1. Art and Diseño

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Migration of Human Monocytes in Response to Vascular Endothelial Growth
Factor (VEGF) Is Mediated via the VEGF Receptor flt-1
By Bernhard Barleon, Silvano Sozzani, Dan Zhou, Herbert A. Weich, Alberto Mantovani, and Dieter Marme
Treatment of human monocytes with vascular endothelial
growth factor (VEGF) isolated from tumor cell supernatants
was reported t o induce monocyte activation and migration.
In this study we show that recombinant human VEGFle6 and
VEGF,, had a maximal effect on human monocyte migration
was
at 65 t o 250 pmol/L. Chemotactic activity of VEGF,,
inhibited by a specific antiserum against VEGF, by heat treatment of VEGFle6, and by protein kinase inhibitors. In addition, we could show that VEGF-stimulated monocyte migration is mediated by a pertussis toxin-sensitive GTP-binding
protein. Placenta growth factor (PIGFIU), a heparin-binding
growth factor related t o VEGF, was also chemotactic for
monocytes at concentrations between 2.5 and 25 pmol/L. In
accordance with these findings, human monocytes showed
specific and saturable binding for '251-VEGFls6 (half-maximal
binding at 1 t o 1.5 nmol/L). Using Northern blot analysis,
we further could show that human monocytes express only
the gene for the VEGF receptor type, flt-1, but not for the
second known VEGF receptor, KDR. Resting monocytes expressed low levels of flt-1 gene only. Brief exposure (2 t o 4
hours) of human monocytes t o lipopolysaccharids, a prototypic monocyte activator, led t o a significant upregulation
of the flt-1 mRNA level. The results presented here suggest
that monocyte chemotaxis in response t o VEGF and most
likely t o PlGF19 is mediated by flt-1 and thus show a possible
function for the VEGF-receptor flt-1.
0 1996 by The American Society of Hematology.
R
ECRUITMENT OF mononuclear phagocytes from the
blood compartment into tissues is a crucial process
in inflammatory reactions and immune response. Monocyte
extravasation involves interaction with the vascular endothelium and response to tissue- and/or vessel wall-derived molecules. The identification of adhesion molecules, whose expression is upregulated on endothelial cells, and of novel
cytokines has considerably improved the understanding of
factors involved in the regulation of monocyte migration,
localization, and activation in tissues.
Vascular endothelial growth factor (VEGF) is a mitogen
specific for endothelial cells. VEGF has been discovered in
and purified from media conditioned by several tumor cell
lines and bovine pituitary folliculo-stellate cells. Molecular
cloning of the cDNA suggests that in human cells at least
four species of VEGF can occur as 121, 165, 189, and 206
amino acid forms, generated by alternative splicing (for a
review, see Senger et all). It has been shown that VEGFlhS
induces monocyte activation manifested by the induction
of tissue factor and monocyte chemotaxis.' Another growth
factor, related to the VEGF family, is the recently identified
placenta growth factor (PIGF), of which two forms, PIGF,,,
and P1GFIs2, are known so far. The overall homology between the amino acid sequences of P1GFlS2and VEGF189is
about 40%, but, if the conservative amino acid changes are
considered, the sequence homology increases to about 67%.
Like VEGF, PlGF is a secreted, dimeric, and N-glycosylated
pr~tein.'.~
Because of the structural and biochemical similarities between VEGF and PlGF, it was suggested that the
latter might also be an angiogenic f a ~ t o r .However,
~
the
physiologic functions of PlGF are almost completely unknown.
High-affinity binding sites for VEGF were mainly found
on endothelial c e k 5 Recently, Shen et al' could show highaffinity binding sites for VEGFlh on human mononuclear
phagocytes indicating the expression of a VEGF receptor
gene. Two receptor tyrosine kinases encoded by the flt- 1
gene7,xand KDWflk-l gene','" have been identified as specific VEGF receptors. Expression of flt-1 and KDFUflk- 1 was
predominantly found in vascular endothelial cells.' ' KDW
flk- 1, when transfected into endothelial cells or nonendothelial cells, mediates proliferating activity.'"," The function of
the second VEGF receptor, flt-1, remains unclear since flt1 transfected cells did not show proliferation or other responses upon exposure to VEGF."." A third member of the
VEGF-receptor subfamily is the recently cloned receptor
tyrosine kinase Flt-4,I4 but the ligand for this receptor is not
yet clearly identified.
