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*
Laboratory of Immunophysiology and
Integrative Biology, Department of Animal Sciences, and
College of Medicine, Department of Pathology, University of Illinois, Urbana, IL 61801; and
Institut National de la Recherche Agronomique-Institut National de la Santé et de la Recherche Médical, Unité 394, Unité 74 de Recherches de Neurobiologie, Institute François Magendie, Bordeaux, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The heterotetramer IL-10R is composed of two ligand binding subunits
(IL-10R
and IL-10R1) and two accessory signaling subunits (IL-10R
and IL10R2) (14, 15, 16). Following ligand binding to the
IL-10R, Jak1 and Tyk2, which are constitutively associated with IL-10R1
and IL-10R2, respectively (3, 14), are phosphorylated on
tyrosine (17). The phosphorylated IL-10R/Jak1/Tyk2 complex
serves as a docking site for the transcription factors Stat-1 and
Stat-3 (11, 18, 19). Jak1, which is required for the
biological activity of IL-10 (20), also tyrosine
phosphorylates the insulin receptor substrate-1
(IRS-1)3 (21, 22) docking molecule. We and others have shown that activation
of IL-4 (23) and IFN- (24, 25, 26) receptors
stimulates tyrosyl phosphorylation of IRS docking proteins, which
depends upon Jak, rather than Stat, family members (21).
Phosphotyrosine-containing IRS proteins recruit the p85 regulatory
subunit of class Ia phosphatidylinositol 3'-kinases (PI 3-kinase) to
the plasma membrane by binding to its two SH2 domains (reviewed in
Refs. 27, 28), which leads to activation of survival
enzymes such as Akt. Activation of PI 3-kinase by IL-10 is required to
promote cell proliferation, but not for IL-10 to suppress synthesis of
the proinflammatory cytokine TNF- (29). More recently,
IL-10 was shown to promote the survival of astrocytes by a mechanism
that depends upon activation of PI 3-kinase (13).
Although the IL-10R is well accepted to activate the Jak/Stat pathway, it is unknown whether IL-10 can also activate IRS-2, as does IL-4. Here we establish that progenitor myeloid cells express IL-10Rs, and that activation of these receptors inhibits the apoptotic death of myeloid progenitors following withdrawal of survival factors. Stimulation of IL-10Rs activates Jak1, Tyk2, and Stat-3 and leads to tyrosyl phosphorylation of IRS-2. This is accompanied by an increase in the enzymatic activity of both PI 3-kinase and Akt. Inhibition of PI 3-kinase not only blocks the ability of IL-10 to promote the survival of promyeloid cells, but also totally inhibits IL-10-induced the activation of extracellular signal-related kinase (ERK) 1/2. Direct inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK)-1 significantly reduces the anti-apoptotic activity of IL-10. These data establish that IL-10 depends upon activation of an IRS-2/PI 3-kinase/Akt pathway to promote the survival of myeloid progenitors by a downstream mechanism that involves ERK1/2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 (Sigma, St. Louis, MO) was prepared with 2.2 g/l sodium bicarbonate, 100 U/ml penicillin, and 60 µg/ml streptomycin (Sigma). Factor-dependent cell progenitor-1/Mac-1 cells (FDCP; a gift from Dr. L. Rohrschneider, Fred Hutchinson Cancer Center, Seattle, WA) were cultured in RPMI 1640 medium containing 10% heat-inactivated equine serum (HyClone Laboratories, Logan, UT) and 2.5 U/ml recombinant murine IL-3 (BioSource International, Camarillo, CA). N13 microglial cells were a gift from Dr. P. Ricciardi-Castagnoli (University of Milan, Milan, Italy) and were maintained in RPMI 1640 medium containing 10% FBS. All cells were maintained at 37°C at 95% humidity and 7% CO2. In experiments that measured apoptotic populations or intracellular kinase activities, cells were washed three times with serum-free RPMI 1640 and incubated with growth factors for the indicated times. An azide-free, IgG fraction of a goat anti-IL-10R1 neutralizing Ab (R&D Systems, Minneapolis, MN) or a control goat IgG were cultured with FDCP cells to test the in vivo effects of IL-10 on apoptosis. Specific Abs that were used for immunoprecipitation and Western blotting were as follows: Akt and phospho-Akt (Ser473), Stat-3, and phospho-Stat-3 (Tyr705) Abs were purchased from Cell Signaling (Beverly, MA); anti-IRS-2 and Jak1 Abs were obtained from Upstate Biotechnology (Lake Placid, NY); the anti-IL-10R1 C terminus Ab C-20, and anti-Tyk2, Stat-1, phospho-Stat-1 (Tyr701), ERK1, and phospho-ERK (Tyr204) Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); and the anti-phosphotyrosine mAb PY20 was obtained from Transduction Laboratories (Lexington, KY). Wortmannin was purchased from Sigma, LY294002 was purchased from Alexis Biochemicals (Pittsburgh, PA), and PD98059 was obtained from Cell Signaling.
