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Stat3 Activation Is Responsible for IL-6-Dependent T Cell Proliferation Through Preventing Apoptosis: Generation and Characterization of T Cell-Specific Stat3-Deficient Mice¹

Kiyoshi Takeda^*, Tsuneyasu Kaisho^*, Nobuaki Yoshida, Junji Takeda, Tadamitsu Kishimoto^§ and Shizuo Akira^2,*

^* Department of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Research Institute, Osaka Maternal and Child Health Center, Izumi, Osaka, Japan; Department of Environmental Medicine, Osaka University Medical School, Osaka, Japan; and ^§ Osaka University, Suita, Osaka, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stat3, a member of STAT, is activated by a variety of cytokines such as IL-6 family of cytokines, granulocyte CSF, epidermal growth factor, and leptin. A recent study with mice genetically deficient in the Stat3 gene has revealed its important role in the early embryogenesis. To assess the function of Stat3 in adult tissues, we disrupted the Stat3 gene specifically in T cells by conditional gene targeting using Cre-loxP system. In Stat3-deficient T cells, IL-6-induced proliferation was severely impaired. IL-6 did not enhance cell cycle progression, but prevented apoptosis of normal T cells. In contrast, IL-6 did not prevent apoptosis of Stat3-deficient T cells. Antiapoptotic protein, Bcl-2, was normally up-regulated in response to IL-6 even in Stat3-deficient T cells. These results demonstrate that Stat3 activation is involved in IL-6-dependent T cell proliferation through prevention of apoptosis independently of Bcl-2.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Amember of STAT is a latent cytoplasmic transcription factor that mediates cytokine signal transduction (1, 2, 3, 4). Recent studies using mice deficient in STAT family members have revealed the essential role of STAT proteins in cytokine-mediated biologic functions (5, 6, 7, 8, 9, 10, 11, 12, 13). STAT proteins were recognized initially as transcription factors that were involved in expressions of specific genes, but not required for cell proliferation (3, 14). However, Stat4- and Stat6-deficient mice were severely impaired in IL-12- and IL-4-induced proliferation of T cells, respectively. In addition, studies with the mutated erythropoietin receptor and the dominant negative Stat5 protein demonstrated that Stat5 is crucial for erythropoietin- and IL-3-dependent cell proliferation, respectively (15, 16, 17). Stat5a-deficient mice were defective in IL-2-induced T cell proliferation due to the impaired expression of the IL-2R -chain (18). Stat1 has been shown to be involved in cell cycle arrest through induction of p21^WAF1 (19). Stat6 and Stat4 have been shown to be involved in cell cycle control by regulating p27^Kip1 expression. (20). Furthermore, Stat1, Stat3, and Stat5 have been shown to be constitutively phosphorylated in Src-transformed cell lines (21, 22). Thus, several lines of evidences indicate that STAT proteins are involved in cell proliferation.

Stat3 was identified originally as an acute phase response factor that was tyrosine phosphorylated by IL-6 (23, 24). Stat3 has been shown to be activated by a variety of cytokines. These include IL-6 family of cytokines, granulocyte CSF, epidermal growth factor, IFN-, leptin, IL-2, and IL-10 (1, 25). Constitutive activation of Stat3 has been observed in several T cell leukemias, indicating the important role of Stat3 in T cell oncogenesis or proliferation (26, 27, 28, 29, 30). However, it remains unclear how Stat3 activation contributes to proliferation of T cells.

We previously intended to generate Stat3-deficient mice to examine the in vivo role of Stat3. Stat3-deficient mice unexpectedly died during their early embryogenesis (31). Therefore, in an attempt to assess the role of Stat3 in T cells, we have utilized Cre-loxP recombination system to generate mice in which Stat3 is deficient in T cell-specific manner (32, 33, 34, 35). We first generated floxed-Stat3 mice, in which two loxP sites were introduced into 5' and 3' of exon encoding tyrosine residue critical for STAT activation. Floxed-Stat3 mice were mated with transgenic mice expressing Cre recombinase specifically in T cells under the control of Lck promoter (33, 36). T cells from these mice displayed the severely impaired proliferative response to IL-6 plus costimuli. The severely impaired IL-6-induced T cell proliferation was due to the profound defect in IL-6-mediated prevention of apoptosis in Stat3-deficient T cells. However, antiapoptotic protein, Bcl-2, was normally expressed in Stat3-deficient T cells. These data indicate that IL-6-induced Stat3 activation is indispensable for antiapoptotic signal independently of Bcl-2, resulting in enhanced T cell proliferation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of the targeting vector

