| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RI/CD64) Gene and STAT Protein Binding to the IFN- Response Region (GRR) Are Regulated Differentially in Human Neutrophils and Monocytes by IL-101
Departments of
*
General Pathology and
Biochemistry, University of Verona, Verona, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI/CD64) in human monocytes, we
examined whether the cytokine exerts a similar action toward
polymorphonuclear neutrophils (PMN). Unexpectedly, we found that in
neutrophils, IL-10 failed to induce either the mRNA accumulation or the
surface expression of FcRI. Consistent with these findings,
stimulation of PMN with IFN-, but not with IL-10, resulted in the
induction of specific DNA-binding activities to the IFN- response
region (GRR), a regulatory element located in the FcRI gene
promoter, required for transcriptional activation. In electrophoretic
mobility shift assays (EMSAs), we confirmed that in PBMC, IL-10 induces
the binding to the GRR of both STAT1 and STAT3, two members of the STAT
family. In neutrophils, however, these activators did not bind to the
GRR in response to IL-10, despite the fact that both STAT1 and STAT3
are expressed in these cells. On the other hand, IFN- was an
efficient inducer of STAT1 binding to the GRR in both PMN and PBMC. The
lack of inducible GRR-binding activity in IL-10-treated PMN could not
be ascribed to a lack of IL-10R, and did not appear to reflect an
inhibitory effect of the cytokine. Taken together, our data suggest
that IL-10 is unable to induce FcRI gene expression in neutrophils
because the intracellular signaling pathway triggered by the cytokine
is impaired at the level of, or upstream of, STAT1 and/or STAT3
activation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI/CD64) stands out as a particularly important
molecule (4, 5), in view of its role in mediating Ab-dependent cellular
cytotoxicity and the release of several inflammatory mediators (6).
While FcRI is expressed constitutively in mononuclear phagocytes, it
is usually undetectable on the surface of resting PMN (4, 5). However,
treatment of neutrophils in vitro and in vivo with IFN- (4, 5, 7, 8, 9), or with granulocyte CSF (G-CSF) (10), results in a dramatic
induction of expression of FcRI to the cell surface. In recent
years, the signaling pathway used by IFN- to induce the FcRI gene
in monocytic cells has been analyzed extensively (11, 12, 13, 14). Monocytic
cell stimulation by IFN- results in the activation of two
receptor-associated tyrosine kinases belonging to the Janus kinase
(JAK) family, namely JAK1 and JAK2 (15), which in turn catalyze the
tyrosine phosphorylation and concurrent activation of a latent
cytoplasmic transcription factor, STAT1 (reviewed in Refs. 16 and 17).
Activated STAT1 molecules dimerize and translocate to the nucleus,
where they can bind to the IFN- response region (GRR) located in the
FcRI gene promoter, thereby stimulating gene transcription (11, 12, 13, 14).
Promoter deletion studies have indeed defined the GRR as a 39-bp region
that is necessary and sufficient for the IFN--dependent activation
of FcRI (11). Remarkably, the GRR contains a 9-bp core element that
bears a striking similarity to the IFN--activated consensus sequence
(GAS), an element that is typically found in IFN--inducible genes
(16). Recent investigations have demonstrated that in human monocytes
and murine macrophages, FcRI gene and surface expression can also be
up-regulated by IL-10, both in vitro and in vivo (18, 19, 20, 21). Accordingly,
IL-10 was found to induce GRR DNA-binding activities in mononuclear
phagocytes (19). Thus, from a general standpoint, the action of IL-10
toward FcRI gene expression resembles that of IFN-. However, the
mechanisms underlying this action of IL-10 differ from those used by
IFN-. Indeed, IL-10 promotes the activation of the JAK1 and TYK2
tyrosine kinases, leading to the tyrosine phosphorylation of STAT1 and
STAT3 (22, 23); the resulting STAT1 and STAT3 homo- and heterodimers
can subsequently bind to GAS sequences in the regulatory regions of
target genes (16, 17). Since neutrophils represent one of the cellular
targets of IL-10 (24), we investigated whether IL-10 might affect the
expression of FcRI, as previously shown for monocytic cells. We now
demonstrate that IL-10 fails to stimulate either the mRNA accumulation
or the surface expression of FcRI in human neutrophils. Accordingly,
no GRR DNA-binding activity is induced in IL-10-treated neutrophils. In
contrast, IFN- functions as a potent inducer of GRR activity in PMN,
as it does in monocytic cells. This therefore suggests that in human
neutrophils, the IL-10 signaling pathway leading to FcRI gene
induction is functionally impaired at the level of, or upstream of,
STAT1 and/or STAT3 activation. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Highly purified (>99.5%) PMN and PBMC were isolated under
LPS-free conditions from buffy coats of healthy donors by
centrifugation on a Ficoll-Hypaque gradient, as previously described
(25). Monocytes (60–80% purity) were isolated from PBMC after
centrifugation over Percoll gradients, as described earlier (26).