Measurement of apoptosis by flow cytometry
Flow cytometry (Moflo cytometer; Cytomation, Fort Collins, CO) was used to determine the proportion of Hoechst 33342-positive, propidium iodide (PI; Sigma)-negative, apoptotic FDCP cells, as previously described (30, 31). The cells were incubated in serum-free medium in the presence or the absence of IL-10 (0.1, 1, 10, or 50 ng/ml) or the positive controls, consisting of either IL-3 (25 U/ml) or insulin-like growth factor I (IGF-I; 100 ng/ml). Cells were preincubated with pharmacological inhibitors for 30 min before addition of cytokines. After an additional 11.5-h incubation, 1 ml of cells from each treatment were added to 1.5-ml microfuge tubes. Hoechst 33342 (1 µg/ml; Sigma) was incubated with the cells for 5 min at room temperature, and the tubes were then placed on ice. Immediately before flow cytometry, 1 µg/ml PI was added. A total of 104 cells were analyzed for each sample. Apoptotic FDCP cells were measured as the proportion of cells that excluded PI and were positive for Hoechst 33342 when plotted against the forward angle light scatter of the cells.
The apoptotic population of N13 microglial cells was determined by intracellular flow cytometry using the TUNEL (Phoenix Flow Systems, San Diego, CA) method, as previously described by our laboratory (32). Following fixation and permeabilization of N13 microglial cells, cells were fixed and permeablized, and bromolated dUTP (BRDU) was added in the presence or the absence of TdT. A directly conjugated FITC Ab directed against BRDU was added, and 1 x 104 cells were analyzed on an EPICS XL flow cytometer (Coulter, Miami, FL).
Lipid phosphorylation to quantitate PI 3'-kinase activity
FDCP cells (5 x 107) were cultured
in serum-free medium as described above and then incubated with 50
ng/ml IL-10. Cells were homogenized in 1 ml of lysis buffer containing
1% Nonidet P-40, 50 mM Tris-HCl, 100 mM NaCl, 50 mM NaF, 10 mM
tetrasodium pyrophosphate, 2 mM
Na3VO4, 2.5 mM benzamidine,
and 1 mM PMSF, pH 7.4. Clarified lysates (10 min, 14,000 x
g) were added to 50 µl of a 50% suspension of protein
G-Sepharose (Pharmacia Biotech, Uppsala, Sweden) conjugated with 2 µg
of anti-phosphotyrosine Ab PY-20 (Transduction
Laboratories). Lipid kinase activity was directly measured
in these immunoprecipitates, as we previously described (30, 33). Briefly, the precipitated complex was incubated in a
reaction mixture containing
L--phosphatidylinositol (0.33 mg/ml; Sigma),
20 mM HEPES, 0.4 mM EGTA, 0.4 mM NaPO4, 10 mM
MgCl2, 5 µmol of ATP, and 6 µCi of
[
-32P]ATP/reaction (6,000 Ci/mmol; Amersham,
Arlington Heights, IL). Phosphoinositides were separated by TLC using a
chloroform/methanol/ammonium hydroxide (75/58/17, v/v/v) running
buffer. The plates were then exposed to phosphorimager screens. A
series 400 PhosphorImager using ImageQuant 3.2 software (Molecular
Dynamics, Sunnyvale, CA) was used to quantify the phosphorylation of
-phosphatidylinositol.