A targeting vector was constructed to delete exon 22, which encodes tyrosine residue essential for STAT activation. The SpeI-HindIII-digested 3-kb fragment of the Stat3 genomic DNA containing exon 21 was inserted between two loxP sites of the vector pKSTKNEOLOXP, which has herpes simplex virus thymidine kinase and loxP-flanked pGK-neo. Then the 1-kb genomic fragment 5' upstream of exon 21 and 3' downstream HindIII-HindIII 5-kb genomic fragment were inserted.

Generation of floxed-Stat3 mice

The linearized targeting vector was electroporated into E14.1 embryonic stem (ES)³ cells. The doubly resistant clones to G418 and ganciclovir were screened for homologous recombination by PCR. The correctly targeted clones were confirmed by Southern blot analysis with the probe indicated in Figure 1A. One of the targeted clones was then transiently transfected with pIC-Cre to delete pGK-neo gene flanked by two loxP sites (32). The clones that became sensitive to G418 were subjected to Southern blot analysis to detect Stat3 flox and Stat3 clones. Three Stat3 flox ES clones were microinjected into C57BL/6 blastocysts to generate chimeric mice. Two chimeric mice successfully transmitted the germline. (Stat3^flox/+)F₁ mice were intercrossed to generate Stat3^flox/flox mice.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Generation of T cell-specific Stat3-deficient mice. A, Construction of the targeting vector. a, Genomic structure of the mouse Stat3 gene. b, The targeting vector. Exon 21 was flanked by two loxP sequences. Third loxP sequence was introduced to flank neo gene. c, The targeted allele contained three loxP sequences and neo gene. Cre-mediated deletion was expected to produce Stat3flox allele (d) and Stat3 allele (e). Solid triangle denotes loxP sequence. E, EcoRI; H, HindIII. B, Southern blot analysis of thymocytes. DNA was extracted from thymocytes from the indicated mice, electrophoresed, transferred to nylon membrane, and hybridized with the probe indicated in A. C, Western blot analysis of thymocytes and liver cells. Lysates from 1 x 107 cells stimulated with IL-6 for 30 min were immunoprecipitated with anti-Stat3 Ab, and immunoblotted with anti-phosphotyrosine Ab (-PY) or anti-Stat3 Ab. D, Nucleotide sequence and predicted amino acid pattern of the RT-PCR products from thymocytes of Lck-Cre/Stat3flox/- mice.

Lck-Cre transgenic mice (36) were bred with formerly generated Stat3-deficient mice to generate mice carrying lck-cre and heterozygous Stat3 (Lck-Cre/Stat3^+/-) genes. These mice were mated with Stat3^flox/flox or Stat3^flox/+ mice. Offspring carrying lck-cre and floxed Stat3 genes (Lck-Cre/Stat3^flox/+ or Stat3^flox/-) and wild-type Stat3 gene (Lck-Cre/Stat3^+/+ or Lck-Cre/Stat3^+/-) were used for further analysis.

Western blot analysis

Thymocytes or Con A-activated splenic T cells (2 x 10⁷) were stimulated with the indicated cytokines for 30 min. IL-2, IL-6, and IL-7 were purchased from Genzyme (Cambridge, MA). Leukemia-inhibitory factor (LIF) was from Life Technologies (Grand Island, NY), and oncostatin M was from R&D Systems (Minneapolis, MN). Cells were solubilized with ice-cold lysis buffer containing 0.5% Nonidet P-40, 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, 1 mM Na₃VO₄, and 5 µg/ml aprotinin. Whole cell lysates were incubated with Ab to Stat3 (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA) or Stat5b (C-17; this Ab recognizes both Stat5a and Stat5b) (Santa Cruz Biotechnology) and protein A-Sepharose (Pharmacia, Uppsala, Sweden) for 4 h at 4°C. Immunoprecipitates were separated on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with anti-phosphotyrosine mAb (4G10; Upstate Biotechnology, Lake Placid, NY). Bound Ab was visualized with an enhanced chemiluminescence system (DuPont, Boston, MA).

