Angiogenic Effects of Interleukin 8 (CXCL8) in Human Intestinal Microvascular Endothelial Cells Are Mediated by CXCR2*

Jan HeidemannDagger , Hitoshi OgawaDagger , Michael B. Dwinell§, Parvaneh Rafiee, Christian Maaser||, Henning R. Gockel||, Mary F. Otterson, David M. Ota, Norbert Lügering||, Wolfram Domschke||, and David G. BinionDagger **

From the Departments of Dagger  Medicine, § Microbiology and Molecular Genetics, and  Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the || Department of Medicine B, University of Münster, Münster 48149, Germany

Received for publication, August 12, 2002, and in revised form, December 17, 2002

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Angiogenesis plays a critical role in metastasis and tumor growth. Human tumors, including colorectal adenocarcinoma, secrete angiogenic factors, inducing proliferation and chemotaxis of microvascular endothelial cells, eventually leading to tumor neovascularization. The chemokine interleukin 8 (IL-8; CXCL8) exerts potent angiogenic properties on endothelial cells through interaction with its cognate receptors CXCR1 and CXCR2. As CXCR1 and CXCR2 expression is differentially regulated in tissue-specific endothelial cells and effects of IL-8 on intestinal endothelial cells are not defined, we characterized the potential IL-8-induced angiogenic mechanisms in primary cultures of human intestinal microvascular endothelial cells (HIMEC) and IL-8 receptor expression in human intestinal microvessels. CXCR1 and CXCR2 expression on HIMEC were defined using reverse transcriptase-PCR, immunohistochemistry, flow cytometry, and Western blot analysis. IL-8-induced downstream signaling events were assessed using immunoblot analysis and immunofluorescence. The angiogenic effects of IL-8 on HIMEC were determined using proliferation and chemotaxis assays. HIMEC responded to IL-8 with rapid stress fiber assembly, chemotaxis, enhanced proliferation, and phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2). HIMEC express CXCR2, but not CXCR1. Neutralizing antibodies to CXCR2 diminished IL-8-induced chemotaxis and stress fiber assembly. Specific inhibitors of ERK 1/2 and phosphoinositide 3-kinase abrogated endothelial tube formation and IL-8-induced chemotaxis in HIMEC. IL-8 elicits angiogenic responses in microvascular endothelial cells isolated from human intestine by engaging CXCR2. We confirmed tissue expression of CXCR2 in human intestinal microvessels. Supported by the notion that malignant colonic epithelial cells overexpress IL-8, CXCR2 blockade may be a novel target for anti-angiogenic therapy in colorectal adenocarcinoma.

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Angiogenesis, the formation of new vessels from existing capillary beds, is a crucial process involved in various physiological and pathophysiological conditions, including embryonal development, wound healing, chronic inflammation, and tumor growth. It comprises a multistep sequence of basement membrane proteolysis, endothelial cell (EC)1 proliferation and chemotaxis, and organization and maturation of tubular structures. Angiogenesis results from an increase in angiogenic mediators (such as vascular endothelial growth factor (VEGF)) or a reduction in angiostatic factors, including interferon (IFN)-gamma , as well as the release of proteases, which degrade the extracellular matrix (1, 2). Interleukin-8 (IL-8, CXCL-8), an ELR (Glu-Leu-Arg) motif positive (ELR+) CXC chemokine (3, 4), has been shown to exert direct angiogenic effects on EC in vitro and in vivo (5-7). IL-8 exerted a more pronounced chemotactic effect on dermal microvascular EC compared with macrovascular EC (2). In corneal micropocket assays in rats (4, 5, 7), angiogenesis was strongly induced by local administration of IL-8 and variably accompanied by an inflammatory infiltrate, raising the possibility of an indirect angiogenic mechanism by IL-8-mediated release of angiogenic mediators (7).

IL-8 was initially identified as a major proinflammatory cytokine. In Crohn's disease and ulcerative colitis, total mucosal IL-8 mRNA and protein are significantly up-regulated in the colonic mucosa (8-11), in direct proportion with the degree of inflammation (12). IL-8 acts as a potent chemoattractant for neutrophils, the major cellular component of acute inflammatory infiltrates (8). In addition to this proinflammatory function, there is growing evidence that IL-8 exerts effects on nonimmune cells, including the vascular endothelium. Studies from human gastrointestinal carcinomas have suggested a pivotal role for IL-8 in tumor angiogenesis and tumor growth. IL-8 expression correlates with vascularity in gastric carcinomas (13), and microvessel counts in histologically normal tissue adjacent to colonic adenocarcinoma indicated distant angiogenic effects mediated by soluble factors (14). Notably, IL-8 is highly expressed in hyperplastic mucosa adjacent to colon cancer, supporting an indirect angiogenic effect of colon cancer cells (15). Moreover, IL-8 is apparently involved in development of distant metastasis from colorectal cancer (16). Interestingly, both colonic epithelial cells (17) and microvascular EC of human gut secrete IL-8 in a regulated fashion in vitro (18), suggestive of a cross-talk between those cells in the human intestinal mucosa (19). Substantial evidence exists that IL-8 is a critical factor in angiogenesis in a multitude of human tumors. Strategies blocking the angiogenic activity of IL-8 have proven effective to inhibit angiogenesis, metastasis, and tumor progression in human tumors in murine models (20-28).

