©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Functional Role of the ELR Motif in CXC Chemokine-mediated Angiogenesis (*)

(Received for publication, July 7, 1995; and in revised form, August 11, 1995)

Robert M. Strieter (1)(§) Peter J. Polverini (3) Steven L. Kunkel (2) Douglas A. Arenberg (1) Marie D. Burdick (1) James Kasper (6) Judith Dzuiba (7) Jo Van Damme (4) Alfred Walz (5) David Marriott (8) Sham-Yuen Chan (8) Steven Roczniak (6) Armen B. Shanafelt (6)

From the  (1)Departments of Internal Medicine (Division of Pulmonary and Critical Medicine) and (2)Pathology, the University of Michigan Medical School, Ann Arbor, Michigan 48109-0360, the (3)University of Michigan Dental School, Section of Oral Pathology, Ann Arbor, Michigan 48109, the (4)Rega Institute, University of Leuven, B-3000 Leuven, Belgium, the (5)Theodor Kocher Institut, University of Bern, CH-3000, Bern 9, Switzerland, the (6)Institute of Molecular Biologicals and (7)Institute of Research Technologies, Bayer Corporation, West Haven, Connecticut 06516, and the (8)Department of Molecular and Cellular Biology, Bayer Corporation, Berkeley, California 94701

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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), (^1)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.


MATERIALS AND METHODS

Reagents

Human recombinant IP-10 (lyophilized protein with no additives) was purchased from Pepro Tech Inc. (Rocky Hill, NJ). IP-10 was >98% pure by SDS-PAGE analysis. Human recombinant bFGF (lyophilized protein with no additives) was purchased from R& Systems Inc. (Minneapolis, MN). bFGF was >97% pure, as determined by NH(2) terminus analysis and SDS-PAGE. Recombinant human PF4, natural NH(2)-terminal truncated forms of platelet basic protein (connective tissue activating protein-III, beta-thromboglobulin, and neutrophil-activating protein-2), recombinant IL-8 (72 amino acids), recombinant human growth-related oncogene (GRO-alpha), recombinant human GRO-beta, recombinant human GRO-, and recombinant human epithelial neutrophil activating protein-78 (ENA-78) were provided by A. Walz. These chemokines were lyophilized proteins with no additives and were >97% pure, as determined by NH(2) terminus analysis and SDS-PAGE. Natural granulocyte chemotactic protein-2 (GCP-2; (34) ) was provided by J. Van Damme and was >98% pure, as determined by NH(2) terminus analysis and SDS-PAGE. Endotoxin levels were less than 0.1 ng/µg for the above cytokines. The proteins were either reconstituted in Dulbecco's modified Eagle's medium with 0.1% bovine serum albumin for analysis in endothelial cell chemotaxis assays, Hanks' balanced salt solution with calcium/magnesium for analysis in neutrophil cell chemotaxis assays, or 1 times PBS for the corneal micropocket model of angiogenesis.

Bacterial Host Strains and Vectors

The Escherichia coli K12 strain DH5alphaF` (Life Technologies, Inc.) was used as host for the propagation and maintenance of M13 DNA, and for expression of IL-8 and MIG proteins. Strain CJ236 was used to prepare uracil-DNA for use in site-directed mutagenesis(37) . pGEX 4T-1 (Pharmacia Biotech Inc.) was used as the expression vector for all MIG cDNAs(38) . pMAL-c2 (New England BioLabs) was used as the expression vector for all IL-8 cDNAs.

