1Department of Physiology and 2Clinical Hematology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
Submitted 11 September 2003 ; accepted in final form 16 January 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
monocytes; granulocyte colony-stimulating factor; interleukin-10; lipopolysaccharide; tumor necrosis factor-; signal transducer and activator of transcription 3
It has been reported that the functions of human monocytes and lymphocytes are directly affected by G-CSF itself or indirectly affected by G-CSF via activated neutrophils or other mechanisms (5, 34, 39, 41, 45). A recent study shows that G-CSF may affect human monocytes directly to inhibit LPS-induced TNF- production (5). More recently, it has been shown that dendritic cells differentiated from monocytes treated with G-CSF lose the ability to produce interleukin (IL)-12 in response to CD40 ligand or LPS, suggesting that G-CSF directly affects monocytes (49). However, the signals provoked by the binding of G-CSF to its receptors on human monocytes as well as the mechanisms by which G-CSF modulates monocyte functions are largely unknown. We studied the signaling pathways activated in human monocytes stimulated by G-CSF and the mechanisms by which G-CSF inhibits LPS-induced TNF-
production. The results show that G-CSF, like IL-10, may inhibit LPS-induced TNF-
production in human monocytes through selective activation of signal transducer and activator of transcription 3 (STAT3).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of cells. Human neutrophils and mononuclear cells were prepared from healthy adult donors as described previously (19, 29) by using dextran sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis of contaminated erythrocytes. Neutrophil fractions contained >98% neutrophils. Monocytes were further purified from mononuclear cells by centrifugal elutriation in a Hitachi SRR6Y elutriation rotor (Hitachi, Tokyo, Japan) (55). Monocyte fractions contained 9095% monocytes and 510% lymphocytes. Cells were suspended in Hanks' balanced salt solution (HBSS) containing 10 mM HEPES (pH 7.4). For experiments with cell cultivation, cells were suspended in RPMI 1640 supplemented with 10% fetal calf serum (FCS).
Determination of TNF- production.
For the experiment with TNF-
production, monocytes (3 x 106 cells/ml) were suspended in RPMI 1640 supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 1% human serum. The monocyte suspension (1 ml) was placed in a polypropylene tube (Falcon no. 2059; Falcon Labware, Becton Dickinson, NJ) and were cultivated with or without LPS (1 µg/ml) in 5% CO2-95% humidified air at 37°C. When required, G-CSF or IL-10 was added to the culture medium. In some experiments, monocytes were pretreated with PD-98059 (50 µM), SB-203580 (10 µM), SP-600125 (5 or 25 µM), AG-490 (50 µM), or SN-50 (100 µg/ml) for 1 h at 37°C before stimulation with LPS. After incubation, the amount of TNF-
in the cell-free culture supernatants was determined by the TNF-
-specific ELISA kit (R&D Systems, Minneapolis, MN), which can detect >0.18 pg/ml TNF-
.
Western blotting. Human neutrophils or monocytes suspended in HBSS were prewarmed for 10 min at 37°C and were then stimulated with cytokines or LPS for 10 min at 37°C. When required, cells were pretreated with AG-490 (50 µM) for 1 h at 37°C before stimulation with cytokines. The reactions were terminated by rapid centrifugation, and the pellets were frozen in liquid nitrogen after aspiration of the supernatant. The cell pellets were resuspended in ice-cold solution containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, and 10 µg/ml leupeptin and were lysed for 10 min at 4°C. After rapid centrifugation, the supernatant was mixed 1:1 with 2x sample buffer [4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% mercaptoethanol, and a trace amount of bromphenol blue dye in 125 mM Tris·HCl, pH 6.8], heated at 100°C for 5 min, and then frozen at 80°C until use. Samples were subjected to 10% SDS gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol at 2 mA/cm2 for 1.5 h at 25°C. Residual binding sites on the membrane were blocked by incubating the membrane in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 2 h at 25°C. The blots were washed in Tris-buffered saline containing 0.1% Tween 20 and then incubated with appropriate antibody overnight at 4°C. After washing, the membrane was incubated with anti-rabbit IgG antibody conjugated with horseradish peroxidase, and the antibody complexes were visualized by the ECL detection system as directed by the manufacturer. Immunoreactive bands were quantified with the use of a NIH Image program on a Macintosh computer.
