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IL-12 Provides Proliferation and Survival Signals to Murine CD4⁺ T Cells Through Phosphatidylinositol 3-Kinase/Akt Signaling Pathway¹

National Laboratory of DNA Medicine, Division of Molecular and Life Science, Pohang University of Science and Technology, Hyoja Dong, Pohang, Korea

	Abstract

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IL-12 is a pleiotropic cytokine that plays an important role in innate and adaptive immunity. IL-12 induces T cell proliferation and IFN- secretion from activated T cells. It was also reported that IL-12 prevents apoptosis of CD4⁺ T cells. However, the signaling mechanism that regulates these IL-12-induced responses is poorly understood yet. In this study, we demonstrated that IL-12 activates phosphatidylinositol 3-kinase (PI3K)/Akt pathway in murine CD4⁺ T cells, and that this signaling pathway is required for IL-12-induced T cell proliferation and antiapoptotic function, but not for IFN- induction. Through PI3K/Akt pathway, IL-12 up-regulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins-2, followed by down-regulation of active caspase-3. These results suggest that PI3K/Akt pathway is critical for mediating IL-12-induced CD4⁺ T cell responses such as T cell proliferation and survival.

	Introduction

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Interleukin-12 is a pleiotropic cytokine that is secreted by activated professional APCs (1, 2, 3). IL-12 can induce Th1-type cellular immune responses, T cell proliferation, and IFN- secretion from activated T and NK cells (1, 2, 3, 4, 5). In addition, it has been reported that IL-12 prevents apoptotic cell death of the activated human and mouse peripheral T cells (6, 7, 8, 9).

IL-12R is composed of two subunits, designated 1 and 2. IL-12 activates the Janus family of protein tyrosine kinases (Jaks)³ such as Jak2 and Tyk2 through IL-12R (10). These activated Jaks phosphorylate tyrosine residues on IL-12R2, providing docking sites for the Src homology 2 domain of STAT-4, and then STAT-4 is phosphorylated and dimerized to bind to the IL-12-responsive genes (11). STAT-4 appears to be an important mediator in IL-12R signaling, because IL-12-induced IFN- secretion and T cell proliferation are impaired in STAT-4^-/- mice (5). Besides the Jak-STAT pathway, IL-12 was also shown to activate mitogen-activated protein kinase (MAPK) kinase (MKK) 6/p38 MAPK pathway, but not extracellular signal-regulated kinases (Erk) 1/2 or c-Jun N-terminal kinase pathway (12, 13). In particular, MKK 6/p38 MAPK pathway is required to promote IL-12-induced IFN- secretion and STAT-4 serine phosphorylation on serine 721, but not T cell proliferation (12, 13, 14, 15). These results suggest that IL-12 might activate multiple signaling pathways for a variety of IL-12-induced T cell responses.

Many cytokines and growth factors activate phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in T cells (16). In mammalian cells, multiple isoforms of PI3K were found and subdivided into three classes. Among them, class I PI3Ks are generally coupled to extracellular stimuli and transmit signals to induce protein synthesis, cytoskeletal changes, cell cycle regulation, and apoptosis regulation (17). In the case of IL-2R signaling, it was reported that PI3K/Akt pathway activated by IL-2 regulates cell cycle-related molecules such as cyclin D3 and p27^kip1 to induce T cell proliferation (18). Recently, it was also shown that PI3K/Akt pathway activated by IL-2 family cytokines such as IL-2, IL-4, and IL-15 promotes T cell survival through the modulation of an antiapoptotic molecule such as Bcl-2 (19). However, it is not clear yet whether IL-12 activates the PI3K/Akt pathway, leading to the modulation of IL-12-induced T cell responses. In this study, we clearly demonstrated that IL-12 stimulates the PI3K/Akt signaling pathway in activated CD4⁺ T cells, and that this signaling pathway is critical for IL-12-induced T cell proliferation and inhibition of apoptosis. In particular, IL-12 modulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins (cIAP)-2 through PI3K/Akt pathway.