The biological activity of IL-8 is mediated by binding to two highly related receptors, CXCR1 and CXCR2, on target cells. The chemokine receptor CXCR2 promiscuously binds all known angiogenic ELR+ CXC chemokines, including IL-8, the growth-regulated oncogene family members (GRO-alpha , -beta , and -gamma ), NAP-2, GCP-2, and ENA-78 with high affinity (29, 30). In contrast, CXCR1 specifically binds only IL-8 and GCP-2 (30, 31). Both CXCR1 and CXCR2 are members of the seven-transmembrane domain rhodopsin-like G protein-coupled receptor superfamily and share 78% amino acid sequence homology (32).

The detection of the IL-8 receptors, CXCR1 and CXCR2 in cultured EC, by means of RNA message and functional protein expression, has demonstrated conflicting results (33), as the degree of monolayer confluence and the extracellular matrix likely regulate CXC receptor expression in EC (34). In addition, several cytokines, including tumor necrosis factor (TNF)-alpha are potent inducers of CXCR2 on human microvascular EC (35). EC subsets from different vascular beds demonstrate heterogeneous responsiveness to cytokines and differentially express adhesion molecules and secretory products (36-39). Therefore, we sought to define the expression of IL-8 receptors in human intestinal microvessels and characterize the functional angiogenic response of human intestinal microvascular EC (HIMEC) to IL-8.

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Antibodies and Reagents-- Monoclonal anti-CXCR1 (Clone 5A12) and anti-CXCR2 (Clone 6C6) antibodies were purchased from BD Pharmingen (San Diego, CA). Mouse IgG (R&D Systems, Minneapolis, MN) served as an isotype control. Recombinant human IL-8 and cytokines were purchased from R&D Systems. Endothelial cell growth supplement was from Upstate Biotech Inc. (Lake Placid, NY). MCDB-131 medium, porcine heparin, bacterial lipopolysaccharide (LPS) (Escherichia coli O111:B4), concanavalin A, trypsin inhibitor (type II-S), and PSF (penicillin/streptomycin/fungizone) were from Sigma. Fetal bovine serum (FBS) and RPMI 1640 medium were obtained from BioWhittaker (Walkersville, MD). Bovine serum albumin (BSA, Fraction V) was obtained from Fisher Scientific (Fair Lawn, NJ). Human plasma fibronectin was purchased from Chemicon International (Temecula, CA). The specific inhibitors of p42/44 mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide 3-kinase (wortmannin and LY294002) were purchased from Calbiochem (La Jolla, CA), and MatrigelTM was obtained from BD Discovery Labware (Bedford, MA).

Cell Culture-- Macroscopically normal intestinal specimens for HIMEC isolation were obtained from patients undergoing scheduled bowel resection. The use of human tissues was approved by the Institutional Review Board of the Medical College of Wisconsin. HIMEC were isolated as previously described (40). In brief, mucosal strips from resected normal colon were washed, minced, and digested in collagenase type II solution (Worthington, Lakewood, NJ; 2 mg/ml). EC were extruded by mechanical compression and plated onto fibronectin-coated tissue culture dishes in growth medium (MCDB-131 medium supplemented with 20% (v/v) FBS and endothelial cell growth supplement, porcine heparin (130 mg/ml), and 2.5% (v/v) PSF solution). After 7-10 days of culture, microvascular EC clusters were physically isolated, and a pure culture was obtained. HIMEC cultures were recognized by microscopic phenotype, modified lipoprotein uptake (Dil-ac-LDL, Biomedical Technology, Inc., Stoughton, MA), and expression of Factor VIII-associated antigen. All experiments were carried out using HIMEC cultures between passages 8 and 12.

Peripheral blood mononuclear cells (PBMC) from healthy donors were purified by Ficoll-Hypaque (Amersham Biosciences) density centrifugation. Whole blood samples were aseptically drawn into heparinized vials, mixed (1:1) with phosphate-buffered saline (PBS, pH 7.4), and centrifuged over a Ficoll density gradient. Cell viability was >= 95% as assessed by trypan blue exclusion. PBMC were washed, plated, and cultured overnight in RPMI 1640 containing 2 mM glutamine, 25 mM HEPES, 2 µg/ml LPS, 4 µg/ml concanavalin A, and 5% (v/v) FBS. Cells were washed and subjected to RNA isolation.

PCR Primers-- Unlabeled oligonucleotides with the following sequences were purchased from Operon (Alameda, CA): CXCR1 sense 5'-GGGGCCACACCAACCTTC-3' and antisense 5'-AGTGCCTGCCTCAATGTCTCC-3' (product size 363 bp; GenBankTM accession number: BC028221); CXCR2 sense 5'-GGGCAACAATACAGCAAACT-3' and antisense 5'-GCACTTAGGCAGGAGGTCTT-3' (499 bp; accession number: NM_001557); beta -actin sense 5'-CCAGAGCAAGAGAGGCATCC-3' and antisense 5'-CTGTGGTGGTGAAGCTGTAG-3' (436 bp; accession number: BC016045).

RNA Isolation and cDNA Synthesis-- Total RNA was isolated from subconfluent HIMEC cultures by a single step guanidinium thiocyanate/phenol-chloroform extraction using TRIzol reagent (Invitrogen, Carlsbad, CA). Genomic DNA was subsequently removed by DNase I (Amplification Grade, Invitrogen). For reverse transcription, cDNA was generated using 1 µg of total RNA according to the manufacturer's protocol (Superscript II reverse transcription kit, Invitrogen) in a total volume of 20 µl.