Analysis of Akt enzymatic activity
Akt activity was analyzed using a commercially available Akt kit
(Upstate Biotechnology). FDCP cells were cultured in serum-free RPMI
1640 medium for 4 h. The cells (5 x
107) were treated with IL-10 at 50 ng/ml for the
indicated times and lysed as described above. An anti-Akt PH domain
IgG (4 µg; Upstate Biotechnology) was conjugated to protein G-agarose
(25 µl) for 90 min at room temperature. This Ab/protein G conjugate
was incubated with equal amounts of clarified lysates for 90 min at
4°C. Following this immunoprecipitation, the precipitates were washed
three times with 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1 mM EDTA, 1 mM
EGTA, 0.5 mM sodium orthovanadate, and 0.1% 2-ME, followed by two
washes with 100 mM MOPS (pH 7.2), 125 mM -glycerolphosphate, 25 mM
EDTA, 5 mM sodium orthovanadate, and 5 mM DTT. Kinase activity of Akt
in the immunoprecipitates was measured for 10 min at 30°C in 40 µl
of kinase buffer (100 mM MOPS (pH 7.2), 125 mM -glycerolphosphate,
25 mM EGTA, 7.5 mM magnesium chloride, 5 mM sodium orthovandadate, 5 mM
DTT, 10 µM protein kinase A inhibitor, and 50 µM ATP) in the
presence of 10 µCi [-32P]ATP (6000 Ci/mM;
Amersham) and 100 µM Akt-specific peptide substrate. The kinase
reaction was stopped with 20 µl of 40% TCA, followed by transfer of
40 µl of the final reaction volume to p80 cellulose paper. The
phosphocellulose disks were washed three times with 0.75% phosphoric
acid and once with acetone, and 32P that was
incorporated into the substrate was determined in a Beckman LS60001C
scintillation counter (Palo Alto, CA).
Immunoprecipitation and Western blotting
FDCP cells were plated at a density of 5 x 107 cells/ml in serum-free medium and incubated for 4 h. Cells were then treated with cytokines for the indicated times. In experiments that used pharmacological inhibitors, cells were pretreated with these inhibitors for 30 min before cytokine induction. The cells were then homogenized in lysis buffer (1% Nonidet P-40, 50 mM Tris, 100 mM NaCl, 1 mM PMSF, 48 trypsin inhibitor unit aprotinin, and 40 nM leupeptin). The amount of protein was determined using a Bio-Rad DC protein assay kit (Hercules, CA). Proteins (50–75 µg) were prepared for Western blotting by heating in SDS-PAGE loading buffer for 5 min, followed by separation on either 7.5 or 10% polyacrylamide gels as indicated. The separated proteins were then transferred to Transblot polyvinylidene difluoride (PVDF) membranes (Bio-Rad) that were blocked with 5% BSA or 5% skim milk for 1 h at room temperature. PVDF membranes were then incubated overnight at 4°C with the appropriate Abs, which were diluted with 1 or 5% BSA in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20 as recommended by the manufacturer. Blots were incubated with HRP-labeled anti-rabbit or anti-mouse IgG (1/2000), developed with ECL substrate (Amersham), and subsequently exposed to autoradiographic film (Eastman Kodak, Rochester, NY). Autoradiograms were scanned using an Agfa Duosacan T1200 scanner, and band intensities were quantified using GelExpert 3.5 software (NucleoTech, San Mateo, CA).
Proteins were immunoprecipitated in some experiments before SDS-PAGE. In some cases supernatants from clarified cell lysates were precleared for 1 h at 4°C with an isotype-specific Ab and protein G-Sepharose beads (Pharmacia Biotech), followed by centrifugation at 14,000 x g. Precleared or clarified cell lysates from 5 x 107 cells were then immunoprecipitated with 2–4 µg of specific Abs and protein G-Sepharose beads overnight at 4°C as described above. The protein bound on the beads was washed three times with 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20 and heated with 50 µl of loading buffer before electrophoresis.
