GCSF deficiency attenuates nonalcoholic fatty liver disease through regulating GCSFR-SOCS3-JAK-STAT3 pathway and immune cells infiltration.

Granulocyte colony stimulating factor (GCSF) is a cytokine with immunomodulation effects. However, little is known about its role in metabolic diseases. In the current study, we aimed to explore the role of GCSF in nonalcoholic fatty liver disease (NAFLD). Male GCSF-/- mice were used to investigate the function of GCSF in vivo after high-fat diet (HFD). Primary hepatocytes were used for evaluating the function of GCSF in vitro. Liver immune cells were isolated and analyzed by flow cytometry. Our results showed that GCSF administration significantly increased serum triglyceride (TG) levels in patients. Circulating GCSF was markedly elevated in HFD-fed mice. GCSF-/- mice exhibited alleviated HFD-induced obesity, insulin resistance, and hepatic steatosis. Extra administration of GCSF significantly aggravated palmitic acid (PA)-induced lipid accumulation in primary hepatocytes. Mechanically, GCSF could bind to granulocyte colony stimulating factor receptor (GCSFR) and regulate suppressors of cytokine signaling 3, Janus kinase, signal transducer and activator of transcription 3 (SOCS3-JAK-STAT3) pathway. GCSF also enhanced hepatic neutrophils and macrophages infiltration, thereby modulating NAFLD. These findings suggest that GCSF plays an important regulatory role in NAFLD and may be a potential therapeutic target for NAFLD.NEW & NOTEWORTHY We found GCSF was involved in lipid metabolism and NAFLD development. GCSF administration increased serum triglyceride levels in patients. GCSF deficiency alleviated HFD-induced insulin resistance and hepatic steatosis in mice. GCSF could directly act on hepatocytes through GCSFR-SOCS3-JAK-STAT3 pathway, and regulate the infiltration of immune cells into the liver to indirectly modulate NAFLD. Our finding indicates that GCSF may provide new strategies for the treatment of NAFLD.

Neutrophils promote clearance of nuclear debris following acid-induced lung injury.

Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared with wild type. Size analysis of DNA debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation, they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intratracheal administration of DNase I. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.

The Arp2/3 complex binding protein HS1 is required for efficient dendritic cell random migration and force generation.

Dendritic cell migration to the T-cell-rich areas of the lymph node is essential for their ability to initiate the adaptive immune response. While it has been shown that the actin cytoskeleton is required for normal DC migration, the role of many of the individual cytoskeletal molecules is poorly understood. In this study, we investigated the contribution of the Arp2/3 complex binding protein, haematopoietic lineage cell-specific protein 1 (HS1), to DC migration and force generation. We quantified the random migration of HS1-/- DCs on 2D micro-contact printed surfaces and found that in the absence of HS1, DCs have greatly reduced motility and speed. This same reduction in motility was recapitulated when adding Arp2/3 complex inhibitor to WT DCs or using DCs deficient in WASP, an activator of Arp2/3 complex-dependent actin polymerization. We further investigated the importance of HS1 by measuring the traction forces of HS1-/- DCs on micropost array detectors (mPADs). In HS1 deficient DCs, there was a significant reduction in force generation (3.96 ± 0.40 nN per cell) compared to WT DCs (13.76 ± 0.84 nN per cell). Interestingly, the forces generated in DCs lacking WASP were only slightly reduced compared to WT DCs. Taken together, these findings show that HS1 and Arp2/3 complex-mediated actin polymerization are essential for the most efficient DC random migration and force generation.

HS1 deficiency impairs neutrophil recruitment in vivo and activation of the small GTPases Rac1 and Rap1.

Neutrophil extravasation is a critical step of the innate immune system's response to inflammation. This multistep process is tightly regulated by adhesion and signaling molecules in the endothelium and neutrophils. Activation of the ß2 integrin LFA-1 is critical for adhesion of leukocytes to postcapillary venules. This step requires coordinated activation of signaling pathways in chemokine-stimulated neutrophils, including GTPase activation and cytoskeletal remodeling, leading to conformational changes in LFA-1. Hematopoietic cell-specific lyn substrate 1 (HS1) is a cortactin-related and leukocyte-specific actin-binding protein (ABP) that regulates several processes in various immune cells. It has been shown in vitro that HS1 is important for neutrophil chemotaxis and transendothelial migration of NK cells, but its role in neutrophil extravasation in vivo has not been investigated yet. Intravital microscopy of CXCL1-stimulated cremaster venules revealed an increased rolling velocity and reduced neutrophil adhesion and transmigration in HS1 knockout (KO) mice. CXCL1-induced rapid neutrophil arrest in vivo and adhesion under flow conditions in vitro were also reduced significantly. Whereas random motility of neutrophils was unaffected, chemotaxis toward a CXCL1 gradient was reduced in the absence of HS1. Further analysis of the underlying mechanisms demonstrated that HS1 controls CXCL1-induced activation of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap1), thus supporting LFA-1-mediated neutrophil adhesion. Importantly, with the use of Rac1 KO neutrophils, we could show that Rac1 acts upstream of Rap1. Our results establish HS1 as an important regulator of proper Rac1 and Rap1 activation and neutrophil extravasation.