Reverse transcriptase-polymerase chain reaction analysis. Total RNA was isolated from monocytes and neutrophils with the RNeasy mini kit (Qiagen, Hilden, Germany). To generate cDNA, we used 500 ng of RNA for each reaction. The reaction mixtures (10 µl) contained RNA, random primer pd(N)6 (1 µM), RNase inhibitor (0.5 U/µl; Roche Molecular Biochemicals, Mannheim, Germany), dNTP mixture (500 µM of each dNTP), and Omniscript reverse transcriptase (0.2 U/µl; Qiagen), and the reaction mixtures were incubated for 60 min at 37°C. Semiquantitative RT-PCR analysis was performed by using GeneAmp PCR system model 9700 (Perkin Elmer, Norwalk, CT). PCR reaction mixtures (25 µl) contained cDNA, dNTP mixture (200 µM of each dNTP), MgCl2 (1.5 mM), Taq DNA polymerase (0.02 U/µl; Fermentas AB, Vilnius, Lithuania), and the forward and reverse primers.
The following primer pairs were used. For each set, the forward and reverse primers as well as the accession number are listed: TNF-, 5'-AGA GGG AAG AGT TCC CCA GGG AC-3', 5'-TGA GTC GGT CAC CCT TCT CCA G-3', M10988
[GenBank]
; suppressor of cytokine signaling 3 (SOCS3), 5'-CTC GCC ACC TAC TGA ACC CTC-3', 5'-AAG CGG GGC ATC GTA CTG GT-3', AF159854
[GenBank]
;
-actin, 5'-CCA ACC GCG AGA AGA TGA-3', 5'-GGA AGG AAG GCT GGA AGA GT-3', X00351
[GenBank]
; and
2-microglobulin, 5'-GCT ATG TGT CTG GGT TTC-3', 5'-TAC ATG TCT CGA TCC CAC-3', V00567
[GenBank]
. For the analysis of G-CSF receptor (accession no. M59818), three sets of overlapping primers were used (4). For each set, the forward and reverse primers are listed: primer A (nt 20062351), 5'-GTC CTC ACC CTG ATG ACC TTG-3' and 5'-CAT AGG TCT GGA CCA GAG TGG-3'; primer B (nt 22292550), 5'-GCC TTG GCA CGC CAC CCA TCA-3' and 5'-GCC TGG AAC CAG AGG TTC TCA-3'; and primer C (nt 24612740), 5'-AGG GCA CTA TCT CCG CTG TGA-3' and 5'-TCT TCT CCA GCT AGC TCA GGC-3'.
The conditions for PCR amplification were as follows: denaturation for 30 s at 94°C, annealing for 30 s at 55°C, and elongation for 30 s at 72°C with 26 cycles for TNF-, SOCS3,
-actin, and
2-microglobulin; and denaturation for 30 s at 94°C, annealing for 30 s at 60°C, and elongation for 30 s at 72°C with 35 cycles for G-CSF receptor. The PCR products were analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide. The PCR products were purified, and the nucleotide sequence was analyzed for confirmation.
Statistical analysis. An ANOVA followed by a multiple comparison test (Bonferroni method) was used to determine statistical significance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Selective activation of STAT3 in monocytes stimulated by G-CSF.
To elucidate the mechanisms by which G-CSF inhibits LPS-induced TNF- production in monocytes, the signaling pathways activated in monocytes stimulated by G-CSF were explored. As shown in Fig. 3, STAT3, especially STAT3
, was strongly tyrosine-phosphorylated by stimulation with G-CSF. STAT3 was also tyrosine-phosphorylated by stimulation with IL-10 and was serine-phosphorylated by stimulation with TNF-
or IL-10 but not with G-CSF (11, 15). The potency of these cytokines to induce tyrosine phosphorylation of STAT3 was IL-10 > G-CSF. ERK was phosphorylated in monocytes stimulated by TNF-
but not by G-CSF or IL-10. No significant phosphorylation of ERK was detected in G-CSF-stimulated monocytes even when the incubation time was prolonged to 90 min or when a high concentration of G-CSF (500 ng/ml) was employed (Fig. 3C). Phosphorylation of p38 was induced by stimulation with TNF-
but not with G-CSF or IL-10 (Fig. 3, A and B). TNF-
also induced phosphorylation of JNK in monocytes. G-CSF-induced tyrosine phosphorylation of STAT3 in monocytes was already detected at 5 min after stimulation with 50 ng/ml G-CSF, and the maximal level was obtained at 10 min, followed by a gradual decline of the level (Fig. 3C). Tyrosine phosphorylation of STAT3 was dependent on the concentration of G-CSF used as stimulus. A significant effect was detected at 5 ng/ml G-CSF, and the optimal effect was obtained at 50 ng/ml (Fig. 3C).
