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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3169-3177
Granulocyte-Macrophage Colony-Stimulating Factor Induces Expression
of Heparin-Binding Epidermal Growth Factor-Like Growth
Factor/Diphtheria Toxin Receptor and Sensitivity to Diphtheria Toxin in
Human Neutrophils
By
Fabrizio Vinante,
Martina Marchi,
Antonella Rigo,
Patrizia Scapini,
Giovanni Pizzolo, and
Marco A. Cassatella
From the Department of Clinical and Experimental Medicine, Section of
Hematology, and the Department of Pathology, Section of General
Pathology, University of Verona, Verona, Italy.
 |
ABSTRACT |
Heparin-binding epidermal growth factor-like growth factor (HB-EGF)
is a widely expressed EGF superfamily member that induces mitogenic
and/or chemotactic activities toward different cell types through
binding to EGF receptors 1 or 4. Membrane-bound HB-EGF exerts growth
activity and adhesion capabilities and possesses the unique property of
being the receptor for diphtheria toxin (DT). Using molecular and
functional techniques, we show that human polymorphonuclear
granulocytes (PMN), which did not express HB-EGF in resting conditions,
expressed it at mRNA and protein level, following incubation with
granulocyte-macrophage colony-stimulating factor (GM-CSF). Other
classic agonists for PMN (including lipopolysaccharide, phagocytable
particles, tumor necrosis factor- , or G-CSF) failed to induce
HB-EGF. The effects of GM-CSF on HB-EGF mRNA levels were
concentration-dependent, reached a plateau after 1 to 2 hours of
stimulation, and did not require protein synthesis. After GM-CSF treatment, membrane-bound HB-EGF was detected by flow cytometry. At the
same time, PMN acquired sensitivity to the apoptosis-promoting effect
of DT, which, moreover, specifically suppressed the GM-CSF-induced priming of
formyl-methionyl-leucyl-phenylalanine-stimulated
superoxide anion release. Finally, soluble HB-EGF was detected in the
PMN culture medium by a specific enzyme-linked immunosorbent assay. Thus, we provide evidence that HB-EGF is specifically inducible by
GM-CSF in PMN and represents a novel peptide to be included in the
repertoire of PMN-derived cytokines.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
NEUTROPHIL POLYMORPHONUCLEAR granulocytes
(PMN) act as the first line of defense against invading
micro-organisms, representing the predominant infiltrating cell type in
the cellular phase of the acute inflammatory response.1
Although mature PMN are terminally differentiated cells and have
generally been considered as lacking RNA/protein synthesis capacity,
convincing studies have clearly shown that PMN are capable of producing
a variety of cytokines under appropriate circumstances.2
Human PMN may release pro- and anti-inflammatory cytokines, including the interleukin-1 (IL-1) and IL-1 , tumor necrosis factor- (TNF-), IL-1 receptor antagonist (IL-1ra), and chemokines such as
IL-8, macrophage inflammatory protein-1 (MIP-1) and MIP-1, growth-related gene product- (GRO), and others.3
Remarkably, PMN have also been shown to express cytokines involved in
processes such as angiogenesis, including vascular endothelial growth
factor (VEGF),4-6 GRO,7 and interferon-
inducible protein-10 (IP-10),8 as well as in cell
proliferation and fibrosis, such as transforming growth factor-
(TGF),9 and TGF1,10,11 less
conventionally related to PMN functions. On the whole, these and other
data present PMN as candidate regulatory cells in conditions such as
wound healing, neoplastic growth, and even degenerative
lesions.3
Heparin-binding EGF-like growth factor (HB-EGF) is a heavily
glycosylated EGF superfamily member of approximately 22 kD, originally identified in human macrophages and U937
monocytic cell line conditioned medium,12,13 and expressed
in a wide range of cell types, including monocytes,13,14
CD4+ lymphocytes,15 eosinophils,16
myeloid leukemia blasts,17 vascular smooth muscle cells
(SMC),18 and endothelial19 and normal20 or neoplastic epithelial cells.12,21
Although HB-EGF can be released from the cell membrane through
proteolytic mechanisms,22 multiple mRNA species for HB-EGF
are produced,23 including transcripts corresponding to a
short HB-EGF form lacking intramembrane and intracytoplasmic
domains.24 Membrane-bound and soluble HB-EGF bind to EGF
receptors 1 (HER-1) and 4 (HER-4),13,14,25 eliciting different biological responses,12,25 including adhesion
activities,12 both mitogenic and chemotactic effects on
fibroblasts15 and SMC,12,26,27 chemotaxis on
endothelial cells27 and astrocytes,28 and
growth activity for some epithelial cells.12,20,21
Interestingly, membrane-bound HB-EGF has the unique property of acting
as the receptor for the diphtheria toxin (DT),29 a protein
synthesis inhibitor capable of triggering apoptotic death on target
cells.30 CD9 coexpression enhances the mitogenic activity
of membrane-bound HB-EGF31 as well as the sensitivity to
DT.32
In this study, we analyzed whether human PMN express HB-EGF. We show
that, after incubation with GM-CSF, PMN express amounts of HB-EGF mRNA,
synthesize the related protein, and produce both the membrane-bound and
soluble HB-EGF forms. We also provide evidence for a functional
significance of PMN-derived HB-EGF.
| |
MATERIALS AND METHODS |
Cell purification and culture.
Highly purified granulocytes (>99.5%) and peripheral blood
mononuclear cells (PBMC) were isolated under endotoxin-free conditions from buffy coats of healthy donors, as previously
described.33 The granulocyte populations usually contained
less than 4% eosinophils, as shown by May-Grünwald-Giemsa
staining. In selected experiments, granulocytes were depleted of
eosinophils according to the method described by Koenderman et
al.34 Immediately after purification, cells were suspended
in RPMI-1640 medium supplemented with 10% low-endotoxin (<6 pg/mL)
fetal calf serum (FCS) (Seromed; Biochrom KG, Berlin, Germany) and
treated with one of the following: 25 ng/mL
granulocyte-macrophage colony-stimulating factor (GM-CSF) (Genetics
Institute, Boston, MA), 100 ng/mL lipopolysaccharide (LPS) (from
Escherichia coli, serotype 026:B6; Sigma, St Louis, MO), 5 ng/mL TNF- (Peprotech, Rocky Hill, NJ), heat-killed yeast particles
opsonized with IgG (Y-IgG) at a particle/cell ratio of 2:1, 10 nmol/L
formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma),
1,000 U/mL G-CSF (Granulokine; Hoffmann-La Roche, Basel, Switzerland),
100 U/mL interferon- (IFN-) (Hoffmann-La Roche), 1,000 U/mL
IFN- (Peprotech), 15 ng/mL IL-3 (Peprotech), 10 ng/mL IL-5
(Peprotech), 100 U/mL IL-10 (kindly provided by Dr K. Moore, DNAX and
Schering-Plough, Palo Alto, CA), 100 ng/mL IL-12 (Peprotech), 100 ng/mL
IL-15 (Genzyme, Cambridge, MA), 300 nmol/L staurosporin (Sigma), 100 µmol/L dexamethasone (DEX) (Sigma), or 20 µg/mL cycloheximide (CHX)
(Sigma). In many experiments we also stimulated PMN with optimal
concentrations (0.5 ng/mL) of GM-CSF purchased from Peprotech. All
reagents used were the highest available grade and were dissolved in
pyrogen-free water for clinical use. Cells were plated either in
24-well tissue culture plates (Nunc, Roskilde, Denmark) at 3 × 106/mL, or at 5 to 8 × 106/mL in
polystyrene flasks (Greiner, Nurtingen, Germany), or at 3 × 106/mL in 96-well tissue culture plates (Greiner) and
subsequently incubated at 37°C, 5% CO2 atmosphere.