Mutagenesis, Recombinant DNA, and Sequencing Protocols

Site-directed mutagenesis was followed the protocol described by Kunkel et al.(37) . Individual clones were sequenced using the dideoxynucleotide method (39) with modifications described in the Sequenase® (U. S. Biochemical Corp.) protocol. M13 (replicative form) DNA (40) containing confirmed MIG mutations was cleaved with BamHI and XbaI (New England Biolabs) and subcloned into pGEX 4T-1. A 197-base pair Sac I (New England Biolabs) fragment from pMAL.hIL-8 (maltose binding protein-Ile-Glu-Gly-Arg-human IL-8 fusion protein expression vector) containing the coding sequence for the NH(2)-terminal 49 amino acids of the 72-amino acid form of human IL-8 sequence was subcloned to pUC118 (ATCC) digested with SacI for site-directed mutagenesis. Clones containing confirmed IL-8 mutations were cleaved with SacI and subcloned into pMAL.hIL-8 digested with SacI.

Cloning, Expression, and Purification of Human MIG

The open reading frame of human MIG (38) was amplified from cDNA generated from interferon -stimulated (1000 units/ml for 16 h) THP-1 cells by polymerase chain reaction. The 5`-primer used, 5`-CAAGGTGGATCCATGAAGAAAAGTGGTGTTC-3`, encodes a BamHI restricition site immediately upstream of the ATG start site. The 3`-primer, 5`-GCAAGCTCTAGATTATGTAGTCTTCTTTTGACGAGAACG-3`, encodes a XbaI restriction site immediately downstream of the TAA stop codon. The 402-base pair fragment was subcloned to M13mp19 and was confirmed as the human MIG open reading frame by sequencing. Thr of the open reading frame sequence is the predicted NH(4)-terminal amino acid of the mature, secreted MIG protein (38) and will be referred to here as amino acid position 1. Amino acids Lys^6 and Gly^7 were modified to Glu and Leu, respectively, by site-directed mutagenesis, generating the MIG mutein ELR-MIG. A BamHI restriction site was introduced overlapping Gly and Thr^1 by site-directed mutagenesis(37) , resulting in mutein MIG or ELR-MIG cDNAs encoding a Thr^1 to Ser substitution. 324-base pair fragments obtained from correct M13 RF clones digested with BamHI/XbaI were subcloned to pGEX 4T-1 to generate glutathione S-transferase-MIG fusion DNAs (GST-MIG or GST-ELR-MIG). The sequence encoded by these DNAs contains the thrombin recognition sequence LVPRGS between the GST and MIG sequences. Digestion of GST-MIG fusion protein with thrombin is predicted to release MIG protein having an NH(4)-terminal sequence Gly-Ser-Pro, versus the predicted nonmodified NH(4)-terminal sequence Thr-Pro.

Cultures of E. coli strain DH5alphaF` 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-beta-D-galactoside and continued incubation at 22 °C for 5-6 h. After induction, the cells were harvested by centrifuging at 6,000 times 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 times 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 geq95% 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(4), 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.

Cloning, Expression, and Purification of Human IL-8

The 72amino acid mature form of IL-8 was amplified using polymerase chain reaction from an IL-8 cDNA in pET3a (kindly provided by I. U. Schraufstatter, Scripps Clinic). The 5`-primer used, 5`-AGTGCTAAAGAACTTAGATG-3`, encodes the beginning reading frame of IL-8, and the 3` primer, 5`-GGGATCCTCATGAATTCTC-3`, contains a BamHI restriction site immediately after the stop codon. The 220-base pair PCR product was purified by gel electrophoresis, digested with BamHI (New England Biolabs), subcloned into pMal-c2 previously digested with XmnI and BamHI (New England Biolabs) to generate pMal.hIL-8. Clones containing inserts were confirmed by sequencing. Site-directed mutagenesis was used to modify amino acids Glu^4-Leu^5-Arg^6 to Thr-Val-Arg or Asp-Leu-Gln, generating TVR-IL-8 or DLQ-IL-8, respectively. Correct clones were identified by sequencing and subcloned as SacI fragments from pUC118 into pMal.hIL-8 digested with SacI.

Cultures of E. coli strain DH5alphaF` 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-beta-D-galactoside and continued incubation at 37 °C for 2 h. Cells were harvested by centrifuging at 5800 times 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 times g, the supernatant was diluted 5-fold in 10 mM NaPO(4), 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(4), 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(4), 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 geq95% 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%.