Neutrophils promote alveolar epithelial regeneration by enhancing type II pneumocyte proliferation in a model of acid-induced acute lung injury.

Alveolar epithelial regeneration is essential for resolution of the acute respiratory distress syndrome (ARDS). Although neutrophils have traditionally been considered mediators of epithelial damage, recent studies suggest they promote type II pneumocyte (AT2) proliferation, which is essential for regenerating alveolar epithelium. These studies did not, however, evaluate this relationship in an in vivo model of alveolar epithelial repair following injury. To determine whether neutrophils influence alveolar epithelial repair in vivo, we developed a unilateral acid injury model that creates a severe yet survivable injury with features similar to ARDS. Mice that received injections of the neutrophil-depleting Ly6G antibody had impaired AT2 proliferation 24 and 72 h after acid instillation, which was associated with decreased reepithelialization and increased alveolar protein concentration 72 h after injury. As neutrophil depletion itself may alter the cytokine response, we questioned the contribution of neutrophils to alveolar epithelial repair in neutropenic granulocyte-colony stimulating factor (G-CSF)-/- mice. We found that the loss of G-CSF recapitulated the neutrophil response of Ly6G-treated mice and was associated with defective alveolar epithelial repair, similar to neutrophil-depleted mice, and was reversed by administration of exogenous G-CSF. To approach the mechanisms, we employed an unbiased protein analysis of bronchoalveolar lavage fluid from neutrophil-depleted and neutrophil-replete mice 12 h after inducing lung injury. Pathway analysis identified significant differences in multiple signaling pathways that may explain the differences in epithelial repair. These data emphasize an important link between the innate immune response and tissue repair in which neutrophils promote alveolar epithelial regeneration.

Prevention of an increase in cortical ligand binding to AMPA receptors may represent a novel mechanism of endogenous brain protection by G-CSF after ischemic stroke.

PURPOSE: Using G-CSF deficient mice we recently demonstrated neuroprotective properties of endogenous G-CSF after ischemic stroke. The present follow-up study was designed to check, whether specific alterations in ligand binding densities of excitatory glutamate or inhibitory GABAA receptors may participate in this effect. METHODS: Three groups of female mice were subjected to 45 minutes of MCAO: wildtype, G-CSF deficient and G-CSF deficient mice substituted with G-CSF. Infarct volumes were determined after 24 hours and quantitative in vitro receptor autoradiography was performed using [3H]MK-801, [3H]AMPA and [3H]muscimol for labeling of NMDA, AMPA and GABAA receptors, respectively. Ligand binding densities were analyzed in regions in the ischemic core, peri-infarct areas and corresponding contralateral regions. RESULTS: Infarct volumes did not significantly differ between the experimental groups. Ligand binding densities of NMDA and GABAA receptors were widely in the same range. However, AMPA receptor binding densities in G-CSF deficient mice were substantially enhanced compared to wildtype mice. G-CSF substitution in mice lacking G-CSF largely reversed this effect. CONCLUSIONS: Although infarct volumes did not differ 24 hours after ischemia the increase of AMPA receptor binding densities in G-CSF deficient mice may explain the bigger infarcts previously observed at later time-points with the same stroke model.

Olfactomedin 4 deficiency promotes prostate neoplastic progression and is associated with upregulation of the hedgehog-signaling pathway.