Immediately after purification, cells were suspended in RPMI 1640
supplemented with 10% low endotoxin FCS (<0.006 ng/ml; HyClone
Laboratories, Logan, UT), and treated with or without 100 U/ml IFN-
(endotoxin free; kindly provided by Dr. G. Garotta, Hoffman-La Roche,
Basel, Switzerland) (8, 9), or up to 2000 U/ml IL-10 (kindly provided
by Dr. K. Moore, DNAX, Palo Alto, CA). Identical results were obtained
when IL-10 (20 ng/ml) purchased from Peprotech (Rocky Hill, NJ) was
used. Cells thus treated were cultured at 37°C under a 5%
CO2 humidified atmosphere, in six-well tissue culture
plates (Nunc, Roskilde, Denmark) for antigenic or RNA studies, or at
room temperature in polypropylene tubes (Greiner, Nürtingen,
Germany) for EMSA studies. HL-60 cell line was grown in RPMI 1640
containing 10% FCS. For cell cultures, all reagents were of the
highest available grade, and all buffers were prepared using
pyrogen-free water for clinical use.
Immunofluorescence assays
After a 20-h (or 44-h) incubation in the presence or absence of
IFN- or IL-10, cells were washed and treated (30 to 60 min at
+4°C) with appropriate dilutions of the different mAbs (in the
presence of 5% human serum, except when mAbs B137.17 were used) and
then with FITC-labeled goat F(ab')2 anti-mouse Ig
(Cappel Laboratories, Cochranville, PA) preadsorbed with human IgG, as
previously described (8). Murine mAbs 32.2 (IgG directed against
FcRI) were purchased from Medarex (Annandale, NJ). B137.17 (IgG2a,
which specifically binds, via Fc, to the FcRI) (27) and OKMI (IgG2b,
which reacts with the CR3) were kindly provided by Dr. G. Trinchieri
(Wistar Institute, Philadelphia, PA). Cytofluorographic analyses (using
104 cells/sample) were performed on a FACScan (Becton
Dickinson, Mountain View, CA) coupled to a Hewlett Packard 9000 system
computer. The fluorescence intensity of cells stained with irrelevant
Abs and/or FITC-conjugated anti-mouse IgG was the same. To measure
the IL-10R, we used the Fluorokine kit (NF100; R&D Systems,
Minneapolis, MN), according to the manufacturer’s instructions.
Northern blot and RT-PCR analyses
Total RNA was extracted from PMN or monocytes by the guanidinium
isothiocyanate method, and processed for Northern blot analysis, as
already described (25). The various mRNA species were detected by
autoradiography after hybridization of nylon filters with
32P-labeled cDNA probes (Ready-to-go DNA labeling kit;
Pharmacia, Uppsala, Sweden) (8, 9, 25). The following full-length cDNA
fragments, excised from their respective vectors, were used: the p135
probe encoding the FcRI gene A (kindly provided by Dr. B. Seed,
Harvard University, Boston, MA); p47-phox, encoding the 47-kDa
nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase component
(kindly provided by Dr. H. Malech, National Institutes of Health,
Bethesda, MD); class II/DRß (kindly provided by Dr. R. Accolla, CBA,
Genova, Italy); MIP-1
(kindly provided by Dr. B. Sherry, Picower
Institute, Manhasset, NY); and actin and glyceraldehyde-3-phosphate
dehydrogenase (GAPD) (kindly provided by Dr. G. Trinchieri, Wistar
Institute). For RT-PCR, total RNA extracted by the guanidinium
isothiocyanate method was reverse transcribed by Superscript II RT
(Life Technologies, Paisley, U.K.) for 50 min at 42° in a 20-µl
reaction mix containing oligo(dT) primers (1 µg). Ten microliters of
the same cDNA preparations were amplified in a 50-µl vol of final
reaction mix in a Perkin-Elmer (Norwalk, CT) 4800 thermal cycler, with
25 pmol of primers specific for the IL-10R (sense 5'-3', region
1426–1443; antisense 5'-3', region 2215–2198) (kindly provided by Dr.
A. Pinto, Centro di Riferimento Oncologico, Aviano, Italy), and murine
actin (sense 5'-3', region 224–244; antisense 5'-3', region 411–433).
PCR conditions for IL-10R were 3 min at 94°C, followed by 35 cycles
of 30 s at 94°C, 150 s at 68°, and a final extension of 5
min at 72°C. Fifteen microliters of amplified cDNA were run in 1.5%
agarose gels and visualized by ethidium bromide staining.
Cellular extracts
After stimulation with IFN- or IL-10 for the indicated times,
neutrophils (1–2 x 108/condition) or
monocytes/PBMC (0.3–1 x 108/condition) were diluted
in ice-cold PBS and centrifuged twice at 500 x g for 5
min at 4°C. The cells were then resuspended in 1 ml of relaxation
buffer (10 mM PIPES, 3.5 mM MgCl2, 3 mM NaCl, 100 mM KCl,
1.25 mM EGTA, 1 mM NaVO4, 50 mM NaF, 5 µg/ml leupeptin, 5
µg/ml pepstatin A, 33 µg/ml aprotinin, 1 mM PMSF, and 3 mM
diisopropyl fluorophosphate), before being disrupted in a nitrogen bomb
(Parr Instruments, Mobile, IL), as previously described (28, 29).
