SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis

To determine the importance of suppressor of cytokine signaling-3 (SOCS3) in the regulation of hematopoietic growth factor signaling generally, and of G-CSF-induced cellular responses specifically, we created mice in which the Socs3 gene was deleted in all hematopoietic cells. Although normal until young adulthood, these mice then developed neutrophilia and a spectrum of inflammatory pathologies. When stimulated with G-CSF in vitro, SOCS3-deficient cells of the neutrophilic granulocyte lineage exhibited prolonged STAT3 activation and enhanced cellular responses to G-CSF, including an increase in cloning frequency, survival, and proliferative capacity. Consistent with the in vitro findings, mutant mice injected with G-CSF displayed enhanced neutrophilia, progenitor cell mobilization, and splenomegaly, but unexpectedly also developed inflammatory neutrophil infiltration into multiple tissues and consequent hind-leg paresis. We conclude that SOCS3 is a key negative regulator of G-CSF signaling in myeloid cells and that this is of particular significance during G-CSF-driven emergency granulopoiesis.     

G-CSF: a key regulator of neutrophil production, but that's not all!

G-CSF is a major extracellular regulator of haemopoiesis and the innate immune system. Named for its relatively specific stimulation of the growth of neutrophil progenitor cells in vitro in semi-solid cultures (Burgess and Metcalf 1980, Nicola et al. 1983), G-CSF influences the survival, proliferation and differentiation of all cells in the neutrophil lineage, from haemopoietic stem cell through to mature neutrophil. Further, G-CSF influences the function of mature neutrophils. These actions underpin its rapid uptake into clinical medicine as a drug that increases the production of neutrophils in patients with chemotherapy-induced neutropenia.Ongoing research has uncovered initially unsuspected polyfunctionality for G-CSF. G-CSF is well recognised as a potent mobiliser of haemopoietic stem cells from the bone marrow into the blood, and now is being increasingly accepted as a regulator of immune responses. These two "new" actions of G-CSF first came to light through observations made during clinical trials of G-CSF. Subsequent investigations into the cellular and molecular basis for this polyfunctionality have generated exciting new knowledge about the biology of G-CSF. This review emphasises recent advances in knowledge about G-CSF signalling, mechanisms of G-CSF-induced stem cell mobilisation, and how G-CSF influences T-cell function and dendritic cell activation. An attempt is made to link the current issues about the biology of G-CSF with its clinical uses, both present and future.     

G-CSF: A key regulator of neutrophil production,but that's not all!

G-CSF is a major extracellular regulator of haemopoiesis and the innate immune system. Named for its relatively specific stimulation of the growth of neutrophil progenitor cells in vitro in semi-solid cultures (Burgess and Metcalf 1980, Nicola et al. 1983), G-CSF influences the survival, proliferation and differentiation of all cells in the neutrophil lineage, from haemopoietic stem cell through to mature neutrophil. Further, G-CSF influences the function of mature neutrophils. These actions underpin its rapid uptake into clinical medicine as a drug that increases the production of neutrophils in patients with chemotherapy-induced neutropenia.

Ongoing research has uncovered initially unsuspected polyfunctionality for G-CSF. G-CSF is well recognised as a potent mobiliser of haemopoietic stem cells from the bone marrow into the blood, and now is being increasingly accepted as a regulator of immune responses. These two “new” actions of G-CSF first came to light through observations made during clinical trials of G-CSF. Subsequent investigations into the cellular and molecular basis for this polyfunctionality have generated exciting new knowledge about the biology of G-CSF. This review emphasises recent advances in knowledge about G-CSF signalling, mechanisms of G-CSF-induced stem cell mobilisation, and how G-CSF influences T-cell function and dendritic cell activation. An attempt is made to link the current issues about the biology of G-CSF with its clinical uses, both present and future.     

GRK6 deficiency is associated with enhanced CXCR4-mediated neutrophil chemotaxis in vitro and impaired responsiveness to G-CSF in vivo

The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling pathway is thought to play an important role in the induction of neutrophil mobilization from the bone marrow in response to granulocyte-colony stimulating factor (G-CSF) treatment. CXCR4 belongs to the family of G protein-coupled receptors. Multiple members of this receptor family are desensitized by agonist-induced G protein-coupled receptor kinase (GRK)-mediated phosphorylation. Here, we demonstrate that in vitro SDF-1-induced chemotaxis of bone marrow-derived neutrophils from GRK6-deficient mice is significantly enhanced and that desensitization of the calcium response to SDF-1 is impaired in GRK6-/- neutrophils. CXCR4 activation by SDF-1 provides a key retention signal for hematopoietic cells in the bone marrow. It is interesting that we observed that in the absence of GRK6, the G-CSF-induced increase in circulating neutrophils is profoundly impaired. Three days after injection of pegylated-G-CSF, significantly lower numbers of circulating neutrophils were observed in GRK6-/- as compared with wild-type (WT) mice. In addition, early/acute neutrophil mobilization in response to G-CSF (3 h after treatment) was also impaired in GRK6-/- mice. However, blood neutrophil levels in untreated GRK6-/- and WT mice were not different. Moreover, the percentage of neutrophils in the bone marrow after G-CSF treatment was increased to the same extent in WT and GRK6-/- mice, indicating that neutrophil production is normal in the absence of GRK6. However, the increased chemotactic sensitivity of GRK6-/- neutrophils to SDF-1 was retained after G-CSF treatment. In view of these data, we suggest that the impaired G-CSF-induced neutrophil mobilization in the absence of GRK6 may be a result of enhanced CXCR4-mediated retention of PMN in the bone marrow.     