Endothelial Cell Chemotaxis

Endothelial cell chemotaxis was performed in 48-well chemotaxis chambers (Nucleopore Corp.) as described previously(28, 42) . Briefly, bovine adrenal gland capillary endothelial cells were suspended at a concentration of 10^6 cells/ml in Dulbecco's modified Eagle's medium with 0.1% bovine serum albumin and placed into each of the bottom wells (25 µl). Nucleopore chemotaxis membranes (5-µm pore size) were coated with 0.1 mg/ml gelatin. The membranes were placed over the wells and the chambers were sealed, inverted, and incubated for 2 h to allow cells to adhere to the membrane. The chambers were then reinverted; 50 µl of sample (containing media alone, ELR-CXC chemokines, non-ELR-CXC chemokines, bFGF, or combinations of ELR-CXC and non-ELR-CXC chemokines or non-ELR-CXC chemokines and bFGF) was dispensed into the top wells and reincubated for an additional 2 h. Membranes were then fixed and stained with Diff-Quik staining kit (American Scientific Products), and cells that had migrated through the membrane were counted in 10 high power fields (HPF; 400times). Results were expressed as the number of endothelial cells that migrated per HPF after subtracting the background (unstimulated control) to demonstrate specific migration. Each sample was assessed in triplicate. Experiments were repeated at least three times.

Neutrophil Chemotaxis

Heparinized venous blood was collected from healthy volunteers and mixed 1:1 with 0.9% saline, and mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation. Human neutrophils were then isolated by sedimentation in 5% dextran, 0.9% saline (Sigma) and separated from erythrocytes by hypotonic lysis. After washing twice, neutrophils were suspended in Hanks' balanced salt solution with calcium/magnesium (Life Technologies, Inc.) at a concentration of 2 times 10^6 cells/ml. Neutrophils were >95% viable as determined by trypan blue exclusion. Neutrophil chemotaxis was performed as described previously(43, 44) . 150 µl of sample (ELR-CXC, non-ELR-CXC, or combination of ELR-CXC and non-ELR-CXC chemokines), 1 times 10M formylmethionyleucylphenylalanine (Sigma), or Hanks' balanced salt solution (Life Technologies, Inc., Grand Island, NY) alone were placed in duplicate bottom wells of a blind well chemotaxis chamber. A 3-µm pore size polycarbonate filter (polyvinylpyrrolidone-free, Nucleopore Corp.) was placed in the assembly, and 250 µl of human neutrophil was suspension placed in each of the top wells. Chemotaxis chamber assemblies were incubated at 37 °C in humidified 95% air, 5% CO(2) for 60 min. The filters were removed, fixed in absolute methanol, and stained with 2% toluidine blue (Sigma). Neutrophils that had migrated through to the bottom of the filter were counted in 10 HPF (400times) using a Javelin chromachip camera (Javelin Electronics, Japan) attached to a Olympus BH-2 microscope interfaced with a MacIntosh II computer containing an Image Capture 1000 frame grabber (Scion Corp., Walkersville, MD) and NIH Image, version 1.40 software (National Institutes of Health Public Software, Bethesda, MD). Each sample was assessed in triplicate. Experiments were repeated at least three times.