Briefly, neutrophils (108 cells/ml) were pressurized under
an N2 atmosphere (350 psi, 20 min at 4°C) with constant
stirring. Cavitates were spun at 1000 x g (10 min,
4°C), to pellet unbroken cells and intact nuclei, and the
supernatants were recentrifuged (1000 x g, 10 min,
4°C), to remove remaining nuclei and unbroken cells. Both pellets
were combined, resuspended in relaxation buffer containing the
antiprotease mixture, and washed twice (1000 x g, 10
min, 4°C). Pellets were gently resuspended in ice-cold relaxation
buffer containing the antiprotease mixture; NaCl was then added to
yield a final concentration of 420 mM. Following a 30-min incubation on
ice (with occasional mixing), samples thus treated were spun at
12,000 x g (30 min, 4°C), and the resulting
supernatants (the nuclear extracts) were aliquoted and immediately
stored at -80°C. The 1,000 x g (postnuclear)
cavitate supernatants were centrifuged at 12,000 x g
(30 min, 4°C) to pellet neutrophil granules. The resulting
12,000 x g supernatants are referred to as cytoplasmic
fractions, even though they do contain plasma membranes in addition to
cytosol.
Alternatively, PBMC or purified monocytes were resuspended in a high salt buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT) supplemented with the aforementioned antiprotease and antiphosphatase mixture, and lysed by three freeze-and-thaw cycles in liquid nitrogen. After a 30-min incubation on ice, insoluble material was removed by centrifugation (30 min at 12,000 x g at 4°C); the resulting supernatants are referred to as whole cell extracts. Small aliquots of the various extracts were processed routinely for protein content determination, by using a protein assay kit (Bio-Rad, Hercules, CA).
Electrophoretic mobility shift assays (EMSA)
Protein-DNA complexes were detected by EMSA analysis of the
various extracts, as described previously (30), with the following
modifications. Usually, 10 to 40 µg of cytoplasmic or 5 to 20 µg of
nuclear extracts were 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, and 10% glycerol, followed by
addition of a 32P-labeled double-stranded oligonucleotide
probe corresponding to the GRR element located within the promoter of
the FcRI/CD64 gene (5'-CTT TTC TGG GAA ATA CAT CTC AAA TCC TTG AAA
CAT GCT-3') (11), for 15 min. In competition experiments,
double-stranded oligonucleotides corresponding to different GAS
consensus sequences were used, i.e., the high affinity synthetic
derivative of the c-sis-inducible element (SIE), hSIE/m67
(5'-gtc gaC ATT TCC CGT AAA TCg-3') (31), guanylate binding protein GAS
(5'-AGT TTC ATA TTA CTC TAA ATC-3') (32), IRF1 IFN-responsive
element (5'-CCT GAT TTC CCC GAA ATG ATG-3') (33), as well as an
unrelated oligonucleotide corresponding to the murine leukemia virus
upstream conserved region (UCR) (5'-CTG CAG TAA CGC CAT TTT GCA AGG CAT
GAA-3') (34). Supershift experiments were performed by incubating the
proteins with 0.5 µg, or 1/25 dilutions, of the various anti-STAT
Abs, for 30 min at room temperature, before adding the labeled probe.
Four different anti-STAT1 Abs were used: N1 (raised against amino
acids 1–194) was purchased from Transduction Laboratories (Memphis,
TN); C1 and C2, raised against amino acids 741–750 and 712–728,
respectively, were a generous gift from Dr. K. Ozato (National
Institute of Child Health and Human Development, National Institutes of
Health) (35); and C3 (E-23, raised against amino acids 688–710) was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The
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) Abs were purchased
from Santa Cruz Biotechnology. Supershift experiments with
anti-phosphotyrosine Abs were performed by incubating the proteins
with 5 µg of 4G10 (Upstate Biotechnology, Lake Placid, NY) for 3
h at 4°C, before adding the labeled probe for 15 min at room
temperature.
Western blot analyses
For immunoblotting experiments, cellular extracts obtained from PMN, PBMC, and HL-60 cells were electrophoresed on 9% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Hybond ECL; Amersham Little Chalfont, U.K.) at 100 V for 1 h in a Transblot Transfer Cell (Bio-Rad) in a 25 mM Tris, pH 8.3, 192 mM glycine, 20% methanol buffer; transfer efficiency was visualized by reversible Ponceau Red staining. The membranes were first blocked overnight at 4°C in TBS/T (25 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.2% Tween-20) containing 7.5% BSA, and further incubated (60 min, 37°C) in the presence of the desired primary Ab. Ab dilutions were as follows: 1/1000 of anti-IL-10R (984; Santa Cruz Biotechnology); 1/1000 of purified rabbit IgG (15006; Sigma Chemical Co., St. Louis, MO); and 1/2000 of anti-STAT1 (C1). The membranes were then washed thrice with 150 ml of TBS/T, and incubated for 45 min at room temperature in TBS/T containing an appropriate 1:10,000 horseradish peroxidase-linked anti-rabbit secondary Ab. After three washes, the signal was revealed with the ECL reagent, according to the manufacturer’s instructions.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
on FcRI surface and mRNA expression
in neutrophils and monocytes
To test whether IL-10 can induce FcRI surface expression in
human neutrophils, cells were cultured for 20 h in the presence or
absence of the cytokine, and then subjected to indirect
immunofluorescence flow cytometry; as a positive control, neutrophils
were also cultured in presence of 100 U/ml of IFN-. For comparative
purposes, autologous monocytes were also examined under identical
experimental conditions. As shown in Figure 1
, 100 U/ml of IL-10 failed to stimulate
FcRI surface expression in neutrophils, whereas IFN- proved to be
an efficient inducer; by comparison, the surface expression of CR3
(CD11b/CD18) was not affected significantly by either cytokine under
the same conditions (Fig. 1). In the same cell populations, however,
IL-10 decreased the extracellular release of IL-8 in neutrophils
stimulated with 1 µg/ml of LPS, from 2983 to 358 pg/ml, in agreement
with our previous findings (3, 24). Results identical to those depicted
in Figure 1 were obtained when PMN were incubated with higher doses of
IL-10 (up to 2000 U/ml) or for longer periods (up to 48 h), or
when mAb B137.17, which binds FcRI through its Fc portion (27), was
used in the FACS analyses (data not shown). The inability of IL-10 to
stimulate FcRI surface expression in PMN did not appear to involve
the mobilization of an inhibitory pathway by the cytokine, since the
IFN-- or G-CSF-elicited expression of FcRI to the cell surface
was unaffected by costimulation of the cells with IL-10 (data not
shown). In contrast to neutrophils, both IL-10 and IFN- efficiently
up-regulated FcRI surface expression in monocytes, in agreement with
previous studies (4, 5, 18).
