(Received for publication, July 7, 1995; and in revised form, August 11, 1995)
From the
In this study, we demonstrate that the CXC family of chemokines
displays disparate angiogenic activity depending upon the presence or
absence of the ELR motif. CXC chemokines containing the ELR motif
(ELR-CXC chemokines) were found to be potent angiogenic factors,
inducing both in vitro endothelial chemotaxis and in vivo corneal neovascularization. In contrast, the CXC chemokines
lacking the ELR motif, platelet factor 4, interferon -inducible
protein 10, and monokine induced by
-interferon, not only failed
to induce significant in vitro endothelial cell chemotaxis or in vivo corneal neovacularization but were found to be potent
angiostatic factors in the presence of either ELR-CXC chemokines or the
unrelated angiogenic factor, basic fibroblast growth factor.
Additionally, mutant interleukin-8 proteins lacking the ELR motif
demonstrated potent angiostatic effects in the presence of either
ELR-CXC chemokines or basic fibroblast growth factor. In contrast, a
mutant of monokine induced by
-interferon containing the ELR motif
was found to induce in vivo angiogenic activity. These
findings suggest a functional role of the ELR motif in determining the
angiogenic or angiostatic potential of CXC chemokines, supporting the
hypothesis that the net biological balance between angiogenic and
angiostatic CXC chemokines may play an important role in regulating
overall angiogenesis.
Angiogenesis, characterized by the neoformation of blood vessels, is an essential biological event encountered in a number of physiological and pathological processes, such as embryonic development, the formation of inflammatory granulation tissue during wound healing, chronic inflammation, and the growth of malignant solid tumors(1, 2, 3, 4, 5) . Neovascularization can be rapidly induced in response to diverse pathophysiologic stimuli. Under conditions of homeostasis, the rate of capillary endothelial cell turn-over is typically measured in months or years(6, 7) . However, the process of angiogenesis during normal wound repair is rapid, transient, and tightly controlled. During neovascularization, normally quiescent endothelial cells are stimulated, degrade their basement membrane and proximal extracellular matrix, migrate directionally, divide, and organize into new functioning capillaries invested by a basal lamina(1, 2, 3, 4, 5) . The abrupt termination of angiogenesis that accompanies the resolution of the wound repair suggests two possible mechanisms of control: a marked reduction in angiogenic mediators coupled with a simultaneous increase in the level of angiostatic factors that inhibit new vessel growth(8) . In contrast to neovascularization of normal wound repair, tumorigenesis is associated with exaggerated angiogenesis, suggesting the existence of augmented angiogenic and reduced levels of angiostatic mediators(3, 9) . Although most investigations studying angiogenesis have focused on the identification and mechanism of action of angiogenic factors, recent evidence suggests that angiostatic factors may play an equally important role in the control of neovascularization(8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) .
Recently, platelet factor 4 (PF4), ()a member of the CXC
chemokine family, has been found to be an inhibitor of angiogenesis (27) . In contrast, interleukin-8 (IL-8), another member of the
CXC chemokine family, has been shown to have potent angiogenic
properties (28, 29, 30) . Although these CXC
chemokines have significant homology on the amino acid level, one of
the major differences between IL-8 and PF4 is the presence in IL-8 of
the sequence Glu-Leu-Arg (the ELR motif), which is not found in
PF4(31, 32, 33, 34) . These three
amino acids appear to be important in ligand/receptor interactions on
neutrophils (35, 36) and are highly conserved in all
members of the CXC chemokine family that demonstrate biological
activation of neutrophils(35, 36) .
In this study,
we demonstrate that members of the CXC chemokine family that contain
the ELR motif, as compared with members that lack these three amino
acids, are potent inducers of angiogenic activity. In addition, we show
that CXC chemokines that lack the ELR motif, PF4, interferon
-inducible protein 10 (IP-10), and monokine induced by
-interferon (MIG) are potent inhibitors of both CXC (ELR)
chemokine and basic fibroblast growth factor (bFGF)-induced
angiogenesis. Moreover, substitution of the ELR motif in IL-8 generated
proteins that antagonized the angiogenic effects of ELR-CXC chemokines
and bFGF, while a mutant of MIG containing the ELR motif was
angiogenic. These results suggest that the presence or absence of the
ELR motif in CXC chemokines functionally defines the angiogenic (ELR
containing) or angiostatic (non-ELR) characteristics of these proteins.