After culture for the appropriate times (see below), cells were either
extracted for total RNA or used for functional assays. Cell-free
supernatants (SN) were harvested and stored at  20°C. In
selected experiments, the U937 cell line (monocytic
leukemia-derived)35 was also used.
RNA isolation and Northern blot analysis.
Total RNA from PMN and PBMC was extracted and analyzed by Northern blot
(10 µg of RNA per lane) as previously described.17,36 Filters were hybridized using an HB-EGF cDNA probe obtained as described below, IL-1ra, IL-6, and -actin cDNA fragments labeled with 32P using a Ready-to-go DNA labeling kit (Pharmacia,
Uppsala, Sweden).
HB-EGF and HER-4 RT-PCR. HB-EGF probe generation.
A quantity of 4 µg of RNA from the cells of interest was reverse
transcribed as previously described.17,37 cDNA was
amplified using the following primers (Genenco, M-medical, Florence,
Italy). (1) HB-EGF sense 5'-TGGTGCTGAAGCTCTTTCTGG-3' and
antisense 5'-GTGGGAATTAGTCATGCCCAA-3'; these primers were
designed to give a fragment of 605 bp (complete form of
HB-EGF)23 or a fragment of 605 + 94 bp (short form of HB-EGF).24 (2) HER-4 sense
5'-AGATGGAGGTTTTGCTGCTGAACA-3' and antisense
5'-TTACACCACAGTATTCCGGTGTCT-3' (726-bp
fragment).38 (3) Vimentin sense
5'-GCTCAGATTCAGGAACAGCAT-3' and antisense
5'-TAAGGGCATCCACTTCACAGG-3' (266-bp fragment). The cDNA was
denatured for 5 minutes at 94°C before 35 runs in a thermal cycler
(GeneAmp PCR System 2400; Perkin Elmer, Norwalk, CT) using 1.25 U of
Taq polymerase (Perkin Elmer, Branchburg, NJ) in 50 µL (94°C 40 seconds, 57°C 40 seconds, 72°C 50 seconds) followed by 5 minutes at 72°C. The PCR products were separated by electrophoresis
on 1.5% agarose gel. HB-EGF cDNA amplified from the U937 cell line
using the primers specified above was analyzed for the SmaI
(Life Technologies, Rockville, MD) restriction site (which gave the
expected HB-EGF fragments of 388 and 217 bp), and was sequenced
(Sequenase 2.0 sequencing kit; USB, Cleveland, OH) as a plasmid insert
(TA cloning kit; Invitrogen, San Diego, CA), from which the HB-EGF
probe was generated for Northern blot analysis.
Flow cytometric analysis.
A quantity of 1 × 106/mL PMN cultured for 21 hours in
the presence or absence of GM-CSF or LPS was washed and incubated in 100 µL of phosphate-buffered saline (PBS) for 30 minutes at 4°C with 5% human serum and stained with 10 µL of 100 µg/mL purified rabbit anti-HB-EGF polyclonal H6 antibody (kindly provided by Dr S. Higashiyama, Osaka, Japan)32 for 1 hour at 4°C followed by a biotinylated second antibody [goat F(ab')2
anti-rabbit IgG (Caltag, Burlingame, CA) preadsorbed with human IgG]
for 30 minutes at 4°C and, after washing, by phycoerythrin
(PE)-conjugated streptavidin (Becton Dickinson, Sunnyvale, CA) for 15 minutes at 4°C. Freshly isolated PMN (3 × 106/mL)
were incubated with 10 µL PE-conjugated anti-CD9 (SBA, Birmingham, AL) or fluorescein isothiocyanate (FITC)-conjugated anti-HER-1 (Medac,
Hamburg, Germany) monoclonal antibodies (MoAbs) for 30 minutes at
4°C. Irrelevant purified rabbit Ig or isotype FITC- or
PE-conjugated (Immunotech, Westbrook, MA) MoAbs were used as controls.
Flow cytometry analysis was performed on a FACScan (Becton Dickinson,
Mountain View, CA).
DT-induced apoptosis.
Aliquots of 1 × 106/mL PMN were incubated for the
times indicated with or without GM-CSF in the presence or absence of
10 11 to 108 highly purified DT
(kindly provided by Dr E. Papini, CNR Center for Biomembranes, Padua,
Italy) and then examined for cell apoptosis by two distinct methods.
(1) Analysis of apoptotic (hypodiploid) nuclei by flow cytometry, as
described by others.39 PMN were harvested, washed twice
with PBS, and suspended in 1.5 mL hypotonic fluorochrome solution
(propidium iodide 50 µg/mL in 0.1% sodium citrate and 0.1% Triton
X-100). The mixture was placed in the dark overnight at 4°C. The
fluorescence of each nucleus was measured using an XL-Coulter flow
cytometer (Coulter, Hialeah, FL). (2) Analysis of apoptotic cell
morphology. A quantity of 0.5 × 106 PBS-washed PMN
were centrifuged for cytospin preparations and stained using
May-Grünwald-Giemsa. Cells were scored as apoptotic versus
nonapoptotic, based on diminution of cell volume and chromatin condensation yielding fragmented homogeneously stained
nuclei.40
Measurement of superoxide anion (O2)
generation.
This was performed as previously described.41 Briefly, 100 µL of PMN suspension (3 × 106/mL) containing or not
GM-CSF or LPS in the presence or absence of 108
mol/L DT were added to tissue-culture polystyrene 96-well plates. After
a 2- or 21-hour incubation, 100 µL of Hanks'
Balanced Salt Solution (HBSS), containing 1 mmol/L CaCl2,
10 mmol/L glucose, 4 mmol/L NaN3, and 160 µmol/L
cytochrome C, with or without stimulus (100 nmol/L fMLP) in the
presence or not of superoxide dismutase (SOD), were added on top to
each well. Plates were then incubated at 37°C in an automated EL34
microplate reader (Biotec Instruments, Highland Park, UT) to record
absorbance at 550 and 468 nm. Nanomoles of
O2 were calculated using an extinction
coefficient of 24.5 mmol/L.42 In selected experiments for
neutrophil-derived superoxide anion, 0.5 to 1 ng/mL HB-EGF (R&D System,
Minneapolis, MN) was used either as a direct triggering agonist or as a
priming agent.