PCR Amplification-- PCR was performed using RoboCycler (Stratagene, La Jolla, CA). For each reaction, 1.25 µl of cDNA was amplified using specific primers (25 pmol) in a final volume of 50 µl. For CXCR1, PCR was performed for 35 cycles of 94 °C (45 s), 62 °C (30 s), and 72 °C (60 s) followed by a final 72 °C step (6 min). For CXCR2 and beta -actin, the samples were subjected to 35 cycles of 94 (60 s) and 62 °C (150 s), followed by a final 72 °C step (6 min). PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining. DNase-treated total RNA samples (no reverse transcriptase) were used as a negative control, whereas cDNA of stimulated PBMC served as a positive control. Equal loading of cDNA was confirmed by amplification of beta -actin.

Flow Cytometry-- HIMEC monolayers were washed and detached with ice-cold 20 mM EDTA (pH 7.6), PBS and resuspended in fixation buffer (PBS containing 0.05% (w/v) NaN3, 0.5% (w/v) BSA, and 1% (w/v) paraformaldehyde). After washing in wash buffer (fixation buffer without paraformaldehyde), cells were incubated with primary antibodies (10 µg/ml), washed, incubated with fluorescein isothiocyanate-conjugated goat anti-mouse antibody (BD Biosciences) in the dark and analyzed (FACScan; BD Biosciences). Mouse isotype IgG served as a negative control (R&D Systems).

Immunoblot Analysis-- Neutrophils from healthy donors were purified as described previously (41). Cell viability and purity was >= 95% as assessed by trypan blue exclusion and DiffQuik staining (Baxter Scientific, McGraw, IL). Neutrophils and HIMEC from two different isolations were lysed in lysis buffer (50 mM Tris-Cl, 2 mM EDTA, 2 mM EGTA, 75 mM NaCl, 25 mM NaF, 25 mM beta -glycerophosphate, 1 mM Na3VO4, 1 mM Na2MoO4, 1 mM phenylmethanesulfonyl fluoride, 1% Nonidet P-40, and 5 µg/ml of each leupeptin, aprotinin, and pepstatin (all from Sigma) on ice. Lysates were cleared by centrifugation, and total protein concentration was determined by Bradford assay (Bio-Rad). 10 µg of neutrophil protein and 30 µg of HIMEC extracts were separated by SDS-PAGE, blotted onto nitrocellulose, and blocked for 1 h at room temperature in 3% (w/v) BSA, 3% (w/v) nonfat dry milk in Tris-buffered saline (50 mM Tris-HCl, pH 7.4) containing 0.1% (v/v) Tween 20 (TBS-T). After washing in TBS-T, blots were incubated with polyclonal rabbit anti-human anti-CXCR2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Immunodetection was performed using horseradish peroxidase-conjugated goat anti-rabbit antibodies (Zymed Laboratories Inc., South San Francisco, CA) and enhanced chemiluminescence (ECL, Amersham Biosciences). For detection of extracellular signal-regulated kinase (ERK) 1/2, subconfluent HIMEC monolayers were stimulated with recombinant human IL-8 (100 ng/ml) at different time points. HIMEC were washed with ice-cold PBS and lysed as described above. 15 µg of total protein per lane was separated and blotted, and blocked blots were incubated with antibodies specific for phosphorylated and total ERK 1/2 (Cell Signaling Tech., Beverly, MA) at 4 °C overnight. Immunodetection was performed as described above.

Immunofluorescence-- F-Actin polymerization was assessed in subconfluent HIMEC seeded on fibronectin-coated glass chamber slides (LabTek; Nalge Nunc, Naperville, IL). Cells were cultured in MCDB-131 containing 10% FBS, stimulated with 100 ng/ml recombinant human IL-8 (15 min), and fixed with 4% formaldehyde in PBS for 20 min. Cells were washed and permeabilized with Triton X-100 (0.2% (v/v) in PBS) for 10 min, blocked with 2.5% (w/v) BSA/PBS, and stained with fluorescein phalloidin (Molecular Probes, Eugene, OR). After washing, slides were air dried and mounted with Fluromount-G (Southern Biotechnology, Birmingham, AL) and examined with a fluorescence microscope (Olympus BX-40) using a fixed shutter speed to allow for comparison of fluorescence intensity. In some experiments, stress fiber assembly was blocked by preincubation of cells with 10 µg/ml neutralizing CXCR2 antibody (30 min at 37 °C).

Endothelial Cell Chemotaxis Assay-- Polycarbonate filters (8 µm pore size, BD Biosciences) were coated with human fibronectin (10 µg/ml) at 4 °C overnight. HIMEC were trypsinized, washed with chemotaxis buffer (MCDB-131 + 1% (w/v) BSA) containing soybean trypsin inhibitor (10 mg/ml), and resuspended in chemotaxis buffer. 5 × 105 cells were added to the upper chamber, and chemotaxis buffer (1000 µl) containing VEGF (50 ng/ml) or recombinant human IL-8 were filled into the lower compartment of the 12-well plates. After 3 h of incubation at 37 °C (5% CO2), cell culture inserts were removed and the upper side of the membrane was gently wiped. Filters were stained with DiffQuik (Baxter Scientific, McGraw, IL), air dried, and mounted onto glass slides. Migrated HIMEC adherent to the lower side of the membrane were counted (10 random high-power fields (×200) per condition). In inhibition studies, resuspended cells were incubated with neutralizing anti-CXCR2 antibodies (4 µg/ml) for 30 min at 37 °C, isotype control antibodies, or inhibitors of MAPK kinase and PI3K as indicated in the figure legends. Control cells remained free of inhibitors. Cell viability was >95% as assessed by trypan blue exclusion. Each condition was assessed in triplicate.