Loss of olfactomedin 4 (OLFM4) gene expression is associated with the progression of human prostate cancer, but its role and the molecular mechanisms involved in this process have not been completely understood. In this study, we found that Olfm4-knockout mice developed prostatic intraepithelial neoplasia and prostatic adenocarcinoma. Importantly, we found that the hedgehog-signaling pathway was significantly upregulated in the Olfm4-knockout mouse model. We also found that restoration of OLFM4 in human prostate-cancer cells that lack OLFM4 expression significantly downregulated hedgehog signaling-pathway component expression. Furthermore, we demonstrated that the OLFM4 protein interacts with sonic hedgehog protein, as well as significantly inhibits GLI-reporter activity. Bioinformatic and immunohistochemistry analyses revealed that decreased OLFM4 and increased SHH expression was significantly associated with advanced human prostate cancer. Thus, olfactomedin 4 appears to play a critical role in regulating progression of prostate cancer, and has potential as a new biomarker for prostate cancer.

Postinfarction Cardiac Remodeling Proceeds Normally in Granulocyte Colony-Stimulating Factor Knockout Mice.

Treatment with granulocyte colony-stimulating factor (G-CSF) reportedly mitigates postinfarction cardiac remodeling and dysfunction. We herein examined the effects of G-CSF knockout (G-CSF-KO) on the postinfarction remodeling process in the hearts of mice. Unexpectedly, the acute infarct size 24 hours after ligation was similar in the two groups. At the chronic stage (4 weeks later), there was no difference in the left ventricular dimension, left ventricular function, or histological findings, including vascular density, between the two groups. In addition, expression of vascular endothelial growth factor (VEGF) was markedly up-regulated in hearts from G-CSF-KO mice, compared with wild-type mice. Microarray failed in detecting up-regulation of VEGF mRNA, whereas G-CSF administration significantly decreased myocardial VEGF expression in mice, indicating that G-CSF post-transcriptionally down-regulates VEGF expression. When G-CSF-KO mice were treated with an anti-VEGF antibody (bevacizumab), cardiac remodeling was significantly aggravated, with thinning of the infarct wall and reduction of the cellular component, including blood vessels. In the granulation tissue of bevacizumab-treated hearts 4 days after infarction, vascular development was scarce, with reduced cell proliferation and increased apoptosis, which likely contributed to the infarct wall thinning and the resultant increase in wall stress and cardiac remodeling at the chronic stage. In conclusion, overexpression of VEGF may compensate for the G-CSF deficit through preservation of cellular components, including blood vessels, in the postinfarction heart.

Pten loss in the bone marrow leads to G-CSF-mediated HSC mobilization.

The phosphatase and tumor suppressor PTEN inhibits the phosphoinositol-3-kinase (PI3K) signaling pathway and plays a key role in cell growth, proliferation, survival, and migration. Pten conditional deletion using MxCre or Scl-CreER(T) leads to splenomegaly and leukemia formation, which occurs after the relocation of normal hematopoietic stem cells (HSCs) from the bone marrow to the spleen. Unexpectedly, dormant HSCs in the bone marrow do not enter the cell cycle upon Pten loss, they do not lose self-renewal activity, and they are not exhausted. Instead, Pten deficiency causes an up-regulation of the PI3K pathway in myeloid cells, but not in HSCs. Strikingly, myeloid cells secrete high levels of G-CSF upon Pten loss, leading to the mobilization of HSCs from the bone marrow and accumulation in the spleen. After deletion of Pten in mice lacking G-CSF, the splenomegaly, myeloproliferative disease, and splenic HSC accumulation are rescued. Our data show that although PTEN has little if any role in normal HSCs, it is essential to prevent overt G-CSF production by myeloid and stromal cells which otherwise causes HSCs to relocate to the spleen followed by lethal leukemia initiation.

Olfactomedin 4 inhibits cathepsin C-mediated protease activities, thereby modulating neutrophil killing of Staphylococcus aureus and Escherichia coli in mice.

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4⁻/⁻ mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4⁻/⁻ mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4⁻/⁻ mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4⁻/⁻ neutrophils. The bacterial killing and clearance capabilities observed in OLFM4⁻/⁻ mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.

Granulocyte colony-stimulating factor protects mice during respiratory virus infections.

A burst in the production of pro-inflammatory molecules characterizes the beginning of the host response to infection. Cytokines, chemokines, and growth factors work in concert to control pathogen replication and activate innate and adaptive immune responses. Granulocyte colony-stimulating factor (G-CSF) mobilizes and activates hematopoietic cells from the bone marrow, and it has been shown to mediate the generation of effective immunity against bacterial and fungal infections. G-CSF is produced at high levels in the lungs during infection with influenza and parainfluenza viruses, but its role during these infections is unknown. Here we show that during infection of mice with a non-lethal dose of influenza or Sendai virus, G-CSF promotes the accumulation of activated Ly6G+ granulocytes that control the extent of the lung pro-inflammatory response. Remarkably, these G-CSF-mediated effects facilitate viral clearance and sustain mouse survival.

