previously reported.20,22,23,40 In
addition, we show for the first time that stimulation of PBMC with
IL-10 also results in the phosphorylation of STAT3 on serine residues.
Collectively, our data suggest that the inability of IL-10 to induce
the FcRI gene in neutrophils involves a defective postreceptor event
that is upstream from STAT1 and STAT3 phosphorylation. However, the nature of this upstream event remains unclear. Although it can be
envisaged that IL-10 somehow triggers an inhibitory signal in
neutrophils, we tend to exclude this possibility, because costimulation of neutrophils with IL-10 plus either IFN or G-CSF did not influence the IFN-elicited or G-CSF-elicited GRR-binding activities or FcRI gene expression.10 Another explanation could be
that, in PMN, the IL-10 receptor (IL-10R) complex differs from that present in monocytes. A functional IL-10R is in fact a multicomponent structure composed of the IL-10R151 and of the recently
identified CRFB4/CRF-2 molecule,51-54 in which IL-10R1
associates with JAK1 and recruits STAT340 and CRFB4/CRF-2
associates with TYK2.55 We previously assessed the presence
of IL-10R on the neutrophil surface by flow cytometry using
biotinylated IL-10 and showed that freshly isolated neutrophils clearly
possess IL-10 binding sites, albeit to a lesser extent than peripheral
blood monocytes or lymphocytes.10 More recently, we have
also investigated by Northern blot analysis the mRNA expression of
IL-10R1 and CRFB4/CRF-2 and found that the constitutive expression of
IL-10R1 transcripts is higher in PBMC than in neutrophils (our
unpublished observations). However, whether this
differential mRNA expression in neutrophils and PBMC has a functional
consequence remains an open question. For instance, the IL-10 receptor
in neutrophils might lack the intracellular distal portion containing
the docking sites for the recruitment of STAT3.40 In this
regard, it is noteworthy that transfection of the murine IL-10R1 chain
into L929 fibroblasts conferred to these cells the ability to bind
murine IL-10; however, despite this, IL-10 failed to induce STAT
activation in these cells.56 Finally, an obvious reason for
the lack of inducible STAT tyrosine phosphorylation in IL-10-treated
PMN might be that IL-10 fails to activate JAK1 and/or TYK2, the 2 tyrosine kinases previously shown to catalyze the phosphorylation of
STAT1 and STAT3 after cell treatment with IL-10.22,23
Whereas JAK1 and TYK2, as well as JAK2 and JAK3, can all be
detected at low levels in neutrophils by immunoblot,50,57
JAK2 is the only member of the JAK family that to date has been
reported to be activated in response to GM-CSF stimulation of
neutrophils.49,50
In the present work, we also demonstrate that IL-10 selectively
regulates CIS3 mRNA steady-state levels in both neutrophils and PBMC,
in that no effects of IL-10 on CIS1, CIS2, CIS5, and JAB mRNA
expression were observed. In IL-10-treated neutrophils, CIS3 mRNA
accumulation was also found to be transient, peaking after 2 to 3 hours
of incubation. To our knowledge, this constitutes the first
demonstration of the ability of IL-10 to modulate the gene expression
of a CIS family member in neutrophils. In agreement with our data,
IL-10 was shown to upregulate CIS3 mRNA expression in monocytes while
the present manuscript was under revision.58 CIS protein
expression is known to be induced via the JAK-STAT pathway in response
to a wide range of cytokines, including GM-CSF, G-CSF, macrophage
colony-stimulating factor (M-CSF), IL-1, IL-2, IL-3, IL-6, leukemia
inhibitory factor, and IFN,24-31,45 and as such, is
thought to negatively regulate signal transduction. For instance,
expression of CIS1 appears to reduce the proliferative response of
hemopoietic cells to erythropoietin and IL-3,24 whereas JAB
and CIS3 appear to be potent inhibitors of M1 cell differentiation in
response to a number of cytokines.27 Likewise, overexpression of JAB and CIS3, but not of CIS1 or CIS2, inhibits the
ability of growth hormone to regulate gene expression in
adypocites.59 Interestingly, we show here that, similar to
IL-10, LPS also upregulates the expression of CIS3 mRNA in neutrophils
and that it synergizes with IL-10, resulting in a sustained (as opposed
to transient) upregulation of CIS3 mRNA levels. Under the latter
conditions, IL-8 and IL-1ra mRNA expression was modulated exactly as
reported previously,4-7 indicating that IL-10 was acting as
an anti-inflammatory agent. Further investigation showed that IL-10
substantially increased the half-life of CIS3 transcripts in
LPS-treated cells. Consistent with this finding is the finding that a
similar posttranscriptional action of IL-10 has also been reported in
the case of IL-1ra.5 Although other effects of IL-10 at the