|
RI
surface expression in neutrophils and monocytes, we examined the
ability of IL-10 to modulate FcRI mRNA steady state levels in both
cell types, as determined by Northern blot analysis. Figure 2A Shows that in agreement
with our flow-cytometry data, a 3-h treatment of PMN with IFN-, but
not with 100 U/ml of IL-10, resulted in the accumulation of FcRI
mRNA transcripts, whereas in monocytes, FcRI mRNA expression was
clearly increased by either IL-10 or IFN-. As a control, Figure 2A also shows that IFN- increased DRß and p47-phox mRNA
levels in monocytes, while it decreased p47-phox mRNA levels in
neutrophils, in keeping with previous publications (36, 37, 38). By
comparison, no significant effect of IL-10 toward DRß and p47-phox
mRNA levels was observed in either granulocytes or monocytes. Time
course experiments in which PMN were incubated with 100 U/ml of IL-10
for up to 24 h confirmed the inability of the cytokine to affect
FcRI mRNA expression (data not shown). Moreover, costimulation of
PMN with IL-10 and IFN- resulted in an accumulation of FcRI mRNA
transcripts that was comparable with that observed with IFN- alone
(Fig. 2B), in agreement with our FACS data. Finally,
under conditions in which IL-10 failed to induce FcRI mRNA
transcripts, it was nevertheless possible to demonstrate an inhibitory
effect of the cytokine on the LPS-mediated up-regulation of MIP-1
mRNA steady state levels (Fig. 2B).
|
on GRR-binding activities in
neutrophils and PBMC
The differential ability of IL-10 to induce FcRI mRNA and
surface expression in PMN and monocytes prompted us to determine
whether IL-10 might also differentially elicit GRR-binding activities
in both cell types. Because preliminary experiments repeatedly failed
to reveal any difference between purified monocytes and PBMC in terms
of IL-10- or IFN--elicited GRR-binding activities, we indifferently
used monocytes or PBMC in our experiments, to ensure that the few
contaminating PBMC present in our neutrophil suspensions did not
account for the DNA-binding activities detected in our neutrophil
extracts. Neutrophils and autologous PBMC were stimulated for 15 min in
the presence or absence of 1000 U/ml IL-10 or 100 U/ml IFN-, and the
resulting nuclear and cytoplasmic extracts were analyzed in EMSA, using
a labeled GRR probe. In agreement with previous studies (19), distinct
GRR-binding complexes were detected in nuclear extracts from PBMC
stimulated by IFN- or IL-10 (Fig. 3A). By comparison,
stimulation of PMN with IFN- led to the formation of GRR-binding
complexes in nuclear extracts, whereas GRR-binding activities were
consistently undetectable in extracts from IL-10-stimulated cells (Fig. 3A), even when the cells were stimulated with 2000
U/ml of IL-10 for up to 2 h (data not shown). The various
GRR-binding activities observed in PMN nuclear extracts were also
detected in the corresponding cytoplasmic extracts, although this
required the inclusion of three to five times more protein in the
binding reactions. Figure 3B also shows that costimulation
of PMN with IFN- and IL-10 resulted in the induction of GRR-binding
activities that were indistinguishable from those observed in cells
treated with IFN- alone, in keeping with our FACS and Northern blot
data. Similarly, IL-10 failed to affect the G-CSF-elicited formation of
GRR-binding complexes in neutrophils (data not shown). Thus, IL-10 not
only represents an ineffective stimulus for the formation of
GRR-binding complexes in PMN, but also lacks the ability to modulate
the induction of such DNA-binding activities by IFN- or G-CSF.