Corneal Micropocket Model of Angiogenesis

In vivo angiogenic activity was assayed in the avascular cornea of Long Evans rat eyes, as described previously(28, 29, 42) . Briefly, cytokines were combined with sterile Hydron (Interferon Sciences Inc.) casting solution, and 5-µl aliquots were air-dried on the surface of polypropylene tubes. Prior to implantation, pellets were rehydrated with normal saline. Animals were anesthetized with an intraperitoneal injection of ketamine (150 mg/kg) and atropine (250 µg/kg). Rat corneas were anesthetized with 0.5% proparacaine hydrochloride ophthalmic solution followed by implantation of the Hydron pellet into an intracorneal pocket (1-2 mm from the limbus). 6 days after implantation, animals were pretreated intraperitoneally with 1000 units of heparin (Elkins-Sinn, Inc., Cherry Hill, NJ), anesthetized with ketamine (150 mg/Kg), and perfused with 10 ml of colloidal carbon via the left ventricle. Corneas were then harvested and photographed. No inflammatory response was observed in any of the corneas treated with the above cytokines. Positive neovascularization responses were recorded only if sustained directional ingrowth of capillary sprouts and hairpin loops toward the implant were observed. Negative responses were recorded when either no growth was observed or when only an occasional sprout or hairpin loop displaying no evidence of sustained growth was detected.

Statistical Analysis

Data were analyzed by a Macintosh IIfx computer using the Statview II statistical package (Abacus Concepts, Inc., Berkeley, CA). Data were expressed as mean ± S.E. and compared using the nonparametric analysis with the Wilcoxon signed rank test. Data were considered statistically significant if p values were leq0.05.


RESULTS

CXC Chemokines Display Disparate Angiogenic Activity

Endothelial cell chemotaxis was performed in the presence or absence of IL-8, ENA-78, PF4, and IP-10 at concentrations of 50 pM to 50 nM. Both IL-8 and ENA-78 demonstrated a dose-dependent increase in endothelial migration that was significantly greater (p < 0.05) than control (background) at concentrations equal to or above 0.1 and 1 nM, respectively, with evidence of a ``bell-shape'' curve seen with other chemotactic factors (Fig. 1). In contrast, neither PF4 nor IP-10 induced significant (p > 0.05) endothelial cell chemotaxis (Fig. 1). Similar findings were also observed using either human umbilical or dermal microvascular endothelial cells (data not shown). The migration seen in response to IL-8 or ENA-78 was due to chemotaxis, not chemokinesis, as checkerboard analysis demonstrated directed, not random, migration (data not shown). Other CXC chemokines were tested for their ability to induce endothelial cell chemotaxis, including ELR-CXC chemokines IL-8, ENA-78, GCP-2, GRO-alpha, GRO-beta, GRO-, platelet basic protein, connective tissue activating protein-III, and neutrophil-activating protein-2 or the non-ELR CXC chemokines IP-10, PF4, and MIG (Table 1). In a similar fashion to IL-8 or ENA-78, all of the ELR-CXC chemokines tested demonstrated significant (p < 0.05) endothelial cell chemotactic activity over the background control, whereas the endothelial cell chemotactic activity induced by MIG was either similar to background control or to the endothelial cell chemotactic activity seen with either PF4 or IP-10. These findings suggested that CXC chemokines could be divided into two groups with defined biological activities, one that contains the ELR motif and is chemotactic for endothelial cells and the other that lacks the ELR motif and does not induce endothelial chemotaxis.


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.





PF4, IP-10, or MIG Inhibit IL-8-, ENA-78-, or bFGF-induced Angiogenic Activity

While the above experiments suggested that PF4, IP-10, and MIG were not significant chemotactic factors for endothelial cells, we postulated that these CXC chemokines may be potent inhibitors of angiogenesis. To test this hypothesis, endothelial cell chemotaxis was performed in the presence or absence of IL-8 (10 nM), ENA-78 (10 nM), or bFGF (5 nM) with or without varying concentrations of PF4, IP-10, or MIG from 0 to 10 nM (Fig. 2, a-c, respectively). Endothelial cell migration in response to either IL-8, ENA-78, or bFGF was significantly inhibited by PF4, IP-10, or MIG in a dose-dependent manner (Fig. 2). PF4 and IP-10 in a concentration of 50 pM inhibited either IL-8- or ENA-78-induced endothelial chemotaxis by 50%, whereas, PF4 and IP-10 in a concentration of 1 nM attenuated the response to bFGF by 50% (Fig. 2, a and b, and Table 2). MIG at a concentration of 1, 5, and 1 nM inhibited the endothelial cell chemotactic response to IL-8, ENA-78, and bFGF, respectively, by 50% (Fig. 2c and Table 2). Interestingly, while IP-10 and MIG inhibited IL-8-induced endothelial cell chemotactic activity, neither IP-10 nor MIG were effective in attenuating IL-8-induced neutrophil chemotactic activity (p > 0.05) (Table 3).