These findings support the notion that CXC chemokines play an important
role in the regulation of angiogenesis by acting as either angiogenic
or angiostatic factors.
Cultures of E. coli strain DH5F` harboring GST-MIG or GST-ELR-MIG
plasmid were grown in 1 liter of LB media containing 50 µg/ml
ampicillin to A
0.5 at 22 °C with
aeration, and protein expression was induced by the addition of 0.1
mM final isopropyl-1-thio-
-D-galactoside and
continued incubation at 22 °C for 5-6 h. After induction, the
cells were harvested by centrifuging at 6,000
g for 10
min and the pellet was washed once in ice-cold PBS and resuspended in
10 ml of ice-cold 10 mM HEPES, 30 mM NaCl, 10 mM EDTA, 10 mM EGTA, 0.25% Tween 20, 1 mM phenylmethylsulfonyl fluoride (added fresh), pH 7.5 (lysis
buffer). The resulting suspension was quick-frozen in liquid nitrogen.
After thawing, phenylmethylsulfonyl fluoride was again added to yield a
final concentration of 2 mM. The suspension was sonicated
using a Branson Sonifier 250 equipped with a microtip for 2 min at
output setting 5 with a 40% duty cycle. Triton X-100 was added to a
final concentration of 1%, and the lysate was mutated for 30 min at
room temperature to aid in the solubilization of the fusion protein.
The lysate was then centrifuged at 34,500
g for 10
min, and the supernatant was transferred to a fresh tube.
The
GST-MIG protein was purified using the Pharmacia GST purification
module (Pharmacia) essentially as described in the manufacturer's
protocol. GST-fusion protein sonicate was passed over a 2-ml
glutathione-Sepharose 4B column equilibrated in PBS. After washing with
PBS, the GST-fusion protein was eluted with 3 column volumes of 10
mM reduced glutathione, 50 mM Tris-HCL, pH 8.0. 10
units of thrombin/A unit of fusion protein was
added to the eluted GST-MIG or GST-ELR-MIG fusion protein and incubated
at room temperature with occasional gentle mixing for 2-3 h. MIG
or ELR-MIG protein was
95% cleaved from the GST protein under these
conditions as monitered by SDS-PAGE(41) . The pH of the
MIG-containing solution was adjusted to 4.0 using 0.5 M sodium
acetate, pH 4.0, filtered through a cellulose acetate 0.45-µm
filter (Costar) and passed over a Mono S column (Pharmacia)
equilibrated with 20 mM sodium acetate, pH 4.0. MIG protein
was eluted as a single peak using a 0-2 M NaCl gradient,
and dialyzed against 0.5 mM NaPO
, 20 mM NaCl, pH 7.0. Purified MIG and ELR-MIG was obtained endotoxin-free
(<1.0 enzyme units/ml; QCL-1000 test, BioWhittaker), and yields
ranged from 100-200 µg/liter (quantitated by amino acid
analysis) with a purity of >95% (determined by SDS-PAGE, with
apparent molecular mass of 16 kDa; amino acid analysis accuracy >
90%). Mass spectrometry of the purified MIG and ELR-MIG proteins
confirmed their predicted mass.
Cultures of E. coli strain
DH5F` harboring pMal.hIL-8, pMal.TVR-IL-8, or pMal.DLQ-IL-8 were
grown in 1-liter LB media containing 50 µg/ml ampicilin to A
0.5 at 37 °C with aeration, and
protein expression was induced by the addition of 0.3 mM final
isopropyl-1-thio-
-D-galactoside and continued incubation
at 37 °C for 2 h. Cells were harvested by centrifuging at 5800
g for 10 min and the pellet was washed once in
ice-cold PBS and resuspended in 10 ml of ice-cold lysis buffer. The
resulting suspension was quick-frozen in liquid nitrogen.
