The RNA m6A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression.

Emergency granulopoiesis and neutrophil mobilization that can be triggered by granulocyte colony-stimulating factor (G-CSF) through its receptor G-CSFR are essential for antibacterial innate defense. However, the epigenetic modifiers crucial for intrinsically regulating G-CSFR expression and the antibacterial response of neutrophils remain largely unclear. N6-methyladenosine (m6A) RNA modification and the related demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, but their roles in neutrophil production and mobilization are still unknown. We used cecal ligation and puncture (CLP)-induced polymicrobial sepsis to model systemic bacterial infection, and we report that ALKBH5 is required for emergency granulopoiesis and neutrophil mobilization. ALKBH5 depletion significantly impaired the production of immature neutrophils in the bone marrow of septic mice. In addition, Alkbh5-deficient septic mice exhibited higher retention of mature neutrophils in the bone marrow and defective neutrophil release into the circulation, which led to fewer neutrophils at the infection site than in their wild-type littermates. During bacterial infection, ALKBH5 imprinted production- and mobilization-promoting transcriptome signatures in both mouse and human neutrophils. Mechanistically, ALKBH5 erased m6A methylation on the CSF3R mRNA to increase the mRNA stability and protein expression of G-CSFR, consequently upregulating cell surface G-CSFR expression and downstream STAT3 signaling in neutrophils. The RIP-qPCR results confirmed the direct binding of ALKBH5 to the CSF3R mRNA, and the binding strength declined upon bacterial infection, accounting for the decrease in G-CSFR expression on bacteria-infected neutrophils. Considering these results collectively, we define a new role of ALKBH5 in intrinsically driving neutrophil production and mobilization through m6A demethylation-dependent posttranscriptional regulation, indicating that m6A RNA modification in neutrophils is a potential target for treating bacterial infections and neutropenia.

Antibodies to sclerostin or G-CSF receptor partially eliminate bone or marrow adipocyte loss, respectively, following vertical sleeve gastrectomy.

Vertical sleeve gastrectomy (VSG), the most utilized bariatric procedure in clinical practice, greatly reduces body weight and improves a variety of metabolic disorders. However, one of its long-term complications is bone loss and increased risk of fracture. Elevated circulating sclerostin (SOST) and granulocyte-colony stimulating factor (G-CSF) concentrations have been considered as potential contributors to VSG-associated bone loss. To test these possibilities, we administrated antibodies to SOST or G-CSF receptor and investigated alterations to bone and marrow niche following VSG. Neutralizing either SOST or G-CSF receptor did not alter beneficial effects of VSG on adiposity and hepatic steatosis, and anti-SOST treatment provided a further improvement to glucose tolerance. SOST antibodies partially reduced trabecular and cortical bone loss following VSG by increasing bone formation, whereas G-CSF receptor antibodies had no effects on bone mass. The expansion in myeloid cellularity and reductions in bone marrow adiposity seen with VSG were partially eliminated by treatment with Anti-G-CSF receptor. Taken together, these experiments demonstrate that antibodies to SOST or G-CSF receptor may act through independent mechanisms to partially block effects of VSG on bone loss or marrow niche cells, respectively.

Predictive Parameters of Febrile Neutropenia and Clinical Significance of G-CSF Receptor Signaling Pathway in the Development of Neutropenia during R-CHOP Chemotherapy with Prophylactic Pegfilgrastim in Patients with Diffuse Large B-Cell Lymphoma.

PURPOSE: Pegfilgrastim is widely used to prevent chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN) in patients with diffuse large B-cell lymphoma (DLBCL). We investigated the predictive factors affecting CIN and FN incidence in patients with DLBCL receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemotherapy with pegfilgrastim and conducted experiments to find reason for the occurrence of CIN even when pegfilgrastim was used. MATERIALS AND METHODS: We reviewed the CIN and FN events of 200 patients with DLBCL. Based on these data, we investigate the association with predictive factor and the levels of granulocyte-colony stimulating factor (G-CSF) receptor signaling pathway markers (pSTAT3, pAKT, pERK1/2, pBAD, and CXCR4) in bone marrow (BM) samples isolated from patients with DLBCL. RESULTS: FN was significantly associated with stage III/IV (hazard ratio [HR], 12.74) and low serum albumin levels (HR, 3.87). Additionally, patients with FN had lower progression-free survival (PFS; 2-year PFS, 51.1 % vs. 74.0%) and overall survival (OS; 2-year OS, 58.2% vs. 85.0%) compared to those without FN. The occurrence of CIN was associated with overexpression of G-CSF receptor signaling pathway markers, and expression levels of these markers were upregulated in BM cells co-cultured with DLBCL cells. The rate of neutrophil apoptosis was also higher in neutrophils co-cultured with DLBCL cells and was further promoted by treatment with doxorubicin. CONCLUSION: Our findings suggest that high DLBCL burden may alter the BM environment and G-CSF receptor signaling pathway, even in chemotherapy-naïve state, which may increase CIN frequency during R-CHOP chemotherapy.