|
Identical G-CSF receptor is expressed on human neutrophils and monocytes. Differential activation of the signaling pathways in human neutrophils and monocytes stimulated by G-CSF might be ascribed to either a difference in G-CSF receptor isoforms expressed on these cells or the difference of the signaling system from the identical G-CSF receptor. In fact, seven isoforms were reported for G-CSF receptor (4). G-CSF receptor isoforms expressed on neutrophils and monocytes were then analyzed by RT-PCR, using three sets of primers that have been shown to be able to discriminate G-CSF receptor isoforms (4). The results showed that a G-CSF receptor band with an identical size was detected for neutrophils and monocytes when each of these different primer sets was used (data not shown), in agreement with the previous report by Boneberg et al. (5). These findings indicate that an identical G-CSF receptor is primarily expressed on neutrophils and monocytes and suggest that the differential activation of the signaling pathways in these cells may reflect the difference of the signaling system from the identical G-CSF receptor.
G-CSF inhibits LPS-induced TNF- production through activation of the JAK2-STAT3 pathway.
The results depicted in Figs. 1 and 3 show that both G-CSF and IL-10 induced selective phosphorylation of STAT3 and inhibited LPS-induced TNF-
production in monocytes. IL-10 was more potent than G-CSF in both functions. In addition, it has been shown that activation of STAT3 is essential for IL-10-mediated inhibition of LPS-induced TNF-
production (28, 44). Furthermore, G-CSF and IL-10 inhibited LPS-induced TNF-
mRNA expression (Fig. 4C), and IL-10 was more potent than G-CSF in this effect. All of these findings raise the possibility that G-CSF, like IL-10, might inhibit LPS-induced TNF-
production through STAT3 activation. This possibility was explored by using AG-490, a potent inhibitor of JAK2 (15, 24). As shown in Fig. 4A, G-CSF-induced STAT3 phosphorylation in monocytes was significantly inhibited by pretreatment of cells with AG-490, indicating that G-CSF may activate the JAK2-STAT3 pathway in monocytes (15). On the other hand, IL-10-induced STAT3 phosphorylation was unaffected by AG-490. The failure of AG-490 to inhibit IL-10-induced STAT3 phosphorylation may be explained by the fact that Tyk2 and JAK1, but not JAK2, are involved in IL-10-induced STAT3 phosphorylation (10). Consistent with these findings, G-CSF-mediated, but not IL-10-mediated, inhibition of LPS-induced TNF-
production and mRNA expression was significantly prevented by pretreatment of monocytes with AG-490. In the absence of AG-490, G-CSF and IL-10 inhibited LPS-induced TNF-
production by 40.1 ± 7.1 and 94.2 ± 2.2%, respectively, whereas in the presence of AG-490, they inhibited it by 25.5 ± 2.9 and 95.1 ± 3.3%, respectively (n = 5). G-CSF-mediated, but not IL-10-mediated, inhibition of LPS-induced TNF-
production was significantly (P < 0.05) prevented by AG-490 (Fig. 4B). Similarly, the densitometric analysis showed that, in the absence of AG-490, G-CSF and IL-10 inhibited LPS-induced TNF-
mRNA expression by 43.0 ± 10.2 and 85.8 ± 13.9%, respectively, whereas in the presence of AG-490, they inhibited it by 20.3 ± 4.9 and 84.2 ± 13.9%, respectively (n = 3). G-CSF-mediated, but not IL-10-mediated, inhibition of LPS-induced TNF-
mRNA expression was significantly (P < 0.05) prevented by AG-490 (Fig. 4C).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The signaling pathways were differentially activated in neutrophils and monocytes stimulated by G-CSF despite the facts that the identical G-CSF receptor isoform is expressed on both types of cells and that the surface expression of G-CSF receptor on these cells is comparable (5). The differential activation of the signaling pathways in neutrophils and monocytes was also observed when TNF- or IL-10 was used as stimulus. The cell-type-specific activation of a different set of distinct signaling pathways from the identical cytokine receptors may reflect the different role of these cytokines on neutrophils and monocytes (42). For example, Kitagawa and colleagues (15, 35) recently reported that the JAK2-STAT3 pathway activated in human neutrophils stimulated by G-CSF contributes to G-CSF-mediated prolongation of neutrophil survival. The present experiments suggest that the JAK2-STAT3 pathway activated in human monocytes stimulated by G-CSF contributes to G-CSF-mediated inhibition of LPS-induced TNF-
production. In hematopoietic progenitor cells, STAT3 activation has been demonstrated to be required for G-CSF-dependent proliferation and granulocytic differentiation (25, 40, 50), although a recent study suggested that STAT3 is rather a negative regulator of granulopoiesis and is not required for G-CSF-dependent differentiation (22). These findings taken together suggest that STAT3 plays a different role in G-CSF-mediated responses according to the stages of cell differentiation and the cell types.
