Mature Neutrophil Function Research Articles

A curious case of cyclin-dependent kinases in neutrophils.

Neutrophils are terminally differentiated, short-lived white blood cells critical for innate immunity. Although cyclin-dependent kinases (CDKs) are typically related to cell cycle progression, increasing evidence has shown that they regulate essential functions of neutrophils. This review highlights the roles of CDKs and their partners, cyclins, in neutrophils, outside of cell cycle regulation. CDK1-10 and several cyclins are expressed in neutrophils, albeit at different levels. Observed phenotypes associated with specific inhibition or genetic loss of CDK2 indicate its role in modulating neutrophil migration. CDK4 and 6 regulate neutrophil extracellular traps (NETs) formation, while CDK5 regulates neutrophil degranulation. CDK7 and 9 are critical in neutrophil apoptosis, contributing to inflammation resolution. In addition to the CDKs that regulate mature neutrophil functions, cyclins are essential in hematopoiesis and granulopoiesis. The pivotal roles of CDKs in neutrophils present an untapped potential in targeting CDKs for treating neutrophil-dominant inflammatory diseases and understanding the regulation of the neutrophil life cycle.

Prediction of Survival Benefit of Filgrastim in Adult and Pediatric Patients With Acute Radiation Syndrome

Acute exposure to high doses of radiation leads to severe myelosuppression, but few treatments are currently available to treat hematopoietic syndrome of acute radiation syndrome. Granulocyte colony stimulating factors (e.g., filgrastim) stimulate proliferation of neutrophil precursors and enhance mature neutrophil function. Owing to ethical constraints on conducting clinical research in lethally irradiated humans, we developed a model‐based strategy to integrate preclinical experience in irradiated nonhuman primates (NHPs) and other clinical myelosuppressive conditions to inform filgrastim dosing to treat hematopoietic syndrome of acute radiation syndrome. Models predicting neutrophil counts and overall survival based on drug exposures were calibrated and scaled from NHPs to adult and pediatric human subjects. Several scenarios were examined investigating variations in filgrastim doses, dose frequency, treatment initiation, and duration, as well as the effect of age and radiation dose rate. Model‐based simulations and established safety profiles supported that a subcutaneous filgrastim dose of 10 µg/kg once daily provides a significant survival benefit (50%) over placebo in both adults and children, provided that the treatment is initiated within 1–14 days after radiation exposure and lasts 2–3 weeks. For treatment durations of longer than 3 weeks, filgrastim treatment is not expected to provide significantly greater benefit. This survival benefit is expected to hold for the wide range of radiation doses and dose rates (0.01–1,000 Gy/hours) examined.

Neutrophil Maturity in Cancer.

Neutrophils are implicated in almost every stage of oncogenesis and paradoxically display anti- and pro-tumor properties. Accumulating evidence indicates that neutrophils display diversity in their phenotype resulting from functional plasticity and/or changes to granulopoiesis. In cancer, neutrophils at a range of maturation stages can be identified in the blood and tissues (i.e., outside of their developmental niche). The functional capacity of neutrophils at different states of maturation is poorly understood resulting from challenges in their isolation, identification, and investigation. Thus, the impact of neutrophil maturity on cancer progression and therapy remains enigmatic. In this review, we discuss the identification, prevalence, and function of immature and mature neutrophils in cancer and the potential impact of this on tumor progression and cancer therapy.

Functional Secretion of Granulocyte Colony Stimulating Factor in Bacillus subtilis and Its Thermogenic Activity in Brown Adipocytes

Granulocyte colony-stimulating factor (G-CSF) is involved in generation of colonies hematopoietic cells by stimulation of bone marrow cells, resulting in the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. To express and secrete G-CSF in Bacillus system, the G-CSF gene was amplified using gene-specific primers and template DNA (pUNO1-hGCSFb). As a result, the PCR product of 530 bp was obtained, sequenced, and analyzed using the BLAST program. The open reading frame encoded a 204-amino acid polypeptide precursor consisting of the 30-residue signal sequence and a 174-residue mature polypeptide. Recombinant secretion vector (pRBAS-CSF3, 6.96 kb) was constructed using pRBAS vector which contains promoter (0.45 kb) and signal sequence (87 bp) of alkaline protease gene (aprE), ribosome binding site (GGAGAGGG) and transferred to Bacillus subtilis LKS. G-CSF protein was secreted and optimized to increase the productivity (19% of total secreted proteins, 0.12 mg/ml). The culture broth was harvested and followed by metal chelating, ethanol precipitation, and gel chromatography. The purified G-CSF was tested for thermogenic activity in cultured brown adipocytes. G-CSF activated HIB1B brown adipocytes by increasing expression levels of brown fat-specific marker proteins (UCP1 and PRDM16) and genes (Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. This result suggests that G-CSF per se or G-CSF-containing foodstuff can be used to treat obesity.

