IL-10 Inhibits Apoptosis of Promyeloid Cells by Activating Insulin Receptor Substrate-2 and Phosphatidylinositol 3′-Kinase¹

Interleukin-10 was originally characterized for its ability to inhibit the production of cytokines by macrophages (1) and Th1 cells (2). However, IL-10 is now recognized to also directly affect the growth and development of a variety of cells (reviewed in Ref. 3). For example, IL-10 can directly promote the death of inflammatory cells, including activated macrophages (4), neutrophils (5), and T cells (6). In contrast, IL-10 increases the survival of some types of human cancer cells, such as Burkitt lymphoma (7). Non-Hodgkin’s lymphoma cells secrete IL-10, and inhibition of this autocrine IL-10 has recently been shown to promote cell death and reduce expression of the anti-apoptotic protein Bcl-2 (8). Similarly, IL-10 increases the expression of Bcl-x_L in thyrocytes from patients with Graves’ disease (9). The expression of Bcl-2 is also increased by IL-10 in germinal center B cells (10) and in human CD34⁺ cells (11). This anti-apoptotic property of IL-10 is consistent with recent results showing that the death of both primary oligodendrocyte progenitor cells (12) and astrocytes (13) is inhibited by IL-10. These new data indicate that although IL-10 can promote the death of activated inflammatory cells, it also provides a survival signal for nonactivated cells or their progenitors.

The heterotetramer IL-10R is composed of two ligand binding subunits (IL-10Rα and IL-10R1) and two accessory signaling subunits (IL-10Rβ 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-α (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-α (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.

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.

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 × 10⁴ cells were analyzed on an EPICS XL flow cytometer (Coulter, Miami, FL).

FDCP cells (5 × 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 × 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-α-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 [γ-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 α-phosphatidylinositol.

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 × 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 β-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 β-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 [γ-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).

FDCP cells were plated at a density of 5 × 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 × g. Precleared or clarified cell lysates from 5 × 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.

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.

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.

To determine whether activation of these IL-10Rs regulates the survival of myeloid progenitors, we used flow cytometry to measure the proportion of cells that were negative for PI and positive for Hoechst 33342 as previously described (30, 31). In the presence of IL-3, practically no apoptotic cells were detected, whereas ∼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. 1,C). 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. 1,C). 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. 1 D). 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.

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. 2,A) and Tyk2 (Fig. 2,B), and their tyrosyl phosphorylation by IL-10 could be detected as early as 5 min for Jak1 (Fig. 2,A) and 10 min for Tyk2 (Fig. 2,B). 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. 2 C). 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.

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. 3,A). 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. 2,B). 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. 3,C. 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. 3 D), 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.

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.

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. 5,A). 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. 5,B). 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. 5 C). 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.

IL-10 is best known for its ability to inhibit the synthesis of proinflammatory cytokines, but evidence is growing that it directly regulates the growth and survival of noninflammatory cells as well (3). IL-10 promotes survival of CD34⁺ progenitors that develop into myeloid cells (11), and IL-10 has recently been shown to increase survival of oligodendrocyte progenitors (12). Here we extend these findings by establishing that IL-10 specifically inhibits the death of factor-dependent promyeloid cells (Fig. 1). 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-α, 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-x_L (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.