Induction Of Granulocyte Colony-stimulating Factor Research Articles

G-CSF–induced hematopoietic stem cell mobilization from the embryonic hematopoietic niche does not require neutrophils and macrophages

Hematopoietic stem cell transplantation requires the collection of hematopoietic cells from patients or stem cell donors. Granulocyte colony-stimulating factor (G-CSF) is widely used in the clinic to mobilize hematopoietic stem and progenitor cells (HSPCs) from the adult bone marrow niche into circulation, allowing a collection of HSPCs from the blood. The mechanism by which G-CSF acts to mobilize HSPCs is unclear, with some studies showing a direct stimulation of stem cells and others suggesting that myeloid cells are required. In this study, we developed a heat-inducible G-CSF transgenic zebrafish line to study HSPC mobilization in vivo. Live imaging of HSPCs after G-CSF induction revealed an increase in circulating HSPCs, demonstrating a successful HSPC mobilization. These mobilized HSPCs went on to prematurely colonize the kidney marrow, the adult zebrafish hematopoietic niche. We eliminated neutrophils or macrophages using a nitroreductase-based cell ablation system and found that G-CSF still mobilizes HSPCs from the niche. Our findings indicate that neutrophils and macrophages are not required for G-CSF-induced HSPC mobilization from the embryonic hematopoietic niche.

Vitamin D Promotes Trophoblast Cell Induced Separation of Vascular Smooth Muscle Cells in Vascular Remodeling via Induction of G-CSF.

Vitamin D deficiency is associated with complications of pregnancy such as pre-eclampsia, fetal growth restriction, and miscarriage, all of which are also associated with incomplete spiral artery (SpA) remodeling. We have previously shown that both uterine natural killer (uNK) cells and extravillous trophoblast cells (EVT) are required for successful SpA remodeling, but whether their activity in this process is modulated by vitamin D is not known. In the current study, we use a previously described chorionic plate artery (CPA) ex vivo model of vascular remodeling to determine the effects of 1,25(OH)2D treated uNK cell, placental explant (PEx), and uNK/PEx conditioned medium (CM) on vascular smooth muscle cell (VSMC) disorganization and phenotypic switching. Significant results were followed up in VSMCs in vitro. We demonstrate that 1,25(OH)2D can enhance the ability of PEx to induce SpA remodeling, via a mechanism associated with increased secretion of granulocyte-colony stimulating factor (G-CSF). G-CSF appears able to increase VSMC disorganization and phenotypic switching in both an ex vivo vascular model and in vitro VSMC cultures. The clinical relevance of these findings are still to be determined. G-CSF may have differential effects depending on dose and vascular bed, and vitamin D may play a role in potentiating these actions. G-CSF may be an interesting potential therapeutic target for facilitating physiological vascular remodeling for the prevention of adverse obstetric outcomes.

Honey isomaltose contributes to the induction of granulocyte-colony stimulating factor (G-CSF) secretion in the intestinal epithelial cells following honey heating

We have previously discovered that heated honey but not unheated honey could induce the secretion of granulocyte-colony stimulating factor (G-CSF) in the MCE301 intestinal epithelial cells. The objective of this study was to identify compounds in honey that could contribute to this activity. We bought several kinds of commercial honey samples derived from different flowers, as well as corn syrup samples, in the markets of China and Japan, and heated them at 180 °C for 30 min. MCE301 cells were treated with the medium containing the samples, and G-CSF levels in the medium were measured by ELISA. By comparing their activities and sugar contents, we discovered that isomaltose was primarily implicated. The optimum heating conditions for isomaltose were at 180 °C for 60 min or at 200 °C for 15–30 min, and these time- and temperature-dependencies were similar to those of honey in our previous study. When heated isomaltose was partitioned by dialysis, the active ingredients were transferred into a high-molecular-weight fraction. By size-exclusion HPLC analysis, the average molecular weight of heated isomaltose was 790 kDa. When heated isomaltose was hydrolyzed by acids, glucose was subsequently produced. Maltose, sucrose, turanose, and trehalose did not exhibited any activity when heated at 180 °C for 60 min, indicating that the glucose groups with α(1 → 6)-binding in the isomaltose molecule play important roles in its activity when oxidatively polymerized by heat. The stimulating activity of heated isomaltose was inhibited by toll-like receptor 4 (TLR4) inhibitor, suggesting that heated isomaltose activates TLR4 to induce G-CSF. Since G-CSF is clinically used for cancer patients to accelerate their recovery from neutropenia following chemotherapy or accompanied with aplastic anemia, these findings indicate that honey which contains high level of isomaltose could improve immunosuppressive conditions when honey is heated, and that heated isomaltose might be of potential therapeutic use in patients with compromised immunity caused by chemotherapeutic agents.

