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Originally published In Press as doi:10.1074/jbc.C300496200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 8, 6905-6910, February 20, 2004
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SOCS3 Is a Physiological Negative Regulator for Granulopoiesis and Granulocyte Colony-stimulating Factor Receptor Signaling*

Akiko Kimura{ddagger}§, Ichiko Kinjyo{ddagger}, Yumiko Matsumura{ddagger}, Hiroyuki Mori{ddagger}, Ryuichi Mashima{ddagger}, Mine Harada§, Kenneth R. Chien¶, Hideo Yasukawa||, and Akihiko Yoshimura{ddagger}**

From the {ddagger}Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, the §First Department of Internal Medicine, Graduate School of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, theInstitute of Molecular Medicine and Department of Medicine, University of California San Diego, La Jolla, California 92093-0641, and the||Cardiovascular Research Institute and The Third Department of Internal Medicine, Kurume University, 67 Asahi-machi, Kurume 830-0011, Japan

Received for publication, November 16, 2003 , and in revised form, December 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The suppressor of cytokine signaling-3 (SOCS3/CIS3) has been shown to be an important negative regulator of cytokines, especially cytokines that activate STAT3. To examine the role of SOCS3 in neutrophils and the granulocyte colony-stimulating factor (G-CSF) signaling in vivo, we compared neutrophils from two types of conditional knockout mice, LysM-Cre:SOCS3fl/fl mice and Tie2-Cre:SOCS3fl/fl mice, in which the Socs3 gene had been deleted in mature neutrophils and hematopoietic stem cells, respectively. The size of the G-CSF-dependent colonies from Tie2-Cre:SOCS3fl/fl mouse bone marrow was much larger than that of colonies from control wild-type mice, while the size of interleukin-3-dependent colonies was similar. Moreover, LysM-Cre:SOCS3fl/fl mice had more neutrophils than SOCS3fl/fl mice, suggesting that SOCS3 is a negative regulator of G-CSF signaling in neutrophils. Consistent with this notion, G-CSF-induced STAT3 as well as mitogen-activated protein kinase activation was much stronger and prolonged in SOCS3-deficient mature neutrophils than in wild-type neutrophils. The preventive effect of G-CSF on apoptosis was more prominent in SOCS3-deficient mature neutrophils than in control neutrophils. These data indicate that SOCS3 negatively regulates granulopoiesis and G-CSF signaling in neutrophils and may contribute to neutrophilia or neutropenia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Signaling from cytokine receptors is initiated by receptor oligomerization that is induced by cytokine binding, which brings associated Janus kinases (JAKs)1 into close apposition and allows their cross-phosphorylation and activation. The active JAKs phosphorylate tyrosine residues on the receptors, which leads to the recruitment and activation of various signal-transduction proteins, including the signal transducer and activator of transcription (STAT) family of transcription factors (1, 2). The Ras-mitogen-activated protein kinase (ERK) pathway is another major signaling pathway that is downstream of JAKs. The strength of cytokine signals is regulated, in part, by a family of endogenous JAK kinase inhibitor proteins referred to as suppressors of cytokine signaling (SOCS), cytokine-inducible Src homology 2 (SH2) proteins (CIS), or STAT-induced STAT inhibitors (SSI) (3-7). Among these, SOCS3 is strongly induced by a variety of cytokines and other stimulations, including IL-6, IL-10, granulocyte-colony stimulating factor (G-CSF), EPO, EGF, leptin, and LPS (1-10). Both SOCS1 and SOCS3 have an N-terminal kinase inhibitory region and inhibit JAK tyrosine kinase activity. However, SOCS3 inhibits JAKs through binding to cytokine receptor tyrosine residues, while SOCS1 directly binds to JAKs (11). The interaction of SOCS3 with cytokine receptors through its SH2 domain with high affinity probably ensures relatively specific inhibition of a particular cytokine signaling (3).