G-CSF in tumors: Aggressiveness, tumor microenvironment and immune cell regulation.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine most well-known for maturation and mobilization of bone marrow neutrophils. Although it is used therapeutically to treat chemotherapy induced neutropenia, it is also highly expressed in some tumors. Case reports suggest that tumors expressing high levels of G-CSF are aggressive, more difficult to treat, and present with poor prognosis and high mortality rates. Research on this topic suggests that G-CSF has tumor-promoting effects on both tumor cells and the tumor microenvironment. G-CSF has a direct effect on tumor cells to promote tumor stem cell longevity and overall tumor cell proliferation and migration. Additionally, it may promote pro-tumorigenic immune cell phenotypes such as M2 macrophages, myeloid-derived suppressor cells, and regulatory T cells. Overall, the literature suggests a plethora of pro-tumorigenic activity that should be balanced with the therapeutic use. In this review, we present an overview of the multiple complex roles of G-CSF and G-CSFR in tumors and their microenvironment and discuss how clinical advances and strategies may open new therapeutic avenues.

G-CSFR antagonism reduces mucosal injury and airways fibrosis in a virus-dependent model of severe asthma.

BACKGROUND AND PURPOSE: Asthma is a chronic disease that displays heterogeneous clinical and molecular features. A phenotypic subset of late-onset severe asthmatics has debilitating fixed airflow obstruction, increased neutrophilic inflammation and a history of pneumonia. Influenza A virus (IAV) is an important viral cause of pneumonia and asthmatics are frequently hospitalised during IAV epidemics. This study aims to determine whether antagonising granulocyte colony stimulating factor receptor (G-CSFR) prevents pneumonia-associated severe asthma. EXPERIMENTAL APPROACH: Mice were sensitised to house dust mite (HDM) to establish allergic airway inflammation and subsequently infected with IAV (HKx31/H3N2 subtype). A neutralising monoclonal antibody against G-CSFR was therapeutically administered. KEY RESULTS: In IAV-infected mice with prior HDM sensitisation, a significant increase in airway fibrotic remodelling and airways hyper-reactivity was observed. A mixed granulocytic inflammatory profile consisting of neutrophils, macrophages and eosinophils was prominent and at a molecular level, G-CSF expression was significantly increased in HDMIAV-treated mice. Blockage of G-CSFR reduced neutrophilic inflammation in the bronchoalveolar and lungs by over 80% in HDMIAV-treated mice without altering viral clearance. Markers of NETosis (dsDNA and myeloperoxidase in bronchoalveolar), tissue injury (LDH activity in bronchoalveolar) and oedema (total bronchoalveolar-fluid protein) were also significantly reduced with anti-G-CSFR treatment. In addition, anti-G-CSFR antagonism significantly reduced bronchoalveolar gelatinase activity, active TFGß lung levels, collagen lung expression, airways fibrosis and airways hyper-reactivity in HDMIAV-treated mice. CONCLUSIONS AND IMPLICATIONS: We have shown that antagonising G-CSFR-dependent neutrophilic inflammation reduced pathological disruption of the mucosal barrier and airways fibrosis in an IAV-induced severe asthma model.

Granulocyte-colony stimulating factor-producing malignant phyllodes tumor of the breast: a rare case.