	Materials and Methods

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Mice

Five- to 10-wk-old C57BL/6 female mice were purchased from Japan SLC (Shizuoka, Japan) and used for the isolation of lymph nodes (LNs). The mice were maintained in specific pathogen-free conditions and fed autoclaved food and water.

Reagents and Abs

For MACS, anti-mouse CD11b, MHC class II, CD8, and B220 microbeads were obtained from Miltenyi Biotec (Auburn, CA). Purified hamster anti-mouse CD3e and CD28 NA/LE Abs were purchased from Southern Biotechnology Associates (Birmingham, AL). For the flow cytometric analysis and cytokine ELISA, anti-mouse CD4 FITC, active caspase 3 FITC, annexin V FITC, and anti-mouse IFN- capture and detection Abs were purchased from BD PharMingen (San Diego, CA). Recombinant mouse IL-2, IL-4, and IL-12 were all obtained from R&D Systems (Minneapolis, MN) and reconstituted as described in technological notes. General reagents used were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. SB203580 and LY294002 were purchased from Calbiochem (La Jolla, CA). RPMI 1640 and FBS were purchased from Life Technologies (Grand Island, NY) and HyClone Laboratories (Logan, UT), respectively. For immunoblot assay, rabbit Abs against p38 MAPK, Erks, Akt, Thr¹⁸⁰/Tyr¹⁸²-phosphorylated p38 MAPK, Thr²⁰²/Tyr²⁰⁴-phosphorylated Erks, and Ser⁴⁷³/Thr³⁰⁸-phosphorylated Akt were all purchased from New England Biolabs (Beverly, MA). Rabbit Abs against STAT-4 and cIAP-1/2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse Abs against p27^kip1, Bcl-2, and cyclin D3 were also obtained from Santa Cruz Biotechnology. For IFN- response region (GRR) affinity purification, streptavidin-agarose beads were obtained from Sigma-Aldrich.

Preparation of murine primary CD4⁺ T lymphocytes

Mouse naive CD4⁺ T cells were purified from the LNs of C57BL/6 female mice. CD4⁺ T cells were negative selected through VS⁺ columns using the high gradient MACS (Miltenyi Biotec), as described previously (20). Briefly, harvested LN cells were treated with ammonium chloride lysing buffer to clear RBC. Then remaining cells were treated with anti-CD11b, anti-MHC class II, anti-B220, and anti-CD8 microbeads, and purified by negative selection using the MACS Separation System. Purified cell populations were 92% CD4⁺ T cells. The purity was assessed by flow cytometric analysis on a FACScan flow cytometer (BD Biosciences, Heidelberg, Germany) after staining with anti-CD4 FITC Abs.

In vitro activation and culture of CD4⁺ T cells

Purified CD4⁺ T cells were in vitro activated for 72 h with plate-bound anti-CD3 (300 ng/well) and anti-CD28 (100 ng/well) hamster Abs in 96-well plate. T cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS (HyClone Laboratories), 2 mM L-glutamine, 50 mM 2-ME, and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin). The Ab-activated CD4⁺ T cells were harvested and washed twice with complete medium and used for the following experiments.

Preparation of whole cell lysates and immunoblot analysis

The Ab-activated T cells (5 x 10⁶ cells/ml) were pretreated for 20 min with medium alone, 5 µM LY294002, or 10 µM SB203580 (Calbiochem). After pretreatment, T cells were incubated with medium, 20 ng/ml IL-2, 100 ng/ml IL-4, or 10 ng/ml IL-12p70 (R&D Systems) for the indicated times. Then cells were harvested and whole cell lysates were prepared, as described previously (10). To quantify the phosphorylation of Akt, p38 MAPK, and Erks, equal amounts of whole cell lysates were resolved by 10% SDS-PAGE. Then Western blots were immunoanalyzed using specific Abs against the phosphorylated form of each protein. To confirm that the same amount of proteins was loaded in each lane, primary Ab/secondary Ab complex was removed, as described previously (21). The blots were then subjected to autoradiography to confirm that the Ab signal had been removed. After this procedure, the blots were reprobed with the specific Abs against total Akt, p38 MAPK, or Erks. For cell cycle and antiapoptotic molecules, we used each indicated Ab or -actin as a control, followed by goat anti-rabbit or mouse HRP-conjugated Abs.

