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. 2000 Sep;121(3):533–538. doi: 10.1046/j.1365-2249.2000.01272.x

Expression of vascular endothelial growth factor by synovial fluid neutrophils in rheumatoid arthritis (RA)

T Kasama *, K Kobayashi †, N Yajima *, F Shiozawa *, Y Yoda *, H T Takeuchi *, Y Mori *, M Negishi *, H Ide *, M Adachi *
PMCID: PMC1905727  PMID: 10971521

Abstract

Most of the leucocytes infiltrating rheumatoid synovial fluid (SF) are neutrophils capable of producing a variety of inflammatory mediators known to contribute significantly to the disease process during active RA. In the present study, we investigated the contribution made by SF neutrophils to the elevated levels of vascular endothelial growth factor (VEGF) seen in rheumatoid SF. Rheumatoid SF neutrophils were found to contain significantly larger amounts of both VEGF protein and its mRNA than peripheral blood neutrophils from either RA patients or healthy controls. Levels of cell-associated VEGF were well correlated with free VEGF in SF, which was significantly higher than in SF from osteoarthritis patients. Levels of SF neutrophil-associated VEGF also correlated with RA disease activity and cell surface integrin expression. Thus, SF neutrophil-associated VEGF may be considered an indicator of both local and systemic inflammation of RA, contributing to the neovascularization seen during RA synovitis.

Keywords: neutrophil, vascular endothelial growth factor, rheumatoid arthritis, angiogenesis

INTRODUCTION

The progression of RA is characterized by the appearance of inflammatory cells in both the pannus and the joint fluid, resulting in subsequent tissue destruction, and by pronounced tumour-like expansion of the synovium. The latter means that neovascularization may play a pivotal role during the progression of the disease.

Neovascularization is a complex process, involving endothelial cell division, selective degradation of vascular basement membranes and surrounding extracellular matrix, and endothelial cell migration. Several polypeptide growth factors found in the rheumatoid joint have been identified based on their ability to stimulate the proliferation of endothelial cells [1], as have a number of angiogenic factors that may be important in neovascularization. These include tumour necrosis factor-alpha (TNF-α), acidic and basic fibroblast growth factor (FGF) and IL-8 [2]. Another important mediator of neovascularization is vascular endothelial growth factor (VEGF), which is a secreted, heparin-binding, homodimeric glycoprotein with several protein variants resulting from alternative mRNA splicing [3, 4]. In vitro, VEGF acts as an endothelial cell-specific mitogen, whereas in vivo it serves as angiogenic growth factor. VEGF is known to play an important role in female reproductive cycling as well as in such pathological conditions as diabetic retinopathy, certain tumours and RA [5–8].

Neutrophils are known to produce a variety of inflammatory polypeptide mediators [9], and a recent report by Taichman et al. demonstrated that they also secrete VEGF [10]. Because most of the leucocytes infiltrating the synovial fluid (SF) of the rheumatoid joint are neutrophils, this biosynthetically active leucocyte population almost certainly contributes significantly to the disease process during active RA. In fact, we recently showed that freshly isolated SF neutrophils contained substantial concentrations of chemokines, including IL-8 and macrophage inflammatory protein (MIP)-1α, and their expression was well correlated with RA disease activity [11, 12]. In this context, the aim of the present study was to examine the expression of VEGF by SF neutrophils in patients with RA, and to characterize the relationship between levels of neutrophil-derived VEGF and RA disease activity.

PATIENTS AND METHODS

Reagent preparation

Recombinant human TNF-α with a specific activity of 1·6 × 108 U/mg was purchased from Pepro Tech EC (London, UK). Complete medium consisted of Dulbecco's modified Eagle's medium (DMEM; Dai-nippon Pharmaceutical Co. Ltd, Tokyo, Japan) supplemented with 2 mm l-glutamine, 25 mm HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco Labs, Grand Island, NY) and 10% heat-inactivated fetal bovine serum (FBS; Gibco). Monoclonal antibody (clone no. 26503.11) and polyclonal antibody for VEGF were purchased from R&D Systems (Minneapolis, MN).

