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INTRODUCTION |
Interleukin-10 (IL-10)1
is a cytokine produced by Th0 and Th2 CD4+ T cells,
CD5+ B cells, thymocytes, ketatinocytes, and macrophages
(1-5) that regulates the function and/or development of both lymphoid
and myeloid cells (2, 6, 7). One of the most unique actions of this
cytokine is its ability to inhibit production of pro-inflammatory cytokines, such as tumor necrosis factor
(TNF
), IL-1, and IL-12, which are synthesized by macrophages in response to bacterial products,
such as lipopolysaccharide (LPS). This activity results in decreased
IFN
production and inhibition of cell-mediated immune responses
while concomitantly enhancing humoral immunity (6, 8-10).
IL-10 exerts its biologic effects on cells by interacting with a
specific cell surface receptor (1, 11). Functionally active IL-10
receptors are composed of two distinct subunits. Both subunits belong
to the class II cytokine receptor family that also contains the
receptors for IFN
and IFN
(12). The IL-10 receptor
chain is a
110-kDa polypeptide that plays the dominant role in mediating high
affinity ligand binding and signal transduction (1, 11). The IL-10
receptor
subunit (also known as CRF2-4) is predicted to be a
40-kDa polypeptide that is largely required only for signaling (13,
14).
Engagement of the IL-10 receptor has been shown to activate the
JAK-STAT signaling pathway. Specifically, IL-10 effects the activation
of Jak1 (associated with the IL-10 receptor
chain) and Tyk2
(associated with the IL-10 receptor
chain) and induces the
activation of Stat1, Stat3, and, in some cells, Stat5 (15-19). Previous work from our laboratory using cells from mice with disrupted genes for Jak1 and Stat1 has revealed that the characteristic ability
of IL-10 to inhibit TNF
production in LPS-stimulated macrophages
(i.e. anti-inflammatory actions of IL-10) displays an
obligate dependence on Jak1 but does not require the presence of Stat1
(20, 21). Thus, whereas the Janus kinases are clearly required for
promoting the anti-inflammatory effects of IL-10, it remains uncertain
which STAT protein, if any, is involved in mediating these unique
IL-10-induced responses.
Other studies have suggested that Stat3 participates in manifesting at
least some of the biologic effects of IL-10 on B cells and macrophages.
First, Stat3 is directly recruited to two redundant YXXQ
sequences in the intracellular domain of the IL-10 receptor following
ligand binding (18, 22). Receptor mutants lacking these two sequences
fail to activate Stat3 and fail to promote a variety of
IL-10-dependent responses when expressed in the Ba/F3 pro-B
cell line (16, 18). Second, overexpression of a dominant-negative Stat3
mutant protein in the J774 murine macrophage cell line inhibited IL-10-dependent antiproliferative responses and partially
blocked IL-10-induced expression of the CD32/16 Fc
receptor (23).
Importantly, these latter developmental responses are also effected by
other cytokines that activate Stat3, such as IL-6. However, these same studies failed to find a blocking effect of mutant Stat3 proteins on
the anti-inflammatory actions of IL-10. Thus, the existing data suggest
either that the IL-10 receptor utilizes different signaling mechanisms
to manifest anti-inflammatory versus developmental effects
or that different IL-10-induced biologic responses in cells display
differential requirements for Stat3 activation.
Herein we demonstrate that 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. These results thus unequivocally establish the
requirement of Stat3 for the anti-inflammatory functions of IL-10 in
primary cells. Using gain-of-function and loss-of-function receptor
mutants, we also define the functionally critical regions on the IL-10
receptor that regulate developmental versus
anti-inflammatory functions of this cytokine. The developmental functions map to IL-10 receptor regions that are critical for Stat3
recruitment and that are shared by receptors for other cytokines that
activate Stat3, such as IL-6. In contrast, the anti-inflammatory function of IL-10 displays the additional requirement for a
carboxyl-terminal 30-amino acid sequence in the intracellular domain of
the receptor that contains at least one functionally important serine
residue. Thus, the unique anti-inflammatory functions of IL-10 can be
explained by distinctive receptor intracellular domain sequences that
are not shared by other Stat3 activating cytokine receptors.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant murine IL-10 was generated as
described previously (24). Purified recombinant human and murine IFN
were generously provided by Genentech, Inc. (South San Francisco, CA).
Purified recombinant murine IL-3 and IL-6 were purchased from Genzyme
(Cambridge, MA). M-CSF were obtained from R&D Systems (Minneapolis,
MN). Lipopolysaccharide (LPS) derived from Escherichia coli
0127:B8 was purchased from Difco (Detroit, MI).
Antibodies--
GIR-208 is a murine IgG1 monoclonal antibody
specific for the human IFN
receptor (25), and 9E10 is a murine IgG1
monoclonal antibody specific for a 13-amino acid peptide tag derived
from the human c-Myc protein (SMEQKLISEEDLN) (26). These two antibodies were purified and conjugated to biotin using the Enzo biotinylating reagent (Enzo Biochem, Inc.) as described (25).
