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Departments of Pediatrics, Microbiology and Immunology, and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
Correspondence: Dr. Andrew C. Issekutz, Department of Pediatrics, IWK Health Centre, 5850 University Ave., Halifax, Nova Scotia, Canada B3J 3G9. E-mail: Andrew.Issekutz{at}iwk.nshealth.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, C5a, or IL-8. None of these inhibitory effects was observed
with VEGF. The acute effects of bFGF and VEGF may facilitate PMNL
emigration during acute inflammation, but continued bFGF production may
have anti-inflammatory actions during chronic inflammation,
angiogenesis, and tumor defense by inhibition of endothelial activation
for leukocyte recruitment.
Key Words: bFGF • VEGF • leukocyte • endothelium • adhesion molecule
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During inflammation, the cytokines tumor necrosis factor- (TNF-),
interleukin (IL)-1, and interferon-
(IFN) have been shown to
induce leukocyte infiltration in part by regulating the expression of
leukocyte adhesion molecules on vascular endothelial cells
[1
]. It is well established that the recruitment and
emigration of circulating leukocytes are dependent on a multistep
cascade of events involving leukocyte tethering, rolling, firm
adhesion, and emigration that are mediated by distinct adhesion
molecules and activation pathways [1
, 8
].
The selectins (L-, P-, and E-selectin) and 4-integrins (4ß1 and
4ß7) [8
, 9
] mediate rolling of
leukocytes in postcapillary venules of the circulatory system. Firm
adhesion and emigration of rolling leukocytes such as neutrophils
[polymorphonuclear leukocytes (PMNLs)] are mostly dependent on two
members of the CD18 (ß2)-integrin family—CD11a/CD18 [lymphocyte
function-associated antigen-1 (LFA-1)] and CD11b/CD18 (Mac-1)—on the
PMNL surface and intercellular adhesion molecules (ICAM)-1 and ICAM-2
on the endothelium [10
11
12
]. These findings suggest that
the modulation of cell adhesion molecule expression on the endothelium
can influence the trafficking of leukocytes into tissues.
In comparison with our understanding of the mechanisms in normal vessels, relatively little is known about factors that regulate PMNLs or other leukocyte adhesion and transmigration across angiogenic vessels in inflammation or in tumors. Recent studies in vivo have suggested that rolling, adhesion, and transmigration of leukocytes in angiogenic blood vessels may be impaired [13 , 14 ]. Previous studies have shown that many tumor cells produce bFGF and VEGF [15 , 16 ], and the freshly isolated tumor endothelium exhibits decreased expression of ICAM-1 compared with the endothelium in normal tissue [13 ]. Furthermore, endothelial vascular cell adhesion molecule 1 (VCAM-1) expression is suppressed in melanomas and carcinomas [17 ]. High levels of endothelial growth factors are often found in plasma of patients with various kinds of tumors [15 , 16 , 18 ]. The potential impact of these factors on leukocyte-endothelial cell adhesion and emigration has not been fully evaluated. Griffioen et al. [19 ] have observed decreased induction of ICAM-1, VCAM-1, and E-selectin on human umbilical vein endothelial cells (HUVECs) after bFGF or VEGF treatment. However, these studies did not investigate the effects on PMNL adhesion or transendothelial migration (TEM) or whether these effects could be overcome. To better define functional effects of bFGF and VEGF on endothelial cell function in inflammation, we examined the effects of these growth factors on basal and inflammatory cytokine-stimulated leukocyte adhesion molecule expression on HUVECs and the impact of modulated endothelial responses on PMNL adhesion and TEM.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was from Genentech, Inc. (South San Francisco, CA).
