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Published ahead of print on November 7, 2003, doi:10.1165/rcmb.2003-0314OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 729-735, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0314OC

Expression and Function of the Vascular Endothelial Growth Factor Receptor FLT-1 in Human Eosinophils

Clemens Feistritzer, Nicole C. Kaneider, Daniel H. Sturn, Birgit A. Mosheimer, Christian M. Kähler and Christian J. Wiedermann

Division of General Internal Medicine, Department of Internal Medicine, University of Innsbruck, Innsbruck, Austria

Address correspondence to: Prof. Dr. Christian J. Wiedermann, Department of Internal Medicine, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail: christian.wiedermann{at}uibk.ac.at


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vascular endothelial growth factor (VEGF) is highly expressed in the airway of patients with asthma. Whether VEGF affects eosinophil function in vitro and if VEGF receptors are involved was tested. Eosinophils were from venous blood of healthy donors. Cell migration was studied by micropore filter assays. Signaling mechanisms required for VEGF-dependent migration were tested using signaling enzyme blockers. Expression of flt-1 and KDR/flk-1 mRNA in eosinophils was demonstrated in reverse transcriptase–polymerase chain reaction, and receptor expression was investigated by fluorescence-activated cell sorting analysis. Eosinophil cationic protein release was measured in eosinophil supernatants by enzyme-linked immunosorbent assay. VEGF significantly stimulated eosinophil chemotaxis via activation of protein kinase C and phosphatidylinositol 3'-kinase. The effect on migration was reversed by an antibody against VEGF receptor flt-1, but not by an antibody against KDR/flk-1. Expression of VEGF receptor flt-1 mRNA was shown and synthesis of VEGF receptor in eosinophils is suggested by detection of VEGF receptor immunoreactivity on the cell surface. Data suggest that VEGF receptor flt-1 is expressed by eosinophils whose activation with VEGF stimulates directed migration and release of eosinophil cationic protein. Thus, VEGF may play an important role in the modulation of eosinophilic inflammation.

Abbreviations: bovine serum albumin, BSA • bisindolylmaleimide I GF 109203X, GFX • chemotaxis index, CI • eosinophil cationic protein, ECP • ethylenediamietetraacetic acid, EDTA • fluorescence-activated cell sorting, FACS • granulocyte/macrophage colony-stimulating factor, GM-CSF • Hanks' balanced salt solution, HBSS • human umbilical vein endothelial cells, HUVEC • isobutylmethylxanthine, IBMX • magnetic-activated cell sorting, MACS • phosphate-buffered saline, PBS • phosphatidylinositol 3'-kinase, PI3K • protein kinase C, PKC • regulated upon activation, normal T-lymphocytes expressed and secreted, RANTES • recombinant human vascular endothelial growth factor, rhVEGF • reverse transcriptase–polymerase chain reaction, RT-PCR • vascular endothelial growth factor, VEGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The structural alterations in the airway walls of chronic inflammatory diseases include fibrosis, smooth muscle hypertrophy, hyperplasia of mucus-secreting cells, and new vessel formation (1). Increased vascularity of bronchial mucosa is closely related to the expression of angiogenic factors, which contribute to the pathogenesis of diseases like asthma bronchiale (2). Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic cytokines (3). VEGF also increases vascular permeability so that plasma proteins can leak into extravascular space, which leads to edema and profound alterations in the extracellular matrix (4). Patients with asthma showed significantly increased VEGF-expression in cells of the airway mucosa compared with healthy subjects (5). Further clinical studies revealed that asthma is associated with elevated levels of VEGF in induced sputum (6, 7). Recently, Asai and coworkers reported that VEGF levels correlate with the percentage of eosinophils and eosinophil cationic protein (ECP) levels in induced sputum of individuals with asthma (8).

In allergic and parasitic diseases, the cellular infiltrate consists mainly of eosinophils. Selective migration of circulating eosinophils into tissues plays an important role and is under tight regulation by a number of chemoattractants (9). At the site of inflammation, eosinophils release toxic cationic proteins upon stimulation, a process thought to be important in host defense (10). Tissue damage caused by eosinophil granule proteins may also be important in the pathophysiology of asthma, atopic dermatitis, and other chronic allergic diseases (11, 12).