Primers--
The primers listed below were synthesized on an
Oligo 1000 DNA synthesizer (Beckman, Fullerton, CA) and were based on
the nucleotide sequence of either the human IFN
cDNA (27) or the murine IL-10 receptor cDNA (11): primer 100741, 5'-AACATACAGAAGAACTTTCTAG-3'; primer 100341, 5'-CCTGGAATGTCACCATGACGGCTTTTATTACGGTTATGAG-3'; primer 100242, 5'-CTCATAACCGTAATAAAAGCCGTCATGGTGACATTCCAGG-3'; primer 100141, 5'-CATGCCAAGCTTCTAGATTATTCTTCTACCTGCAGGC-3'; primer 5393, 5'-GTCTGTTTCTGGAAGCCCTGGAATGTCACC-3'; primer 5394, 5'-TTCCAGGGCTTCCAGAAACAGACCAGATGG-3'; primer 5395, 5'-CTCCTGTTTCAAGAAACCTGCGGCCAGAGC-3'; primer 5396, 5'-TGGCCGCAGGTTTCTTGAAACAGGAGTCTC-3'; primer 38436, 5'-CATGCCAAGCTTCTAGATTAAGAGCCAAGGCTATCCAGG-3'; primer 38437, 5'-CATGCCAAGCTTCTAGATTACAGAGGGTCAAGTTTATGG-3'; primer 38438, 5'-CATGCCAAGCTTCTAGATTAATCTTCACAGCTAACCACACC-3'; primer 38439, 5'-CATGCCAAGCTTCTAGATTATTGATTCCACTGTCTACTTGG-3' and primer 373356, 5'-TACCCAAGCTTGGGTCATTCTT
CTACCTGCAGGGCGGCGATCAACGGCAGGGTGACCAGGTTAGCGCCAAGGGCATCCAGGAGGCC-3'.
Plasmid Construction--
Plasmids were constructed using
standard procedures (28). The hgr
/IL-10R
chimeric receptor
(hgr
1-434aa/muIL-10R
420-559aa) was created through the use of a
two-step polymerase chain reaction as described previously (29).
Primers 100741 and 100141 were used as the 5'-and 3'-primers, whereas
primers 100341 and 100242 were the internal primers used to generate
the chimeric receptor. The chimeric receptor point mutants
(hgr
/IL-10R
Y427F, hgr
/IL-10R
Y477F, and
hgr
/IL-10R
Y427F/Y477F) were also generated through the use of a
two-step polymerase chain reaction. All chimeric receptor point mutants
utilized primers 100741 and 100141 as the 5'- and 3'-primers.
hgr
/IL-10R
Y427F was constructed using primers 5393 and 5394 as
the internal primers. hgr
/IL-10R
Y477F was created using internal
primers 5395 and 5396. In both cases, a Bluescript plasmid (Stratagene,
La Jolla, CA) that contained the wild type hgr
/IL-10R
chimeric
receptor was used as template. hgr
/IL-10R
Y427F/Y477F was
generated using internal primers 5395 and 5396. A Bluescript plasmid
that contained hgr
/IL-10R
Y427F was used as template. We generated
a series of hgr
/IL-10R
chimeric receptor truncation mutants by
serially truncating 15 amino acids from the carboxyl terminus of the
wild type hgr
/IL-10R
chimeric receptor. All of the receptor
truncation mutants were generated using primer 100741 as the 5'-primer.
The 3'-primers used to construct hgr
/IL-10R
1-4 were 38436, 38437, 38438, and 38439 respectively. The chimeric receptor serine to
alanine mutant (hgr
/IL-10R
S541A/S544A/S553A/S554A) was generated
using primers 100741 and 373356 as the 5'- and 3'-primers, respectively. All previously described constructs were digested with
SmaI/HindIII and subcloned into a Bluescript
plasmid that contained the wild type human IFN
receptor
chain
digested with the same enzymes. All constructs were then subcloned into
the pSR
expression vector. The accuracy of all polymerase chain
reaction-generated DNA was confirmed by automated sequencing
(Perkin-Elmer Corp.).
Cells and Cell Culture--
RAW264.7 cells, a murine
monocyte-macrophage cell line, were obtained from American Type Culture
Collection (Manassas, VA) and maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, 2%
L-glutamine, 1 mM sodium pyruvate, 50 units/ml
penicillin, and 50 µg/ml streptomycin. Ba/F3 cells, a murine pro-B
cell line, were generously provided by Kevin Moore (DNAX, Palo Alto,
CA) and maintained in RPMI 1640 medium supplemented with 10% fetal
calf serum, 1% L-glutamine, 1 mM sodium
pyruvate, 50 units/ml penicillin, 50 µg/ml streptomycin, and 10 ng/ml
muIL-3. Primary macrophages derived from fetal liver or bone marrow
were prepared using CSF-1 as described (21). Cells were then placed in
culture for two days in D-10 supplemented with 5% heat inactivated horse serum. Bone marrow macrophages derived from Stat3-deficient mice
were >99% positive for MAC-1 and Fc
receptor as determined by flow cytometry.