A combined prophylactic modality of podophyllotoxin and rutin alleviates radiation induced injuries to the lymphohematopoietic system of mice by modulating cytokines, cell cycle progression, and apoptosis

The present study was conceptualized to delineate radioprotective efficacy of a formulation G-003M (a combination of podophyllotoxin and rutin) against radiation-induced damage to the lymphohematopoietic system of mice. C57BL/6J mice, treated with G-003M 1 h prior to 9 Gy lethal dose, were assessed for reactive oxygen species (ROS)/nitric oxide (NO) generation, antioxidant alterations, Annexin V/PI and TUNEL staining for apoptosis, modulation of apoptotic proteins, cell proliferation, histological alterations in thymus and cell cycle arrest in bone marrow cells. Induction of granulocyte colony-stimulating factor (G-CSF), granulocytes macrophage colony-stimulating factor (GM-CSF), interleukin-IL-6, IL-10, IL-1α, and IL-1β in response to G-003M was also evaluated in different groups of mice. Haematopoietic reconstitution with G-003M was explored by examining endogenous spleen colony-forming units (CFU-S) in irradiated animals. G-003M significantly inhibited ROS/NO, malondialdehyde (MDA) and restored cellular antioxidant glutathione in the thymus of irradiated animals. G-003M pre-treatment significantly (p < 0.001) restrained apoptosis in thymocytes via upregulation of Bcl2 and down-regulation of Bax, p53 and caspase-3. Stimulation of cell proliferation and inhibition of apoptosis by G-003M, restored architecture of thymus in irradiated animals within 30 days as evaluated by histological analysis. G-003M arrested cells at the G2/M phase by inducing reversible cell cycle arrest. Peak expression of G-CSF (45-fold) and IL-6 (60-fold) as well as moderate induction of GM-CSF, IL-10, IL-1α by G-003M helped in haematopoietic recovery of irradiated mice. A higher number of endogenous CFU-S in G-003M pre-treated irradiated mice suggested haematopoietic recovery. Data obtained from the current study affirms that G-003M can be proved as a potential radioprotective agent against radiation damage.

Deuteration of the farnesyl terminal methyl groups of δ-tocotrienol and its effects on the metabolic stability and ability of inducing G-CSF production

δ-tocotrienol (DT3), a member of vitamin E family, has been shown to have a potent radio-protective effect. However, its application as a radioprotectant is limited, at least in part, by its short plasma elimination half-life and low bioavailability. In an effort to increase the metabolic stability of DT3, a deuterium substituted DT3 derivative, d6-DT3, was designed and synthesized. d6-DT3 showed improved in vitro and in vivo metabolic stability compared to DT3. The unexpected lower potency of d6-DT3 in inducing granulocyte-colony stimulating factor (G-CSF) production in mouse revealed that the metabolite(s) of DT3 might play a major role in inducing G-CSF induction.

