Expression Of G-CSF Research Articles

Reduced expression of G-CSF in intestinal mononuclear cells from non-inflamed regions is associated with inflammatory bowel disease

Reduced expression of G-CSF in intestinal mononuclear cells from non-inflamed regions is associated with inflammatory bowel disease

Frequent in Vivo redundancy of C/EBPβ and C/EBPε during Myeloid Development

CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.

Id1 Provides a Proper Hematopoietic Progenitor Niche Function

Development of hematopoietic stem cells (HSC) and their progeny is maintained by the interaction with cells in the microenvironment. In addition to hematopoietic cells, Id1 is expressed in stromal cells known to support hematopoiesis, and is involved in cell proliferation, differentiation and senescence. Therefore, to investigate the role of Id1 in hematopoiesis, we examined hematologic phenotypes of Id1−/− mice. In this study, we found increased neutrophils and macrophages, and decreased B cells and platelets in peripheral blood, and decreased BM cellularity. While the percentages of hematopoietic stem cells (HSC) in Id1−/− mice were increased relative to the Id1+/+ mice, their total numbers and function appeared normal. For example, Id1 was not required for self-renewal or repopulation of HSC. In contrast, we found that there were increased numbers of hematopoietic progenitor cells (HPC) in S phase of cell cycle in Id1−/− mice BM, suggesting that the loss of Id1 within HPC promotes proliferation. However, purified Id1−/− HPC had the same proliferation potential as Id1+/+ HPC when cultured in vitro. In transplantation experiments, we proved that BM microenvironment in Id1−/− mice is defective by showing that the Id1+/+ HSC showed impaired hematopoietic development in Id1−/− mice, while the Id1−/− HSC had normal repopulation potential in an Id1+/+ microenvironment. In agreement with these findings, Id1−/− BM stromal cell cultures supported enhanced proliferation of hematopoietic progenitors. Furthermore, quantitative PCR showed that SCF, M-CSF, OPN, SDF-1 and TGF-α mRNA expression was decreased in Id1−/− stromal cells relative to Id1+/+ stromal cells, while G-CSF, GM-CSF, and VEGF mRNA expression was significantly increased. Id1−/− BM showed decreased number of mesenchymal stem/progenitor cells. Thus, Id1 does not play a role in maintaining HSC, but is involved in regulating hematopoietic progenitor niche. Funded by NCI contract No. N01-CO-12400.

Expression and Suppressive Effects of Interleukin-19 on Vascular Smooth Muscle Cell Pathophysiology and Development of Intimal Hyperplasia

Anti-inflammatory cytokines may play a protective role in the progression of vascular disease. The purpose of this study was to characterize interleukin (IL)-19 expression and function in the development of intimal hyperplasia, and discern a potential mechanism of its direct effects on vascular smooth muscle cells (VSMCs). IL-19 is an immunomodulatory cytokine, the expression of which is reported to be restricted to inflammatory cells. In the present study, we found that IL-19 is not expressed in quiescent VSMCs or normal arteries but is induced in human arteries by injury and in cultured human VSMCs by inflammatory cytokines. Recombinant IL-19 significantly reduced VSMC proliferation (37.1 +/- 4.8 x 10(3) versus 72.2 +/- 6.1 x 10(3) cells/cm(2)) in a dose-dependent manner. IL-19 adenoviral gene transfer significantly reduced proliferation and neointimal formation in balloon angioplasty-injured rat carotid arteries (0.172 +/- 29.9, versus 0.333 +/- 71.9, and 0.309 +/- 56.6 microm(2)). IL-19 induced activation of STAT3 as well as the expression of the suppressor of cytokine signaling 5 (SOCS5) in VSMCs. IL-19 treatment significantly reduced the activation of p44/42 and p38 MAPKs in stimulated VSMCs. Additionally, SOCS5 was found to interact with both p44/42 and p38 MAPKs in IL-19-treated human VSMCs. This is the first description of the expression of both IL-19 and SOCS5 in VSMCs and of the functional interaction between SOCS5 and MAPKs. We propose that through induction of SOCS5 and inhibition of signal transduction, IL-19 expression in VSMCs may represent a novel, protective, autocrine response of VSMCs to inflammatory stimuli.