In this study we investigated the effects of recombinant human VEGF (rhVEGF) on the chemotaxis and ECP release of eosinophils. Involvement of the flt-1 and KDR/flk-1 VEGF receptor was tested using specific antibodies. Furthermore, we detected the expression of VEGF receptor flt-1 mRNA and synthesis of VEGF receptor on the cell surface of eosinophils.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Materials
All stock solutions were stored at –20°C before use. RPMI 1640 with phenol red was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Bovine serum albumin (BSA) was from Dade Behring (Marburg, Germany). Recombinant human vascular endothelial growth factor (rhVEGF), C5a, dextran, Staurosporine, isobutylmethylxanthine (IBMX), wortmannin, rolipram, gelatin and the mouse monoclonal anti-VEGF receptor-2 (KDR/flk-1) antibody were from Sigma Chemical (St. Louis, MO). Bisindolylmaleimide I GF 109203X (GFX) was from Boehringer Ingelheim KG (Ingelheim am Rhein, Germany). GM-CSF (Leucomax) was from Novartis (Vienna, Austria). Lymphoprep was from Nycomed Pharma AS (Oslo, Norway). Dulbecco's phosphate-buffered saline (PBS) and fetal calf serum was from PAA Laboratories (Linz, Austria), Hanks' balanced salt solution (HBSS) without phenol red from Invitrogen (Carlsbad, CA). The Endothelial Cell Growth Medium was from PromoCell (Heidelberg, Germany). The biotinylated mouse anti-mouse antibody and the IgG isotype control were from eBioscience (San Diego, CA), and streptavidin-PE was from Becton-Dickinson (San Jose, CA). MACS separation columns and microbeads were from Miltenyi Biotech (Auburn, CA). The microchemotaxis chambers were from Neuroprobe (Bethesda, MD), and cellulose nitrate filters were from Sartorius (Goettingen, Germany). Mouse monoclonal to human anti-VEGF receptor 1 (Anti–flt-1) antibodies and rabbit polyclonal to human VEGF A antibody were from Abcam (Cambridge, UK). RNA-Bee was from Tel Test Inc (Friendswood, TX), reverse transcriptase was from Gibco BRL (Life Technologies, Vienna, Austria), hot Star Taq polymerase was purchased from Qiagen Inc (Valencia, CA), and primers were from MWG Biotech (Ebersdorf, Germany). Certified PCR Agarose was from Bio-Rad (Hercules, CA).

Preparation of Human Eosinophils
Granulocytes were obtained from the peripheral blood of healthy donors with no history of atopic or hypereosinophilic conditions by means of dextran sedimentation and centrifugation through Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). This step was repeated once to remove most mononuclear cells, and was followed by hypotonic lysis of contaminating erythrocytes with sodium chloride solution. After washing, cells were resuspended in 50 µl/5 x 107 cells ice-cold MACS buffer (PBS with 5 mmol/liter EDTA and 0.5% BSA); an equal volume of MACS colloidal superparamagnetic microbeads conjugated with monoclonal anti-human CD16 mAb was added and incubated (30 min at 6°C). Recommended volumes of ice-cold MACS buffer were added to the cell–microbead mixture and the cell suspension loaded onto the separation column. The eluate containing CD16– eosinophils was collected, washed, resuspended in RPMI 1640/0.5% BSA, and the separation procedure was repeated to increase purity. Purity of sorted eosinophils was greater than 98%, as determined by means of morphology and fluorescence-activated cell sorting (FACS) analysis. Contaminating cells comprised < 1% lymphocytes, < 1% neutrophils and basophils, and negligible numbers of monocytes-macrophages.

Eosinophil and Endothelial Cell Culture
HUVEC from fresh placenta cords were isolated and grown to confluence in a humidified atmosphere at 37°C. The growth medium was Endothelial Cell Growth Medium (PromoCell) supplemented with 10% fetal calf serum (PAA Laboratories, Linz, Austria). Tissue culture flasks were coated with 0.2% gelatin (Sigma Chemical) before seeding of cells. HUVEC of passage 2 were used for RNA extraction.