Enzyme-linked immunosorbent assay (ELISA) for HB-EGF.
Soluble HB-EGF protein was measured in the cell-free SN using a
specific ELISA developed in our laboratory, according to a method
recently published.43 Briefly, flat-bottomed 96-well plates
(MaxiSorp; Nunc) were coated with 50 µL/well of 2 µg/mL polyclonal
anti-HB-EGF antibody (R&D System) in 50 mmol/L sodium bicarbonate
buffer, pH 8.5 for 8 hours at room temperature, and incubated overnight
at 4°C with 100 µL/well of blocking buffer (20 mmol/L TBS, pH
7.4, 3% bovine serum albumin [BSA]). After extensive washings with
TBS, pH 7.4, 0.05% Tween 20 (washing buffer), 50 µL/well of either
HB-EGF standards (R&D System) or cell-free culture SN were added,
followed by a 2-hour incubation at 37°C. SN
collected after a 21-hour culture of U937 cells or PBMC and PMN
stimulated or not with GM-CSF or LPS were used undiluted. SN of PMN
previously cultured in medium supplemented with 1% low-endotoxin FCS
were used after approximately 80-fold concentration (by the Centricon
Plus 20 device from Amicon Inc, Beverly, MA). Plates were rinsed with
washing buffer before addition of 50 µL/well of biotinylated
anti-HB-EGF antibody (R&D System) (0.5 µg/mL in TBS, 0.05% Tween
20, 0.1% BSA) and incubated for 1.5 hours at room temperature. After
extensive washings of the plates, streptavidin-conjugated alkaline
phosphatase, diluted 1:10,000 (Life Technologies) with TBS containing
0.5% BSA and 0.05% Tween 20, was added and incubated for 1 hour at
room temperature. After washing, a chromogenic reaction was performed
using the ELISA Amplification System (Life Technologies), according to
the manufacturer's protocol. Assays were carried out in duplicate. The
reaction was stopped with 0.3 mol/L H2SO4 and
the absorbance at 490 nm was measured. This ELISA had a detection limit
of 6.25 pg/mL, and did not cross-react with 100 ng/mL IP-10, 100 ng/mL
IL-8, 1 ng/mL MIP-1, 10 ng/mL GRO, 10 ng/mL IFN-, 5 ng/mL
IL-10, 10 ng/mL IL-1, 10 ng/mL TNF-, or 10 ng/mL GM-CSF other
than EGF, TGF, and HGF.43
Statistical analysis.
Student's t-test, the Mann-Whitney U test, and analysis of
variance according to Kruskall-Wallis ANOVA by ranks were used. Differences were considered statistically significant when the P value was <.05.
| |
RESULTS |
GM-CSF-induced HB-EGF mRNA in PMN.
To investigate whether amounts of HB-EGF mRNA were expressed in PMN,
these cells were initially cultured for 2 hours in the presence or
absence of 100 ng/mL LPS and 25 ng/mL GM-CSF, and then total RNA was
processed for Northern blot analysis. Autologous PBMC were also
stimulated under identical experimental conditions, for the purposes of
comparison. Figure 1 shows that resting and LPS-treated PMN did not express detectable HB-EGF mRNA. The
effectiveness of LPS-stimulation was confirmed by its ability to
upregulate the IL-1ra mRNA levels.44 Other PMN agonists,
listed in Table 1, also failed to induce
HB-EGF steady-state mRNA levels in PMN, even if PMN were cultured for
up to 21 hours in the presence or absence of IFN- (data not shown),
a cytokine which usually primes PMN for an enhanced gene
expression.3 By contrast, GM-CSF-treated PMN exhibited a
considerable accumulation of HB-EGF transcripts as well as IL-1ra
mRNA.45
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| Fig 1.
Comparative ability of PMN and PBMC to express HB-EGF
mRNA when stimulated with GM-CSF. GM-CSF induced HB-EGF transcripts in
PMN and, more dramatically, in PBMC, while LPS, which upregulated
IL-1ra in PMN and IL-6 in PBMC, was ineffective on HB-EGF mRNA
production. PBMC presented also a costitutive, low production of HB-EGF
mRNA. Purified populations of PMN and PBMC from the same donor were
cultured with or without 25 ng/mL GM-CSF and 100 ng/mL LPS for 2 hours
and then total RNA was extracted and Northern blot analysis for HB-EGF,
IL-1ra, IL-6, and actin mRNA was performed. Ten micrograms of total RNA
was loaded on each gel lane. The experiment depicted is representative
of 4.
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HB-EGF accumulation pattern in PMN and PBMC.
Kinetic experiments showed that maximal levels of HB-EGF mRNA occurred
after 1 to 2 hours of GM-CSF stimulation
(Fig 2A), whereas dose-response
studies indicated that the highest HB-EGF mRNA accumulation in PMN
could be obtained after stimulation with 25 ng/mL of GM-CSF (Fig 2B). A
different pattern of HB-EGF mRNA accumulation was observed in PBMC,
which not only presented low but constitutive HB-EGF transcripts in all
donors examined (n = 4), but dramatically responded to GM-CSF
stimulation (Fig 1), producing, as observed for other
cytokines,2 more HB-EGF transcripts than PMN did.
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| Fig 2.
HB-EGF mRNA levels in GM-CSF-treated PMN. (A)
Time-course. PMN were incubated with 25 ng/mL GM-CSF. At the
time-points indicated, total mRNA was extracted and analyzed for
HB-EGF, IL-1ra, and actin mRNA expression. The experiment depicted is
representative of 2. (B) Dose-dependence. PMN were stimulated with
increasing doses of GM-CSF for 2 hours and then total RNA was extracted
and analyzed for HB-EGF, IL-1ra, and actin mRNA expression. The
experiment depicted is representative of 2.
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Genuine expression of HB-EGF by PMN.
PBMC expressed several HB-EGF mRNA species, in agreement with earlier
studies.23,46 As illustrated in Figs 2 and
3, at least 2 HB-EGF mRNA species were
induced by GM-CSF in PMN, the most abundant being the 2.7-kb size
transcript, a pattern matching that observed in PBMC. Furthermore, IL-6
mRNA was expressed only in LPS-stimulated PBMC and not in PMN,
according to published data,47,48 thus excluding
contamination with mononuclear cells (Fig 1). Also consistent with the
genuine ability of PMN to express HB-EGF mRNA in response to GM-CSF
were 3 additional experiments in which complete depletion of
eosinophils, which have also been reported to express HB-EGF in a rat
experimental model,16 did not influence the accumulation of
HB-EGF mRNA in GM-CSF-treated PMN (data not shown). Taken together,
our data rule out the possibility that the results obtained in PMN with
respect to HB-EGF mRNA levels can be attributed to contamination with
mononuclear cells or eosinophils.