SOCS3-deficient mice die as a result of placental defects during embryonic development (12-14). Embryonic lethality can be rescued by replacing wild-type placental function, demonstrating the essential role of SOCS3 in placental development and its non-essential function in embryo development (13). Rescued SOCS3-deficient mice show perinatal lethality with cardiac hypertrophy, which suggests that SOCS3 is essential for negative regulation of the leukemia inhibitory factor receptor signaling. The generation of conditional knock-out (KO) mice using the Cre-loxP system revealed that SOCS3 is also essential for the suppression of IL-6/gp130 signaling in macrophages. This is consistent with the high-affinity binding of SOCS3 to the Tyr-759 region of gp130 (6).

The G-CSF is the major regulator of differentiation and activation in the granulocyte lineage. The G-CSF induces activation of the JAK-STAT (9, 10, 15-18) and the Ras-Raf-ERK pathways (18, 19). Overexpression studies have suggested that SOCS3 interacts with the G-CSF receptor and inhibits G-CSF-induced STAT3 activation (9, 20). However, the physiological relevance of these studies has not been shown using SOCS3-deficient animals. In this study, we first demonstrate that SOCS3 is an important negative regulator of G-CSF signaling in neutrophils and regulates the survival as well as growth of neutrophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Animals—Conditional targeting of SOCS3 using the loxP system and crossing with LysM-Cre mice have been described (21, 22). To delete the Socs3 gene in hematopoietic stem cells, Tie2-Cre mice (23) were crossed with SOCS3fl/fl mice because Tie2 is expressed in hemangioblasts. Age-matched SOCS3fl/fl and LysM-Cre:SOCS3fl/fl or Tie2-Cre:SOCS3fl/fl mice were used for analysis.

Hematology—A peripheral blood smear was stained with May-Grünwald-Giemsa. Differential cell counts were scored visually on coded samples or using a K-4500 automated counter.

Preparation of Neutrophils from Peripheral Blood and the Peritoneal Cavity—To mobilize neutrophils into peripheral blood, we injected G-CSF (a gift from the Kirin Pharmaceutical Co., 8 µg/body/day) subcutaneously for 7 consecutive days. Whole peripheral blood was collected 4 h after the last injection from the orbital venus plexus, and erythrocytes were removed by hypotonic lysis. To obtain peritoneal neutrophils, 2 ml of 4% thioglycollate was injected intraperitoneally. After 4 h, peritoneal exudate cells were harvested by phosphate-buffered saline lavage. Granulocytes were enumerated, followed by staining with May-Grünwald-Giemsa. The percentage of neutrophils in white blood cells from the peritoneal cavity was about 80% in both SOCS3fl/fl mice and LysM-Cre:SOCS3fl/fl mice.

The chemotaxis assay for neutrophils was performed as described previously (24-26). Briefly, the lower well (24-well plate) contained 800 µl of Iscove's modified Dulbecco's medium (IMDM) including 1 x 10-8 M fMLP and 10% FBS, and the upper chemotaxicell (Kurabo) contained 400 µl of neutrophil (3 x 106 cells/ml) suspension. After incubation for 1 h at 37 °C, cells that passed through the membrane were counted.

The phagocytic activity of peritoneal neutrophils was assayed by incubating 5 x 104 cells of peritoneal neutrophils with 5 x 106 of Texas Red-conjugated opsonizing zymosan (TR-OZ; Molecular Probes) for 30 min. Phagocytic activity was assessed by flow cytometry (27). Superoxide anion () production was measured using superoxide dismutase-inhabitable reduction of the cytochrome c assay (28, 29).

Western Blotting—Western blotting was performed using peritoneal and peripheral neutrophils as described previously (5). Neutrophils were incubated in IMDM without G-CSF for 6 h and then stimulated with 50 ng/ml G-CSF. Approximately 5 x 105 cells/sample were used for Western blotting using the following antibodies: anti-STAT3 (sc-482; Santa Cruz), anti-tyrosine-phosphorylated STAT3 (number 9131; Cell Signaling), anti-G-CSF receptor (sc-9173; Santa Cruz), SOCS3 (IBL), ERK2 (number 330018; Cell Signaling), and phosphorylated ERK1/2 (number 9101; Cell Signaling).