IFN was from InterMune Pharmaceutical Inc. (Palo Alto, CA). IL-1
was a gift from Immunex Corp. (Seattle, WA). Human serum albumin (HSA)
(pyrogen-free) was from Connaught Laboratories (Downsview, Ontario,
Canada). The antibodies used were mouse monoclonal antibody (mAb) R6.5
to ICAM-1 and mAb CBRM-IC2/2 to ICAM-2 (gifts from T. A. Springer,
Boston, MA), mAb 4B9 to VCAM-1 and mAb BB11 to E-selectin (gifts from
R. Lobb, Biogen Inc., Cambridge, MA), mAb 5H2 to Platelet endothelial
cell adhesion molecule (PECAM)-1 (generated in our laboratory), mAb BV6
human vascular-endothelial (VE)-cadherin (Chemicon Int., Temecula, CA),
and mAb 3H11B9 to pertussis toxin (gift from T. Issekutz, Halifax, Nova
Scotia, Canada). Peroxidase-conjugated goat anti-mouse immunoglobulin
(Ig) G was from Bio-Can Scientific (Mississauga, Ontario, Canada).
Fluorescein isothiocyanate (FITC)-conjugated sheep F(ab)2
anti-mouse IgG was from Sigma Chemical Co. (St. Louis, MO).
Human PMNL purification
Human PMNLs were purified as described previously
[20
] from acid citrate dextrose–heparin-anticoagulated
venous blood of healthy donors. Briefly, red cells were sedimented with
6% dextran-saline (Abbott Laboratories, Montréal, Québec,
Canada), leukocyte-rich plasma was collected, and leukocytes were
labeled with Na251CrO4 (Amersham,
Oakville, Ontario, Canada). The PMNLs were then purified by
discontinuous Percoll gradient centrifugation, washed, and resuspended
to 106/mL in RPMI 1640 supplemented with 0.5% HSA and 10
mM HEPES (pH 7.4). This method yielded PMNLs of 
95% purity, with
essentially no red cell contamination, and 98% cell viability.
Endothelial cell cultures
HUVECs were isolated and cultured in flasks as described
elsewhere [21
] and grown on filters as previously
described [20
]. Briefly, endothelial cells were isolated
from umbilical cords after treatment with 0.5 mg/mL of collagenase
(Cooper Biomedical, Mississauga, Ontario, Canada) in 0.01 M
phosphate-buffered saline (pH 7.4) and grown in basal medium composed
of RPMI 1640 (Sigma) containing 2 mM L-glutamine,
2-mercaptoethanol, sodium pyruvate, and penicillin-streptomycin and
supplemented with 20% heat-inactivated human AB serum. This is
referred to as "basal medium." To establish the cultures,
endothelial cell growth supplement (ECGS) (12.5 µg/mL) (Collaborative
Research, Lexington, MA) and 45 µg/mL of heparin (Sigma) were added
to the basal medium, and this is referred to as "growth medium."
Cells were cultured in 2% gelatin-coated culture flasks (NUNC, Life
Technologies, Mississauga, Ontario, Canada). The HUVECs were harvested
using 0.025% trypsin and 0.01% EDTA (Sigma) and cultured on
polyvinylpyrrolidone-free polycarbonate filters bearing
5-µm-diameter pores in Transwell culture plate inserts
(6.5-mm diameter) (Costar, Cambridge, MA) up to the third passage. The
filters were first prepared by coating with 0.01% gelatin (Difco Inc.,
Detroit, MI) (at 37°C for 18 h) followed by application of 3
µg of human fibronectin (Collaborative Research) in 50 µL of water
at 37°C for 2 h. Fibronectin was then replaced by HUVECs
(1.5x104 or 2.5x104 in basal medium
with or without the specifically indicated growth factor,
respectively), 0.1 mL of HUVECs was added to the compartment above the
filter, and 0.6 mL of basal medium was added to the compartment beneath
the filter. The HUVECs formed a tight permeability barrier in 5–6 days
and were evaluated for barrier function by 125I-labeled HSA
diffusion as previously described [20
]. Under all
conditions, <1.5% of labeled HSA diffused across the HUVEC filter
unit in 45 min with 1-mm positive hydrostatic pressure, but
bare filters showed 30% diffusion of 125I-labeled HSA in
this test.