Freshly isolated eosinophils (1 x 106 cells/ml) were transferred in a 24-well-plate (Falcon, Franklin Lakes, NJ) in RPMI 1640/5% BSA, followed by stimulation with different concentrations of rhVEGF (100 ng/ml–0.1 g/ml) for 45 min at 37°C in a humidified atmosphere (5% CO2). Activation with C5a (10–8) served as positive control. The supernatants were harvested after and were subjected to enzyme-linked immunosorbent assay measuring of ECP levels.

ECP Measurements in Blood Eosinophils
ECP levels were measured in eosinophil supernatants using the Pharmacia UniCAP system for ECP (Pharmacia and Upjohn, Dubendorf, Switzerland) according to the manufacturer's instructions. The lower detection limit was 2 µg/liter.

Eosinophil Migration Assay
Migration assays were performed by using a modified 48-well Boyden microchemotaxis chamber (Neuroprobe) in which a 5-µm-pore-size cellulose nitrate filter separated the upper and the lower chambers. Eosinophils were resuspended in RPMI 1640/0.5% BSA (1 x 106 cells/ml). Fifty microliters of the cell suspension were placed into the upper chamber and were allowed to migrate toward various concentrations of rhVEGF (0.1 pg/ml to 100 ng/ml) placed in the lower chamber for 30 min at 37°C in a humidified atmosphere (5% CO2). After the migration period, the nitrocellulose filters were dehydrated, fixed, and stained with hematoxylin. Migration depth of the cells into the filters was quantified by means of microscopy, measuring the distance (µm) from the surface of the filter to the leading front of three cells. Data are expressed as a chemotaxis index (CI), which is the ratio between the distance of directed and random migration of eosinophils into the nitrocellulose filters.

In some experiments eosinophils were preincubated with either function blocking anti-VEGF receptor-1 (flt-1; 10 µg/ml) or anti-VEGF receptor-2 (KDR/flk-1; 10 µg/ml) antibodies for 30 min, followed by migration toward rhVEGF, as described above. In continuative experiments, function of rhVEGF was inhibited by heat deactivation of rhVEGF or co-incubation of rhVEGF with a neutralizing VEGF antibody (1 µg/ml) before performing chemotaxis experiments.

Intracellular signaling of rhVEGF on eosinophils was tested by pre-incubation of the cells with the intracellular enzyme blockers staurosporine (10 ng/ml) (from streptomyces sp.), bisindolylmaleimide I GF 109203X (GFX) (500 nmol/liter), wortmannin (10 nmol/liter) (from penicillium fumiculosum), rolipram (10 µmol/liter), and IBMX (10 ng/ml). In further experiments, eosinophils were preincubated with different concentrations of pertussis toxin for 30 min at 37°C in a humidified atmosphere (5% CO2). The cells were then washed twice, resuspended in RPMI/0.5%BSA, and tested in the migration assay toward rhVEGF.

Checkerboard Analysis
To ensure that the effect observed was true chemotaxis, checkerboard analyses were performed. Eosinophils were resuspended in RPMI 1640/0.5% BSA containing various concentrations of rhVEGF just before they were transferred to the upper chamber. The same concentrations of rhVEGF remained beneath the filter of the Boyden chamber, thus distinct concentration gradients could be formed. Data are expressed as chemotaxis index within a matrix.

Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was isolated from 107 cells by an acid guanidinium thiocyanate-phenol-chloroform mixture. A reverse transcriptase (RT) reaction was performed on 1 µg of RNA using random hexamers reverse transcriptase. One microgram of the resulting cDNA was then subjected to 35 cycles of polymerase chain reaction (PCR) in a 50-µl reaction mixture containing 1 pmol of sense and antisense primer pairs in a Biometra thermocycler: 95°C for 60 s (denaturation), 58°C for 60 s (annealing), and 72°C for 60 s (extension). Primers were designed to amplify a 557-bp coding sequence of human flt-1 receptor. The sense primer sequence was 5'-CAG CCC ATA AAT GGT CTT TGC C-3'. The antisense primer sequence was 5'-TAA TTT GAC TGG GCG TGG TGT G-3'. For the 384-bp coding sequence of KDR/flk-1, the following primers were used: sense, 5'-GGC CAA GTG ATT GAA GCA GAT G-3'; antisense, 5'-TTC AGA TCC ACA GGG ATT GCT C-3'. The PCR products were subjected to agarose gel analysis. HUVEC were used as positive control, as flt-1 and flt-1 expression on HUVEC has previously been described (13).