In this study, we have investigated the chemotactic properties of VEGFlhSrVEGFlzl,and PlGFls2on human monocytes
and found that all three growth factors induce migration in
a dose-dependent manner. Moreover, we show, for the first
time, that the VEGF-induced chemotactic response on human monocytes is mediated via the VEGF-receptor flt-1.
From the Institute of Molecular Medicine, Tumor Biology Center,
Freiburg, Germany; the Department of Gene Expression, Braunschweig, Germany; and the Institut di Ricerche Farmacologiche
Mario Negri, Milan, Italy.
Submitted June 30, 1995: accepted December 6, 1995.
B.B. and S.S. contributed equally to this work.
Address reprint requests to Dieter Marmi, PhD, Institute of Molecular Medicine, Tumor Biology Center, Breisacherstr. 1 17, D79106 Freiburg, Germany.
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
indicute this fact.
0 1996 bv The American Society of Hematology.
Reagents. Human recombinant VEGF,6s, VEGF,,,, and PIGFls2
were expressed in the baculovirus system, purified, and tested for
biologic activity as described.'.l5 fMLP was from Sigma Chemical
Co (St Louis, MO). C-I was a kind gift from Dr C.E. McCall (Bowman Gray School of Medicine, Winston-Salem, NC). Genistein and
pertussis toxin (PTox) were from Calbiochem (San Diego, CA).
Bacterial lipopolysaccharid (LPS D,, Escherichia coli 055:B5) was
from Difco Labs (Detroit, MI).
Cell lines. Primary human umbilical vein endothelial cells (HUVEC) were purchased from Promocell (Heidelberg, Germany) and
cultured according to the provided protocol. Human smooth muscle
cells were isolated and cultured as described."
Isolation of humun monocytes and neutrophils (PMN). Peripheral blood mononuclear cells (PBMC) were obtained from buffy
coats of blood from normal donors courtesy of the Transfu-
MATERIALS AND METHODS
0006-4971/96/8708-0028$3.00/0
3336
Blood, Vol 87, No 8 (April 15), 1996:pp 3336-3343
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3337
VEGF-INDUCED MIGRATION OF MONOCYTES VIA FLT-1
sionsmedizin, University Hospital (Freiburg, Germany) and the
Centro Transfusionale, Ospedale Sacco (Milan, Italy). Isolation was
performed as described," and the viability of the cells was checked
by trypan blue exclusion immediately before the experiments (>
95%).
Chemotaxis assay. Cell migration was evaluated using a microchamber technique as de~cribed.'~,'~
Chemoattractant (27 ? 1 pL)
in RPMI-1640 with 1% fetal calf serum (FCS) was added to the
lower wells of a chemotaxis chamber (Nucleopore Corp, Pleasanton,
CA). Fifty microliters of cell suspension per well (1.5 X 106/mL
monocytes in PBMC, or PMN) were seeded in the upper wells.
The upper and lower wells were separated by a 5-pm pore size
polycarbonate filter (Nucleopore). Polyvinylpyrrolidone-free polycarbonate filters were used for PMN. The chambers were incubated
at 37°C in air with 5% CO, for 60 and 90 minutes for PMN and
monocytes, respectively. At the end of incubation, filters were removed and stained with Diff-Quik (Harleco, Gibbstown, NJ), and
five high power oil immersion fields were counted. In some experiments, a specific rabbit antipeptide antiserum for VEGF" was used
to adsorb the cytokine. Four hundred microliters of 10% Sepharose
protein G was incubated with 60 pL of anti-VEGF antibody (diluted
1:3 in phosphate-buffered saline [PBS]) at 4°C for 30 minutes. Sepharose was then washed 3 x with PBS and 100 pL of 10 ng/mL
VEGF165was added to the pellet and incubated at 4°C for 1 hour.
The supematants were then collected and used in the chemotaxis
assay. In some experiments, monocytes (1.5 X 106/mL in PBMC)
were preincubated with different concentrations of PTox at 37°C for
90 minutes. The cells were then washed twice, resuspended in RPMI
1% FCS, and tested in the migration assay.