Real-time RT-PCR—RT-PCR was carried out using the one-step RT-PCR kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative real-time RT-PCR was monitored using the ABI PRISM 7700 (PE Applied Biosystems), and the results were analyzed with the accompanying software. SYBR Green PCR Master Mix was used for the detection of SOCS3 and Bcl-XL.

Colony Assay—BM cells (4 x 105) were incubated in a medium (MethoCult M3234, Stem Cell Technology) containing 0.9% methylcellulose in IMDM supplemented with 1% bovine serum albumin, 10-4 M 2-mercaptoethanol, 2 mM L-glutamine, 15% FBS, 10 µg/ml bovine pancreatic insulin, 200 µg/ml human transferrin, and each of following recombinant cytokines: 10 ng/ml of murine IL-3 or 10 or 100 ng/ml of G-CSF. Colonies were counted and photographed after 7-10 days of incubation.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
SOCS3 Negatively Regulates G-CSF-dependent Colony Formation—To examine the role of SOCS3 in neutrophils and G-CSF signaling, we first examined the G-CSF-dependent colony formation of BM cells from Tie2-Cre:SOCS3fl/fl mice. The Tie2-Cre transgenic mouse line has been shown to facilitate gene targeting in endothelial and hematopoietic cells. High efficiency of recombination was found in all endothelial cells and in the majority of hematopoietic cells but not in other tissues (30). As shown in Fig. 1, A and B, efficient Socs3 gene deletion was observed in BM cells from Tie2-Cre:SOCS3fl/fl mice but not in SOCS3fl/fl mice. Granulocyte precursor frequencies were assessed by colony formation assays. BM cells were cultured with G-CSF as the only supportive cytokine to enumerate granulocyte precursors. In this medium, the number of colonies from SOCS3-deficient BM cells was slightly higher than that from wild-type BM cells (control: 229 ± 14 versus KO: 350 ± 20; p = 0.02). We found that the size of G-CSF-dependent colonies from Tie2-Cre:SOCS3fl/fl mice was much larger than that from control mice, while the size of IL-3-dependent colonies was not different (Fig. 1C). These data indicate that SOCS3 negatively regulates G-CSF-dependent granulopoiesis.



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  		FIG. 1.
Deletion of the Socs3 gene in BM cells and morphology of G-CSF-dependent colony formation of BM cells derived from Tie2-Cre:SOCS3fl/fl mice. A, the wild-type Socs3 locus, the targeted Socs3 locus in the SOCS3fl/fl allele, and the deleted Socs3 locus (SOCS3del), as well as the positions of PCR primers a, b, and c, are shown. The loxP sites flanked Exon2 of Socs3. B, PCR analysis of genomic DNA from tails (top) and BM cells (middle and bottom) with the primers indicated in A. Primers a and b were used in the top and middle panels; primers a and c were used in the bottom panel. The PCR product obtained with primers a and b from the wild-type Socs3 locus is 280 bp (WT Socs3), and that from the SOCS3fl/fl allele is 380 bp (Socs3fl). In Tie2-Cre:SOCS3fl/fl mice, a band of 250 bp obtained from primers a and c indicates the Cre-mediated deletion of Socs3 (Socs3del). C, morphology of G-CSF- and IL-3-dependent colonies of BM cells from SOCS3fl/fl and Tie2-Cre:SOCS3fl/fl mice. BM cells were cultured in a semi-solid medium containing 10 ng/ml G-CSF or IL-3 for 7 days.