FACS Analysis
Fluorometric analysis for VEGF-receptor expression was performed on eosinophils. A total of 5 x 105 cells were washed twice in Dulbecco's PBS, containing 0.5% BSA and incubated with 150 µg/ml human IgG for 20 min at 4°C. After pelleting, cells were incubated with 10 µg/ml of either anti–flt-1 (Abcam) or anti–KDR/flk-1 antibody (Sigma Chemical) or the respective isotype matched control IgG (eBioscience) for 30 min at 4°C. After washing, 10 µg/ml biotinylated mouse anti-mouse IgG (eBioscience) was incubated for another 30 min. Cells were washed twice and the eosinophils were subsequently incubated with a 1:25 dilution of streptavidin-PE for 30 min, washed twice, then immediately analyzed on a FACScan (Becton-Dickinson) with CellQuest software.

Statistical Methods
Data are expressed as mean ± SEM. Means were compared by the Mann-Whitney U test, the paired t test, and Kruskal-Wallis ANOVA. A difference with P < 0.05 was considered to be significant. Statistical analyses were performed using the StatView software package (Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of VEGF on Release of Eosinophilic Cationic Protein and on Eosinophil Migration
We first tested the effects of rhVEGF on ECP release from freshly prepared human eosinophils of healthy volunteers. As shown in Figure 1A, significant ECP release was observed when eosinophils were stimulated with rhVEGF at concentrations of 100 ng/ml and 1 ng/ml. Preincubation of rhVEGF (1 ng/ml) with a polyclonal VEGF antibody significantly abrogated the effect of rhVEGF on ECP release of human eosinophils. Stimulation with C5a (10 nM) was used as positive control.



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  		Figure 1. Effects of VEGF on ECP release and migration of human eosinophils. (A) Release of ECP from eosinophils after stimulation with different concentrations of VEGF or concomitantly VEGF (1 ng/ml) with a polyclonal anti-VEGF pAb for 45 min. Stimulation with C5a (10–8 M) served as positive control. Results are given as the mean ± SEM of the index, which is the ratio of the ECP release after stimulation with the test substance and ECP release in medium. Spontaneous ECP release in medium was 32.8 ± 7.92 µg/liter. *P < 0.05, Mann-Whitney U test versus medium or paired t test (VEGF versus VEGF incubation with anti-VEGF pAb) after multiple group comparison by using the Kruskal-Wallis test (n = 4). (B) Direct chemotactic effects with different concentrations of VEGF on human eosinophils. RANTES (10 ng/ml) served as positive control. Chemotaxis experiments were performed in modified Boyden chambers. Results are given as the mean ± SEM of the chemotaxis index, which is the ratio of the distance of migration (in micrometers) toward attractant and the distance toward medium. Mean distance of random migration was 75.8 ± 11.38. *P < 0.05, Mann-Whitney U test versus medium after multiple group comparison by using the Kruskal-Wallis test (n = 4).

 
To explore for chemotactic properties of VEGF in the absence of chemoattractants, freshly prepared eosinophils were allowed to migrate toward different concentrations of rhVEGF (100 ng/ml to 0.1 fg/ml); RANTES (10 ng/ml) was used as a positive control. Concentrations ranging from 100 ng/ml to 10 pg/ml of rhVEGF significantly increased migration in a dose-dependent manner as shown in Figure 1B. rhVEGF stimulated migration maximally at concentrations of 1 ng/ml.

In checkerboard analysis the migratory response was confirmed as true chemotaxis. Maximal induction of migration occurred in the presence of a positive concentration gradient between the two compartments (higher concentration below the filter). In the presence of equal concentrations of rhVEGF above and below the filter or of a negative gradient (higher concentration above the filter), no enhanced migration occurred. These results indicate that VEGF is able to activate a chemotactic response in human eosinophils with no appreciable chemokinetic activity (Table 1).


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  		TABLE 1. Effect of concentration gradients of VEGF on eosinophil migration

 
RhVEGF preabsorbed with polyclonal anti-VEGF antibodies was inactive in inducing eosinophil migration. Heat treatment of rhVEGF significantly reduced the chemotactic properties of VEGF (Table 2).


