In Vivo Treatment with Granulocyte Colony-Stimulating Factor does not Delay Apoptosis in Human Neutrophils by Increasing the Expression of the Vacuolar Proton ATPase
Restricted accessResearch articleFirst published online January, 2002
In Vivo Treatment with Granulocyte Colony-Stimulating Factor does not Delay Apoptosis in Human Neutrophils by Increasing the Expression of the Vacuolar Proton ATPase
Neutrophils die by apoptosis, and in vivo administration of granulocyte colony-stimulating factor (G-CSF) delays this apoptotic cell death. G-CSF administered in vitro correlates delayed apoptosis with upregulation of the vacuolar proton ATPase (v-ATPase). Because this enzyme requires assembly of membrane and cytosolic domains to function, we hypothesized that in vivo G-CSF would increase synthesis and assembly of v-ATPase components to delay apoptosis.
Methods
Volunteers received G-CSF for 5 days, and each had a paired control. Neutrophils were isolated from subjects before the first and after the fifth injection. Proteins from cytosol or plasma membrane or from whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted with antibody to the 33kDa v-ATPase E subunit. Densitometry quantified immunoreactivity.
Results
No significant increase on the E subunit occurred between treated and control groups.
Conclusion
In vivo G-CSF does not increase the amount of v-ATPase in neutrophils. Although G-CSF in vivo delays apoptosis, the mechanism(s) by which this occurs is not known.
ForgacM. Structure and properties of the coated vesicle proton pump. Ann N Y Acad Sci1992;671:273–283.
2.
WilkensS, VasilyevaE, ForgacM. Structure of the vacuolar ATPase by electron microscopy. J Biol Chem1999;274:31804–31810.
3.
NandaA, GukovskayaA, TsengJ, GrinsteinS. Activation of vacuolar-type proton pumps by protein kinase C. J Biol Chem1992;267:22740–22746.
4.
GottliebR, GiesingH, ZhuJ, EnglerR, BabiorB. Cell acidification apoptosis: Granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H+-ATPase. Proc Natl Acad Sci U S A1995;92:5965–5968.
5.
NiessenH, MeisenholderG,Hai-LingL, GluckS, LeeB, BowmanB, EnglerR, BabiorB, GottliebR. Granulocyte colony-stimulating factor upregulates the vacuolar proton ATPase in human neutrophils. Blood1997;90:4598–4601.
6.
LeaveyP, SellinsK, ThurmanG, ElziD, HiesterA, SillimanC, ZerbeG, CohenJ, AmbrusoD. In vivo treatment with granulocyte colony-stimulating factor results in divergent effects on neutrophil functions measured in vitro. Blood1998;92:4366–4374.
7.
RosenthalJ, ThurmanG, CusackN, PetersonV, MalechH, AmbrusoD. Neutrophils from patients after burn injury express a deficiency of the oxidase components p47-phox and p67phox. Blood1996;88:4321–4329.
8.
AmbrusoD, BolscherB, StokmanP, VerhoevenA, RoosD. Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate. J Biol Chem1990;265:924–930.
9.
AmbrusoD, StorkL, GibsonB, ThurmanG. Increased activity of the respiratory burst in cord blood neutrophils: Kinetics of the NADPH oxidase enzyme system in subcellular fractions. Pediatr Res1987;21:205–210.
10.
SquierM, SehnertA, CohenJ. Apoptosis in leukocytes. J Leukoc Biol1995;57:2–10.
11.
WangJM, ChenZG, ColellaS, BonillaMA, WelteK, BordignonC, MantovaniA. Chemotactic activity of recombinant human granulocyte colony-stimulating factor. Blood1988;72:1456–1460.
12.
SullivanGW, CarperHT, MandellGL. The effect of three human recombinant hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and interleukin-3) on phagocyte oxidative activity. Blood1993;81:1863–1870.
13.
InaK, KusugamiK, HosokawaT, ImadaA, ShimizuT, YamaguchiT, OhsugaM, KyokaneK, SakaiT, NishioY, YokoyamaY, AndoT. Increased mucosal production of granulocyte colony-stimulating factor is related to a delay in neutrophil apoptosis in inflammatory bowel disease. J Gastroenterol Hepatol1999;14:46–53.
14.
ErtelW, KeelM, BuergiU, HartungT, ImhofH, TrentzO. Granulocyte colony-stimulating factor inhibits neutrophil apoptosis at the local site after severe head and thoracic surgery. J Trauma1999;46:784–792.
15.
OhtaT, ArakawaH, FutagamiF, FushidaS, KitagawaH, KayaharaM, NagakawaT, MiwaK, KurashimaK, NumataM, KitamuraY, TeradaT, OhkumaS. Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan-1. J Pathol1998;185:324–330.
16.
LongX, CrowM, SollottS, O'NeillL, MeneesD, deLourdes Hipolito MBoluytM, AsaiT, LakattaE.Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes. J Clin Invest1998;101:1453–1461.
17.
GottliebR, GruolD, ShuJ, EnglerR. Preconditioning in rabbit cardiomyocytes. Role of pH, vacuolar proton ATPase, and apoptosis. J Clin Invest1996;97:2391–2398.
18.
Karwatowska-ProkopczukE, NordbergJA, LiHL, EnglerR, GottliebR. Effect of vacuolar proton ATPase on pH, Ca2+, and apoptosis in neonatal cardiomyocytes during metabolic inhibition/recovery. Circ Res1998;82:1139–1144.
19.
DibbertB, WeberM, NikolaizikW, VogtP, SchoniM, BlaserK, SimonH. Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: A general mechanism to accumulate effector cells in inflammation. Proc Natl Acad Sci U S A1999;96:13330–13335.
20.
AkifusaS, OhguchiM, KosekiT, NaraK, SembaI, YamatoK, OkahashiN, MerinoR, NunezG, HanadaN, TakeharaT, NishiharaT. Increase in Bcl-2 level promoted by CD40 ligation correlates with inhibition of B cell apoptosis induced by vacuolar type H+-Atpase inhibitor. Exp Cell Res1998;238:82–89.
21.
RaffM. Cell suicide for beginners. Nature1998;396:119–122.
22.
AravindL, VishvaM, DixitV, KooninE. The domains of death: Evolution of the apoptosis machinery. Trends Biochem Sci1999;24:47–53.
23.
KurokiM, O'FlahertyJ. Extracellular signal-regulated protein kinase (ERK)-dependent and ERK-independent pathways target STAT3 on serine-727 in human neutrophils stimulated by chemotactic factors and cytokines. Biochem J1999;341:691–696.
24.
FraschS, NickJ, FadokV, BrattonD, WorthenG, HensonP. p38 mitogen-activated protein kinase-dependent and -independent intracellular signal transduction pathways leading to apoptosis in human neutrophils. J Biol Chem1998;273:8389–8397.