 
Increased Neutrophil Numbers in LysM-Cre:SOCS3fl/fl Mice—We then examined the role of SOCS3 in mature neutrophils using LysM-Cre:SOCS3fl/fl mice. To confirm the deletion of the Socs3 gene, we extracted genomic DNA from peritoneal neutrophils and analyzed them using PCR. Neutrophils from LysM-Cre:SOCS3fl/fl mice showed a 250-bp band corresponding to the Cre-mediated deletion of the Socs3 gene, while no deletion band was observed in the DNA from SOCS3fl/fl mice (Fig. 2A, lower panel). A small amount of the undeleted Socs3 gene band (380 bp) was also observed in the DNA of peritoneal neutrophils from LysM-Cre:SOCS3fl/fl mice, probably because of contamination of lymphocytes (Fig. 2A, upper panel).



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  		FIG. 2.
Deletion of Socs3 genes in mature neutrophils in LysM-Cre:SOCS3fl/fl mice and normal functions of SOCS3-deficient neutrophils. A, PCR analysis of genomic DNA from peritoneal neutrophils with primers a and b (upper panel) and a and c (lower panel), as indicated in Fig. 1A. B, cytology of peripheral blood derived from SOCS3fl/fl and LysM-Cre:SOCS3fl/fl mice. Peripheral blood was stained with May-Grünwald-Giemsa (upper panels) or myeloperoxidase (lower panels). Neutrophils with high activity of myeloperoxidase were 8.5 ± 3.5% in SOCS3fl/fl mice and 8.5 ± 2.1% in LysM-Cre:SOCS3fl/fl mice. C, chemotactic activity of neutrophils. Peritoneal neutrophils (3 x 106 cells/ml) in the upper chamber were attracted by fMLP in the lower chamber for 1 h at 37 °C. The cells that passed through the membrane were counted and are shown as a chemotactic index. D, phagocytosis analysis. Peritoneal neutrophils from SOCS3fl/fl and LysM-Cre:SOCS3fl/fl mice were incubated with Texas Red-conjugated OZ (OZ-TR) at 37 °C for 30 min. Flow cytometry was carried out after staining with fluorescein isothiocyanate-conjugated anti-Gr1 antibody.

 
We investigated the cytological analysis of peripheral blood cells derived from SOCS3fl/fl and LysM-Cre:SOCS3fl/fl mice (Table I). White blood cells differential counts were done by May-Grünwald-Giemsa staining, and the numbers of neutrophils, lymphocytes, macrophages, and eosinophils were calculated. The number of neutrophils in LysM-Cre:SOCS3fl/fl mice was twice as large as that in SOCS3fl/fl mice, with a statistical significance of p < 0.001.


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  		TABLE I
Complete blood count

Peripheral blood counts from untreated LysM-Cre:SOCS3fl/fl and SOCS3fl/fl mice (n = 22) at age 6-10 weeks. White blood cell differential counts were performed by May-Grünwald-Giemsa staining. ND, not detected.

 
Next, we compared the effect of SOCS3 deletion on neutrophil functions. Myeloperoxidase staining of peripheral blood smears as well as the production of superoxide in peritoneal neutrophils was not significantly different between SOCS3fl/fl mice and LysM-Cre:SOCS3fl/fl mice (Fig. 2B). The chemotactic activity of neutrophils for fMLP was almost identical between wild-type and SOCS3-deficient cells (Fig. 2C). The phagocytic activity of neutrophils for opsonizing zymosan (OZ) was assayed using flow cytometry (Fig. 2D). Texas Red-labeled OZ was incubated with peritoneal neutrophils at 37 °C. After 30 min, about 85% of Gr-1-positive neutrophils from both wild-type and SOCS3-deficient mice phagocytosed OZ. These data indicate that SOCS3-deficient neutrophils increased in number but possessed normal functions.

Stronger STAT3 and ERK Activation in Neutrophils Lacking SOCS3—We then investigated G-CSF-dependent signaling by Western blotting (Fig. 3). Peritoneal neutrophils were induced by intraperitoneal injection of thioglycollate. G-CSF-induced STAT3 activation was much stronger and prolonged in SOCS3-deficient peritoneal neutrophils than in wild-type neutrophils (Fig. 3A). Similar hyperactivation of STAT3 was observed in peripheral neutrophils mobilized by G-CSF in LysM-Cre:SOCS3fl/fl mice (Fig. 3B). Phosphorylation of STAT3 was higher in SOCS3-deficient peripheral neutrophils than in control neutrophils.