A Systemic Combination Therapy with Granulocyte-Colony Stimulating Factor Plus Erythropoietin Aggravates the Healing Process of Balloon-Injured Rat Carotid Arteries

Percutaneous vascular interventions lead inevitably to a destruction of the endothelial lining at the site of injury. There are conflicting data on the therapeutic benefit of hematopoietic growth factors aiming at mobilisation of circulating endothelial cells to accelerate the reendothelialisation process. Aim of our study was to evaluate the impact of a maximised 7 day-combination therapy with G-CSF plus EPO on the healing process following balloon injury of the rat carotid artery. Osmotic pumps to systemically deliver G-CSF and EPO at saturating doses during the first seven days post injury were implanted into the peritoneal cavity of splenectomised male Sprague-Dawley rats. Cytokine treatment resulted in significantly elevated numbers of white blood cells, hematocrit levels, circulating hematopoietic stem cells, and-temporarily-circulating endothelial precursors. Functional reendothelialisation as assessed by Evan's blue staining on day 14 post injury was not affected by the cytokine treatment. The neointimal response was analysed on day 7 and 28 post injury, and was significantly higher at the day 7 timepoint (Cytokines: I/M-ratio 1.10+/-0.09 vs. Saline: 0.36+/-0.06). Cytokine treated rats also displayed higher rates of thrombotic occlusion (Cytokines: 25-33% vs. Saline: 0%). Serum levels of PAI-1, TGFbeta1, and PDGF-BB were not elevated in the cytokine treated rats. The proliferative rates both in the injured vessel wall and the surrounding adventitia as assessed by BrdU incorporation were significantly higher in the cytokine treated animals. The number of CD45(pos) hematopoietic cells was significantly higher in the adventitia of the cytokine treated rats. Vasa vasorum were not found to be significantly different. The increased neointimal response was not due to expression of G-CSF- or EPO-receptors on VSMCs within the vessel wall or myofibroblasts in the adventitia. The systemic administration of G-CSF plus EPO during the first week after balloon-injury impairs the vascular healing process by increasing the neointimal response and the risk for thrombotic occlusion.

Characteristics of transplanted mouse myeloproliferative disease developed after repeated injections of granulocytic colony-stimulating factor

Transplanted myeloproliferative disease developed in mice against the background of repeated injections of granulocytic CSF was characterized using morphological and molecular biological methods. It was demonstrated that transplanted myeloproliferative disease had a non-viral nature and is probably induced by repeated injections of granulocytic CSF. Tumor cells actively populate the liver of sick animals, which leads to their rapid death. Expression of Myc, Abl, G-CSF, and MPO genes is enhanced, which is typical of myeloid neoplastic transformation.

Optimization of the AT-content of Codons Immediately Downstream of the Initiation Codon and Evaluation of Culture Conditions for High-level Expression of Recombinant Human G-CSF in Escherichia coli

Enhanced therapeutic importance of recombinant human granulocyte colony stimulating factor (rhG-CSF) has encouraged us to develop a processing method for its high-level expression in E. coli. In this study, we established a high-yielding clone by incorporation of silent mutations at N-terminal region of human G-CSF gene. We studied and optimized various parameters of culture conditions connected with the expression of rhG-CSF. The maximum expression was obtained in a defined medium supplemented with 1% glucose. The gene in pET-3a vector in E. coli BL21 (DE3) PLysS host strain was induced with 2 mM isopropyl beta-D: -1-thiogalacto pyronoside. The cell growth and productivity was enhanced about 1.6- and 1.5-folds, respectively when inducing the culture at OD(600) value of 6 than 2. The protein expression was significantly increased by addition of rifampicin at concentration of 200 microg/ml. The AT content of 51.8% with suitable codon sequences at N-terminal region and the concentration of rifampicin were identified as the key factors with a significant impact on protein expression. The specific productivity of 104 mg/OD/l (68.7% of total cellular protein) of rhG-CSF was obtained toward the end of the study, which is almost 1.5 times higher yield than reported so far in the literature.