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF)-producing tumors can cause leukocytosis despite an absence of infection. G-CSF-producing tumors have been reported in various organs such as the lung, esophagus, and stomach but rarely in the breast. We report a case of G-CSF-producing malignant phyllodes tumor of the breast. CASE PRESENTATION: An 84-year-old woman visited our hospital complaining of a lump in her left breast without fever and pain. Laboratory tests revealed elevated white blood cell (WBC) count and G-CSF levels. A malignant tumor of the breast was diagnosed by core needle biopsy. We performed a total mastectomy and sentinel lymph node biopsy. The tumor was identified as a G-CSF-producing malignant phyllodes tumor. Within 7 days after surgery, the patient's WBC count and G-CSF level had decreased to normal levels. She is alive without recurrence 13 months after surgery. CONCLUSIONS: We encountered a rare case of G-CSF-producing malignant phyllodes tumor of the breast. PET-CT revealed diffuse accumulation of FDG in the bone. Phyllodes tumors need to be differentiated from bone metastasis, lymphoma, and leukemia. We must be careful to not mistake this type of tumor for bone marrow metastasis.

G-CSF and G-CSFR Modulate CD4 and CD8 T Cell Responses to Promote Colon Tumor Growth and Are Potential Therapeutic Targets.

Cytokines are known to shape the tumor microenvironment and although progress has been made in understanding their role in carcinogenesis, much remains to learn regarding their role in tumor growth and progression. We have identified granulocyte colony-stimulating factor (G-CSF) as one such cytokine, showing that G-CSF is linked with metastasis in human gastrointestinal tumors and neutralizing G-CSF in a mouse model of colitis-associated cancer is protective. Here, we set out to identify the role of G-CSF and its receptor, G-CSFR, in CD4+ and CD8+ T cell responses in the tumor microenvironment. MC38 colon cancer cells were injected into WT, G-CSFR-/- mice, or Rag2-/- mice. Flow cytometry, Real Time PCR and Multiplex cytokine array analysis were used for in vitro T cell phenotype analysis. Adoptive transfer of WT or G-CSFR-/- CD4+ of CD8+ T cells were performed. Mouse tumor size, cytokine expression, T cell phenotype, and cytotoxic activity were analyzed. We established that in G-CSFR-/- mice, tumor growth of MC38 colon cancer cells is significantly decreased. T cell phenotype and cytokine production were also altered, as both in vitro and in vivo approaches revealed that the G-CSF/G-CSFR stimulate IL-10-producing, FoxP3-expressing CD4+ and CD8+ T cells, whereas G-CSFR-/- T cells exhibit increased IFNγ and IL-17A production, leading to increased cytotoxic activity in the tumor microenvironment. Furthermore, peritumoral injection of recombinant IFNγ or IL-17A inhibited colon and pancreas tumor growth compared to controls. Taken together, our data reveal an unknown mechanism by which G-CSF, through its receptor G-CSFR, promotes an inhibitory Treg phenotype that limits tumor immune responses and furthermore suggest that targeting this cytokine/receptor axis could represent a novel therapeutic approach for gastrointestinal, and likely other tumors with high expression of these factors.

Application of the Moran Model in Estimating Selection Coefficient of Mutated CSF3R Clones in the Evolution of Severe Congenital Neutropenia to Myeloid Neoplasia.

Bone marrow failure (BMF) syndromes, such as severe congenital neutropenia (SCN) are leukemia predisposition syndromes. We focus here on the transition from SCN to pre-leukemic myelodysplastic syndrome (MDS). Stochastic mathematical models have been conceived that attempt to explain the transition of SCN to MDS, in the most parsimonious way, using extensions of standard processes of population genetics and population dynamics, such as the branching and the Moran processes. We previously presented a hypothesis of the SCN to MDS transition, which involves directional selection and recurrent mutation, to explain the distribution of ages at onset of MDS or AML. Based on experimental and clinical data and a model of human hematopoiesis, a range of probable values of the selection coefficient s and mutation rate µ have been determined. These estimates lead to predictions of the age at onset of MDS or AML, which are consistent with the clinical data. In the current paper, based on data extracted from published literature, we seek to provide an independent validation of these estimates. We proceed with two purposes in mind: (i) to determine the ballpark estimates of the selection coefficients and verify their consistency with those previously obtained and (ii) to provide possible insight into the role of recurrent mutations of the G-CSF receptor in the SCN to MDS transition.

Engineering an anti-granulocyte colony stimulating factor receptor nanobody for improved affinity.

