Src homology 2 domain of overexpressed Lyn kinase is responsible for the acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation

We have previously shown that the overexpression of a Src family kinase, Lyn, and its kinase-negative form, LynKN, in a granulocyte progenitor cell line, GM-I62M, accelerates neutrophilic nuclear lobulation when the cells are cultured in the presence of granulocyte colony-stimulating factor. In this study, we investigated the role of the Src homology 2 (SH2) and SH3 domains of Lyn in the accelerated induction of nuclear lobulation. In contrast to wild-type Lyn, the overexpression of its SH2 domain mutant did not induce the accelerated nuclear morphological changes, but the overexpressed SH3 domain mutant had the same effects as wild-type Lyn. Therefore, the SH2 domain of Lyn is responsible for the accelerated induction of neutrophilic nuclear lobulation upon G-CSF stimulation.     

Commitment of neutrophilic differentiation and proliferation of HL-60 cells coincides with expression of transferrin receptor. Effect of granulocyte colony stimulating factor on differentiation and proliferation.

To examine the regulatory mechanisms of proliferation and maturation in neutrophilic lineage cells, we have tried to sort dimethyl sulfoxide (Me(2)SO)-treated HL-60 cells into transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells. Differentiated Trf-R(-) cells expressed more formyl-Met-Leu-Phe receptor (fMLP-receptor) and ability of O-(2) genaration, as markers of differentiation, than Trf-R(+) cells, and Trf-R(-) cell differentiation was markedly accelerated by the incubation with granulocyte colony stimulating factor (G-CSF). On the other hand, Trf-R(+) cells had a tendency to proliferate rather than differentiate, and proliferation was enhanced by G-CSF. These results indicate that Trf-R expression coincides with the commitment to proliferate or differentiate of HL-60 cells, and G-CSF accelerates these commitments. G-CSF-induced tyrosine phosphorylation of STAT 3 in Trf-R(-) cells much more than in Trf-R(+) cells. Protein 70 S6 kinase expression was higher in Trf-R(+) cells than in Trf-R(-) cells. Furthermore, p70 S6 kinase was hyperphosphorylated by G-CSF in Trf-R(+) cells, but not in Trf-R(-) cells. Rapamycin, an inhibitor of p70 S6 kinase activity, inhibited G-CSF-dependent proliferation of Trf-R(+) cells and increased fMLP-R expression on these cells. These results suggest that commitment to proliferation and differentiation in Me(2)SO-treated HL-60 cells is preprogrammed and correlated with Trf-R expression, and G-CSF potentiates the cellular commitment. STAT 3 may promote differentiation of Me(2)SO-treated HL-60 cells into neutrophils, while p70 S6 kinase may promote proliferation and negatively regulate neutrophilic differentiation.     

Granulocyte colony-stimulating factor promotes the translocation of protein kinase Ciota in neutrophilic differentiation cells

Previously, we suggested that the phosphatidylinositol 3-kinase (PI3K)-p70 S6 kinase (p70 S6K) pathway plays an important role in granulocyte colony-stimulating factor (G-CSF)-dependent enhancement of the neutrophilic differentiation and proliferation of HL-60 cells. While atypical protein kinase C (PKC) has been reported to be a regulator of p70 S6K, abundant expression of PKCiota was observed in myeloid and lymphoid cells. Therefore, we analyzed the participation of PKCiota in G-CSF-dependent proliferation. The maximum stimulation of PKCiota was observed from 15 to 30 min after the addition of G-CSF. From 5 to 15 min into this lag time, PKCiota was found to translocate from the nucleus to the membrane. At 30 min it re-translocated to the cytosol. This dynamic translocation of PKCiota was also observed in G-CSF-stimulated myeloperoxidase-positive cells differentiated from cord blood cells. Small interfering RNA for PKCiota inhibited G-CSF-induced proliferation and the promotion of neutrophilic differentiation of HL-60 cells. These data indicate that the G-CSF-induced dynamic translocation and activation processes of PKCiota are important to neutrophilic proliferation.     