The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.

It was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). By using human fetal osteoblastic cell line hFOB1.19 as a model of control, we investigated the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured and checked for their morphology, immunophenotype, CFU-F forming capacity and the expression of hematopoietic cytokines. A feeder layer was prepared by osteoblasts induced from 3rd generation of cultured MSCs and treated with mitomycin C. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to co-culture in vitro without exogenous cytokines. FCM revealed that both MSCs and hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed mRNA of SCF, IL-6, IL-11, SDF-1, GM-CSF and G-CSF. MSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and CFU-F forming capacity were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS could sustain GM-CFC survival for at least 3 weeks. The CFU-GM yield from cells in upper layer of co-culture was not different from those of control in hematopoiesis supportive experiments in vitro (P>0.05). RT-PCR clearly demonstrated that the cultured BM-MSCs from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSCs differentiated toward osteoblasts, the expression of G-CSF could be detected, whereas GM-CSF remained undetectable. The same expression profile of above cytokines were also seen in osteoblasts induced from BM-MSCs of MDS patients. In conclusion, osteoblasts may play a pivotal role in hematopoiesis. The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support survival of GM-CFC hematopoietic progenitor cells in vitro, according to their expression of multiple cytokines. These findings suggested that the osteoblasts derived from MDS patients may not be involved in the malignant process.

STAT5 Is an Ambivalent Regulator of Neutrophil Homeostasis

BackgroundAlthough STAT5 promotes survival of hematopoietic progenitors, STAT5−/− mice develop mild neutrophilia.Methodology/Principal findingsHere, we show that in STAT5−/− mice, liver endothelial cells (LECs) autonomously secrete high amounts of G-CSF, allowing myeloid progenitors to overcompensate for their intrinsic survival defect. However, when injected with pro-inflammatory cytokines, mutant mice cannot further increase neutrophil production, display a severe deficiency in peripheral neutrophil survival, and are therefore unable to maintain neutrophil homeostasis. In wild-type mice, inflammatory stimulation induces rapid STAT5 degradation in LECs, G-CSF production by LECs and other cell types, and then sustained mobilization and expansion of long-lived neutrophils.ConclusionWe conclude that STAT5 is an ambivalent factor. In cells of the granulocytic lineage, it exerts an antiapoptotic function that is required for maintenance of neutrophil homeostasis, especially during the inflammatory response. In LECs, STAT5 negatively regulates granulopoiesis by directly or indirectly repressing G-CSF expression. Removal of this STAT5-imposed brake contributes to induction of emergency granulopoiesis.

The Neutrophil/Th1 Lymphocyte Balance and the Therapeutic Effect of Granulocyte Colony-Stimulating Factor in TNBS-Induced Colitis of Rat Strains

Intramucosal neutrophil infiltration is related to the activity of ulcerative colitis, and Th1 immunity is responsible for the onset of Crohn's disease. We examined the therapeutic effects of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) in the two types of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis of five rat strains. SD and DA rats showed much lower mRNA expression levels of endogenous G-CSF in lipopolysaccharide (LPS)-stimulated splenocytes than did Lewis, F344, and BN rats. On day 7 after anal instillation of TNBS, SD and DA rats demonstrated massive lymphocyte infiltration with an interferon-gamma (IFN-gamma) mRNA upregulation, whereas Lewis, F344, and BN rats showed an intense submucosal neutrophil accumulation with high tumor necrosis factor-alpha (TNF-alpha) mRNA levels. A 5-day course of rHuG-CSF pretreatment (250 microg/kg/day, s.c.) reduced the elevated levels of both cytokines. The treatment improved the survival rate of DA and reduced the degree of body weight loss of SD, while not significantly influencing the wasting disease of other strains. Interleukin-10 (IL-10) mRNA levels were highly upregulated by rHuG-CSF treatment on day 1 in the neutrophil-dominant lesions of F344 but not in the Th1-type lesions of SD, and IL-12p35 mRNA levels were downregulated in both. A supply of G-CSF prevents the onset of Th1-type TNBS colitis and does not deteriorate neutrophil-dominant chronic colitis in hosts showing higher expression of endogenous G-CSF.