Finally, it is worthy of mention that the inducible GRR-binding
activities detected in PMN extracts are highly unlikely to reflect a
contamination of our neutrophil suspensions by PBMC, in view of the
fact that IL-10-inducible complexes are undetectable in neutrophil
extracts (Fig. 3A). Consistent with this conclusion
is that when whole cell extracts from IFN--treated PMN were analyzed
in parallel with whole cell extracts from IFN--treated PBMC
corresponding to 100 times less cells (i.e., to mimick the equivalent
of 1% contaminating PBMC), the latter consistently failed to yield any
detectable GRR signal (data not shown).
|
-induced GRR-binding activities in
neutrophils and monocytes
Competition experiments revealed that in both neutrophils
and monocytes, the IFN--inducible GRR-binding complexes were
specific, since they were displaced by a 100-fold molar excess of
unlabeled GRR probe, as well as by various unlabeled oligonucleotides
containing other GAS sequences, but not by a 100-fold molar excess of
UCR, an unrelated oligonucleotide (Fig. 4A). Time course
experiments also showed that in IFN--stimulated neutrophils,
GRR-binding complexes were induced as early as after 10 min, displayed
maximal binding activity by 30 min, and remained detectable for up to
4 h (Fig. 4B). To determine the identity of the
two inducible GRR-binding complexes detected following stimulation of
neutrophils or monocytes with IFN-, we next performed supershift
experiments using a panel of Abs raised against the STAT1 protein.
Figure 4C shows that in both cell types, the two
IFN--inducible complexes were recognized by all Abs. Furthermore,
anti-phosphotyrosine Abs substantially hindered the detection of
the IFN--induced GRR-binding complexes in either PMN or PBMC (Fig. 4D). Taken together, these results demonstrate that
in neutrophils, IFN- stimulation results in the formation of
tyrosine-phosphorylated STAT1-containing complexes that can bind the
GRR, as already shown for monocytes (13).
|
-treated
neutrophils (compare, for instance, Figs. 3
A, 3B,
and 4D). These differences were noted regardless of
whether gel shifts were performed with nuclear, cytoplasmic, or whole
cell extracts. Similar donor-to-donor variation was also observed using
monocyte or PBMC extracts, and has been documented by other
investigators as well (12, 15). Characterization of IL-10-induced GRR-binding activities in PBMC
To gain a better understanding of the signaling pathway required
for FcRI induction by IL-10 in PBMC, as well as to provide
indications as to how this response is impaired in PMN, we
characterized the IL-10-inducible GRR-binding complexes observed in
PBMC. While competition experiments demonstrated that the GRR-binding
complexes induced by IL-10 were specific (data not shown), time course
experiments showed that these various complexes were induced as early
as after 2 min, displayed maximal binding activity by 10 to 20 min, and
(at variable levels depending on the donor) remained detectable at 60
min (Fig. 5A).
Interestingly, supershift experiments revealed that GRR-binding
activities actually consist of three distinct complexes (Fig. 5).
Indeed, anti-STAT3 Abs displaced the species of slower and
intermediate electrophoretic migration, whereas the intermediate and
faster migrating complexes were displaced by an anti-STAT1 (C3) Ab
(Fig. 5B). This indicates that the slower and faster
migrating species represent STAT1 and STAT3 homodimers, respectively,
while the intermediate band represents STAT1/STAT3 heterodimers.
Anti-STAT5 Abs were ineffective toward any of the IL-10-inducible
GRR-binding complexes observed in human PBMC (Fig. 5B), in contrast to a recent study performed using
the BaF3 cell line (39). Conversely, the same Abs effectively displaced
inducible STAT5 GRR-binding activities in BaF3 cells stimulated with
erythropoietin (not shown). The above results are in full agreement
with, and further extend, those recently reported by Finbloom and
Winestock (22).
|
In a final series of experiments, we investigated whether
neutrophils express the IL-10R. For this purpose, total RNA from PMN
and PBMC was assayed initially for IL-10R mRNA expression by RT-PCR. In
these experiments, 970-bp bands corresponding to IL-10R-amplified
products were clearly detectable in both cell types (not shown). The
presence of functional IL-10R on the neutrophil surface was then
assessed by flow cytometry, using biotinylated IL-10. As shown in
Figure 6A, freshly isolated
neutrophils clearly possess IL-10 binding sites, albeit to a lesser
extent than peripheral blood monocytes (Fig. 6A) or
lymphocytes (not shown). Binding of IL-10 to PMN or monocytes was
specific, as it was displaced by an anti-IL-10 Ab (Fig. 6A). In contrast, undifferentiated HL-60 cells were
not stained by biotinylated IL-10 (Fig. 6A).
Together, the expression of IL-10R mRNA and the detection of specific
IL-10 binding sites in neutrophils strongly suggest that these cells
possess the IL-10R. To directly test this hypothesis, immunoblot
experiments were performed using a specific anti-IL-10R Ab. Figure 6B shows that the Ab recognized a 62-kDa immunoreactive band
in both neutrophils and PBMC, but not in undifferentiated HL-60 cells.