VEGF receptor binding assay. Binding experiments were performed with recombinant human VEGF165on human monocytes.
The cells were resuspended in ice-cold binding buffer (Dulbecco's
modified Eagle's medium [DMEM], 25 mmol/L HEPES, 1 mg/mL
bovine serum albumin, pH 7.4) at a density of 2.5 to 6 X IO6 cells/
mL. One hundred microliters of the cell suspension was plated on
a 48-well culture disk (with flat bottom). After adding the labeled
and unlabeled VEGF165rthe final volume was 200 pL per well. The
binding was performed for 3 hours on ice with increasing concentrations of 'Z51-VEGF165.1'
Unspecific binding was determined for each
concentration by the addition of a 100-fold excess of unlabeled
ligand. For competition assays, the cells were incubated with a constant concentration of 1251-VEGF165
(20 ng/mL) and increasing
amounts of purified recombinant human VEGF or PlGF. Cells were
washed extensively with binding buffer and then solubilized with
0.3 m o m NaOH, 0.1% sodium dodecyl sulfate (SDS). Radioactivity
present in the lysates was quantified using a gamma counter (Beckmann, Munchen, Germany). Recombinant human VEGF165was iodinated to 30,000 to 136,000 cpm/ng using the chloramin T method."
Northern blot analysis. Total RNA was prepared from cells and
human placental tissue by a guanidinekaesium chloride centrifugation method'" or by a modification of the acid guanidine thiocyanatephenol-chloroform extraction protocol." Northern blot analysis was
performed as described." For the flt-1 gene, hybridization was performed with a 1.05-kb cDNA fragment corresponding to a part of
the extracellular domain (nucleotide 172-1231) of the human cDNA
For KDR, hybridization was performed with a 1.4-kb
~equence.~."
cDNA fragment corresponding to a part of the extracellular domain
(nucleotide 360-1720) of the human cDNA sequence? For FLT-4,
hybridization was performed with a 1.2-kb cDNA fragment that
covers the transmembrane part, the full kinase domain, and a part
of the C-terminus (nucleotide 2534-3788).14
RESULTS
Chemotactic properties of VEGFIGs, VEGFI2], and
P1GFlS2. A first series of experiments was designed to char-
Table 1. Chemotactic Activity of VEGFla on Human Monocytes
and PMN
Migrated Cells
PMN
Monocytes
Control
Cell No.
96
Cell No.
%
21 t 1
100
40 t 2
100
31 t 3
51 ? 3*
81 -C 4*
71 t 3*
107 t 3*
147
242
385
338
509
40 t 6
41 t 4
63 t 4*
43 t 7
103 ? 2'
100
102
157
107
257
VEGFle5 (ng/mL)
0.1
1
10
100
fMLP ( I O nmol/L)
Different concentrations of VEGFls5 were supplied to the lower compartment of the chemotaxis chamber. The results are mean numbers
(?SDI of migrated cells in five oil fields. Each experiment was performed in triplicate.
P < .05 v migration to control medium.
acterize the chemotactic properties of VEGF165.Table 1
shows that VEGF165was able to induce migration of monocytes across polycarbonate filters in a dose-dependent manner. The maximal response was obtained at a concentration
of 220 pmoVL. At this concentration, the number of migrated
monocytes was about 65% of that observed with an optimal
concentration of fMLP (10 nmoVL) that was used as a reference chemoattractant, active on all leukocyte populations.
Table 1 also shows that, under similar experimental conditions, VEGF165was able to induce migration of PMN. The
dose-response curve obtained with PMN was sharper than
that observed with monocytes, although maximal migration
was obtained at the same concentration. At 220 pmoVL
VEGF165,PMN migration was about 45% of the response
observed in the presence of 10 nmoVL fLMP. Heat-inactivated VEGF165,as well as VEGF165preadsorbed on Sepharose-protein G beads coated with anti-VEGF antiserum, were
inactive (Fig 1A). These results indicate that vEGF165is able
to elicit a migratory response in human monocytes and PMN.