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  		FIG. 3.
Western blotting analysis of SOCS3-deficient neutrophils in response to G-CSF. Peritoneal neutrophils (A) or peripheral neutrophils (B) from SOCS3fl/fl and LysM-Cre:SOCS3fl/fl mice were incubated for 6 h without G-CSF; then, they were stimulated with 50 ng/ml G-CSF for indicated periods and analyzed using Western blotting with indicated antibodies. Data are representative of two independent experiments, with similar results.

 
The basal levels of STAT3 and ERK activation were higher in SOCS3-deficient peritoneal neutrophils (Fig. 3A, time = 0 min). ERK activation was very weak in wild-type neutrophils, while strong ERK activation was observed in SOCS3-deficient neutrophils before G-CSF stimulation. Such hyperactivation of STAT3 and ERK before G-CSF-stimulation in thioglycollate-induced peritoneal neutrophils is probably due to the slower down-regulation of G-CSF signaling in SOCS3-deficient neutrophils than in wild-type neutrophils during cytokine-free preincubation. In contrast, STAT3 activation was not observed before stimulation in either control or SOCS3-deficient peripheral neutrophils (Fig. 3B, time = 0 min). The discrepancy of the basal levels of STAT3 phosphorylation observed in Fig. 3, A and B, is probably due to differences in the naure of neutrophils induced by thioglycollate and G-CSF, such as the maturation stage and the levels of G-CSF receptor or signaling molecules.

To evaluate the stronger G-CSF signaling in SOCS3-deficient neutrophils, we examined the effect of G-CSF on the prevention of apoptosis of neutrophils. Viable cells were counted by the trypan blue exclusion test (Fig. 4A). Both wild-type and SOCS3-deficient neutrophils died rapidly within 24 h in the absence of G-CSF. G-CSF slowed down the apoptosis of neutrophils. The effect of G-CSF on the prevention of apoptosis was much greater in SOCS3-deficient neutrophils than in wild-type neutrophils. Flow cytometry analysis confirmed that SOCS3-deficient neutrophils are more resistant against apoptosis than wild-type neutrophils (Fig. 4B). The prevention of apoptosis was correlated to the G-CSF-dependent induction of Bcl-XL, as shown in Fig. 4C. The Bcl-XL mRNA levels were increased more strongly in SOCS3-deficient neutrophils than in wild-type neutrophils. These data suggest that the increase in the number of peripheral neutrophils in LysM-Cre:SOCS3fl/fl mice could be due to the longer half-life of neutrophils.



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  		FIG. 4.
G-CSF-mediated prevention of apoptosis of neutrophils. A, peripheral neutrophils from peripheral blood of G-CSF-injected mice were cultured in IMDM with 10% FBS in the presence of 10 ng/ml of G-CSF. Cell viability was assessed by the trypan blue exclusion assay on days 0, 1, and 3. B, peripheral neutrophils were cultured in IMDM with 10% FBS and 10 ng/ml of G-CSF. Apoptosis of neutrophils was assessed by flow cytometry after staining with propidium iodide (PI) and Gr1 on days 0 and 2. C, real-time PCR analysis for Bcl-XL and SOCS3. Peripheral neutrophils were cultured in 10 ng/ml G-CSF for indicated periods. Real-time PCR was carried out to detect Bcl-XL and SOCS3 mRNA. The expression level of glyceraldehyde-3-phosphate dehydrogenase-mRNA was used as a reference value for quantification within samples to determine the relative amounts of total mRNA. The relative expression levels against the levels at time 0 in SOCS3fl/fl mice are shown. Data are representative of three independent experiments with similar results.