LPS-induced TNF- production in monocytes was highly dependent on activation of ERK, p38, JNK, and NF-
B, because it was markedly inhibited by specific inhibitors for each signaling pathway. These signaling pathways may regulate TNF-
production at the transcriptional and/or posttranscriptional level. In fact, NF-
B, Egr-1, Sp1, ATF-2 (a p38 or JNK substrate), c-Jun (a JNK substrate), and Ets and Elk-1 (ERK substrates) are recruited to the TNF-
promoter in response to LPS stimulation, and the binding sites for each of these transcription factors are required for LPS-stimulated TNF-
gene expression (46, 53). JNK and p38 may also regulate TNF-
production at the posttranscriptional level (3, 7). However, G-CSF and IL-10 did not affect activation of ERK, p38, JNK, and NF-
B, which constitute the MyD88-dependent signaling pathway (2), suggesting that G-CSF and IL-10 affect the pathway downstream or independently of these signaling molecules. It was recently reported that LPS-induced TNF-
production is abolished in murine macrophages lacking Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-
(TRIF), and LPS stimulation leads to almost normal activation of JNK and NF-
B in TRIF-deficient fibroblasts (52). The TRIF-dependent pathway is known to be independent of MyD88. These findings suggest that both the MyD88-dependent and the TRIF-dependent (MyD88 independent) pathways are required for LPS-induced TNF-
production and raise the possibility that G-CSF and IL-10 might affect the TRIF-dependent pathway to inhibit LPS-induced TNF-
production.
G-CSF, like IL-10, induced selective activation of STAT3 in monocytes. STAT3 has been demonstrated to play an essential role in IL-10-mediated inhibition of LPS-induced TNF- production (28, 32, 44). G-CSF-induced STAT3 phosphorylation and SOCS3 mRNA expression were inhibited by AG-490 (a JAK2 inhibitor), indicating that G-CSF may activate the JAK2-STAT3 pathway in monocytes (15). G-CSF-mediated inhibition of LPS-induced TNF-
production and TNF-
mRNA expression in monocytes was also prevented by AG-490. These findings suggest that the JAK2-STAT3 pathway is involved in G-CSF-mediated inhibition of LPS-induced TNF-
production in monocytes. The specific action of AG-490 on G-CSF-mediated effects was supported by the lack of AG-490 action on IL-10-mediated effects, which may be dependent on Tyk2 and JAK1 but not on JAK2 (10). Both G-CSF and IL-10 inhibited LPS-induced TNF-
mRNA expression as well as TNF-
production in monocytes, a finding consistent with a recent report showing that IL-10 inhibits LPS-induced TNF-
production in human macrophages at the transcriptional as well as posttranscriptional levels (9). In contrast to our present results, it was reported that the level of TNF-
mRNA in whole blood stimulated with LPS is not affected by G-CSF (6). This difference might be ascribed to the difference of samples used; i.e., highly purified monocytes and whole blood. G-CSF-induced STAT3 phosphorylation was significantly attenuated and rapidly declined in the presence of LPS. The attenuation of G-CSF-induced STAT3 phosphorylation by LPS was rapid (within 10 min) and is unlikely to be ascribed to induction of SOCS3 protein by LPS. By contrast, IL-10-induced STAT3 phosphorylation was essentially unaffected and sustained in the presence of LPS (27). In addition, G-CSF was less potent than IL-10 in stimulating STAT3 phosphorylation. These characteristics of G-CSF action on monocytes may partly explain the less potent inhibitory effect of G-CSF than IL-10.