3184 A Case of Alcoholic Hepatitis With Unusual Leukamoid Reaction

INTRODUCTION: Leukemoid reaction is defined by predominantly neutrophilic leukocytosis with a total leukocyte count &gt;50,000 cells/µl, after excluding leukemia. Leukemoid reaction is seen in various conditions. It is very rare in alcoholic hepatitis (AH). Herein, we present a case of a young alcoholic male who was admitted with alcoholic hepatitis and leukemoid reaction. He underwent extensive workup to rule out other etiologies and subsequently improved after glucocorticoid therapy. CASE DESCRIPTION/METHODS: 28-year-old male with history of alcohol abuse presented with a two-week history of abdominal pain, distention, jaundice and vomiting. On exam, the patient had a fever and tachycardia. He was also found to be icteric and had ascites. Investigations revealed a bilirubin of 9.4 mg/dl. WBC of 42,000 cells/µl with a left shift. Ultrasound showed enlarged fatty liver. MDF was 61 and MELD was 37. Septic workup was negative including ascitic fluid cell count and culture. He was started on prednisolone 40 mg. Hematologic workup including peripheral smear ruled out neoplasm. Leukocyte alkaline phosphatase was elevated at 249. The patient was discharged on prednisolone and was followed as an outpatient. He improved after four weeks and his WBC count normalized. DISCUSSION: Leukemoid reaction is a very rare event in AH and is a poor prognostic marker. The pathophysiology is likely due to the increased levels of granulocyte colony stimulating factor (G-CSF) released by the damaged hepatocytes. The release of excessive amounts of tumor necrosis factor-alpha has been postulated to be instrumental in the causation of leukemoid reaction in alcoholic patients as well. However, Arguelles-Grande et al. reported a patient in which the cytokine profile did not demonstrate increased amounts of TNF-a. In their patient, levels of IL-18 and IL-1b were increased. IL-1b stimulates the proliferation and maturation of neutrophils in the bone marrow both directly by the induction of granulocyte-macrophage colony-stimulating factor and G-CSF; thus the postulated pathogenic sequence for LR: increased IL-18 produces increased IL-1b, which along with increased IL-8 produces neutrophilia. In this setting, corticosteroids may play a role by their reported inhibition of IL-1b transcription. Hematologic evaluation in these cases is necessary to rule out underlying neoplasm. If patients with severe alcoholic hepatitis are found to have leukemoid reaction, treatment with steroids should be considered after excluding sepsis and neoplasm.

Succinate ester derivative of \u03b4-tocopherol enhances the protective effects against 60Co \u03b3-ray-induced hematopoietic injury through granulocyte colony-stimulating factor induction in mice

α-tocopherol succinate (α-TOS), γ-tocotrienol (GT3) and δ-tocotrienol (DT3) have drawn large attention due to their efficacy as radioprotective agents. α-TOS has been shown to act superior to α-tocopherol (α-TOH) in mice by reducing lethality following total body irradiation (TBI). Because α-TOS has been shown to act superior to α-tocopherol (α-TOH) in mice by reducing lethality following total body irradiation (TBI), we hypothesized succinate may be contribute to the radioprotection of α-TOS. To study the contributions of succinate and to identify stronger radioprotective agents, we synthesized α-, γ- and δ-TOS. Then, we evaluated their radioprotective effects and researched further mechanism of δ-TOS on hematological recovery post-irradiation. Our results demonstrated that the chemical group of succinate enhanced the effects of α-, γ- and δ-TOS upon radioprotection and granulocyte colony-stimulating factor (G-CSF) induction, and found δ-TOS a higher radioprotective efficacy at a lower dosage. We further found that treatment with δ-TOS ameliorated radiation-induced pancytopenia, augmenting cellular recovery in bone marrow and the colony forming ability of bone marrow cells in sublethal irradiated mice, thus promoting hematopoietic stem and progenitor cell recovery following irradiation exposure. δ-TOS appears to be an attractive radiation countermeasure without known toxicity, but further exploratory efficacy studies are still required.

Polyphenol E Enhances the Antitumor Immune Response in Neuroblastoma by Inactivating Myeloid Suppressor Cells