Statistical analysis
Data were analyzed using one-way ANOVA procedures, and the differences between treatment means were detected with an F-protected t test. Treatment differences were considered significant at the 5% probability level. Data were expressed as the mean ± SEM. All experiments were independently replicated at least three times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-10 inhibits the death of oligodendrocyte precursors
(11) and CD34+ cells
(12), and here we asked whether IL-10 is also capable of
inhibiting apoptosis and rescuing factor-dependent progenitor myeloid
cells following removal of growth factors. We first
confirmed that these cells express IL-10Rs at the protein level by
Western blotting with a specific IL-10R1 Ab. Mature mouse IL-10R1
contains 558 aa residues, but the migration rate of this receptor is
between 90–120 kDa due to glycosylation of the protein (34, 35). We found that FDCP cells express a specific 110-kDa protein
that reacts with an Ab to the IL-10R1 (Fig. 1
A). Identical results were
obtained in both immunoprecipitates and whole cell lysates.
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45%
were apoptotic 12 h after removal of IL-3 (Fig. 1
B).
Increasing concentrations of IL-10 linearly reduced the proportion of
apoptotic promyeloid cells, with the least effective dose of 0.1 ng/ml
(p < 0.05; n = 5). To
determine whether IL-10 also acts as a survival factor in cells that
are not dependent upon exogenous cytokines, we cultured N13 cells in
the absence of serum for 48 h. In control cells <1% of the N13
cells were apoptotic (Fig. 1C). Following serum withdrawal,
the proportion of BRDU+ cells increased to 41%.
Addition of increasing amounts of IL-10 reduced the proportion of
apoptotic N13 cells in a dose-responsive pattern (Fig. 1C). Finally, we used an Ab to the soluble form of the
IL-10R (4 µg/ml) that can fully neutralize the biologic activity of 1
ng/ml IL-10, as recommended by the supplier (R&D Systems). Promyeloid
cells were treated with IL-10 (1 ng/ml) in the presence or
the absence of an affinity-purified goat azide-free IL-10-neutralizing
Ab (50 µg) or a control goat IgG Ab (4 µg/ml). IL-10 inhibited
(p < 0.05; n = 3) the
apoptotic population by 40%, and this inhibition was fully reversed
by the neutralizing IL-10R Ab, but not by the control goat IgG (Fig. 1D). These data establish that IL-10 specifically inhibits
the apoptotic death of both FDCP promyeloid cells and N13 microglial
cells in a dose-dependent manner. Functional IL-10Rs are expressed on myeloid progenitor ells
Activation of IL-10Rs leads to tyrosine phosphorylation of both
Jak1 and Tyk2 within 10 min in pro-B cells transfected with the IL-10R
(36), followed by maximal phosphorylation of the Stat-3
transcription factor in monocytes, macrophages, and T cells (17, 19). Therefore, we examined the possibility that similar
pathways are activated in factor-dependent myeloid progenitor cells
following stimulation with IL-10. Whole cell lysates were prepared
following stimulation with IL-10 (50 ng/ml) for optimal times, as
determined in preliminary experiments (data not shown). Following
immunoprecipitation with specific Abs to either Jak1 or Tyk2, the
proteins were separated by SDS-PAGE, transferred to PVDF membranes, and
blotted with an anti-phosphotyrosine Ab. FDCP cells express
abundant amounts of both Jak1 (Fig. 2A) and Tyk2 (Fig. 2B), and their tyrosyl phosphorylation by IL-10 could be
detected as early as 5 min for Jak1 (Fig. 2A) and 10 min for
Tyk2 (Fig. 2B). Similar experiments were conducted to
determine whether IL-10 activates Stat-3, but in this case a
phosphospecific Ab was used for Western blotting. IL-10 strongly
activated Stat-3, with maximal stimulation at 10 min (Fig. 2C). Although others have shown that IL-10 can activate
Stat-1 in T cells and monocytes (17), we did not detect
the activation of Stat-1 by IL-10 (data not shown). Collectively, these
data extend earlier findings that some progenitor cells express IL-10R
(11, 12) and extend them by showing that IL-10 activates
Jak1, Tyk2, and Stat-3 in promyeloid cells.