Proliferation Assay-- Cellular DNA synthesis was assessed by [3H]thymidine uptake. HIMEC were kept in medium without endothelial cell growth supplement (5% (v/v) FBS) for 48 h, trypsinized, and seeded onto fibronectin-coated 24-well plates (4 × 104 cells per well). After adherence (3 h, 37 °C), cells were stimulated for 18 h with IL-8 (1-100 ng/ml) in medium. Following 12 h of stimulation, cells were pulsed with [3H]thymidine (1 µCi/ml; Amersham Biosciences). After washing twice, cells were fixed for 10 min on ice with 5% (v/v) trichloroacetic acid. DNA was then released from precipitated material by alkaline lysis in 0.5 N NaOH, and supernatants were quantified in a beta -counter.

In Vitro Tube Formation Assay-- Endothelial tube formation was assessed using MatrigelTM, a solubilized extracellular basement membrane matrix extracted from the Engelbreth-Holm-Swarm mouse sarcoma, as described previously (42). Multiwell dishes (24-well) were coated with 250 µl of complete medium containing 5 mg/ml Matrigel and HIMEC resuspended in complete growth medium were seeded at a density of 5 × 104. The growth medium was supplemented with the MAPK kinase inhibitors PD98059 (10 and 20 µM) LY294002 (10 and 20 µM), or wortmannin (50 or 100 nM). Control cells remained free of inhibitors. Cells were cultured on MatrigelTM for 16 h and endothelial tube formation was assessed by inverted phase-contrast microscopy. Five high-power fields per condition were examined and experiments were repeated in two independent HIMEC cultures. Control cells receiving Me2SO only served as a vehicle control (not shown).

Immunohistochemistry-- Full thickness normal colonic and ileal mucosa was fixed in 4% (w/v) paraformaldehyde/PBS overnight, saturated in 20% (w/v) sucrose/PBS, embedded in OCT compound (Sakura, Japan), and snap frozen in liquid nitrogen. 6-µm frozen sections were stained with monoclonal mouse anti-human antibodies (anti-CXCR2 (Clone 6C6)) at 4 °C overnight using the Cell and Tissue Staining kit (R&D Systems). After immunodetection of horseradish peroxidase conjugates with diaminobenzidine, sections were briefly counterstained with Mayer's hematoxylin and mounted. Positive staining is visible as a dark-brown precipitate. Isotype IgG was used as a negative control.

Statistical Analysis-- Statistical analysis was performed by analysis of variance using StatView for Macintosh (version 4.51; Abacus Concepts, Inc., Berkeley, CA). p < 0.05 was considered significant, and data shown are mean ± S.E.

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HIMEC Constitutively Express CXCR2, but Not CXCR1 mRNA-- To assess CXCR1 and CXCR2 expression in cultured HIMEC, we performed reverse transcriptase-PCR using receptor-specific primers (Fig. 1). The mRNA for CXCR2 was detected in unstimulated HIMEC. Moreover, CXCR2 mRNA was markedly increased upon challenge with TNF-alpha /LPS (100 IU/ml and 1 µg/ml, respectively). TNF-alpha is known to up-regulate IL-8 receptors in human microvascular EC (35), providing a possible mechanistic explanation for its in vivo proangiogenic effects (43). Incubation of HIMEC with IFN-gamma , an angiostatic cytokine in vitro (44, 45), led to a slight decrease in detectable mRNA for CXCR2, a receptor shown to mediate angiogenesis in vivo (32, 46). The message for CXCR1 could not be amplified in HIMEC cultures from 6 patients using up to 45 PCR cycles or re-amplification of PCR products (data not shown), whereas stimulated PBMC (positive control) from healthy donors yielded a robust signal. 12-Hour stimulation with proinflammatory stimuli (TNF-alpha , 100 IU/ml; LPS, 1 µg/ml; IFN-gamma , 100 IU/ml; combination of TNF-alpha /LPS and IL-1beta , 100 IU/ml) failed to induce detectable CXCR1 mRNA.


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Fig. 1.   HIMEC express mRNA for CXCR2, but not CXCR1. Detection of CXCR1 and CXCR2 mRNA in HIMEC by semiquantitative reverse transcriptase-PCR using specific primers. HIMEC constitutively express mRNA for CXCR2, but not CXCR1. Stimulation (12 h) of HIMEC with a combination of TNF-alpha (100 IU/ml) and LPS (1 µg/ml) led to marked up-regulation of CXCR2, whereas IFN-gamma (100 IU/ml), IL-1beta (100 IU/ml), and TNF-alpha alone did not. These stimuli failed to up-regulate mRNA for CXCR1. PBMC, positive control; beta -actin, loading control; no reverse transcriptase, negative control. Representative figure for HIMEC isolates from six different patients.

