From the Unit of General Pathology and Immunology,
Department of Biomedical Sciences and Biotechnology, University of
Brescia, Brescia 25123, Italy, the ¶ Istituto Ricerche
Farmacologiche Mario Negri, 20157 Milan, Italy, and the
Department of Immunology, Berlex Biosciences,
Richmond, California 94804
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ABSTRACT |
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The proinflammatory and chemoattractant chemokine interleukin-8 (IL-8) inhibits cell proliferation induced by basic fibroblast growth factor (bFGF) in mouse endothelial cells isolated from subcutaneous sponge implant (sponge-induced mouse endothelial cells) and in bovine aortic endothelial GM 7373 cells. The mechanism of action of IL-8 was investigated in GM 7373 cells. IL-8 did not prevent the binding of bFGF to its tyrosine kinase FGF receptors (FGFRs) nor to cell surface heparan sulfate proteoglycans (HSPGs). A transient interaction of IL-8 with the cell before the addition of the growth factor was sufficient to prevent bFGF activity. The inhibitory activity of IL-8 was abolished by protein kinase C (PKC) inhibitors and was mimicked by the PKC activator 12-O-tetradecanoylphorbol-13-acetate. Accordingly, both IL-8 and 12-O-tetradecanoylphorbol-13-acetate caused a ~60% decrease of the binding capacity of GM 7373 cells due to the down-regulation of FGFRs. Several C-X-C and C-C chemokines exerted an inhibitory action on bFGF activity similar to IL-8. Soluble heparin, 6-O-desulfated heparin, N-desulfated heparin, and heparan sulfate but not 2-O-desulfated heparin, chondroitin-4-sulfate, hyaluronic acid, and K5 polysaccharide abrogated IL-8 inhibitory activity consistently with the presence of low affinity, high capacity HSPG-like chemokine-binding sites on GM 7373 cells. Finally, neovascularization induced by bFGF in murine subcutaneous sponge implants was reduced significantly by IL-8. In conclusion, IL-8 inhibits the mitogenic activity exerted by bFGF on cultured endothelial cells by a PKC-dependent, noncompetitive mechanism of action that causes FGFR down-regulation. This activity is shared by several chemokines and requires endothelial cell surface HSPGs. The endothelial cell line utilized in the present study may help to elucidate the complex interplay among chemokines, HSPGs, growth factors, and receptors in endothelial cells.
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INTRODUCTION |
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The process of angiogenesis, in which new capillary blood vessels are formed, is strictly regulated in the adult, and the proliferation of endothelial cells is low compared with many other cell types. Notable exceptions to this tight regulation of angiogenesis are found in the female reproductive system and during wound healing. The consequences of uncontrolled endothelial cell proliferation manifest themselves in tumor neovascularization and in angioproliferative diseases. The control of angiogenesis is achieved by a balance between the levels of angiogenic and angiostatic factors that modulate neovascularization both in physiological and pathological conditions.
Several molecules including basic fibroblast growth factor (bFGF)1 have been demonstrated to be positive mediators of angiogenesis in vitro and in vivo (1). bFGF belongs to the FGF family that includes at least nine different gene products that share a high affinity for heparin and a partial amino acid sequence homology (2). bFGF exerts angiogenic activity in vivo and induces cell proliferation, chemotaxis, and protease production in cultured endothelial cells (3) by interacting with high affinity tyrosine kinase FGF receptors (FGFRs) and low affinity proteoglycans (HSPGs) containing heparan sulfate (HS) polysaccharides (4, 5). The interaction of bFGF with FGFRs is modulated by HSPGs leading to the formation of bFGF·HSPG·FGFR ternary complexes (5). When the binding of bFGF to low affinity HSPGs is prevented by treating the cells with heparinase or chlorate, the binding of bFGF to FGFRs is also reduced together with its ability to stimulate cell proliferation (6). In addition HSPGs may induce FGF(s) oligomerization, which facilitates FGFR dimerization and signal transduction (7).
Chemokines belong to a superfamily of low molecular weight chemotactic proteins that are active on different leukocyte populations. They are divided into three structural subclasses depending on whether the first two of the four invariant cysteine residues are adjacent (C-C chemokines), separated by an intervening residue (C-X-C chemokines) or whether the first cysteine is missing (C chemokines) (8-10). Moreover, C-X-C chemokines are subdivided further depending on the presence or the absence of the sequence Glu-Leu-Arg (the ELR motif) (8). Chemokine receptors are members of the G protein-coupled seven-transmembrane family, and they bind either C-C or C-X-C chemokines (11). An exception to this ligand specificity is the promiscuous Duffy antigen/erythrocyte chemokine receptor (DARC), which can bind members of both the C-X-C and C-C class (12). So far no physiological function for DARC has been established. Besides a high affinity receptor interaction, chemokines bind sulfated glycosaminoglycans (GAGs) including heparin and HS (13) and thus can interact with HSPGs of the cell membrane and extracellular matrix. HSPGs on endothelial cells have been shown to present some chemokines to leukocytes in the multistep process of recruitment (14).