Preparation of splenic T cells

Splenic T cells were purified as described previously (8). Briefly, splenic T cells were enriched by nylon wool column passage. Enriched T cells were incubated with anti-I-Ab mAb for 30 min on ice, then washed, and incubated with rabbit complement (Low-Tox-M; Cedar Lane, Ontario, Canada) for 30 min at 37°C. Purified T cells were analyzed by staining with FITC-conjugated anti-CD3 mAb (PharMingen, San Diego, CA), and >95% CD3-positive cells were used for experiments.

T cell proliferation assay

Thymocytes (1 x 10⁵) or splenic T cells (5 x 10⁴) were cultured in 96-well plates in the presence of the indicated cytokines and/or mitogens for 72 h. A quantity amounting to 1 µCi of [³H]thymidine was pulsed for the last 12 h. Concentrations of cytokines and mitogens used in this assay were as follows: IL-2, 20 ng/ml; IL-6, 10 ng/ml; IL-7, 10 ng/ml (Genzyme); anti-CD3 Ab, 100 ng/ml (PharMingen); PMA, 10 ng/ml; Con A, 2.5 µg/ml; and calcium ionophore, 10 µM (Sigma, St. Louis, MO).

In the case of inhibition of IL-2-induced proliferation, 20 µg/ml of anti-IL-2-neutralizing Ab (Genzyme, Cambridge, MA) was added in the cell cultures.

Flow-cytometric analysis

Cells were stained with biotin-conjugated anti-IL-6R mAb following FITC-streptavidin, or rat anti-gp130 mAb following FITC-conjugated anti-rat Ig. These Abs were purchased from PharMingen. Rat anti-gp130 mAb was kindly provided by Dr. T. Taga (37). Stained cells were analyzed on FACSCalibur using CellQuest software (Becton Dickinson, Lincoln Park, NJ).

Cell cycle analysis

Splenic T cells were cultured with 10 ng/ml IL-6 and/or 100 ng/ml anti-CD3 Ab for 48 h in the presence or absence of 20 µg/ml anti-IL-2 Ab (Genzyme) plus 10 µg/ml anti-IL-2R Ab (clone 3C7; PharMingen). At 48-h culture period, cells were harvested, washed, and fixed in 70% ethanol for 1.5 h at -20°C. Fixed cells were pelleted, incubated in 1 ml of 50 µg/ml RNase for 20 min at 37°C, and washed. Then cells were suspended with 50 µg/ml propidium iodide (PI), incubated for 5 min at room temperature, and analyzed on FACSCalibur (Becton Dickinson).

Detection of apoptotic cells

Splenic T cells were cultured in RPMI 1640 containing 0.1% FCS with or without 10 ng/ml IL-6 or 100 ng/ml anti-CD3 Ab for 5 h. Cells were harvested, washed, and stained with annexin V and PI, according to the manufacturer’s instructions (R & D Systems). Cells were analyzed on FACSCalibur.

Intracellular staining

Thymocytes or splenic T cells were cultured with or without 10 ng/ml IL-6 for 3 days. Cells were stained with Cy-Chrome-conjugated anti-CD4 mAb and phycoerythrin-conjugated anti-CD8 mAb (PharMingen), and washed. Cells were resuspended with 100 µl of PBS containing 4% paraformaldehyde, incubated for 20 min on ice, and washed. Then cells were resuspended with 50 µl of PBS containing 1% FCS, 0.1% sodium azide, 0.1% saponin (Sigma), and 5 µg/ml hamster anti-Bcl-2 mAb (PharMingen). After 30-min incubation on ice, cells were washed and further stained with FITC-conjugated anti-hamster Ig (Vector, Burlingame, CA) for 30 min on ice. Cells were analyzed on FACSCalibur. In the case of Bcl-xl, cells were incubated with mouse anti-Bcl-xl mAb (Transduction Laboratories, Lexington, KY), followed by FITC-conjugated goat anti-mouse IgG (Immunotech, Marseille, France).