Clinical confirmation to demonstrate similarity for a biosimilar pegfilgrastim: a 3-way randomized equivalence study for a proposed biosimilar pegfilgrastim versus US-licensed and EU-approved reference products in breast cancer patients receiving myelosuppressive chemotherapy

BackgroundChemotherapy-induced neutropenia is a common result of myelosuppressive chemotherapy treatment. Infections such as febrile neutropenia (FN) are sensitive to the duration of neutropenia as well as the depth of absolute neutrophil count (ANC) at nadir. Filgrastim, a granulocyte colony stimulating factor (G-CSF), can stimulate the function of mature neutrophils. Pegfilgrastim, a long-acting form of filgrastim, has been shown to reduce FN to a greater extent compared to filgrastim. G-CSF agents have been recommended for prophylactic administration with chemotherapy. Apotex developed a proposed pegfilgrastim biosimilar. This study was conducted to confirm that no clinically meaningful efficacy or safety differences exist between Apotex’s proposed biosimilar and its reference product.Methods589 breast cancer patients were randomized and dosed with the proposed pegfilgrastim biosimilar, US-licensed pegfilgrastim reference product, or EU-approved pegfilgrastim reference product. The primary endpoint assessed was the duration of severe neutropenia (DSN) and secondary endpoints included rate of FN and ANC nadir.ResultsData showed that the mean DSN, the primary endpoint measured, was comparable across all three treatments. The As Treated arm had a 95% confidence interval within the equivalence range for the proposed pegfilgrastim biosimilar with the US-licensed and EU-approved pegfilgrastim reference products. Secondary endpoints, which included depth and peak of ANC nadir, time to ANC recovery post-nadir and rates of FN, also showed similarity between the three different treatment groups. The adverse event incidence was similar across treatment arms and there were no unexpected safety events.ConclusionsOverall, these results show that the proposed pegfilgrastim biosimilar is similar to Amgen’s US-licensed and EU-approved pegfilgrastim reference products with regard to the clinical efficacy and safety endpoints assessed.Trial registration EMA: European Union Clinical Trials Register: (https://www.clinicaltrialsregister.eu/ctr-search/search?query=eudract_number:2011-002678-21) Eudract # 2011-002678-21 Registered: 01/10/2012

The Effect Of Temperature On Recombinant Human Granulocyte Colony Stimulating Factor Production By Pichia pastoris Expression System

Granulocyte colony stimulating factor (G-CSF) is a haematopoetic growth factor that functions as specific stimulator of the survival, proliferation, and differentiation of precursor cells of the neutrophilic granulocyte cell lineage as well as an activator of mature neutrophil function. The main objective of this work is to compare the effect of different temperature on the production of extracellular recombinant G-CSF in Pichia pastoris. Cells were cultured for 72h in baffled shake-flasks at 20°C, 25°C, and 30°C in two different medium; buffered glycerol/methanol-complex medium (BMGY/BMMY) and buffered minimal glycerol/methanol (BMGH/BMMH) after methanol induction every 12h. Expressed recombinant hG-CSF in the methylotrophic yeast P. pastoris was analyzed with SDS-PAGE. The 23 kDa protein was secreted into the culture supernatant when induced with methanol. Production of recombinant G-CSF protein in P. pastoris at 30°C at 48h incubation after methanol induction every 12h is the highest in both complex and minimum medium.

The impact of androgens on neutrophils and melanoma tumor burden.