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  		TABLE 2. Effect of anti-VEGF pAb and heat treatment on VEGF-induced eosinophil migration

 
Effects of Antibodies to flt-1 and KDR/flk-1 on the Chemotactic Response of Eosinophils to VEGF
Because we could demonstrate a chemotactic effect of VEGF on eosinophils, which was abrogated by denaturation of the protein and by VEGF-blocking antibodies, and because it is known that different specific VEGF receptors can be expressed on various human cells, chemotaxis experiments with VEGF were performed in the presence of antibodies to VEGF receptor-1 (flt-1) or VEGF receptor-2 (KDR/flk-1). Cells were pretreated with the two antibodies and then allowed to migrate toward rhVEGF (1 ng/ml). Antibodies to flt-1, but not to the KDR/flk-1 receptor inhibited directed migration of eosinophils toward rhVEGF in a concentration-dependent manner. RANTES-induced chemotaxis was not influenced after pretreatment with these antibodies (Figures 2A and 2B).



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  		Figure 2. Effects of (A) VEGF receptor-1 mAb (flt-1) or (B) VEGF receptor-2 mAb (KDR/flk-1) on VEGF-induced eosinophil migration Eosinophils were incubated with different concentrations of either anti–flt-1 mAb or anti–KDR/flk-1 mAb for 30 min. Cells were washed twice, resuspended in RPMI 1640/0.5% BSA, and chemotaxis toward VEGF (1 ng/ml; solid circles) and RANTES (10 ng/ml; open squares) was monitored. Isotype-matched IgG served as positive control. Results are given as the mean ± SEM of the chemotaxis index, which is the ratio of the distance of migration (in micrometers) toward attractant and the distance toward medium. Mean distance of random migration was 55.2 ± 5.72. *P < 0.05, Mann-Whitney U test versus medium incubation toward VEGF, respectively, RANTES after multiple group comparison by using the Kruskal-Wallis test (n = 4).

 
Blocking of Intracellular Signaling Enzymes in VEGF-Induced Chemotaxis of Eosinophils
To elucidate signaling pathways involved in transmitting VEGF effects in eosinophil migration, different intracellular enzyme blockers were used and compared with effects in RANTES-induced migration of eosinophils. Eosinophils were freshly isolated from the same donor. The blockers staurosporine, GFX, IBMX, wortmannin, and rolipram were used at established signaling blocking concentrations, but showed no detectable influence on basal random migration of the cells. Staurosporine, which is a nonspecific inhibitor of protein kinase (PK) C that also affects PKA signaling, the specific PKC inhibitor bisindolylmaleimide (GFX) and wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, decreased rhVEGF-induced chemotaxis in eosinophils. Rolipram, a selective inhibitor of cAMP-specific phosphodiesterase, and the phosphodiesterase inhibitor IBMX did not affect VEGF-induced eosinophil migration (Table 3).


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  		TABLE 3. Effect of blocking of signaling enzymes on eosinophil migration toward VEGF and RANTES

 
In further experiments, eosinophils were preincubated with different concentrations of pertussis toxin to study if Gi proteins are involved in flt-1–mediated eosinophil migration in response to VEGF. Chemotaxis experiments revealed a significant dose-dependent inhibition of eosinophil migration after incubation with pertussis toxin toward rhVEGF (Figure 3).



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  		Figure 3. Effect of pertussis toxin pretreatment on eosinophil chemotaxis in response to VEGF and RANTES. Eosinophils were incubated with different concentrations of pertussis toxin for 30 min. Cells were washed twice, resuspended in RPMI 1640/0.5%BSA, and chemotaxis toward VEGF (1 ng/ml; solid circles) and RANTES (10 ng/ml; open squares) was monitored. Results are given as the mean ± SEM of the chemotaxis index, which is the ratio of the distance of migration (in micrometers) toward attractant and the distance toward medium. Mean distance of random migration was 60.7 ± 4.12 µm. *P < 0.05, Mann-Whitney U test versus medium incubation toward VEGF, respectively, RANTES after multiple group comparison by using the Kruskal-Wallis test (n = 3).

