ACTIVATED HUMAN NEUTROPHILS EXPRESS VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)

ACTIVATED HUMAN NEUTROPHILS EXPRESS VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) Nicholas J.A. Webb,1,2 Clare R. Myers,1 Carolyn J. Watson,1 Martyn J. Bottomley,1 Paul E.C. Brenchley1 The neutrophil (PMN) influx in the acute inflammatory response is associated with a local increase in vascular permeability and oedema. Vascular endothelial growth factor (VEGF), a growth factor known to have potent vascular permeability-enhancing properties in addition to being an endothelial cell mitogen and a chemo-attractant for mononuclear cells, has previously been shown to be expressed by mononuclear cells and platelets, though PMN VEGF expression has not been reported. PMNs isolated from healthy adult volunteers (n = 16) were incubated for 4 h at 37°C in the presence of tumour necrosis factor a (TNF-a) (5 ng/ml) and serum opsonized zymozan (SOZ) (500 mg/ml). Supernatant VEGF levels were measured using a sandwich antibody capture immuno-assay. Median (interquartile range) VEGF levels were significantly increased in PMN supernatants following stimulation with both TNF-a [347 pg/ml (264–385 pg/ml)] and SOZ [506 pg/ml (407–593 pg/ml)] compared with control values [78 pg/ml (78–87 pg/ml)]. Time course experiments with SOZ stimulated PMNs showed that the majority of VEGF production occurred within the first hour (1 h mean VEGF level 318 pg/ml, 4 h mean VEGF level 451 pg/ml). RT-PCR studies showed that PMNs express mRNA for the two common VEGF splice variants, VEGF121 and VEGF165. PMN VEGF production may be central to the classic acute phase response to injury and the chemo-attraction of other leukocytes to the source of injury. 7 1998 Academic Press Limited

The initial inflammatory cell influx in acute tissue injury consists predominantly of neutrophils (PMNs), which orchestrate the recruitment of monocytes and the activation of lymphocytes required for the maturation of the inflammatory response. Often this PMN influx is associated with a local increase in vascular permeability and oedema. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is an endothelial cell mitogen1 which promotes angiogenesis, is chemoattractant for monocytes and is a potent enhancer of vascular permeability.2 These biological functions are mediated by interaction with two specific tyrosine kinase receptors on endothelial cells; flt-1 (fms-like

From the 1Manchester Renal Research Group, Manchester Royal Infirmary and University Department of Medicine, Manchester M13 OJH, UK; 2Department of Paediatric Nephrology, Royal Manchester Children’s Hospital, Pendlebury, Manchester M27 4HA, UK Correspondence to Nicholas J.A. Webb, Department of Paediatric Nephrology, Royal Manchester Children’s Hospital, Pendlebury, Manchester M27 4HA, UK Received 14 July 1997; accepted for publication 13 October 1997 7 1998 Academic Press Limited 1043–4666/98/040254 + 04 $25.00/0/ck970297 KEY WORDS: VEGF/ VPF/ neutrophils/ immunoassay 254

tyrosine kinase)3 and KDR (kinase insert domain containing receptor),4 also known as VEGFR1 and VEGFR2, respectively. The human gene for VEGF is organized into eight exons.5 Alternate splicing of exons 6 and 7 of VEGF mRNA results in the generation of four protein species consisting of 121, 165, 189 and 206 amino acids (VEGF121, VEGF165, VEGF189 and VEGF206).6 mRNA for a fifth variant, VEGF145, has been detected in human placental tissue,7 and in cell lines derived from carcinomas of the female reproductive tract.8 VEGF121 lacks the residues encoded by exons 6 and 7, and is an acidic protein which binds poorly to heparin and is freely secreted by the cell. VEGF165, the most abundant form in virtually all human tissues, lacks the amino acids encoded by exon 6 and is a basic protein with some heparin-binding capacity, though around 70% is secreted by the cell. The larger two splice variants, VEGF189 and VEGF206, are highly basic polypeptides which bind heparin avidly, and after production are held at the cell surface bound to heparan sulfate rather than being secreted.9 Changes in vascular permeability with oedema are characteristic early features associated with ischaemia– reperfusion injury at a time coincident with increased PMN involvement and influx.10 Similarly, in the inflamed joints of patients with rheumatoid arthritis, CYTOKINE, Vol. 10, No. 4 (April), 1998: pp 254–257