To clarify whether the migration of monocytes across filters depends on the presence of a VEGF165gradient between
the lower and the upper compartment, we performed checkerboard experiments using polycarbonate filters with the
number of migrated cells as the end point. As shown in
Table 2, maximal induction of migration occurred in the
presence of a positive concentration gradient between the
two compartments (higher concentration below the filter). In
the presence of equal concentrations of VEGF,,, above and
below the filter or of a negative gradient (higher concentration above the filter), no enhanced migration occurred. These
results indicate that VEGF165is able to activate a chemotactic
response in human monocytes with no appreciable chemokinetic activity.
The mechanism(s) involved in the induction of monocyte
chemotaxis after VEGF165stimulation is unknown. The
involvement of protein kinases was investigated by using
inhibitors. Figure 1B shows that both C-I and genistein,
inhibitors of threoninelserine kinases and tyrosine kinases,
respectively, cause a dose-dependent inhibition of monocyte
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3338
BARLEON ET AL
A
I
b C-I
1001
40
0
c
0
20
Control
I-I
Anti-VEGF
"
Preimmune
serum
Boiled VEGF
0
.1
1
10
100
1000
Inhibitors, uM
Fig 1. Effect of anti-VEGF antiserum, heat treatment, and protein kinase inhibitors on the monocyte chemotactic response to VEGFls6. (A)
Monocytes (1.5 x 10'lmL) were teated for their ability to migrate in response to 10 nglmL VEGFlffi preincubated with Sepharose-protein G
beads precoated with rabbit anti-VEGF antiserum (column 2) or preimmune serum (column 3). In column 4 the response to 10 ng/mL VEGFlw
boiled for 30 minutes is shown. Chemotactic activity is expresred as a percentage of monocyte migration at 10 ng/mL VEGFlw (column 1).
(B) Monocytes were preincubated at 37°C for 10 minutes with different concentrations of the inhibitors and then tested for their ability to
migrate in response to 10 nglmL VEGFlffi. Values represent the mean of three separate experiments ( S D ) and are expressed as a percentage
of the migration observed in the absence of inhibitors and in the presence of the respective solvents (saline and 0.01% DMSO for C-l and
genidein, respectively). Cell viability at the end of the assay was greater than 90%.
migration. Half-maximal inhibition was observed at 25
pmol/L for C-I and 0.4 pmol/L for genistein. Chemotaxis
induced by fLMP was blocked by similar concentrations of
both inhibitors (data not shown). Stimulation with KMP,
which acts through a seven-transmembrane domain receptor,23induces tyrosine phosphorylation, and this event appears to be important for the induction of cell migrati~n.'~
It was recently reported that the bFGF-induced chemotactic response in bovine aortic endothelial cells is mediated by
a pertussis toxin-sensitive GTP-binding protein.25We investigated the effect of PTox on VEGF-mediated human monocyte migration. Cells were preincubated with increasing concentrations of PTox and stimulated with optimal
concentration of chemoattractants. As shown in Fig 2,
VEGF- and fMLP-induced chemotactic responses were
blocked in a dose-dependent manner by PTox, whereas that
Table 2. Checkerboard Analysis of VEGFlw Stimulation of
Monocvte Migration
Lower
Compartment
Upper Compartment
VEGFqS6Ing/mL)
None
None
0.1
1
10
37 2 2
72 2 5*
79 2 2 t
11423t
0.1
38 2
43 2
41 2
46t
2
6
3
2t
1
10
36 c 2
3922
3422
3623
29 2 2*
3123
3255
37 2 4
Different concentrations of VEGF,= (in nanograms per milliliter)
were supplied to the upper and/or lower compartment of the chemoof migrated cells
taxis chamber. The results are mean numbers (+SD)
in five oil fields. Each experiment was performed in triplicate.
* P < .05 v migration to control medium (above and below the
filter).
t P c .01 v migration to control medium (above and below the
filter).
of phorbol 12-myristate 13-acetate (PMA) was unaffected.
This result suggests that the VEGF-induced migration of
human monocytes is mediated via a pertussis toxin-sensitive
GTP-binding protein.