The identity of this band was ascertained in control experiments by
pretreating the anti-IL-10R Ab with the peptide used to generate
it, which prevented the subsequent immunoblot detection of the 62-kDa
band; conversely, using a purified rabbit IgG as a primary Ab failed to
yield a detectable signal in immunoblot (not shown). On a final note,
PBMC were found to express more IL-10R protein than an equivalent
number of neutrophils (Fig. 6B), in agreement with
our FACS data.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IL-10 fails
to induce FcRI/CD64 surface expression in human neutrophils; by
comparison, both cytokines up-regulated FcRI surface expression in
autologous monocytes. These data confirm recent observations made in
murine neutrophils (40). By taking advantage of the known sequence of
events triggered by IFN- and IL-10 to induce FcRI expression in
monocytic cells (11, 12, 13, 14, 15, 19, 22), we attempted to understand the
reasons for which PMN are unresponsive to IL-10 with respect to FcRI
expression. We initially found that IL-10, unlike IFN-, does not
induce FcRI mRNA accumulation in neutrophils, suggesting a lack of
transcriptional activation. In support of this view, EMSA experiments
demonstrated that no GRR-binding complexes were induced by IL-10 in
neutrophils, whereas three distinct DNA-binding activities were
detected in monocytes/PBMC following IL-10 stimulation. In the latter
instance, supershift experiments showed that these IL-10-inducible
GRR-binding complexes contain STAT1 and STAT3, but no detectable STAT5,
as opposed to other cell types such as BaF3 cells (39). Thus, it
appears that while the ability of IL-10 to induce GRR-binding complexes
can differ widely depending upon the cell type (as shown herein for
human PMN vs monocytes), the qualitative composition of IL-10-inducible
GRR-binding complexes (in terms of their constituent STAT proteins) may
also vary significantly between different cell types. Taken together,
our data suggest that the FcRI gene is not induced by IL-10 in PMN
because the signaling pathway leading to STAT1 and STAT3 binding to GRR
is somehow impaired. Noteworthy is that this cannot be ascribed to the
absence of STAT1 and/or STAT3 in PMN, since both proteins are expressed
constitutively in neutrophils. Similarly, both STAT1 and STAT3 appear
to be functional in PMN, as GRR-binding activities containing these
proteins can be induced following appropriate stimulation of the cells,
as shown herein for STAT1 in IFN--treated PMN, or previously for
both STAT1 and STAT3 in G-CSF-treated PMN (41).
The exact reasons for which IL-10 does not activate GRR-binding
activities in neutrophils remain unclear. However, some hypotheses can
be put forward. In view of the essential role of tyrosine
phosphorylation for the DNA-binding activity of STAT proteins, it is
conceivable that the absence of detectable GRR-binding complexes in
IL-10-treated neutrophils might be related to a deficient induction of
STAT1 and STAT3 tyrosine phosphorylation. However, this is unlikely to
reflect a general inability of PMN to phosphorylate STAT1 or STAT3 on
tyrosine residues in response to cell stimulation. Indeed, we showed
herein that in IFN--treated neutrophils, anti-STAT1 Abs
supershift the inducible GRR-binding complexes, while
anti-phosphotyrosine Abs substantially hinder their detection.
Similarly, we recently reported that STAT3 becomes tyrosine
phosphorylated in response to G-CSF stimulation of neutrophils, and
that GRR-binding activities containing STAT1 and STAT3 are
concomitantly induced (41). In keeping with our observations, Brizzi
and colleagues recently reported that both STAT1 and STAT3 become
tyrosine phophorylated following granulocyte-macrophage CSF stimulation
of neutrophils (42). Another explanation might be that an unidentified
component of the IL-10 signaling pathway (such as a protein tyrosine
phosphatase) might become activated in PMN, which could prevent a
stable activation of either the STAT proteins or the upstream JAK
kinases, thereby impeding the formation of GRR-binding complexes and
subsequent transcriptional activation. However, the fact that
costimulation of neutrophils with IL-10 and either IFN- or G-CSF
neither influences the IFN-- or G-CSF-mediated STAT protein
activation and binding to GRR, nor the FcRI mRNA accumulation or
surface expression, argues against the activation of an inhibitory
pathway by IL-10 in PMN. Yet another explanation might be that in PMN,
the IL-10R differs from that present in monocytes. For instance, it
could lack an associated polypeptide chain required for the generation
of an intracellular signal leading to STAT protein activation.
Alternatively, the intracellular domain of the IL-10R in PMN could lack
a docking site for the recruitment of JAK/STAT proteins.
Known effects of IL-10 toward PMN consist in the modulation of cytokine
production in cells stimulated by LPS, TNF-, or phagocytic particles
(3, 24, 43, 44). These modulatory effects can be generally described as
anti-inflammatory, since IL-10 inhibits the LPS-induced production
of proinflammatory cytokines, such as TNF-, IL-1ß, IL-8, IL-12p40,
growth related peptide alpha, and MIP-1/ß, while it potentiates
the LPS-elicited secretion of IL-1R antagonist (45). Very recently,
other effector functions of PMN were shown to be modulated by IL-10,
such as platelet-activating factor production, phagocytic and
bactericidal activities, Ab-dependent cellular cytotoxicity, and
oxidative metabolism (46, 47, 48, 49). Moreover, a few direct effects of IL-10
were reported in PMN, namely, the rapid and transient induction
(peaking at 8 min) of superoxide production (46), as well as a slight
decrease in CD11b surface expression within 30 min of incubation (47).