RT-PCR analysis

Splenic T cells (1 x 10⁷) were cultured with or without 100 ng/ml of IL-6 for 3 h. Total RNA was extracted with TRIzol reagent (Life Technologies), reverse transcribed with Superscript II (Life Technologies), and amplified by PCR using primers specific for the mouse Bcl-2, Bcl-xl, BAG-1, and ß-actin gene.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of floxed-Stat3 mice

Inactivation of the Stat3 gene in ES cells by the classical targeting method led to early embryonic lethality and could not reveal its role in adult tissues or organs (31). To clarify the function of Stat3 in T cells, we utilized the Cre-loxP system for disruption of the Stat3 gene (32, 33). A target vector was constructed so that Stat3 tyrosine phosphorylation site could be deleted by expression of Cre protein. The neomycin resistance (neo) gene flanked by two loxP sites were introduced into intron 21 of the Stat3 gene (Fig. 1A). An additional loxP site was inserted into intron 22. The herpes simplex virus thymidine kinase was ligated at the 5' end of the homologous region. ES cells were electroporated with this vector and selected in the presence of G418 and ganciclovir. Homologous recombinant clones were identified by PCR and confirmed by Southern blot analysis.

Cre protein was transiently expressed in the targeted ES clones to delete the loxP-flanked neo gene. Two types of clones, Stat3 flox and Stat3, were expected from ES clones that became sensitive to G418 (Fig. 1A). However, Stat3 clones could not be obtained, although Stat3 flox ES clones could be easily obtained. Several G418-sensitive ES clones showed the apparently differentiated morphology, and most of them displayed Stat3 genotype by Southern blot analysis, supporting the previous finding that the Stat3 protein lacking the tyrosine phosphorylation site acts as a dominant negative form and induces differentiation of ES cells even in the presence of LIF (38, 39).

The Stat3 flox ES clones were used to generate chimeric mice, which successfully contributed the germline transmission. Mice homozygous for the loxP-flanked (floxed) Stat3 gene (Stat3^flox/flox) were born at the expected Mendelian ratios and presented no obvious abnormalities.

Generation of T cell-specific Stat3-deficient mice

To generate mice in which Stat3 was deficient in T cell-specific manner, Stat3^flox/flox mice were mated to mice carrying both the Cre transgene under the lck promoter and the heterozygous Stat3 gene (Lck-Cre/Stat3^+/-). Mice with Lck-Cre and the floxed-Stat3 gene (Lck-Cre/Stat3^flox/+ or Lck-Cre/Stat3^flox/-) were born alive and grew healthy. To evaluate the efficiency of the Cre-mediated deletion of the floxed gene, we performed Southern blot analysis of thymocytes (Fig. 1B). In thymocytes from nontransgenic floxed-Stat3 mice, 8-kb band was detected. In thymocytes from Lck-Cre/Stat3^flox/+ or Lck-Cre/Stat3^flox/- mice, the 8-kb band disappeared, but a 4.1-kb band corresponding to Stat3 allele appeared. Thus, complete Cre-mediated deletion occurred in thymocytes of Lck-Cre/floxed-Stat3 mice at DNA level. We next analyzed the Cre-mediated deletion at protein level (Fig. 1C). Thymocytes were stimulated with IL-6 for 30 min, lysed, and incubated with anti-Stat3 Ab, which recognizes C-terminal portion of Stat3 protein. Immunoprecipitants were Western blotted by anti-phosphotyrosine or anti-Stat3 Ab. Stat3 protein was readily detected in thymocytes from mice carrying wild-type allele of Stat3 locus (lck;+/+ and lck;flox/+). However, only a little amount of Stat3 protein was observed in thymocytes from Lck-Cre/Stat3^flox/- mice. In addition, a protein band with slightly decreased m.w. was detected in Lck-Cre/Stat3^flox/- or Lck-Cre/Stat3^flox/+ mice (Fig. 1C, middle). To precisely analyze this protein, we performed RT-PCR analysis with total RNA from thymocytes of Lck-Cre/Stat3^flox/- mice. The RT-PCR products from thymocytes of Lck-Cre/Stat3^flox/- mice consisted of two bands with different sizes. The length of the longer band corresponds to normal Stat3 protein. Sequence analysis of the shorter band revealed the lack of the region encoding exon 21 and a part of exon 22 from normal Stat3 cDNA (Fig. 1D). Thus, Cre-mediated deletion led to the production of a truncated form of Stat3 protein (Stat3 protein). This Stat3 protein contained no tyrosine residue and mitogen-activated protein/kinase recognition site, both of which are important for STAT activation.