 
Expression of VEGF flt-1 Receptor in Eosinophils
Because VEGF-induced effects on ECP release and chemotaxis of eosinophils may be mediated by its binding to and activation of the VEGF receptor-1 (flt-1), surface expression of flt-1 receptor on these cells and mRNA content of eosinophils was tested. In FACS analysis, a significant shift of fluorescence in eosinophils was observed by anti-VEGF flt-1 antibody, but not by an anti–KDR/flk-1–antibody, indicative of cell surface presence of VEGF receptor 1. An IgG antibody was used as negative control. To determine whether flt-1 or KDR/flk-1 receptor mRNA is expressed in eosinophils, RT-PCR analysis was performed. Data confirm that flt-1 but not KDR/flk-1 mRNA can be found in human peripheral blood eosinophils from healthy donors (Figure 4).



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  		Figure 4. RT-PCR and FACScan analysis of VEGF receptors in eosinophils. VEGF receptor flt-1 mRNA in eosinophils and HUVEC. One microgram of total RNA from each sample was reverse-transcribed into cDNA and amplified for the (A) flt-1 or (B) KDR/fkl-1 gene using PCR. Flt-1 and KDR/flk-1 are represented by the 557-bp and by the 344-bp product, respectively. FACS analysis of (C) anti–flt-1 mAb or (D) anti–KDR/flk-1 mAb binding to eosinophils. Fluorescence analysis used a FACScan Flow cytometer, and a histogram of phycoerythrin fluorescence is shown. Cells were either incubated with isotype matched control IgG or anti–flt-1 mAb, respectively, anti–KDR/flk-1 mAb and stained with phycoerythrin-conjugated streptavidin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies provide evidence that VEGF may play an important role in the pathogenesis of bronchial diseases (2, 5–8, 14, 15). Eosinophil infiltration is a characteristic feature of allergic inflammation, as seen in asthma bronchiale. The present study implies that VEGF affects eosinophils via the VEGF receptor 1 (flt-1). In our experiments rhVEGF stimulated migration and induced ECP release of eosinophils. This effect could be reversed by an anti–VEGF receptor 1 antibody, but not by an antibody to VEGF receptor 2. Expression of VEGF receptor 1 in eosinophils is confirmed by means of identification of expression of mRNA and detection of cell surface VEGF-receptor by FACS analysis.

VEGF is an endothelial cell–specific mitogen that has been shown to play a key role in vasculogenesis and angiogenesis. VEGF expression patterns coincide spatially and temporally with blood vessel growth under physiologic and pathologic conditions. VEGF was purified on the basis of its ability to induce transient vascular leakage, vasodilatation, and endothelial migration (16). Studies indicate that VEGF binds to high-affinity cell surface receptors, flt-1, KDR/flk-1, or Flt-4, which are predominantly expressed in endothelial cells (17–19). Cellular sources of VEGF may also be released by eosinophils (20). However, many types of cells in the airway other than eosinophils were reported to produce VEGF, e.g., epithelial cells (21), mast cells (22), macrophages (2), neutrophils (23), myofibroblasts, and smooth muscle cells (24).

Accumulation of a high number of eosinophils in the lungs of patients with asthma and release of toxic granule proteins, oxygen free radicals, eicocosanoids (sulfido-peptide leukotrienes), Th2-like cytokines, and growth factors by eosinophils are believed to be important in the pathogenesis of asthma. Levels of VEGF correlate well with the percentage of eosinophils and eosinophil cationic protein in induced sputum of individuals with asthma (8). Airway hyperresponsiveness and eosinophil infiltration could be reduced by administration of a VEGF receptor antibody in a murine model of toluene diisocyanate–induced asthma (14). Therefore, effects of VEGF on eosinophils in vitro and possible involvement of VEGF receptors were explored in more detail.

As an in vitro model of eosinophil degranulation, we used peripheral blood eosinophils of healthy volunteers stimulated with different concentrations of rhVEGF or C5a as described previously (25). Eosinophils showed an rhVEGF dose-dependent release of ECP with maximal response in the range of 100 ng/ml and 1 ng/ml (Figure 1A), which is the amount of VEGF found in plasma and in induced sputum of human subjects (8, 26). Effects of rhVEGF could be abrogated by coincubation of VEGF with a neutralizing polyclonal anti-VEGF anti-body, indicating a specific effect of VEGF on ECP release of eosinophils.