Regulation of granulocyte and macrophage populations of murine bone marrow cells by G-CSF and CD137 protein

Background

Granulocytes and monocytes/macrophages differentiate from common myeloid progenitor cells. Granulocyte colony-stimulating factor (G-CSF) and CD137 (4-1BB, TNFRSF9) are growth and differentiation factors that induce granulocyte and macrophage survival and differentiation, respectively. This study describes the influence of G-CSF and recombinant CD137-Fc protein on myelopoiesis.

Methodology/Principal Findings

Both, G-CSF and CD137 protein support proliferation and survival of murine bone marrow cells. G-CSF enhances granulocyte numbers while CD137 protein enhances macrophage numbers. Both growth factors together give rise to more cells than each factor alone. Titration of G-CSF and CD137 protein dose-dependently changes the granulocyte/macrophage ratio in bone marrow cells. Both factors individually induce proliferation of hematopoietic progenitor cells (lin^-, c-kit⁺) and differentiation to granulocytes and macrophages, respectively. The combination of G-CSF and CD137 protein further increases proliferation, and results in a higher number of macrophages than CD137 protein alone, and a lower number of granulocytes than G-CSF alone demonstrating that CD137 protein-induced monocytic differentiation is dominant over G-CSF-induced granulocytic differentiation. CD137 protein induces monocytic differentiation even in early hematopoietic progenitor cells, the common myeloid progenitors and the granulocyte macrophage progenitors.

Conclusions/Significance

This study confirms earlier data on the regulation of myelopoiesis by CD137 receptor - ligand interaction, and extends them by demonstrating the restriction of this growth promoting influence to the monocytic lineage.     

SOCS3 is a physiological negative regulator for granulopoiesis and granulocyte colony-stimulating factor receptor signaling

The suppressor of cytokine signaling-3 (SOCS3/CIS3) has been shown to be an important negative regulator of cytokines, especially cytokines that activate STAT3. To examine the role of SOCS3 in neutrophils and the granulocyte colony-stimulating factor (G-CSF) signaling in vivo, we compared neutrophils from two types of conditional knockout mice, LysM-Cre:SOCS3(fl/fl) mice and Tie2-Cre:SOCS3(fl/fl) mice, in which the Socs3 gene had been deleted in mature neutrophils and hematopoietic stem cells, respectively. The size of the G-CSF-dependent colonies from Tie2-Cre:SOCS3(fl/fl) mouse bone marrow was much larger than that of colonies from control wild-type mice, while the size of interleukin-3-dependent colonies was similar. Moreover, LysM-Cre:SOCS3(fl/fl) mice had more neutrophils than SOCS3(fl/fl) mice, suggesting that SOCS3 is a negative regulator of G-CSF signaling in neutrophils. Consistent with this notion, G-CSF-induced STAT3 as well as mitogen-activated protein kinase activation was much stronger and prolonged in SOCS3-deficient mature neutrophils than in wild-type neutrophils. The preventive effect of G-CSF on apoptosis was more prominent in SOCS3-deficient mature neutrophils than in control neutrophils. These data indicate that SOCS3 negatively regulates granulopoiesis and G-CSF signaling in neutrophils and may contribute to neutrophilia or neutropenia.     

Dynamic regulation of neutrophil survival through tyrosine phosphorylation or dephosphorylation of caspase-8

Efficient expression of innate immunity is critically dependent upon the capacity of the neutrophil to be activated rapidly in the face of an acute threat and to involute once that threat has been eliminated. Here we report a novel mechanism regulating neutrophil survival dynamically through the tyrosine phosphorylation or dephosphorylation of caspase-8. Caspase-8 is tyrosine-phosphorylated in freshly isolated neutrophils but spontaneously dephosphorylates in culture, in association with the progression of constitutive apoptosis. Phosphorylation of caspase-8 on Tyr-310 facilitates its interaction with the Src-homology domain 2 containing tyrosine phosphatase-1 (SHP-1) and enables SHP-1 to dephosphorylate caspase-8, permitting apoptosis to proceed. The non-receptor tyrosine kinase, Lyn, can phosphorylate caspase-8 on Tyr-397 and Tyr-465, rendering it resistant to activational cleavage and inhibiting apoptosis. Exposure to lipopolysaccharide reduces SHP-1 activity and binding to caspase-8, caspase-8 activity, and rates of spontaneous apoptosis. SHP-1 activity is reduced and Lyn increased in neutrophils from patients with sepsis, in association with profoundly delayed apoptosis; inhibition of Lyn can partially reverse this delay. Thus the phosphorylation and dephosphorylation of caspase-8, mediated by Lyn and SHP-1, respectively, represents a novel, dynamic post-translational mechanism for the regulation of neutrophil apoptosis whose dysregulation contributes to persistent neutrophil survival in sepsis.     