Recent work indicates that various chemokines may affect endothelial
cell function. In particular, interleukin-8 (IL-8) and other
C-X-C chemokines that contain the ELR motif
(ELR+ C-X-C chemokines) have been reported to
induce endothelial cell chemotaxis and/or proliferation in
vitro and angiogenesis in vivo (15-17). In contrast,
platelet factor-4 (PF-4) and other C-X-C chemokines lacking
the ELR motif (ELR C-X-C chemokines) exert an
angiostatic activity in vivo and inhibit bFGF-induced
proliferation and migration in cultured endothelial cells (17, 18).
However, the molecular basis underlying the pro- and anti-angiogenic
activity of chemokines has not been clearly established, and some
contradictory findings have been reported (see "Discussion"). The
high degree of heterogeneity observed for endothelial cells of
different tissue origins isolated from different animal species and the
significant differences between large vessel and microvascular
endothelium (3, 4) may explain, at least in part, these discrepancies.
Also, the use of human umbilical vein endothelial cells (HUVECs)
obtained from different individuals and cultured under different
conditions probably underlies the conflicting and confusing evidence
available on the interaction of chemokines with endothelial cells (19).
Unclear results have been obtained also concerning the expression and
function of chemokine-binding sites in endothelial cells. DARC has been
detected in endothelial cells of post-capillary venules and splenic
sinusoids (20), whereas the presence of high affinity binding sites for
125I-IL-8, together with IL-8 receptor type II
mRNA expression, has been reported in human vascular endothelial
cells (21) but not confirmed by others (22). The capacity of
interferon-
-inducible protein-10 (IP-10) to share with PF-4 a
specific HSPG-binding site in SV-40-transformed murine endothelial
cells has been also demonstrated (23).
In this study we demonstrate that IL-8 inhibits bFGF-induced cell
proliferation in two out of six endothelial cell types tested. The bFGF
antagonist activity exerted by IL-8 on fetal bovine aortic endothelial
GM 7373 cells was due to a protein kinase C
(PKC)-dependent, noncompetitive mechanism of action whose
activation causes FGFR down-regulation. Various ELR+
C-X-C, ELR C-X-C, and C-C
chemokines share with IL-8 the capacity to inhibit bFGF activity in GM
7373 cells. A promiscuous HSPG-like chemokine-binding site was
identified on these cells by binding assays with radiolabeled IL-8,
growth-related oncogene-
/melanoma growth stimulating activity (GRO
/MGSA), and RANTES and by competition experiments with free GAGs. Finally, neovascularization induced by bFGF in murine
subcutaneous sponge implants was reduced significantly by IL-8, in
keeping with its capacity to inhibit the mitogenic activity exerted
in vitro by bFGF on mouse endothelial cells isolated from
these sponges.
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EXPERIMENTAL PROCEDURES |
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Materials
Human recombinant bFGF was expressed and purified from transformed Escherichia coli cells by heparin-Sepharose affinity chromatography (24). Phorbol esters, epidermal growth factor, dibutyryl cAMP, and 5'-methyl-thioadenosine were from Sigma. Suramin was from Bayer AG (Leverkuse, Germany). H-7 and H-8 were from Siekagaku America (Ijamsville, MD). Bisindolylmaleimide GF 109203X was a gift of Dr. M. Pizzi (Institute of Pharmacology, University of Brescia, Brescia, Italy). Chemokines were from PeproTech Inc. (Rocky Hill, NC). Heparin was obtained from a commercial batch preparation of unfractionated sodium heparin from beef mucosa (1131/900 from Laboratori Derivati Organici S.p.A., Milan, Italy). Type I heparan sulfate (HS) was from Opocrin (Corlo, Italy). K5 polysaccharide was a gift of Dr. G. Zoppetti (Glycosaminoglycan Consultants, Milan, Italy). Chondroitin-4-sulfate and hyaluronic acid were a gift of Dr. M. Del Rosso (University of Florence, Florence, Italy). 2-O-desulfated, 6-O-desulfated, totally O-desulfated, and N-desulfated heparins were provided by Dr. B. Casu (Ronzoni Institute, Milan, Italy). The characteristics of the GAGs utilized in the present study have been described elsewhere (25).