It has been reported that the JAK1-STAT3 pathway is necessary but not sufficient for IL-10-mediated inhibition of TNF- production, and an additional signaling pathway derived from the COOH-terminal domain of IL-10 receptor may also be required for this effect (32). In this regard, it is of interest that a high concentration of G-CSF (>100 ng/ml) was required to inhibit LPS-induced TNF-
production, although an almost maximal level of STAT3 phosphorylation was obtained at the lower concentration (50 ng/ml) of G-CSF. These findings suggest that, besides STAT3 activation, an additional signaling pathway activated by a high concentration of G-CSF also may be required for efficient inhibition of LPS-induced TNF-
production by G-CSF. In fact, it has been shown that the signaling pathways could be differentially activated by G-CSF, depending on G-CSF concentration (51).
The precise mechanisms by which STAT3 activation inhibits TNF- production remain to be elucidated. The coactivator proteins cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 play an important role in integrating multiple transcription factors in various cell systems (38), and CBP/p300 is involved in LPS-induced TNF-
gene expression (46). In addition, it has been shown that CBP/p300 interacts physically with STAT3 in certain cell lines (31, 37). Furthermore, interferon regulatory factor 3 (IRF3), a transcription factor downstream of TRIF, also interacts with CBP/p300 (12). These findings raise the possibility that the inhibition of TNF-
gene expression by G-CSF or IL-10 might be caused by increased competition for limiting amounts of CBP/p300 by STAT3.
LPS-induced TNF- production in monocytes was inhibited by G-CSF in a dose-dependent manner at a concentration range from 100 to 500 ng/ml. This finding is consistent with a recent report showing that G-CSF inhibits LPS-induced TNF-
production in whole blood in a dose-dependent manner at a concentration range from 10 to 300 ng/ml (5). These findings indicate that a higher concentration of G-CSF may be required for inhibition of LPS-induced TNF-
production compared with that required for efficient stimulation of granulopoiesis and activation of neutrophil functions (8, 19, 29). It has been reported that endogenous plasma G-CSF levels are 0.020.09 ng/ml in healthy persons and increase to 0.84 ng/ml upon bacterial infections. Therapeutic intravenous administration of G-CSF (3.4511.5 µg/kg) gives a plasma concentration of 20400 ng/ml (16, 48). Our previous study shows that intravenous administration of 200 µg/m2 G-CSF gives a plasma concentration of 100 ng/ml (29). When G-CSF-mobilized PBSC were harvested, donors received much higher doses of G-CSF; i.e., subcutaneous administration of 200 µg/m2 G-CSF twice daily (15). These findings suggest that therapeutic administration of G-CSF could give a plasma concentration of G-CSF sufficient for inhibition of LPS-induced TNF-
production in monocytes. Such a high concentration of endogenous G-CSF also might be produced locally at the inflammatory sites. On the other hand, it has been reported that endogenous plasma IL-10 levels are 0.10.15 ng/ml upon endotoxemia (30), and IL-10 inhibited LPS-induced TNF-
production in monocytes by >90% at high concentration (10 ng/ml). These findings taken together suggest that G-CSF at the moderate concentrations potentiates the host defense against invading microorganisms through increased production of neutrophils and activation of neutrophil functions, and G-CSF at the high concentrations exerts the anti-inflammatory or immunomodulatory effect by inhibiting the overproduction of proinflammatory cytokines from monocytes. This concentration-dependent dual action of G-CSF may be physiologically relevant and important in maintaining the host-defense mechanism. Furthermore, the results presented here indicate that the immunomodulation observed in vivo by G-CSF administration (18, 33, 49) may be partly ascribed to the direct effect of G-CSF on monocyte functions, which may be mediated through selective activation of STAT3.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Barton GM and Medzhitov R. Toll-like receptor signaling pathways. Science 300: 15241525, 2003.
3. Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, Bhagwat SS, Manning AM, and Anderson DW. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 98: 1368113686, 2001.
4. Bernard T, Gale RE, and Linch DC. Analysis of granulocyte colony stimulating factor receptor isoforms, polymorphisms and mutations in normal haemopoietic cells and acute myeloid leukaemia blasts. Br J Haematol 93: 527533, 1996.[CrossRef][ISI][Medline]
5. Boneberg EM, Hareng L, Gantner F, Wendel A, and Hartung T. Human monocytes express functional receptors for granulocyte colony-stimulating factor that mediate suppression of monokines and interferon-. Blood 95: 270276, 2000.