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| Fig 3.
Effects of CHX on HB-EGF mRNA levels in GM-CSF-treated
PMN. CHX did not inhibit, but overinduced, the GM-CSF-dependent
upregulation of HB-EGF mRNA, showing that protein synthesis was not
required. PMN were pretreated with 20 µg/mL CHX before stimulation
with 25 ng/mL GM-CSF for 2 hours. Total RNA was then extracted and
analyzed for HB-EGF and actin mRNA levels. The experiment depicted is
representative of 2.
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Protein-synthesis-independent induction of HB-EGF mRNA in PMN.
We also examined whether de novo protein synthesis was necessary for
the GM-CSF-driven induction of HB-EGF mRNA in PMN. As shown in Fig 3,
this was not the case. The inhibitor of protein synthesis CHX not only
did not inhibit, but actually superinduced the GM-CSF-dependent
upregulation of HB-EGF mRNA, in a similar way to previous observations
in monocytes stimulated with LPS for 1 hour.46
Membrane-bound HB-EGF demonstration by flow cytometry on
GM-CSF-treated PMN.
To determine whether GM-CSF-treated PMN expressed the membrane-bound
HB-EGF molecule, they were investigated by flow cytometry using a
specific purified anti-HB-EGF polyclonal antibody. As shown in
Fig 4A, resting PMN were negative for HB-EGF.
GM-CSF proved to be an efficient inducer of membrane-bound HB-EGF
surface expression in cultured PMN (Fig 4B), while LPS was not (as
expected from the Northern blot data) (Fig 4C). Surface HB-EGF
expression, however, was detectable when PMN were cultured with GM-CSF
for longer than 6 hours. GM-CSF also efficiently upregulated HB-EGF surface expression in monocytes (data not shown).
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| Fig 4.
Effect of GM-CSF and LPS on the surface expression of
HB-EGF molecule in PMN. PMN were cultured in the absence (A) or
presence of 25 ng/mL GM-CSF (B) or 100 ng/mL LPS (C) for 21 hours. Only
GM-CSF-treated PMN acquired membrane expression of the HB-EGF molecule
(B). Membrane-bound HB-EGF expression was examined by indirect
immunofluorescence analysis using the polyclonal H6 antibody (kindly
provided by Dr S. Higashiyama), followed by a human-IgG preadsorbed
biotinylated second antibody and by PE-conjugated streptavidin. Cells
were also stained with irrelevant antibodies as controls. The
expression patterns presented in this figure were reproduced in 5 independent experiments.
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DT-induced apoptosis in GM-CSF-treated PMN.
To evaluate whether the membrane-bound HB-EGF molecule expressed on
GM-CSF-treated PMN was functionally active, we tested PMN sensitivity
to DT. DT represents a ligand of membrane-bound HB-EGF and induces
apoptosis in a dose-dependent manner on HB-EGF-expressing cells.29,30 Although PMN undergo constitutive apoptosis
when aged ex vivo, evidence suggests that this process may be
substantially delayed if cells are cultured in the presence of GM-CSF,
LPS, IFN-, and other cytokines, or, conversely, that it may be
accelerated if cells are incubated, for instance, with anti-CD95
antibodies.49 Therefore, we sought to determine whether the
presence of DT in the cultures of GM-CSF-treated PMN inhibited the
protective effect of GM-CSF on PMN apoptosis50 as a
consequence of the GM-CSF-induced surface expression of HB-EGF acting
as a DT receptor. To test this hypothesis, we measured the rates of PMN
apoptosis by analyzing apoptotic (hypodiploid) nuclei and cell
morphology. Figure 5
illustrates a representative experiment performed using flow cytometry
to quantify the apoptotic nuclei of PMN, while
Table 2 summarizes the data from all our
experiments. Apoptosis of PMN after a 44-hour culture period was 34% ± 9% (n = 5), and this percentage was not significantly affected
by the presence of DT (35.7% ± 15%, n = 5) in the
culture medium. IFN- and, more effectively, GM-CSF were found to
exert a significant protective effect on PMN apoptosis. By contrast,
108 mol/L DT completely suppressed (P < .05) the protective effect of GM-CSF (Fig 5 and Table 2), but not that
of IFN- (Table 2). Preliminary dose-response experiments in the
range of 1011 to 108 mol/L DT
concentrations showed that 108 mol/L DT displayed
maximal suppression of the protective GM-CSF-mediated effect on PMN
apoptosis (data not shown). Therefore, 108 mol/L DT
was used in all subsequent experiments. GM-CSF-treated PMN also
displayed sensitivity to the apoptosis-promoting effects of DT at
morphological examination (Fig 6).
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| Fig 5.
Effect of DT on the development of apoptotic nuclei in
PMN maintained in culture with GM-CSF. The presence of DT (kindly
provided by Dr E. Papini) in the cultures inhibited specifically the
protective effect of GM-CSF on PMN apoptosis. PMN suspensions, cultured
for 44 hours alone or with 25 ng/mL GM-CSF in the presence or absence
of 108 mol/L DT, were processed for DNA content analysis
by propidium iodide staining and flow cytometry analysis. Data were
plotted as red fluorescence intensity versus number of nuclei with a
given DNA content as determined in each experimental condition. Numbers
reported indicate the percentage of hypodiploid (apoptotic) nuclei.
Similar results were observed in PMN isolated from 3 independent
donors.
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| Fig 6.
Morphologic features of PMN cultured with GM-CSF in the
presence or absence of DT. Only after GM-CSF treatment, PMN acquired
sensitivity to DT, which induced typical apoptotic features. Cytospin
preparations of PMN were stained using the May-Grünwald-Giemsa
method after incubation in vitro for 21 hours in the presence or
absence of 0.5 ng/mL GM-CSF with or without 108 mol/L
DT. The experiment depicted is representative of 2.
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DT-mediated suppression of GM-CSF priming on PMN respiratory burst
capacity.
To determine whether the apoptosis-promoting effects of DT were
selective or accompanied by other biological effects, additional experiments were performed to assess the respiratory burst capacity of
PMN cultured with GM-CSF. Like IFN- or LPS,33 GM-CSF is known to greatly potentiate the ability of PMN to release reactive oxygen intermediates (ROI).51 To this end, PMN were
incubated with GM-CSF or LPS for 2 and 21 hours in the presence or
absence of 108 mol/L DT, and then stimulated with
100 nmol/L fMLP, a bacteria-derived chemotactic peptide routinely used
to stimulate ROI release in PMN.41 Both GM-CSF and
LPS-treatment resulted in a dramatic upregulation of fMLP-stimulated,
SOD-inhibitable superoxide anion (O2)
release from PMN, at both 2- (not shown) and 21-hour incubation (Fig 7). While DT failed to influence the
constitutive ability of PMN to produce
O2 in response to fMLP at both 2- (not
shown) and 21-hour incubation, it significantly suppressed (by
approximately 60%) the priming effect of GM-CSF at 21 hours, but was
largely ineffective toward LPS (Fig 7). Interestingly, DT did not
affect the priming effect of GM-CSF at 2-hour incubation (data not
shown), which is consistent with a lack of surface expression of
membrane HB-EGF at that time-point (see above).