Neuroblastoma is a rare childhood cancer whose high risk, metastatic form has a dismal outcome in spite of aggressive therapeutic interventions. The toxicity of drug treatments is a major problem in this pediatric setting. In this study, we investigated whether Polyphenon E, a clinical grade mixture of green tea catechins under evaluation in multiple clinical cancer trials run by the National Cancer Institute (Bethesda, MD), has anticancer activity in mouse models of neuroblastoma. We used three neuroblastoma models: (i) transgenic TH-MYCN mouse developing spontaneous neuroblastomas; (ii) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenotransplanted with human SHSY5Y cells; and (iii) A/J mice transplanted with syngeneic Neuro 2A cells. Mice were randomized in control and Polyphenon E-drinking groups. Blood from patients with neuroblastoma and normal controls was used to assess the phenotype and function of myeloid cells. Polyphenon E reduced the number of tumor-infiltrating myeloid cells, and inhibited the development of spontaneous neuroblastomas in TH-MYCN transgenic mice. In therapeutic models of neuroblastoma in A/J, but not in immunodeficient NOD/SCID mice, Polyphenon E inhibited tumor growth by acting on myeloid-derived suppressor cells (MDSC) and CD8 T cells. In vitro, Polyphenon E impaired the development and motility of MDSCs and promoted differentiation to more neutrophilic forms through the 67 kDa laminin receptor signaling and induction of granulocyte colony-stimulating factor. The proliferation of T cells infiltrating a patient metastasis was reactivated by Polyphenon E. These findings suggest that the neuroblastoma-promoting activity of MDSCs can be manipulated pharmacologically in vivo and that green tea catechins operate, at least in part, through this mechanism.

Direct Actions of Granulocyte-Colony Stimulating Factor on Human Neuronal and Monocytic Cell Lines

Introduction: Granulocyte colony stimulating factor (G-CSF) administration produces beneficial effects in rodent models of stroke, trauma and neurodegenerative diseases by acting on both bone marrow-derived and neuronal cells. The aim of the study was to elucidate cellular mechanism(s) of G-CSF action by direct application to neuronal and monocytic cell lines. Method: Cell culture models of monocytes (THP-1) and neurons (SH-SY5Y) cells were incubated with G-CSF. The following parameters were measured: G-CSF receptor binding kinetics; DNA synthesis; signal transduction, in particular expression of alternatively spliced protein kinase C (PKCδVIII) and the anti-apoptotic protein Bcl-2; changes in adhesiveness and migratory properties induced by G-CSF in the monocytic cells. Results: G-CSF receptor binding kinetics in the two lines differed, with Kd in the neuronal line being significantly higher than that of the monocytic cells. Despite higher affinity of G-CSF for receptors on the monocytic cells, G-CSF treatment increased Bcl-2 expression in the neuronal line at lower concentrations than that required in the monocytic cell line. G-CSF did not increase either cellular adhesiveness or migration through a semi-permeable membrane, whereas monocyte chemotactic protein (MCP-1) significantly improved migration. Conclusions: The cellular and molecular responses to G-CSF treatment of monocytic cells suggest that neither changes in adhesiveness nor migratory capacity are responsible for the beneficial effects of G-CSF administration in models of neurologic diseases. G-CSF induction of anti-apoptotic signaling in neurons is an important component of its neuroprotective effects in models of brain injury.

Identification of Granulocyte Colony-Stimulating Factor and Interleukin-6 as Candidate Biomarkers of CBLB502 Efficacy as a Medical Radiation Countermeasure

Given an ever-increasing risk of nuclear and radiological emergencies, there is a critical need for development of medical radiation countermeasures (MRCs) that are safe, easily administered, and effective in preventing and/or mitigating the potentially lethal tissue damage caused by acute high-dose radiation exposure. Because the efficacy of MRCs for this indication cannot be ethically tested in humans, development of such drugs is guided by the Food and Drug Administration's Animal Efficacy Rule. According to this rule, human efficacious doses can be projected from experimentally established animal efficacious doses based on the equivalence of the drug's effects on efficacy biomarkers in the respective species. Therefore, identification of efficacy biomarkers is critically important for drug development under the Animal Efficacy Rule. CBLB502 is a truncated derivative of the Salmonella flagellin protein that acts by triggering Toll-like receptor 5 (TLR5) signaling and is currently under development as a MRC. Here, we report identification of two cytokines, granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), as candidate biomarkers of CBLB502's radioprotective/mitigative efficacy. Induction of both G-CSF and IL-6 by CBLB502 1) is strictly TLR5-dependent, 2) occurs in a CBLB502 dose-dependent manner within its efficacious dose range in both nonirradiated and irradiated mammals, including nonhuman primates, and 3) is critically important for the ability of CBLB502 to rescue irradiated animals from death. After evaluation of CBLB502 effects on G-CSF and IL-6 levels in humans, these biomarkers will be useful for accurate prediction of human efficacious CBLB502 doses, a key step in the development of this prospective radiation countermeasure.