IL-6 stimulation of thymocytes from Lck-Cre/Stat3^flox/- mice did not induce phosphorylation of Stat3 protein, although a small amount of wild-type Stat3 protein was detected (Fig. 1C, top). In addition, activation of Stat3 was severely reduced in thymocytes of Lck-Cre/Stat3^flox/+ mice, in which wild-type Stat3 protein abundantly existed, indicating that the truncated Stat3 protein acts as a dominant negative form for activation of Stat3. Production or phosphorylation of Stat3 protein in liver cells was not affected in Lck-Cre/Stat3^flox/- mice, indicating that Cre-mediated deletion of the floxed gene occurred specifically in T cells (Fig. 1C, bottom).

We next analyzed whether dominant negative Stat3 protein affects activation of Stat5 protein. Thymocytes from control and mutant mice were stimulated with IL-2 or IL-7, and Stat5 activation was analyzed. As shown in Figure 2A, Stat5 was equally phosphorylated in response to IL-2 or IL-7 in thymocytes of Lck-Cre/Stat3^flox/+ or Lck-Cre/Stat3^flox/- mice when compared with nontransgenic mice, demonstrating that Stat5 proteins were normally activated in these mice (Fig. 2A).

View larger version (50K): [in this window] [in a new window]   FIGURE 2. Western blot analysis. A, Thymocytes from the indicated mice were stimulated with IL-2 or IL-7 for 30 min, lysed, and incubated with anti-Stat5 Ab, which recognizes both Stat5a and Stat5b. Immunoprecipitates were blotted with anti-phosphotyrosine Ab (-PY), and reblotted with anti-Stat5 Ab. B, Thymocytes were stimulated with the indicated cytokines for 30 min. Splenic T cells were cultured with 2.5 µg/ml of Con A for 3 days, starved in 0.1% FCS medium for 2 h, and stimulated with IL-2 for 30 min. Cells were lysed, incubated with anti-Stat3 Ab, and immunoblotted with anti-phosphotyrosine Ab.

We examined whether the abrogation of Stat3 activation affects T cell development. Total nucleated cell numbers of lymphoid organs such as thymus, spleen, and lymph nodes in Lck-Cre/Stat3^flox/- mice were almost the same as those in Lck-Cre;+/+ mice (data not shown). In addition, flow-cytometric analysis of these organs revealed that CD3-positive T cell population and CD4/CD8 ratios in T cells of Lck-Cre/Stat3^flox/- mice were not altered as compared with Lck-Cre;+/+ mice (data not shown). These results indicate that Stat3 activation is not involved in development and intrinsic maintenance of T cells.

Growth of Stat3-deficient T cells in response to cytokines

We analyzed Stat3 activation by cytokines in T cells (Fig. 2B). Stat3 was phosphorylated by IL-6 family of cytokines such as IL-6, LIF, and oncostatin M in thymocytes. In addition, Stat3 was phosphorylated in response to IL-7, which has been shown to activate Stat5. Although IL-2-induced Stat3 phophorylation was faint in freshly isolated thymocytes or splenic T cells, IL-2-induced phosphorylation of Stat3 was more clearly observed in Con A-activated splenic T cells. Thus, Stat3 is demonstrated to be activated by IL-6, LIF, oncostatin M, IL-2, and IL-7 in T cells.

We then examined proliferation of T cells in response to these cytokines in Lck-Cre/Stat3^flox/- mice. We first examined the effect of Stat3 deficiency upon IL-6-induced T cell proliferation. Thymocytes from Lck-Cre;+/+ mice displayed the increased proliferation in response to stimulation with IL-6 plus anti-CD3 Ab. However, thymocytes from Lck-Cre/Stat3^flox/- mice did not show any enhanced proliferation in response to IL-6 plus anti-CD3 Ab (Fig. 3A). Similarly, splenic T cells from Lck-Cre;+/+ mice showed the increased proliferative response when stimulated with IL-6 plus Con A, whereas proliferation of splenic T cells from Lck-Cre/Stat3^flox/- mice in response to IL-6 plus Con A was severely impaired (Fig. 3B). Thus, IL-6-induced proliferation was severely impaired in T cells from Lck-Cre/Stat3^flox/- mice.

View larger version (43K): [in this window] [in a new window]   FIGURE 3. Cytokine-induced proliferation of T cells. A, Thymocytes were cultured with the indicated stimulation for 72 h. [3H]Thymidine was pulsed for the last 12 h. Concentration of each stimulation was indicated in Materials and Methods. B, Splenic T cells were cultured with the indicated stimulation for 72 h. 3H uptake was measured.