level of gene transcription cannot be excluded, it should be
interesting to know in this context whether CIS3 contains the AU-rich
sequences in its 3'-untranslated regions that are believed to be
involved in the regulation of mRNA stability.60
The function of CIS3 was examined using the murine M1 myeloid cell line
stably expressing CIS3. Our data showed that CIS3 transfection in M1
monocytic cells suppresses LPS-induced growth arrest, macrophage-like
differentiation, and NO synthesis, but not IL-6 mRNA expression or
TNF production. Because LPS induces IL-6 in M1 cells, it is highly
probable that an autocrine/paracrine activation of the JAK/STAT pathway
plays an essential role in the LPS-induced
differentiation.26,27,46 At the light of the established
ability of CIS3 to bind JAKs and inhibit their protein kinase
activity,46 it is tempting to speculate that the mechanism whereby CIS3 transfection blocks LPS-induced differentiation and NO
synthesis occurs at the JAK level. The idea is consistent with a
previous report showing that tyrosine kinase inhibitors suppress LPS-induced NO synthesis in macrophages,61 but it is in
contrast with a more recent study showing that CIS3 was unable to
inhibit JAK kinase activity in vitro.48 Although M1 cells
are not responsive to IL-10 (A. Yoshimura, personal communication,
April 1999), our data suggest that, at least in this
cellular experimental model, CIS3 negatively modulates specific
LPS-elicited responses. Although the data raise the possibility that
CIS3 may act as an anti-inflammatory mediator induced by IL-10, the
question of whether CIS3 induction negatively affects the signaling
pathways mobilized by IL-10 or whether CIS3 might mediate the
modulation by IL-10 of LPS-elicited responses in neutrophils remains to
be elucidated. However, whatever the case may be, the fact that CIS3
transfection does not influence IL-6 mRNA expression or TNF
production in LPS-treated cells might indicate that CIS3 is not
involved in the mechanisms by which IL-10 modulates cytokine expression.
Our finding that IL-10 enhances CIS3 mRNA expression represents the
first molecular demonstration of a direct effect of IL-10 towards
neutrophils. Importantly, our study also makes it clear that the
activation of STAT1 and STAT3 via tyrosine or serine phosphorylation is
not required for CIS3 induction by IL-10 in neutrophils. By inference,
our data raise the possibility that the modulation by IL-10 of cytokine
production in human neutrophils3,62 and, similarly, in
other cell types,1,2 might occur independently of STAT
protein activation. In support of this notion, it has been shown that,
in STAT1-deficient mice, IL-10 retains the ability to inhibit TNF
production in LPS-stimulated macrophages and to induce the formation of
DNA-binding complexes consisting of STAT3 homodimers.63
Furthermore, transfection studies using a truncated STAT3 acting as a
dominant negative mutant showed that the inhibition of monokine
production by IL-10 is similarly independent of STAT3 activation.64 However, it should be pointed out that 2 studies performed in the mouse instead support a critical role of the JAK1-STAT3 pathway in the IL-10-mediated deactivation of macrophages and neutrophils. In one of them, macrophages derived from
JAK1-deficient mice were shown to be unresponsive to IL-10 in terms of
inhibition of LPS-induced TNF production,65 thereby
highlighting an obligatory role for JAK1 in mediating this action of
IL-10. In the other one,66 macrophages and neutrophils
derived from mice engineered to express a genetic STAT3 deficiency in
the myeloid cell compartment failed to respond to IL-10 and secrete
high levels of TNF upon stimulation with IL-10 plus LPS. The
discrepancies between human and murine neutrophils might be partially
explained by our preliminary experiments, in which IL-10 induced STAT3
tyrosine phosphorylation in murine neutrophils (Gasperini et al,
unpublished data, May 1999). These considerations
indicate that human neutrophils could represent an interesting cellular
model for the study of the IL-10R signaling occurring independently
from STAT activation.
| |
ACKNOWLEDGMENT |
The authors thank Dr P.P. McDonald for his invaluable criticisms and suggestions.
| |
FOOTNOTES |
Submitted December 22, 1998; accepted June 21, 1999.
Supported by grants from MURST (40%, 60% funds, and
"cofinanziamento MURST-Universita"), AIRC, and "Progetto
Sanità, Fondazione CARI-VR-VI-BL-AN."
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Marco A. Cassatella, MD, Department
of Pathology, Strada Le Grazie 4, I-37134 Verona, Italy;
e-mail: MCNCSS{at}borgoroma.univr.it.
| |
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ABSTRACT
INTRODUCTION