6. Boneberg EM and Hartung T. Granulocyte colony-stimulating factor attenuates LPS-stimulated IL-1 release via suppressed processing of proIL-1
, whereas TNF-
release is inhibited by the level of proTNF-
formation. Eur J Immunol 32: 17171725, 2002.[CrossRef][ISI][Medline]
7. Dean JLE, Brook M, Clark AR, and Saklatvala J. p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J Biol Chem 274: 264269, 1999.
8. Demetri GD and Griffin JD. Granulocyte colony-stimulating factor and its receptor. Blood 78: 27912808, 1991.[ISI][Medline]
9. Denys A, Udalova IA, Smith C, Williams LM, Ciesielski CJ, Campbell J, Andrews C, Kwaitkowski D, and Foxwell BM. Evidence for a dual mechanism for IL-10 suppression of TNF- production that does not involve inhibition of p38 mitogen-activated protein kinase or NF-
B in primary human macrophages. J Immunol 168: 48374845, 2002.
10. Donnelly RP, Dickensheets H, and Finbloom DS. The interleukin-10 signal transduction pathway and regulation of gene expression in mononuclear phagocytes. J Interferon Cytokine Res 19: 563573, 1999.[CrossRef][ISI][Medline]
11. Finbloom DS and Winestock KD. IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1 and STAT3 complexes in human T cells and monocytes. J Immunol 155: 10791090, 1995.[Abstract]
12. Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenblock DT, Coyle AJ, Liao SM, and Maniatis T. IKK and TBK1 are essential components of the IRF3 signaling pathway. Nat Immun 4: 491496, 2003.[CrossRef][ISI]
13. Görgen I, Hartung T, Leist M, Niehorster M, Tiegs G, Uhlig S, Weitzel F, and Wendel A. Granulocyte colony-stimulating factor treatment protects rodents against lipopolysaccharide-induced toxicity via suppression of systemic tumor necrosis factor-. J Immunol 149: 918924, 1992.
14. Hartung T, Döcke WD, Gantner F, Krieger G, Sauer A, Stevens P, Volk HD, and Wendel A. Effect of granulocyte colony-stimulating factor treatment on ex vivo blood cytokine response in human volunteers. Blood 85: 24822489, 1995.
15. Hasegawa T, Suzuki K, Sakamoto C, Ohta K, Nishiki S, Hino M, Tatsumi N, and Kitagawa S. Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia. Blood 101: 11641171, 2003.
16. Hasenclever D and Sextro M. Safety of AlloPBPCT donors: biometrical considerations on monitoring long term risks. Bone Marrow Transplant 17: S28S33, 1996.[ISI][Medline]
17. Hörtner M, Nielsch U, Mayr LM, Johnston JA, Heinrich PC, and Haan S. Suppressor of cytokine signaling-3 is recruited to the activated granulocyte-colony stimulating factor receptor and modulates its signal transduction. J Immunol 169: 12191227, 2002.
18. Joshi SS, Lynch JC, Pavletic SZ, Tarantolo SR, Pirruccello SJ, Kessinger A, and Bishop MR. Decreased immune functions of blood cells following mobilization with granulocyte colony-stimulating factor: association with donor characteristics. Blood 98: 19631970, 2001.
19. Kitagawa S, Yuo A, Souza LM, Saito M, Miura Y, and Takaku F. Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide. Biochem Biophys Res Commun 144: 11431146, 1987.[ISI][Medline]
20. Krebs DL and Hilton DJ. SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19: 378387, 2001.
21. Kutsuna H, Suzuki K, Kamata N, Kato T, Hato F, Mizuno K, Kobayashi H, Ishii M, and Kitagawa S. Actin reorganization and morphological changes in human neutrophils stimulated by TNF, GM-CSF and G-CSF: the role of MAP kinases. Am J Physiol Cell Physiol 286: C55C64, 2004.
22. Lee C, Raz R, Gimeno R, Gertner R, Wistinghausen B, Takeshita K, DePinho RA, and Levy DE. STAT3 is a negative regulator of granulopoiesis but is not required for G-CSF-dependent differentiation. Immunity 17: 6372, 2002.[ISI][Medline]
23. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, Strickler JE, McLaughlin MM, Siemens IR, Fisher SM, Livi GP, White JR, Adams JL, and Young PR. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372: 739746, 1994.[CrossRef][ISI][Medline]
24. Levitzki A. Protein tyrosine kinase inhibitors as novel therapeutic agents. Pharmacol Ther 82: 231239, 1999.[CrossRef][ISI][Medline]
25. McLemore ML, Grewal S, Liu F, Archambault A, Poursine-Laurent J, Haug J, and Link DC. STAT-3 activation is required for normal G-CSF-dependent proliferation and granulocytic differentiation. Immunity 14: 193204, 2001.[ISI][Medline]
26. Nick JA, Avdi NJ, Young SK, Lehman LA, McDonald PP, Frasch SC, Billstrom MA, Henson PM, Johnson GL, and Worthen GS. Selective activation and functional significance of p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils. J Clin Invest 103: 851858, 1999.