Involvement of TNF Receptor-Associated Factor 6 in IL-25 Receptor Signaling

IL-25 (IL-17E) induces IL-4, IL-5, and IL-13 production from an unidentified non-T/non-B cell population and subsequently induces Th2-type immune responses such as IgE production and eosinophilic airway inflammation. IL-25R is a single transmembrane protein with homology to IL-17R, but the IL-25R signaling pathways have not been fully understood. In this study, we investigated the signaling pathway under IL-25R, especially the possible involvement of TNFR-associated factor (TRAF)6 in this pathway. We found that IL-25R cross-linking induced NF-kappaB activation as well as ERK, JNK, and p38 activation. We also found that IL-25R-mediated NF-kappaB activation was inhibited by the expression of dominant negative TRAF6 but not of dominant negative TRAF2. Furthermore, IL-25R-mediated NF-kappaB activation, but not MAPK activation, was diminished in TRAF6-deficient murine embryonic fibroblast. In addition, coimmunoprecipitation assay revealed that TRAF6, but not TRAF2, associated with IL-25R even in the absence of ligand binding. Finally, we found that IL-25R-mediated gene expression of IL-6, TGF-beta, G-CSF, and thymus and activation-regulated chemokine was diminished in TRAF6-deficient murine embryonic fibroblast. Taken together, these results indicate that TRAF6 plays a critical role in IL-25R-mediated NF-kappaB activation and gene expression.

Murine serum obtained from bone marrow-transplanted mice promotes the proliferation of hematopoietic stem cells by co-culture with MS-5 murine stromal cells

To examine whether serum obtained from bone marrow-transplanted mice can selectively expand hematopoietic stem cells (HSCs) among whole bone marrow cells in vitro, whole bone marrow cells were cultured with or without MS-5 murine stromal cells in the presence of serum obtained from transplanted mice on day 3 (day 3 serum) or serum from normal mice for 7 days. When whole bone marrow cells and MS-5 cells were co-cultured in day 3 serum for 7 days, the c-kit-positive, Sca-1-positive, lineage marker-negative cells (KSL cells) expanded approximately 25 times; however, when they were co-cultured in normal serum for 7 days, the KSL cells expanded approximately 1.3 times. Direct contact between the whole bone marrow cells and MS-5 cells was essential for expansion of KSL cells in the co-culture, and it upregulated the expression of some cytokines in MS-5. Above all, the day 3 serum specifically upregulated the expression of SCF, SDF-1 alpha, G-CSF, IL-11 and IL-6 in MS-5. The level of testosterone in the day 3 serum was higher than normal serum and the addition of the testosterone in the culture expanded the KSL cells among whole bone marrow cells on MS-5 cells and also upregulated the expression of SDF-1 alpha, IL-11 and IL-6 in MS-5. These data indicates that the serum of bone marrow-transplanted mice contains a factor(s) that induced changes in the expression levels of various cytokines in MS-5 stromal cells and enabled the MS-5 cells to expand the KSL cells among whole bone marrow cells.