Cell Cultures
Fetal bovine aortic endothelial GM 7373 cells, corresponding to the described BFA-1c 1BPT clone (26), were obtained from the National Institute of General Medical Sciences, Human Genetic Mutual Cell Repository (Camden, NJ) and grown in Eagle's minimal essential medium (MEM) containing 10% fetal calf serum (FCS), vitamins, and essential and nonessential amino acids. Adult bovine aortic endothelial cells (BAECs) were grown in MEM containing 10% FCS and were utilized at passages 10 and 11. HUVECs were grown in M199 containing 100 µg/ml heparin, 150 µg/ml bovine brain extract, and 20% FCS and were utilized at passages 3 and 4. Balb/c mouse brain microvascular endothelial 10027 cells (MBECs) were provided by Dr. R. Auerbach (University of Wisconsin) and were grown in Dulbecco's modification of MEM added with 10% FCS (27). 1G11 cells, an endothelial cell line cloned from the lung of C57BL/6NCrLBR mice (28) and microvascular endothelial cells isolated from subcutaneous sponges implanted in mice of the same strain (SIECs) (28) were grown in Dulbecco's MEM supplemented with 1 mM glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, and 20% FCS in the presence of 10 ng/ml bFGF and 10 µg/ml heparin on gelatin-coated dishes.
Cell Proliferation Assays
GM 7373 Cells-- The short term cell proliferation assay was performed as described (29, 30). Briefly, 70,000 cells/cm2 were seeded in 96-well dishes. After overnight incubation, the cells were incubated for 24 h in fresh medium containing 0.4% FCS and 10 ng/ml bFGF in the presence of increasing concentrations of different chemokines. At the end of the incubation, the cells were trypsinized and counted in a Burker chamber. During this time period, control cultures incubated with no addition or with 10 ng/ml bFGF undergo 0.1-0.2 and 0.7-0.8 cell population doublings, respectively. Cells grown in 10% FCS undergo 1.0 cell population doublings (30). For long term proliferation studies, GM 7373 cells were seeded in 24-well plates at 10,000 cells/cm2. After 16 h, cells were incubated in fresh medium with 5% calf serum in the presence of 10 ng/ml bFGF and/or 100 ng/ml IL-8. Medium was changed every 72 h, and cells were trypsinized and counted after 6 days of treatment.
BAECs-- Cells were seeded in 35-mm dishes at 2,000 cells/cm2. After 16 h, cells were incubated in fresh medium with 5% calf serum in the presence of 10 ng/ml bFGF and/or 100 ng/ml IL-8. Then medium with or without the growth factor and chemokine was changed every other day. After 6 days, cells were trypsinized and counted.
HUVECs-- Cells were seeded in 96-well plates at 6,250 cells/cm2. After 16 h, cells were incubated in fresh medium with 5% calf serum in the presence of 10 ng/ml bFGF and/or 100 ng/ml IL-8. Then medium with or without the growth factor and chemokine was changed every other day. After 6 days, cells were trypsinized and counted.
MBECs-- Cells were seeded in 24-well plates at 25,000 cells/cm2. After 16 h, cells were incubated in fresh medium with 10% FCS in the presence of 10 ng/ml bFGF and of 100 ng/ml IL-8. Then medium with or without the growth factor and chemokine was changed every other day. After 3 days, cells were trypsinized and counted (27).
1G11 Cells-- They were seeded in gelatin-coated 48-well plates at 8,000 cells/cm2. After 16 h, cells were incubated in fresh medium without heparin containing 20% FCS in the presence of 10 ng/ml bFGF and 100 ng/ml IL-8. After 2 days, cells were trypsinized and counted.
SIECs-- Cells were seeded in gelatin-coated 48-well plates at 10,000 cells/cm2. After 16 h, cells were incubated in fresh medium without heparin containing 20% FCS in the presence of 10 ng/ml bFGF and increasing concentrations of IL-8. After 3 days, cells were trypsinized and counted.
125I-bFGF Binding Assay
Human recombinant bFGF was labeled with 125I (37 GBq/ml; Amersham International) using Iodogen (Pierce) as described (31) at a specific radioactivity equal to 600 cpm/fmol. GM 7373 cells were seeded in 24-well dishes at the density of 75,000 cells/cm2. After 24 h, cells were washed three times with cold PBS and incubated for 2 h at 4 °C in binding medium (serum-free medium containing 0.15% gelatin, 20 mM Hepes buffer, pH 7.5) with 10 ng/ml 125I-bFGF in the presence of increasing concentrations of the chemokine under test. After a PBS wash, cells were then washed twice with 2 M NaCl in 20 mM Hepes buffer (pH 7.5) to remove 125I-bFGF bound to low affinity HSPGs and twice with 2 M NaCl in 20 mM sodium acetate (pH 4.0) to remove 125I-bFGF bound to high affinity FGFRs (32). Nonspecific binding was measured in the presence of a 100-fold molar excess of unlabeled FGF-2 and subtracted from all the values.