HIMEC Express CXCR2 Protein-- We confirmed CXCR2 expression in HIMEC by flow cytometry. Unstimulated HIMEC expressed low levels of CXCR2 when compared with isotype control antibody (Fig. 2A). Staining with anti-CXCR1 antibody did not produce any signal above background (data not shown). Furthermore, we confirmed CXCR2 expression by Western blot analysis of total HIMEC lysates with a polyclonal anti-CXCR2 antibody, demonstrating a specific band in lysates of two different HIMEC populations. Neutrophil lysates from healthy donors served as a positive control (Fig. 2B).


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Fig. 2.   HIMEC express CXCR2 receptors. CXCR2 expression on cultured HIMEC was detected by flow cytometry (A) and Western blot analysis (B). CXCR2 is localized to the HIMEC cell surface (A, solid black), as compared with an isotype control antibody (A, empty) using flow cytometry. Staining with anti-CXCR1 antibodies did not produce any signal above background (data not shown). Total lysates of two different HIMEC isolates (HIMEC 1, HIMEC 2; 30 µg of total protein each) were used to detect CXCR2 in Western blot analysis (B). NP, neutrophil lysate (positive control; 15 µg of total protein).

Human Mucosal Microvessels Express CXCR2 in Vivo-- To further assess expression of CXCR2, the putative receptor for ELR+ chemokine-mediated CXCR2 angiogenesis in vivo, we performed immunostaining. CXCR2 was detected by immunohistochemistry of both colonic (Fig. 3A) and small intestinal (Fig. 3B) normal full thickness specimens. Microvessels in close proximity to colorectal adenocarcinoma were similarly stained as was normal colon (not shown). Isotype controls did not produce detectable specific signal (Fig. 3, C and D).


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Fig. 3.   CXCR2 is expressed in human intestinal microvessels in vivo. CXCR2 detection by immunostaining of frozen sections of colonic (A) and small intestinal (B) microvessels from normal full-thickness specimens. A positive signal is visible in mucosal and submucosal microvessels as a dark brown precipitate (diaminobenzidine; arrows; bright field microscopy; original magnification, ×400). Isotype controls did not produce any specific signal (C and D). Representative figures shown are from three independent experiments.

IL-8 Induces Stress Fibers in HIMEC-- Chemokines typically elicit activation and migration of target cells. Having shown that HIMEC express an IL-8 receptor, we next assessed induction of stress fibers as a biological function for chemokine receptor signaling by incubation with fluorescein phalloidin. In unstimulated HIMEC, F-actin staining was limited to the cell periphery and intercellular junctions (arrow, Fig. 4A). IL-8 (100 ng/ml) lead to a rapid (starting at ~1 min) and sustained (30 min) F-actin reorganization in HIMEC, showing prominent stress fiber bundles (arrows, Fig. 4B) and a marked increase in fluorescence intensity. In addition, cellular retraction could be observed at the periphery (arrowheads) of some cells, leading to gaps in the monolayer (Fig. 4B). Stress fiber induction in response to IL-8 utilizes CXCR2, as preincubation of HIMEC with neutralizing anti-CXCR2 antibodies markedly attenuated IL-8-induced F-actin polymerization (Fig. 4C).


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Fig. 4.   Stress fiber assembly in HIMEC by IL-8 is attenuated by anti-CXCR2. Effect of IL-8 on stress fiber polymerization in HIMEC as assessed by fluorescence staining with fluorescein phalloidin, a substance specifically detecting F-actin. Confluent monolayers of HIMEC were grown on fibronectin-coated glass chamber slides, stimulated with IL-8 (100 ng/ml, 15 min) prior to staining. A, minimal stress fibers in unstimulated HIMEC (negative control). B, IL-8, a CXCR2 ligand, strongly increases stress fiber assembly (arrows denote stress fiber bundles). At some intercellular junctions, a marked retraction (arrowheads) of endothelial cells is notable, leading to scattered interruptions of the continuity of the endothelial cell monolayer. Preincubation of HIMEC with neutralizing anti-CXCR2 antibodies (10 µg/ml, 15 min, 37 °C) markedly attenuated stress fiber assembly by IL-8 (note less fluorescence intensity and less pronounced stress fiber induction, C). Fluorescence microscopic images; original magnification, ×400; fixed shutter speed. Figure shows representative images from one of three independent experiments.

IL-8 Is a Potent Chemoattractant for HIMEC Migration-- Having demonstrated IL-8 activation of stress fibers in HIMEC, we assessed its ability to stimulate cell migration/chemotaxis using fibronectin-coated polycarbonate filters (Transwell). IL-8 concentrations as low as 1 ng/ml induced maximal chemotactic responses of HIMEC, comparable with those induced by the potent angiogenic factor VEGF (50 ng/ml). In accordance with observations on human dermal microvascular EC (2), there is an optimal concentration for IL-8-induced chemotaxis of human microvascular EC, as the number of cells migrating across the filter decreases with higher concentrations of IL-8 (Fig. 5A). Consistent with our findings in F-actin polymerization, preincubation of HIMEC with neutralizing antibodies to CXCR2 (4 µg/ml) significantly decreased chemotactic activity (Fig. 5B), as compared with incubation with isotype control antibodies (4 µg/ml).