We further compared the chemotactic properties of
VEGFIZland of the VEGF-related growth factor P1GFjS2
with those of VEGF165described above. Table 3 shows that
VEGF,,, as well as P1GF152were able to induce migration
of monocytes. The maximal response to VEGF,,, was observed at 65 pmol/L and to P1GF152at 7 pmoVL. At these
concentrations, the percentage of migrated monocytes was
about 50% for VEGF,,, and about 40% for P1GF152of that
observed at 10 nmol/L d M P . Although the maximal effects
to VEGFIzIand P1GF152were observed at lower concentrations as compared with VEGF165(65 pmol/L and 7 pmol/L,
respectively, as compared with 220 pmol/L), both factors
had a lower efficacy than VEGF165in that they induced the
migration of less cells.
Expression of VEGF receptors in human monocytes.
The presence of specific binding sites for '*'I-VEGFlh5on
human monocytes was investigated. In six different experiments we observed specific binding of VEGF165in the range
of 60% to 80% of total binding (Fig 3). Half-maximal binding occured at 1 to 1.5 nmol/L, which is 20-to 25-fold lower
than that observed for endothelial cells and threefold to fivefold lower than that observed on human monocytes by Shen
et al.h Because VEGF binds to and induces migration of
monocytes, it was of interest which of the two known VEGF
receptors is involved. Therefore, we performed Northern blot
analysis of total RNA from human monocytes and PMNs
using human cDNA sequences appropriate for the two VEGF
receptors flt-1 and KDR as well as for the related receptor,
FLT-4. The result is shown in Fig 4. Resting human monocytes express detectable levels of flt-1 mRNA but not of
KDR and Flt-4. The expression pattern of flt-1 is slightly
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3339
VEGF-INDUCED MIGRATION OF MONOCYTES VIA FLT-1
Table 3. Chemotactic Activity of VEGFlzl and PIGFlE2
on Human Monocytes
I
~
Migrated Cells
Cell No.
Control
VEGF,,, (nglmL)
1
3
10
50
PIGFIs2 (ng/mL)
0.03
0.1
0.3
1
fMLP (10 nmol/L)
"0
10
1
100
1000
PTox (ng/ml)
Fig 2. Effect of PTox pretreatment on monocyte chemotaxis in
response to VEGF,,. Monocytes (1.5 x 106/mL in PBMC) were incubated with different concentrations of PTox at 37°C for 90 minutes.
Cells were then washed twice, resuspendend in RPMl 1% FCS, and
tested in the migration assay. Chemoattractantr were used at their
optimal concentration: 220 pmol/L VEGFlss, 10 nmol/L fMLP, and 16
nmol/L PMA. Migration of monocytes incubated in the absence of
PTox was assumed to be 100% (42 2.90 3, and 51 f 4for VEGF,,,
fMLP, and PMA, respectively). Values are the means (+SD) of three
seperate experiments (each performed in triplicate) and are expressed as a percentage of activity.
*
*
different from that obtained with placental tissue and with
HUVECs." Three mRNA species of 7.5, 3.4, and 2.7 kb,
similar to those from placenta, could be detected. However,
in contrast to human placental tissue, in which the 3.4-kb
mRNA species is the dominant one, in monocytes the 2.7kb and 7.5-kb transcripts are the dominant mRNA species,
with a similar expression level. The 2.7-kb mRNA transcript
is too short to code for the full-length receptor mRNA and
probably encodes a truncated form of flt-l.26 Similar to
monocytes, PMNs also express only the gene for the flt-1
receptor type, but the expression is much weaker than that
observed in monocytes. PMNs show flt-1 transcripts of 7.5
kb and 2.7 kb but lack the 3.4-kb mRNA species (Fig 4).
PlCF is believed to act via the VEGF receptor flt-1. It has
been shown that PlGF binds to flt-127.28
and competes with
VEGF binding to flt-l",28 (Barleon and Martiny-Baron, unpublished data). We performed competition experiments
with 1251-VEGF16s
and P1GFIs2on human monocytes. Figure
5 shows the results. P1GF152is able to compete for the binding of 1251-VEGF165
with a similar efficiency as unlabeled
VEGF16s. Taken together, these data indicate that the VEGF
receptor flt-1 is expressed in human monocytes and PMNs
and that both VEGF and PlGF bind to this receptor. Thus,
it is likely that both factors induce the chemotactic response
by activating the VEGF receptor flt-1.