 
Other researchers as well as our group have observed that G-CSF induced SOCS3 mRNA in wild-type neutrophils (31). Previously, STAT3 was shown to be dispensable for granulocyte proliferation, differentiation, and function but required for homeostasis (10). Socs3 is proposed to be a candidate gene that negatively regulates G-CSF signaling downstream of STAT3. Our data strongly support this hypothesis. However, a major signaling pathway for granulopoiesis elicited by G-CSF has not been clarified. Our study strongly suggests that ERK is a plausible candidate for such G-CSF-mediated signaling responsible for anti-apoptosis and proliferation. This is consistent with a recent report indicating that Tyr-764 of the G-CSF receptor is the most important element for G-CSF-induced proliferation, which was reverted by inhibition of ERK activity (20). Furthermore, Bcl-XL, an anti-apoptotic factor, could be a downstream target of such G-CSF-mediated signaling.

The enhanced production of neutrophils in the absence of SOCS3 is reminiscent of the phenotype derived from the G-CSF receptor mutant found in human severe congenital neutropenia (32, 33). The hypergranulopoiesis of mutant mice bearing this receptor correlated with the prolonged activation of STAT1, STAT3, and STAT5 in response to G-CSF treatment (34). These mutated receptors lacked the SOCS3 binding site, Tyr-729. Interestingly, in the absence of SOCS3, prolonged activation of STAT3 and ERK was also observed after G-CSF stimulation. Therefore, the loss of the Socs3 gene in stem cells may contribute to congenital or sporadic neutrophilia.

Under certain stressful situations, such as response to pathogen infection, rapid expansion of neutrophil populations can be achieved through a process known as emergency granulopoiesis, which is accompanied by high levels of G-CSF (35-37). Although the basal levels of neutrophil contents were higher in LysM-Cre:SOCS3fl/fl mice than in wild-type mice, no significant difference was observed after administration of a high dose of G-CSF (data not shown). This suggests that SOCS3-deficient mature neutrophils are more sensitive to homeostatic low levels of G-CSF but that the SOCS3 may not suppress G-CSF signaling efficiently in emergency granulopoiesis with a high dose of G-CSF. This is consistent with the fact that STAT3 activation by the G-CSF receptor can be achieved through both receptor tyrosine-dependent and -independent mechanisms, depending on the G-CSF concentration (35). SOCS3 may not be involved in the negative regulation of tyrosine-independent G-CSF receptor signaling at extremely high levels of G-CSF.


   FOOTNOTES
 
* This work was supported by special grants-in-aid from the Ministry of Education, Science, Technology, Sports, and Culture of Japan, the Japan Health Science Foundation, the Human Frontier Science Program, the Mochida Memorial Foundation, and the Uehara Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Division of Molecular and Cellular Immunology, Medical Inst. of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: 81-92-642-6823; Fax: 81-92-642-6825; E-mail: yakihiko{at}bioreg.kyushu-u.ac.jp.

1 The abbreviations used are: JAK, Janus kinase; SOCS, suppressor of cytokine signaling; STAT, signal transducers and activators of transcription; ERK, extracellular signal-regulated kinase; SH, Src homology; CIS, cytokine-inducible SH2 protein; SSI, STAT-induced STAT inhibitors; IL, interleukin; G-CSF, granulocyte colony-stimulating factor; IMDM, Iscove's modified Dulbecco's medium; fMLP, formyl-methionyl-leucyl-phenylalanine; FBS, fetal bovine serum; TR-OZ, Texas Red-conjugated opsonizing zymosan; RT, reverse transcriptase. Back


   ACKNOWLEDGMENTS
 
We thank Y. Kawabata-Honda for technical assistance, Drs. Nonami and Shimoda (Kyushu University) for discussion, N. Arifuku and F. Yamaura for manuscript preparation, Dr. R. Fukunaga (Osaka University) for G-CSF receptor cDNA, and Dr. A. P. Koni (Medical College of Georgia) for Tie2-Cre mice.



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 RESULTS AND DISCUSSION
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