Down-regulation of FGFRs
GM 7373 cells were plated in 24-well dishes at a density of 70,000 cells/cm2. After 24 h, cells were incubated at 37 °C for 6 h with 100 ng/ml IL-8 or TPA. At the end of the incubation, the cells were washed twice with ice-cold 2.0 M NaCl in 20 mM sodium acetate, pH 4.0, and three times with ice-cold PBS and then incubated at 4 °C in binding medium containing increasing concentrations of 125I-bFGF. After 2 h, the amount of 125I-bFGF bound to high and low affinity sites was measured as described above. Binding data were analyzed by the Scatchard plot procedure (33).
Western Blot Analysis of Extracellular Signal-regulated Kinase-2 (ERK-2)
GM 7373 cells were grown at subconfluence in 60-mm dishes in Dulbecco's MEM containing 10% FCS. Then cells were treated for 20 min with fresh medium with or without 10 ng/ml bFGF in the absence or in the presence of 100 ng/ml IL-8. Western blot analysis of the cell extracts was performed exactly as described (34) using anti-ERK-2 antibodies (kindly provided by Dr. Y. Nagamine, Friedrich Miescher Institute, Basel, Switzerland). Phosphorylation of ERK-2 was evidenced as a mobility shift on the gel (34).
Chemokine Binding Assays
GM 7373 cells (1 × 106 cells/ml) were incubated in suspension with 125I-labeled ligands (0.5 nM) and varying concentrations of unlabeled ligands at 4 °C for 1 h. The incubation was terminated by removing aliquots from the cell suspension and separating cells from buffer by centrifugation through a silicone/paraffin oil mixture as described previously (35). Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. The binding data were curve fit with the computer program IGOR (Wavemetrics) to determine the affinity (KD), the number of sites, and nonspecific binding.
Subcutaneous Sponge Assay
Fragments (about 1 cm3) of sponge (Spongostan Dental, Ferrosan, Denmark) hydrated for 2 h at 4 °C in saline alone or saline containing 100 ng of bFGF, 1 µg of IL-8, or bFGF plus IL-8 (200 µl/sponge) were inserted subcutaneously in the flank of anesthetized Crl:CD1(ICR)BR mice (Charles River, Milan, Italy). All sponges also contained 200 µg of heparin to stabilize the bFGF molecule in vivo (36). After 7 days sponges were removed, and neovascularization was quantified by measuring their hemoglobin content as described (36).
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RESULTS |
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IL-8 Inhibits the Mitogenic Activity of bFGF-- A high degree of heterogeneity has been observed in endothelial cells originating from different tissues, vessels of different caliber, or different animal species (37). The effect of IL-8 on endothelial cell proliferation was therefore evaluated on cells of different origin. Primary HUVEC, BAEC, 1G11, and SIEC cultures and immortalized MBEC and fetal bovine aortic endothelial GM 7373 cell lines were incubated with the chemokine in the absence or in the presence of 10 ng/ml bFGF. As shown in Fig. 1, 100 ng/ml IL-8 did not affect the proliferation of HUVECs, BAECs, MBECs, and 1G11 cells when administered either in the absence or in the presence of bFGF. In contrast, IL-8 abolished the mitogenic activity exerted by bFGF in SIECs and GM 7373 cells with an ID50 of 30 ng/ml both in short term and in long term cell proliferation assays (Fig. 1). Heat inactivation abolished the capacity of IL-8 to inhibit the mitogenic activity of bFGF (data not shown), indicating that an appropriate three-dimensional conformation is required for the chemokine to elicit its inhibitory activity. No morphologic signs of apoptosis, including cell rounding and detachment, were detected even after 6 days of IL-8 treatment in all cell types investigated. These observations prompted us to more carefully characterize the mechanism of action of IL-8. To this purpose, the short term (24 h) cell proliferation assay performed on the fetal bovine aortic endothelial GM 7373 cell line was used routinely.
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Role of Protein Kinase C in the Inhibitory Activity of IL-8 on GM 7373 Cells-- The above data indicate that a 3-h interaction of IL-8 with GM 7373 cells is sufficient to prevent bFGF-induced cell proliferation and that the presence of the chemokine is not required during the incubation with the growth factor, further supporting the hypothesis that the mechanism of inhibition of IL-8 is not competitive. We took advantage of these characteristics to attempt to define the mechanism of action of IL-8 by incubating GM 7373 cells with putative inhibitors of IL-8 activity during a 3-h period of preincubation at 37 °C with the chemokine. Then cell cultures were washed extensively to remove the chemokine and its inhibitor before the 24-h incubation with bFGF. Under these experimental conditions, the inhibitors tested were anticipated to affect only IL-8 activity without affecting bFGF activity.