Role of radiation-induced granulocyte colony-stimulating factor in recovery from whole body gamma-irradiation

The purpose of this study was to further elucidate the radioprotective role of granulocyte colony-stimulating factor (G-CSF) induced in response to irradiation. The induction of G-CSF and interleukin-6 (IL-6) in response to radiation exposure was evaluated in mice. The level of cytokine in serum was determined by multiplex Luminex. The role of G-CSF on survival and tissue injury after total body gamma-irradiation was evaluated by administration of neutralizing antibody to G-CSF before radiation exposure. An isotype control was used for comparison and survival was monitored for 30d after irradiation. Jejunum samples were used for immunohistochemistry. Ionizing radiation exposure induced significant levels of the hematopoietic cytokines G-CSF and IL-6, in mice receiving 9.2Gy radiation. Maximal levels of G-CSF were observed in peripheral blood of mice 8h after irradiation. IL-6 levels were maximum at 12h after irradiation. Administration of G-CSF antibody significantly enhanced mortality in irradiated mice. G-CSF antibody-treated mice had higher numbers of CD68+ cells and apoptotic cells in intestinal villi. Our results confirm that radiation exposure induces elevations of circulating G-CSF and IL-6. Neutralizing antibody to G-CSF exacerbates the deleterious effects of radiation, indicating that G-CSF induced in response to irradiation plays an important role in recovery.

IL-17A stimulates granulocyte colony-stimulating factor production via ERK1/2 but not p38 or JNK in human renal proximal tubular epithelial cells

We investigated the potential role of IL-17A in the induction of granulocyte colony-stimulating factor (G-CSF), a critical granulopoietic growth factor, in human renal proximal tubular epithelial cells. Human renal proximal tubular cells (HK-2, ATCC) were used to characterize the effects of IL-17A or IL-17F on G-CSF production, using ELISA, real-time RT-PCR, and immunoblotting. The cell surface expression of IL-17 receptors (IL-17Rs) was analyzed by flow cytometry. IL-17A stimulation of proximal tubular cells led to a dose- and time-dependent increase in secreted G-CSF. This effect was dependent on mRNA transcription and protein translation. Real-time RT-PCR demonstrated that G-CSF mRNA expression reached a maximum level at 6 h following IL-17A stimulation and that this increase was dose dependent. Both IL-17RA and IL-17RC were expressed on proximal tubular cells. IL-17A also enhanced TNF-α- or IL-1β-mediated G-CSF secretion from cells. Additionally, IL-17A induced MAPK (ERK1/2 but not p38 MAPK or JNK) activation, and pharmacological inhibitors of MEK1/2 (U0126) but not of p38 MAPK (SB203580) or JNK (SP600125), significantly blocked the IL-17A-mediated G-CSF release. We demonstrated the potential ability of IL-17A to induce G-CSF in renal proximal tubular cells. It is proposed that IL-17A may play an important role in neutrophil transmigration and activation via stimulation of G-CSF in tubular injury.

Radioprotective efficacy of tocopherol succinate is mediated through granulocyte-colony stimulating factor