Different angiogenic phenotypes in primary and secondary glioblastomas

Primary and secondary glioblastomas (pGBM, sGBM) are supposed to evolve through different genetic pathways, including EGF receptor and PDGF and its receptor and thus genes that are involved in tumor-induced angiogenesis. However, whether other angiogenic cytokines are also differentially expressed in these glioblastoma subtypes is not known so far, but this knowledge might be important to optimize an antiangiogenic therapy. Therefore, we studied the expression of several angiogenic cytokines, including VEGF-A, HGF, bFGF, PDGF-AB, PDGF-BB, G-CSF and GM-CSF in pGBMs and sGBMs as well as in gliomas WHO III, the precursor lesions of sGBMs. In tumor tissues, expression of all cytokines was observed albeit with marked differences concerning intensity and distribution pattern. Quantification of the cytokines in the supernatant of 30 tissue-corresponding glioma cultures revealed a predominant expression of VEGF-A in pGBMs and significantly higher expression levels of PDGF-AB in sGBMs. HGF and bFGF were determined in nearly all tumor cultures but with no GBM subtype or malignancy-related differences. Interestingly, GM-CSF and especially G-CSF were produced less frequently by tumor cells. However, GM-CSF secretion occurred together with an increased number of simultaneously secreted cytokines and correlated with a worse patient prognosis and may thus represent a more aggressive angiogenic phenotype. Finally, we confirmed an independent contribution of each tumor-derived cytokine analyzed to tumor-induced vascularization. Our data indicate that an optimal antiangiogenic therapy may require targeting of multiple angiogenic pathways that seem to differ markedly in pGBMs and sGBMs.

Comparative methods for multiplex analysis of cytokine protein expression in plasma of lipopolysaccharide-treated mice

Changes in circulating cytokines might serve as predictors of compound-evoked inflammatory responses. CD-1 mice were treated with lipopolysaccharide (LPS; 0.2 ml of 0.25 mg/ml, intraperitoneal) for subsequent expression measurement of plasma cytokine protein expression at 24-h post-treatment using multiple antibody Western blot, and at both 2-h and 24-h post-treatment using antibody array and suspension bead array. Antibody array provided a semi-qualitative assessment and suggested significantly increased expression of GCSF at 2-h post-treatment and GCSF, IL-6, IL-12, MCP-1, MCP-5, RANTES and sTNFR1 at 24-h post-treatment. Densitometric analysis of multiple antibody Western blots provided a semi-quantitative assessment and indicated significantly increased expression of IL-6, IL-12, IL-17, GCSF, eotaxin, and MCP-2 at 24-h post-treatment. The suspension bead array yielded statistically significant cytokine protein expression increases for IL-6, IL-10, IFNγ and TNFα at both 2-h and 24-h post-treatments, while significant expression at 24-h post-treatment only was noted for IL-1β, IL-5, IL-12 and GM-CSF. Suspension bead array provided the greatest range of detection, revealing subtle increased expression of GM-CSF, IL-1β, IL-5, IL-10, TNFα and IFNγ at 24-h post-treatment, not detected by antibody array or multiple antibody Western blot. Suspension bead array proved to be the best method for detection of LPS-evoked changes in plasma cytokine levels.

Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell

Mesenchymal stem cells (MSCs), which are adherent stromal cells of a nonhematopoietic origin, have the ability to give rise to various differentiated cell types. MSCs regulate localization, self-renewal and differentiation of hematopoietic stem cells (HSCs) due to MSCs' secretion of cytokines and growth factors, the cell-to-cell interactions and the influence of the extracellular matrix proteins. Using RT-PCR analysis, we examined the expression levels of cytokines and growth factors from MSCs and their differentiated cell types, including osteoblasts, adipocytes and endothelial cells. Cytokine and growth factor genes, including IL-6, IL-8, IL-11, IL-12, IL-14, IL-15, LIF, G-CSF, GM-CSF, M-SCF, FL and SCF, were found to be expressed in the MSCs. In contrast, there was no IL-1α, IL-1β, or IL-7 expression observed. The IL-12, IL-14, G-CSF, and GM-CSF mRNA expression levels either disappeared or decreased after the MSCs differentiated into osteoblasts, adipocytes, and endothelial cells. Among the differentiated cells derived from MSCs, osteoblasts, adipocytes, and endothelial cells expressed the osteopontin, aP 2, and the VEGFR-2 gene, respectively. These profiles could help determine future clinical applications of MSCs and their derivatives for cell therapy.