27. Niemand C, Nimmesgern A, Haan S, Fischer P, Schaper F, Rossaint R, Heinrich PC, and Muller-Newen G. Activation of STAT3 by IL-6 and IL-10 in primary human macrophages is differentially modulated by suppressor of cytokine signaling 3. J Immunol 170: 32633272, 2003.
28. O'Farrell AM, Liu Y, Moore KW, and Mui ALF. IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for STAT3-dependent and -independent pathways. EMBO J 17: 10061018, 1998.
29. Ohsaka A, Kitagawa S, Sakamoto S, Miura Y, Takanashi N, Takaku F, and Saito M. In vivo activation of human neutrophil functions by administration of recombinant human granulocyte colony-stimulating factor in patients with malignant lymphoma. Blood 74: 27432748, 1989.[Abstract]
30. Pajkrt D, Camoglio L, Tiel-van Buul MCM, de Bruin K, Cutler DL, Affrime MB, Rikken G, van der Poll T, ten Cate JW, and van Deventer SJH. Attenuation of proinflammatory response by recombinant human IL-10 in human endotoxemia. J Immunol 158: 39713977, 1997.[Abstract]
31. Paulson M, Pisharody S, Pan L, Guadagno S, Mui AL, and Levy DE. Stat protein transactivation domains recruit p300/CBP through widely divergent sequences. J Biol Chem 274: 2534325349, 1999.
32. Riley JK, Takeda K, Akira S, and Schreiber RD. Interleukin-10 receptor signaling through the JAK-STAT pathway: requirement for two distinct receptor-derived signals for anti-inflammatory action. J Biol Chem 274: 1651316521, 1999.
33. Ringdén O, Remberger M, Runde V, Bornhauser M, Blau IW, Basara N, Holig K, Beelen DW, Hagglund H, Basu O, Ehninger G, and Fauser AA. Peripheral blood stem cell transplantation from unrelated donors: a comparison with marrow transplantation. Blood 94: 455464, 1999.
34. Saito M, Kiyokawa N, Taguchi T, Suzuki K, Sekino T, Mimori K, Suzuki T, Nakajima H, Katagiri YU, Fujimura J, Fujita H, Ishimoto K, Yamashiro Y, and Fujimoto J. Granulocyte colony-stimulating factor directly affects human monocytes and modulate cytokine secretion. Exp Hematol 30: 11151123, 2002.[CrossRef][ISI][Medline]
35. Sakamoto C, Suzuki K, Hato F, Akahori M, Hasegawa T, Hino M, and Kitagawa S. Anti-apoptotic effect of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and cyclic AMP on human neutrophils: protein synthesis-dependent and protein synthesis-independent mechanisms and role of Janus kinase-STAT pathway. Int J Hematol 77: 6070, 2003.[ISI][Medline]
36. Scherle PA, Jones EA, Favata MF, Daulerio AJ, Covington MB, Nurnberg SA, Magolda RL, and Trzaskos JM. Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes. J Immunol 161: 56815686, 1998.
37. Schuringa JJ, Schepers H, Vellenga E, and Kruijer W. Ser727-dependent transcriptional activation by association of p300 with STAT3 upon IL-6 stimulation. FEBS Lett 495: 7176, 2001.[CrossRef][ISI][Medline]
38. Shikama N, Lyon J, and La Thangue NB. The p300/CBP family: integrating signals with transcription factors and chromatin. Trends Cell Biol 7: 230236, 1997.[CrossRef][ISI]
39. Sloand EM, Kim S, Maciejewski JP, van Rhee F, Chaudhuri A, Barrett J, and Young NS. Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo. Blood 95: 22692274, 2000.