All of these observations point to the presence of a sufficient number
of functional IL-10R in PMN. In the current study, we confirmed that
neutrophils constitutively express the IL-10R, albeit on a smaller
scale than monocytes or lymphocytes. Thus, the inability of IL-10 to
induce GRR-binding activities in PMN cannot be attributed to a lack of
receptors. Whereas the mechanisms underlying the various actions of
IL-10 are understood only partially (22), our current data strongly
suggest that they are independent of STAT1 or STAT3 activation. Further
support for this conclusion is that in STAT1-deficient mice, IL-10
retained the ability to inhibit LPS-stimulated TNF- production, and
to induce the formation of DNA-binding complexes consisting of STAT3
homodimers (50). Thus, an obligatory role for STAT1 in mediating the
effects of IL-10 on cytokine production can be excluded. Collectively,
these considerations indicate that IL-10R signaling for certain
cellular functions involves a pathway that is distinct from STAT
protein activation. It follows that neutrophils could represent an
interesting cellular model for the study of STAT-independent IL-10R
signaling.
The biologic significance of the ability of IL-10 to induce FcRI
expression in monocytes, but not in PMN, remains to be elucidated. In
this context, it is perhaps relevant that the biologic effects of IL-10
are known to vary depending upon the cell target. For instance, IL-10
induces the expression of Fc
RII/CD23 in the myelomonocytic cell
line, U937, whereas it down-regulates CD23 expression in peripheral
blood monocytes (51). This not only suggests that the differences in
IL-10 responsiveness may be linked to the stage of cell differentiation
and maturation, but further supports the view that the responsiveness
of neutrophils and monocytes (or other cell types) to IL-10 with
respect to selected cellular functions is mediated by multiple and
distinct signaling pathways. In contrast to IL-10, the action of
IFN- toward FcRI gene and surface expression was found to be
similar in both PMN and monocytes. In keeping with the role of STAT1 in
the IFN--induced transcriptional activation of the FcRI gene
previously shown in monocytes, we were able to demonstrate that in PMN,
IFN- also induces GRR-binding complexes that contain
tyrosine-phosphorylated STAT1. Similarly, IFN- treatment of
neutrophils resulted in the binding of a STAT1-containing complex to an
m67/hSIE oligonucleotide probe (52, and our unpublished observations).
On a final note, the demonstration that neutrophils express detectable
amounts of transcription factors, such as STAT1, STAT3, and STAT5 (our
unpublished data), further emphasizes the potential ability of these
cells to be functionally regulated at the level of gene
transcription.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: AIDS Immunopathogenesis Unit, DIBIT, San Raffaele Scientific Institute, Milan, Italy.
3 Address correspondence and reprint requests to Dr. Marco A. Cassatella, Istituto di Patologia Generale, Strada Le Grazie, 8, I-37134 Verona, Italy. E-mail address:
4 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; EMSA, electrophoretic mobility shift assay; G-CSF, granulocyte colony-stimulating factor; GAS, interferon--activated consensus sequence; GRR, interferon- response region; JAK, Janus kinase; MIP, macrophage-inflammatory protein; RT, reverse transcriptase; SIE, c-sis inducible element; UCR, upstream conserved region.
Received for publication February 28, 1997. Accepted for publication September 30, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
transcriptionally modulates the expression of the genes for the high affinity IgG-Fc receptor and the 47-kDa cytosolic component of NADPH oxidase in human polymorphonuclear leukocytes. J. Biol. Chem. 266:22079.-dependent gene expression of NADPH oxidase components (gp91-phox, p47-phox, and p22-phox) and high-affinity receptor for IgG (FcR-I) in human polymorphonuclear leukocytes. Blood 79:735. receptor I expression can be used as marker for biologic activity of human recombinant interferon- in vivo. Blood 77:2088.R-I after stimulation with granulocyte colony stimulating factor (G-CSF). J. Leukocyte Biol. 57:455.[Abstract]
-interferon is mediated through common DNA response elements. Proc. Natl. Acad. Sci. USA 88:11305. rapidly induces in human monocytes a DNA-binding factor that recognizes the response region within the promoter of the gene for the high affinity Fc receptor. Proc. Natl. Acad. Sci. USA 89:11964.-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3. Proc. Natl. Acad. Sci. USA 90:4314.) activation of the human high affinity Fc receptor gene: a novel IFN regulatory mechanism. Mol. Cell. Biol. 13:2182.-induced transcription of the FcR-I gene in human monocytes by preventing the tyrosine phosphorylation of the p91 (Stat1) transcription factor. J. Immunol. 154:318.[Abstract]
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]
responsive promoter elements. J. Immunol. 153:165.[Abstract]
R-I/CD64 triggers enhanced antibody responses in transgenic mice. J. Clin. Invest. 97:331.[Medline]
and STAT3 complexes in human T cells and monocytes. J. Immunol. 155:1079.[Abstract]
and lipopolysaccharide enhancement of phagocyte respiratory burst capability: studies on the gene expression of several NADPH oxidase components. J. Biol. Chem. 265:20241.