THE GRANULOPOIETIC CYTOKINE RESPONSE AND ENHANCEMENT OF GRANULOPOIESIS IN MICE DURING ENDOTOXEMIA

In response to bacterial infection, the production of neutrophils by the bone marrow is accelerated. This study investigated the granulopoietic cytokine response and granulopoiesis during endotoxemia. Male Balb/c mice were intravenously challenged with lipopolysaccharide (LPS, 20 microg in 100 microL of saline per mouse). Control animals received saline alone. In a separate set of experiments, i.v. murine granulocyte colony-stimulating factor (G-CSF; 20 microg/kg) or vehicle (5% dextrose) was administered to mice. Endotoxemia caused a marked increase in G-CSF, keratinocyte-derived chemokine (KC), and macrophage inflammatory protein-2 (MIP-2) in the plasma and bone marrow between 1 and 4 h after the challenge. G-CSF, KC, and MIP-2 mRNA expression was also upregulated in the lung, liver, spleen, and bone marrow between 1 and 4 h after i.v. LPS. Intravenous administration of G-CSF caused a significant increase in G-CSF concentration in the plasma and bone marrow without upregulating G-CSF mRNA expression in the bone marrow. The levels of phospho-signal transducers and activators of transcription 3 and phospho-p44/42 mitogen-activated protein kinase were elevated in bone marrow cells at 30 min and 4 h after i.v. G-CSF and LPS, respectively. Granulocyte-macrophage colony-forming unit counts were significantly increased in the bone marrow, spleen, and blood at 48 h post-i.v. LPS or G-CSF. These data show that extramedullary organs produce granulopoietic cytokines in response to LPS. Because the tissue mass in extramedullary organs far exceeds that in the bone marrow, extramedullary production of these cytokines likely play a critical role in the regulation of the host's granulopoietic response to endotoxemia.

Role of transcription factor Egr-1 in liver injury following hemorrhagic shock and resuscitation

Objective: To investigate the role of transcription factor Egr-1 in liver injury following hemorrhagic shock (HS) /resuscitation (R). Methods: Both Egr-1 knockout (KO) and wild-type (WT) mice were subjected to HS and HSR injuries. The expressions of TNF-α, IL-6, G-CSF and ICAM-1 mRNAs in the liver were examined by RT-PCR, and their serum levels were measured by ELISA. The liver inflammatory infiltration and liver injury in both Egr-1 WT and KO mice following HS/R were evaluated by liver MPO content, serum ALT level and histological examination. Results: Egr-1 inhibition resulted in less mRNA expression of TNF-α, IL-6 , G-CSF and ICAM-1 in the liver, and lower serum levels of TNF-α, IL-6, G-CSF and ICAM-1 antigens in Egr-1 KO mice following HS/R. The liver inflammatory infiltration and liver injury were less severe in Egr-1 KO mice following HS/R, as evidenced by lower serum ALT level, lower hepatic MPO content and histological manifestations. Conclusion: Our data suggest that transcription factor Egr-1 is involved in regulating the expression of inflammatory response genes and plays a role in liver injury following HS/R.

Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation

The aim of this study was to investigate the stimulating effect of low-dose radiation (LDR) on bone marrow hematopoietic progenitor cell (HPC) proliferation and peripheral blood mobilization. Mice were exposed to 25- to 100-mGy x-rays. Bone marrow and peripheral blood HPCs (BFU-E, CFU-GM, and c-kit+ cells) were measured, and GM-CSF, G-CSF, and IL-3 protein and mRNA expression were detected using ELISA, slot blot hybridization, and Northern blot methods. To functionally evaluate LDR-stimulated and -mobilized HPCs, repopulation of peripheral blood cells in lethally irradiated recipients after transplantation of LDR-treated donor HPCs was examined by WBC counts, animal survival, and colony-forming units in the recipient spleens (CFUs-S). 75-mGy x-rays induced a maximal stimulation for bone marrow HPC proliferation (CFU-GM and BFU-E formation) 48 hours postirradiation, along with a significant increase in HPC mobilization into peripheral blood 48 to 72 hours postradiation, as shown by increases in CFU-GM formation and proportion of c-kit+ cells in the peripheral mononuclear cells. 75-mGy x-rays also maximally induced increases in G-CSF and GM-CSF mRNA expression in splenocytes and levels of serum GM-CSF. To define the critical role of these hematopoietic-stimulating factors in HPC peripheral mobilization, direct administration of G-CSF at a dose of 300 microg/kg/day or 150 microg/kg/day was applied and found to significantly stimulate GM-CFU formation and increase c-kit+ cells in the peripheral mononuclear cells. More importantly, 75-mGy x-rays plus 150 microg/kg/day G-CSF (LDR/150-G-CSF) produced a similar effect to that of 300 microg/kg/day G-CSF alone. Furthermore, the capability of LDR-mobilized donor HPCs to repopulate blood cells was confirmed in lethally irradiated recipient mice by counting peripheral WBC and CFUs-S. These results suggest that LDR induces hematopoietic hormesis, as demonstrated by HPC proliferation and peripheral mobilization, providing a potential approach to clinical application for HPC peripheral mobilization.

Expression of G-CSF and GM-CSF in human meningiomas correlates with increased tumor proliferation and vascularization.

The hematopoietic growth factors granulocyte- and granulocyte-macrophage colony stimulating factor (G-CSF and GM-CSF) are nowadays widely used in routine cancer therapies as potent factors to control radiation and chemotherapy induced neutropenia, a side effect that frequently endangers the success of tumor therapies. However, there is little information about the role of G-CSF and GM-CSF for tumor growth or progression. We were interested in the expression and potential role of both factors in human meningiomas, tumors of arachnoidal origin that account for about 20% of all primary intracranial tumors. Therefore, we analyzed immunohistochemically the protein expression of G-CSF, GM-CSF and their respective receptors in 30 meningioma tissues of different malignancy and histopathological type. Both factors and receptors were not expressed in the corresponding normal tissue. In contrast, G-CSF, GM-CSF and their receptors were expressed to a varying degree in human meningiomas. Increasing expression of both factors and receptors correlated significantly with enhanced proliferation in the tumor and thus with higher malignancy. In addition, a strong perivascular expression of G-CSF was associated with a highly vascularized tumor type. Thus, expression of both G-CSF and GM-CSF is associated with the expression of proliferation vascularization, two markers of an increasingly malignant tumor phenotype, suggesting a contribution of both factors to tumor progression.

AIF-1 expression modulates proliferation of human vascular smooth muscle cells by autocrine expression of G-CSF.

Allograft inflammatory factor-1 (AIF-1) is associated with vascular smooth muscle cell (VSMC) activation and vascular injury. The purpose of this study was to characterize the molecular mechanism of AIF-1 growth-enhancing effects in human VSMC. Primary human VSMCs were stably transduced with AIF-1 retrovirus (RV). Impact on cell growth was evaluated by the increase in cell number, and the effects on gene expression were determined by cDNA microarray analysis. AIF-RV overexpressing cells grew significantly more rapidly than empty-RV control cells in growth medium and serum-reduced medium (P<0.01 and 0.02, respectively). cDNA microarray analysis and Western blotting on serum-starved AIF-1-transduced VSMCs identified increased mRNA expression of several cell cycle proteins and, surprisingly, the cytokine G-CSF. Addition of G-CSF caused a 75% increase in proliferation of VSMCs in the absence of serum growth factors. The proliferative effects of AIF-1 were abrogated by neutralizing antibodies to G-CSF (P<0.05), and AIF-1-transduced VSMCs are chemotactic for human monocytes. Increased expression of G-CSF and colocalization with AIF-1 positive cells were seen in diseased, not normal human coronary arteries. This study indicates that AIF-1 enhances VSMC growth by autocrine production of G-CSF, and AIF-1 expression may influence VSMC-inflammatory cell communication.