The purpose of this study was to elucidate the role of granulocyte colony-stimulating factor (G-CSF) induced by α-tocopherol succinate (TS) in protecting mice from total-body irradiation. CD2F1 mice were injected with a radioprotective dose of TS and the levels of cytokine in serum induced by TS were determined by multiplex Luminex. Neutralization of G-CSF was accomplished by administration of a G-CSF antibody and confirmed by cytokine analysis. The role of G-CSF on gastrointestinal tissue protection afforded by TS after irradiation (11Gy, 0.6Gy/min of 60Co γ-radiation) was determined by analysis of jejunum histopathology for crypt, villi, mitotic figures, apoptosis, and cell proliferation. Our results demonstrate that TS protected mice against high doses of radiation-induced gastrointestinal damage and TS also induced very high levels of G-CSF and keratinocyte-derived chemokine (KC) production in peripheral blood 24h after subcutaneous administration. When TS-injected mice were administered a neutralizing antibody to G-CSF, there was complete neutralization of G-CSF in circulating blood, and the protective effect of TS was significantly abrogated by G-CSF antibody. Histopathology of jejunum from TS-injected and irradiated mice demonstrated protection of gastrointestinal tissue, yet the protection was abrogated by administration of a G-CSF antibody. In conclusion, our current study suggests that induction of G-CSF resulting from TS administration is responsible for protection from 60Co γ-radiation injury.

TNF-α–dependent loss of IKKβ-deficient myeloid progenitors triggers a cytokine loop culminating in granulocytosis

Loss of IκB kinase (IKK) β-dependent NF-κB signaling in hematopoietic cells is associated with increased granulopoiesis. Here we identify a regulatory cytokine loop that causes neutrophilia in Ikkβ-deficient mice. TNF-α-dependent apoptosis of myeloid progenitor cells leads to the release of IL-1β, which promotes Th17 polarization of peripheral CD4(+) T cells. Although the elevation of IL-17 and the consecutive induction of granulocyte colony-stimulating factor compensate for the loss of myeloid progenitor cells, the facilitated induction of Th17 cells renders Ikkβ-deficient animals more susceptible to the development of experimental autoimmune encephalitis. These results unravel so far unanticipated direct and indirect functions for IKKβ in myeloid progenitor survival and maintenance of innate and Th17 immunity and raise concerns about long-term IKKβ inhibition in IL-17-mediated diseases.

Rapamycin inhibits lipopolysaccharide induction of granulocyte-colony stimulating factor and inducible nitric oxide synthase expression in macrophages by reducing the levels of octamer-binding factor-2

This article reports an inhibitory effect of rapamycin on the lipopolysaccharide (LPS)-induced expression of both inducible nitric oxide synthase (iNOS) and granulocyte-colony stimulating factor (G-CSF) in macrophages and its underlying mechanism. The study arose from an observation that rapamycin inhibited the LPS-induced increase in octamer-binding factor-2 (Oct-2) protein levels through a mammalian target of rapamycin (mTOR)-dependent pathway in mouse RAW264.7 macrophages. As both iNOS and G-CSF are potential Oct-2 target genes, we tested the effect of rapamycin on their expression and found that it reduced the LPS-induced increase in iNOS and G-CSF mRNA levels and iNOS and G-CSF protein levels. Blocking of mTOR-signaling using a dominant-negative mTOR expression plasmid resulted in inhibition of the LPS-induced increase in iNOS and G-CSF protein levels, supporting the essential role of mTOR. Forced expression of Oct-2 using the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LPS-induced increase in iNOS and G-CSF mRNA levels. Chromatin immunoprecipitation assays showed that LPS enhanced the binding of Oct-2 to the iNOS and G-CSF promoters and that this effect was inhibited by pretreatment with rapamycin. Moreover, RNA interference knockdown of Oct-2 reduced iNOS and G-CSF expression in LPS-treated cells. The inhibitory effect of rapamycin on the LPS-induced increase in Oct-2 protein levels and on the iNOS and G-CSF mRNA levels was also detected in human THP-1 monocyte-derived macrophages. This study demonstrates that rapamycin reduces iNOS and G-CSF expression at the transcription level in LPS-treated macrophages by inhibiting Oct-2 expression.