PKC participates in chemokine-dependent signal transduction (11) and plays a role in the IL-8-dependent inhibition of IL-4-induced growth of human B cells (42). On this basis, we evaluated the effect of different protein kinase inhibitors on IL-8-mediated inhibition of GM 7373 cell proliferation. As shown in Fig. 4A, the PKC inhibitors H-7 (43) and bisindolylmaleimide GF 109203X (GF 109) (44) abolished the inhibitory effect of IL-8 preincubation on bFGF mitogenic activity. In contrast, the PKA inhibitor N-[2-methylamino)ethyl]-5-isoquinoline-sulfonamide (H-8) (43) and the tyrosine kinase inhibitors genistein (45, 46) and 5'-methylthioadenosine (47) were ineffective. It must be pointed out that none of the inhibitors were able to affect the mitogenic activity of bFGF when administered to GM 7373 in the absence of IL-8 under the same experimental conditions. Conversely, the PKC activator phorbol ester TPA was able to mimic the activity of IL-8. Indeed, as observed for IL-8, 3 h of preincubation with 100 ng/ml TPA fully inhibited the mitogenic activity of bFGF in GM 7373 cells, whereas the inactive phorbol esters
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IL-8 Pretreatment Decreases the Binding Capacity of FGFR-- Transmodulation of tyrosine kinase receptors by PKC activation has been reported (48, 49). To assess whether the inhibitory activity of IL-8 on bFGF mitogenic activity could be explained by a PKC-dependent decrease in FGFR binding capacity, GM 7373 cells were preincubated for 6 h at 37 °C with 100 ng/ml IL-8 or TPA. After extensive washing of the cell monolayers, cells were further incubated for 2 h at 4 °C with 30 ng/ml 125I-bFGF. As anticipated, both molecules caused a significant decrease in the amount of 125I-bFGF bound to high affinity FGFRs (45 and 57% inhibition for IL-8 and TPA, respectively) without affecting the ability of 125I-bFGF to bind to low affinity HSPGs (Fig. 5A). Scatchard plot analysis of the 125I-bFGF binding data, obtained when the experiment was repeated in the presence of increasing concentrations of the radiolabeled ligand, indicated that IL-8 pretreatment caused a significant decrease (~60%) in the number of FGFRs present on the surfaces of GM 7373 cells without affecting their affinity for 125I-bFGF. IL-8 pretreatment did not cause any significant modifications in the number of 125I-bFGF-binding cell surface HSPGs and in their affinity for the growth factor (Table I).
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Both C-X-C and C-C Chemokines Inhibit bFGF Mitogenic Activity in GM
7373 Cells--
IL-8 belongs to the ELR+ subclass of the
C-X-C chemokines. To assess whether the capacity to inhibit
the mitogenic activity exerted by bFGF in GM 7373 was shared by other
chemokines, the ELR+ C-X-C chemokines GRO,
GRO
/MGSA, the GRO
mutant E6A (50), the ELR
C-X-C chemokine PF-4, and the C-C chemokines RANTES,
monocyte chemoattractant protein-1, and macrophage inflammatory
protein-1
(MIP1
) were compared with IL-8 in the short term
proliferation assay (Fig. 6). Like IL-8,
most of the chemokines tested inhibit bFGF activity with an
ID50 equal to 20-40 ng/ml (
3 nM), the
exceptions being MIP1
and monocyte chemoattractant protein-1 that
half-maximally inhibit cell proliferation at 100 and 1000 ng/ml,
respectively. It is interesting to note that despite their similar
ID50 values, IL-8, GRO
/MGSA, GRO
, PF-4, and RANTES
cause 80-95% inhibition of GM 7373 cell proliferation at 1000 ng/ml,
whereas cells treated with the same dose of MIP1
or with the GRO
E6A mutant still retain 40% of their proliferative response.
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Identification of a Promiscuous Chemokine-binding Site in GM 7373 Cells--
The capacity of both C-X-C and C-C chemokines to
exert a similar inhibitory effect on endothelial GM 7373 cells pointed
to the possibility that these cells express DARC, as observed for post-capillary venule and splenic sinusoid endothelial cells (20). However, Northern blot analysis failed to detect DARC mRNA in GM
7373 cells (data not shown). Also, GM 7373 cells do not express the
C-X-C receptors IL-8-A and IL-8RB (11) as assessed by
Northern blot analysis using the corresponding cDNA
probes.2 These data prompted
us to study the IL-8 binding properties of this endothelial cell line
(Fig. 7A). Scatchard plot
analysis indicates that 125I-IL-8 binding to GM 7373 cells
is both saturable and linear, consistent with a single class of binding
sites (900,000 sites/cell) having a KD of 132 ± 16 nM, significantly higher than the
KD of the C-X-C receptors IL-8RA and
IL-8RB (3-5 nM, Ref. 11) Analysis performed with the
C-X-C chemokine 125I-GRO/MGSA confirmed the
presence of a single class of binding sites on the GM 7373 cell surface
(KD equal to 150 ± 13 nM) ranging
from 600,000 to 1,000,000 sites/cell. Scatchard analysis with
125I-RANTES yielded similar results (data not shown). The
ability of unlabeled chemokines of the different subclasses to displace 125I-IL-8 from GM 7373 cell surface was then explored. To
this purpose, the C-X-C chemokines GRO
/MGSA and
neutrophil-activating peptide-2 and the C-C chemokines RANTES,
MIP1
, and MIP1
were utilized. As shown in Fig.