B pathway by inflammatory stimuli in human neutrophils. Blood 89:3421.-regulated DNA-binding factor. EMBO J. 10:927.[Medline]
-gene regulatory elements. Cell 54:903.[Medline]
, in vitro and in vivo, on mRNA levels of phagocyte oxidase components. J. Leukocyte Biol. 60:716.[Abstract]
to activate superoxide anion and hydrogen peroxide release by human macrophages. Immunol. Lett. 28:1.[Medline]
RI/CD64 transgenic model for in vivo analysis of (bispecific) antibody therapeutics. J. Hematother. 4:351.[Medline]
-response region within the promoter of the FcRI/CD64 gene in human neutrophils. FEBS Lett. 386:239.[Medline]
production in human granulocytes. J. Inflamm. 45:143.[Medline]
This article has been cited by other articles:
|
|
 |
|
  W.-D. Docke, K. Asadullah, G. Belbe, M. Ebeling, C. Hoflich, M. Friedrich, W. Sterry, and H.-D. Volk Comprehensive biomarker monitoring in cytokine therapy: heterogeneous, time-dependent, and persisting immune effects of interleukin-10 application in psoriasis J. Leukoc. Biol., March 1, 2009; 85(3): 582 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 D. Spight, B. Zhao, M. Haas, S. Wert, A. Denenberg, and T. P. Shanley Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L251 - L265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Al-Mohanna, S. Saleh, R. S. Parhar, and K. Collison IL-12-dependent nuclear factor-{kappa}B activation leads to de novo synthesis and release of IL-8 and TNF-{alpha} in human neutrophils J. Leukoc. Biol., November 1, 2002; 72(5): 995 - 1002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bovolenta, E. Pilotti, M. Mauri, B. Panzeri, M. Sassi, P. Dall'Aglio, U. Bertazzoni, G. Poli, and C. Casoli Retroviral Interference on STAT Activation in Individuals Coinfected with Human T Cell Leukemia Virus Type 2 and HIV-1 J. Immunol., October 15, 2002; 169(8): 4443 - 4449. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Crepaldi, L. Silveri, F. Calzetti, C. Pinardi, and M. A. Cassatella Molecular basis of the synergistic production of IL-1 receptor antagonist by human neutrophils stimulated with IL-4 and IL-10 Int. Immunol., October 1, 2002; 14(10): 1145 - 1153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bovolenta, E. Pilotti, M. Mauri, M. Turci, P. Ciancianaini, P. Fisicaro, U. Bertazzoni, G. Poli, and C. Casoli Human T-cell leukemia virus type 2 induces survival and proliferation of CD34+ TF-1 cells through activation of STAT1 and STAT5 by secretion of interferon-gamma and granulocyte macrophage-colony-stimulating factor Blood, January 1, 2002; 99(1): 224 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Crepaldi, S. Gasperini, J. A. Lapinet, F. Calzetti, C. Pinardi, Y. Liu, S. Zurawski, R. de Waal Malefyt, K. W. Moore, and M. A. Cassatella Up-Regulation of IL-10R1 Expression Is Required to Render Human Neutrophils Fully Responsive to IL-10 J. Immunol., August 15, 2001; 167(4): 2312 - 2322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Xu, T. Yen, and C. L. Geczy IL-10 Up-Regulates Macrophage Expression of the S100 Protein S100A8 J. Immunol., May 15, 2001; 166(10): 6358 - 6366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Elbim, H. Reglier, M. Fay, C. Delarche, V. Andrieu, J. El Benna, and M.-A. Gougerot-Pocidalo Intracellular Pool of IL-10 Receptors in Specific Granules of Human Neutrophils: Differential Mobilization by Proinflammatory Mediators J. Immunol., April 15, 2001; 166(8): 5201 - 5207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Lapinet, P. Scapini, F. Calzetti, O. Perez, and M. A. Cassatella Gene Expression and Production of Tumor Necrosis Factor Alpha, Interleukin-1beta (IL-1beta ), IL-8, Macrophage Inflammatory Protein 1alpha (MIP-1alpha ), MIP-1beta , and Gamma Interferon-Inducible Protein 10 by Human Neutrophils Stimulated with Group B Meningococcal Outer Membrane Vesicles Infect. Immun., December 1, 2000; 68(12): 6917 - 6923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Okayama, A. S. Kirshenbaum, and D. D. Metcalfe Expression of a Functional High-Affinity IgG Receptor, Fc{gamma}RI, on Human Mast Cells: Up-Regulation by IFN-{gamma} J. Immunol., April 15, 2000; 164(8): 4332 - 4339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bovolenta, L. Camorali, A. L. Lorini, S. Ghezzi, E. Vicenzi, A. Lazzarin, and G. Poli Constitutive Activation of STATs Upon In Vivo Human Immunodeficiency Virus Infection Blood, December 15, 1999; 94(12): 4202 - 4209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Cassatella, S. Gasperini, C. Bovolenta, F. Calzetti, M. Vollebregt, P. Scapini, M. Marchi, R. Suzuki, A. Suzuki, and A. Yoshimura 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 Blood, October 15, 1999; 94(8): 2880 - 2889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Thivierge, J.-L. Parent, J. Stankova, and M. Rola-Pleszczynski Modulation of Formyl Peptide Receptor Expression by IL-10 in Human Monocytes and Neutrophils J. Immunol., March 15, 1999; 162(6): 3590 - 3595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bovolenta, A. L. Lorini, B. Mantelli, L. Camorali, F. Novelli, P. Biswas, and G. Poli A Selective Defect of IFN-{gamma}- But Not of IFN-{alpha}-Induced JAK/STAT Pathway in a Subset of U937 Clones Prevents the Antiretroviral Effect of IFN-{gamma} Against HIV-1 J. Immunol., January 1, 1999; 162(1): 323 - 330. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