DNA Transfection--
Cells (1 × 107) were
transfected with 50 µg of plasmid by electroporation at 320 V and 960 µF (RAW264.7 cells) or at 400 V and 960 µF (Ba/F3 cells) on a
Bio-Rad gene pulser. Cotransfections were carried out using 5 µg of
the pMon1118 plasmid (hygromycin resistance) and 45 µg of the
expression plasmid. Selection with G418 and hygromycin B was begun
48 h after transfection. After selection was completed, cells were
sorted based on their expression of both the hgr
/IL-10R
chimeric
receptor and the human IFN
receptor
chain. Cell lines were
subsequently cloned by limiting dilution. Similar results were obtained
with bulk-transfected populations.
Both RAW264.7 and Ba/F3 cells were stably transfected with a plasmid
(pSR
, which confers neomycin resistance) (30) encoding the wild type
human IFN
receptor
chain (tagged at the amino terminus with a
13-amino acid peptide derived from c-Myc) (30). These cells were then
stably transfected with a plasmid encoding either wild type or mutant
forms of the hgr
/IL-10R
chimeric receptor and the plasmid
pMON1118, which confers hygromycin resistance (30). RAW264.7 cells
transfected with different forms of the chimeric receptor and the human
IFN
receptor
chain were selected on G418 (Life Technologies,
Inc., 1.0 mg/ml active compound) and 0.5 mg/ml of hygromycin B
(Calbiochem). Ba/F3 cells transfected with receptor constructs were
selected using G418 (1.0 mg/ml active compound) and 1.3 mg/ml
hygromycin B.
Demonstration of Receptor Expression in Transfected Cell
Lines--
Flow cytometry for the hgr
/IL-10R
chimeric receptor
and receptor mutants or the human IFN
receptor
chain was
conducted using the biotinylated forms of GIR-208 (anti-human IFN
receptor) and 9E10 (anti-c-Myc peptide) respectively and
streptavidin-phycoerythrin conjugate (Chromoprobe, Redwood City, CA) as
described (31). Cells were analyzed on a Becton Dickinson FACScan.
Electrophoretic Mobility Shift Assay--
Cells (5 × 106) were washed once and then resuspended in 500 µl of
medium. 1000 units/ml murine or human IFN
or 200 ng/ml muIL-10 was
added to the cells and incubated for 7 min at 37 °C. The cells were
analyzed by electrophoretic mobility shift assay as described
previously (32) using an 18-base pair oligonucleotide probe that
contained the gamma response region of the Fc
RI gene.
Ba/F3 Proliferation Assay--
Ba/F3 cells were washed twice in
supplemented RPMI 1640 medium that lacked muIL-3. The cells were seeded
in a 96-well plate at a density of 2 × 104 cells/well
and rested for 3 h in the absence of muIL-3. The cells were then
incubated with varying amounts of huIFN
or muIL-10 in a total volume
of 150 µl of medium. Cells were incubated for 48 h at 37 °C
and an 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
assay was performed as described previously (33). Experiments were
performed at least three times using multiple clones of each
transfected cell line. Similar results were obtained using transfected
bulk populations.
RAW264.7 Bioassay--
RAW264.7 cells were washed once, seeded
in 96-well plates at a density of 5 × 104 cells/well,
and incubated with varying amounts of huIFN
, muIL-6, or muIL-10 in a
total volume of 150 µl supplemented Dulbecco's modified Eagle's
medium. Cells were incubated for 1 h at 37 °C and then treated
with 10 ng of LPS/well. The cells were placed at 37 °C for 24 h, at which point the culture supernatants were harvested, and TNF
levels were quantitated via a TNF
ELISA (34). Experiments were
performed at least three times using multiple clones of each
transfected cell line. Similar results were obtained using transfected
bulk populations.
Peritoneal exudate cells (5 × 104) derived from naive
female BALB/c ByJ mice were plated in each well of a 96-well tissue
culture plate. Cells were allowed to adhere for 3 h. Adherent
peritoneal exudate cells were then washed twice with warm medium and
treated as described above.
Macrophages derived from Stat3-, Stat1-, and Jak1-deficient mice were
cultured as described above. Cells were incubated with different doses
of muIL-10 for 12 h. LPS was added at a final concentration of 2 µg/ml, and the cells were cultured for and additional 24 h.
Culture supernatants were harvested, and TNF
levels were quantitated
via a TNF
ELISA.