AIMS: Granulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is overexpressed in several malignant tumors and blocking its binding to the receptor can lead to significant decrease in tumor growth, vascularization and metastasis. Furthermore, targeting G-CSF receptor has shown therapeutic benefit in other diseases such as rheumatoid arthritis, progressive neurodegenerative disorder and uveitis. Camelid single-chain antibodies (nanobodies) have exceptional properties making them appropriate for tumor imaging and therapeutic application. In this study we aim to use the rational design approach to engineer a previously described G-CSF-R targeting nanobody (VHH1), to improve its affinity toward G-CSF-R. MAIN METHODS: We redesigned the complementary determining region 3 (CDR3) domain of the VHH1 nanobody to mimic G-CSF interaction to its receptor and developed five new engineered nanobodies. Binding affinity of the engineered nanobodies was evaluated by ELISA (Enzyme-linked immunosorbent assay) on NFS60 cells. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) confirmed the specificity of the engineered nanobodies and ELISA-based determination of affinity revealed that two of the engineered nanobodies (1c and 5a) bind to G-CSF-R on the surface of NFS60 cells in a dose-dependent manner and with a higher potency compared to the parental nanobody. SIGNIFICANCE: Additional studies are required to better characterize these nanobodies and assess their interaction with G-CSF-R in vitro and in vivo. These newly developed nanobodies could be beneficial in tumor imaging and therapy and make a basis for development of additional engineered nanobodies.

Impairment of G-CSF receptor on granulocytic progenitor cells causes neutropenia in protein malnutrition.

OBJECTIVE: It is well known that protein malnutrition (PM) states can affect hematopoiesis, leading to severe leukopenia and reduced number of granulocytes, which act as the first line of defense, and are important to the innate immune response. The aim of this study was to elucidate some of the mechanisms involved in the impairment of granulopoiesis in PM. METHODS: Male C57BL/6 mice were submitted to PM with a low-protein diet containing 2% protein. Control mice were fed a 12% protein-containing diet. Bone marrow histology and the percentage of granulocytic progenitors were evaluated after in vivo granulocyte-colony stimulating factor (G-CSF) stimulus. Cell proliferation, STAT3 signaling, and the expression of G-CSF receptor were evaluated in hematopoietic progenitor cells. RESULTS: Malnourished animals presented with leukopenia associated with reduced number of granulocytes and reduced percentage of granulocytic progenitors; however, no differences were observed in the regulatory granulopoietic cytokine G-CSF. Additionally, the malnourished group presented with impaired response to in vivo G-CSF stimulus compared with control animals. PM was implicated in decreased ability of c-Kit+ cells to differentiate into myeloid progenitor cells and downregulated STAT3 signaling. Furthermore, the malnourished group exhibited reduced expression of G-CSF receptor on granule-monocytic progenitors. This reduced expression was not completely reversible with G-CSF treatment. CONCLUSIONS: This study implies that PM promotes intrinsic alterations to hematopoietic precursors, which result in hematologic changes, mainly neutropenia, observed in peripheral blood in PM states.

Granulocyte-colony stimulating factor enhances load-induced muscle hypertrophy in mice.

Granulocyte-colony stimulating factor (G-CSF) is a cytokine crucially involved in the regulation of granulopoiesis and the mobilization of hematopoietic stem cells from bone marrow. However, emerging data suggest that G-CSF exhibits more diverse functions than initially expected, such as conferring protection against apoptosis to neural cells and stimulating mitogenesis in cardiomyocytes and skeletal muscle stem cells after injury. In the present study, we sought to investigate the potential contribution of G-CSF to the regulation of muscle volume. We found that the administration of G-CSF significantly enhances muscle hypertrophy in two different muscle overload models. Interestingly, there was a significant increase in the transcripts of both G-CSF and G-CSF receptors in the muscles that were under overload stress. Using mutant mice lacking the G-CSF receptor, we confirmed that the anabolic effect is dependent on the G-CSF receptor signaling. Furthermore, we found that G-CSF increases the diameter of myotubes in vitro and induces the phosphorylation of AKT, mTOR, and ERK1/2 in the myoblast-like cell line C2C12 after differentiation induction. These findings indicate that G-CSF is involved in load-induced muscle hypertrophy and suggest that G-CSF is a potential agent for treating patients with muscle loss and sarcopenia.

Neuroblastoma pathogenesis: deregulation of embryonic neural crest development.