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| Fig 7.
Effect of DT on the respiratory burst of PMN maintained
in culture with GM-CSF. DT specifically suppressed the priming effect
of GM-CSF, but was largely ineffective toward LPS (P < .05).
PMN (3 × 105/well) were cultured for 21 hours in the
presence or absence of 0.5 ng/mL GM-CSF with or without
108 mol/L DT before stimulation with 100 nmol/L fMLP.
Data represent the mean values (±SD) of SOD-inhibitable
O2 release from triplicate assays for each
condition. Similar results were obtained in 3 separate experiments.
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Release of membrane-bound HB-EGF into SN.
Finally, we investigated whether GM-CSF-treated PMN, in addition to
expressing the membrane-bound form, could also release HB-EGF protein
into the SN. To this end, we developed a specific ELISA, according to
the indications by Yamada et al.43 The threshold sensitivity of this ELISA was 6.25 pg/mL and its validity was demonstrated by the fact that it was able to detect substantial amounts
of soluble HB-EGF in U937 cell-conditioned medium12,14 (Fig 8A). No extracellular production of
soluble HB-EGF was detected in cell-free SN harvested from either
resting or GM-CSF- and LPS-treated PMN, whereas significant levels of
soluble antigenic HB-EGF were measured in SN harvested from autologous
PBMC in resting conditions, which increased (P < .05) after
treatment with GM-CSF (Fig 8A). If SN from GM-CSF-treated PMN were
previously concentrated up to 80-fold, then detectable yields of
soluble HB-EGF could be specifically measured (Fig 8B).
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| Fig 8.
Release of the soluble form of HB-EGF by PMN and PBMC.
GM-CSF treatment induces release of the HB-EGF molecule into the
culture medium (P < .05). (A) PMN and PBMC were cultured
alone or in the presence of either 0.5 ng/mL GM-CSF or 100 ng/mL LPS.
Cell-free SN were collected after 21 hours, and the levels of soluble
HB-EGF protein were measured by a specific ELISA. The levels of soluble
HB-EGF spontaneously released by the U937 cell line were also
determined for comparison. (B) In selected experiments, PMN-derived SN
were concentrated approximately 80-fold before ELISA detection. Values
represent means (±SD) of duplicate determinations calculated from 3 independent experiments.
|
|
Lack of HER-1, HER-4, and CD9 expression in PMN: Inefficacy of
HB-EGF.
Because HB-EGF binds to HER-1 or HER-4,13,14,25 and CD9
behaves as a coreceptor of membrane-bound HB-EGF,31,32 we
investigated whether these molecules were expressed by PMN. We failed
to detect the expression of either HER-1 or CD9 molecules, or of HER-4
mRNA in PMN, as investigated by flow cytometry analysis and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. In line
with the lack of the related receptors, stimulatory experiments using
HB-EGF did not induce any modifications in PMN. Interestingly, HER-4
mRNA was detectable in U937 cells in basal conditions (data not shown).
| |
DISCUSSION |
HB-EGF is a member of the EGF superfamily, which also includes
epidermal growth factor, TGF, amphiregulin, betacellulin, epiregulin, neuregulin-1 and -2, and vaccinia growth
factor.12,52 These are growth and differentiation factors,
some of which, especially HB-EGF, have been shown to actively
participate in important tissue-modeling phenomena, involving autocrine
or paracrine regenerative and neoplastic growth and highly complex
activities such as angiogenesis and blastocyst
implantation.12,52 Although PMN have been found to
infiltrate a number of proliferative or degenerative lesions, their
ability to release cytokines of the EGF family has rarely been
considered. For instance, attention has only recently focused on
PMN-produced TGF.9 In general, the role of PMN
infiltrating tissue lesions is far from clear and there is currently a
great deal of interest in studying PMN-derived cytokines and especially the role they play in cell proliferation and angiogenesis.3
This study provides the first demonstration that human PMN, under
particular stimulatory conditions, are capable of producing, bearing on
their membrane, and releasing the important EGF superfamily cytokine,
HB-EGF. Our findings, therefore, document a mechanism whereby PMN might
directly influence tissue regeneration and even cancer progression.
Although negative in basal conditions, PMN expressed HB-EGF mRNA upon
treatment with GM-CSF, as shown by both Northern blot analysis (Fig 1)
and RT-PCR (data not shown). Classic agonists (listed in Table 1) for
PMN all failed to induce HB-EGF, despite the presence in the culture
medium of IFN-, which usually acts as a PMN priming factor for gene
expression.3 The effects of GM-CSF on HB-EGF mRNA levels
were concentration-dependent, reached a plateau after 1 to 2 hours of
stimulation, and were independent of protein synthesis. Because it has
been shown that HB-EGF may be induced via the Ras
pathway12,53,54 and that GM-CSF actually activates the
Ras and Raf-1 and the MAP-kinase signaling pathways by
binding to the beta subunit of its receptor,55,56 the
Ras pathway may therefore be a likely candidate for the
GM-CSF-mediated induction of HB-EGF in PMN. By contrast, NFkB are
unlikely to play a role in PMN HB-EGF upregulation. Although putative
binding sites for NFkB have been identified in the HB-EGF
promoter12,23 and NFkB can be mobilized through the GM-CSF
receptor in many cell types,55,56 recent studies of ours
have failed to demonstrate activation of NFkB in GM-CSF-stimulated
PMN.57
The surface expression and structural integrity of membrane-bound
HB-EGF were detected by flow cytometry and by the demonstration that
PMN acquired sensitivity to the apoptosis-promoting effect of
DT29,30 after GM-CSF treatment. DT-induced PMN cytolysis was associated with the development of hypodiploid nuclei and apoptotic
cellular morphology. We did not, however, find complete killing of PMN
after exposure to DT. DT receptor density or expression only in a
subset of cells, lack of coreceptors such as CD9,32 efficiency of DT internalization, and, in general, the equilibrium status of apoptotic pathways in terms of overexpression of
anti-apoptotic factors, possibly induced by GM-CSF itself, may
influence PMN sensitivity to DT at any given time. The suppression of
the priming effect of GM-CSF on fMLP-stimulated, SOD-inhibitable
superoxide anion release was another effect of DT internalization. This
effect coincided with the surface expression of membrane-bound HB-EGF, because it was not observed at 2 hours of incubation with GM-CSF. Whether the suppressive action on the respiratory burst is attributable to DT-mediated inhibition of protein synthesis,22 and
specifically to the synthesis of the various NADPH oxidase
components,33 is an intriguing possibility, whose further
investigation may help to clarify the molecular basis of cytokine
enhancement of the phagocyte respiratory burst capability.