After
thawing, the suspension was sonicated using a Branson Sonifier 250
equipped with a microtip for 2 min at output setting 5 with a 40% duty
cycle. The suspension was clarified by centrifugation at 9000 g, the supernatant was diluted 5-fold in 10 mM NaPO
, 500 mM NaCl, 1 mM EGTA, 0.25%
Tween 20, pH 7.0 (column buffer), and loaded onto a 10-ml amylose resin
(New England Biolabs) affinity column. After extensive washing with
column buffer, the maltose binding protein fusion protein was eluted
with column buffer containing 10 mM maltose. Mutein or
wild-type IL-8 proteins were released by incubation with 1 µg of
Factor Xa (New England Biolabs)/A
maltose
binding protein fusion protein at room temperature overnight and were
then passed over a Mono S column (Pharmacia) equilibrated in 10
mM NaPO
, pH 6.2, and eluted in a 0-1 M NaCl gradient. 1 ml of amylose resin was added to fractions
containing mutant or wild-type IL-8 protein to remove residual free
maltose binding protein by incubation for 30 min at room temperature
with gentle shaking. The resin was removed by centrifugation, and the
supernatant was dialyzed against 0.5 mM NaPO
, 20
mM NaCl, pH 7.5. Yields were ranged from 0.2 to 3.5 mg for
wild-type or mutant IL-8 proteins and were
95% pure as assessed by
SDS-PAGE and endotoxin-free (<1.0 enzyme units/ml). Proteins were
quantitated by amino acid analysis, and had accuracies between
88-93%.
Figure 1: Endothelial cell chemotaxis in response to CXC chemokines (50 pM to 50 nM). To demonstrate specific migration, background (unstimulated control) migration (cells/HPF) was subtracted.
Figure 2: Endothelial cell chemotaxis in response to IL-8 (10 nM), ENA-78 (10 nM), and bFGF (5 nM) in the presence of varying concentrations PF4 (50 pM to 10 nM; part a), IP-10 (50 pM to 10 nM; part b), and MIG (500 pM to 10 nM; part c). To demonstrate specific migration, background (unstimulated control) migration (cells/HPF) was subtracted.
The rat corneal micropocket model of neovascularization was used to
determine whether IP-10 or MIG could inhibit the angiogenic activity of
either the ELR-containing CXC chemokines or bFGF in vivo. Hydron pellets alone, pellets containing IL-8, ENA-78, GRO-,
GCP-2, IP-10, MIG, or bFGF in a concentration of 10 nM, or
pellets containing combinations of 10 nM each of IL-8 +
IP-10, ENA-78 + IP-10, GRO-
+ IP-10, GCP-2 + IP-10,
IL-8 + MIG, ENA-78 + MIG, bFGF + IP10, or bFGF +
MIG were embedded into the normally avascular rat cornea and assessed
for a neovascular response (Fig. 3, a-c). The CXC chemokines (IL-8, ENA-78,
GRO-
, or GCP-2) or bFGF-induced positive corneal angiogenic
responses in six of six corneas, without evidence for significant
leukocyte infiltration (assessed by light microscopy). In contrast,
hydron pellets alone (n = 6 corneas) or pellets
containing either IP-10 or MIG (10 nM) (n = 6
corneas for each chemokine) only resulted in a positive neovascular
response in less than one of six corneas tested for each variable. When
IP-10 was added in combination with the ELR-CXC chemokines (IL-8,
ENA-78, GRO-
, or GCP-2) or bFGF (Fig. 3, a and c, respectively), IP-10 significantly abrogated the ELR-CXC
chemokine and bFGF-induced angiogenic activity in five of six corneas (n = 6 corneas for each manipulation). In addition, MIG
inhibited IL-8, ENA-78, and bFGF-induced corneal angiogenic activity in
a similar manner to IP-10 (Fig. 3, b and c).