Neuroblastoma (NB) is an aggressive pediatric cancer that originates from neural crest tissues of the sympathetic nervous system. NB is highly heterogeneous both from a clinical and a molecular perspective. Clinically, this cancer represents a wide range of phenotypes ranging from spontaneous regression of 4S disease to unremitting treatment-refractory progression and death of high-risk metastatic disease. At a cellular level, the heterogeneous behavior of NB likely arises from an arrest and deregulation of normal neural crest development. In the present review, we summarize our current knowledge of neural crest development as it relates to pathways promoting 'stemness' and how deregulation may contribute to the development of tumor-initiating CSCs. There is an emerging consensus that such tumor subpopulations contribute to the evolution of drug resistance, metastasis and relapse in other equally aggressive malignancies. As relapsed, refractory disease remains the primary cause of death for neuroblastoma, the identification and targeting of CSCs or other primary drivers of tumor progression remains a critical, clinically significant goal for neuroblastoma. We will critically review recent and past evidence in the literature supporting the concept of CSCs as drivers of neuroblastoma pathogenesis.

Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models.

Autocrine protective mechanisms of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells after optic nerve crush.

This study investigated the role of autocrine mechanisms in the anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells (RGCs) after optic nerve (ON) crush. We observed that both G-CSF and G-CSF receptor (G-CSFR) are expressed in normal rat retina. Further dual immunofluorescence staining showed G-CSFR immunoreactive cells were colocalized with RGCs, Müller cells, horizontal and amacrine cells. These results confirm that G-CSF is an endogenous ligand in the retina. The semi-quantitative RT-PCR finding demonstrated the transcription levels of G-CSF and G-CSFR were up-regulated after ON crush injury. G-CSF treatment further increased and prolonged the expression level of G-CSFR in the retina. G-CSF has been shown to enhance transdifferentiation of the mobilized hematopoietic stem cells into tissue to repair central nervous system injury. We test the hypothesis that the hematopoietic stem cells recruited by G-CSF treatment can transdifferentiate into RGCs after ON crush by performing sublethal irradiation of the rats 5 days before ON crush. The flow cytometric analysis showed the number of CD34 positive cells in the peripheral blood is significantly lower in the irradiated, crushed and G-CSF-treated group than the sham control group or crush and G-CSF treated group. Nevertheless, the G-CSF treatment enhances the RGC survival after sublethal irradiation and ON crush injury. These data indicate that G-CSF seems unlikely to induce hematopoietic stem cell transdifferentiation into RGCs after ON crush injury. In conclusion, G-CSF may serve an endogenous protective signaling in the retina through direct activation of intrinsic G-CSF receptors and downstream signaling pathways to rescue RGCs after ON crush injury, exogenous G-CSF administration can enhance the anti-apoptotic effects on RGCs.

Protective role of G-CSF in dextran sulfate sodium-induced acute colitis through generating gut-homing macrophages.

Granulocyte colony-stimulating factor (G-CSF) is a pleiotropic cytokine best known for its role in promoting the generation and function of neutrophils. G-CSF is also found to be involved in macrophage generation and immune regulation; however, its in vivo role in immune homeostasis is largely unknown. Here, we examined the role of G-CSF in dextran sulfate sodium (DSS)-induced acute colitis using G-CSF receptor-deficient (G-CSFR(-/-)) mice. Mice were administered with 1.5% DSS in drinking water for 5days, and the severity of colitis was measured for the next 5days. GCSFR(-/-) mice were more susceptible to DSS-induced colitis than G-CSFR(+/+) or G-CSFR(-/+) mice. G-CSFR(-/-) mice harbored less F4/80(+) macrophages, but a similar number of neutrophils, in the intestine. In vitro, bone marrow-derived macrophages prepared in the presence of both G-CSF and macrophage colony-stimulating factor (M-CSF) (G-BMDM) expressed higher levels of regulatory macrophage markers such as programmed death ligand 2 (PDL2), CD71 and CD206, but not in arginase I, transforming growth factor (TGF)-ß, Ym1 (chitinase-like 3) and FIZZ1 (found in inflammatory zone 1), and lower levels of inducible nitric oxide synthase (iNOS), CD80 and CD86 than bone marrow-derived macrophages prepared in the presence of M-CSF alone (BMDM), in response to interleukin (IL)-4/IL-13 and lipopolysaccharide (LPS)/interferon (IFN)-γ, respectively. Adoptive transfer of G-BMDM, but not BMDM, protected G-CSFR(-/-) mice from DSS-induced colitis, and suppressed expression of tumor necrosis factor (TNF)-α, IL-1ß and iNOS in the intestine. These results suggest that G-CSF plays an important role in preventing colitis, likely through populating immune regulatory macrophages in the intestine.