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the association of joint swelling, increased PMN counts in synovial fluid and high levels of VEGF have been reported.11,12 Whilst human circulating mononuclear cells and platelets have been shown to express VEGF mRNA,13,14 mRNA expression or VEGF protein production by PMNs has not previously been reported. The aims of this study were to determine whether human peripheral blood PMNs express VEGF mRNA and are capable of producing VEGF protein, and to investigate the ability of various known PMN stimulants to modulate PMN VEGF production.

RESULTS Fluorescence-activated cell sorting (FACS) analysis showed the purity of the PMN preparations to be high [median (interquartile range) purity 98.3% (97.6–99.2%)], therefore excluding a significant contribution from peripheral mononuclear cells to any observed effect of stimulation. No platelets were visible in the PMN preparations. Supernatant VEGF levels at 4 h were significantly higher in PMNs stimulated with tumour necrosis factor a (TNF-a) and serum opsonized zymozan (SOZ) compared with unstimulated control cells (Table 1). Time course experiments with PMNs stimulated with SOZ showed that stimulated PMNs produced VEGF early, with levels being significantly increased at 1 h compared with unstimulated samples, this difference persisting at 2 and 4 h (Table 2). RT-PCR of mRNA from pure populations of freshly isolated human PMNs from two normal adults showed detectable PCR product corresponding with mRNA for the two common VEGF splice variants, VEGF121 and VEGF165 (Fig. 1).

TABLE 2. VEGF production by isolated PMNs stimulated with serum opsonized zymozan (SOZ) over a 0–4 h period VEGF (pg/ml)

1h 2h 4h

SOZ

Control

318 ( 2 43.3) 378 ( 2 45.8) 451 ( 2 67.9)

93 ( 2 9.7) 125 ( 2 16.1) 136 ( 2 31.5)

P = 0.02* P = 0.02* P = 0.02*

Isolated PMNs (5 × 106/ml) resuspended in Iscoves/10% autologous serum were stimulated with serum opsonized zymozan (SOZ) (500 mg/ml). Unstimulated PMNs acted as control samples. Cells were incubated at 37°C, 5%CO2 for varying time points between 0 and 4 h. Supernatant VEGF levels were measured by ELISA and mean ( 2 SEM) values are shown). *Paired t-test.

MIP-1,18 which are involved in the activation of the classic acute phase response to injury and the chemoattraction of other leukocytes to the source of injury. This paper shows for the first time that activated PMNs both express mRNA for the two common VEGF splice variants and secrete significantly increased levels of VEGF within 4 h of culture with TNF-a and SOZ. As the assay, in common with other immunoassays for VEGF, recognizes both soluble secreted forms, VEGF121 and VEGF165, it is not possible to determine which is the predominantly expressed form. Time course studies of PMN VEGF production following stimulation with SOZ revealed 1 VEGF

2

3

4

5

6

7

8 607 bp 535 bp 403 bp

DISCUSSION PMNs are known to produce a number of inflammatory cytokines following activation including interleukin 1b (IL-1b) and IL-6,15 TNF-a,16 IL-817 and

β-actin

322 bp

TABLE 1. VEGF production by isolated PMNs stimulated with TNF-a and serum opsonized zymozan (SOZ) Stimulant TNF-a

SOZ

Control

No. of subjects (n) 14 16 16 VEGF (pg/ml) 347 (264–385) 506 (407–593) 78 (78–87) Significance vs control* P = 0.0002 P Q 0.0001 Isolated PMNs (5 × 106/ml) resuspended in Iscoves/10% autologous serum were stimulated with TNF-a (5 ng/ml) or serum opsonized zymozan (SOZ) (500 mg/ml) for 4 h at 37°C, 5%CO2. Unstimulated PMNs acted as control samples. Supernatant VEGF levels were measured by ELISA and median (interquatile ranges) are shown. *Wilcoxon matched pairs test.