Cytokine ELISA and intracellular staining

Murine IFN- ELISA was performed as described in the recommended protocol (BD PharMingen).

Ab-activated T cells were harvested and treated with indicated inhibitors and cytokines. At 36 h after treatment, cells were collected and washed twice with PBS. Cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD PharMingen) for 20 min at 4°C, and then washed with Perm/Wash buffer (BD PharMingen). Permeabilized cells were resuspended with the wash buffer containing FITC-conjugated rabbit anti-mouse active caspase-3 Abs (BD PharMingen), and incubated for 30 min at 4°C. After incubation, cells were harvested and resuspended with PBS containing 0.5% FBS and 0.1% sodium azide. Intracellular active caspase-3 levels were assessed by flow cytometry.

Oligonucleotide affinity purification and immunoblotting

Oligonucleotide affinity purification of STAT-4 proteins was performed using a 5'-biotinylated double-stranded oligonucleotide corresponding to the GRR of mouse FcRI gene (22, 23), coupled to streptavidin-agarose. Briefly, 20 min after cytokine stimulation, whole cell lysates were prepared by lysis of cells in a lysis buffer comprised of 50 mM Tris-Cl (pH 8.0), 1% Nonidet P-40, 15 mM NaCl, 0.1 mM EDTA, 10 mM NaF, 10% glycerol, 1 mM Na₃VO₃, 1 mM PMSF, 1 mM DTT, protease inhibitors, and 0.1% Triton X-100. The oligonucleotide sequence used was GTATTTCCCAGAAAAGGAAC. Cell lysates were incubated with GRR-bound agarose beads, which were then washed and subjected to SDS-PAGE. Oligo-precipitated proteins were electrophoretically transferred to nitrocellulose transfer membrane and immunoblotted with anti-STAT-4 Abs (Santa Cruz Biotechnology).

	Results

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IL-12 directly activates the PI3K/Akt signaling pathway

To investigate which signaling pathways are activated by IL-12, we examined the activation of STAT-4 molecule and several kinases such as PI3K/Akt, p38 MAPK, and Erk1/2 by immunoblot analysis. As expected, IL-12 activated STAT-4 and induced their binding to the GRR DNA (Fig. 1A). When STAT-4 is activated by IL-12, it is translocated to the nucleus and regulates the transcription of the IL-12-responsive genes through the binding of conserved DNA sequences, GRR (23, 24). In addition, the treatment of IL-12 induced the phosphorylation of p38 MAPK, which was significantly inhibited by 10 µM p38 MAPK inhibitor, SB203580. However, Erk1/2 were not activated by the treatment of IL-12. These results agree well with the previous reports that IL-12 stimulates MKK6/p38 MAPK, but not Erk1/2 signaling pathway in activated CD4⁺ T cells (12, 13). Particularly, IL-12 induced Akt phosphorylation, which was significantly blocked by 3.5 IC₅₀ (5 µM) of PI3K-specific inhibitor, LY294002 (25) (Fig. 1B). As reported previously (16, 19), IL-2 family cytokines such as IL-2 and IL-4 also induced Akt phosphorylations, which appeared to be less than those by IL-12 (Fig. 1C). Therefore, we demonstrated that, in addition to STAT-4 and p38 MAPK, IL-12 directly activates the PI3K/Akt signaling pathway in murine CD4⁺ T cells.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. IL-12 activates the PI3K/Akt signaling pathway. Murine naive CD4+ T cells were prepared and stimulated, as described in Materials and Methods. A, Cells were harvested after in vitro stimulation, and then treated for 20 min with medium alone or IL-12 (10 ng/ml). Oligonucleotide affinity purification of STAT-4 and immunoblotting were performed as described in Materials and Methods. B, Ab-activated T cells were rested in serum-free medium for 24 h, followed by stimulation with or without IL-12 (10 ng/ml). p38 MAPK inhibitor SB203580 (10 µM) or PI3K-specific inhibitor LY294002 (5 µM) was added and incubated for 20 min before IL-12 treatment. Cells were harvested at the indicated times, and equal amounts of whole cell lysates were resolved by SDS-PAGE, followed by immunoblot analysis, as described in Materials and Methods. C, Activated CD4+ T cells were treated with each indicated cytokine for 20 min, and then cells were harvested and resolved by SDS-PAGE, followed by immunoblot analysis using a set of Abs. Data are representative of two independent experiments.