Activation of granulocyte cytotoxic function by purified mouse colony-stimulating factors

Highly purified mouse colony-stimulating factors (CSF) were tested for their effect on neutrophil cytotoxic function in a homologous antibody-dependent cell-mediated cytotoxicity (ADCC) assay in which TNP-coupled mouse thymoma cells coated with mouse anti-TNP antibodies were used as targets, and purified normal mouse bone marrow neutrophils or induced peritoneal neutrophils were used as effector cells. Biochemically pure granulocyte-macrophage (GM)- and granulocyte (G)-CSF enhanced the cytotoxic activity of neutrophils obtained from both sources, allowing them to kill target cells at low antibody concentrations. Furthermore, GM- and G-CSF showed an additive effect, suggesting either the presence of separate receptors for GM- and G-CSF or of separate subsets of neutrophils. Induced peritoneal neutrophils showed a higher level of basal cytotoxic activity than did bone marrow neutrophils, suggesting neutrophil activation in vivo, but both reached similar levels of cytotoxicity upon maximal stimulation with CSF. In addition, CSF was found to be cross-reactive between mouse and human species in their enhancement of neutrophil cytotoxicity. By testing purified mouse CSF on human neutrophils, it could be shown that G-CSF and GM-CSF are functionally distinct molecules, because only G-CSF enhanced ADCC by human neutrophils. These experiments show that the purified factors that control the production of neutrophils by progenitor cells in vitro also activate differentiated neutrophils to carry out their cytotoxic activity in a more effective manner.     

Phosphorylation of Ser2078 modulates the Notch2 function in 32D cell differentiation

Notch signaling is involved in the regulation of many cell fate determination events in both embryonic development and adult tissue homeostasis. We previously demonstrated that Notch1 and Notch2 molecules inhibit myeloid differentiation in a cytokine-specific manner and that the Notch cytokine response domain is necessary for this functional specificity. We have now investigated the putative role of phosphorylation in the activity of Notch in response to cytokine signals. Our results show that the granulocyte colony-stimulating factor (G-CSF) stimulation of 32D cells expressing the intracellular Notch2 protein induces phosphorylation at specific sites of this molecule, rendering the molecule inactive and permitting differentiation of these cells. In contrast, when cells are stimulated with granulocyte macrophage colony-stimulating factor (GM-CSF), intracellular notch2 is not phosphorylated at these residues and differentiation is inhibited. We also show that deletion of the Ser/Thr-rich region between amino acids 2067 and 2099 abrogates G-CSF-induced phosphorylation and results in a molecule that inhibits differentiation in response to either G-CSF or GM-CSF. Our results further indicate that Ser(2078) is a critical residue for phosphorylation and modulation of Notch2 activity in the context of G-CSF-induced differentiation of 32D cells.     

Substrate recognition by the Lyn protein-tyrosine kinase. NMR structure of the immunoreceptor tyrosine-based activation motif signaling region of the B cell antigen receptor

The immunoreceptor tyrosine-based activation motif (ITAM) plays a central role in transmembrane signal transduction in hematopoietic cells by mediating responses leading to proliferation and differentiation. An initial signaling event following activation of the B cell antigen receptor is phosphorylation of the CD79a (Ig-alpha) ITAM by Lyn, a Src family protein-tyrosine kinase. To elucidate the structural basis for recognition between the ITAM substrate and activated Lyn kinase, the structure of an ITAM-derived peptide bound to Lyn was determined using exchange-transferred nuclear Overhauser NMR spectroscopy. The bound substrate structure has an irregular helix-like character. Docking based on the NMR data into the active site of the closely related Lck kinase strongly favors ITAM binding in an orientation similar to binding of cyclic AMP-dependent protein kinase rather than that of insulin receptor tyrosine kinase. The model of the complex provides a rationale for conserved ITAM residues, substrate specificity, and suggests that substrate binds only the active conformation of the Src family tyrosine kinase, unlike the ATP cofactor, which can bind the inactive form.     