40. Steinman RA and Iro A. Suppression of G-CSF-mediated Stat signalling by IL-3. Leukemia 13: 5461, 1999.[CrossRef][ISI][Medline]
41. Sunami K, Teshima T, Nawa Y, Hiramatsu Y, Maeda Y, Takenaka K, Shinagawa K, Ishimaru F, Ikeda K, Niiya K, and Harada M. Administration of granulocyte colony-stimulating factor induces hyporesponsiveness to lipopolysaccharide and impairs antigen-presenting function of peripheral blood monocytes. Exp Hematol 29: 11171124, 2001.[CrossRef][ISI][Medline]
42. Suzuki K, Hino M, Hato F, Tatsumi N, and Kitagawa S. Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-. Blood 93: 341349, 1999.
43. Suzuki K, Hino M, Kutsuna H, Hato F, Sakamoto C, Takahashi T, Tatsumi N, and Kitagawa S. Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1. J Immunol 167: 59405947, 2001.
44. Takeda K, Clausen BE, Kaisho T, Tsujimura T, Terada N, Forster I, and Akira S. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10: 3949, 1999.[ISI][Medline]
45. Terashima T, Soejima K, Waki Y, Nakamura H, Fujishima S, Suzuki Y, Ishizaka A, and Kanazawa M. Neutrophils activated by granulocyte colony-stimulating factor suppress tumor necrosis factor- release from monocytes stimulated by endotoxin. Am J Respir Cell Mol Biol 13: 6973, 1995.[Abstract]
46. Tsai EY, Falvo JV, Tsytsykova AV, Barczak AK, Reimold AM, Glimcher LH, Fenton MJ, Gordon DC, Dunn IF, and Goldfeld AE. A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo. Mol Cell Biol 20: 60846094, 2000.
47. Van der Bruggen T, Nijenhuis S, van Raaij E, Verhoef J, and van Asbeck BS. Lipopolysaccharide-induced tumor necrosis factor alpha production by human monocytes involves the Raf-1/MEK1-MEK2/ERK1-ERK2 pathway. Infect Immun 67: 38243829, 1999.
48. Vincent ME, Foote M, and Morstyn G. Pharmacology of filgrastim (r-metHuG-CSF). In: Filgrastim (r-metHuG-CSF) in Clinical Practice, edited by Morstyn G and Dexter TM. New York: Dekker, 1994, p. 3350.
49. Volpi I, Perruccio K, Tosti A, Capanni M, Ruggeri L, Posati S, Aversa F, Tabilio A, Romani L, Martelli MF, and Velardi A. Postgrafting administration of granulocyte colony-stimulating factor impairs functional immune recovery in recipients of human leukocyte antigen haplotype-mismatched hematopoietic transplants. Blood 97: 25142521, 2001.
50. Wang L, Rudert WA, Loutaev I, Roginskaya V, and Corey SJ. Repression of c-Cbl leads to enhanced G-CSF Jak-STAT signaling without increased cell proliferation. Oncogene 21: 53465355, 2002.[CrossRef][ISI][Medline]
51. Ward AC, Hermans MHA, Smith L, van Aesch YM, Schelen AM, Antonissen C, and Touw IP. Tyrosine-dependent and independent mechanisms of STAT3 activation by the human granulocyte colony-stimulating factor (G-CSF) receptor are differentially utilized depending on G-CSF concentration. Blood 93: 113124, 1999.
52. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, and Akira S. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301: 640643, 2003.
53. Yao J, Mackman N, Edgington TS, and Fan ST. Lipopolysaccharide induction of the tumor necrosis factor- promoter in human monocytic cells: regulation by Egr-1, c-Jun, and NF-
B transcription factors. J Biol Chem 272: 1779517801, 1997.
54. Yasukawa H, Ohishi M, Mori H, Murakami M, Chinen T, Aki D, Hanada T, Takeda K, Akira S, Hoshijima M, Hirano T, Chien KR, and Yoshimura A. IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat Immun 4: 551556, 2003.[CrossRef][ISI]
55. Yuo A, Kitagawa S, Motoyoshi K, Azuma E, Saito M, and Takaku F. Rapid priming of human monocytes by human hematopoietic growth factors: granulocyte-macrophage colony-stimulating factor (CSF), macrophage-CSF, and interleukin-3 selectively enhance superoxide release triggered by receptor-mediated agonists. Blood 79: 15531557, 1992.[Abstract]
56. Ziegler-Heitbrock HWL, Sternsdorf T, Liese J, Belohradsky B, Weber C, Wedel A, Schreck R, Bauerle P, and Strobel M. Pyrrolidine dithiocarbamate inhibits NF-B mobilization and TNF production in human monocytes. J Immunol 151: 69866993, 1993.