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RESULTS |
Stat3 Is Necessary but Not Sufficient for
IL-10-dependent Inhibition of TNF
Production by
Macrophages--
To determine whether Stat3 is required for the
anti-inflammatory actions of IL-10, we monitored the ability of this
cytokine to inhibit LPS-induced TNF
production in macrophages
derived from mice with a genetic deficiency of Stat3 targeted to
myeloid cells (35). IL-10 treatment of wild type macrophages resulted in a dose-dependent inhibition of LPS-induced TNF
production that reached maximal levels at a 1 ng/ml dose of IL-10 (Fig.
1). In contrast, macrophages derived from
mice with a myeloid cell Stat3 deficiency were unable to inhibit
LPS-induced TNF
production at any dose of IL-10 used. As additional
controls, macrophages from Jak1
/
and
Stat1
/
mice were also tested in these experiments for
IL-10 sensitivity (21, 36). In agreement with our previous reports (20,
21), Jak1
/
macrophages were unresponsive to IL-10,
whereas the IL-10 response in Stat1
/
macrophages was
indistinguishable from that of wild type mice. Thus, Stat3, together
with Jak1, is obligatorily required for IL-10-dependent
inhibition of TNF
production in LPS-stimulated macrophages.

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Fig. 1.
Stat3 and Jak1 but not Stat1 are required for
IL-10-dependent inhibition of TNF
production by macrophages. Macrophages derived from wild
type, Stat1 / , Stat3 / , and
Jak1 / mice were pretreated with varying concentrations
of muIL-10 for 12 h. The cells were treated with 2 µg/ml LPS and
incubated for an additional 24 h. Supernatants were harvested, and
TNF levels were quantitated via ELISA. Percentage of inhibition of
TNF production upon IL-10 stimulation is indicated.
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Whereas these results demonstrated that Stat3 was required for
mediating the anti-inflammatory actions of IL-10, they did not reveal
whether it was sufficient to induce these functions. To address this
issue, we compared the anti-inflammatory action of IL-10 to that of
IL-6, a cytokine that utilizes a distinct receptor system but that also
activates Stat3 and Jak1. Pretreatment of RAW264.7 cells with
increasing doses of IL-10 resulted in a dose-dependent
inhibition of TNF
production, reaching a maximal level of 75% at a
dose of 200 ng of the cytokine (Fig.
2A). In contrast, RAW264.7
cells pretreated with IL-6 were only slightly inhibited in their
ability to produce TNF
(maximal level of inhibition, 30% at a
200-ng dose of IL-6). We also compared the anti-inflammatory effects of
IL-10 and IL-6 on primary murine macrophages. Here again, whereas IL-10
displayed potent inhibitory activity on TNF
production, IL-6 did not
(Fig. 2B). These functional differences could not be
ascribed to different levels of Stat3 activation because the kinetics
and ultimate magnitude of Stat3 activated by IL-10 and IL-6 in either
RAW264.7 cells or primary macrophages was identical (data not shown).
Thus, the JAK-STAT pathway is required but not sufficient for
IL-10-dependent inhibition of LPS-induced TNF
production.

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Fig. 2.
The JAK/STAT pathway is necessary but not
sufficient for the anti-inflammatory actions of IL-10.
A, RAW264.7 cells were pretreated with varying doses of
either muIL-10 or muIL-6 for 1 h. Subsequently, the cells were
treated with 60 ng/ml LPS and incubated for 24 h. Supernatants
were harvested, and TNF levels were quantitated by ELISA. The graph
indicates percentage of inhibition of TNF production. B,
peritoneal exudate cells were isolated from naive female BALB/c ByJ
mice. Cells were plated and allowed to adhere for 4 h. The cells
were then washed once and pretreated for 1 h with various doses of
either muIL-10 or muIL-6. The cells were stimulated with LPS (60 ng/ml)
for 24 h. TNF levels in the supernatant were quantitated by
ELISA.
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Demonstration That IL-10-dependent Biologic Responses
in Macrophages and B Cells Can Be Manifest by a Gain of Function
hgr
/IL-10R
Chimeric Receptor--
To identify functionally
critical amino acid residues within the intracellular domain of the
muIL-10 receptor ligand binding chain involved in mediating the
anti-inflammatory versus developmental effects of IL-10, we
generated a chimeric receptor that consisted of the human IFN
receptor
chain extracellular and transmembrane domains and the
first 184 amino acids of the intracellular domain (i.e.
truncated just above the Stat1 docking site) attached to a 140-amino
acid region of the murine IL-10 receptor carboxyl terminus, which
contains the Stat3 docking sites (Fig.
3). The resulting chimeric receptor thus
consists of the human IFN
R
chain that retained the Jak1 binding
site and lacked the Stat1 docking site but now contained the two
redundant IL-10 receptor Stat3 docking sites. The chimeric polypeptide
was then expressed in murine RAW264.7 macrophages or Ba/F3 pro-B cells
that had been engineered to also stably express the human IFN
receptor
chain (Table I).

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Fig. 3.