Figure 3:
Rat cornea neovascularization in response
to ELR-CXC chemokines, non-ELR-CXC chemokines, bFGF, or combinations of
these cytokines. Part a, panels A, B, C, E, G, and I, respectively,
represent the corneal neovascular response to a hydron pellet alone
(vehicle control), IP-10 (10 nM), IL-8 (10 nM),
ENA-78 (10 nM), GRO- (10 nM), or GCP-2 (10
nM); part a, panels D, F, H, and J, respectively, represent the combination of IL-8 with IP-10,
ENA-78 with IP-10, GRO-
with IP-10, or GCP-2 with IP-10. Part
b, panels A-D, respectively, represent the corneal
neovascular response to a hydron pellet alone (vehicle control), MIG
(10 nM), IL-8 (10 nM), or ENA-78 (10 nM); part b, panels E and F, respectively,
represents the corneal neovascular response to the combination of IL-8
with MIG or ENA-78 with MIG. Part c, panels
A-D, respectively, represents the corneal
neovascular response to a hydron pellet alone (vehicle control), bFGF
(5 nM), MIG (10 nM), or IP-10 (10 nM); part c, panels E and F, respectively,
represents the corneal neovascular response to the combination of bFGF
with MIG or bFGF and IP-10. All panels are at 25
magnification.
Figure 4: Endothelial cell chemotaxis in response to the presence or absence of varying concentrations of IL-8 and IL-8 muteins, TVR-IL-8, and DLQ-IL-8. Panel A is endothelial cell chemotaxis in response to the presence or absence of varying concentrations of IL-8 (1-10 nM), TVR-IL-8 (10 nM), or in combination of varying concentrations of IL-8 with TVR-IL-8 (10 nM). Panel B is endothelial cell chemotaxis in response to the presence or absence of varying concentrations of IL-8 (1-10 nM), DLQ-IL-8 (10 nM), or in combination of varying concentrations of IL-8 with DLQ-IL-8 (10 nM). To demonstrate specific migration, background (unstimulated control) migration (cells/HPF) was subtracted.
Using the in vivo rat cornea micropocket model of neovascularization, TVR-IL-8 (10 nM) alone did not induce a positive neovascular response in any of the six corneas tested. However, TVR-IL-8 (10 nM) in combination with either IL-8 (10 nM) or ENA-78 (10 nM) resulted in 83% reduction (only one of six corneas positive) in the ability of either IL-8 or ENA-78 to induce cornea neovascularization, as compared with 100% (six of six) of the corneas positive in the presence of either IL-8 or ENA-78 alone (Fig. 5). Moreover, the angiostatic activity of the IL-8 muteins was not only unique to inhibition of ELR-CXC chemokine-induced angiogenic activity, as TVR-IL-8 (10 nM) inhibited both bFGF-induced (10 nM) maximal endothelial cell chemotaxis by 65% (p < 0.05) (Fig. 6a) and corneal neovascularization (five of six corneas; n = 6 corneas for each cytokine) (Fig. 6b). Endothelial cell viability, as determined by the exclusion of trypan blue, was unchanged in the presence or absence of the TVR-IL-8 mutant (data not shown). In addition, ELR-MIG (10 nM) induced angiogenic responses in 8 of 10 corneas, as compared with wild-type MIG, which induced an angiogenic response in only 1 of 7 corneas (Fig. 7, A-D). Interestingly, MIG (10 nM) inhibited the angiogenic response of ELR-MIG in five of six corneas (Fig. 7, E and F). These data further support the importance of the ELR motif as a domain for mediating angiogenic activity. Similar to the synthetic ELR-IP-10(36) , ELR-MIG in a concentration of 10 pM to 100 nM failed to induce neutrophil chemotaxis (data not shown).
Figure 5:
Rat cornea neovascularization in response
to the IL-8, ENA-78, the IL-8 mutein (TVR-IL-8), and combinations of
ENA-78 and TVR-IL-8 or IL-8 and TVR-IL-8. Panels A-D represent a hydron pellet alone, TVR-IL-8 (10 nM), ENA-78
(10 nM), and IL-8 (10 nM), respectively. Panels E and F represent the combination of ENA-78 and TVR-IL-8
and of IL-8 and TVR-IL-8, respectively. All panels are at 25
magnification.
Figure 6:
Endothelial chemotaxis (part a)
and rat cornea neovascularization (part b) in response to the
presence or absence of varying concentrations of bFGF and the IL-8
mutein, TVR-IL-8 (10 nM). Part a is the endothelial
chemotaxis in response to the presence or absence of varying
concentrations of bFGF (1-10 nM), TVR-IL-8 (10
nM), or in combination of varying concentrations of IL-8 with
TVR-IL-8 (10 nM). To demonstrate specific migration,
background (unstimulated control) migration (cells/HPF) was subtracted. Part b, panels A-C is rat cornea
neovascularization in response to bFGF (10 nM), TVR-IL-8 (10
nM), and the combination of bFGF and TVR-IL-8 at 25
magnification, respectively.