Finally, we have also shown that GM-CSF-treated PMN may release the
HB-EGF molecule into the culture medium. Because neither HER-1 nor
HER-4 expression was detected and HB-EGF did not induce any
modifications in PMN, we are inclined to rule out the possibility that
soluble HB-EGF might act in an autocrine manner.
In conclusion, HB-EGF is a novel protein that can now be included in
the repertoire of PMN-derived cytokines. It is not expressed in resting
conditions, but is specifically inducible by GM-CSF, a factor involved
in modulating a number of PMN functions.1,3 The role, if
any, played by HB-EGF expressed in GM-CSF-stimulated PMN is less
intuitive. Nevertheless, the fact that PMN produce HB-EGF is of
interest, considering that a relationship has been detected between
HB-EGF and pivotal biological activities12,53 which, up
until very recently, were rarely or never associated with PMN. HB-EGF
has been reported as playing a role in reproductive biology,58,59 wound healing,60 atheromatous
phenomena,31,61 angiogenesis,27 and epithelial
neoplastic growth.21 Remarkably, a number of epithelial
neoplasias, for which HB-EGF is an autocrine growth factor and,
possibly, an angiogenetic factor through VEGF induced in vascular
SMC,27 are capable of producing and releasing GM-CSF62-64 and are often infiltrated by PMN, especially
when necrosis occurs.65 Thus, although the relationship
between infiltrating PMN and neoplastic cells is fairly elusive, PMN
may provide an unexpected source of a proliferative, angiogenetic
factor such as HB-EGF, thus participating in the progression of
cancer.21 Similar findings have been reported with regard
to neoplasia-infiltrating T lymphocytes that have been shown to produce
HB-EGF, when, as has been reported, exposed to a nonsupporting
environment.21 In PMN, however, we have documented a
specific mechanism of activation for HB-EGF that may match the specific
biological properties of some cancers.62-65 Anyway, the
fact that PMN can synthesize, store, and release a substantial array of
cytokines, including HB-EGF, lends further support to the suggestion
that the role of PMN in physiopathology needs to be redefined.
| |
ACKNOWLEDGMENT |
The authors thank Dr E. Papini for kindly providing highly purified DT,
Dr S. Higashiyama for his generous gift of anti-HB-EGF antibodies, and
F. Calzetti for her excellent technical assistance.
| |
FOOTNOTES |
Submitted April 2, 1999; accepted July 7, 1999.
Supported by grants from MURST (60% funds and "cofinanziamento
MURST-Università 40%"), Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano, Italy) and Progetto Sanità 96/97,
Fondazione Cassa di Risparmio VR-VI-BL-AN (Verona, Italy).
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 Fabrizio Vinante, MD, Cattedra
di Ematologia, Policlinico GB Rossi, 37134 Verona, Italy; e-mail:
VINANTE{at}borgoroma.univr.it; Marco A. Cassatella, MD, Patologia
Generale, 37134 Verona, Italy; e-mail: MCNCSS{at}BORGOROMA.UNIVR.IT.
| |
REFERENCES |
1.
Edwards S:
Biochemistry and Physiology of the Neutrophil. Cambridge, UK, Cambridge University Press, 1994
2.
Cassatella MA:
The production of cytokines by polymorphonuclear neutrophils.
Immunol Today
16:21, 1995[Medline]
[Order article via Infotrieve]
3.
Cassatella MA:
Cytokines Produced by Polymorphonuclear Neutrophils: Molecular and Biological Aspects. Austin, TX, RG Landes Co, 1996
4.
Taichman NS, Young S, Cruchley AT, Taylor P, Paleolog E:
Human neutrophils secrete vascular endothelial growth factor.
J Leukoc Biol
62:397, 1997[Abstract]
5.
Gaudry M, Bregerie O, Andrieu V, El Benna J, Pocidalo MA, Hakim J:
Intracellular pool of vascular endothelial growth factor in human neutrophils.
Blood
90:4153, 1997[Abstract/Free Full Text]
6.
Webb NJ, Myers CR, Watson CJ, Bottomley MJ, Brenchley PE:
Activated human neutrophils express vascular endothelial growth factor.
Cytokine
10:254, 1998[Medline]
[Order article via Infotrieve]
7.
Iida N, Grotendorst GR:
Cloning and sequencing of a new gro transcript from activated human monocytes: Expression in leukocytes and wound tissue.
Mol Cell Biol
10:5596, 1990[Abstract/Free Full Text]
8.
Cassatella MA, Gasperini S, Calzetti F, Bertagnin A, Luster AD, McDonald PP:
Regulated production of the interferon-gamma-inducible protein-10 (IP-10) chemokine by human neutrophils.
Eur J Immunol
27:111, 1997[Medline]
[Order article via Infotrieve]
9.
Calafat J, Janssen H, Stahle-Backdahl M, Zuurbier AE, Knol EF, Egesten A:
Human monocytes and neutrophils store transforming growth factor- in a subpopulation of cytoplasmic granules.
Blood
90:1255, 1997[Abstract/Free Full Text]
10.
Grotendorst GR, Smale G, Pencev D:
Production of transforming growth factor by human peripheral blood monocytes and neutrophils.
J Cell Physiol
140:396, 1989[Medline]
[Order article via Infotrieve]
11.
Fava RA, Olsen NJ, Postlethwaite AE, Broadley KN, Davidson JM, Nanney LB, Lucas C, Townes AS:
Transforming growth factor 1 (TGF1) induced neutrophil recruitment to synovial tissues: Implications for TGF-driven synovial inflammation and hyperplasia.
J Exp Med
173:1121, 1991[Abstract/Free Full Text]
12.
Raab G, Klagsbrun M:
Heparin-binding EGF-like growth factor.
Biochim Biophys Acta
1333:F179, 1997[Medline]
[Order article via Infotrieve]
13.
Higashiyama S, Abraham JA, Miller J, Fiddes JC, Klagsbrun M:
A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF.
Science
251:936, 1991[Abstract/Free Full Text]
14.
Higashiyama S, Lau K, Besner GE, Abraham JA, Klagsbrun M:
Structure of heparin-binding EGF-like growth factor. Multiple forms, primary structure, and glycosylation of the mature protein.
J Biol Chem
267:6205, 1992[Abstract/Free Full Text]
15.
Blotnick S, Peoples GE, Freeman MR, Eberlein TJ, Klagsbrun M:
T lymphocytes synthesize and export heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor, mitogens for vascular cells and fibroblasts: Differential production and release by CD4+ and CD8+ T cells.
Proc Natl Acad Sci USA
91:2890, 1994[Abstract/Free Full Text]
16.
Powell PP, Klagsbrun M, Abraham JA, Jones RC:
Eosinophils expressing heparin-binding EGF-like growth factor mRNA localize around lung microvessels in pulmonary hypertension.