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Fig. 5.   IL-8 is a potent chemoattractant for HIMEC migration in vitro. Effect of IL-8 on HIMEC migration assessed by a Transwell chemotaxis assay. HIMEC migrating across fibronectin-coated polycarbonate filters (pore size, 8 µm) were quantified using modified Wright's stain and bright field microscopy. IL-8 elicits strong chemotactic properties in HIMEC, comparable with VEGF (50 ng/ml; positive control). Note the biphasic dose response, as higher concentrations of IL-8 (100 ng/ml and higher) do not exhibit marked chemotaxis in HIMEC (A). The inhibitory effects of anti-CXCR2 antibodies on HIMEC were studied at 10 ng/ml IL-8, a concentration shown to effectively evoke a chemotactic response in HIMEC. HIMEC were preincubated with neutralizing anti-CXCR2 antibodies (4 µg/ml, 30 min, 37 °C) or isotype control antibodies (4 µg/ml). Preincubation with anti-CXCR2 significantly inhibited IL-8-induced chemotactic response in HIMEC (B). *, p < 0.01 versus positive control. No IL-8, negative control; IL-8 (10 ng/ml), positive control. All conditions were assessed in triplicate, and data are expressed as mean number of migrated cells per high-power field (×200) ± S.E.

IL-8 Enhances Proliferation Rate of HIMEC-- Angiogenesis requires the directed migration and increased cellular proliferation of EC. Proliferation rates of HIMEC, assessed by [3H]thymidine uptake, were significantly higher following 12 h of IL-8 stimulation (Fig. 6). Comparable with our chemotaxis data, IL-8-induced proliferation was maximal at concentrations of 10 ng/ml. Thus, IL-8 was able to produce a marked increase in proliferation rates, suggesting a pivotal role for IL-8-induced EC proliferation in vivo.


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Fig. 6.   IL-8 enhances the proliferation rate of HIMEC in vitro. IL-8-induced HIMEC proliferation was assessed by [3H]thymidine uptake. 4 × 104 HIMEC/well were stimulated for 12 h with increasing concentrations of IL-8 (0, 1, 10, and 100 ng/ml) and pulsed for 6 h with 1 µCi/ml [3H]thymidine. [3H]Thymidine uptake in total cell lysates was measured in a beta -counter. IL-8 enhanced HIMEC proliferation in a biphasic fashion, with an apparent maximum at 10 ng/ml IL-8. A concentration of 100 ng/ml IL-8 did not result in a significant increase in proliferation. All conditions were assessed in triplicate, and data are expressed as percent of control [3H]thymidine uptake (0 ng/ml IL-8) ± S.E. *, p < 0.05 versus control.

IL-8 Enhances Phosphorylation of ERK 1/2 (p42/44 MAPK)-- Chemokine activation of MAPK phosphorylation in target cells has been demonstrated previously (47). We next assessed the ability of IL-8 to induce MAPK phosphorylation by immunoblot analysis. IL-8 (100 ng/ml) leads to a marked phosphorylation of extracellular signal-regulated kinase (ERK 1/2, p42/44 MAPK), reaching its maximum after 30 min of stimulation (Fig. 7). This phosphorylation is not transient, as ERK 1/2 remains strongly phosphorylated after 60 min of stimulation (Fig. 4). Equal loading of protein was assured using the Bradford assay and Coomassie staining of the polyacrylamide gel after separation of proteins.


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Fig. 7.   IL-8 enhances phosphorylation of ERK 1/2 (p42/44 MAPK) in HIMEC. Activation of ERK 1/2, a member of the MAPK superfamily, in HIMEC by IL-8 was assessed using Western blot analysis. Total cell lysates from cultured HIMEC stimulated with IL-8 (100 ng/ml) were subjected to SDS-PAGE and immunoblotted with phospho-specific anti-ERK 1/2 antibodies. IL-8 enhances phosphorylation of ERK 1/2 in a time-dependent fashion. Note the strong and sustained increase of phosphorylated ERK 1/2 at 15 min of stimulation with IL-8, as compared with total ERK. Coomassie staining of the polyacrylamide gel confirms equal amounts of total protein loaded. The representative figure shown is from one of three independent experiments.

In Vitro Tube Formation of HIMEC Requires Activation of ERK 1/2 and PI3K-- The role of ERK 1/2 and PI3K in in vitro angiogenesis of HIMEC was assessed by tube formation assays. HIMEC in complete growth medium were seeded onto a three-dimensional extracellular matrix preparation (MatrigelTM) and incubated for 16 h at 37 °C. Where indicated, HIMEC were incubated with inhibitors of MAPK kinase (PD98059) and PI 3-kinase (wortmannin and LY294002), respectively. Naive HIMEC seeded onto Matrigel in complete growth medium display formation of robust tube-like structures within 8 h (not shown), with further maturation after 16 h (Fig. 8, panel A). Inhibitors of both MAPK kinase and PI3K at concentrations specific for their signaling targets exhibited a marked inhibitory effect on the formation of tube-like structures by HIMEC, visible by the disruption of tube-like structures and cells remaining coherent in spherical clusters (Fig. 8, panels B-H). These results indicate that activation of both ERK 1/2 and PI3K are required for endogenous in vitro tube formation in HIMEC, defining the crucial role of both signaling pathways in functional angiogenesis of HIMEC.