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-alpha, 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-alpha + 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-alpha, 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-alpha, 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-alpha (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-alpha 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 25times magnification.



ELR Muteins of IL-8 and MIG Generate Angiostatic and Angiogenic Proteins, Respectively

Muteins of IL-8 lacking the ELR motif and a mutant of MIG containing the ELR motif were generated to delineate its functional role in CXC chemokine-induced angiogenesis. The ELR motif in wild-type IL-8 was mutated to either TVR (TVR-IL-8; corresponding IP-10 sequence) or DLQ (DLQ-IL-8; corresponding to PF4 sequence) by site-directed mutagenesis and expressed in E. coli. TVR-IL-8 and DLQ-IL-8 alone failed to induce endothelial cell chemotactic activity (Fig. 4, A and B, respectively), yet these muteins inhibited the maximal endothelial chemotactic activity of wild-type IL-8 by 83 and 88% (p < 0.05), respectively (Fig. 4, A and B). Endothelial cell viability, as determined by the exclusion of trypan blue, was unchanged in the presence or absence of either of the IL-8 muteins (data not shown). Neither TVR-IL-8 nor DLQ-IL-8 induced neutrophil chemotaxis, nor were they effective in attenuating neutrophil chemotaxis in response to IL-8 (data not shown).


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 25times 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 25times 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 50times, respectively. Panels C and D represent the cornea neovascular response to MIG (10 nM) at 25 and 50times magnification, respectively. Panels E and F represent the cornea neovascular response to the combination of ELR-MIG and MIG at 25 and 50times magnification, respectively.




DISCUSSION

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(2)-terminal truncated forms of platelet basic protein (connective tissue activating protein-III, beta-thromboglobulin, neutrophil-activating protein-2), IL-8, GRO-alpha, GRO-beta, 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-alpha, IFN-beta, 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-alpha, IFN-beta, and IFN- are potent inhibitors of the production of monocyte-derived IL-8, GRO-alpha, and ENA-78(51, 52) , supporting the notion that IFN-alpha, IFN-beta, 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(2)-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-alpha, 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.


FOOTNOTES

*
This work was supported, in part, by National Institutes of Health Grants HL50057, CA66180, and 1P50HL46487 (to R. M. S.), HL39926 (to P. J. P.), and HL31693 and HL35276 (to S. L. K.) and by the General Savings and Retirement Fund (ASLK) Cancer Foundation, Belgium (to J. V. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Internal Medicine, Div. of Pulmonary and Critical Care, Box 0360, University of Michigan Medical Center, 3916 Taubman Dr., Ann Arbor, MI 48109-0360. Tel.: 313-764-4554; Fax: 313-764-4556.

(^1)
The abbreviations used are: PF4, platelet factor 4; IL-8, interleukin-8; IP-10, interferon -inducible protein 10; MIG, monokine induced by -interferon; bFGF, basic fibroblast growth factorbasic fibroblast growth factor; PAGE, polyacrylamide gel electrophoresis; GRO, growth-related oncogene; ENA-78, epithelial neutrophil activating protein-78; GCP-2, granulocyte chemotactic protein-2; PBS, phosphate-buffered saline; GST, glutathione S-transferase; HPF, high power field(s); IFN, interferon.


ACKNOWLEDGEMENTS

We thank Carla Forte (Bayer Corp., West Haven, CT) for technical help during the course of this work and Ghislain Opdenakker (University of Leuven, Leuven, Belgium) for critical review of this manuscript.


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