To investigate whether flt-1 gene expression in human
monocytes is upregulated by LPS, a prototypic monocyte
%
28 t 4
100
38
45
40
38
t 3'
t 2t
2 2
135
160
142
135
43 c 3 t
46 t 2 t
37 2 I t
29 2 3
94 c 2
153
164
132
103
335
2 4
Different concentrations of VEGF,,, and PIGFlS2 were supplied t o
the lower compartment of the chemotaxis chamber. The results are
means number (cSD) of migrated cells in five oil fields. Each experiment was performed i n triplicate.
* P > .01.
t P > .05.
activator:' monocytes were exposed to 100 ng/mL LPS for
2 to 4 hours. Figure 6 shows that flt-1 transcripts increase
after just 2 hours of stimulation; this stimulation was dose
dependent (data not shown). In preliminary experiments, we
observed that 16 hours of incubation of monocytes with 100
nglmL LPS resulted in a 1.5-fold to twofold increase in
specific binding of L251-VEGF165
as compared with untreated
cells. The calculated half-maximal binding was 0.4 to 0.6
nmol/L for LPS-treated and 1 to 1.5 nmoVL for untreated
monocytes. In addition, LPS-incubated monocytes showed
an increased ability to migrate in response to suboptimal
concentrations of VEGF165(data not shown).
Taken together, our results indicate that, in human monocytes, the migration signal induced by VEGF and most prob-
total binding
150A
0
specific binding
unspecific binding
C
1)
gs
m F j 1000,x
% E
82
,$ 5 0 c?
0
0
50
100
'2sI-VEGF16s
150
(ng/ml)
200
Fig 3. '261-VEGFl~
binding to human monocytes.Bindingwas performed on 6 x 10' cells in 48-well plat- for 3 houm at 4°C in the
presence of increaaing concentrationsof '9-VEGFl,. The nonspocific
binding was determined by adding 100-fold excess of unbbeled
VEGF,,.
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BARLEON ET AL
3340
A
I
(kW
7.5
*
28s
-
3.4
2.7
18s
(kb)
Otb)
5.8
*
28 s -
4.5
*
*
-
18s-
Fig 4. Expression of VEGF receptors in human monocytes and PMNs. Five micrograms of total RNA per lane was separated on an agarose
gel, transferred t o a nitrocellulose filter, and hybridized with specific cDNA probes. Lane 1, human neutrophils (PMNs); lane 2,human monocytes;
lane 3, human placenta; lane 4, HUVEC; lane 5, human smooth muscle cells (SMC). (A) For flt-1, hybridization was performed with a 1.05-kb
cDNA fragment. (B) For KDR, hybridization was performed with a 1.4-kb cDNA fragment. (C) For FLT-4, hybridization was performed with a
1.2-kb cDNA fragment. Exposure time was 7 days for flt-1 and 9 days for KDR and FLT-4 (with amplifier screen).
ably also that induced by PIGFIs2is mediated via the flt-l
receptor and, secondly, activation of human monocytes by
LPS causes a significant upregulation of the flt-1 mRNA
level and an increase of specific binding for VEGF.
DISCUSSION
Wound repair and immune response are essential biologic
processes that are driven by the cooperative interaction of
multiple cell types and mediator systems. The main cell
types seen in inflammatory responses are polymorphonuclear
neutrophilic leukocytes together with macrophages and their
precursor monocytes. Although numerous cell types are involved, monocyte-derived macrophages appear to play a piv-
I
20‘
0
100 150 200 250
Factor (fold-excess)
50
Fig 5. Competition of ’261-VEGF,ss binding t o human monocytes
by VEGFlss and PIGFlS2. Binding was performed on 2.5 x lo5 cells
in 24-well plates for 3 hours at 4°C in the presence of a constant
concentrations of “51-VEGFlss (20 ng/mL) and increasing amounts of
recombinant VEGFlss and PIGF152.