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IL-4 was the first cytokine receptor that was established to
tyrosyl phosphorylate IRS-2 (37). Many cytokine receptors
are now recognized to stimulate tyrosine phosphorylation of IRS
proteins through a Jak-dependent mechanism, which leads to activation
of the PI 3-kinase and Akt pathway (reviewed in Ref. 38).
Therefore, we explored the possibility that IL-10Rs activate the major
IRS protein in myeloid progenitor cells, IRS-2 (32). Whole
cell lysates were prepared following treatment with IL-10 (50 ng/ml)
and were immunoprecipitated with anti-IRS-2, separated on SDS-PAGE,
and blotted with an anti-phosphotyrosine Ab or anti-IRS-2 Ab
(Fig. 3A). Activation of
IL-10Rs on factor-dependent promyeloid cells led to tyrosyl
phosphorylation of IRS-2 within 2 min. A densitometric summary and
statistical analysis of independent experiments showed that IL-10
increased (p < 0.05; n = 3)
IRS-2 tyrosyl phosphorylation at 2, 5, and 10 min, with the maximal
increase in tyrosyl phosphorylation at 5 min (Fig. 2B).
Enzymatic activity of PI 3-kinase was then measured in FDCP cells
following incubation with IL-10, and a representative TLC autoradiogram
is shown in Fig. 3C. An increase in PI 3-kinase activity was
detected within 5 min following addition of IL-10, and this elevation
in PI 3-kinase activity remained (p < 0.05;
n = 3) for at least 10 min. Finally, we
immunoprecipitated Akt from whole cell lysates of promyeloid cells that
were incubated with IL-10 (50 ng/ml) for various times. The maximal
increase in Akt activity occurred at 10 min (p
< 0.01; n = 4; Fig. 3D), in a time frame
consistent with the increase in PI 3-kinase activity. These experiments
establish that stimulation of the IL-10R on promyeloid cells rapidly
leads to tyrosyl phosphorylation of IRS-2 as well as activation of the
downstream survival enzymes PI 3-kinase and Akt.
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The survival of hemopoietic myeloid progenitors is maintained by
at least two different intracellular signaling pathways, one that
requires PI 3-kinase and one that does not (30, 39, 40).
To determine whether activation of PI 3-kinase is involved in the
ability of IL-10 to promote survival of promyeloid cells, we incubated
FDCP cells with wortmannin (500 nM). This fungal metabolite
irreversibly binds the p110 catalytic subunit of PI 3-kinase, and we
have shown that it fully blocks the activation of PI 3-kinase in FDCP
cells (31). As previously reported (30), IL-3
promotes cell survival in the absence or the presence of wortmannin
(Fig. 4). In contrast, inhibition of PI
3-kinase activity with wortmannin blocked (p <
0.01; n = 3) the protective effects of the survival
factor IGF-I, which was used as a positive control. We then incubated
promyeloid cells with IL-10 in the presence or the absence of
wortmannin. The proportion of apoptotic cells was reduced from 45 to
15% by IL-10. When wortmannin was added, the ability of IL-10 to
promote cell survival was completely blocked
(p < 0.01; n = 3). These data
establish that IL-10, which acts like both IGF-I and IL-4 to
activate the IRS proteins, similarly promotes cell survival
by a mechanism that is dependent upon the activation of PI
3-kinase.