IL-12 has been known as a cytokine that induces the proliferation of activated CD4⁺ T cells (3, 5). In addition, it has also been reported that the PI3K/Akt pathway is an important cellular signaling pathway that regulates multiple cellular responses, one of which is to induce cellular proliferation through the modulation of cell cycle-related molecules such as cyclin D3 and cyclin-dependent kinase (cdk) inhibitor, p27^kip1 (17, 18). As shown in Fig. 2A, the proliferation of CD4⁺ T cells was enhanced by IL-12 treatment compared with the cells treated with medium alone. This IL-12 effect was completely inhibited by LY294002, but not by SB203580, suggesting that PI3K/Akt, but not p38 MAPK pathway is critical for IL-12-induced CD4⁺ T cell proliferation.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. IL-12 regulates the expression of cyclin D3 to induce T cell proliferation through PI3K/Akt signaling pathway. A, Seventy-two hours after Ab stimulation, cells (1 x 105 cells/sample) were harvested and stimulated with medium alone or IL-12 (10 ng/ml) for the indicated times. PI3K-specific inhibitor LY294002 (5 µM) or p38 MAPK-specific inhibitor SB203580 (10 µM) was added and incubated for 20 min before IL-12 treatment. T cell proliferation was measured through the counting of total cell numbers at the indicated times. Data are representative of three independent experiments with similar results. B, Ab-activated T cells were incubated with LY294002 (5 µM) or SB203580 (10 µM) for 20 min, and then stimulated with medium alone or IL-12 (10 ng/ml) for 20 h. Total cell lysates were resolved by SDS-PAGE, followed by immunoblot with each indicated Ab. Data are representative of two independent results.

The PI3K/Akt pathway via IL-12 is not required for IL-12-induced IFN- production

One of the important properties of IL-12 is its ability to induce the production of IFN- from activated T cells (3). As expected, IL-12 induced large amounts of IFN- production from activated CD4⁺ T cells, which was inhibited by the treatment of SB203580 (Fig. 3). This result is consistent with previous reports that p38 MAPK pathway is required for IL-12-induced IFN- production from activated T cells (14, 15). In contrast, the treatment of LY294002 did not significantly inhibit the IL-12-induced IFN- production, indicating that PI3K/Akt pathway is not required for IL-12-induced IFN- production from activated CD4⁺ T cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. The PI3K/Akt signaling pathway is not involved in IL-12-induced IFN- secretion from CD4+ T cells. Ab-stimulated T cells were preincubated with each indicated inhibitor, as described in Fig. 2A, and then treated with medium alone or IL-12 (10 ng/ml) for 24 h. Cell culture supernatants were harvested and tested for murine IFN- production by cytokine ELISA. Data are shown as the mean ± SD of triplicate cultures and representative of two independent experiments.

Because IL-12 has been shown to prevent the apoptosis of activated CD4⁺ T cells in various conditions (3, 6, 7, 8, 9), we tested whether PI3K/Akt pathway is involved in antiapoptotic effects of IL-12 in passive cell death.