Generation of a chimeric cytokine receptor
containing portions of the human IFN
receptor chain and the murine IL-10
receptor ligand binding chain. To generate the hgr /IL-10R
chimeric receptor, we truncated the human IFN receptor chain
(IFNGR1) at position 435 (i.e. five amino acids prior to
Tyr-440, the Stat1 docking site) and attached to it a 140-amino acid
region of the IL-10 receptor consisting of residues 420-559, which
contains the Stat3 recruitment sites. Thus, the resulting chimeric
receptor retains the Jak1 binding site, lacks the Stat1 docking site,
and now contains the two redundant Stat3 docking sites from the muIL-10
receptor. All cells transfected with the chimeric receptor were
cotransfected with the human IFN receptor chain (IFNGR2).
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Engagement of the endogenous IL-10 receptor on RAW264.7 cells with
muIL-10 led to a dose-dependent inhibition of TNF
production in LPS-treated cells (Fig.
4A). HuIFN
has no effect on
these cells because of the strict species specificity that governs the interaction of IFN
with its receptor. Treatment of RAW264.7 cells expressing the hgr
/IL-10R
chimeric receptor with human IFN
induced a dose-dependent inhibition of
LPS-dependent TNF
production in a manner that was
quantitatively identical to that induced by the activated, endogenous
IL-10 receptor. In contrast, treatment of wild type or transfected
RAW264.7 cells with murine IFN
caused a dose-dependent
increase of TNF
production (data not shown). In a similar manner,
Ba/F3 engineered to express the wild type muIL-10 receptor proliferated
in the presence of muIL-10 (Fig. 4B) but did not respond to
human IFN
. In contrast, Ba/F3 cells bearing the hgr
/IL-10R
chimeric receptor proliferated upon exposure to human IFN
. Thus,
transfer of IL-10 receptor sequences containing the Stat3 recruitment
sites to the IFN
receptor results in the generation of a modified
IFN
receptor that promotes IL-10-dependent rather than
IFN
-dependent responses in murine macrophages and B
cells exposed to human IFN
.

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Fig. 4.
Demonstration that
IL-10-dependent biologic responses in macrophages and B
cells can be manifest by a gain of function
hgr /IL-10R chimeric
receptor. A, RAW264.7 cells bearing the wild type IL-10
receptor, the intact chimeric receptor, or distinct chimeric receptor
intracellular domain mutants were pretreated with various doses of
IL-10 or huIFN for 1 h and subsequently treated with 60 ng/ml
of LPS for 24 h. Again, TNF concentrations were determined by
ELISA. The graph depicts percentage of inhibition of LPS-induced TNF
production. B, Ba/F3 cells bearing the wild type IL-10
receptor, the intact chimeric receptor, or distinct chimeric receptor
intracellular domain mutants were were rested in medium lacking muIL-3
for 3 h. The cells were then treated with increasing doses of
either muIL-10 or huIFN , respectively. The cells were incubated for
48 h, and subsequently, 50 µl of 3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide at a concentration of
2.5 mg/ml was added to the cells. The cells were then incubated at
37 °C for 2 h. The dark blue formazan crystals were dissolved
by adding 100 µl of lysis solution. The figure represents viable
cells as determined by A570.
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Tyrosine Residues 427 and 477 within the Intracellular Domain of
the Chimeric hgr
/IL-10R
Are Redundantly Required for the
Development of IL-10-like Effects on Macrophages and B Cells--
In a
previous report, we showed that tyrosine residues at positions 427 and
477 in the intact murine IL-10 receptor played redundant roles in
recruiting Stat3 to the activated receptor (18). In addition, we found
that Stat3 recruitment was required for development of IL-10-induced
proliferative responses (i.e. developmental responses) in B
cells. To examine whether the anti-inflammatory functions of IL-10 also
show the same requirement for these Stat3 recruitment sites and to test
whether the functionally redundant tyrosines act in a similar manner in
the chimeric receptor, we generated a set of tyrosine to phenylalanine
mutant chimeric receptors, expressed them in macrophages and B cells
(Table I) and examined their capacity to promote IL-10 like biologic
effects on the transfected cells.
RAW264.7 cells stably expressing single Tyr
Phe chimeric receptor
mutants (hgr
/IL-10R
Y427F or hgr
/IL-10R
Y477F) were responsive to huIFN
and produced approximately 80% less TNF
than
controls when challenged with LPS (Fig. 4A). Thus, the
receptors containing single tyrosine-directed point mutations behaved
in a manner that was analogous to the chimeric receptor containing wild
type IL-10 receptor sequences. These mutants also activated Stat3 upon
binding ligand (data not shown). In contrast, RAW264.7 cells expressing
a chimeric receptor that lacked both Tyr-427 and Tyr-477
(hgr
/IL-10R
Y427F/Y477F) were unresponsive to huIFN
(Fig.
4A) and failed to activate Stat3 (data not shown).