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Overexpression of a constitutively active form of MEK or ERK2 has
recently been shown to increase cell survival, whereas a dominant
negative form of MEK promotes apoptosis (41). Therefore,
we tested the possibility that IL-10 activates the MAPK pathway by
measuring activation of ERK1/2 in FDCP cells. FDCP cells were cultured
with IL-10 (50 ng/ml) for 10 min, and activation of ERK1/2 was measured
by blotting whole cell lysates with an
anti-Tyr204 Ab. A representative
autoradiogram shows that IL-10 increased the amount of activated ERK1/2
(Fig. 5A). The increase in
activity of ERK1/2 was inhibited in a dose-dependent manner by a 30-min
pretreatment of FCPC cells with the PI 3-kinase inhibitors, wortmannin,
or LY294002. A densitometric summary of autoradiograms from three
independent experiments demonstrated that the 50- and 500-nM
concentrations of wortmannin and the 10-µM concentration of LY294002
inhibited (p < 0.05; n = 3)
activation of ERK1/2 (Fig. 5B). The inhibitory effects of
both wortmannin and LY294002 were specific because they did not affect
the activation of Stat-3 by IL-10 (data not shown). These results
suggested that inhibition of ERK1/2 activation might reduce the ability
of IL-10 to promote cell survival. To test this possibility we
incubated myeloid progenitor cells in the presence or the absence of
the MEK inhibitor PD98059 at a concentration that fully
inhibits activation of ERK1/2 (10 µg/ml; data not shown). Although
this MEK inhibitor did not affect the ability of IL-3 to promote cell
survival, it partially, but significantly (p <
0.05; n = 3), reversed the anti-apoptotic property
of IL-10 (Fig. 5C). These experiments establish that IL-10
activates ERK1/2 in a PI 3-kinase sensitive manner and that the
MEK-dependent activation of ERK1/2 contributes to the
survival-promoting activity of IL-10.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). IL-10-induced
activation of Jak1 and Tyk2 family members leads not only to tyrosyl
phosphorylation of the transcription factor Stat-3 (Fig. 2), but also
to tyrosyl phosphorylation of the large m.w. docking molecule, IRS-2
(Fig. 3). The subsequent activation of PI 3-kinase is required for
IL-10 to promote promyeloid survival (Fig. 4), which acts by a
mechanism that at least partially depends upon increased activity of
the survival-promoting property of ERK1/2 (Fig. 5). These data
establish that functional IL-10Rs are expressed on myeloid progenitor
cells and show that activation of these receptors inhibits the death of
these cells. This anti-apoptotic property of IL-10 occurs directly
by activating an IRS-2/PI 3-kinase/Akt survival pathway. IL-10 has been well characterized for its ability to suppress the synthesis of proinflammatory cytokines. The synthesis of IL-10 is increased in mycobacterial (42) and influenza (43) infections, and at least some of its anti-inflammatory properties in vivo result from activation of the hypothalamic-pituitary adrenal axis (44). Progenitor CD34+ cells that develop into myeloid cells and mature macrophages express IL-10Rs, so we were not surprised to find a 110-kDa IL-10R1 on myeloid progenitor cells. However, the function of these IL-10Rs was not known, because promyeloid cells are not a major source of proinflammatory cytokines. IL-10 has been recently established to promote the survival of oligodendrocyte progenitor cells (12), so we tested the hypothesis that IL-10 might serve as a survival signal for myeloid progenitor cells. We found that IL-10 concentrations as low as 1 ng/ml significantly inhibit the apoptotic death of factor-deprived promyeloid cells. Although the anti-apoptotic activity of IL-10 is not as great as that of IL-3, the survival-promoting activity of IL-10 is specifically blocked by a neutralizing IL-10R Ab. These data are consistent with the idea that IL-10 regulates cellular activities in addition to inhibiting the synthesis of proinflammatory cytokines. In the case of myeloid progenitor cells, activation of IL-10Rs leads directly to a reduction in their apoptotic death.