otal role in modulating the repair process. Although the role
of macrophages in wound repair is certainly multi-functional, several lines of evidence show that they are key regulators of new capillary growth or angiogenesis.’”,”
Previous studies have shown that VEGF165 induces directed migration of mononuclear phagocytes across an endothelial cell monolayer as well as their activation based on
expression of tissue factor procoagulant activity.’.‘ For
VEGFlh5,we observed a maximal response in migration of
human monocytes at 220 pmol/L, which is in the same range
as reported by Clauss et al’ and Shen et a1.6 Checkerboard
experiments with VEGFI6.(clearly showed that the migration
depends on the presence of a VEGFlhs gradient. VEGFlos
also induced a chemotactic response in PMNs but seems to
be less active on these cells. PMNs are another nonspecific
inflammatory cell type attracted in the early phase of an acute
inflammatory process. These results indicate that VEGF is
able to act on the two main cell types seen in inflammatory
responses. The mechanism(s) involved in the migratory response of leukocytes is not well understood. The current
view of chemoattractant signalling indicates a substantial
diversity in the structural elements and pathways involved.”
Shen et a16 showed that VEGFlh5,like most of the known
monocyte chemotactic factors, induces a transient increase
in cytosolic Ca”, which peaks at 30 seconds. In the present
study, we show that, as with other chemotactic agonists,”
the signal transduction pathway of VEGF16sin human monocytes involves both threoninekerine as well as tyrosine
kinases. Inhibitors of both types of kinases exhibit a dosedependent attenuation of monocyte migration. It was recently shown that PTox inhibits the bFGF-induced chemotactic response in bovine aortic endothelial cells, whereas
the bFGF-induced mitogenic response was unaffected.*’ This
result shows that distinct intracellular signalling mechanisms
are involved in the migratory and proliferative responses of
endothelial cells to bFGF and that the migratory signal is
mediated by a pertussis toxin-sensitive GTP-binding protein.
Heterotrimeric GTP-binding proteins are generally coupled
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3341
VEGF-INDUCED MIGRATION OF MONOCYTES VIA FLT-1
A
28 s-
B
-
flt-l
18 S -
28 s-
Eth.Br.
18 sFig 6. Influence of LPS on the flt-1 mRNA level in human monocytes. Human monocytes from two different donors (A and 6 ) were
plated at a density of 2.5 x l o 6 cells/mL and incubated with 100 ng/
mL LPS for 2 and 4 hours. Five micrograms of total RNA per lane
was separatedon an agarose gel, transferredto a nitrocellulosefilter,
and hybridizedwith a specific cDNA probe for the human flt-1 receptor gene (see the Materials and Methods). The ethidium bromidestained gel is also shown. The exposure time was 7 days (with amplifier screen).
to receptors containing seven transmembrane regions, eg,
the rhodopsin and adrenergic receptors3' However, recent
evidence has indicated that other types of receptors. such as
the insulin."' epidermal growth factor.'5 and the transforming
growth factor fl I '"receptors, are also coupled to pertussis
toxin-sensitive. 40- to 4 I -kD Gi-like G-proteins. In preliminary experiments, we could show that this is also the case for
the VEGF receptor flt- I , at least in monocytes. The VEGFlhSinduced chemotactic response was inhibited in a dose-dependent manner by PTox. We are currently investigating
whether the VEGF-induced migratory response in vascular
endothelial cells is also blocked by PTox.
The biologic functions of PlGF are still unclear. It has
been shown by some investigators that PlGF induces a mitogenic response in endothelial cells.',J This could not be confirmed by others'7 (Barleon, unpublished data). It was recently suggested that PlGF could amplify the action of
VEGF by displacing it from flt-l binding sites and thus
promoting binding to KDR." On the other hand, it was
shown that PIGF,,, and PIGF15?induce a chemotactic response in HUVECs with similar potency as VEGF1(,,." We
show here that PIGFIS2induces human monocyte migration
across polycarbonate filters in a dose-dependent manner. The
maximal response occured at concentrations somewhat
lower than those obtained with VEGF,,, (7 p m o l n for
P1GF152and 220 pmol/L for VEGF16,). However, the efficacy
of PlGF appears to be lower, inducing migration of less cells.
These results and those obtained by Weindel et aIJ7 show
that PlGF is able to induce a chemotactic response in human
monocytes as well as in endothelial cells, indicating clearly
a direct biologic activity of the PlGF protein.
The next question we addressed was which of the two
known VEGF receptors, KDR or flt-I, is responsible for
VEGF-induced activation of human monocytes and PMNs.
