Distinctive effects of G-CSF, GM-CSF and TNFalpha on neutrophil apoptosis in systemic lupus erythematosus.

OBJECTIVE: To investigate the influence of culture with G-CSF GM-CSF and TNFalpha on neutrophil apoptosis, comparing neutrophils from SLE patients with rheumatoid arthritis (RA) patients and healthy control subjects. METHODS: Neutrophils were isolated from SLE (n= 10), RA (n= 10) and healthy control subjects (n= 10), and cultured with two different concentrations of G-CSF, GM-CSF and TNFalpha. Proportion of apoptotic neutrophils at T=0, T=2hrs and T=24hrs was measured using FITC-labelled annexinV and flow cytometry. RESULTS: Significantly more neutrophils were apoptotic at T=0 in the SLE subjects than in the other groups (median, range--Control 3.5% (0.3-7.9) SLE 9.5% (2.9-29.1) RA 3.0% (0.4-23.0) p

The role of the granulocyte colony-stimulating factor receptor (G-CSF-R) in disease

Granulocyte colony-stimulating factor (G-CSF) is a key regulator of granulopoiesis via stimulation of a specific cell-surface receptor, the G-CSF-R, found on hematopoietic progenitor cells as well as neutrophilic granulocytes. It is perhaps not surprising, therefore, that mutations of the G-CSF-R has been implicated in several clinical settings that affect granulocytic differentiation, particularly severe congenital neutropenia, myelodysplastic syndrome and acute myeloid leukemia. However, other studies suggest that signalling via the G-CSF-R is also involved in a range of other malignancies. This review focuses on the molecular mechanisms through which the G-CSF-R contributes to disease.

G-CSF: function and modes of action (Review)

Since the observations in the 1960s that granulocyte-colony stimulating factor (G-CSF) stimulated the proliferation of granulocytic cells in semisolid cultures of bone marrow cells, G-CSF has established itself as a useful clinical agent for increasing levels of neutrophilic granulocytes. However, these early findings did not firmly establish whether G-CSF is a genuine regulator of granulocyte formation under normal physiological conditions or rather acts as an emergency regulator, playing an important role only under stress conditions. The advent of <gene-knockout technology> has allowed us to evaluate these questions in a physiological setting through analysis of mice with a targeted mutation of G-CSF or its receptor, while the development of relevant cell models has enabled us to dissect the molecular basis of G-CSF action. This review discusses our current state of knowledge regarding the role of G-CSF in granulopoiesis.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Y704, Y729, Y744, Y764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation and cell survival. However, it is unclear whether these tyrosines are equally important under more physiological conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated GCSF- R deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (mO), single tyrosine "add back" mutants or wild type (WT) receptors. G-CSFinduced responses were determined in primary colony assays, serial replatings and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Y764 strongly enhanced proliferativeresponses, which was reverted by inhibition of ERK activitity. Y729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing mO gradually dropped compared to WT. The presence of Y729, but also Y704 and Y744, both involved in activation of STAT3, further reduced replating
efficiencies. Conversely, Y764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a >104–fold increase of colony forming cells over mO after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

C/EBPÁ and G-CSF receptor signals cooperate to induce the myeloperoxidase and neutrophil elastase genes

To assess cooperation between G-CSF signals and C/EBP, we characterized Ba/F3 pro-B cell lines expressing C/EBPWT-ER and the G-CSF receptor (GCSFR). In these lines, GCSFR signals can be evaluated independent of their effect on C/EBP levels. G-CSF alone did not induce the MPO, NE, LF, or PU.1 RNAs, and C/EBPWT-ER alone stimulated low-level MPO and high-level PU.1 expression. Simultaneous activation of the GCSFR and C/EBPWT-ER markedly increased MPO and NE induction at 24 h, and LF mRNA was detected at 48 h. G-CSF did not increase endogenous GCSFR, endogenous C/EBP or exogenous C/EBPWT-ER levels, and C/EBPWT-ER did not induce endogenous or exogenous GCSFR. Several GCSFR mutants were also co-expressed with C/EBPWT-ER. Mutation of all four cytoplasmic tyrosines prevented NE induction but enhanced MPO induction. Mutation of Y704 was required for increased MPO induction. Consistent with this finding, removing IL-3 without G-CSF addition enabled MPO, but not NE, induction by C/EBPWT-ER. GCSFR signals or related signals from other receptors may cooperate with C/EBP to direct differentiation of normal myeloid stem cells.

Receptor activation and 2 distinct COOH- terminal motifs control G-CSF receptor distribution and internalization kinetics.

We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position –4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.

Somatostatin modulates G-CSF-induced but not interleukin-3-induced proliferative responses in myeloid 32D cells via activation of somatostatin receptor subtype 2

Somatostatin, originally identified as a peptide involved in neurotransmission, functions as an inhibitor of multiple cellular responses, including hormonal secretion and proliferation. Somatostatin acts through activation of G-protein-coupled receptors of which five subtypes have been identified. We have recently established that human CD34/c-kit expressing hematopoietic progenitors and acute myeloid leukemia (AML) cells exclusively express SSTR2. A major mechanism implicated in the antiproliferative action of somatostatin involves activation of the SH2 domain-containing protein tyrosine phosphatase SHP-1. While 0.1-1 x 10(-9) M of somatostatin, or its synthetic stable analog octreotide, can inhibit G-CSF-induced proliferation of AML cells, little or no effects are seen on GM-CSF- or IL-3-induced responses.
MATERIALS AND METHODS: To study the mechanisms underlying the antiproliferative responses of myeloblasts to somatostatin, clones of the IL-3-dependent murine cell line 32D that stably express SSTR2 and G-CSF receptors were generated. RESULTS: Similar to AML cells, octreotide inhibited G-CSF-induced but not IL-3-induced proliferative responses of 32D[G-CSF-R/SSTR2] cells. Somatostatin induced SHP-1 activity and inhibited G-CSF-induced, but not IL-3-induced, activation of the signal transducer and activator of transcription proteins STAT3 and STAT5.
CONCLUSION: Based on these data and previous results, we propose a model in which recruitment and activation of the tyrosine phosphatase SHP-1 by SSTR2 is involved in the selective negative action of somatostatin on G-CSF-R signaling.

Role and regulation of G-CSF and its receptor in muscle

Granulocyte-Colony Stimulating Factor (G-CSF) is a commercially available drug with research linking it to favourable muscle adaptations, post trauma. Molecular techniques were used to identify the G-CSF receptor in skeletal muscle and G-CSF treatment was used to determine the molecular mechanisms by which G-CSF enhances muscle growth and regeneration.

G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle

Granulocyte-colony stimulating factor (G-CSF) increases recovery of rodent skeletal muscles after injury, and increases muscle function in rodent models of neuromuscular disease. However, the mechanisms by which G-CSF mediates these effects are poorly understood. G-CSF acts by binding to the membrane spanning G-CSFR and activating multiple intracellular signaling pathways. Expression of the G-CSFR within the haematopoietic system is well known, but more recently it has been demonstrated to be expressed in other tissues. However, comprehensive characterization of G-CSFR expression in healthy and diseased skeletal muscle, imperative before implementing G-CSF as a therapeutic agent for skeletal muscle conditions, has been lacking. Here we show that the G-CSFR is expressed in proliferating C2C12 myoblasts, differentiated C2C12 myotubes, human primary skeletal muscle cell cultures and in mouse and human skeletal muscle. In mdx mice, a model of human Duchenne muscular dystrophy (DMD), G-CSF mRNA and protein was down-regulated in limb and diaphragm muscle, but circulating G-CSF ligand levels were elevated. G-CSFR mRNA in the muscles of mdx mice was up-regulated however steady-state levels of the protein were down-regulated. We show that G-CSF does not influence C2C12 myoblast proliferation, differentiation or phosphorylation of Akt, STAT3, and Erk1/2. Media change alone was sufficient to elicit increases in Akt, STAT3, and Erk1/2 phosphorylation in C2C12 muscle cells and suggest previous observations showing a G-CSF increase in phosphoprotein signaling be viewed with caution. These results suggest that the actions of G-CSF may require the interaction with other cytokines and growth factors in vivo, however these data provides preliminary evidence supporting the investigation of G-CSF for the management of muscular dystrophy.