The role of granulocyte-colony stimulating factor (G-CSF) in the healthy brain: a characterization of G-CSF-deficient mice.

Granulocyte-colony stimulating factor (G-CSF) is a hematopoietic growth factor that controls proliferation and differentiation of neural stem cells. Although recent studies have begun to explore G-CSF-related mechanisms of action in various disease models, little is known about its function in the healthy brain. In the present study, the effect of G-CSF deficiency on memory formation and motor skills was investigated. The impact of G-CSF deficiency on the structural integrity of the hippocampus was evaluated by analyzing the generation of doublecortin-expressing cells, the amount of bromodeoxyurine-labeled cells, the dendritic complexity in hippocampal neurons, the binding densities of NMDA and GABA(A) receptors and the induction of long-term potentiation (LTP). G-CSF deficiency caused a disruption in memory formation and in the development of motor skills. These impairments were associated with reduced ligand binding densities of NMDA receptors in hippocampal subfields CA3 and the dentate gyrus. The reduced excitation was potentiated by increased ligand binding densities of GABA(A) receptors resulting in a relative shift in favor of inhibition and impaired behavioral performance. These alterations were accompanied by impaired induction of LTP in the CA1 region. Moreover, G-CSF deficiency led to decreased dendritic complexity in hippocampal neurons in the dentate gyrus and the CA1 region. G-CSF deficiency also caused a reduction of neuronal precursor cells in the dentate gyrus. These findings confirm G-CSF as an essential neurotrophic factor, and point to a role in the proliferation, differentiation and functional integration of neural cells necessary for the structural and functional integrity of the hippocampal formation.

Hematopoietic lineage cell-specific protein-1 (HS1) regulates PAR-mediated ERK activation and thromboxane generation in platelets.

Thrombin-induced platelet activation leads to tyrosine phosphorylation of hematopoietic lineage cell-specific protein-1 (HS1), a 75 kDa adapter protein expressed exclusively in cells of hematopoietic lineage. We have shown HS1 to be a functionally important signaling molecule downstream of PAR-4 and GPVI collagen receptor. We have thus begun to elucidate PAR signaling pathway of HS1 phosphorylation, and its functional implications. PAR-1 and PAR-4 activating peptides (SFLLRN and AYPGKF, respectively) induced HS1 phosphorylation in a Gq-dependent manner as shown by incubation with the Gq inhibitor, YM254890. Consistently, HS1 phosphorylation was abolished in platelets from Gq deficient mice upon AYPGKF stimulation. Treatment with ADP receptor antagonists did not affect HS1 phosphorylation. Pretreatment of platelets with Src kinase inhibitors abolished HS1 phosphorylation. Further Syk activation, as measured by tyrosine phosphorylation of Syk (residues 525/526), in response to PAR activation was abolished in the presence of Src inhibitors. HS1 null mice show inhibition of PAR-mediated thromboxane A2 generation compared to wild type littermates. Phosphorylation of Erk, a key signaling molecule in thromboxane generation, was also diminished in HS1 null mice platelets. Based on these findings, we conclude that tyrosine phosphorylation of HS1 occurs downstream of both PAR-1 and PAR-4. HS1 phosphorylation is a Gq mediated response regulated by Src kinases. Thus, HS1 may mediate PAR-induced thromboxane generation through regulation of Erk phosphorylation.

IL6/sIL6R complex contributes to emergency granulopoietic responses in G-CSF- and GM-CSF-deficient mice.

Mice defective in both granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have severely impaired neutrophil production and function, yet these mice respond to acute pathogen challenge with a significant neutrophil response. We have recently reported the development of an in vitro system to detect granulopoietic cytokines secreted from cells isolated from G-CSF, GM-CSF double knockout mice. The conditioned media produced by these cells after stimulation with lipopolysaccharide or Candida albicans supports the production and differentiation of granulocytes (ie, the conditioned media contains neutrophil promoting activity [NPA]). We now show that the NPA in the G-CSF(-/-)/GM-CSF(-/-) conditioned media requires interleukin-6 (IL6), is abolished by soluble gp130, and can be specifically immunodepleted by an anti-IL6R antibody. NPA effects on bone marrow cells are also mimicked by Hyper-IL6, and the soluble IL6R is present in NPA. These results show that the IL6/sIL6R complex is the major effector of NPA. NPA production by mice defective for both G-CSF and GM-CSF uncovers an alternative pathway to granulocyte production, which is activated after exposure to pathogens.