Figure 1. RT-PCR analysis of VEGF and b-actin expression by human PMNs. RT-PCR products were electrophoresed through 2% agarose gels and stained with ethidium bromide. Lanes 1 and 8 contain 1 mg of 100-bp DNA ladder (Life Technologies); lanes 2, 3, 4 and 5 represent RT-PCR products following amplification of PMN cDNA from two different donors: lanes 2 and 4 are 4-h unstimulated control PMNs and lanes 3 and 5 are 4-h TNF-stimulated PMNs. Lane 6 is a positive control (normal paediatric kidney) and lane 7 is a negative control (no cDNA template). Bands at 403, 535 and 607 bp correspond with VEGF121, VEGF165 and VEGF189, respectively.

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that the majority of VEGF production occurs within the first hour of stimulation, with only relatively small amounts of protein being produced thereafter. The production of VEGF by PMNs in vivo may have significant implications for inflammatory pathologies involving modulation of vascular permeability as seen in ischaemia–reperfusion injury, PMN-mediated glomerulonephritis and rheumatoid arthritis. Furthermore, the chemoattractant properties of PMN secreted VEGF for monocytes19 may reinforce the recruitment of monocytes to sites of acute inflammation following the initial influx of PMNs.

MATERIALS AND METHODS Recombinant (baculovirus-derived) VEGF165 and mouse anti-VEGF mAb 4.6.1 were kind gifts from Dr N. Ferrara (Genentech, USA). Rabbit polyclonal anti-VEGF was purchased from Serotec (Kidlington, UK). Peroxidaseconjugated goat anti-mouse antibody was purchased from Jackson ImmunoResearch (Luton, UK). TNF-a was purchased from R&D Systems (Abingdon, UK) and zymozan was purchased from Sigma Chemical Co. (Poole, UK). Taq DNA polymerase, SuperscriptTM reverse transcriptase, RNase inhibitor, oligo (dT)12–18, 100-bp DNA ladder, and dNTP solutions were purchased from Life Technologies (Paisley, UK). PMNs were isolated from whole blood collected from healthy adult volunteers (n = 16) using the Haslett modification20 of the discontinuous plasma-percoll gradient method described by Danpure et al.21 This produces a highly pure, platelet-free preparation of PMNs with minimal mononuclear cell contamination. Briefly, venous blood samples (36 ml) were collected into containers containing 4 ml of 3.8% sodium citrate and centrifuged at 300 × g for 20 min. The plasma was then removed and dextran (6 ml of 6% solution) was added to the blood cells. The volume was then made up to a total of 50 ml by the addition of 0.9% saline which had been prewarmed to 37°C. This was allowed to sediment and the supernatant was then removed and centrifuged for 6 min at 275 × g. The resultant cell pellet was then resuspended in 2 ml of autologous plasma and carefully layered over a Percoll gradient, prepared using a 90% Percoll/10% saline stock solution to make a 2-ml 51% layer overlaid by a 42% Percoll layer made up in autologous plasma. Centrifugation at 275 × g for 10 min produced a discreet band of highly pure PMNs at the interface of the 51% and 42% Percoll layers. These cells were then aspirated and washed in autologous plasma. Contaminating red cells were lysed by osmotic shock and the cells washed again in Hanks balanced salt solution without calcium or magnesium. The purity of the PMN preparation was assessed by fluorescence-activated cell sorting (FACS) using mAb to CD15 and CD11b as PMN markers and anti-CD14 to detect mononuclear cell contamination. Isolated PMNs were resuspended at 5 × 106/ml in Iscoves/10% autologous serum and aliquots of 1 ml were