G-CSF – A double edge sword in neutrophil mediated immunity

Neutrophils are vital for the innate immune system’s control of pathogens and neutrophil deficiency can render the host susceptible to life-threatening infections. Neutrophil responses must also be tightly regulated because excessive production, recruitment or activation of neutrophils can cause tissue damage in both acute and chronic inflammatory diseases. Granulocyte colony stimulating factor (G-CSF) is a key regulator of neutrophil biology, from production, differentiation, and release of neutrophil precursors in the bone marrow (BM) to modulating the function of mature neutrophils outside of the BM, particularly at sites of inflammation. G-CSF acts by binding to its cognate cell surface receptor on target cells, causing the activation of intracellular signalling pathways mediating the proliferation, differentiation, function, and survival of cells in the neutrophil lineage. Studies in humans and mice demonstrate that G-CSF contributes to protecting the host against infection, but conversely, it can play a deleterious role in inflammatory diseases. As such, neutrophils and the G-CSF pathway may provide novel therapeutic targets. This review will focus on understanding the role G-CSF plays in the balance between effective neutrophil mediated host defence versus neutrophil-mediated inflammation and tissue damage in various inflammatory and infectious diseases.     

Effects of granulocyte colony-stimulating factor on the proliferation and cell-fate specification of neural stem cells

Granulocyte-colony stimulating factor (G-CSF) is a growth factor that regulates proliferation, differentiation and survival of hematopoietic progenitor cells. There is growing evidence to suggest that G-CSF exerts a powerful neuroprotective effect in different neurological disorders. However, it has remained to be elucidated if G-CSF has a direct effect on neural stem cells (NSCs). Here, we show that G-CSF could stimulate the proliferation of NSCs and promote their differentiation in vitro. Additionally, we have shown that G-CSF-induced proliferation of NSCs is associated with phosphorylation of STAT3, and the differentiation is linked to altered expression of differentiation-related genes. Remarkably, G-CSF could not initiate the differentiation of NSCs. The added roles of G-CSF in regulating proliferation and differentiation of NSCs as shown in this study would serve as a useful reference in designing new stem cell therapy strategies for promoting brain recovery and repair.     

STAT3 is a negative regulator of granulopoiesis but is not required for G-CSF-dependent differentiation

STAT3 has been described as an essential component of G-CSF-driven cell proliferation and granulopoiesis. This notion was tested by conditional gene ablation in transgenic mice. Contrary to expectation, granulocytes developed from STAT3 null bone marrow progenitors, and STAT3 null neutrophils displayed mature effector functions. Rather than a deficit in granulopoiesis, mice lacking STAT3 in their hematopoietic progenitors developed neutrophilia, and bone marrow cells were hyperresponsive to G-CSF stimulation. These studies provide direct evidence for STAT3-independent granulopoiesis and suggest that STAT3 directs a negative feedback loop necessary for controlling neutrophil numbers, possibly through induced expression of the signaling inhibitor, SOCS3.     

miR126 positively regulates mast cell proliferation and cytokine production through suppressing Spred1

The protein known as Spred1 (Sprouty-related Ena/VASP homology-1 domain-containing protein) has been identified as a negative regulator of growth factor-induced ERK/mitogen-activated protein kinase activation. Spred1 has also been implicated as the target of microRNA-126 (miR126), a miRNA located within the Egfl7 gene, and is involved in the regulation of vessel development through its role in regulating VEGF signaling. In this study, we examined the role of miR126 and Spred1 in the hematopoietic system, as miR126 has been shown to be overexpressed in leukemic cells. miR126 levels were down-regulated during mast cell differentiation from bone marrow cells, whereas Spred1 expression was inversely up-regulated. Overexpression of miR126 suppressed Spred1 expression and enhanced ERK activity in primary bone marrow cells and MC9 mast cells, which were associated with elevated FcεRI-mediated cytokine production. To confirm the effect of Spred1 reduction in vivo, we generated hematopoietic cell-specific Spred1-conditional knockout mice. These mice showed increased numbers of mast cells, and Spred1-deficient bone marrow-derived mast cells were highly activated by cross-linking of Fcε-R stimulation as well as by IL-3 and SCF stimulation. These results suggest that Spred1 negatively regulates mast cell activation, which is modulated by miR126.     