IL-6 is not directly involved in cell cycle progression in T cells

We next investigated the molecular mechanism of impaired IL-6-induced proliferation of T cells from Lck-Cre/Stat3^flox/- mice. We first examined the expression of the receptors for IL-6 on the surface of T cells. Flow-cytometric analysis of splenic T cells was performed using mAb against IL-6R and gp130, a signal transducer of IL-6. Expression of IL-6R and gp130 was not altered between T cells from Lck-Cre;+/+ and Lck-Cre/Stat3^flox/- mice (Fig. 4). In addition, in vitro IL-6 stimulation did not increase the expression of both receptors in Lck-Cre;+/+ and Lck-Cre/Stat3^flox/- T cells (data not shown). These indicate that lack of IL-6 response in Stat3-deficient T cells is not a result of the reduced expression of the receptors. We next examined whether proliferation of T cells in response to IL-6 plus anti-CD3 Ab or Con A is due to the increased production of IL-2, a potent growth factor for T cells. As shown in Figure 5A, treatment with anti-IL-2 Ab did not reduce the proliferative response to IL-6 plus anti-CD3 Ab, indicating that IL-6-induced T cell proliferation is not a result of the increased IL-2 production from T cells.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Normal expression of the IL-6R complex on T cells from Lck-Cre/Stat3flox/- mice. Splenic T cells from wild-type and Lck-Cre/Stat3flox/- mice were stained with anti-IL-6R Ab (left) or anti-gp130 Ab (right), followed by FITC-conjugated second Ab.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Involvement of IL-6 in T cell proliferation. A, Wild-type splenic T cells were cultured with the indicated stimulation in the presence or absence of 20 µg/ml anti-IL-2-neutralizing Ab. 3H uptake was measured. B, Wild-type splenic T cells were cultured with 10 ng/ml IL-6 and/or 100 ng/ml anti-CD3 Ab for 48 h in the presence or absence of anti-IL-2 Ab plus anti-IL-2R Ab. Then cells were fixed and stained with PI. Percentage of the population in S phase and G2/M phase was shown.

IL-6 does not prevent apoptosis of Stat3-deficient T cells

We next analyzed whether IL-6 could rescue apoptosis of T cells by staining with both PI and annexin V (Fig. 6). Apoptotic cells become annexin V positive at its early phase due to the loss of cell membrane phospholipid asymmetry, and then become PI positive. In vitro 5-h culture of Lck-Cre;+/+ T cells in 0.1% FCS showed about 40% (33 + 6.2%) of annexin V-positive apoptotic cells. Treatment with anti-CD3 Ab did not cause a significant change. In contrast, addition of IL-6 significantly decreased the population of annexin V-positive cells. This demonstrates that IL-6 rescues in vitro cultured T cells from apoptotic cell death. However, addition of IL-6 did not lead to any reduction of annexin V-positive T cells from Lck-Cre/Stat3^flox/- mice, demonstrating that Stat3-deficient T cells are devoid of IL-6-mediated antiapoptotic response. Thus, our results demonstrate that IL-6-induced Stat3 activation is essential for IL-6-mediated antiapoptotic response.

View larger version (45K): [in this window] [in a new window]   FIGURE 6. IL-6-induced prevention of T cell apoptosis was impaired in Lck-Cre/Stat3flox/- mice. Splenic T cells from wild-type and Lck-Cre/Stat3flox/- mice were cultured in 0.1% FCS culture medium in the presence of 10 ng/ml IL-6 and/or 100 ng/ml anti-CD3 Ab for 5 h. Then cells were stained with FITC-annexin V and PI.