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incubated for 4 h at 37°C/5% CO2 in the presence of TNF-a (5 ng/ml) and SOZ (500 mg/ml). Unstimulated PMNs acted as a control group. Time course experiments were also performed in five volunteers using SOZ (500 mg/ml) as a stimulant under the same experimental conditions, with incubation times of 1, 2 and 4 h. Following incubation, the cell suspensions were microfuged for 6 min at 13 000 rpm. The cell pellets were frozen for RNA extraction for RT-PCR studies and the supernatants submitted for measurement of VEGF protein levels. VEGF levels were measured using an in-house sandwich ELISA. Rabbit polyclonal anti-VEGF (5 mg/ml in 0.05 M carbonate buffer pH 9.6, 100 ml/well) was adsorbed onto 96-well plates (Immulon II, Dynatech Laboratories USA) for a minimum of 16 h at 4°C. The plates were washed 10 times in wash buffer [(0.1 M PBS) pH 7.2, 0.05% Tween 20] before and after being blocked with assay buffer (5% BSA in PBS, 150 ml/well) for 2 h on an agitator at room temperature. The wells were then filled with either samples, standards or controls (100 ml/well). The standard curve was generated using rVEGF diluted in assay buffer and ranged from 19 pg/ml to 20 ng/ml in nine serial two-fold steps. Background wells contained assay buffer alone. rVEGF previously diluted to 1 ng/ml and 2 ng/ml in 5% BSA and frozen in multiple aliquots at −80°C acted as a positive control for ascertainment of inter- and intra-plate variability. Plates were incubated for 16 h at 4°C after which they were washed 10 times in wash buffer. Mouse monoclonal anti-VEGF (mAb 4.6.1) (0.82 mg/ml in assay buffer, 100 ml/well) was then added for 4 h on an agitator at room temperature followed by a further 10 washes. Peroxidaseconjugated goat anti-mouse IgG (heavy and light chain specific) and Dako anti-human secretory component (both diluted 1:2000 in assay buffer, 100 ml/well) was then added and the plate was incubated at room temperature on an agitator for 4 h followed by a further 10 washes. The plate was then developed by adding ABTS (1 ml in 50 ml citrate phosphate buffer plus 5 ml hydrogen peroxide [100 ml per well)]. The plate was allowed to develop prior to the optical density at 405 nm being measured using a Molecular Devices microplate reader. Plate values were taken when the top standard gave an absorbance of approximately 1. This assay is sensitive to 78 pg/ml and is specific for VEGF with no cross-reaction with a large range of other recombinant cytokines/growth factors. Total RNA was isolated from PMN cell pellets using a modification of the guanidinium thiocyanate–phenol–chloroform extraction protocol.22 cDNA was synthesised from 2 mg of total RNA using SuperscriptTM reverse transcriptase and oligo (dT)12–18 according to the manufacturers instructions. PCR amplification was performed using previously described primers known to amplify all reported VEGF splice variants9 and b-actin primers designed to amplify cDNA sequence and not related processed pseudogene sequences (forward primer 5'-GCC GTC TTC CCC TCC AT C-3'; reverse primer 5'-TAG CAA CGT ACA TGG CTG GGG-3', product size 322 bp). PCR amplification was carried out in a Perkin-Elmer 480 DNA thermal cycler in 20-ml reactions overlaid with paraffin oil containing 4% of the cDNA reaction; 10 pmol of each primer; 0.75 mM of each dNTP; 10% DMSO; 3.5 mM MgCl2; 16.6 mM (NH4)2SO4; 67 mM Tris–HCl pH 8.0;

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85 mg/ml BSA and 0.5 units of Taq DNA polymerase. Thermal cycling conditions consisted of an initial denaturation step at 94°C for 3 min followed by 35 cycles for VEGF cDNA amplification at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min; and 30 cycles for b-actin cDNA amplification at 94°C for 1 min, 65°C for 1 min, 72°C for 1 min; and a final extension step at 72°C for 5 min. PCR products and a 100-bp DNA ladder (Life Technologies) were electrophoresed through 2% agarose gels, stained with ethidium bromide and visualised and photographed under UV illumination.

Statistical analysis Paired t-tests were used where data were determined to be normally distributed using the Kolmogorov–Smirnov test. The Wilcoxon matched pairs test was used for paired non-parametric data. Calculations were performed using GraphPad PrismTM, GraphPad Software Inc., USA.

Acknowledgements This work was funded by grants from the North West Kidney Assoociation, the Wellcome Trust and the Arthritis and Rheumatism Council, UK. The authors would like to thank Dr. N. Ferrara of Genentech for the generous gifts of recombinant VEGF and mAb 4.6.1.

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