Complement anaphylatoxin C5a contributes to hemodialysis-associated thrombosis

AbstractThrombosis is a common complication of end-stage renal disease, particularly in patients on hemodialysis. Although substantial progress has been made in preventing thrombotic complications in various other groups of patients, the mechanisms of thrombosis during hemodialysis require clarification. In this report, we demonstrate that complement activation triggered by hemodialysis biomaterials, and the subsequent generation of the complement anaphylatoxin C5a, results in the expression of functionally active tissue factor (TF) in peripheral blood neutrophils. Because TF is a key initiator of coagulation in vivo, we postulate that the recurring complement activation that occurs during long-term hemodialysis contributes to thrombosis in dialyzed end-stage renal disease patients. Furthermore, we found that complement contributed to the induction of granulocyte colony-stimulating factor, which has been implicated in the pathogenesis of thrombosis in patients treated with the recombinant form of this molecule. Importantly, the inhibition of complement activation attenuated the TF expression and granulocyte colony-stimulating factor induction in blood passing through a hemodialysis circuit, suggesting that the complement system could become a new therapeutic target for preventing thrombosis in patients with chronic renal failure who are maintained on hemodialysis.

Complement anaphylatoxin C5a contributes to hemodialysis-associated thrombosis

Thrombosis is a common complication of end-stage renal disease, particularly in patients on hemodialysis. Although substantial progress has been made in preventing thrombotic complications in various other groups of patients, the mechanisms of thrombosis during hemodialysis require clarification. In this report, we demonstrate that complement activation triggered by hemodialysis biomaterials, and the subsequent generation of the complement anaphylatoxin C5a, results in the expression of functionally active tissue factor (TF) in peripheral blood neutrophils. Because TF is a key initiator of coagulation in vivo, we postulate that the recurring complement activation that occurs during long-term hemodialysis contributes to thrombosis in dialyzed end-stage renal disease patients. Furthermore, we found that complement contributed to the induction of granulocyte colony-stimulating factor, which has been implicated in the pathogenesis of thrombosis in patients treated with the recombinant form of this molecule. Importantly, the inhibition of complement activation attenuated the TF expression and granulocyte colony-stimulating factor induction in blood passing through a hemodialysis circuit, suggesting that the complement system could become a new therapeutic target for preventing thrombosis in patients with chronic renal failure who are maintained on hemodialysis.

Abstract 5196: Granulocyte-colony stimulating factor (G-CSF) plays a causative role for H-Ras-mediated invasion and migration of human breast epithelial cells

Abstract The whole human genome microarray on normal MCF10A, N-Ras-transformed, non-invasive MCF10A and H-Ras-transformed invasive MCF10A human breast epithelial cells revealed that the granulocyte-colony stimulating factor (G-CSF) was most highly upregulated in an H-Ras-specific manner. The present study investigated whether G-CSF plays a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, siRNA-mediated knockdown of G-CSF expression significantly reduced H-Ras-induced matrix metalloproteinase(MMP)-2 expression as well as invasion/migration. Activations of Rac1, MKK3/6 and p38 MAPK were inhibited by siRNA-knockdown of G-CSF. The induction of G-CSF expression conferred the invasive/migratory phenotype to parental MCF10A cells. Expression of MMP-2 and activation of signaling molecules Rac1, MKK3/6 and p38 MAPK were increased by G-CSF induction. Taken together, this study revealed G-CSF gene as a candidate marker for metastatic potential of breast epithelial cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5196.

A Novel Role of CD40/CD40Ligand Dyad in Regulation of Bone Marrow Granulopoiesis.