Similar results were obtained when the three mutant chimeric receptor
proteins were expressed in Ba/F3 cells. Ba/F3 cells expressing either
chimeric receptor point mutant proliferated in response to huIFN
(Fig. 4B) and activated Stat3 (data not shown) in a manner
that was comparable to or greater than that of the unaltered chimeric
receptor. In contrast cells bearing the double point mutant chimeric
receptor failed to manifest proliferative responses to huIFN
and
failed to activate Stat3. Thus, the IL-10 receptor residues Tyr-427 and
Tyr-477 function in a redundant manner to induce both IL-10 like
anti-inflammatory responses in macrophages as well as developmental
responses in B cells. These results demonstrate that the chimeric
receptor faithfully recapitulates the known functions of the
full-length IL-10 receptor and thereby validates the use of the
chimeric receptor as a suitable experimental model system.
Distinct Residues within the Carboxyl Terminus of the IL-10
Receptor Are Required to Inhibit LPS-induced TNF
Production but Not
for the Induction of an IL-10 Proliferative Response--
The data
presented this far show that the JAK-STAT signaling pathway and
specifically Stat3 and Jak1 are required for induction of
IL-10-dependent anti-inflammatory and developmental
responses in macrophages and B cells. However, they did not reveal a
mechanism by which IL-10 could uniquely manifest its anti-inflammatory
effects. Thus, at least one signal in addition to IL-10 must arise from the activated IL-10 receptor in stimulated macrophages. To explore this
possibility, we generated a family of progressive deletion mutants of
the hgr
/IL-10R
chimeric receptor such that each sequential mutant
was 15 amino acids shorter at the carboxyl terminus compared with its
predecessor. Each receptor was expressed in RAW264.7 and Ba/F3 cells
(Table I and Fig. 3). Expression of each receptor in the resulting
stably transfected cell populations was confirmed by FACS analysis, and
several clones of each transfected cell line were chosen for further
analysis based on their expression of comparable levels of receptor.
HuIFN
treatment of RAW264.7 cells bearing either intact
hgr
/IL-10R
or the chimeric receptor lacking the most
carboxyl-terminal 15 residues (hgr
/IL-10R
1) produced a
dose-dependent inhibition of LPS-stimulated TNF
production (Fig. 5A). However,
RAW264.7 cells bearing receptor mutants lacking additional
carboxyl-terminal sequence (hgr
/IL-10R
2-hgr
/IL-10R
4) were unable to mediate this response. In contrast, all of the receptor
carboxyl-terminal deletion mutants were able to support IL-10-dependent proliferative responses in Ba/F3 cells
(Fig. 5B). Thus, additional residues within the carboxyl
terminus are used by the IL-10 receptor to inhibit TNF
production in
macrophages but not proliferative responses in B cells.

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Fig. 5.
Distinct residues within the carboxyl
terminus of the IL-10 receptor are required to inhibit LPS-induced
TNF production but not for the induction of an
IL-10 proliferative response. A, RAW264.7 cells bearing
the wild type muIL-10 receptor, the intact hgr /IL-10R chimeric
receptor, or the chimeric receptor carboxyl-terminal truncation mutants
were pretreated with various doses of muIL-10 or huIFN for 1 h.
The cells were then treated with 60 ng/ml LPS and incubated for an
additional 24 h. TNF concentrations were quantified via ELISA.
This panel depicts percentage of inhibition of LPS-induced TNF
production. B, Ba/F3 cells bearing the wild type muIL-10
receptor, the intact hgr /IL-10R chimeric receptor, or the
chimeric receptor carboxyl-terminal truncation mutants were rested in
medium lacking muIL-3 for 3 h. The cells were then treated with
increasing doses of either muIL-10 or huIFN . The cells were
incubated for 48 h, and subsequently, 50 µl of 3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide at a concentration of
2.5 mg/ml was added to the cells. The cells were then incubated at
37 °C for 2 h. The dark blue formazan crystals were dissolved
by adding 100 µl of lysis solution. The figure represents viable
cells as determined by A570.
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The basis for the activity differences observed with the various
deletion mutants in RAW264.7 versus Ba/F3 cells was not due to differences in Stat3 activation. As determined by electrophoretic mobility shift assay analysis, huIFN
induced comparable levels of
Stat3 homodimers (slower migrating band) and Stat1:Stat3 heterodimers (faster migrating band) in each cell type regardless of the presence of
the carboxyl-terminal 60 amino acids of the chimeric receptor (Fig.
6, A and B).

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Fig. 6.
Carboxyl-terminal chimeric receptor
truncation mutants maintain huIFN -induced STAT
activation. A, RAW264.7 cells bearing the wild type
murine IL-10 receptor, the intact chimeric receptor or chimeric
receptor truncation mutants were stimulated with medium, 200 ng/ml
muIL-10, or 25 ng/ml huIFN for 7 min at 37 °C. The cells were
then lysed, and the nuclear extracts were generated. 5 µg of extract
was incubated with the gamma response region of the Fc RI gene probe
for 30 min. The protein/DNA binding complexes were imaged by
audioradiography. B, Ba/F3 cells bearing the wild type
murine IL-10 receptor, the intact chimeric receptor, or chimeric
receptor truncation mutants were treated as described above, and the
protein/DNA binding complexes were imaged by audioradiography.