Abstract Background Aged men have increasingly higher incidence of melanoma compared with women and continuously decreasing testosterone (T). Investigation into the mechanism underlying the sex disparity is warranted as there is a low survival rate for metastatic melanoma patients, especially in men. Methods We used B16 and YUMM melanoma lung metastatic models. Neutrophils or NK depletion and ovariectomy or castrations were performed. Neutrophils from prostate cancer patients were analyzed. Data Compared with males, female mice with melanoma have higher lung tumor burden, reduced neutrophil infiltration, and decreased NK cell activation. While ovariectomy did not affect tumor burden, castrated males had increased lung metastasis and reduced survival compared with sham, suggesting a protective role for T. Further, T replacement in castrated mice reduced metastasis to sham levels. Depletion of NK cells increased tumor burden in male and female mice in a sex independent manner. In contrast, neutrophil depletion increased tumor burden only in males, suggesting a sex difference in neutrophils. More immature neutrophils with impaired function were observed in both castrated mice and prostate cancer patients on androgen ablation therapy. Conclusion Our data indicate that innate immunity is important for the response against melanoma colonization of the lung. Neutrophil depletion and castration data suggest a sex specific response which may contribute to the sex disparity observed in human melanoma incidence and survival. Finally, prostate cancer patients on androgen ablation therapy show a similar decrease in mature neutrophils and impaired neutrophil function, which may put these patients at a higher risk for infection and melanoma.

Therapeutic Targeting of the G-CSF Receptor Reduces Neutrophil Trafficking and Joint Inflammation in Antibody-Mediated Inflammatory Arthritis.

G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF- and G-CSF receptor-deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti-G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti-G-CSF receptor-treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti-G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1β, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti-G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis.

In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a four-stage differentiation protocol involving: embryoid body (EB) formation (stage-1); EB culture with hematopoietic cytokines (stage-2); HSPC expansion (stage-3); and neutrophil maturation (stage-4). CD34(+) CD45(-) putative hemogenic endothelial cells were observed in stage-3 cultures, and expressed VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24, miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34(+) CD45(-) cells generated CD34(+) CD45(+) HSPCs that produced hematopoietic CFUs. Mid-stage-3 CD34(+) CD45(+) HSPCs exhibited increased expression of GATA-2, AML1/RUNX1, SCL/TAL1, C/EBPα, and PU.1 transcription factors, but exhibited decreased expression of HSPC-associated microRNAs, and failed to engraft in immune-deficient mice. Mid-stage-3 CD34(-) CD45(+) cells maintained PU.1 expression and exhibited increased expression of hematopoiesis-associated miR-142-3p/5p and a trend towards increased miR-223 expression, indicating myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBPɛ expression were observed, with 25%-65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. Stem Cells 2016;34:1513-1526.

Differential Expression of Neutrophil Granule Protein Genes in Bone Marrow Myeloid Cells at the Peak and Nadir of Neutrophil Counts in Cyclic Neutropenia

Cyclic neutropenia (CyN) is a hematologic disorder in which peripheral-blood neutrophil counts show cycles at approx. 21-days intervals. The majority of CyN patients (ca. 90 %) harbor inherited mutations in the ELANE gene. The mechanism of cycling hematopoiesis downstream of ELANE mutations is unclear. In the present study we aimed to identify if there is a geterogeniety of bone marrow (BM) myeloid progenitors and granulocytic cells at the peak and nadir of the cycle of neutrophil counts.We performed FACS analysis of BM populations in CyN patient at the peak and nadir of the cycle and revealed reduced number of CD33high promyelocytes at the peak, as compared to the nadir neutrophil counts (6% vs 47%). Morphological examination of BM smears confirmed this observation. These data suggest differences in myeloid differentiation potential of hematopoietic cells of CyN patient during cycle. To compare the myeloid differentiation of BM cells at the peak and nadir, we performed CFU assay using BM cells isolated at these two different time points. Indeed, we found diminished capacity to produce CFU-G colonies at the peak of cycle, in comparison to the nadir (50 vs 68). This difference might be explained by the presence of different sub-populations of myeloid cells during the cycle. It was shown that the neutrophil populations can be distinguished by membrane expression of CD177, which is GPI-linked neutrophil antigen, localized primarily to the membrane of specific granules and to the plasma membrane. The proportion of CD177+ cells increased during neutrophil maturation in BM. Interestingly, in healthy individuals the fraction of CD177+ cells appeared to be constant in each individual. We evaluated the differences of CD177+ cell populations in CyN patients at the peak and nadir of cycle by FACS. We found that numbers of CD33+ CD177+ and CD16+ CD177+ populations were different during the cycle. At the peak we measured 7,1% of CD33+ CD177+ cells and 83% of CD16+ CD177+ cells. At the nadir 3,78% of cells were CD33+ CD177+ and 69% were CD16+ CD177+.We further performed mRNA expression analysis of CD33+ BM cells isolated from CyN patient at the peak and nadir of cycle and compared it to healthy individuals. We found lower mRNA expression (more than 10-fold) of CRISP3, ELANE, OLFM4, CEACAM6, MMP8, DEFA4 and LCN2 in CD33+ cellsat the peak of the cycle comparing to the nadir. These genes encode for neutrophil granule proteins, playing an important role in the developement and function of mature neutrophils. We further confirmed differential expression of these factors in CFU colonies using BM of CyN patient isolated at the peak and nadir of the cycle: CFU-G colonies grown from cells taken at the peak of the cycle expressed less mRNA levels of granula proteins than CFU-G colonies grown from cells taken at the nadir of the cycle.In summary, we hypothesize that the differential expression of the granule proteins is involved in the regulation of the cycle in myeloid cells in CyN. At the peak and nadir of neutrophil counts different populations (based on CD177 expression) of myeloid progenitors and neutrophils are present in the CyN BM. DisclosuresNo relevant conflicts of interest to declare.