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Fig. 8.   In vitro tube formation of HIMEC requires activation of ERK 1/2 and PI3K. In vitro endothelial tube formation assays employing MatrigelTM as a three-dimensional extracellular matrix. HIMEC in complete growth medium (5 × 104) were seeded onto 24-well plates containing MatrigelTM (5 mg/ml). Where indicated, the medium was supplemented with specific inhibitors of MAPK kinase (PD98059, 10 and 20 µM) or PI3K (wortmannin, 50 and 100 nM; or LY294002, 10 and 20 µM), respectively. Control cells receiving Me2SO only served as a vehicle control (not shown). Naive HIMEC seeded in complete growth medium display robust tube formation (A). All three inhibitors markedly abrogated spontaneous tube formation of HIMEC seeded in complete growth medium. Note the cells remaining adherent in spherical clusters, lacking mature tube-like structures (B-H).

Inhibitors of MAPK Kinase and PI3K Elicit Inhibitory Effects on IL-8-induced Chemotaxis in HIMEC-- To specifically assess the role of ERK 1/2 and PI3K in the propagation of the intracellular signal evoked by IL-8, HIMEC were incubated with inhibitors of MAPK kinase (PD98059; 10 and 20 µM) and of PI3K (wortmannin; 50 and 100 nM) and subjected to a chemotaxis assay using 10 ng/ml IL-8, a concentration shown to evoke a robust chemotactic response in HIMEC. Both inhibitors used at target-specific concentrations resulted in a dose-dependent decrease in the chemotactic activation of HIMEC by IL-8, as compared with the vehicle control (Me2SO) (Fig. 9). Supported by the notion that HIMEC require activation of both ERK 1/2 and PI3K for in vitro angiogenesis, these data indicate that IL-8, a major angiogenic CXC chemokine, utilizes the ERK 1/2 and PI3K signaling pathways to exhibit its biological effect in HIMEC.


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Fig. 9.   IL-8-induced chemotaxis in HIMEC is diminished by specific inhibitors of ERK 1/2 and PI3K. Chemotaxis assay using 10 ng/ml IL-8 and specific inhibitors of ERK 1/2 and PI3K. As shown above, IL-8 acts as a potent chemoattractant on HIMEC. This chemotactic response was potently diminished by PD98059 (10 and 20 µM) and wortmannin (50 and 100 nM) in a dose-dependent fashion. *, p < 0.05 versus positive control; n.s., not significant. No IL-8, negative control; IL-8 (10 ng/ml), positive control. All conditions were assessed in triplicate, and data are expressed as mean number of migrated cells per high-power field (×200) ± S.E.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recently, angiogenesis has evoked intense clinical interest. Novel anti-angiogenic drugs that potentially could help in the prevention ("angioprevention") (48) or treatment of advanced tumor stages by inducing tumor regression or inhibiting tumor progression have undergone clinical investigation. In colorectal adenocarcinoma, angiogenesis is linked to prognostic parameters, including recurrence rate, lymph node metastasis, and disease-free survival (49-51). However, most multicenter trials (phase II and III) investigating angiogenesis inhibitors, many of them focusing on VEGF and its receptors (52), have failed to improve the clinical course. SU5416 (semaxanib), a VEGF receptor inhibitor, recently failed in phase III studies in the treatment of advanced colorectal cancer. Other phase III failures were observed for matrix metalloproteinase inhibitors (BB2516, AG3340, and Bay-12-9566) in the treatment of various solid tumors.

Although VEGF and its receptors may be important in the development of new blood vessels, these trials suggest that malignancy-associated neovascularization in humans is dependent on multiple angiogenic and angiostatic factors. Additional mechanisms to be considered in the inhibition of tumor neovascularization will involve angiostatin (53), endostatin (54), and cyclooxygenase (55). Accordingly, as a plausible mechanism for the failure of VEGF-based anti-angiogenesis trials, a local decrease in angiostatic factors, as well as an increase in established angiogenic factors, including ELR+ CXC chemokines such as IL-8, may contribute to the development of malignancy-associated neovascularization. Our study demonstrates that human intestinal microvascular EC do not express the high specificity/affinity IL-8 receptor (CXCR1), and IL-8-induced angiogenesis in the human intestine is likely dependent on CXCR2, a receptor targeted by multiple angiogenic ELR+ CXC chemokines. This is in accordance with the observations of Addison et al. (32) that CXCR2, not CXCR1 is the receptor most likely responsible for ELR+ CXC chemokine-induced angiogenesis in microvascular EC.

VEGF, a major angiogenic factor, was recently shown to utilize several intracellular signaling cascades, including ERK 1/2, p70 S6 kinase, and PI3K (phosphoinositide 3-OH kinase) through its receptor Flk-1/KDR in human EC (56, 57). Recent investigation has focused on the role of PI3K in the migration of various cell types (58), including EC (59, 60). Specifically, PI3K is crucial for mitogen-induced cell motility responses of EC, including actin reorganization and chemotaxis (61). Wortmannin, a multicyclic fungal metabolite and a selective inhibitor of PI3K, is a potent inhibitor of angiogenesis in vivo (62) and EC chemotaxis (61). In turn, the intracellular lipid kinase PI3K is now appreciated as a critical mediator of angiogenesis through its downstream targets PKB (63) and Akt, a serine/threonine protein kinase (64), as overexpression of the catalytic subunit of PI3K as well as of constitutively active Akt has revealed increased in vivo angiogenesis in the chicken chorioallantoic membrane assay (65). Activation of ERK 1/2 by chemokines seems to be a signaling event downstream of PI3K, because phosphorylation of ERK 1/2 in response to several chemokines can be inhibited by PI3K inhibitors (58, 66). In addition, PI3K is believed to exert oncogenic properties involved in cellular transformation, aberrant cell cycle progression, and tumorigenesis (68).