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Serine Residues within the Carboxyl Terminus of the muIL-10
Receptor Are Required for Inhibition of TNF
Production by
Macrophages--
In an attempt to identify specific amino acids in the
carboxyl-terminal region of the IL-10 receptor that are required for inhibition of TNF
production, we generated one final
hgr
/IL-10R
chimeric receptor mutant in which all of the
carboxyl-terminal serine residues (i.e. those residing at
positions 541, 544, 553, and 554) were mutated to alanines and stably
expressed it in RAW264.7 cells (Table I). HuIFN
treatment of
RAW264.7 cells bearing this mutant failed to inhibit LPS-mediated
TNF
production. The minimal levels of TNF
inhibition manifest by
this mutant were indistinguishable from those produced by IL-6 (Fig.
7A). Electrophoretic mobility shift assay analysis showed comparable Stat3 activation by huIFN
treated RAW264.7 cells bearing either full-length chimeric receptor or
the receptor lacking the carboxyl-terminal serines (Fig.
7B). Thus, IL-10-dependent inhibition of
LPS-induced TNF
production in macrophages requires the concomitant
presence of Stat3 docking sites on the receptor and a restricted
carboxyl-terminal portion of the receptor that contains at least one
functionally important serine residue.

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Fig. 7.
Serine residues within the carboxyl terminus
of the muIL-10 receptor are required for inhibition of
TNF production by macrophages.
A, RAW264.7 cells bearing the wild type murine IL-10
receptor, the intact chimeric receptor, or chimeric receptor serine to
alanine mutant were pretreated with various doses of muIL-10, muIL-6,
or huIFN . The cells were then treated with 60 ng/ml LPS and
incubated for an additional 24 h. TNF concentrations were
quantified via ELISA. This panel depicts percentage of inhibition of
LPS-induced TNF production. B, an electrophoretic
mobility shift assay was performed on these cells. 5 µg of extract
was incubated with the gamma response region of the Fc RI gene probe
for 30 min. The protein-DNA binding complexes were imaged by
audioradiography.
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 |
DISCUSSION |
The focus of this study was to characterize the mechanism that
underlies the selective ability of IL-10 to inhibit TNF
production by LPS-stimulated macrophages. Using cells from mice with a genetic Stat3 deficiency in the myeloid cell compartment, we demonstrated that
Stat3 is obligatorily required for expression of the inhibitory functions of IL-10 on TNF
production. Taking into account our previous study (21), which showed an obligate role for Jak1 in this
process, we can now unequivocally conclude that the JAK-STAT signaling
pathway is required for expression of the anti-inflammatory actions of
IL-10. In addition, we find that the developmental versus
anti-inflammatory actions of IL-10 are distinguishable by their
requirements for different regions of the IL-10 receptor
chain
intracellular domain. For the former, the functionally critical
receptor region maps selectively to a redundant set of Stat3 docking
sites residing at positions 427-430 and 477-480. In contrast, for the
latter, the additional presence on the receptor of a carboxyl-terminal
sequence that contains at least one functionally important serine
residue is also required. Our results thus provide a molecular
explanation for the unique biologic actions of IL-10 that are not
expressed by other cytokine receptors, such as the IL-6 receptor, that
activate similar patterns of JAKs and STATs.
Based on previous work from our laboratory and others (15, 16, 18), the
IL-10 receptor was known to activate Stat3, Jak1 and Tyk2. At least two
of these signaling components (i.e. Stat3 and Jak1) are also
activated by other cytokine receptors, such as those that belong to the
IL-6 receptor family. In fact, IL-6 induces many of the same biologic
responses in cells as are induced by IL-10. Specifically, both induce
proliferative responses in B cells and antiproliferative responses in
macrophages as well as inducing expression of a variety of cell surface
markers on these cells, such as HSA and CD32/16, respectively (37-42).
Induction of these functions has been shown to require Stat3
recruitment and activation by the ligand assembled IL-6 and IL-10
receptors. In contrast, as shown in the current study, the ability of
IL-10 to inhibit LPS-induced TNF
production in macrophages is not
significantly shared by IL-6. Thus the latter represents a biologic
response that is IL-10-specific.