Am J Pathol
143:784, 1993[Abstract]
17.
Vinante F, Rigo A, Papini E, Cassatella MA, Pizzolo G:
Heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor expression by acute myeloid leukemia cells.
Blood
93:1715, 1999[Abstract/Free Full Text]
18.
Dluz S, Higashiyama S, Damm D, Abraham JA, Klagsbrun M:
Heparin-binding epidermal growth factor-like growth factor expression in cultured fetal human vascular smooth muscle cells. Induction of mRNA levels and secretion of active mitogen.
J Biol Chem
268:18330, 1993[Abstract/Free Full Text]
19.
Yoshizumi M, Kourembanas S, Temizer DH, Cambria RP, Quertermous T, Lee ME:
Tumor necrosis factor increases transcription of the heparin-binding epidermal growth factor-like growth factor gene in vascular endothelial cells.
J Biol Chem
267:9467, 1992[Abstract/Free Full Text]
20.
Hashimoto K, Higashiyama S, Asada H, Hashimura E, Kobayashi T, Sudo K, Nakagawa T, Damm D, Yoshikawa K, Taniguchi N:
Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes.
J Biol Chem
269:20060, 1994[Abstract/Free Full Text]
21.
Peoples GE, Blotnick S, Takahashi K, Freeman MR, Klagsbrun M, Eberlein TJ:
T lymphocytes that infiltrate tumors and atherosclerotic plaques produce heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor: A potential pathologic role.
Proc Natl Acad Sci USA
92:6547, 1995[Abstract/Free Full Text]
22.
Dethlefsen SM, Raab G, Moses MA, Adam RM, Klagsbrun M, Freeman MR:
Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C.
J Cell Biochem
69:143, 1998[Medline]
[Order article via Infotrieve]
23.
Fen Z, Dhadly MS, Yoshizumi M, Hilkert RJ, Quertermous T, Eddy RL, Shows TB, Lee ME:
Structural organization and chromosomal assignment of the gene encoding the human heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor.
Biochemistry
32:7932, 1993[Medline]
[Order article via Infotrieve]
24.
Loukianov E, Loukianova T, Wiedlocha A, Olsnes S:
Expression of mRNA for a short form of heparin-binding EGF-like growth factor.
Gene
195:81, 1997[Medline]
[Order article via Infotrieve]
25.
Elenius K, Paul S, Allison G, Sun J, Klagsbrun M:
Activation of HER-4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation.
EMBO J
16:1268, 1997[Medline]
[Order article via Infotrieve]
26.
Higashiyama S, Abraham JA, Klagsbrun M:
Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: Dependence on interactions with cell surface heparan sulfate.
J Cell Biol
122:933, 1993[Abstract/Free Full Text]
27.
Abramovitch R, Neeman M, Reich R, Stein I, Keshet E, Abraham JA, Solomon A, Marikovsky M:
Intercellular communication between vascular smooth muscle and endothelial cells mediated by heparin-binding epidermal growth factor-like growth factor and vascular endothelial growth factor.
FEBS Lett
425:441, 1998[Medline]
[Order article via Infotrieve]
28.
Faber-Elman A, Solomon A, Abraham JA, Marikovsky M, Schwartz M:
Involvement of wound-associated factors in rat brain astrocyte migratory response to axonal injury: In vitro simulation.
J Clin Invest
97:162, 1996[Medline]
[Order article via Infotrieve]
29.
Naglich JG, Metherall JE, Russell DW, Eidels L:
Expression cloning of a diphtheria toxin receptor: Identity with a heparin-binding EGF-like growth factor precursor.
Cell
69:1051, 1992[Medline]
[Order article via Infotrieve]
30.
Chang MP, Bramhall J, Graves S, Bonavida B, Wisnieski BJ:
Internucleosomal DNA cleavage precedes diphtheria toxin-induced cytolysis. Evidence that cell lysis is not a simple consequence of translation inhibition.
J Biol Chem
264:15261, 1989[Abstract/Free Full Text]
31.
Ouchi N, Kihara S, Yamashita S, Higashiyama S, Nakagawa T, Shimomura I, Funahashi T, Kameda-Takemura K, Kawata S, Taniguchi N, Matsuzawa Y:
Role of membrane-anchored heparin-binding epidermal growth factor-like growth factor and CD9 on macrophages.
Biochem J
328:923, 1997
32.
Iwamoto R, Higashiyama S, Mitamura T, Taniguchi N, Klagsbrun M, Mekada E:
Heparin-binding EGF-like growth factor, which acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9, which up-regulates functional receptors and diphtheria toxin sensitivity.
EMBO J
13:2322, 1994[Medline]
[Order article via Infotrieve]
33.
Cassatella MA, Bazzoni F, Flynn RM, Dusi S, Trinchieri G, Rossi F:
Molecular basis of interferon- and LPS enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components.
J Biol Chem
265:20241, 1990[Abstract/Free Full Text]
34.
Koenderman L, Kok PT, Hamenlink ML, Verhoeven AJ, Bruijnzeel PL:
An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals.
J Leukoc Biol
44:79, 1998[Abstract]
35.
McDonald PP, Cassatella MA, Bald A, Maggi E, Romagnani S, Gruss HJ, Pizzolo G:
CD30 ligation induces nuclear factor-kappa B activation in human T cell lines.
Eur J Immunol
25:2870, 1995[Medline]
[Order article via Infotrieve]
36.
Vinante F, Rigo A, Vincenzi C, Ricetti MM, Marrocchella R, Chilosi M, Cassatella MA, Bonazzi L, Pizzolo G:
IL-8 mRNA expression and IL-8 production by acute myeloid leukemia cells.
Leukemia
7:1552, 1993[Medline]
[Order article via Infotrieve]
37.
Vinante F, Rigo A, Tecchio C, Morosato L, Nadali G, Ricetti MM, Krampera M, Zanolin E, Locatelli F, Gallati H, Chilosi M, Pizzolo G:
Serum levels of p55 and p75 soluble TNF receptors in adult acute leukaemia at diagnosis: Correlation with clinical and biological features and outcome.
Br J Haematol
102:1025, 1998[Medline]
[Order article via Infotrieve]
38.
Mograbi B, Rochet N, Imbert V, Bourget I, Bocciardi R, Emiliozzi C, Rossi B:
Human monocytes express amphiregulin and heregulin growth factors upon activation.
Eur Cytokine Netw
8:73, 1997[Medline]
[Order article via Infotrieve]
39.
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi CA:
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
J Immunol Methods
139:271, 1991[Medline]
[Order article via Infotrieve]
40.
Wyllie AH, Kerr JF, Currie AR:
Cell death: The significance of apoptosis.
Int Rev Cytol
68:251, 1980[Medline]
[Order article via Infotrieve]
41.