7B, 125I-IL-8 is similarly displaced by a
100-fold excess of unlabeled IL-8, GRO
/MGSA, neutrophil-activating
peptide-2, or RANTES, whereas MIP1
competes much less effectively
and MIP1
does not compete. Similar results were obtained when
unlabeled chemokines were evaluated for their capacity to displace
125I-GRO
/MGSA from GM 7373 cell surface (data not
shown).
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IL-8 Inhibits bFGF Angiogenic Activity in Vivo-- The capacity of IL-8 to inhibit the mitogenic activity of bFGF in mouse SIECs isolated from neovascularized subcutaneous sponges (see Fig. 1) prompted us to assess whether the chemokine was also able to affect the angiogenic activity exerted in vivo by the growth factor in this experimental model. To this purpose, sponges were adsorbed with bFGF alone (100 ng/sponge) or with bFGF added with a molar excess of IL-8 (1 µg/sponge) and were implanted into the flank of anesthetized mice. After 7 days sponges were removed and neovascularization was compared with that measured in implants adsorbed with saline or with IL-8 alone. Despite a certain variability within experimental groups, the results indicate that IL-8 causes a significant decrease of the angiogenic activity of bFGF (Table II). Also, IL-8 alone does not exert a significant angiogenic response in this experimental model at the dose tested.
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DISCUSSION |
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This work demonstrates that IL-8 inhibits the mitogenic response elicited by bFGF on certain endothelial cell types and significantly affects its angiogenic activity in vivo. At variance with previous results on the inhibitory activity exerted by PF-4 on bFGF (51), several lines of evidence indicate that the mechanism of action of IL-8 is not competitive: (i) the inhibitory activity of IL-8 on cultured endothelial GM 7373 cells is not overcome by increasing concentrations of the growth factor; (ii) IL-8 does not compete for the binding of 125I-bFGF to high affinity FGFRs and low affinity HSPGs; and (iii) a short interaction of IL-8 with GM 7373 cells, followed by the removal of the chemokine before the addition of bFGF, is sufficient to prevent the mitogenic response. The demonstration that the inhibitory activity of IL-8 is abolished by the PKC inhibitors H-7 and GF 109 and mimicked by the PKC activator TPA points instead to a role for PKC in mediating IL-8 action on GM 7373 cells. To our knowledge, this is the first evidence for the chemokine-dependent activation of a signal transduction pathway that modulates the response of endothelial cells to angiogenic growth factor(s).
PKC activation has been demonstrated to attenuate signaling from
tyrosine kinase growth factor receptors (48). We have found that IL-8
does not affect the capacity of bFGF to activate ERK-2, an early
downstream signal that follows receptor tyrosine phosphorylation (34),
thus indicating that IL-8 does not affect the tyrosine kinase activity
of FGFR. However, IL-8 causes a 60% down-regulation of FGFRs that
may explain, at least in part, the inhibitory action of the chemokine.
The observation that a partial down-regulation of FGFRs is sufficient
to abolish the capacity of the target cell to respond to bFGF is not
surprising and may reflect the existence of threshold effects in the
long term FGFR signaling required to sustain bFGF-induced cell
proliferation (30). Similar conclusions were drawn for TPA-treated
HUVECs in which a partial decrease in FGFR binding capacity caused a
complete suppression of the mitogenic activity of acidic FGF (49), a
member of the FGF family that shares with bFGF similar receptor binding
and biological activity (2).
Previous observations had shown that ELR+ and
ELR C-X-C chemokines exert opposite effects on
angiogenesis in vitro and in vivo (17). However,
contradictory results about the chemokine action on endothelial cells
exist. For instance, the responsiveness of HUVECs to IL-8 has been
subject to conflicting reports, perhaps reflecting differences in
culture conditions (19). In addition, the anti-angiogenic chemokine
IP-10 was reported in one study to inhibit bFGF-induced proliferation
in HUVECs (23) but did not affect their proliferation in another (52).