Bcl-2 has been shown to protect T cells against several apoptotic stimuli. Indeed, Bcl-2-deficient mice displayed the shortened life span of T cells (40). Therefore, we examined whether expression of Bcl-2 is defective in Stat3-deficient T cells. Thymocytes or splenic T cells were cultured with or without IL-6 for 3 days, and Bcl-2 expression was analyzed by intracellular staining with anti-Bcl-2 Ab. Bcl-2 expression was up-regulated by IL-6 in CD4^- single-positive, CD8^- single-positive, and double-negative thymocytes of Lck-Cre;+/+ mice (Fig. 7A). Bcl-2 expression was also up-regulated by IL-6 in splenic T cells of Lck-Cre;+/+ mice (Fig. 7A). Interestingly, IL-6 stimulation of thymocytes and splenic T cells from Lck-Cre/Stat3^flox/- mice normally up-regulated Bcl-2 expression. Thus, Stat3-deficient T cells displayed normal IL-6-induced Bcl-2 expression, although these cells were devoid of IL-6-induced enhancement of cell survival. Other Bcl-2-related proteins, such as Bcl-xl and BAG-1, are also shown to be responsible for the prevention of apoptosis of lymphocytes (41, 42, 43). Flow-cytometric analysis revealed that expression of Bcl-xl was not enhanced in splenic T cells cultured in medium alone or IL-6, although dramatically enhanced by stimulation with IL-2 in wild-type mice (Fig. 7B). RT-PCR analysis demonstrated that mRNA expression of BAG-1 and Bcl-xl was not augmented by IL-6 in splenic T cells from wild-type and Lck-Cre/Stat3^flox/- mice (Fig. 7C). Thus, IL-6 did not enhance the expression of Bcl-xl or BAG-1 in T cells, indicating that both proteins are not involved in IL-6-mediated antiapoptotic activity. Taken together, our results indicate that IL-6-induced Stat3 activation is responsible for antiapoptotic signals independently of Bcl-2 or its related proteins.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Normal IL-6-induced Bcl-2 expression in T cells of Lck-Cre/Stat3flox/- mice. A, Thymocytes or splenic T cells were cultured with or without 10 ng/ml IL-6 for 72 h. Cells were intracellularly stained for Bcl-2, as described in Materials and Methods. B, Wild-type splenic T cells were cultured with 10 ng/ml IL-6 or IL-2 for 72 h. Cells were stained for Bcl-xl and analyzed by flow cytometry. C, Splenic T cells were cultured with or without 100 ng/ml IL-6 for 3 h. Total RNA was extracted, reverse transcribed, and PCR amplified with primers specific for the indicated genes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-6-induced Stat3 activation is responsible for antiapoptotic signal in T cells

Costimulation with IL-6 and anti-CD3 Ab did not enhance proliferation of Stat3-deficient T cells. Although IL-6 has been shown to be a costimulant for T cell proliferation, IL-6 itself did not have a mitogenic activity for T cells (46). Indeed, IL-6 did not induce any progression of cell cycle in T cells. We first suspected that the severely impaired IL-6-induced proliferation of Stat3-deficient T cells might be due to the impaired IL-2 production, because anti-CD3 Ab- or Con A-stimulated T cells produce IL-2. However, anti-IL-2-neutralizing Ab did not reduce proliferation of T cells stimulated with IL-6 and anti-CD3 Ab, demonstrating that the impaired IL-6-induced proliferation of Stat3-deficient T cells was not a result of the impaired IL-2 production. In addition, two components for the IL-6R complex, IL-6R and gp130, were normally expressed on the surface of Stat3-deficient T cells, indicating that the impaired IL-6-induced T cell proliferation was not a result of defective IL-6R expression. This is unlike the case in Stat5a-deficient mice, which were defective in IL-2-induced T cell proliferation due to the impaired IL-2-induced IL-2R -chain (18). Recently, IL-6 has been shown to inhibit apoptosis of resting T cells (47). These facts prompted us to hypothesize that IL-6-induced prevention of T cell apoptosis contributes to the enhanced proliferation of anti-CD3 Ab-stimulated T cells. As expected, IL-6 rescued in vitro cultured T cells from apoptosis. Moreover, IL-6 did not prevent apoptosis of Stat3-deficient T cells. Thus, our results demonstrate that IL-6-induced Stat3 activation is critically involved in prevention of T cell apoptosis, and further indicate that the activity of IL-6 as a costimulant for T cell proliferation attributes to the prevention of T cell apoptosis through Stat3 activation.