Macrophage-colony stimulating factor is required for the production of neutrophil-promoting activity by mouse embryo fibroblasts deficient in G-CSF and GM-CSF.

G-CSF and GM-CSF play important roles in regulating neutrophil production, survival, differentiation, and function. However, we have shown previously that G-CSF/GM-CSF double-deficient [knockout (KO)] mice still develop a profound neutrophilia in bone marrow and blood after infection with Candida albicans. This finding suggests the existence of other systems, which can regulate emergency neutrophil production. We have now developed an "in vitro" technique to detect and characterize a neutrophil-promoting activity (NPA) in the media conditioned by mouse embryonic fibroblasts (MEFs) derived from G-CSF(-/-)/GM-CSF(-/-) mice. NPA is produced in vitro by the MEFs after stimulation with LPS or heat-inactivated C. albicans. Although M-CSF added directly to bone marrow cultures does not sustain granulocyte production, our studies indicate that production of NPA requires activation of the M-CSF receptor (c-fms). First, G-CSF(-/-)/GM-CSF(-/-) MEFs produce high levels of NPA after stimulation with LPS or C. albicans, and G-CSF/GM-CSF/M-CSF triple-KO MEFs do not. Second, the production of NPA by the G-CSF(-/-)/GM-CSF(-/-) MEFs is reduced significantly upon incubation with neutralizing antibodies to M-CSF or c-fms. Third, NPA production by G-CSF(-/-)/GM-CSF(-/-)/M-CSF(-/-) fibroblasts is enhanced by supplementing culture medium with M-CSF. Thus, stimulation of c-fms by M-CSF is a prerequisite for the production of NPA.

Hematopoietic lineage cell specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation.

Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell-specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors. Coimmunoprecipitation experiments revealed that HS1 associates with Syk and phosphatidylinositol 3-kinases. HS1-null mice displayed increased bleeding times and increased time to occlusion in the FeCl(3) in vivo thrombosis model compared with their wild-type littermates. In addition, aggregation and secretion responses were diminished in HS1-null mouse platelets after stimulation of GPVI and protease-activated receptor 4 (PAR-4) agonists compared with wild-type littermate mouse platelets. Finally, Akt phosphorylation was diminished after GPVI or PAR-4 stimulation in platelets from HS1-null mice compared with their wild-type littermates. These results demonstrate that phosphorylation of the HS1 protein occurs downstream of GPVI stimulation and that HS1 plays a significant functional role in platelet activation downstream of GPVI and PARs.

Elevated interleukin-6 and G-CSF in human pancreatic cancer cell conditioned medium suppress dendritic cell differentiation and activation.

Although dendritic cell (DC) function is impaired in pancreatic cancer patients, the underlying mechanisms are unknown. This study analyzed the soluble factors released by pancreatic cancer cells responsible for inhibiting DC differentiation and activation. Medium conditioned by a highly metastatic human pancreatic cancer cell line BxPC-3 [BxPC-3 conditioned medium (BxCM)] was mainly used for the study. Both CD34+ hematopoietic progenitor cell-derived and CD14+ monocyte-derived immature DCs and mature DCs (mDCs) were inhibited by BxCM. Allostimulation of CD4+ and CD8+ T cells by BxCM-treated mDCs was inefficient and resulted in production of lower levels of Th1 and Th2 cytokines. Antigen-specific T-cell activation capability was also reduced in BxCM-treated mDCs. Addition of exogenous interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), which were present in high amounts in BxCM, mimicked the inhibitory effect of BxCM on DC differentiation and maturation. IL-6 was able to suppress DC differentiation and G-CSF mainly acted on the suppressing allostimulatory capacity of DCs. In addition, pancreatic cancer patient sera were able to inhibit DC differentiation of CD14+ monocytes obtained from healthy donors. Depleting IL-6 or G-CSF from BxCM could reverse the DC-inhibitory properties of BxCM. Furthermore, BxCM, IL-6, or G-CSF led to the activation of signal transducer and activator of transcription 3 (STAT3) in CD14+ monocytes to different degrees. Blocking BxCM-induced STAT3 activation also reversed the inhibitory effect of BxCM on DC differentiation. Therefore, IL-6 and G-CSF in BxCM represent two main factors responsible for suppression of DC differentiation, maturation, and antigen presentation, and this suppression of DC functions may be due to the aberrant activation of STAT3 by BxCM.