Granulocyte colony-stimulating factor-induced activation of protein kinase-C in myeloid cells

Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, differentiation, and activation of myeloid cells. It binds to a high affinity receptor (G-CSF-R) expressed on myeloid cells, for which the signal transduction mechanisms other than protein tyrosine kinase (PTK) activation have not been completely identified. We explored the potential involvement of protein kinase-C (PKC) in G-CSF-R signal transduction. In this report, we provide direct evidence of PKC activation by G-CSF-R. G-CSF treatment of peripheral blood neutrophils, granulocytic cell lines (HL-60, NFS-60, KG-1), and monocytic cell lines (WEHI-3B,U-937) resulted in PKC activation. Chelerythrine chloride and HA-100, an isoquinolinesulfonamide derivative, the specific inhibitors of PKC, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), a chelator of intracellular calcium, and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8), an inhibitor of intracellular calcium release, blocked G-CSF-induced PKC activation in HL-60 cells, and reduced CD11b upregulation in neutrophils, but did not affect ligand-binding or down-modulation of G-CSF-R. Methyl 2,5-dihydroxycinnamate (MDHC), a potent inhibitor of protein tyrosine kinases (PTK), also inhibited PKC activation in response to G-CSF treatment, suggesting that PKC activation may occur downstream of PTK activation. Our results demonstrate the involvement of PKC in G-CSF-R signal transduction, and suggest a common signaling pathway in myeloid cells of granulocytic and monocytic lineages. J. Cell. Biochem. 66:286–296, 1997. © 1997 Wiley-Liss, Inc.     

Actin reorganization and morphological changes in human neutrophils stimulated by TNF,GM-CSF,and G-CSF: the role of MAP kinases

Stimulation of human neutrophils with tumor necrosis factor- (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) resulted in decreased fluorescence intensity of FITC-phalloidin (actin depolymerization) and morphological changes. Cytokine-induced actin depolymerization was dependent on the concentration of cytokines used as stimuli. The maximal changes were detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation with G-CSF. Cytokine-induced actin depolymerization was sustained for at least 30 min after stimulation. In contrast, N-formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly (within 45 s) induced an increase in the fluorescence intensity of FITC-phalloidin (actin polymerization) and morphological changes. TNF- and GM-CSF-induced actin depolymerization and morphological changes, but not FMLP-induced responses, were partially inhibited by either PD-98059, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase, or SB-203580, an inhibitor of p38 MAPK, and were almost completely abolished by these inhibitors in combination. G-CSF-induced responses were almost completely abolished by PD-98059 and were unaffected by SB-203580. These findings are consistent with the ability of these cytokines to activate the distinct MAPK subtype cascade in human neutrophils. Phosphorylated ERK and p38 MAPK were not colocalized with F-actin in neutrophils stimulated by cytokines or FMLP. Furthermore, FMLP-induced polarization and actin polymerization were prevented by cytokine pretreatment. These findings suggest that TNF, GM-CSF, and G-CSF induce actin depolymerization and morphological changes through activation of ERK and/or p38 MAPK and that cytokine-induced actin reorganization may be partly responsible for the inhibitory effect of these cytokines on neutrophil chemotaxis. neutrophil; actin reorganization; cytokines; mitogen-activated protein kinase; tumor necrosis factor-; granulocyte-macrophage colony stimulating factor     

Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which acts primarily to stimulate the proliferation, differentiation and activation of committed progenitor cells of the neutrophil–granulocyte lineage into functionally mature neutrophils. The traditional biological assays employed to detect G-CSF are a myeloid bone marrow colony assay, a factor-dependent cell line specific for G-CSF and commercially available immunoassays. However, these methods will not distinguish between glycosylated and non-glycosylated forms of the molecule. In this study high-performance capillary electrophoresis (HPCE) was used to analyse glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (r-met-hG-CSF). Glycosylated G-CSF preparations contained human serum albumin (HSA), added as a protein carrier. Glycosylated and non-glycosylated G-CSFs were prepared in 40 mM Na₂HPO₄ buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 mM Na₂HPO₄ buffer, pH 9.0. Glycosylated G-CSF could be separated into two distinct glycoform populations at the lower pH studied. Differences in migration time and peak shape between glycosylated and non-glycosylated G-CSF were demonstrated. HPCE analysis of G-CSF produced using a baculovirus expression vector system revealed a further distinct G-CSF glycoform and demonstrated the resolving power of the technique.     