In further experiments it was observed that rhVEGF stimulates migration of eosinophils in a VEGF receptor 1–dependent fashion because the chemotactic effect could be reversed with an anti–flt-1 antibody. rhVEGF was able to enhance eosinophil migration in a dose-dependent manner (Figure 1B). Maximal response could be seen in the range of 100 ng/ml and 1 ng/ml, which is the same quantity as demonstrated in the ECP release assays. Previous studies have shown that VEGF induces directed migration of mononuclear cells across an endothelial cell monolayer (27). Barleon and colleagues demonstrated a strong chemotactic effect of VEGF on monocytes, but only minor effects on neutrophil migration (18). In our study checkerboard analysis confirmed the activity of rhVEGF on eosinophils as chemotactic. Checkerboard experiments clearly showed that the eosinophil migration depends on the presence of a VEGF concentration gradient. Results of the checkerboard analyses were internally consistent with that of migration assays, and demonstrated gradient-dependent effects of rhVEGF on eosinophils with significant responses at nanomolar concentrations. VEGF levels measured at this range previously in induced sputum of patients with asthma support a possible physiologic relevance of our observation (14). Moreover, a nearly complete abrogation of rhVEGF-stimulated migration was seen after rhVEGF blocking with neutralizing polyclonal anti-VEGF antibody or heat deactivation of VEGF.

VEGF functions are mediated for the most part by two receptor tyrosine kinases, flt-1 and KDR/flk-1. Previous studies have demonstrated that in endothelial cells transcripts of both main VEGF receptors flt-1 and KDR/flk-2 can be detected (13), whereas other cells like monocytes and neutrophils only express the flt-1 receptor (18). Thus, flt-1 may also be involved in the eosinophil's response to VEGF. A blocking antibody against flt-1 was able to diminish effects of VEGF on eosinophil migration. An antibody which binds to KDR/flk-1 and a control IgG antibody did not inhibit VEGF-induced chemotaxis. As expected, all tested antibodies failed to affect RANTES-induced eosinophil migration. These results clearly indicate that ligation of the VEGF receptor-1 (flt-1) for VEGF is required for the functional response to occur. Data show that eosinophils express the flt-1 but not the KDR/flk-1 gene (Figure 4). Expression of flt-1 on the cell surface of eosinophils was also seen by FACS analysis, thus confirming functional and molecular data.

Various effects of VEGF on endothelial cells are mediated through PI3K (28), PKC, and phospholipase C pathways (29). To analyze which of these pathways mediates the chemotactic response of eosinophils toward rhVEGF, cells were incubated with different enzyme blockers. The PKC inhibitors staurosporine and GFX, as well as specific inhibitor of phosphatidylinositol 3-kinase wortmannin, significantly inhibited VEGF-induced chemotaxis in eosinophils, whereas the phosphodiesterase inhibitors rolipram and IBMX did not affect VEGF-induced eosinophil migration (Table 3). These results indicate that beside effects on HUVEC proliferation, PKC and PI3K are also involved in VEGF-induced eosinophil migration.

It was recently shown that pretreatment with pertussis toxin inhibited VEGF-stimulated macrophage migration. Based on the fact that macrophages express flt-1 but not KDR/flk-1, it is suggested that pertussis toxin–sensitive G proteins were involved in flt-1–mediated macrophage migration in response to VEGF (18). Our chemotaxis experiments revealed a significant dose-dependent inhibition of eosinophil migration toward VEGF after incubation with pertussis toxin. Similar effects of pertussis toxin could be seen for RANTES, which activates at nanomolar concentrations a heterotrimeric Gi protein–coupled signaling pathway upon binding to any one of its known seven-transmembrane, G protein–coupled receptors (30). Thus, results indicate involvement of pertussis toxin–sensitive G proteins in flt-1–induced eosinophil migration.

In our experiments, exogenous VEGF has been applied to normal eosinophils and the response was found. This does not mean that in vivo VEGF has been shown to play a role in the eosinophil response. The answer to this question requires further studies.

Taken together, these results show that VEGF enhances the release of ECP and stimulates directed migration of eosinophils via activation of VEGF receptor flt-1. However, VEGF induced effects on eosinophil function might play an important role in modulation of allergic inflammation.


   Acknowledgments
 
This study was supported by the "Verein zur Förderung von Forschung und Fortbildung in klinischer Kardiologie und Intensivmedizin – Innsbruck."

Received in original form August 22, 2003

Received in final form October 13, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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