Quantification of adhesion molecule expression on HUVECs by ELISA
and flow cytometry
The expression of ICAM-1, VCAM-1, and E-selectin on HUVECs was
determined with whole-cell enzyme-linked immunosorbent assay (ELISA) as
described previously with minor modifications [22
].
Briefly, HUVEC monolayers in 96-well plates were incubated with
(1.2x104 cells/well) or without (2x104
cells/well) a specific growth factor at various concentrations for
various periods. In some experiments, the HUVEC monolayers were treated
with TNF- or IL-1 for 4 h. The stimuli were then removed by
washing, and 100 µL of RPMI 1640-5% fetal calf serum (FCS)-0.1%
NaN3 containing mAb to ICAM-1, to VCAM-1, to E-selectin, or
control mAb was added. After 60 min (37°C in 5% CO2),
the monolayers were washed four times, and then 100 µL of
peroxidase-conjugated goat anti-mouse IgG (1:4,000 in RPMI 1640-5%
FCS) was added for 60 min (37°C in 5% CO2). The
monolayers were washed four times, and then 100 µL of substrate
[o-phenylendiamine, 12.5 mg/mL; 0.1 M citrate-phosphate
buffer (pH 5); and 0.012% H2O2] were added.
The enzyme reaction was stopped by adding 100 µL of 4N
H2SO4, and absorbance at 490 nm was measured.
Results are expressed as optical density (OD) x 1,000.
The expression of endothelial adhesion molecules was also determined by immunofluorescence flow cytometry using a standard immunofluorescence protocol [23 ]. Briefly, HUVECs were detached by brief treatment with 0.01% trypsin and 0.02% EDTA. Cell surface expression of ICAM-1, VCAM-1, and E-selectin was assessed using mAbs R6.5, 4B9, and BB11 (5µg/mL each), respectively. Binding was assessed by secondary detection with FITC conjugated to sheep F(ab)2 anti-mouse IgG. Nonspecific fluorescence was assessed by substituting a nonbinding isotype-matched control mAb (3H11B9) for the primary mAb. Analysis was performed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The results are expressed as fluorescence histograms plotted on a log scale.
PMNL adhesion and TEM assay
HUVECs were cultured on gelatin- and fibronectin-coated
polycarbonate filters with (1.5x104cells/well) or without
(2.5x104 cells/well) a growth factor for 6 days. Migration
assays were performed as described previously [20
,
22
]. Briefly, HUVEC monolayers on the filters and the
lower compartments were washed with RPMI 1640 and then were transferred
to a new, clean well (lower compartment). To this well, 0.6 mL of RPMI
1640 supplemented with 10 mM HEPES and 0.5% HSA containing the
chemotactic stimulus [complement fragment 5a (C5a) or IL-8] were
added. Before immersion of the HUVEC filter unit, 0.1 mL of medium
containing 105 labeled PMNLs was added above the HUVECs.
After incubation (75 min at 37°C in 5% CO2), migration
was stopped by washing the upper compartment twice with 0.1 mL of RPMI
1640 to remove nonadherent PMNLs. The undersurface of the filter was
wiped with a cotton swab saturated with an ice-cold phosphate-buffered
saline-0.2% EDTA solution, and this was added to the lower
compartment. The cells that spontaneously detached from the
undersurface of the filter or were removed by the swab were lysed by
adding 0.5% Triton X-100, and all the 51Cr released in the
lower compartment and on the swab was quantified with an LKB 1280
spectrometer (Fisher Scientific Co., Dartmouth, Nova Scotia, Canada).
The results are expressed as the percentage of the total
51Cr-labeled PMNLs added above the HUVECs that migrated
through the HUVEC filter unit.
The PMNL adhesion to HUVECs was quantified by lysis with 0.5 N NaOH of the 51Cr-labeled PMNLs that remained on the HUVEC monolayer after three washes of the monolayer/filter unit with warm RPMI 1640. The 51Cr in this NaOH lysate was quantified and expressed as the percentage of the total 51Cr-labeled PMNLs added above the HUVECs that adhered on the HUVEC monolayer. All experiments were performed in triplicate.