Figure 7:
Rat cornea neovascularization in response
to the MIG mutein, ELR-MIG, MIG, and the combination of ELR-MIG and
MIG. Panels A and B represents the cornea neovascular
response to ELR-MIG (10 nM) at 25 and 50, respectively. Panels C and D represent the cornea neovascular
response to MIG (10 nM) at 25 and 50
magnification,
respectively. Panels E and F represent the cornea
neovascular response to the combination of ELR-MIG and MIG at 25 and
50
magnification, respectively.
The CXC chemokine family of chemotactic cytokines are
polypeptide molecules that appear, in general, to have proinflammatory
activities. In monomeric forms, they range from 7 to 10 kDa and are
characteristically basic heparin-binding proteins. They display four
highly conserved cysteine amino acid residues with the first two
cysteines separated by a nonconserved amino acid residue (the CXC
cysteine motif). The CXC chemokines are all clustered on human
chromosome 4 (q12-q21), and exhibit between 20 and 50% homology on the
amino acid level(31, 32, 33, 34) .
Over the last 2 decades, several human CXC chemokines have been
identified, including PF4, NH-terminal truncated forms of
platelet basic protein (connective tissue activating protein-III,
-thromboglobulin, neutrophil-activating protein-2), IL-8,
GRO-
, GRO-
, GRO-
, ENA-78, GCP-2, IP-10, and
MIG(31, 32, 33, 34, 38) .
The ubiquitous nature of CXC chemokine production by a variety of cells
suggest that these cytokines may play a role in mediating biological
events other than leukocyte chemotaxis.
We hypothesized that members
of the CXC chemokine family may exert disparate effects in mediating
angiogenesis as a function of the presence or absence of the ELR motif
for primarily four reasons. First, members of the CXC chemokine family
that display binding and activation of neutrophils share the highly
conserved ELR motif that immediately precedes the first cysteine amino
acid residue, whereas, PF4, IP-10, and MIG lack this
motif(35, 36) . Second, IL-8 (contains ELR motif)
mediates both endothelial cell chemotactic and proliferative activity in vitro and angiogenic activity in
vivo(28) , and, in addition, endogenous IL-8 has been
found to represent a major angiogenic factor that mediates net
angiogenic activity of human nonsmall cell lung cancer(42) . In
contrast, PF4 (lacking the ELR motif) has been shown to have
angiostatic properties(27) , and attenuates growth of tumors in vivo(45) . Third, the interferons (IFN-,
IFN-
, and IFN-
) are all known inhibitors of wound repair,
especially
angiogenesis(18, 46, 47, 48, 49) .
These cytokines, however, up-regulate IP-10 and MIG from a number of
cells, including keratinocytes, fibroblasts, endothelial cells, and
mononuclear phagocytes(38, 50) . Finally, we and
others have found that IFN-
, IFN-
, and IFN-
are potent
inhibitors of the production of monocyte-derived IL-8, GRO-
, and
ENA-78(51, 52) , supporting the notion that IFN-
,
IFN-
, and IFN-
may shift the biological balance of ELR- and
non-ELR-CXC chemokines toward a preponderance of angiostatic (non-ELR)
CXC chemokines.
In this study, we demonstrated that the members of the CXC chemokine family behave as either angiogenic or angiostatic factors, depending upon the presence or absence of the ELR motif, respectively. This was supported using both in vitro (endothelial cell chemotaxis) and in vivo (rat cornea neovascularization) analyses. The evidence in vitro of directed (chemotaxis not chemokinesis by checkerboard analysis) migration in response to varying concentrations of ELR-CXC chemokines, IL-8, ENA-78, and the MIG mutein ELR-MIG, and the absence in vivo of leukocyte infiltration in the rat cornea during ELR-CXC chemokine-induced neovascularization, supports the direct role ELR-containing CXC chemokines play in mediating angiogenic activity. In contrast, CXC chemokines lacking the ELR motif, PF4, IP-10, MIG, and the two IL-8 muteins DLQ-IL-8 and TVR-IL-8, behave as potent angiostatic regulators of neovascularization, inhibiting not only the angiogenic activity of ELR-CXC chemokines, but also the structurally unrelated angiogenic factor, bFGF. Thus, the ELR motif appears to be essential for dictating the angiogenic activity of the CXC chemokines.