The ELR+ C-X-C chemokines GRO
/MGSA and
GRO
, but not GRO
, have been shown to inhibit the mitogenic
activity of bFGF in bovine capillary endothelial cells (53). In the
same study GRO
reduced bFGF-stimulated mouse corneal
neovascularization and tumor growth. In contrast, a second study (17)
has shown that GRO
/MGSA, GRO
, and GRO
can stimulate chemotaxis
in bovine capillary endothelial cell and neovascularization of the rat
cornea (limited to GRO
/MGSA). Others have failed to observe any
activity of GRO
/MGSA on endothelial cells (14).
Here we report that various ELR+ and ELR
C-X-C chemokines, as well as C-C chemokines, share with IL-8
the capacity to inhibit bFGF activity in GM 7373 cells. A promiscuous,
low affinity, high capacity chemokine-binding site was identified on
these cells by binding assays with 125I-IL-8,
125I-GRO
/MGSA, and 125I-RANTES. These data,
together with the ability of soluble heparin and HS to overcome the
inhibitory activity of IL-8, suggest that cell surface HSPGs play an
important role in mediating chemokine activity in GM 7373 cells.
Similar conclusions were drawn for some chemokines on different
endothelial cell types. For instance, IP-10 and PF-4, which share a
specific HSPG-binding site in SV-40-transformed murine endothelial
cells, cause inhibition of bFGF-induced proliferation in HUVECs that is
abrogated by soluble heparin (23). Similarly, inhibition of bFGF
activity by PF-4 was overcome by exogenous heparin in 3T3 fibroblasts
(51). However, this was not the case for the bFGF antagonist action of
GRO
/MGSA and GRO
on bovine capillary endothelial cells, for which
heparin is ineffective (53).
IL-8 exerts half-maximal inhibition of bFGF mitogenic activity at concentrations equal to 1-3 nM (see Fig. 1). At these doses, approximately 5,000 HSPG-like binding sites/cell are occupied by the chemokine on GM 7373 cells (KD equal to 132 nM). By assuming a tight coupling for these receptors, as observed for instance for IL-1 receptor (54), IL-8 receptor occupancy may be adequate for signaling to occur. Northern blot analysis has shown that endothelial GM 7373 cells express the transmembrane HSPG syndecan-1.3 Four tyrosine residues are highly conserved in the C-terminal of all the members of the syndecan family, and one of them fits a consensus sequence for tyrosine phosphorylation (55). Recently, tyrosine phosphorylation of the intracellular domain of syndecan-1 by cytoplasmic tyrosine kinases has been described in intact cells (56), thus supporting the hypothesis of HSPG involvement in signal transduction. Accordingly, endothelial HSPGs have been directly implicated in mediating the inhibitory activity of PF-4 and IP-10 (23). On the other hand, HSPGs have been demonstrated to mediate the binding of various heparin-binding growth factors to their high affinity receptors, including bFGF, vascular endothelial growth factor, and hepatocyte growth factor (5, 57, 58), and to facilitate receptor dimerization and signal transduction (7). Thus, even though we failed to detect the expression of the C-X-C receptors IL8-RA and IL-8RB mRNAs in GM 7373 cells,2 we cannot rule out the hypothesis that IL-8-HSPG interaction may present the chemokine to an as yet uncharacterized high affinity promiscuous chemokine receptor present at very low abundance on the cell surface. This receptor, undetected in the Scatchard plot analysis because of the high capacity of HSPGs, could be responsible for PKC activation, leading to down-regulation of FGFRs and eventually to the inhibition of bFGF activity. In keeping with this hypothesis is the capacity of HS to potentiate receptor-dependent neutrophil responses to IL-8, including [Ca2+]i increase and chemotaxis (38). In both cases, our data point to a role for endothelial cell HSPGs in mediating the antagonist activity of IL-8.