20: Neutralising anti-G-CSFR monoclonal antibody blocks neutrophil-driven inflammatory joint disease

Granulocyte colony-stimulating factor (G-CSF) is a haemopoietic cytokine first identified for its role in steady state granulopoiesis but is now also recognised as a proinflammatory mediator of mature neutrophil activation and function. G-CSF binds to the G-CSF receptor (G-CSFR), which is present on neutrophils and haemopoietic progenitors. G-CSF-deficient mice are protected from disease in several models of rheumatoid arthritis, and blockade of G-CSF is also protective in those models. To further investigate the actions of blocking G-CSF/G-CSFR signalling in inflammatory disease, we developed a neutralising monoclonal antibody to mouse G-CSFR which prevents the binding of G-CSF and inhibits G-CSFR signalling. In the anti-collagen antibody induced arthritis model, anti-G-CSFR treatment rapidly halted the progression of established disease. This therapeutic effect was observed at low dose and was associated with transient suppression of peripheral blood neutrophilia. Anti-G-CSFR treatment significantly inhibited neutrophil accumulation in the joints without rendering animals neutropenic, suggesting an effect of G-CSFR blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti-G-CSFR treated mice showed alterations in homing receptors, with reduced CXCR2 and increased CD62L expression. Microarray analysis of neutrophils from joints and peripheral blood showed a common up-regulation of 6 genes and down-regulation of 22 genes following anti-G-CSFR treatment. Blocking neutrophil traffic to joints with anti-G-CSFR suppressed local production of proinflammatory cytokines (IL-1b and IL-6) and chemokines (KC and MCP-1) known to drive tissue damage. These data show for the first time the effect of blockade of G-CSFR in a therapeutic model of inflammation and provide further support for utilising this approach in the treatment of inflammatory diseases such as rheumatoid arthritis.

Innate Immune Functions of Immature Neutrophils in Patients With Sepsis and Severe Systemic Inflammatory Response Syndrome*

A hallmark of sepsis and severe systemic inflammatory response syndrome (SIRS) is the massive recruitment of immature neutrophils from the bone marrow into the circulation (left shift, band forms). Their capacity to participate in innate defense against bacteria is ill defined. We aimed at comparing various innate immune functions of mature vs. immature neutrophils circulating during sepsis and SIRS. Prospective, observational cohort study. Tertiary level ICU and associated research laboratory. : Thirty-three ICU patients with sepsis; 12 ICUs with SIRS; 32 healthy volunteers. Twenty milliliters of whole heparinized blood was used for in vitro studies including neutrophil viability and apoptosis, surface expression of CD16, Toll-like receptors () 4 and TLR2, CD14, MD-2, HLA-DP,-DQ and -DR, and CXCR2, chemotaxis, phagocytosis, bacterial killing, and tumor necrosis factor-α/interleukin-10 baseline intracellular cytokine levels. Immature neutrophils were capable of mediating important innate immune functions such as bacterial phagocytosis and killing via the production of reactive oxygen species, although less efficiently than mature neutrophils. Immature neutrophils had a longer life span and resistance to spontaneous apoptosis, and could mature ex vivo. They expressed lower levels of receptors for bacterial molecules such as CD14 and MD-2 and migrated less efficiently than mature granulocytes. Immature neutrophils had higher basal intracellular tumor necrosis factor-α/interleukin-10 ratio than that of mature neutrophils, suggesting a proinflammatory phenotype. No significant differences were observed between immature neutrophils isolated from patients with sepsis and those from patients with severe SIRS. Despite their "immaturity", band forms are capable of mediating crucial innate immune functions during severe infections and sepsis. Their fate and capacity to mature in vivo remain to be determined.