Northern blot analysis showed that both cell types express
only the gene for the flt-l receptor type but not for KDR.
This is in marked contrast to human vascular endothelial
cells, in which transcripts for both receptor types could be
detected." We suggest that the VEGF- and most probably
also the PIGF-induced migration of human monocytes is
mediated via the VEGF receptor flt- 1. This is supported by
the following findings. ( I ) PIGFlll and PIGFls2 bind with
high affinity to the flt-l receptor but not to the KDR receptor
t y ~ e ' ~ (Barleon
.'~
and Martiny-Baron, unpublished data). (2)
PIGFlzzis able to compete for "51-VEGF16sbinding to human
monocytes. (3) There exists only one high-affinity binding
site for VEGFlh5on human monocytes.h These results clearly
indicate that the migration signal of VEGF and most probably of PIGFls2 is mediated via the flt-l receptor in human
monocytes. However, the existence of another yet unidentified receptor for VEGF and/or PlGF in these cells cannot be
excluded.
There is unequivocal evidence that the KDR receptor type
is a signal transducing molecule mediating a mitogenic response,"'.'' whereas the function of the flt-I receptor type
remains unclear."." Transfected porcine aortic endothelial
cells (PAEC)." CHO cells, and NIH3T3 cellsX~"'expressing
flt-l do not show any mitogenic response upon exposure to
VEGF. In contrast, transfection of PAECs with human KDR
cDNA or transfection of NIH3T3 with the mouse homolog
flk-I cDNA resulted in VEGF-induced receptor autophosphorylation and a mitogenic
The results presented here show for the first time that, in human monocytes,
flt-l mediates the chemotactic response upon activation by
VEGF and PIGF, showing a function for the flt-l receptor.
Because corneal endothelial cells (BCECs) also express the
flt-l receptor type and respond to VEGF with chemotaxis,''"
it is suggested that the flt-l receptor might be responsible for
mediating the chemotactic signal in some cells (eg, BCECs,
monocytes, and PMN). whereas the KDR receptor type may
be responsible for the mitogenic and chemotactic response
in vascular endothelial cells. However, additional studies are
needed to support this hypothesis.
Another interesting point is the significant upregulation
of the flt-l mRNA level by LPS. a prototypic monocyte
activator.'" Whether this increase in flt- 1 transcripts is due
to a higher transcription rilte or to an increase in flt-1 mRNA
stability is not yet known. In preliminary experiments, we
found that the upregulation of the flt-I mRNA level by LPS
seems also to result in an increase in the specific binding
of 1'51-VEGF16sto human monocytes and in a potentiated
chemotactic response.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
3342
BARLEON ET AL
A role for macrophages in the vascularization and growth
of tumors is supported by a number of lines of evidence.
For instance, macrophages are known to infiltrate murine
and human tumors and mice depleted of monocytes show
a strong reduction of tumor vascularization i n implanted
syngenic fibrosarcomas.'" Tumor-associated macrophages
(TAMS) also elicite complex functions in the immunobiology of neoplastic t i s s ~ e sand
~ ~ have
. ~ ~ angiogenic activity.&
In addition, at least in certain tumors in which TAMS have
procoagulant activity, they provide a scaffold for formation
of new blood ves~els.4~
The results presented here show that
VEGF, commonly expressed in tumors, may contribute to
TAM recruitment and that activation signals (eg, LPS) can
upregulate the flt-1 receptor. Thus, the endothelial growth
factor VEGF and the putative endothelial growth factor PlGF
would contribute to angiogenesis of neoplastic and inflammatory tissues by acting directly on endothelial cells as well
as indirectly by favoring macrophage recruitment.
ACKNOWLEDGMENT
We thank Dr Bruce Terman for the human KDR probe, Dr Avnar
Yayon for the human flt-l probe, and Prof Kari Alitalo for the human
FLT-4probe. We also thank Drs Karin Weindel, Georg MartinyBaron, and Russ Butcher for carefully and critically reading the
manuscript.
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1996 87: 3336-3343
Migration of human monocytes in response to vascular endothelial
growth factor (VEGF) is mediated via the VEGF receptor flt-1
B Barleon, S Sozzani, D Zhou, HA Weich, A Mantovani and D Marme
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