Apoptotic T cell death was measured through flow cytometric analysis stained with both annexin V and propidium iodide (PI). Preactivated CD4⁺ T cells showed spontaneous apoptosis over time, but the addition of IL-12 significantly reduced apoptosis in a dose-dependent manner (Fig. 4A), which is consistent with previous reports (7, 27). As shown in Fig. 4, B and C, LY294002 inhibited the antiapoptotic effect of IL-12 in a dose-dependent manner, but SB203580 did not, suggesting that IL-12 induces CD4⁺ T cell survival through PI3K/Akt pathway, but not through p38 MAPK pathway.

View larger version (53K): [in this window] [in a new window]   FIGURE 4. PI3K/Akt, but not p38 MAPK pathway is critical for the survival of CD4+ T cells by IL-12 in passive cell death. A, 72 h after in vitro stimulation, Ab-activated CD4+ T cells were harvested and treated with medium alone or each indicated concentration of IL-12 (0.1, 1, 10 ng/ml). At the indicated times, cells were harvested and incubated with annexin V FITC in a buffer containing PI and analyzed by flow cytometry. Annexin V- and PI-double-negative cells were regarded as viable cells, annexin V-positive but PI-negative cells as apoptosis-undergoing cells, and annexin V- and PI-double-positive cells as dead cells. The sum of percentages of both apoptosis-undergoing and dead cell population is presented in each FACS profile. The result shown here is representative of more than five independent experiments with similar results. B and C, Ab-activated CD4+ T cells were harvested and incubated for 20 min with indicated concentrations of LY294002 (B) or SB203580 (C). After preincubation, cells were treated with IL-12 (10 ng/ml) for additional 48 h and then cells were assayed for cell death by flow cytometry, as described in A. The percentage of live cells before IL-12 treatment was regarded as 100%. The percentage of relative live cells of each sample was calculated and shown in histogram. Data are representative of five independent experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 5. IL-12 up-regulates the expression of Bcl-2 and cIAP-2 molecules, but down-regulates active caspase-3 through PI3K/Akt pathway. Ab-activated CD4+ T cells were incubated with LY294002 (5 µM) or SB203580 (10 µM) for 20 min and then treated with medium alone or IL-12 (10 ng/ml) for additional 36 h. A, Total cell lysates were resolved by SDS-PAGE, and followed by immunoblot with each indicated Ab. Data are representative of two independent experiments. B, Intracellular staining for the detection of active caspase-3 was performed as described in Materials and Methods. Active caspase-3-positive populations were distinguished from negative populations by using isotype control Ab. Each percentage of the active caspase-3-positive and caspase-3–negative population is presented in histograms. Data are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has been known that multiple cytokines and growth factors such as IL-2, IL-4, and insulin-like growth factors activate PI3K/Akt signaling pathway (16). In this study, we demonstrated that like IL-2 family cytokines, IL-12 activates the PI3K/Akt signaling pathway in murine CD4⁺ T cells, which is essential for IL-12-induced T cell proliferation and survival. On the contrary, Veronica et al. (24) recently reported that IL-12 could not activate PI3K/Akt pathway in human PBL T cells. This discrepancy might be due to the difference of origin of cells that were harvested, or the difference of experimental conditions in which T cells were prestimulated.