Cassatella MA, Bazzoni F, Ceska M, Ferro I, Baggiolini M, Berton G:
Interleukin 8 production by human polymorphonuclear leukocytes. The chemoattractant Formyl-methionyl-Leucyl-Phenylalanine induces the gene expression and release of interleukin 8 through a pertussis toxin sensitive pathway.
J Immunol
148:3216, 1992[Abstract]
42.
Bellavite P, Dri P, Della Bianca V, Serra MC:
The measurement of superoxide anion production by granulocytes in whole blood. A clinical test for evaluation of phagocyte function and serum opsonic capacity.
Eur J Clin Invest
13:363, 1983[Medline]
[Order article via Infotrieve]
43.
Yamada A, Kawata S, Tamura S, Kiso S, Higashiyama S, Umeshita K, Sakon M, Taniguchi N, Monden M, Matsuzawa Y:
Plasma heparin-binding EGF-like growth factor levels in patients after partial hepatectomy as determined with an enzyme-linked immunosorbent assay.
Biochem Biophys Res Commun
246:783, 1998[Medline]
[Order article via Infotrieve]
44.
Cassatella MA, Meda L, Gasperini S, Calzetti F, Bonora S:
Interleukin 10 (IL-10) upregulates IL-1 receptor antagonist production from lipopolysaccharide-stimulated human polymorphonuclear leukocytes by delaying mRNA degradation.
J Exp Med
179:1695, 1994[Abstract/Free Full Text]
45.
McColl SR, Paquin R, Menard C, Beaulieu AD:
Human neutrophils produce high levels of the interleukin 1 receptor antagonist in response to granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-.
J Exp Med
176:593, 1992[Abstract/Free Full Text]
46.
Nakano T, Raines EW, Abraham JA, Klagsbrun M, Ross R:
Lysophosphatidylcholine upregulates the level of heparin-binding epidermal growth factor-like growth factor mRNA in human monocytes.
Proc Natl Acad Sci USA
91:1069, 1994[Abstract/Free Full Text]
47.
Bazzoni F, Cassatella MA, Laudanna C, Rossi F:
Phagocytosis of opsonized yeast induces TNF mRNA accumulation and protein release by human polymorphonuclear leukocytes.
J Leukoc Biol
50:223, 1991[Abstract]
48.
Wang P, Wu P, Anthes JC, Siegel MI, Egan RW, Billah MM:
Interleukin-10 inhibits interleukin-8 production in human neutrophils.
Blood
83:2678, 1994[Abstract/Free Full Text]
49.
Liles WC, Klebanoff SJ:
Regulation of apoptosis in neutrophils Fast track to death?
J Immunol
155:3289, 1995[Medline]
[Order article via Infotrieve]
50.
Cassatella MA, Berton G:
Modulation of neutrophil functions by IFN, in
Baron S
(ed):
Interferon Principles and Application. Galveston, TX, University of Texas Medical Branch, 1992, p 387
51.
Mege JL, Gomez-Cambronero J, Molski TF, Becker EL, Sha'afi RI:
Effect of granulocyte-macrophage colony-stimulating factor on superoxide production in cytoplasts and intact human neutrophils: Role of protein kinase and G-proteins.
J Leukoc Biol
46:161, 1989[Abstract]
52.
Davis-Fleischer KM, Besner GE:
Structure and function of heparin-binding EGF-like growth factor (HB-EGF).
Front Biosci
3:288, 1998
53.
McCarthy SA, Samuels ML, Pritchard CA, Abraham JA, McMahon M:
Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes.
Genes Dev
9:1953, 1995[Abstract/Free Full Text]
54.
Kerkhoff E, Rapp UR:
Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1.
Mol Cell Biol
17:2576, 1997[Abstract]
55.
Kwon EM, Sakamoto KM:
The molecular mechanism of action of granulocyte-macrophage colony-stimulating factor.
J Invest Med
44:442, 1996[Medline]
[Order article via Infotrieve]
56.
Armitage JO:
Emerging applications of recombinant human granulocyte-macrophage colony-stimulating factor.
Blood
92:4491, 1998[Free Full Text]
57.
McDonald PP, Bald A, Cassatella MA:
Activation of the NF-kB pathway by inflammatory stimuli in human neutrophils.
Blood
89:3421, 1997[Abstract/Free Full Text]
58.
Zhang Z, Funk C, Glasser SR, Mulholland J:
Progesterone regulation of heparin-binding epidermal growth factor-like growth factor gene expression during sensitization and decidualization in the rat uterus: Effects of the antiprogestin, ZK 98.299.
Endocrinology
135:1256, 1994[Abstract]
59.
Das SK, Wang XN, Paria BC, Damm D, Abraham JA, Klagsbrun M, Andrews GK, Dey SK:
Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: A possible ligand for interaction with blastocyst EGF-receptor in implantation.
Development
120:1071, 1994[Abstract]
60.
Marikovsky M, Breuing K, Liu PY, Eriksson E, Higashiyama S, Farber P, Abraham JA, Klagsbrun M:
Appearance of heparin-binding EGF-like growth factor in wound fluid as a response to injury.
Proc Natl Acad Sci USA
90:3889, 1993[Abstract/Free Full Text]
61.
Miyagawa J, Higashiyama S, Kawata S, Inui Y, Tamura S, Yamamoto K, Nishida M, Nakamura T, Yamashita S, Matsuzawa Y, Taniguchi N:
Localization of heparin-binding EGF-like growth factor in the smooth muscle cells and macrophages of human atherosclerotic plaques.
J Clin Invest
95:404, 1995
62.
Yoshida M, Matsuzaki H, Sakata K, Takeya M, Kato K, Mizushima S, Kawakita M, Takatsuki K:
Neutrophil chemotactic factors produced by a cell line from thyroid carcinoma.
Cancer Res
52:464, 1992[Abstract/Free Full Text]
63.
Pak AS, Wright MA, Matthews JP, Collins SL, Petruzzelli GJ, Young MRI:
Mechanisms of immune suppression in patients with head and neck cancer: Presence of CD34(+) cells which suppress immune functions within cancers that secrete granulocyte-macrophage colony-stimulating factor.
Clin Cancer Res
1:95, 1995[Abstract/Free Full Text]
64.
Lahn M, Fisch P, Kohler G, Kunzmann R, Hentrich I, Jesuiter H, Behringer D, Muschal B, Veelken H, Kulmburg P, Ikle DN, Lindemann A:
Pro-inflammatory and T cell inhibitory cytokines are secreted at high levels in tumor cell cultures of human renal cell carcinoma.
Eur Urol
35:70, 1999[Medline]
[Order article via Infotrieve]
65.
Moon DC, Nakayama J, Urabe A, Terao H, Kinoshita N, Hori Y:
Immunohistochemical characterization of cellular infiltrates in epidermal tumors induced by two-stage and complete chemical carcinogenesis in mouse skin.
Dermatology
19:146, 1992
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ABSTRACT
INTRODUCTION