Binding of IL-10 to IL-10R1, coupled with the accessory IL-10R2
signaling subunit, is well known to cause tyrosyl phosphorylation of
the Jak kinase family members Jak1 and Tyk2 (3). Jak1 is
required for the biologic activity of IL-10, because macrophages from
Jak1-/- mice do not respond to IL-10
(20). Activation of Jak family members leads to the
recruitment and tyrosyl phosphorylation of the Stat family of latent
transcription factors, primarily Stat-3. However, a variety of cytokine
receptors that activate the Jak/Stat signaling pathway are now known to
cause tyrosyl phosphorylation of the large IRS family of docking
proteins (26). For example, Jak1 is activated following
stimulation with a variety of cytokines, such as IL-4, IFN-, or
oncostatin M, and Jak1 also activates the IRS and PI 3-kinase signaling
pathways (21). Here we extend this concept to the
endogenously expressed IL-10R on promyeloid cells. In addition to
activation of Jak1 and Stat-3, we demonstrate that IL-10 leads to
tyrosyl phosphorylation of IRS-2. The activation of IRS-2 is
accompanied by an increase in the enzymatic activity of PI 3-kinase. As
expected, PI 3-kinase causes recruitment and activation of the survival
enzyme Akt, and inhibition of PI 3-kinase completely eliminates the
anti-apoptotic activity of IL-10. These data establish that IL-10
activates the IRS-2/PI 3-kinase/Akt signaling pathway in myeloid
progenitor cells. Similar to IL-4 and IGF-I, but unlike IL-3 (31, 39), this pathway is necessary for the survival-promoting
activity of IL-10.
Activation of PI 3-kinase in factor-dependent promyeloid cells regulates the activity of several enzymes, including Akt. The enzymatic activity of Akt leads to the phosphorylation of the proapoptotic protein Bcl-2 antagonist of cell death on Ser112 and Ser136, causing it to associate with the adaptor protein 14-3-3 and disassociate with Bcl-xL (45). In hemopoietic cells a variety of cytokines activate Akt, including IL-4, insulin, stem cell factor, IL-3, and GM-CSF (46). However, this activation of Akt is not related to either phosphorylation of BAD or cell survival (47). Similarly, PI 3-kinase increases the enzymatic activity of p70 S6-kinase in myeloid progenitors, but inhibition of S6 kinase does not affect cell survival (31). Consistent with our results reported here, inhibition of PI 3-kinase has recently been shown to inhibit activation of ERK1/2 in endothelial (48) and osteoblastic (49) cells. Phosphorylation of ERK1/2 has also been recently demonstrated to directly protect cells from proapoptotic signals generated by Fas, TNF, and TNF-related apoptosis-inducing ligand receptors (50). Similarly, overexpression of the upstream ERK1/2 kinase, MEK1, significantly promotes cell survival (41). Protective effects of ERKs have also been reported in neurons (reviewed in Ref. 51). Our data are consistent with these findings by showing that IL-10 increases the activation of ERK1/2. Inhibition of PI 3-kinase activity, which abrogates the anti-apoptotic activity of IL-10, blocks tyrosine phosphorylation of ERK1/2. Direct inhibition of ERK1/2 with the MEK inhibitor PD98059 significantly, although partially, impaired the ability of IL-10 to increase the survival of myeloid progenitors. These data are consistent with a role for ERK1/2 in promoting cell survival following IL-10-induced activation of IRS-2 and PI 3-kinase.
In summary, these experiments demonstrate a previously unrecognized role for IL-10 in promoting the survival of myeloid progenitor cells. Binding of IL-10 to its receptor on these cells leads to activation not only of the Jak1/Tyk2/Stat-3 pathway but also of the IRS-2/PI 3-kinase/Akt pathway. Inhibition of PI 3-kinase abrogates the anti-apoptotic activity of IL-10, which is associated with a reduction in IL-10 activation of ERK1/2. These data indicate that the survival-promoting activity of IL-10 is caused by stimulation of the IRS-2/PI 3-kinase/Akt pathway, which increases the survival-promoting activity of ERK1/2.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Keith W. Kelley, Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois, 207 Edward R. Madigan Laboratory, 1207 West Gregory Drive, Urbana, IL 61801. E-mail address: kwkelley{at}uiuc.edu
3 Abbreviations used in this paper: IRS-2, insulin receptor substrate-2; BRDU, bromolated dUTP; FDCP, factor-dependent cell progenitor 1/Mac-1; IGF-I, insulin-like growth factor I; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; MEK, MAPK/ERK kinase; PI, propidium iodide; PI 3-kinase, phosphatidylinositol 3'-kinase; PVDF, polyvinylidene difluoride.