The PI3K/Akt signaling pathway is associated with cellular proliferation in T cells. It was reported that IL-2 induces T cell proliferation by modulation of both cyclin D3 and p27^kip1 through PI3K/Akt pathway (18). Similarly, IL-12 appeared to regulate these two molecules to induce CD4⁺ T cell proliferation. However, the expression of cyclin D3, but not that of p27^kip1, appeared to be regulated via PI3K/Akt signaling pathway. Previously, it was reported that the regulation of p27^kip1 by IL-12 is mediated through STAT-4-dependent pathway (30). In addition to p27^kip1, p21^WAF₁/cip1 has been known as a cdk inhibitor molecule that inhibits cellular proliferation (26). In contrast to p27^kip1 regulation, the expression of p21^WAF₁/cip1 was up-regulated by IL-12 (data not shown). This result was consistent with previous reports that p21^WAF₁/cip1 is up-regulated following lymphocyte activation, and that the regulation of p21^WAF₁/cip1 is not required for cytokine-induced T cell proliferation (31, 32). Taken together, it is likely that IL-12 induces CD4⁺ T cell proliferation through PI3K/Akt pathway-dependent modulation of cyclin D3, a cell cycle-positive regulator molecule, and presumably through STAT-4 pathway-dependent modulation of p27^kip1, a cell cycle-negative regulator molecule.

Recently, it has been reported that IL-12 suppresses apoptotic cell death of activated T cells, but the molecular mechanisms are not clear yet (3, 6, 7, 8, 9, 33). In this study, we demonstrated that IL-12 provides survival signals to CD4⁺ T cells through the up-regulation of Bcl-2 and cIAP-2, and the down-regulation of active caspase-3 via PI3K/Akt pathway. Our results are similar to previous reports that PI3K/Akt signaling pathway via IL-2 family cytokines promotes T cell survival through the modulation of antiapoptotic molecule, Bcl-2 (19, 28). In addition, the activation of PI3K/Akt pathway was reported to prevent the apoptosis of T cells via NF-B activation, which was known to induce the expression of cIAP molecules (34, 35, 36). Bcl-2 eventually regulates caspase-3 activation through modifying the mitochondrial membrane potential, and cIAP-2 is potent inhibitor of caspases (29). Therefore, it is likely that IL-12 inhibits caspase-3 activation by up-regulation of both Bcl-2 and cIAP-2 molecules through PI3K/Akt pathway. Estaquier et al. (6) showed that IL-12 prevents apoptosis of CD4⁺ T cells induced by anti-Fas or anti-CD3. cIAP-2 has also been reported to inhibit death receptor-mediated apoptosis through suppression of caspase-8 activation (35, 36). Therefore, it is possible that IL-12 may prevent the death receptor-mediated apoptosis by up-regulation of antiapoptotic molecules such as cIAP-2 through PI3K/Akt pathway.

Even if p38 MAPK pathway is not involved in IL-12-induced T cell proliferation and antiapoptotic function, this pathway is required for IL-12-induced IFN- production from activated CD4⁺ T cells (14, 15). It has also been reported that MKK6/p38 MAPK pathway is involved in IL-12-induced STAT-4 serine phosphorylation that is important for the trans-activation activity of STAT-4 (12, 13). Although we have demonstrated that the PI3K/Akt pathway is not critical for IL-12-induced IFN- induction, it should be further studied whether this pathway is associated with STAT-4 serine phosphorylation. Consequently, these results suggest that IL-12 differently employs signaling pathways such as PI3K/Akt, MKK6/p38 MAPK, and Jak/STAT to perform diverse functions of activated CD4⁺ T cells.

	Acknowledgments

 
We thank Sang Chun Lee for devoted animal care and Chang Geun Lee and Kwan Hyuck Baek for technical assistance.

	Footnotes

 
¹ This work was supported by a National Research Laboratory grant from the Korea Institutes of Science and Technology Evaluation and Planning (2000-N-NL-01-C-202).

² Address correspondence and reprint requests to Dr. Young Chul Sung, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang, 790-784, Korea. E-mail address: ycsung{at}postech.ac.kr

³ Abbreviations used in this paper: Jak, Janus kinase; cdk, cyclin-dependent kinase; cIAP, cellular inhibitor of apoptosis protein; Erk, extracellular signal-regulated kinase; GRR, IFN- response region; LN, lymph node; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase.

Received for publication April 17, 2002. Accepted for publication July 25, 2002.

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