Based on this argument, we considered two possibilities that could
explain the distinctive actions of IL-10. The first is that the
JAK-STAT signaling pathway is required but not sufficient for
expression of IL-10-dependent anti-inflammatory actions and that at least one additional signal must be concomitantly delivered to
the cell through the ligated IL-10 receptor. The second is that the
anti-inflammatory actions of this cytokine are induced through a
different signaling pathway. The latter possibility has been ruled out
by the current study, which shows that the ability of IL-10 to inhibit
LPS-induced TNF
production in macrophages requires the concomitant
presence in cells of Stat3 and Jak1. Moreover, as presented elsewhere
(35), mice lacking Stat3 in the myeloid compartment display enhanced
spontaneous inflammatory responses similar to those observed in mice
lacking either IL-10 (43) or the CRF2-4 component of the IL-10
receptor (13). These mice develop chronic enterocolitis and exhibit
exaggerated Th1 responses characterized by a 4-5-fold increase in
IFN
production as compared with control animals. Thus, taken
together, these results show that Stat3 is required for expression of
IL-10 anti-inflammatory functions both in vitro and in
vivo. In addition, the data presented in the current manuscript
directly support the possibility that an accessory signaling pathway in
addition to the JAK-STAT pathway is required for expression of the
anti-inflammatory actions of IL-10. Specifically we show that a second
region of the IL-10 receptor intracellular domain is required in
addition to the Stat3 recruitment sites on the receptor to mediate the
capacity of IL-10 to inhibit LPS-induced TNF
production in macrophages.
This conclusion is thus in conflict with that of O'Farrell et
al. (25), who reported that IL-10-dependent inhibition
of TNF
production occurs in a Stat3-independent manner. The
conclusions reached in the latter study were derived from two types of
experiments. First, macrophages were engineered to express a tagged
form of Stat3 containing the bacterial gyrase B protein (gyrB-Stat3)
that could be inducibly dimerized within the cell following addition of
the antibiotic coumermycin (44). Whereas this manipulation led to
inhibition of macrophage proliferation, it did not effect LPS-induced
TNF
production. However, based on the data presented in our study,
we now know that Stat3 is required but not sufficient for induction of
the anti-inflammatory actions of IL-10, and thus the coumermycin
experiments delivered only one of the two signals needed to inhibit
TNF
production. Second, J774 macrophages were engineered to
overexpress a dominant negative mutant form of Stat3 lacking the
carboxyl terminus. Whereas these cells became either totally or
partially insensitive to many of the actions of IL-10, they still
displayed IL-10-induced inhibition of LPS-dependent TNF
production. However, the cells that overexpressed mutant Stat3 were not
completely blocked in their capacity to activate (i.e.
phosphorylate) their own endogenously expressed Stat3. Thus, the lack
of effect of the dominant negative Stat3 mutant on the anti-inflammatory functions of IL-10 may reflect the incomplete blockade of Stat3 activation and different threshold requirements for
activated Stat3 for induction of various biologic responses. It is also
possible that for induction of IL-10 anti-inflammatory responses, Stat3
may function as an adapter protein that facilitates recruitment of
another signaling component to the activated receptor. In this case, a
Stat3 mutant that could not be tyrosine-phosphorylated maintained a
functional SH2 domain but could, through its ability to bind to the
activated IL-10 receptor, still be able to fulfill a role as an adapter protein.
A key approach used in our present study was the employment of a
gain-of-function chimeric cytokine receptor in which the murine IL-10
receptor Stat3 recruitment sites and carboxyl-terminal region were
substituted for the Stat1 recruitment site on the human IFN
receptor. We specifically chose the human IFN
receptor
chain as
the recipient of the IL-10 receptor sequence for three reasons. First,
the
chains of the IFN
receptor and the IL-10 receptor belong to
the same class II cytokine receptor family. Second, although the
polypeptides belong to the same receptor family, they differ not only
in the STATs they recruit but also in the biologic responses they
induce. Third, the use of the huIFN
receptor extracellular domain
allowed us to take advantage of the strict species-specific receptor
binding activity exhibited by human and murine IFN
, thus providing
us with a mechanism to selectively monitor the activity of the chimeric
receptor expressed on murine cells through the addition of
huIFN
.
Based on the data presented herein, we envision two possible models by
which the different regions of the IL-10 receptor may function. In the
first, the receptor may deliver two distinct signals that arise
independently through interaction of different proteins with the two
functionally important regions of the receptor intracellular domain. In
this model, one of these signals is Stat3, which binds to the Stat3
docking sites on the IL-10 receptor, becomes tyrosine-phosphorylated,
dimerizes, and then translocates to the nucleus, where it functions as
a transcriptional activator. The second signal is then derived from an
additional protein that docks at the carboxyl terminus of the IL-10
receptor, possibly by binding to serine or phosphoserine residues. In
the second model, the association of Stat3 with the ligand-induced
Stat3 docking site on the receptor may result in the formation of a binding site for another protein that also requires interaction with
the carboxyl-terminal sequence of the IL-10 receptor. In this model,
Stat3 would function as an adapter protein. An adapter role for Stat3
has been previously reported for signaling by the IFN
/
receptor,
where Stat3 binding to the IFNAR1 provides a binding site on the
receptor for phosphatidylinositol 3-kinase. Our current efforts are
focused on defining the nature of the second signal needed for
induction of IL-10-dependent anti-inflammatory responses
and in determining the mechanism by which it is recruited to the
activated IL-10 receptor.