K562 leukemia cells transfected with the human c-fes gene acquire the ability to undergo myeloid differentiation

Expression of the proto-oncogene p93c-fes and its associated tyrosine kinase activity is marked in mature granulocytes, monocytes, differentiated HL-60 leukemia cells, and leukemia cell lines KG-1, THP-1, HEL, and U-937, which can be induced to differentiate along the granulocyte/monocyte pathway. Conversely, p93-c-fes expression is absent in the K562 cell line, which is resistant to myeloid differentiation. Upon transfection and clonal selection of K562 cells using a mammalian expression vector containing the 13-kilobase pair c-fes gene, c-fes mRNA was transcribed and p93-c-fes tyrosine activity kinase was expressed. Clones expressing c-fes underwent myeloid differentiation as assessed by the appearance of phagocytic activity, Fc receptors, nitro blue tetrazolium reduction, Mac-1 immunofluorescence, and lysozyme production. These results indicate that the expression of the c-fes protooncogene and its associated tyrosine kinase activity plays a major role in the initiation of myeloid differentiation.     

Accelerated clearance of Escherichia coli in experimental peritonitis of histamine-deficient mice

IL-5 plays a pivotal role in growth and differentiation of eosinophils. The signal transduction mechanism of IL-5Ralpha is largely unknown. We have demonstrated that IL-5 induces tyrosine phosphorylation of IL-5Ralpha in eosinophils. To identify IL-5Ralpha-associated tyrosine kinases, we have examined the expression of Src family tyrosine kinases in eosinophils. Among the Src family members, Lyn, Hck, Fgr, and Lck are present in eosinophils, and, among these four kinases, only Lyn is associated with the IL-5Ralpha under basal conditions. We also confirm the association of Janus kinase (Jak)2 with IL-5Ralpha. Lyn kinase phosphorylates both IL-5Ralpha and betacR in vitro. The importance of Lyn kinase for eosinophil differentiation was studied using antisense oligodeoxynucleotides. Lyn antisense oligodeoxynucleotide blocks eosinophil differentiation from stem cells in a dose-dependent manner. The Jak2 inhibitor tyrphostin AG490 also inhibits eosinophil differentiation. The importance of Lyn for eosinophil differentiation was further studied using Lyn knockout mice. The IL-5-stimulated eosinophil differentiation from bone marrow cells is significantly inhibited in Lyn(-/-) mice as compared with that in control mice. We conclude that both Lyn and Jak2 play an essential role in IL-5Ralpha signaling, leading to eosinophil differentiation. The effect of Lyn appears to be relatively specific for the eosinophilic lineage.     

The mechanism of granulocyte nuclear shape determination: possible involvement of the centrosome

Mature blood neutrophils (polymorphonuclear granulocytes) have characteristically complex nuclear shapes. The human neutrophil nucleus generally possesses 3-4 lobes; the mouse neutrophil nucleus frequently resembles a twisted toroid with a central hole. Myeloid tissue culture systems (e.g., human HL-60 and murine MPRO) can be induced to differentiate in vitro towards neutrophils by addition of retinoic acid, exhibiting the characteristic nuclear shape changes. Confocal immunostaining and thin-section transmission electron microscopic image data from differentiated HL-60 and MPRO cells clearly demonstrate proximity of the centrosomal region (containing dynein, gamma-tubulin and C-Nap1) to regions of granulocytic nuclear indentations. In addition, the centrosomal region, flanked by the Golgi apparatus, is shown to be present within the central hole of the toroidal mouse granulocyte nucleus. A role for the centrosomal region and associated microtubules in molding granulocytic nuclear shape is suggested.     

Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK.