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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1261-1272
Primitive Myeloid Cells Express High Levels of Phospholipase
A2 Activity in the Absence of Leukotriene Release:
Selective Regulation by Stem Cell Factor Involving the MAP Kinase
Pathway
By
Pamela J. Roberts,
Elahe Mollapour,
Michael J. Watts, and
David C. Linch
From the Department of Haematology, University College London Medical
School, London, UK.
 |
ABSTRACT |
The activation of phospholipase A2 (PLA2)
with release of eicosanoids and prostanoids in mature myeloid cells and
the augmentation (priming) of this activity by cytokines such as
granulocyte-macrophage colony-stimulating factor (GM-CSF) are central
to the inflammatory process. Yet, there are few data concerning
PLA2 activity and its regulation by growth factors in
primary hematopoietic cells. We therefore analyzed the PLA2
activity of mobilized human CD34 antigen-positive (CD34+)
stem cells by quantitation of the extracellular release of
3H-arachidonate. The PLA2 activity of
CD34+ cells stimulated with calcium ionophore (A23187)
was of similar magnitude to that of mature neutrophils and monocytes.
Preincubation of CD34+ cells with stem cell factor (SCF)
before A23187-stimulation resulted in primed PLA2 activity,
whereas interleukin-3 (IL-3), GM-CSF, and tumor necrosis factor  had
no significant effect. When CD34+ cells were induced to
differentiate, PLA2 activity remained responsive to SCF for
several days, but after 8 days, at which stage morphological and
functional evidence of maturation was occurring, priming of PLA2 by SCF could no longer be elicited, whereas responses
to GM-CSF and IL-3 had developed. The further metabolism of arachidonic acid to eicosanoids by CD34+ cells was not detected by
either thin-layer chromatography, enzyme immunoassay, or differential
spectroscopy. SCF stimulated the rapid but transient activation of ERK2
(p42 MAP kinase) in CD34+ cells, and we used the MAP
kinase kinase inhibitor, PD 098059, which at 30 µmol/L blocks ERK2
activation in CD34+ cells, to investigate whether
SCF-mediated priming of arachidonate release was mediated by this
kinase. PD 098059 only partially inhibited A23187-stimulated
PLA2 activity primed by SCF, suggesting the involvement of
ERK2 and possibly a further signal transduction pathway. Methyl
arachidonyl fluorophosphonate (5 µmol/L), a dual inhibitor of i and
cPLA2 isoforms, completely inhibited arachidonate release
without affecting ERK2 activation, demonstrating the lack of cellular
toxicity. These data provide the first evidence that primitive myeloid
cells have the capacity to release arachidonate, which is regulated by
an early acting hematopoietic growth factor important for the growth
and survival of these cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PRIMITIVE MYELOID CELLS have a blastic
appearance and a characteristic immunophenotype that includes the
expression of the CD34 antigen.1,2 These cells are able to
undergo a series of amplification divisions within the bone marrow that are associated with phenotypic differentiation and the acquisition of
mature phagocytic cell functions such as respiratory burst activity.3,4 Many of the growth factors that induce
proliferation of immature cells also modulate the function of the
mature end cells. Granulocyte-macrophage colony-stimulating factor
(GM-CSF), for instance, not only stimulates proliferation of neutrophil and monocyte progenitor cells, but also augments (primes) many of the
agonist-induced functions of the mature end-cells, such as phagocytosis
and killing of micro-organisms.5
Phospholipase A2 (PLA2) has a key role in
mature phagocytes in releasing arachidonic acid from cell membranes to
serve as substrate for the production of the eicosanoids and
prostanoids.6,7 These molecules are important in
host-defense and inflammation, because they regulate the migration and
activation of phagocytic cells. However, arachidonate itself is
important as a signal transduction molecule in raising intracellular
calcium levels,8,9 activating enzymes such as protein
kinase C,10 MAP kinase,11 and neutral sphingomyelinase12 and modulating GTP protein
function13 and gene expression,14 and might
also serve these functions in immature cells. The PLA2
enzymes form a superfamily comprising at least 9 groups,15
which can be divided into 3 mechanistic classes, the low-molecular
weight secretory group of PLA2 enzymes that require calcium
for catalytic activity (sPLA2)16; the 80-kD cytosolic PLA2 that does not require calcium for catalysis
but for translocation to the location of its substrate in cellular membranes (cPLA2)17,18; and the
calcium-independent enzymes (iPLA2).19 The
expression of the different PLA2 isoforms varies according
to cell type, and some cells express multiple isoforms, such as
macrophages that express group II sPLA2, group IV
cPLA2, and group VI iPLA2.20
Stimulated PLA2 activity in phagocytes can be increased by
short-term incubation with inflammatory mediators such as tumor necrosis factor  (TNF)21 and GM-CSF22-24
during the process known as cell priming that is not dependent on new
protein synthesis. At present, it is unclear which PLA2
isoforms are activated during cytokine-mediated priming of arachidonate
release, although it was recently reported that both group IV
cPLA2 and group II sPLA2 are activated during
the TNF-mediated priming of neutrophils.21 The mechanism
of cytokine-mediated regulation of the different PLA2
enzymes by cytokines has not been fully elucidated. Activation of
cPLA2 is associated with translocation of cPLA2
to cell membranes in response to an increase in intracellular calcium
and is mediated by its CalB/C2 domain25,26; however, many
growth factors do not stimulate a calcium flux. Serine phosphorylation
is reported to be important for activating cPLA2
activity,27 and several serine kinases that are activated
by growth factors may be involved, including protein kinase
C,28 mitogen-activated protein kinase (p42/Erk-2 MAP
kinase),29,30 and stress-activated protein kinase (p38).31 Ser-505 in the cPLA2 sequence is a
major phosphorylation site29 and lies within an MAPK
consensus sequence. However, inhibition of p38 kinase was shown to only
partially inhibit the phosphorylation of both Ser-505 and Ser-727;
therefore, another kinase may be involved downstream of
p38.31
Many of the studies of primitive myeloid cells and the differentiation
process have, in the past, been restricted to growth factor-independent
leukemic cell lines because of the unavailability of adequate numbers
of the rare primary cells. Cell lines such as HL-60 and U-937 express
cPLA2 protein,32,33 which is active in in vitro
assays, yet intact cells release barely detectable amounts of AA when
stimulated via functional receptors or receptor-independent pathways.32,34-36 During in vitro differentiation of HL-60
cells, the capacity to release AA after stimulation increases
concomitantly with the capacity to synthesize
eicosanoids,37,38 and this is in accord with a pivatol role
for AA production in the effector functions of mature phagocytic cells.
However, it must be borne in mind that these cell lines are highly
abnormal, eg, both HL-60 and U937 cells are completely
factor-independent for proliferation.
There is conflicting evidence that leukotrienes can stimulate the
proliferation of immature myeloid cells,39-42 and it is
unclear whether primary immature cells have functional PLA2
and are able to synthesize eicosanoids. A prelimary study reports the
presence of cPLA2, cyclooxygenase, and 5-lipoxygenase mRNA
in CD34 antigen-positive (CD34+) cells mobilized into the
peripheral blood43; however, another study found no
cPLA2 mRNA in CD34+ cells derived from cord
blood.44 Advances in the ability to mobilize human
hematopoietic progenitor/precursor cells from the marrow into the
blood45,46 and in the techniques used to collect and
isolate reasonably pure primitive cell populations on the basis of CD34
antigen expression47 now make it more readily possible to
study such primary cells. Freshly purified peripheral blood
CD34+ cells are in a quiescent G0 stage of the cell
cycle48,49 but can be induced to cycle in vitro by a range
of growth factors.49,50 With continued culture in the
presence of appropriate factors, these cells then lose expression of
the CD34+ antigen and begin to acquire the appearance and
functional properties of mature phagocytes.3 The aim of
this study was to determine whether PLA2 activity is
present in very primitive myeloid cells, and if so, to determine its
cytokine dependence.
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MATERIALS AND METHODS |
Growth factors, agonists, and inhibitors.
Stock solutions of recombinant human GM-CSF (rhGM-CSF; expressed in
Escherichia coli; Hoechst UK/Behringwerke, Marburg, Germany), recombinant human stem cell factor (rhSCF; expressed in E
coli), recombinant human interleukin-3 (rhIL-3), and rhIL-6 (all
from R&D Systems, Europe, Abingdon, Oxfordshire, UK) were prepared in
sterile phosphate-buffered saline (PBS; pH 7.4) containing 1%
(vol/vol) fetal calf serum (FCS) and stored at  20°C. MK886 was a gift from Merck-Frosst Canada Inc (Pointe Claire-Dorval, Quebec,
Canada). A 100 µmol/L stock solution in dimethyl sulphoxide (DMSO)
was prepared immediately before use. 12-O-tetradecanoylphorbol 13-acetate (TPA), calcium ionophore (A23187), and indomethacin were
from Sigma Chemical Co (Poole, Dorset, UK). Stock solutions of A23187,
prepared in DMSO at 5 mg/mL and stored at 20°C, were diluted
to 100 µmol/L in PBS immediately before the experiment. A stock
solution of PD 098059 (Alexis Corp [UK] Ltd, Nottingham, UK) at 30 mmol/L DMSO was stored at 20°C and diluted 1,000-fold into
final reaction mixtures. Methyl arachidonyl fluorophosphonate (MAFP)
was supplied in solution in methyl acetate (Cayman Chemical, Ann Arbor,
MI). The solvent was evaporated under nitrogen and MAFP was
reconstituted with DMSO at 50 mmol/L and stored at 80°C. It
was further diluted with DMSO on the day of the assay.
Purification of CD34+ progenitor cells mobilized into
the peripheral blood.
CD34+ peripheral blood progenitor cells (PBPC)
were purified from 26 patients (4 with Hodgkin's disease, 10 with
non-Hodgkin's lymphoma, and 12 with myeloma) and 10 healthy normal
donors after obtaining informed consent. Progenitor cells were first
mobilized from the bone marrow of patients with either cyclophosphamide (1.5 g/m2) followed by daily granulocyte colony-stimulating
factor (G-CSF; 10 mg/kg/d filgrastim or 263 µg/d lenograstim) for 10 days or an ESHAP regime followed by G-CSF (lenograstim) as described
before.47 Normal donors received G-CSF alone. White blood
cells were isolated from the peripheral blood by apheresis and
CD34+ cells were purified either on a CEPRATE SC
immunoaffinity column (Cell Pro Inc, Bothwell, WA) using a biotinylated
mouse-antihuman CD34 monoclonal antibody and avadin-coated
beads47 or with an AmCell Clinimacs device and
anti-CD34+ antibody-coated microbeads (Miltenyi Biotech
Ltd, Bisley, Surrey, UK). Progenitor cell purity was assessed by flow
cytometry using an anti-CD34 antibody HPCA (Becton Dickinson, Franklin
Lakes, NY) and by morphological analysis.51 The purity of
CD34+ cells after affinity purification on CEPRATE columns
was 82% ± 10% (mean ± 1 SD, n = 19) with 2% ± 1%
recognizable neutrophil precursors, but no mature neutrophils were
seen. The major cell contaminants were lymphocytes (6% ± 2%) and
monocytes (10% ± 2%), which were removed, respectively, with
anti-CD3 and CD19 magnetic beads according to the manufacturer's
instructions (Dynal, Bromborough, UK) or by adherence to plastic. The
purity of CD34+ cells after Clinimacs separation was 98%,
with less than 2% contamination with lymphocytes. Contamination with
monocytes of the final cell suspension used for experimentation was 5% ± 1% for CEPRATE-separated cells and 0.8% ± 0.1% for
Clinimacs cells, and the respective viabilities of these cells were
93% ± 3% and 94% ± 2%. CD34+ cells were used
for experimentation when the number obtained exceeded that required for
rapid and sustained hematological engraftment.47 Mobilized
CD34+ cells were either cultured overnight in Iscove's
Minimal Essential Medium (IMEM; GIBCO-BRL, Paisley, UK) supplemented
with 20% (vol/vol) FCS (GIBCO-BRL) in a humidified atmosphere of 5%
carbon dioxide in air (day 1 cells) or for up to 8 days in IMEM/20%
FCS supplemented with rhSCF, IL-3, and IL-6 (all at 10 ng/mL) and
antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Before
analysis of cellular responses to cytokines, cells were harvested from
the culture medium by centrifugation, followed by 3 large volume washes to remove ambient growth factors, and incubated for 18 hours in IMEM/20% FCS to allow re-expression of surface receptors. The morphological maturity of cultured cells was assessed from
cytocentrifuge preparations stained with Leishman's stain. Functional
maturity was assessed by the nitroblue tetrazolium (NBT) reduction test of cells stimulated with TPA (1 µg/mL), as previously
described.35
Purification of neutrophils and monocytes.
Neutrophils were purified from peripheral blood anticoagulated with 2 mmol/L EDTA (pH 7.4), by dextran sedimentation, by centrifugation through Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden), and by
hypotonic lysis as described previously,52 using sterile preparations and procedures to minimize contact of cells with endotoxin
and reduce inadvertant priming. Platelet-free preparations of monocytes
were obtained by centrifugation of whole blood through 12%
Ficoll-Paque/Dulbecco's PBS without calcium and magnesium (PBS ; GIBCO-BRL). The platelet-rich supernatant was
removed and the platelet-depleted blood was layered over a single
cushion of Ficoll-Paque and centrifuged as described before. T and B
lymphocytes were removed from the monocytes using magnetic beads coated
with CD19 and CD3 according to the manufacturers' instructions
(Dynal). Purified cells were resuspended in PBS supplemented with 0.9 mmol/L calcium and 0.5 mmol/L magnesium (PBS+) and 5 mmol/L glucose.
Cell lines.
The HL-6053 and TF-1 myeloid cell lines54 were
grown in Roswell Park Memorial Institute (RPMI) medium (GIBCO-BRL)
supplemented with 10% FCS at concentrations not exceeding 5 × 105/mL. Cultures of TF-1 cells were additionally
supplemented with 5 ng/mL GM-CSF every 2 days. Cells were harvested and
resupended in PBS+ supplemented with 10 mmol/L glucose.
Before analysis of responses to cytokines, the cells were removed from
medium containing growth factor and incubated for 18 hours to allow
re-expression of GM-CSF receptors.
3H-Arachidonate release from intact cells.
Cells were incubated with [5,6,8,9,11,12,14,15-3H]AA
(specific activity, 7.33 TBq/mmol; 202 Ci/mmol; Amersham
International, Amersham, Buckinghamshire, UK) at a final concentration
of 0.5 µCi/mL for 2 hours with occasional mixing. Purified
neutrophils (5 × 106/mL PBS+/5 mmol/L
glucose/0.1% vol/vol FCS) were incubated at room temperature. Mobilized CD34+ cells (2.5 × 106/mL 50%
IMEM/50% PBS/10 mmol/L glucose/0.1% vol/vol FCS) and TF-1 cells (2.5 × 106/mL 50% RPMI/50% PBS+/10 mmol/L
glucose/0.1% vol/vol FCS) were incubated at 37°C for 1 hour
followed by 1 hour at room temperature. The radiolabeled cells were
centrifuged (300g for 7 minutes) and the supernatants were
removed. The cell pellets were washed a further 3 times in PBS+G and finally were resuspended at 5 × 105 cells/mL PBS+G for CD34+ cells
and at 2 × 106 cells/mL for all other cell types.
Aliquots (0.5 mL) of these cell suspensions were equilibrated to
37°C. Neutrophil samples were incubated at this stage for 5 minutes
with 200 nmol/L MK886 to inhibit the conversion of AA to leukotrienes
via the action of 5-lipoxygenase.55 MK886 was not added to
samples of immature cells, because pilot experiments showed that these
cells did not release leukotrienes. All samples were then incubated
with the following cytokines and growth factors for 10 minutes: TNF
(500 U/mL), IL-3 or GM-CSF (both at 10 ng/mL), SCF (100 ng/mL), or growth factor diluent (0.01% FCS; final concentrations); or with TPA
(500 ng/mL) or DMSO diluent (0.01%). Replicate samples were then
stimulated for 20 minutes with or without 1 µmol/L calcium ionophore,
A23187, in the presence of fatty acid-free bovine serum albumin (BSA;
final concentration, 1 mg/mL), which was added to trap the AA that was
released and to prevent re-esterification. The reaction was terminated
by placing the samples on ice. The samples were then centrifuged
(12,000g for 4 minutes) and 0.4-mL aliquots of the supernatants
were assayed for radioactivity by liquid scintillation spectroscopy.
Duplicate samples containing 0.4 mL of the initial radiolabeled cell
suspension were used to estimate the total amount of incorporated
radioactivity, and arachidonate release was expressed as a percentage
of this.
Measurement of AA release from CD34+ cells by thin-layer
chromatography (TLC).
Freshly purified PBPC were incubated at 2.5 × 105/mL
for 24 hours with IL-3, IL-6, and SCF, followed by removal of growth
factors and overnight starvation as described above. On day 2, cells
were harvested from culture, resuspended at 2.5 × 106/mL of 50% IMEM/50% PBS/10 mmol/L glucose/0.1%
vol/vol FCS, and radiolabeled with 3H-AA (1 µCi/mL) as
described above, followed by washing to remove unincorporated isotope.
PBPC were resuspended in PBS+/10 mmol/L glucose in two 1-mL
aliquots of 5.6 × 106 cells and equilibrated to
37°C. MAFP,56 the dual inhibitor of c and
iPLA2 isoforms,19 was added at a final
concentration of 5 µmol/L to one aliquot and DMSO diluent (0.1%
vol/vol) was added to the replicate sample for 15 minutes, followed by
100 ng/mL SCF to both samples for 10 minutes. Both samples were
stimulated with 1 µmol/L calcium ionophore in the presence of 2 mg/mL
BSA for 30 minutes. The samples were chilled on ice and then
centrifuged (12,000g for 4 minutes). The supernatant was taken
into glass centrifuge tubes (15 × 100 mm) and 100 µg AA was
added to act as carrier. Lipids were extracted by the addition of 3 mL
of ice-cold chloroform:methanol: 1/10 formic acid (1:2:0.15). The
samples were vortex mixed and left on ice for 15 minutes. One
milliliter of ice-cold distilled water and 2 mL of chloroform were
added to achieve phase separation. The samples were vortex-mixed, left on ice for 15 minutes, and then centrifuged at 1,800g for 10 minutes at 4°C. Samples were taken from the aqueous and organic
phases as well as the protein interface to determine their
radioactivity. The organic layer was dried in a vacuum evaporator and
then taken up in 10 mL chloroform:methanol, 9:1. Lipids were separated
by TLC on silica gel 60 plates (Merck, Darmstadt, Germany) using as the
mobile phase, the upper phase from a mixture of ethyl
acetate:iso-octane (2,2,4-trimethylpentane):water:acetic acid
(4.5:2.5:5:1) as described previously.32 A standard
composed of a mixture of 3H-AA and nonlabeled AA (100 µg)
was extracted and run on the TLC plate in parallel with the cell
samples. The lanes on the TLC plate were marked with a pencil and
divided into 16 × 1 cm fractions starting from the origin. The
fractions were scraped from the plate, and their radioactivity was
determined by  -scintillation spectroscopy. Daily measurements of the
radioactivity in the scintillation vials showed that at least 3 days
were required before the radioactive lipids had eluted fully from the silica.
Measurement of leukotriene production.
Leukotriene B4 (LTB4), LTC4,
LTD4, and LTE4 were detemined by
enzymeimmunoassay using Biotrak kits, RPN 223 (LTB4) and
RPN 224 (LTC4, LTD4, and LTE4;
Amersham International) following the manufacturer's instructions.
LTB4 production by neutrophils stimulated with 1 µmol/L
A23187 and peptidoleukotriene production by monocytes also stimulated
with A23187 were used as positive controls. Eicosanoid production was
also measured by difference spectroscopy of supernatants from
107 cells, using a dual-beam spectrophotometer (Unicam UV2
spectrometer; ATI Unicam, Cambridge, UK) with matched quartz cuvettes.
The concentration of LTB4 was calculated from the height of
the 270 nm peak (absorption maximum), taking the extinction coefficient
of LTB4 as E270 nm = 50 mmol1 cm1. Confirmation of the
absorbance spectrum of LTB4 was by comparison with
authentic LTB4 and its omega oxidation metabolites as well as the peptidoleukotrienes, LTC4, LTD4, and
LTE4 (Cascade Biochem Ltd, Reading, Berkshire, UK).
Measurement of MAP kinase (p42/Erk2) activation by gel retardation
assay.
Cells (1.3 × 107/mL PBS+G) were stimulated with
either cytokine diluent (0.01%), SCF (100 ng/mL), GM-CSF (10 ng/mL),
or TPA (500 ng/mL). At timed intervals, 30-µL samples (5 × 105 cells) were taken into 30 µL lysis buffer
supplemented with protease inhibitors (20 µg/mL leupeptin, 20 µg/mL
pepstatin, 20 µg/mL aprotinin, 1 µmol/L
phenylmethylsulfonylfluoride, and 1 mmol/L diisopropylfluorophosphate), phosphatase inhibitors (50 mmol/L sodium orthovanadate, 1 mmol/L glycerophosphate, 5 mmol/L pyrophosphate, and 50 mmol/L
sodium fluoride), and 2 mmol/L n-ethylmaleimide. Sixty microliters of boiling Laemmli sample buffer was added and the samples were heated at
100°C for 3 minutes. Proteins from 1 to 2 × 105
cells were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE; 15% acrylamide:0.075% bisacrylamide) and
immunoblotted first with anti-MAP kinase antibody (0.2 µg/mL, Erk-2
C-14; Santa Cruz Biotechnology, Santa Cruz, CA) and second with a
peroxidase-conjugated goat-antirabbit antibody (at 1:2,000 dilution;
Dako Ltd, High Wycombe, Buckinghamshire, UK), followed by enhanced
chemiluminescence and autoradiography for detection (Amersham International).
Data analysis.
Unless otherwise stated, the data are the mean ± 1 SE of the number
of experiments given in the text.
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RESULTS |
Detection of PLA2 activity in undifferentiated
hematopoietic cells.
PLA2 activity was quantitated by measuring the release of
radioactivity into the supernatant from cells that had been
radiolabeled with 3H-arachidonic acid (AA) and then
stimulated with either 500 ng/mL TPA or 1 µmol/L A23187 or by the
sequential addition of these 2 agonists. The radioactivity released was
then expressed as a percentage of total cell radioactivity to control
for any differences in the amount of 3H-AA incorporated by
the different cell types. Initial studies performed on cells from the
immature erythroleukemia cell line, TF-1, showed levels of arachidonate
release in these primitive cells that were comparable to that seen in
mature neutrophils, whereas promyelocytic HL-60 cells released very
little arachidonate under any condition
(Table 1). Further studies were then
performed in primary human hematopoietic cells, namely purified
CD34+ cells, mobilized into the circulation by a
combination of chemotherapy and G-CSF.47 The levels of
arachidonate release were again comparable with that seen in mature
neutrophils and monocytes (Table 1).
Regulation of PLA2 activity by growth factors in
undifferentiated hematopoietic cells.
We and others have previously shown that PLA2 activity in
mature phagocytes stimulated with either receptor-dependent agonists or
receptor-independent stimuli such as A23187 is primed by short-term incubation (10 to 20 minutes) with growth factors such as
GM-CSF.22-24 We therefore explored the effect on AA release
of prior exposure to various growth factors in the TF-1 cell line and
primary CD34+ cells, in comparison with purified
neutrophils from peripheral blood. The effect of preincubation with
optimal concentrations of the growth factors, GM-CSF (10 ng/mL), IL-3
(10 ng/mL), TNF (500 U/mL), and SCF (100 ng/mL) on
ionophore-stimulated AA release was measured. To compare the magnitude
of cytokine-mediated priming between cell types, arachidonate release
in resting cells (no A23187 stimulation) was subtracted from the
A23187-stimulated value and the data for cytokine-primed cells were
then expressed as a percentage of the respective FCS diluent control.
As shown in Fig 1, each cell type had a
different pattern of response to the cytokines. In the primary
CD34+ cells (Fig 1A), SCF was the major priming agent of AA
release and IL-3 had a weak but statistically significant effect,
whereas neither GM-CSF nor TNF did so. The CD34+ cells
used in this study were from patients who had received both
chemotherapy and G-CSF during the mobilization procedure as well as
normal donors who received G-CSF alone. No statistically significant
differences were seen in the CD34+ cell responses to
cytokines between patients and normal donors (data not shown). In
cultures of CD34+ TF-1 cells (Fig 1B), AA release was
equally primed by GM-CSF and IL-3, whereas SCF had a small but
significant effect and TNF had no significant effect. In neutrophils
(Fig 1C), GM-CSF had the greatest priming effect and TNF had a minor
but statistically significant effect, whereas neither IL-3 nor SCF had
any significant effect, which may reflect the low expression of IL-3
and SCF receptors on these cells. Monocytes purified from peripheral
blood were also tested, because these cells were present as very minor
contaminants (2% ± 1%, n = 9) in the CD34+ cell
preparations used for the experiments in Fig 1. SCF failed to affect AA
release in monocytes, with the priming being 99% ± 19% of the FCS
control, whereas GM-CSF-mediated priming was 140% ± 22% of
control (P = .03, n = 10).
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| Fig 1.
Effect of growth factors on A23187-stimulated AA release
in CD34+ cells compared with other cell types.
Suspensions of (A) day 1 CD34+ cells, (B) TF-1 cells, and
(C) neutrophils were incubated for 10 minutes with either growth factor
diluent (0.01% vol/vol FCS), GM-CSF (10 ng/mL), IL-3 (10 ng/mL),
TNF (500 U/mL), or SCF (100 ng/mL) and then stimulated with 1 µmol/L A23187. Arachidonate release was measured as described in
Materials and Methods and expressed as a percentage of growth factor
diluent control. The number of replicate experiments performed are
indicated in the figure. The statistical significance of the
differences between cytokine- and diluent-mediated priming are shown
(*.05 > P > .01; **.01 > P > .001;
***.001 > P; Student's paired t-test). The absolute
values of the background release of AA was 4.6% ± 0.4%, 3.1% ± 0.3%, and 2.3% ± 0.2% of total cell radioactivity for
CD34+ cells, TF-1 cells, and neutrophils, respectively.
Similarly, A23187-stimulated AA release was 7.2% ± 0.8%, 5.6% ± 0.6%, and 6.9% ± 1.1% in the FCS-primed samples.
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The effect of in vitro culture with IL-3, IL-6, and SCF on
arachidonate release from primary CD34+ cells.
Freshly purified CD34+ cells were grown for up to 8 days in
medium supplemented with a cytokine cocktail (IL-3, IL-6, and SCF, all
at 10 ng/mL), which induced proliferation and partial differentiation, as was previously reported.50,57 As shown in
Fig 2B, morphological maturation was
apparent at day 8, and a proportion of cells acquired the functional
characteristics of fully mature cells (11% ± 2% NBT-positive
cells on day 8 [n = 8]; 2% ± 1% NBT positive on day 1 [n = 12]).
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| Fig 2.
Regulation of PLA2 activity during
differentiation of CD34+ cells. (A) Freshly purified
CD34+ cells were incubated for up to 8 days with IL-3,
IL-6, and SCF (all at 10 ng/mL). After 18 hours of incubation without
growth factors, cells were primed for 10 minutes with either TNF
(500 U/mL), IL-3 (10 ng/mL), GM-CSF (10 ng/mL), SCF (100 ng/mL), or
diluent (0.01% vol/vol FCS), followed by activation with 1 µmol/L
A23187 for 20 minutes, and arachidonate release was measured. Basal
release in unstimulated samples was subtracted from the
A23187-stimulated values and the data are expressed as a percentage of
total cell radioactivity. The data shown are the mean ± 1 SE of 3 to
8 experiments. The statistical significance of the differences between
cytokine and diluent-treated cells at each time interval are given
(Student's paired t-test). (B) Morphological analysis of
cytospin preparations from the cultures as used in (A).
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Samples were taken for analysis of AA release on days 1, 2, and 8. Before analysis, cells were washed free of growth factor and incubated
for 18 hours in growth factor-free medium to allow the re-expression of
receptors that may have been downregulated during culture. Thus, the
cells analyzed on day 1 were not exposed to the cytokine cocktail.
3H-AA release from radiolabeled cells was measured as
described above in cells preincubated for 10 minutes with either TPA or growth factors before stimulation with A23187. Basal AA release from
cells stimulated with PBS instead of A23187 was measured in parallel
samples and these values were subtracted from the A23187-stimulated
values to give the increment in AA release due to A23187.
Maximal stimulation of AA release by TPA + A23187 decreased during the
culture period, with levels of AA release being 36% ± 4%, 25% ± 5%, and 23% ± 1% of cell radioactivity on days 1, 2, and
8, respectively (the significance of difference between day 1 and
either day 2 or 8 was P = .014 [n = 6]; Wilcoxon's signed rank test). The differential effects of acute stimulation with growth
factors on calcium-stimulated AA release is shown in Fig 2A. The
release of AA from A23187-stimulated cells that were preincubated with
growth factor diluent (FCS) remained relatively constant over the
culture period. The effect of SCF declined concomitantly with the
reduction in the proportion of cells with a blast-like morphology in
the cultures (Fig 2B), and by day 8 no significant SCF responses were
seen. In contrast, the priming effect of GM-CSF and IL-3 on
A23187-stimulated PLA2 activity increased during the culture period to levels that were significantly greater than those
seen with diluent. The increase in GM-CSF and IL-3 responses coincided
with an increase in the proportion of more mature cells (promyelocytes,
myelocytes, and metamyelocytes constituted about 60% of cells on day
8). TNF had little effect on AA release at any stage of culture.
Analysis by TLC of tritiated products released from SCF-primed
CD34+ cells.
In 3 experiments, primary CD34+ cells were labeled with
3H-AA and incubated with SCF, followed by stimulation with
ionophore as described above, and the tritiated lipids released into
the supernatant were analyzed by TLC (see Materials and Methods). It
was not possible to analyze AA release using mass measurements because
of the limited number of primary cells available. The distribution of
radiolabel in the different phases of the lipid extraction was 91% ± 1% (mean ± 1 SD) in the organic phase, 9% ± 1% in the
aqueous phase, and 1% ± 1% in the BSA protein layer. The recovery
of radioactivity from the cell supernatants in the organic phase was
97% ± 5%, and this confirmed the efficiency of the extraction
procedure. A sample of the 3H-AA that was used for cell
labeling was also extracted and subject to TLC in parallel with the
cell supernatants. Figure 3 shows that the
profile of the radioactive lipids released from cells primed with SCF
and stimulated with A23187 was similar to the 3H-AA
standard. These data demonstrated that AA was the predominant lipid
released from SCF-stimulated CD34+ cells and validated the
radiometric AA release assay.
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| Fig 3.
Analysis of arachidonate release by TLC. Day-1
CD34+ cells labeled with 3H-arachidonic acid
were incubated with either 5 µmol/L MAFP or DMSO diluent before
priming for 10 minutes with 100 ng/mL SCF, followed by activation with
1 µmol/L A23187 for 20 minutes. The lipids were extracted, spotted
onto the origin (0) of the TLC plate, and separated as described in
Materials and Methods. The distribution of radioactivity in 1-cm
fractions scraped from the plate is shown. The data shown are from a
single experiment representative of 3 that were performed.
|
|
Studies with the PLA2 inhibitor, MAFP.
To confirm that the arachidonate released from primitive myeloid cells
was attributable to PLA2 activity, studies were performed with MAFP, which acts as a transition state analogue at the active site
of PLA2 and is a dual inhibitor of both cPLA2
and iPLA2.19 Primary CD34+ cells
were preincubated with 5 µmol/L MAFP or DMSO diluent for 10 minutes,
and AA release in response to priming with TPA, SCF, and GM-CSF,
followed by stimulation with A23187, was measured with the radiometric
assay. The data from 3 experiments are shown in
Fig 4. In control samples incubated with
DMSO diluent, TPA and SCF increased the cells' responses to A23187
above that seen with growth factor diluent controls, whereas GM-CSF had
no enhancing effect, which confirms the selective effect of SCF in
priming AA release shown in Figs 1 and 2. Preincubation with 5 µmol/L MAFP inhibited AA release from the CD34+ cells,
irrespective of the priming agent used. The arachidonate released from
the primary CD34+ cells that were incubated with and
without 5 µmol/L MAFP, followed by priming with SCF and stimulation
with ionophore, was analyzed by TLC as described above. The data
shown in Fig 3 confirm that MAFP inhibited AA release to near basal
levels. MAFP at 5 µmol/L also inhibited A23187-stimulated AA release
from CD34+ cells that had been cultured for 8 days with
IL-3, IL-6, and SCF (94% inhibition compared with DMSO control; single
experiment) as well as AA release from peripheral blood neutrophils
(98% and 92% inhibition compared with DMSO control; data from 2 separate experiments). There is always concern that the effects of an
inhibitor represent nonspecific toxicity to the cell, but it should be
noted that 5 µmol/L MAFP had no inhibitory effect on TPA- or
SCF-mediated MAP kinase (ERK2) activity in CD34+ cells (see
below, Fig 6A). The data shown in Figs 3 and 4 provide evidence for
PLA2 activity being the major enzyme responsible for
cytokine-primed AA release.
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| Fig 4.
The effect of MAFP on arachidonic acid release. Day-2
cultured CD34+ cells were incubated with either 5 µmol/L MAFP or DMSO diluent for 15 minutes, followed by priming with
either 0.01% FCS diluent, 100 ng/mL SCF, or 500 ng/mL TPA, and
activated with either 1 µmol/L A23187 or PBS. The data shown are the
mean ± 1 SE of 3 experiments. The significance of the difference
between MAFP- and DMSO-treated samples is shown (Student's paired
t-test).
|
|
Investigation of eicosanoid release by primary CD34+
cells and their in vitro progeny.
Experiments were performed to determine whether the arachidonate
produced by the CD34+ cells was metabolised by
5-lipoxygenase or cyclo-oxygenase. In initial experiments, cells
radiolabeled with 3H-AA were preincubated with either DMSO
diluent or 200 nmol/L MK886 (a specific inhibitor of
5-lipoxygenase)55 or 20 µmol/L indomethacin to inhibit
cyclo-oxygenase, before stimulation with A23187. The radioactivity
released into the extracellular medium was counted. No delipidated
albumin was added to the reaction mixtures (such as was used in the
assays of arachidonate release), because this would trap arachidonate
and prevent its metabolism to eicosanoids. In these experiments, the
radioactivity released from TF-1 cells or freshly purified
CD34+ cells after stimulation with A23187 remained at basal
levels, and no effect of MK886 or indomethacin was seen. In contrast, there was significant radioactive release from neutrophils stimulated with A23187 (11.7% ± 0.9% of total cell radioactivity, n = 17), which was inhibited to basal levels by preincubation with MK886 (1.1% ± 0.2%). In 2 experiments with monocytes, A23187 stimulated the
release of 10.3% and 10.4% of cell radioactivity and this was
inhibited to 5.1% and 5.4%, respectively, by indomethacin. These data
suggested that there was little lipoxygenase or cyclo-oxygenase activity present in the primitive cells.
The data from the radiometric assays was confirmed by
enzymeimmunoassays of nonradiolabeled cells. In 3 experiments, the
amount of LTB4 released from day 1 CD34+ cells
was 8 ± 2, 6 ± 1, and 6 ± 2 pg/5 × 105
cells when these were stimulated with diluent, 100 ng/mL SCF, or SCF + 1 µmol/L A23187, respectively. Similar data were obtained with TF-1
cells. In comparison, neutrophils stimulated with A23187 produced
approximately 2,000 times more LTB4 (12.3 ng/5 × 105 cells). Production of the peptidoleukotrienes
LTC4, LTD4, and LTE4 in
CD34+ cells stimulated as described before with either SCF
or SCF + A23187 was less than 1 pg/5 × 105 cells,
whereas monocytes stimulated with A23187 produced more than 1.2 ng/5 × 105 cells. As a further confirmation of these data,
eicosanoid production by neutrophils primed with GM-CSF and by day 1 CD34+ cells primed with SCF was analyzed by difference
spectroscopy. In these experiments, the absorption spectrum of
supernatants from cells stimulated with A23187 was measured after
subtraction of the spectrum of supernatants from unstimulated cells.
Figure 5 (trace 1) shows the characteristic
absoption spectrum of LTB4 produced by neutrophils after
stimulation with A23187, with peaks at 260, 270, and 280 nm. In 4 experiments, the mean production of LTB4 and its 20 omega
hydroxy- and carboxy-metabolites by neutrophils was determined from its
extinction coefficient at 270 nm and was 24.6 ± 2.6 ng/106 cells, which is in close agreement with the
concentration of LTB4 measured by the enzyme immunoassays.
The absoption spectrum of LTB4 was not visible in trace 2 when neutrophils had been preincubated with 5-lipoxygenase inhibitor,
MK886. In contrast, no leukotriene peaks were seen in SCF-primed
CD34+ cells stimulated with A23187 (trace 3) or
unstimulated control cells (trace 4).
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| Fig 5.
Analysis of eicosanoid production by difference
spectroscopy. Neutrophils (top panel) and day 1 CD34+
cells (bottom panel), both at 1 × 107 cells/mL, were
incubated, respectively, with 10 ng/mL GM-CSF or 100 ng/mL SCF,
followed by 1 µmol/L A23187 or PBS diluent as indicated in the inset,
and the absorption spectrum of the supernatants was measured in a
dual-beam spectrometer with the cuvettes arranged as indicated.
|
|
Further studies were then performed on primary CD34+ cells
that had been cultured for 8 days and were at an intermediate stage of
differentiation, at which time they had lost CD34 antigen expression and were beginning to acquire functional respiratory burst activity. However, no leukotriene production was detected in response to stimulation with A23187 (data not shown).
Activation of p42/Erk2 MAP kinase in CD34+ cells.
A possible mechanism by which growth factors can prime
cPLA2 activity is by phosphorylation and activation of MAP
kinase (ERK2) with subsequent phosphorylation of the cPLA2
isoform on serine 505, resulting in increased enzyme
activity.28,29 This has been previously suggested as a
mechanism of GM-CSF-mediated priming of PLA2 activity in
neutrophils.23 We therefore sought to determine whether
SCF, GM-CSF, and TPA could activate MAP kinase in primary CD34+ cells. Cells were stimulated with either 500 ng/mL
TPA, 10 ng/mL GM-CSF, or 100 ng/mL SCF, and activation
(phosphorylation) of p42/Erk2 MAP kinase was measured by gel
retardation assay and Western blotting. The results from 3 experiments
show that both TPA and SCF activated MAP kinase, but with different
kinetics (Fig 6). MAP kinase activation by
SCF and TPA was rapid, being apparent within 1 minute and maximal
within 2 to 3 minutes of stimulation (Fig 6A). However, responses to
SCF were transient and were virtually undetectable at 11 minutes
poststimulation (Fig 6B), whereas the responses to TPA persisted at
these time intervals. In contrast, no response to GM-CSF was apparent
in the day 1 CD34+ cells (Fig 6B), in confirmation of our
previous study.57 These data are consistent with the
ability of TPA and SCF and the inability of GM-CSF to prime
PLA2 activity in these primitive cells. However, GM-CSF
activated ERK2 in day-8 cultured cells (Fig
7A), as was previously reported.57
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| Fig 6.
Activation of p42/Erk2 MAP kinase in primary
CD34+ cells measured by gel retardation assay and Western
blotting (see Materials and Methods). (A) Cells were stimulated with
either cytokine diluent (0.001% FCS), SCF (100ng/mL), or TPA (500 ng/mL) for the times indicated. Replicate samples were preincubated
with either DMSO diluent or the PLA2 inhibitor MAFP (5 µmol/L) for 15 minutes before cytokine stimulation. (B) Cells were
incubated with cytokines as in (A) as well as GM-CSF (10 ng/mL) for the
times indicated, and Erk2 activation was measured as in (A).
|
|
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| Fig 7.
Effect of the MEK1 inhibitor, PD 098059, on
CD34+ cells. (A) Cells cultured for 8 days in IL-3, IL-6,
and SCF (day-8 cells) were incubated for 18 hours in the absence of
growth factors to allow re-expression of growth factor receptors. Cells
were then preincubated with 30 µmol/L PD 098059 or DMSO diluent for
30 minutes before stimulation with GM-CSF for 7 minutes, and p42/ERK2
activation was measured by gel retardation assay. Data from 2 separate
experiments are shown. (B) Effect of PD 098059 on PLA2
activity of primary CD34+ cells (day-1 cells, n = 5) or
day-8 cultured cells (n = 3). Cells were incubated for 30 minutes
with 30 µmol/L PD 098059 or DMSO diluent, and arachidonate release
was measured after 10 minutes of priming with 100 ng/mL SCF, 10 ng/mL
GM-CSF, or 500 ng/mL TPA as indicated, followed by activation with 1 µmol/L A23187 for 20 minutes. Basal release of arachidonate in
unstimulated samples was subtracted from the A23187-stimulated values,
and the data are expressed as the percentage of incorporated cellular
radioactivity. The statistical significance of the difference between
DMSO and PD-treated cells is shown: *.05 > P > .01; **.01 > P > .001 (Student's paired t-test).
|
|
Effect of the MEK1 kinase inhibitor, PD 098059, on PLA2
activity.
To test whether activation of MAP kinase was required for
PLA2 activity in CD34+ cells, we sought to
block MAP kinase activity and look at the effect on AA release. PD
098059 is a selective inhibitor of MAP kinase kinase (MEK1) and is
therefore an upstream inhibitor of MAP kinase (p42 ERK2).58
Figure 7A confirms that 30 µmol/L PD 098059 inhibited the activation
of ERK2. CD34+ cells were then incubated for 15 minutes
with either 30 µmol/L PD 098059 or DMSO diluent control, and
arachidonate release after priming with growth factors and activation
with ionophore was measured (see Materials and Methods). Figure 7B
shows that PD 098059 partially inhibited AA release in day 1 cells
primed with either growth factor diluent (to 84% ± 36% of no PD
control; n = 5), SCF (to 46% ± 5% of control; .01 > P > .001), or TPA (to 31% ± 10% of control; .01 > P > .001). Similarly, in 3 experiments, PD 098059 partially
inhibited AA release in day-8 cells primed by diluent (to 66% ± 8% of no PD control), GM-CSF (to 45% ± 8% of control; .01 > P > .001), and TPA (to 57% ± 8% of control).
| |
DISCUSSION |
This study has demonstrated that primitive CD34+
hematopoietic cells express PLA2 activity, which was
measured in intact cells by the extracellular release of arachidonate
using a radiometric assay and confirmed by TLC. The magnitude of
PLA2 activity in CD34+ cells stimulated by
calcium ionophore was similar to that of peripheral blood neutrophils
and monocytes. This degree of PLA2 activity in
hematopoietic progenitor cells was relatively unexpected, because
immature cells from the promyelocytic HL-60 line only reach their full
capacity to release AA when they are stimulated to
differentiate.34,37 It should be borne in mind that the CD34+ cells had been mobilized into the peripheral blood by
in vivo G-CSF, and this may have altered the PLA2 activity
of the cells in some way. However, in confirmation of the data with
primary CD34+ cells, we found that primitive
CD34+ TF-1 cells also express levels of PLA2
activity that are comparable with those of mature cells.
Regulation of AA release by myeloid cell-specific growth factors such
as GM-CSF has been demonstrated in mature human phagocytic cells.5,22-24 In the current study, we found that
A23187-stimulated AA release from freshly purified CD34+
cells was selectively enhanced by preincubation with SCF and to a minor
degree by IL-3, but not significantly by GM-CSF or TNF. This
differential activation by SCF was confirmed in experiments in which
SCF was able to activate p42 MAP kinase, whereas no responses to GM-CSF
were seen. The activation of CD34+ cells by SCF at this
early stage of differentiation is consistent with SCF being an early
acting factor for hematopoiesis.60 Preliminary experiments
with other early acting factors such as Flt-3 ligand and thrombopoietin
failed to detect enhancement of PLA2 activity, suggesting
that SCF has a specific role in this respect PLA2 (data not
shown). At the present time, the difference in the ability of Flt-3 and
SCF to mobilize AA is not understood. Both bind to receptors that are
members of the tyrosine kinase-containing family of receptors, but they
may have more subtle differences in signal transduction pathways; this
is an area for future work.