Plasma G-CSF and GM-CSF Concentration and Amount of Their Receptors on the Granulocyte in Kawasaki Disease

PURPOSE: This study aimed to demonstrate the possible pathogenesis of granulopoiesis in patients of Kawasaki disease(KD) using quantitative analysis of G-CSF, GM-CSF and their CSFr. METHODS: The plasma levels of G-CSF, GM-CSF, G-CSFr and GM-CSFr were studied in 14 patients in the acute phase of KD; 13 children with normal peripheral white blood cell counts were used as the normal control group. The plasma concentration of G-CSF, GM-CSF were analyzed by ELISA. The G-CSFr and GM-CSFr on the peripheral granulocytes were analyzed by a quantitative flow cytometric assay and QuantiBRITE, and the quantitative changes of receptors which did not combine with G-CSF and GM-CSF were measured. RESULTS: The total number of leukocytes in KD was similar to normal control group, but the leukocytes increased according to the number of neutrophils. The plasma concentration of G-CSF were decreased similar to normal control group(P=0.133), but that of GM-CSF decreased more than the normal control group(P=0.227). The quantity of G-CSFr, GM-CSFr were revealed to be no less than the normal control(P=0.721, P=0.912). After incubation with excessive G-CSF, the expressed G-CSFr on the neutrophils were decreased in both groups(P=0.554). The quantities of expressions of GM- CSFr on the neutrophil after incubation with the excessive GM-CSF were always increased in both groups(P=0.255). The amount of GM-CSFr of neutrophils are in proportion to total white blood cells (r=0.788, P=0.035), but it wasn't in the case of KD(P=0.644). CONCLUSION: The leukocytosis in KD that mediated by increasing neutrophil was not correlated with the plasma concentrations of G-CSF and GM-CSF, and the amount of expression of G-CSFr and GM-CSFr on granulocyte. It is possible that the reduction of concentration of GM-CSF results by increasing the active GM-CSFr.

Plasma G-CSF and GM-CSF Concentrations and Expression of their Receptors on the Granulocyte in Children with Leukocytosis

PURPOSE: Granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF) are principal cytokines in granulopoiesis and their physiologic effects are mediated through binding to specific cell surface receptors. Although it is known that the level of serum G-CSF and GM-CSF, and presentation of the receptors are increased in infectious diseases, there have been no studies to find the correlation between the granulopoiesis and leukocytosis. This study was designed to measure G-CSF and GM-CSF in leukocytosis and in control and to demonstrate the possible pathogenesis of granulopoiesis in leukocytosis using quantitative analysis of G- CSF, GM-CSF and their CSFr. METHODS: The plasma levels of G-CSF, GM-CSF of 13 children without leukocytosis and 14 children with leukocytosis were measured. Counts of cell surface G-CSFr and GM-CSFr were measured by combining anti G-CSFr and anti GM-CSFr monoclonal antibodies to their respective receptors by using quantitative flow cytometric assay. RESULTS: There was no significant difference betweeen the plasma concentration of G-CSF and GM-CSF in acute leukocytosis and in the control group. However, levels of G-CSFr in acute leukocytosis decreased significantly compared to the control(P=0.012) and the levels of GM-CSFr in both groups revealed no significant difference. CONCLUSION: Increase in the number of leukocyte in leukocytosis was mediated by increasing the number of neutrophil, and increased plasma concentration of G-CSF may be the cause of neutrophilia. But GM-CSF did not have any influence on leukocytosis.

Expression of Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) and Clinical Correlates in Acute Leukemia / 대한혈액학회지