We further examined whether IL-6-mediated Stat3 activation induces expression of the antiapoptosis-related genes. Bcl-2 is a well-characterized protein that prevents apoptosis in lymphocytes. Bcl-2 is shown to be expressed in peripheral T cells, CD4⁺, CD8⁺, and CD4^-CD8^- thymocytes, but not in CD4⁺CD8⁺ thymocytes, which are exposed to the programmed cell death under negative selection of T cell development (48, 49). T cells from Bcl-2-deficient mice were highly sensitive to apoptosis (40, 50, 51). In addition, decreased Bcl-2 expression was observed in IL-7- or IL-7R-deficient mice, which displayed the profound decrease in T and B cells (52). From these findings, we speculated that cytokine-induced Bcl-2 expression is important for prevention from apoptosis of in vitro cultured T cells. Indeed, stimulation with IL-2, IL-7, or IL-6 up-regulated Bcl-2 expression and increased the survival of T cells from wild-type mice (data not shown). Surprisingly, IL-6 normally up-regulated Bcl-2 expression even in Stat3-deficient T cells, in which IL-6-induced prevention of apoptosis was not observed. These findings imply that Stat3 activation is involved in IL-6-mediated antiapoptotic activity independently of Bcl-2. Expression of other Bcl-2-related antiapoptotic proteins, such as Bcl-xl and BAG-1, was not induced by IL-6 stimulation. Therefore, IL-6-mediated Stat3 activation may induce expression of as yet unidentified antiapoptotic genes.

STAT activation in lymphocyte proliferation

The involvement of STAT proteins in lymphocyte proliferation has been discussed in several knockout mice of STAT family (7, 8, 9, 10, 11, 18, 20). First, proliferation of lymphocytes in responses to IL-4 and IL-12 has been shown to be impaired in Stat6- and Stat4-deficient mice, respectively (7, 8, 9, 10, 11). But, it remained controversial how STAT proteins are involved in proliferation. However, recent studies have revealed the mechanisms for the regulation of cell proliferation by STAT proteins. Stat5a-deficient mice have been shown to be defective in IL-2-mediated T cell proliferation due to the impaired IL-2-induced IL-2R -chain expression (18). This study implies that STAT proteins indirectly control lymphocyte proliferation through expressions of genes that are necessary for cell proliferation. In contrast, the study using Stat4- and Stat6-deficient mice has demonstrated that these mice were not defective in expression of the receptor, but impaired in IL-12- and IL-4-induced down-regulation of expression of p27^Kip1, one of the inhibitors of cyclin-dependent kinases (CDK) that progress cell cycle (20). Similarly, Stat1 has been shown to regulate cell cycle machinery in nonlymphoid cells through IFN-induced expression of p21^WAF1, another inhibitor of CDK (19). These studies imply that STAT proteins control cell cycle through regulating expression of CDK inhibitors. Our present study demonstrates that IL-6-induced Stat3 activation does not directly progress cell cycle; however, it is indispensable for prevention of apoptosis, resulting in enhanced cell proliferation. Thus, studies using knockout mice have established that STAT proteins regulate cell proliferation through distinct mechanisms, such as gene expressions responsible for cell proliferation, regulation of CDK inhibitor expression, and prevention of apoptosis.

In addition to impaired IL-6-induced proliferation, T cells from Lck-Cre/Stat3^flox/- mice displayed partial defect in IL-2-induced T cell proliferation. We observed partial impairment in IL-2-induced IL-2R expression in Lck-Cre/Stat3^flox/- T cells (unpublished data). This indicates that the defective IL-2-induced T cell proliferation is due to the defective IL-2-induced IL-2R expression, like the case of Stat5a-deficient mice (18). However, high concentration of IL-2 induced normal expression of IL-2R in Lck-Cre/Stat3^flox/- T cells, and these T cells proliferated as well as wild-type T cells in response to IL-2 (unpublished data). These may explain the normal population of peripheral T cells in Lck-Cre/Stat3^flox/- mice.

Constitutive activation of Stat3 was observed in several T cell leukemias, such as adult T cell leukemia, Sezary syndrome, and mycosis fungoides (26, 27, 28, 29, 30). Stat3 activation in T cells, as demonstrated in this study, may contribute to immortalization of T cells and finally to T cell oncogenesis. Inhibition of Stat3 activation may be an effective therapeutic maneuver for these T cell leukemias.

	Acknowledgments

 
We thank T. Taga for providing anti-gp130 mAb, W. Reith for providing pKSTKNEOLOXP plasmid, Y. Kataoka for technical assistance, and T. Aoki for secretarial assistance.

	Footnotes

 
¹ This work was supported by CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST) and by grants from the Ministry of Education of Japan.

² Address correspondence and reprint requests to Dr. Shizuo Akira, Department of Biochemistry, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, 663-8501, Japan. E-mail address:

³ Abbreviations used in this paper: ES, embryonic stem; CDK, cyclin-dependent kinase; LIF, leukemia-inhibitory factor; PI, propidium iodide.

Received for publication April 6, 1998. Accepted for publication June 29, 1998.

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