Statistical analysis
A one-way analysis of variance, Student’s t-test, or
paired t-test was used for statistical analysis of the data
as indicated. P values of >0.05 were not considered
significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, or
IFN stimulation of the endothelial cells in vitro and in vivo is
well documented [1
, 2
]. We therefore
examined the effect of these angiogenic factors on up-regulation of
adhesion molecules on HUVECs by these proinflammatory cytokines. HUVECs
were treated with 20 ng/mL of bFGF or VEGF for 3 days until they were
confluent, and then the cells were stimulated with 50 U/mL of TNF-
or 0.25 ng/mL of IL-1 for 4 h or with 300 U/mL of IFN for
20 h. As shown in Figure 3
, bFGF significantly inhibited the up-regulation of ICAM-1, VCAM-1,
and E-selectin on TNF--, IL-1-, or IFN-stimulated HUVECs. The
suppressive effect of bFGF on TNF-- or IL-1-induced adhesion
molecule expression was overcome by high concentrations of TNF- (200
U/mL) or IL-1 (1 ng/mL) (data not shown). In contrast to bFGF, high
or low doses of VEGF had no effect on up-regulation of these adhesion
molecules (data not shown).
|
Flow cytometry analysis of adhesion molecule expression on bFGF-
and VEGF-treated endothelial cells
Adhesion molecule expression on HUVECs was also quantified by
immunofluorescence flow cytometry to allow analysis of expression on
individual cells rather than on the whole HUVEC population as in the
ELISA system. The effect of bFGF and VEGF on expression of ICAM-1,
VCAM-1, and E-selectin on resting as well as cytokine-stimulated HUVECs
was determined as shown in Figure 4
. For each assay, 5,000 cells were gated. The results confirmed the
ELISA results and indicated that not the cell number but rather the
level of ICAM-1, VCAM-1, and E-selectin expression on the whole cell
population is altered by bFGF or VEGF treatment of HUVECs (Fig. 4)
.
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- or IL-1-stimulated endothelial monolayers. After 6 days’
pretreatment of HUVECs with bFGF, there was significant inhibition of
IL-8 (1.2x10-9 M)- and C5a (5x10-10
M)-enhanced PMNL migration across TNF- (25 U/mL)- or IL-1 (0.25
ng/mL)-activated HUVECs (Fig. 7
). VEGF was not observed to elicit such an effect (data not shown).
Treatment with bFGF or VEGF for 18 h did not have a significant
effect on TNF-- or IL-1-induced PMNL adhesion or TEM (data not
shown).
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. Figure 8
shows that on resting HUVECs (with no IL-1 or TNF-
stimulation), increasing the concentrations of IL-8 and C5a could
normalize PMNL TEM. Figure 9
shows similarly that at submaximal concentrations of IL-8 and C5a,
increased TNF- stimulation at concentrations of 100 U/mL overcame
the bFGF-induced inhibition of PMNL TEM.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated endothelium (Fig. 5)
and a
marked inhibition of PMNL transmigration across resting or IL-1- or
TNF--activated HUVECs, even in response to potent chemotactic
factors (IL-8 and C5a) (Fig. 6
and 7)
. These effects of bFGF were
unrelated to its endothelium-growth-promoting effects because prolonged
VEGF stimulation did not affect adhesion molecule expression or PMNL
TEM, despite the fact that VEGF and bFGF had similar initial effects on
these parameters (Fig. 1)
and growth-promoting effects (data not
shown). These effects of bFGF and VEGF on endothelial adhesion
molecules and PMNL TEM were likely observed because the HUVECs used in
the assays were grown without supplemental growth factors such as ECGS,
which is commonly used to culture HUVECs. This was possible because
HUVECs cultured in 20% human AB serum at an 
50% confluent seeding
density did not require additional growth factors to attain confluence,
something which could not be consistently achieved in 20% FCS
(unpublished observations). We observed that culture with ECGS caused
many of the same effects on adhesion molecule expression and TEM as did
bFGF (H. Zhang and A. C. Issekutz, unpublished results).