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) is commonly used to reduce leukopenic period during treatment of malignancy including acute leukemia. Leukemic blasts expressing granulocyte colony-stimulating factor receptor (G-CSFR) were reported and also may proliferate in response to therapeutic administration of G-CSF. However, it is not clear whether G-CSFR expression on leukemic blasts is related to clinical outcome such as leukocyte recovery or leukemia relapse. Current study evaluated expression of G-CSFR in acute leukemia and correlated with hematologic and clinical parameters. METHODS: Peripheral blood or bone marrow aspirate was evaluated from 20 patients with acute myelogenous leukemia (AML) and 10 with acute lymphoblastic leukemia (ALL), 2 with acute undifferentiated leukemia (AUL), 1 with acute biphenotypic leukemia (ABL), 1 with acute mixed-lineage leukemia (AMLL). G-CSFR expression was analyzed using flow cytometry and was correlated with immunophenotype and response for chemotherapy. RESULTS: More than 20% of blasts were positive for G-CSFR in 65% (13/20) of AML, 40% (4/10) of ALL, and all negative in ABL, AMLL, and AUL. Except that all 6 monocytic lineage leukemias (M4, M5) and all three cases of ALL with CD33 expression were positive, no consistent correlation was observed among G-CSFR expression pattern, type of acute leukemia, response to induction therapy and relapse (P>0.05). CONCLUSION: Current study revealed G-CSFR was expressed on not only myelogenous leukemic cells but also lymphoid ones. Although our data suggest G-CSFR expression does not affect therapeutic outcome, it remains to be determined whether G-CSF therapy is safe in G-CSFR-positive acute leukemia.

Quantities of Receptor Molecules for Colony Stimulating Factors on Leukocytes in Measles

We analyzed the comparative amounts of granulocyte-colony stimulating factor (G-CSFr) and granulocyte macrophage CSF (GM-CSFr) receptors expressed on neutrophils and monocytes in measles patients to investigate the role of these CSFrs in the development of leukopenia including neutropenia and monocytopenia in measles. EDTA-anticoagulated peripheral blood of 19 measles patients, 10 children with other infections showing leukopenia and 16 children with normal complete blood cell counts (CBC)s were analyzed using flow cytometry and QuantiBRITE. The leukocyte (5260 +/- 2030/uL vs. 9900 + 2680/uL, p=0.000), neutrophil (2580 +/- 960/uL vs. 4250 +/- 2750/uL, p=0.024) and the lymphocyte counts of measles patients (1810 +/- 1430/uL vs. 4530 +/- 3450/uL, p= 0.006) were lower than in the normal controls. The neutrophils of measles patients expressed similar amounts of G- CSFr (1858 +/- 355) as normal children (1764 +/- 477, p= 0.564) and leukopenic patients (1773 +/- 673, p=0.713), but lower levels of GM-CSFr (535 +/- 118) than normal children (957 +/- 344, p=0.000) and leukopenic patients (832 +/- 294, p=0.002). The monocytes of measles patients expressed similar amounts of G-CSFr (916 +/- 336) and GM-CSFr (3718 +/- 906) as normal children (1013 +/- 391 and 4125 (2645, p > 0.05) but less than leukopenic patients (1454 +/- 398 and 5388 +/- 806, p > 0.05). The neutrophil and monocyte counts of measles patients did not correlate with the amount of G-CSFr or GM-CSFr expressed on neutrophils or monocytes (p > 0.05), but in the normal children, the monocyte count correlated with the levels of GM-CSFr on monocytes (r=0.951, p=0.049). In conclusion, neutropenia is one of the more important characteristics of measles patients, which could be due to the decreased GM-CSFr expression on neutrophils. However, the monocytopenia found in measles patients is not due to the decreased expression of CSFr on the monocytes.

Transcriptional activation function of intracellular domain of EPOR and G-CSFR protein in yeast: a comparative study / 解放军医学杂志

Objective To investigate the feasibility of searching for proteins which interact with intracellular domain of hematopoietic growth factor receptors using yeast two-hybrid system. Methods RT-PCR method was performed to amplify the genes of intracellular domains of G-CSF receptor and EPO receptor in NFS-60 and BET-2 cells of mice. The genes were cloned into yeast expression plasmid pGBKT7 vector,and then transformed into yeast AH109. The yeast proteins were isolated and analyzed with Western blotting. Transcriptional activation was analyzed by the ?-galactosidase colony-lift filter assay. Results The intracellular domains of G-CSF receptor and EPO receptor genes were successfully cloned into pGBKT7 vector. The results of Western blotting assay showed that both proteins were expressed in the yeast cells. The ?-galactosidase colony-lift filter assay demonstrated that G-CSF receptor alone had no activity of transcriptional activation,while the EPO receptor alone could activate transcription. Conclusion The findings suggested that intracellular domain of G-CSF receptor could be used as a bait to find interacting proteins using yeast two-hybrid system,while that of the EPO receptor could not. Therefore the system could not be applied to all hematopoietic factor receptor.