It was previously shown that differentiation of myeloid
CD34+ cells can be achieved by in vitro culture with IL-3,
IL-6, and SCF, resulting in both morphological and partial functional
maturation,57 and we therefore investigated whether changes
in PLA2 activity also occurred under these conditions.
There was a minor decrease over time in the responses to TPA + A23187
(which elicit maximal PLA2 responses in intact
cells),59 which contrasts with the significant increase in
PLA2 activity during HL-60 cell differentiation that was
previously reported.34,37 When CD34+ cells were
induced to differentiate, the pattern of responsiveness of
PLA2 to growth factors also changed, in that
A23187-stimulated PLA2 activity ceased to be enhanced by
SCF, but was able to be upregulated by both IL-3 and GM-CSF, whereas
the lack of response to TNF remained unchanged. The
differentiation-dependent changes in factor-enhanced PLA2
activity may reflect either a change in receptor expression or changes
in signaling pathways. The responsiveness to SCF during the first 2 days of culture is in accord with the report that 80% of progenitor
cells freshly purified from peripheral blood express c-kit, the
receptor for SCF,61 and the reduction in PLA2
responses to SCF that we saw after 8 days in culture is in accord with
the reported gradual loss of SCF receptors from progenitor cells
induced to differentiate down the granulocytic pathway.61-63 Morphological analysis of the cultures
confirmed that the loss of SCF responsiveness was associated with the
reduction in the proportion of blast cells and development of
promyelocytes, myelocytes, and metamyelocytes. In contrast, we recently
reported that freshly purified CD34+ cells express very few
high-affinity GM-CSF receptors, but these increased in number after
culture in SCF, IL-3, and IL-6.57 However, the limiting
factor for the acquisition of PLA2 responses to GM-CSF by
cultured cells may not reside at the level of receptor expression,
because our previous study also showed that there was a
differentiation-linked increase in GM-CSF-mediated signaling down the
JAK2, ERK2, and STAT5 transduction pathways that reached maximal levels
well before there was any increase in the number of high-affinity
GM-CSF receptors.57
At this stage, we do not know which isoform of PLA2
mediates the capacity for AA release in CD34+ cells. AA
release was enhanced by an increase in intracellular calcium stimulated
by the calcium ionophore, A23187, and this was further increased by
preincubation of the cells with the phorbol ester, TPA. These are
characteristics shared by the 80- to 110-kD cytosolic isoform of
PLA2,59 whose presence in mobilized peripheral blood CD34+ cells was recently detected at the mRNA level
by RT-PCR.43 However, this group recently reported that the
iPLA2 isoform was also expressed in stem
cells.64 We also showed that the PLA2 activity
could be enhanced by growth factors, which is again characteristic of
cPLA2. SCF was previously shown to stimulate
cPLA2 phosphorylation and arachidonate release within 10 minutes in mouse bone marrow-derived mast cells65 as well
as increasing the expression of cPLA2 at the protein and
mRNA level over a longer time course.66 Our experiments
with MAFP, which is reported to be a dual inhibitor of both
cPLA2 and iPLA2,19 show inhibition
of both unprimed and growth factor-primed AA release and suggest that
either of these PLA2 isoforms are candidates. When primary
CD34+ cells were incubated with the MEK1 inhibitor, PD
098059,58 PLA2 activity, whether in SCF-primed
or unprimed cells, was partially inhibited (to 30% to 50% of control
responses), suggesting the involvement of MEK1 and thus most probably
ERK2 in PLA2 activation. This evidence increases the
likelihood that the isoform in CD34+ cells is
cPLA2, because this isoform can be phosphorylated and activated by ERK2.28,29 However, inhibition of
PLA2 by the MEK1 inhibitor was only partial, and it is
possible that there is another pathway of activation. Indeed, recent
evidence from platelet studies suggests that p38 and other kinases have
a role in the activation of cPLA2 in certain cell
types.30 Although not previously reported, it is possible
that ionophore stimulation of PLA2 may also be partially
mediated by ERK2.
The kinetics of activation of p42 MAP kinase by SCF were extremely
rapid and transient as measured by a gel retardation assay (optimal at
2 minutes and decreasing by 5 to 11 minutes). The first demonstration
of the activation of MAP kinase by SCF in human myeloid MO7 cells
showed reponses as sustained as 15 minutes poststimulation.67 However, our data are in accord with a
previous study using human fetal liver cells that showed similarly
rapid and transient activation of JAK-2 kinase by SCF.68
Rapid and transient ERK2 activation appears to be a feature of growth
factor signaling in immature myeloid cells, because previous work with both HL-60 cells69,70 and CD34+
cells57 show a conversion from transient to prolonged ERK2 activation after differentiation induction.
Although there is preliminary evidence that CD34+ cells
express mRNA for both prostaglandin H synthase I and II, as well as 5-lipoxygenase,43 we have shown that stimulation of
PLA2 activity was not associated with the release of
leukotrienes. This is in contrast with mature phagocytes that
metabolize endogenously generated AA to eicosanoid molecules that are
important for regulating host defense. The apparent lack of
5-lipoxygenase activity in these progenitor cells is consistent with
the observation that the acquisition of 5-lipoxygenase activity depends
on the synthesis of the 5-lipoxygenase activating protein later during
myeloid maturation.38 There are conflicting reports that
myeloid cell proliferation is regulated by
eicosanoids.39-42 Because we observed the proliferation and differentiation of myeloid stem cells during in vitro culture in the
absence of eicosanoid production, our data suggest that proliferation
of CD34+ cells may not depend on endogenous eicosanoid production.
This study presents evidence for active PLA2 in very
primitive myeloid cells. Its regulation by SCF, which promotes the
maintenance and proliferation of these early cells, supports the notion
that PLA2 activity has a role in the physiology of human
stem cells. This report has also shown that AA release is under the
regulation of later growth factors in a time-dependent pattern of
regulation during CD34+ cell differentiation and suggests
that AA may be needed at several specific stages of myeloid cell
differentiation. Further work is underway to investigate its role in
regulating both proliferation and differentiation of human
hematopoietic cells.
| |
ACKNOWLEDGMENT |
The authors are grateful to Stuart J Ings for preparation of
CD34+ cells.
| |
FOOTNOTES |
Submitted October 23, 1998; accepted April 20, 1999.
Supported by the Kay Kendall Leukaemia Fund (P.J.R.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Pamela J. Roberts, PhD, Department of
Haematology, University College London Medical School, 98 Chenies Mews,
London WC1E 6HX, UK; e-mail: pamela.roberts{at}ucl.ac.uk.
| |
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