Specific inhibitors of both ERK 1/2 and PI3K activation displayed a marked inhibitory effect on biological endothelial functions in HIMEC. In an in vitro angiogenesis assay employing Matrigel as an extracellular matrix, the PI3K inhibitors wortmannin and LY294002 as well as the MAPK kinase inhibitor, PD98059, markedly abrogated the spontaneous tube formation of HIMEC. Similarly, inhibitors of both mentioned pathways suppressed the chemotactic activity of IL-8 (10 ng/ml) in a dose-dependent fashion. Given the notion that major proangiogenic factors share intracellular signaling pathways required for angiogenesis, the development of tailored endothelium-specific drugs inhibiting MAPK kinase and PI3K may emerge as novel pharmacologic strategies to prevent tumor-associated angiogenesis.

The importance of IL-8 in tumor progression, metastasis, and angiogenesis has been demonstrated by several groups using neutralizing anti-IL-8 antibodies in the treatment of human adenocarcinomas transplanted into athymic nude mice (22, 23, 26). However, it is important to consider variation in the chemokine biology of rodents compared with humans, where mice express IL-8 receptors, but a murine analog of IL-8 has not yet been identified. Models of tumorigenesis in mice cannot as of yet directly assess the contribution of IL-8 through gene targeted deletion. Therefore, utilization of human cell systems will remain an essential component of defining receptor-mediated growth pathways involving IL-8.

The biological effects of IL-8 on EC are concentration dependent in a biphasic fashion, as higher concentrations of IL-8 reduce its chemotactic properties. This observation was demonstrated in human dermal microvascular cells and human macrovascular EC, although human macrovascular EC displayed lower overall levels of chemotactic response toward different concentrations of IL-8 (2). Similarly, EC proliferation rates induced by IL-8 revealed a biphasic pattern, a phenomenon previously described for human macrovascular EC (69). The biphasic functional response to IL-8 was not unexpected, as it has been shown for other chemokines, including chemotactic response to the ELR- CXC chemokine stromal cell-derived factor-1 (70).

Another possible IL-8 receptor, the Duffy antigen, was originally identified as a blood group antigen required for invasion by the malarial parasites, Plasmodium vivax and Plasmodium knowlesi (71-73). Further studies revealed that a chemokine-binding protein expressed on red blood cells is identical to the Duffy antigen, which was subsequently renamed Duffy antigen receptor for chemokines (DARC) (74). DARC nonselectively binds the ELR+ CXC chemokines IL-8, GRO-alpha (MGSA), and NAP-2 as well as the nonangiogenic CC chemokines RANTES (regulated on activation normal T cell expressed and secreted) and MCP-1 with comparably high affinity (75). DARC does not couple to G-proteins, and binding of chemokines to DARC does not evoke any intracellular signal (76). Although DARC is expressed on microvascular EC of Duffy-negative individuals (77), its molecular role in neovascularization is unknown (32, 46, 78). Consistent with this, it has been suggested that DARC might act as a chemokine sink to bind excess chemokines during inflammatory responses (32), thus sustaining chemokine action in a buffering fashion. Alternatively, DARC might serve as a competing decoy receptor for chemokines, limiting excessive response to high amounts of chemokines in inflammation (46).

CXCR2 apparently plays a major role in IL-8-induced signaling, as different biological effects of IL-8 on intestinal EC can be markedly attenuated by preincubation with neutralizing anti-CXCR2 antibodies. These data suggest that in addition to VEGF signaling, which frequently has been evaluated as a single target in recent anti-angiogenesis trials, there are additional pathways of tumor-induced angiogenesis in human cancers. Targeting multiple receptors with redundant function may be essential for the inhibition of angiogenesis clinically. Blocking of CXCR2 signaling by antibodies, or delivery of selective CXCR2 antagonistic compounds (79) or peptides (67) may emerge as an additional therapeutic approach in anti-angiogenesis treatment of colonic adenocarcinoma.

    ACKNOWLEDGEMENT

We thank H. Brandenburg for expert assistance in preparation of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK056234, DK057139 (to D. G. B.), and DK02808 (to M. B. D.) and grants from the Deutsche Gesellschaft für Verdauungs- und Stoffwechselkrankheiten (to J. H.), the Crohn's and Colitis Foundation of America (to D. G. B.), the Cancer Center (to D. G. B., P. R., and M. B. D.), and the Digestive Disease Center (to D. G. B., P. R., and M. F. O.) of the Medical College of Wisconsin.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: Division of Gastroenterology and Hepatology, Dept. of Medicine, Medical College of Wisconsin, 9200 W. Wisconsin Ave., Milwaukee, WI 53226. Tel.: 414-456-6845; Fax: 414-456-6214; E-mail: dbinion@mcw.edu.

Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M208231200

    ABBREVIATIONS

The abbreviations used are: EC, endothelial cell; ERK, extracellular signal-regulated kinase; HIMEC, human intestinal microvascular endothelial cells; IL-8, interleukin 8; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; VEGF, vascular endothelial growth factor; TNF-alpha , tumor necrosis factor alpha ; IFN, interferon; LPS, lipopolysaccharide; FBS, fetal bovine serum; BSA, bovine serum albumin; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; DARC, Duffy antigen receptor for chemokines.

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