Evaluation of serum granulocyte colony stimulating factor levels in infants of preeclamptic mothers.

In this study we aimed to evaluate the relationship between serum granulocyte colony stimulating factor (G-CSF) levels and absolute neutrophil counts (ANC) in infants of preeclamptic mothers. The study group consisted of 31 infants of preeclamptic mothers while the control group consisted of 24 gestational age-adjusted infants of normotensive mothers. G-CSF levels were determined by enzyme-linked immunosorbent assay (ELISA). The mean G-CSF level was 981.8 +/- 1682.5 (25.7-5924) pg/ml in the study group and 770.8 +/- 1779 (18-8526) pg/ml in control group (p > 0.05). There was no correlation between G-CSF levels and absolute or total neutrophil counts on the 1st, 2nd and 7th days in infants of preeclamptic mothers. There were positive correlations between G-CSF levels and ANC on the 1st and 7th days of life in infants of normotensive mothers. Neutropenia developed in 42.3% of the study group and in 21.7% of the control group on the 1st day of life (p > 0.05). On the 2nd day, neutropenia was observed in 61.5% of the study group and 26.1% of the control group (p = 0.013). Serum G-CSF levels were not low in neutropenic babies of preeclamptic mothers. In contrast, higher G-CSF levels in neutropenic infants suggest impaired G-CSF response in infants of preeclamptic mothers.

Mice lacking three myeloid colony-stimulating factors (G-CSF, GM-CSF, and M-CSF) still produce macrophages and granulocytes and mount an inflammatory response in a sterile model of peritonitis.

To assess the combined role of G-CSF, GM-CSF, and M-CSF in myeloid cell production, mice deficient in all three myeloid CSFs were generated (G-/-GM-/-M-/- mice). G-/-GM-/-M-/- mice share characteristics found in mice lacking individual cytokines: they are toothless and osteopetrotic and furthermore acquire alveolar proteinosis that is more severe than that found in either GM-/- or G-/-GM-/- mice. G-/-GM-/-M-/- mice have a significantly reduced lifespan, which is prolonged by antibiotic administration, suggesting compromised ability to control bacterial infection. G-/-GM-/-M-/- mice have circulating neutrophils and monocytes, albeit at significantly reduced numbers compared with wild-type mice, but surprisingly, have more circulating monocytes than M-/- mice and more circulating neutrophils than G-/-GM-/- mice. Due to severe osteopetrosis, G-/-GM-/-M-/- mice show diminished numbers of myeloid cells, myeloid progenitors, and B lymphocytes in the bone marrow, but have significantly enhanced compensatory splenic hemopoiesis. Although G-/-GM-/-M-/- mice have a profound deficiency of myeloid cells in the resting peritoneal cavity, the animals mount a moderate cellular response in a model of sterile peritonitis. These data establish that in the absence of G-CSF, GM-CSF, and M-CSF, additional growth factor(s) can stimulate myelopoiesis and acute inflammatory responses.

G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth.

Granulocyte colony-stimulating factor (G-CSF) drives the production, survival, differentiation, and inflammatory functions of granulocytes. Reactive oxygen species (ROSs) provide a major thrust of the inflammatory response, though excessive ROSs may be deleterious. G-CSF stimulation showed a time- and dose-dependent increase in ROS production, correlating with activation of Lyn and Akt. Inhibition of Lyn, PI3-kinase, and Akt abrogated G-CSF-induced ROS production. This was also blocked by DPI, a specific inhibitor of NADPH oxidase. Following G-CSF stimulation, neutrophils from Lyn-/- mice produced less ROSs than wild-type littermates. G-CSF induced both serine phosphorylation and membrane translocation of p47phox, a subunit of NADPH oxidase. Because patients with a truncated G-CSF receptor have a high risk of developing acute myeloid leukemia (AML), we hypothesized that dysregulation of ROSs contributes to leukemogenesis. Cells expressing the truncated G-CSF receptor produced more ROSs than those with the full-length receptor. G-CSF-induced ROS production was enhanced in bone marrow-derived neutrophils expressing G-CSFRdelta715, a truncated receptor. The antioxidant N-acetyl-L-cysteine diminished G-CSF-induced ROS production and cell proliferation by inhibiting Akt activation. These data suggest that the G-CSF-induced Lyn-PI3K-Akt pathway drives ROS production. One beneficial effect of therapeutic targeting of Lyn-PI3K-kinase-Akt cascade is abrogating ROS production.