Endothelial cells are characterized by a high degree of heterogeneity, depending on tissue type, vessel caliber, and animal species from which they originated (37, 59). IL-8 exerts a bFGF antagonist action in two out of the six endothelial cell types here investigated, thus supporting previous observations on the different capacity of endothelial cells of different origin to respond to certain cytokines (discussed in Ref. 59). At present, it is not possible to anticipate how and whether a certain endothelial cell type will respond to IL-8. This may represent a serious limitation for studies aimed to elucidate the role of modulators in angiogenesis where in vivo data are frequently compared with in vitro observations obtained with large vessel endothelium originating from different animal species. A typical example is represented by the comparison of the results obtained in chick embryo chorioallantoic membrane, rabbit cornea, and/or rat cornea angiogenesis assays to observations on cultured endothelium isolated from bovine aorta and/or human umbilical vein. Among the endothelial cell types studied, we have investigated the effect exerted by IL-8 on murine endothelial cells (SIECs) isolated from the same anatomical district utilized for in vivo angiogenesis assays. We have observed that IL-8 inhibits the mitogenic activity of bFGF in SIECs isolated from neovascularized subcutaneous sponge implants and significantly reduces the in vivo angiogenic response in these bFGF-adsorbed implants. These data indicate that the bFGF-stimulated murine endothelium of this anatomical district is susceptible to the inhibitory activity of IL-8 both in vitro and in vivo. In apparent contrast with our observations, IL-8 has been demonstrated to promote new blood vessel formation in different experimental models, including rat corneal pocket (15), rabbit corneal pocket (16), rat mesenteric window (60), and rat sponge (61) angiogenesis assays, even though dose-response experiments have shown that angiogenesis occurs only over a narrow range of doses of the chemokine (15, 60). More data on the effect of IL-8 on endothelial cells isolated from these anatomical sites and animal species are required to clarify whether the observed IL-8-induced neovascularization depends on a direct action of the chemokine on endothelium or on secondary effects consequent to inflammatory cell recruitment.
Recent observations have shown that heparin-HS interaction of various angiogenic growth factors depends on specific structural characteristics of the polysaccharide chain (62). Also, heparin-HS subpopulations with chemokine binding selectivity exist (13). We have found that, at variance with heparin and HS, chondroitin-4-sulfate and nonsulfated hyaluronic acid and K5-polysaccharide were unable to overcome IL-8 activity, suggesting that the backbone structure, sulfation, and the arrangement of the charges are of importance in determining the capacity of GAGs to bind IL-8 and to modulate its activity on endothelial GM 7373 cells. The more significant effect of selective 2-O-desulfation in preventing the anti-bFGF activity of IL-8, when compared with 6-O- and N-desulfation, confirms this hypothesis. Thus, chemokines may bind heparin-HS in a distinct and possibly specific manner. Even though the specific chemokine-binding sequence(s) may be hidden in heparin because of its high degree of sulfation, the high heterogeneity in HS structure may allow a more refined tailoring of selective binding regions in different endothelial cell types or under different cell culture conditions that may influence the biological activity and bioavailability of HS-binding chemokines, thus explaining some of the contradictory results about chemokine action on endothelial cells.
In conclusion, a complex interplay occurs among chemokines, angiogenic growth factors, their cognate receptors, and cell surface HSPGs. Our data suggest that the different, possibly contrasting effects of various chemokines on endothelial cells of different origin may depend both on the type of chemokine-binding sites expressed and on the interplay between the activated chemokine- and growth factor-dependent signal transduction pathways. The stable endothelial GM 7373 cell line utilized in the present study may represent a useful tool to elucidate this interplay.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Rusnati for 125I-bFGF binding studies and helpful discussion and Dr. S. Ramponi for the in vivo angiogenesis assays.
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FOOTNOTES |
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* This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (Special Project on Angiogenesis), by the Ministero Superiore della Sanità (AIDS and Oncology Projects), by the Centro por lo Studio del Trattamento dello Scompenso Cardiaco, and by MURST (Project Inflammation, Biology, and Clinics; to M. P. and A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: General Pathology, School of Medicine, via Valsabbina 19, 25123 Brescia, Italy. Tel.: 39-30-3715315; Fax: 39-30-3701157; E-mail: presta{at}med.unibs.it.
1
The abbreviations used are: bFGF, basic
fibroblast growth factor; FGFR, tyrosine kinase FGF receptor; HS,
heparan sulfate; HSPG, heparan sulfate proteoglycan; GAG,
glycosaminoglycan; FCS, fetal calf serum; PBS, phosphate-buffered
saline; BAEC, adult bovine aortic endothelial cell; MBEC, mouse brain
microvascular endothelial cell; HUVEC, human umbilical vein endothelial
cell; SIEC, sponge-induced mouse endothelial cell; DARC, Duffy
antigen/erythrocyte chemokine receptor; ERK-2, extracellular
signal-regulated kinase-2; PKC, protein kinase C; IL-8, interleukin-8;
PF-4, platelet factor-4; GRO, growth-related oncogene; MGSA, melanoma
growth stimulating activity; IP-10, interferon--inducible
protein-10; MIP, macrophage inflammatory protein; TPA,
12-O-tetradecanoylphorbol-13-acetate; H-7,
1-(5-isoquinolynsulfonyl)-2-methylpiperazine; H-8,
N-[2-methylamino)ethyl]-5-isoquinoline-sulfonamide; GF
109, bisindolylmaleimide GF 109203X; RANTES, regulated on activation normal T cell expressed and secreted; MEM, Eagle's minimal essential medium.
2 R. Horuk, unpublished observations.
3 M. Presta and P. Dell'Era, unpublished observations.
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REFERENCES |
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