Dasatinib inhibits proinflammatory functions of mature human neutrophils

AbstractDasatinib is a tyrosine kinase inhibitor used to treat imatinib-resistant chronic myeloid leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia. At present, little is known about how dasatinib influences nonmalignant cells. In the present study, we tested the effect of dasatinib on functional responses of normal mature human neutrophils. Dasatinib completely blocked integrin- and Fc-receptor–mediated neutrophil functions, with the lowest IC50 values below 10nM under serum-free conditions. Dasatinib caused a partial inhibition of neutrophil responses triggered by G-protein–coupled receptors and had a moderate effect on neutrophil responses triggered by microbial compounds. Whereas dasatinib inhibited neutrophil chemotaxis under static conditions in 2 dimensions, it did not affect migration under flow conditions or in 3-dimensional environments. Dasatinib did not have any major effect on phagocytosis or killing of bacteria by neutrophils. Adhesion of human neutrophils in the presence of whole serum was significantly inhibited by 50-100nM dasatinib, which corresponds to the reported serum concentrations in dasatinib-treated patients. Finally, ex vivo adhesion of mouse peripheral blood neutrophils was strongly reduced after oral administration of 5 mg/kg of dasatinib. Those results suggest that dasatinib treatment may affect the proinflammatory functions of mature neutrophils and raise the possibility that dasatinib-related compounds may provide clinical benefit in neutrophil-mediated inflammatory diseases.

PRELIMINARY STUDY ON PRESERVATION OF RECOMBINANT hG-CSF BY LYOPHILIZATION

hG-CSF is a cytokine that stimulates the proliferation, differentiation, function of mature neutrophils and is generally used for treatment of neutropenia in cancer patients under chemotherapy or other diseased patients. After production process, E. coli-derived recombinant hGCSF is usually available as non-glycosylated, liquid protein, so that it’s often unstable, easy to lose their biological activity. In this study, we report the results of using lyophilization to storage recombinant hG-CSF. We found out the freezing conditions and the suitable protein volumes for freezedrying process. Furthermore, we also studied the effects of lyophilization on this protein by testing the structure with Native-PAGE, RP-HPLC, testing biological activity on M-NFS-60 cell lines and in mouse (Mus musculus).

Pharmacokinetics and Pharmacodynamics of Pegfilgrastim

Pegfilgrastim is a sustained-duration form of filgrastim, a recombinant methionyl form of human granulocyte colony-stimulating factor (G-CSF), to which a 20 kDa polyethylene glycol molecule is covalently bound to the N-terminal methionine residue. Similar to filgrastim, pegfilgrastim increases the proliferation and differentiation of neutrophils from committed progenitor cells, induces maturation, and enhances the survival and function of mature neutrophils, resulting in dose-dependent increases in neutrophils. After subcutaneous administration, pegfilgrastim exhibits nonlinear pharmacokinetics and exposure to pegfilgrastim increases in more than a dose-proportional manner, suggesting that the clearance of pegfilgrastim decreases with increased dosing. Filgrastim is primarily eliminated by the kidney and neutrophils/neutrophil precursors; the latter presumably involves binding of the growth factor to the G-CSF receptor on the cell surface, internalization of the growth factor-receptor complexes via endocytosis, and subsequent degradation inside the cells. Pegylation of filgrastim renders renal clearance insignificant, which was demonstrated in bilaterally nephrectomized rats and confirmed in subjects with renal impairment. As a result, the neutrophil-mediated clearance is the predominant elimination pathway for pegfilgrastim. During chemotherapy-induced neutropenia, the clearance of pegfilgrastim is significantly reduced and the concentration of pegfilgrastim is sustained until onset of neutrophil recovery. Pegfilgrastim concentrations are sustained longer in patients with profound neutropenia. Evidence supports the use of a postnadir absolute neutrophil count (ANC) of ≥ 1 × 109/L as a surrogate marker threshold for the clearance of pegfilgrastim to subtherapeutic levels. After repeated administration of pegfilgrastim, the peak concentrations of pegfilgrastim decrease, likely due to increased neutrophil and neutrophil precursor mass. A pharmacokinetic-pharmacodynamic model was developed to describe the pharmacokinetic and ANC profiles of pegfilgrastim; the analysis supported that 100 μg/kg was an adequate weight-based dose of pegfilgrastim and predicted that 6 mg would be an optimal fixed dose of pegfilgrastim to simplify treatment. Data from a pivotal study confirmed that a once-per-chemotherapy-cycle injection of pegfilgrastim at 6 mg was as safe and effective as 11 daily injections of filgrastim at 5 μg/kg in reducing neutropenia and its complications in patients with breast cancer receiving four cycles of doxorubicin/docetaxel chemotherapy. Because of the highly efficient regulation of pegfilgrastim clearance via neutrophils and neutrophil precursors, a single fixed dose of pegfilgrastim can be given once per chemotherapy cycle in conjunction with a variety of myelosuppressive chemotherapy regimens.