Granulocyte-colony stimulating factor-mobilized mesenchymal stem cells: A new resource for rapid engraftment in hematopoietic stem cell transplantation

The bone marrow (BM) microenvironment plays an important role in regulating hematopoietic stem cell self-renewal and differentiation. Mesenchymal stem cells (MSCs), which constitute approximately 0.01-0.0001% of the nucleated cells in the adult human BM, are an important component of the BM stroma that supports hematopoiesis. The BM stroma system is often damaged in patients who have undergone high-dose chemotherapy and/or radiation treatment. Thus, the BM stroma should be reconstructed during hematopoietic stem cell transplantation (HSCT). Granulocyte-colony stimulating factor (G-CSF) is a potent hematopoietic cytokine that regulates neutrophil generation within the BM by modulating the mobility, proliferation and maturation of neutrophil progenitor cells. The results from our study here show that G-CSF markedly increased the number of donor-derived MSCs in the BM and the peripheral blood. Engraftment was faster in HSCTs with bone marrow that was treated with G-CSF (G-BM) or with G-BM- and G-CSF-treated peripheral blood stem cells compared to stead-state bone marrow (SS-BM). Based on these findings, we hypothesize that G-CSF-mobilized treatment of MSCs may accelerate engraftment in HSCT.     

α-Tocopherol induces hematopoietic stem/progenitor cell expansion and ERK1/2-mediated differentiation

Tocopherols promote or inhibit growth in different cell types. In the hematopoietic system, the radioprotective property of tocopherols is thought to act through the expansion of primitive hematopoietic cells. However, the mechanisms activated by tocopherols and which HPs are affected remain poorly understood. To better address these questions, mice were treated with α-tocopherol, and its effects were investigated in the BM microenvironment. α-Tocopherol induced increased proliferation in HSC/HP cells, leading to BM hyperplasia. In addition, differentiation to the granulocytic/monocytic lineage was enhanced by α-tocopherol treatment. α-Tocopherol treatment resulted in decreased basal phosphorylation of ERK1/2, PKC, and STAT-5 in HSC/HP cells. In contrast, α-tocopherol enhanced ERK1/2 activation in response to IL-3 stimulation in HSC/HP cells without altering the expression of IL-3Rs. Moreover, α-tocopherol-induced differentiation and ERK1/2 activation were abolished in mice pretreated with a MEK inhibitor (PD98059); however, pretreatment with PD98059 did not reduce the α-tocopherol-mediated increase in HSC/HP cells but instead, further enhanced their proliferation. Therefore, α-tocopherol induces expansion of HSC/HP cells by a nonidentified intracellular pathway and granulocytic/monocytic differentiation through ERK1/2 activation.     

Conditional overexpression of Stat3alpha in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern

Normal neutrophil development requires G-CSF signaling, which includes activation of Stat3. Studies of G-CSF-mediated Stat3 signaling in cell culture and transgenic mice have yielded conflicting data regarding the role of Stat3 in myelopoiesis. The specific functions of Stat3 remain unclear, in part, because two isoforms, Stat3alpha and Stat3beta, are expressed in myeloid cells. To understand the contribution of each Stat3 isoform to myelopoiesis, we conditionally overexpressed Stat3alpha or Stat3beta in the murine myeloid cell line 32Dcl3 (32D) and examined the consequences of overexpression on cell survival and differentiation. 32D cells induced to overexpress Stat3alpha, but not Stat3beta, generated a markedly higher number of neutrophils in response to G-CSF. This effect was a result of decreased apoptosis but not of increased proliferation. Comparison of gene expression profiles of G-CSF-stimulated, Stat3alpha-overexpressing 32D cells with those of cells with normal Stat3alpha expression revealed novel Stat3 gene targets, which may contribute to neutrophil expansion and improved survival, most notably Slc28a2, a purine nucleoside transporter, which is critical for maintenance of intracellular nucleotide levels and prevention of apoptosis, and Gpr65, an acid-sensing, G protein-coupled receptor with pro-oncogenic and antiapoptotic functions.     

Enhanced neutrophil motility by granulocyte colony-stimulating factor: the role of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase

The effect of granulocyte colony-stimulating factor (G-CSF) on human neutrophil motility was studied using videomicroscopy. Stimulation of neutrophils with G-CSF resulted in enhanced motility with morphological change and increased adherence. Enhanced neutrophil motility was detected within 3-5 min after G-CSF stimulation, reached a maximum at 10 min, and was sustained for approximately 35 min. The maximum migration rate was 84.4 +/- 2.9 microm/5 min. A study using the Boyden chamber method revealed that G-CSF-stimulated neutrophils exhibited random migration but not chemotaxis. Enhanced neutrophil motility and morphological change were inhibited by MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] inhibitors (PD98059 and U0126), and a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), but not by a p38 MAPK inhibitor (SB203580). These findings are consistent with the fact that G-CSF selectively activates MEK/ERK and PI3K, but not p38, in neutrophils. MEK/ERK activation was associated with G-CSF-induced redistribution of F-actin and phosphorylated myosin light chain. Enhanced neutrophil motility was observed even in the presence of neutralizing anti-CD18 antibody, which prevented cell adherence. These findings indicate that G-CSF induces human neutrophil migration via activation of MEK/ERK and PI3K.