Stimulation of endothelial cells with TNF- or IL-1 increases PMNL
adhesion and TEM due to up-regulated expression of ICAM-1 and
E-selectin [26
, 27
]. Our results suggest
that bFGF decreases the sensitivity of the endothelium to these
inflammatory cytokines, because TNF-- or IL-1-induced ICAM-1,
VCAM-1, and E-selectin expression was always diminished by the presence
of bFGF for longer than 24 h. These observations are in agreement
with previous studies [19
]. However, we also showed that
even the low-level constitutive expression of these molecules was
markedly suppressed by bFGF (Fig. 3
and 4)
. Such an effect of bFGF,
which is produced by many tumors [31
, 32
],
may interfere with immune and inflammatory/effector cell infiltration
and attack on tumor cells. Our results indicate that the potent effect
of bFGF in impairing PMNL TEM can be overcome by increasing
concentrations of chemoattractant or TNF- (Fig. 8
and 9)
, suggesting
that the antitumor effect of TNF- may be partially due to TNF-’s
antagonism of this anti-inflammatory effect. This concept is supported
by the in vivo observation in tumor microcirculation that the levels of
leukocyte rolling and adhesion were significantly lower in tumor
vessels than in normal vessels, even after stimulation with the
chemoattractant, formyl-Met-Leu-Phe or endotoxin (lipopolysaccharide)
[33
, 34
]. Likewise, in a murine model of
tumor angiogenesis, leukocytes failed to roll in significant numbers in
tumor vessels of nude mice, while activation with TNF- resulted in
an increase in leukocyte rolling in these vessels [35
].
Borgstrom et al. [14
] made similar observations and
demonstrated that lymphotoxin plus leukotriene B4
stimulation could induce leukocyte adhesion and emigration. However, a
comparison with the response in normal vessels was not made. These
effects may be explained by our results indicating that high doses of
TNF- (200 U/mL) could overcome the bFGF-induced down-regulation of
E-selectin, ICAM-1, and VCAM-1. Previous studies have shown that
freshly isolated endothelia derived from the vasculature of human solid
tumors exhibit a decreased expression of ICAM-1 compared with
endothelial cells derived from normal tissue vessels
[13
], and endothelial VCAM-1 expression is suppressed by
melanoma and carcinoma [17
]. VCAM-1 expression was also
noted to be diminished by tumors on small but not on large vessels
[36
], which is relevant because leukocyte emigration
occurs primarily in the small postcapillary venules. Thus, the high
circulating level of bFGF detected in patients with different cancers
may well have an anti-inflammatory and antitumor role by the
suppression of endothelial adhesion molecule expression and leukocyte
TEM, as shown here and suggested in the above-mentioned studies
[15
, 16
, 18
, 31
,
32
, 37
, 38
].
In conclusion, our results show that rapid transient up-regulation of ICAM-1 and VCAM-1 induced by bFGF and VEGF could promote PMNL recruitment but that bFGF also has a biphasic effect, resulting in a strong inhibition of basal and stimulated endothelial cell expression of ICAM-1, VCAM-1, and E-selectin. This is accompanied by a marked decrease in endothelial cell-PMNL adhesive interaction and the ability of the endothelium to facilitate PMNL transmigration. These results emphasize the potent modulatory effect of bFGF in regulating leukocyte recruitment at sites of tumor growth, acute or chronic inflammation, and angiogenesis. A better understanding of regulation of the endothelium by growth factors may facilitate development of novel therapeutic strategies in cancer as well as inflammatory and vascular diseases.
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ACKNOWLEDGEMENTS |
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Received December 10, 2000; revised April 4, 2001; accepted April 5, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3-mediated human endothelial cell adhesion and calcium signaling Exp. Cell Res. 251,57-66[Medline]
4 Integrins mediate lymphocyte attachment and rolling under physiologic flow Cell 80,413-422[Medline]
Immunology 79,600-607[Medline]
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