These findings are compatible with the ability of ELR-containing CXC chemokines to bind to both endothelial cells and neutrophils. However, the non-ELR muteins of wild-type IL-8, as well as IP-10 and MIG, inhibited ELR-CXC chemokine-induced angiogenesis but not neutrophil chemotaxis. The finding that IP-10 and MIG block other ELR-CXC chemokine-induced functions, i.e. angiogenesis, is unprecedented(53) . Moreover, the muteins of wild-type IL-8, as well as IP-10 and MIG, also inhibited the angiogenic activity of the unrelated cytokine, bFGF, suggesting that a receptor system(s) other than the IL-8 receptor may be operative on endothelial cells, which allows the angiostatic CXC chemokines to regulate both ELR-CXC chemokine and bFGF-induced angiogenic activity. This contention is further supported with the evidence that equimolar concentrations of mutant and wild-type IL-8 do not result in a 50% restoration of the endothelial cell chemotactic effect. This response is most likely due to the use of another ``receptor'' by the angiostatic CXC chemokines. While the Duffy antigen receptor for chemokines has been identified on post-capillary venule endothelial cells(54) , this receptor binds not only ELR-CXC chemokines, but also MCP-1 and RANTES(55) . We have found that these latter two CC chemokines are not chemotactic for endothelial cells (data not shown).
While
the NH-terminal ELR motif appears to be essential for
angiogenic activity of CXC chemokines, it is uncertain whether the
angiostatic properties of the non-ELR-CXC chemokines tested are due to
the absence of the ELR motif. In particular, bFGF binds to low affinity
cell surface receptors on endothelia that appear to be sulfate
proteoglycans(47, 56) , and IL-8 specific binding to
endothelial cells can be inhibited by preincubation with either heparin
or heparan sulfate(57) . One can speculate that, in the absence
of the ELR motif, a potential mechanism exists by which another amino
acid domain, perhaps within the COOH terminus of PF4, IP-10, MIG,
TVR-IL-8, and DLQ-IL-8 may compete with either ELR-CXC chemokines or
bFGF for proteoglycan binding sites and thus prevent endothelial cell
activation and angiogenesis. It also possible, however, that the
angiostatic effects of CXC chemokines lacking the ELR motif are not
directly competitive in nature, but are rather mediated through an
independent receptor system. Studies in our laboratories are currently
addressing these issues.
The interferons have been shown to inhibit
wound repair and tumorigenesis through a presumed antiproliferative and
angiostatic
mechanism(46, 47, 48, 49) . While
the expression of IL-8, GRO-, and ENA-78 can be induced by a
variety of factors, including TNF and IL-1, these chemokines are
down-regulated by IFN-
(51, 52) . In contrast,
IP-10 and MIG expression is up-regulated by
IFN-
(38, 50) . This suggests that the disparate
activity of the CXC chemokines as angiogenic or angiostatic factors may
be physiologically relevant. The finding that IP-10 and MIG are potent
angiostatic factors suggests that IFN-
, in part, may mediate its
angiostatic activity through the local stimulation of production of
IP-10 and MIG and by down-regulation of the expression of the
angiogenic CXC chemokines, such as IL-8 and ENA-78. This suggests that
the magnitude of local IFN-
expression by mononuclear cells during
wound repair, chronic inflammation, or tumorigenesis may be a pivotal
event in regulating both angiogenic (through negative feedback) and
angiostatic (through positive feedback) CXC chemokine production.
Thus, our findings suggest that the ELR motif is the functional domain that dictates the angiogenic activity of the CXC chemokines, and supports the contention that members of the CXC chemokine family may exert disparate effects in mediating angiogenesis. The magnitude of the expression and relative concentrations of either angiogenic or angiostatic CXC chemokines during neovascularization may thus significantly contribute to the regulation of net angiogenesis during either wound repair, chronic inflammation, or tumorigenesis.