CLONING AND EXPRESSING HG-CSF (HUMAN GRANULOCYTE COLONY STIMULATING FACTOR) BEING FUSED WITH FERRITIN HEAVY-CHAIN IN E. COLI

G-CSF is a cytokine that stimulates the proliferation, differentiation, function of mature neutrophils and is generally used for treatment of neutropenia in cancer patients under chemotherapy. In this study, we report results on the cloning and expression of hG-CSF being fused with the heavy-chain (FTN-H) of human ferritin, which had been showed to be capable of facilitate the folding of several human protein expressed in E. coli. The hg-csf gene and gene encoding FTN-H were inserted into plasmid pET28a to form expression vector PET-FHG. 6xHis tag and TEV sequence (being recognized and cleaved by TEV protease) was added between of G-CSF and FTN-H to facilitate G-CSF purification and recovery afterwards. PET-FHG was transformed into E. coli BL21(DE3). The expression of hG-CSF was induced by IPTG and confirmed by SDS-PAGE and Western blot using anti-hG-CSF antibody.

STAT3 governs distinct pathways in emergency granulopoiesis and mature neutrophils

AbstractGranulocyte colony-stimulating factor (G-CSF) is essential for the host response to bacterial infection by controlling neutrophil production in the bone marrow. The G-CSF receptor (G-CSFR) activates the Jak/STAT pathway, although little is understood about how these signals regulate basal and stress-induced granulopoiesis. We examined STAT3 function in granulocytes using a bone marrow conditional knockout mouse model. Our results show that STAT3 has a crucial role in emergency granulopoiesis and mature neutrophil function. STAT3-deficient mice have an aberrant response to G-CSF in vivo, characterized by failure to accumulate immature granulocytes and an increased ratio of mature to immature neutrophils in the bone marrow, peripheral blood, and spleen. Acute neutrophil mobilization is impaired in STAT3-deficient mice as judged by their failure to up-regulate circulating neutrophils following short-term G-CSF exposure. STAT3 also controls neutrophil chemotactic responses to natural ligands for CXCR2 and regulates the magnitude of chemoattractant-induced actin polymerization. These functions of STAT3 are independent of its principal target gene Socs3, which encodes a crucial feedback inhibitor of cytokine signaling. Our results demonstrate the existence of distinct STAT3 target pathways in neutrophils required for granulopoiesis and innate immunity.

Regulation of Neutrophil Functions by Proinflammatory Cytokines

Various functions of mature human neutrophils are activated or potentiated by hematopoietic growth factors or proinflammatory cytokines such as granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and interleukin 1beta. The major signaling pathways activated in human neutrophils stimulated by proinflammatory cytokines include mitogen-activated protein kinases, Janus kinase/signal transducer and activator of transcription, phosphatidylinositol 3-kinase, and nuclear factor kappaB. These signaling pathways are involved in cytokine-mediated regulation of neutrophil functions in a cytokine-specific manner.

CFTR Expression in Human Neutrophils and the Phagolysosomal Chlorination Defect in Cystic Fibrosis

Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.

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.