Received for publication June 4, 2001. Accepted for publication August 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII) whose engagement blocks the cell death programme and facilitates differentiation. Immunology 102:331.[Medline]
cytotoxicity in oligodendroglial cells: role of nitric oxide and protection by the anti-inflammatory cytokine IL-10. Eur J. Neurosci. 13:493.[Medline]
) is a shared component of both IL-10 and IL-TIF (IL-22) receptor complexes. J. Biol. Chem. 16:16.
and STAT3 complexes in human T cells and monocytes. J. Immunol. 155:1079.[Abstract]
receptors. Blood 90:2574.. J. Immunol. 158:2390.[Abstract]
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A. A. R. Rahimi, K. Gee, S. Mishra, W. Lim, and A. Kumar STAT-1 Mediates the Stimulatory Effect of IL-10 on CD14 Expression in Human Monocytic Cells J. Immunol., June 15, 2005; 174(12): 7823 - 7832. [Abstract] [Full Text] [PDF]
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 B. Halvorsen, T. Waehre, H. Scholz, O. P. Clausen, J. H. von der Thusen, F. Muller, H. Heimli, S. Tonstad, C. Hall, S. S. Froland, et al. Interleukin-10 enhances the oxidized LDL-induced foam cell formation of macrophages by antiapoptotic mechanisms J. Lipid Res., February 1, 2005; 46(2): 211 - 219. [Abstract] [Full Text] [PDF]
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 S. Bhattacharyya, P. Sen, M. Wallet, B. Long, A. S. Baldwin Jr, and R. Tisch Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the PI3K/Akt pathway and of I{kappa}B kinase activity Blood, August 15, 2004; 104(4): 1100 - 1109. [Abstract] [Full Text] [PDF]
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L. Williams, L. Bradley, A. Smith, and B. Foxwell Signal Transducer and Activator of Transcription 3 Is the Dominant Mediator of the Anti-Inflammatory Effects of IL-10 in Human Macrophages J. Immunol., January 1, 2004; 172(1): 567 - 576. [Abstract] [Full Text] [PDF]
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 B. McMorran, L. Town, E. Costelloe, J. Palmer, J. Engel, D. Hume, and B. Wainwright Effector ExoU from the Type III Secretion System Is an Important Modulator of Gene Expression in Lung Epithelial Cells in Response to Pseudomonas aeruginosa Infection Infect. Immun., October 1, 2003; 71(10): 6035 - 6044. [Abstract] [Full Text] [PDF]
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C. Niemand, A. Nimmesgern, S. Haan, P. Fischer, F. Schaper, R. Rossaint, P. C. Heinrich, and G. Muller-Newen Activation of STAT3 by IL-6 and IL-10 in Primary Human Macrophages Is Differentially Modulated by Suppressor of Cytokine Signaling 3 J. Immunol., March 15, 2003; 170(6): 3263 - 3272. [Abstract] [Full Text] [PDF]
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M. M. Monick, P. K. Robeff, N. S. Butler, D. M. Flaherty, A. B. Carter, M. W. Peterson, and G. W. Hunninghake Phosphatidylinositol 3-Kinase Activity Negatively Regulates Stability of Cyclooxygenase 2 mRNA J. Biol. Chem., August 30, 2002; 277(36): 32992 - 33000. [Abstract] [Full Text] [PDF]
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 W.-H. Shen, J.-H. Zhou, S. R. Broussard, G. G. Freund, R. Dantzer, and K. W. Kelley Proinflammatory Cytokines Block Growth of Breast Cancer Cells by Impairing Signals from a Growth Factor Receptor Cancer Res., August 15, 2002; 62(16): 4746 - 4756. [Abstract] [Full Text] [PDF]
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