G-CSF treatment can attenuate dexamethasone-induced reduction in C2C12 myotube protein synthesis

Granulocyte-colony stimulating factor (G-CSF) has been demonstrated to enhance skeletal muscle recovery following injury and increases muscle function in the context of neuromuscular disease in rodent models. However, understanding of the underlying mechanisms used by G-CSF to mediate these functions remains poor. G-CSF acts on responsive cells through binding to a specific membrane spanning receptor, G-CSFR. Recently identified, the G-CSFR is expressed in myoblasts, myotubes and mature skeletal muscle tissue. Therefore, elucidating the actions of G-CSF in skeletal muscle represents an important prerequisite to consider G-CSF as a therapeutic agent to treat skeletal muscle. Here we show for the first time that treatment with moderate doses (4 and 40ng/ml) of G-CSF attenuates the effects of dexamethasone in reducing protein synthesis in C2C12 myotubes. However, a higher dose (100ng/ml) of G-CSF exacerbates the dexamethasone-induced reduction in protein synthesis. In contrast, G-CSF had no effect on basal or dexamethasone-induced protein degradation, nor did G-CSF influence the phosphorylation of Akt, STAT3, Erk1/2, Src, Lyn and Erk5 in C2C12 myotubes. In conclusion, physiologically relevant doses of G-CSF may attenuate reduced skeletal muscle protein synthesis during catabolic conditions, thereby improving recovery.

Recovery of pulmonary structure and exercise capacity by treatment with granulocyte-colony stimulating factor (G-CSF) in a mouse model of emphysema

Emphysema is a chronic obstructive pulmonary disease characterized abnormal dilatation of alveolar spaces, which impairs alveolar gas exchange, compromising the physical capacity of a patient due to airflow limitations. Here we tested the effects of G-CSF administration in pulmonary tissue and exercise capacity in emphysematous mice. C57Bl/6 female mice were treated with elastase intratracheally to induce emphysema. Their exercise capacities were evaluated in a treadmill. Lung histological sections were prepared to evaluate mean linear intercept measurement. Emphysematous mice were treated with G-CSF (3 cycles of 200 μg/kg/day for 5 consecutive days, with 7-day intervals) or saline and submitted to a third evaluation 8 weeks after treatment. Values of run distance and linear intercept measurement were expressed as mean ± SD and compared applying a paired t-test. Effects of treatment on these parameters were analyzed applying a Repeated Measures ANOVA, followed by Tukey's post hoc analysis. p < 0.05 was considered statistically significant. Twenty eight days later, animals ran significantly less in a treadmill compared to normal mice (549.7 ± 181.2 m and 821.7 ± 131.3 m, respectively; p < 0.01). Treatment with G-CSF significantly increased the exercise capacity of emphysematous mice (719.6 ± 200.5 m), whereas saline treatment had no effect on distance run (595.8 ± 178.5 m). The PCR cytokines genes analysis did not detect difference between experimental groups. Morphometric analyses in the lung showed that saline-treated mice had a mean linear intercept significantly higher (p < 0.01) when compared to mice treated with G-CSF, which did not significantly differ from that of normal mice. Treatment with G-CSF promoted the recovery of exercise capacity and regeneration of alveolar structural alterations in emphysematous mice. © 2013.

Treatment of adriamycin-induced nephropathy with erythropoietin and G-CSF

Background: Granulocyte colony-stimulating factor (G-CSF) and Erythropoietin (EPO) are known to stimulate the growth and differentiation of progenitor cells to prevent acute renal injury. This study aimed to assess the use of growth factors to mobilize stem cell in a mouse model of adriamycin-induced chronic kidney disease. Methods: All animals were injected with adriamycin for kidney injury and allocated into three treatment groups (G-CSF, EPO and G-CSF + EPO), and a control group (adriamycin alone). Results: Number of atrophic sites, glomerulosclerosis rate and interstitial fibrosis severity score were assessed in all groups. In all treatment groups, histologic parameters did not significantly differ, but were lower than in the control group (P<.001). Scal and CD34 expressions among treatment groups showed no statistically significant difference, but were higher than in the control group (P<.0001). CD105 expression was higher in EPO and G+EPO as compared to G-CSF and the control group (P<.0001), with no statistically significant difference between the latter two groups (P = NS). Conclusion: G-CSF and EPO had a histologic protective effect, while treatment with EPO + G-CSF had no additive effects in a model of adriamycin-induced chronic kidney disease. © 2013 Societá Italiana di Nefrologia.