Abstract 4556Members of the tumor necrosis factor / tumor necrosis factor receptor (TNF/TNFR) superfamily are involved in bone marrow (BM) homeostasis by regulating the survival, apoptosis, proliferation, and differentiation of hematopoietic progenitor and precursor cells. The CD40 Ligand (CD40L)/CD40 molecules belong to the TNF/TNFR superfamily; we have recently showed that the CD40/CD40L interaction on BM CD34+ cells accelerates the apoptotic cell death. The aim of the present study is to investigate the distribution and function of the CD40/CD40L molecules in the BM granulocytic progenitor and precursor cell populations as well as the effect of their interactions on the stromal release of cytokines related to granulopoiesis. BM samples were obtained from posterior iliac crest aspirates from 19 hematologically healthy subjects after informed consent. We evaluated: (a) the surface expression of CD40 and CD40L on immunomagnetically sorted CD34+, CD34-/CD33+, CD34-/CD33-/CD15+ cells representing sequential stages of the granulocytic development, using flow cytometry in steady state conditions and following 20-hour incubation with 25ng/ml recombinant-human (rh) TNFα; (b) the proportion of apoptotic cells and the level of caspase-3 activation following CD40 engagement in the above cell populations using flow cytometry and a chromo-enzymatic method, respectively; (c) the production of granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) by long-term BM culture (LTBMC) stromal cells upon induction with rhCD40L (1μg/ml), using an enzyme-linked immunoabsorbent assay (ELISA) in culture supernatants. We found that the basal expression of CD40 and CD40L on the CD34+, CD34-/CD33+, CD34-/CD33-/CD15+ cells was 3.34%±3.90% and 1.8%±1.03%, 16.74%±14.69% and 6.96%±4.68%, 3.22%±3.36% and 2.26%±2.71%, respectively. Following incubation with rhTNFα a substantial increase was obtained in the expression of CD40 in all the above cell populations compared to baseline (37.18%±12.51%, 29.25%±14.81%, 15.84%±6.28%, respectively) (p<0.0001, p=0.0003, p=0.0002 respectively). Upon engagement of the TNF-induced CD40 in the CD34+, CD34-/CD33+, CD34-/CD33-/CD15+ cells, a significant increase was obtained in the proportion of apoptotic cells (13.98%±10.43%, 27.24%±19.30%, 25.64%±14.82%, respectively) in comparison to TNF-induced cells without CD40 ligation (5.25%±4.04%, 11.6%±9.04%, 12.42%±5.22%, respectively) (p=0.0071, p=0.0296, p=0.0169, respectively). In keeping with this finding was the significant increase in the caspase-3 activation in all the above populations upon ligation of CD40 compared to baseline (p<0.0001, p=0.0281, p=0.0046, respectively). CD40 was expressed in both CD45+ (8.25%±8.31%) and CD45- (8.46%±4.93%) adherent cell populations (hematopoietic and non hematopoietic, respectively) of confluent LTBMCs (week-3). Following 48-hour incubation of confluent LTBMC stromal layers with rhCD40L, a significant increase was obtained in the production of G-CSF and GM-CSF (2171±2161 pg/ml and 84.10±84.25 pg/ml, respectively) compared to unstimulated cultures (885±1099 pg/ml and 26.57±28.25 pg/ml, respectively) (p=0.0041 and p=0.0074, respectively). These data suggest that the CD40/CD40L interactions, although minimal under steady state conditions, display a dual role in regulation of granulopoiesis under inflammatory conditions by affecting both the BM granylocytic progenitor/precursor cells and the BM microenvironmental cells. More specifically, the expression of CD40 is upregulated in all stages of granulocytic development under the influence of TNFα and upon ligation with CD40L results in acceleration of the apoptotic cell death. The induction of G-CSF and GM-CSF production by BM stroma may represent a compensatory mechanism to rescue granulocytic progenitor/precursor cells from apoptosis. These mechanisms may have a role in the pathogenesis of chronic idiopathic neutropenia where there is evidence for both increased BM TNFα and serum CD40L levels. Disclosures:No relevant conflicts of interest to declare.

Induction of granulocyte colony-stimulating factor by globular adiponectin via the MEK–ERK pathway

Adiponectin, an adipocyte-derived cytokine, affects a number of physiological processes, including immune function and inflammation. We investigated whether globular adiponectin (gAd) affects the expression of inflammation-related genes in murine macrophages (RAW264 cells). DNA microarray analysis indicated that granulocyte colony-stimulating factor (G-CSF) showed the largest increase in expression in gAd-stimulated RAW264 cells. The gAd-induced secretion of G-CSF increased in a time- and dose-dependent manner. U0126 (MEK1/2 inhibitor) and PD98059 (MEK1 inhibitor) reduced the gAd-induced G-CSF mRNA expression and G-CSF protein production. gAd induced the phosphorylation of MEK1/2 and ERK1/2 in RAW264 cells. In addition, the gAd-induced phosphorylation of MEK1/2 and ERK1/2 was dramatically reduced by PD98059 and U0126, respectively. Collectively, these results suggest that MEK1/2-ERK1/2 signaling is involved in the adiponectin-induced secretion of G-CSF.