Isolation and culture conditions of peripheral blood and SF neutrophils

RA SF neutrophils, RA peripheral blood (PB) and normal PB neutrophils were obtained from knee puncture of 19 patients with RA who fulfilled the 1987 ACR criteria [13], and from 12 out of 19 patients with RA and 15 age- and sex-matched healthy individuals, by Ficoll–Hypaque (Pharmacia LKB Biotechnology Inc., Piscataway, NJ). Most RA patients were receiving a non-steroidal anti-inflammatory drug (NSAID) and a slow-acting anti-rheumatic drug. Of those receiving a slow-acting anti-rheumatic drug, two were taking methotrexate, four were taking d-penicillamine, six were taking sulfasalazine, and three were taking gold. No patient was receiving either > 5 mg oral prednisolone/day or intra-articular injection of glucocorticoids within 1 month from SF sample aspiration. The recovered neutrophils were washed three times and resuspended at a density of 5 × 106 cells/ml in complete medium. The final cell preparation contained > 98% neutrophils by morphology and viability was > 98% by trypan blue dye exclusion, and monocyte contamination was < 2% by non-specific esterase staining. To assess cell-associated cytokine, neutrophil lysates were obtained by addition of same volume of complete medium to the cells, followed by three freeze/thaw cycles. Furthermore, SF samples were aspirated from patients with RA or osteoarthritis (OA) under aseptic conditions, and then centrifuged (1000 g, 10 min) to remove cell debris. The supernatants were immediately frozen.

C-reactive protein (CRP) was measured by the nephelometric standard method.

All human experiments were performed in accordance with protocols approved by the Human Subjects Research Committee at our Institution, and informed consent was obtained from all patients and volunteers.

Assessment of VEGF levels by specific ELISA

Specific neutrophil-derived VEGF was quantified using a modification of a double ligand ELISA method. Murine anti-human VEGF MoAb (2 μg/ml) as first antibody and polyclonal goat anti-VEGF antibody (1 μg/ml) as second antibody were used. Monoclonal and polyclonal antibodies for VEGF used in our ELISA have captured either VEGF121 or VEGF165 form, as well as natural human VEGF, and were not cross-reactive with other human cytokines or growth factors. Standards were dilutions of recombinant human VEGF (Pepro Tech EC) from 50 ng/ml to 7·5 pg/ml. This ELISA method consistently detected VEGF in a linear fashion between 30 pg/ml and 10 ng/ml.

Immunohistochemistry

Neutrophil-derived VEGF was visualized by immunohistochemistry as previously described [12, 14]. Briefly, neutrophils were deposited on a glass slide using a Cytospin II (Shandon Southern Instruments, Inc., Sewickley, PA). The slides were incubated with polyclonal goat anti-VEGF antibody (1:500 dilution) or preimmune goat IgG. Biotinylated rabbit anti-goat IgG and peroxidase-conjugated streptavidin (BioGenex Inc., San Ramon, CA) were used as second and third reagents, respectively. The substrate for red colour reaction was 3-amino-9-ethylcarbazole in N,N-dimethylformamide. After rinsing with distilled water, the slides were stained with Mayer's haematoxylin.

Isolation of total RNA and reverse transcription-polymerase chain reaction

Total cellular RNA from neutrophils was isolated as previously described [14, 15]. Briefly, samples were dispersed in a solution of 25 mm Tris pH 8·0, which contained 4·2 m guanidine isothiocyanate, 0·5% Sarkosyl, and 0·1 m 2-mercaptoethanol. The RNA was further extracted with the use of chloroform-phenol and alcohol precipitated.

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as previously described [16]. Briefly, 5 μg of total RNA was reverse transcribed into cDNA utilizing M-MLV reverse transcriptase (Gibco BRL). The PCR reaction was performed with the primers 5′-GAG-TGT-GTG-CCC-ACT-GAG-GAG-TCC-AAC (sense) and 5′-CTC-CTG-CCC-GGC-TCA-CCG-CCT-CGG-CTT (antisense) for VEGF [17], and 5′-GGC-ATC-CGG-ACG-TTC-TAC-GG (sense) and 5′-ATG-GTG-AAG-GTC-GGT-GTG-AAC (antisense) for GAPDH [18], as an internal control. The PCR reactions were separated on 2% agarose gel containing 0·3 μg/ml of ethidium bromide, and were visualized and photographed using UV transillumination.

Flow cytometric analysis

Neutrophils were incubated with anti-CD11b or CD18 MoAb (5 μg/ml), purchased from Ancell Corporation (Bayport, MN), for 30 min on ice, and then were washed extensively and incubated with FITC-conjugated goat anti-mouse IgG for 30 min on ice. The stained cells were washed and resuspended in PBS containing 1% FBS, and analysed immediately by a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Background or non-specific staining was determined by using isotype-matched irrelevant antibodies. Data were expressed by the mean fluorescence intensity (MFI).

Statistical analysis

Data were analysed by Power Macintosh computer using a statistical software package (Statview 4.5; Abacus Concept, Inc., Berkeley, CA) and expressed as the mean ± s.e.m. Data were evaluated by Mann–Whitney test and the Spearman rank correlation coefficient where appropriate. P < 0·05 was considered significant.

RESULTS

Expression of cell-associated VEGF by freshly isolated neutrophils and VEGF concentrations in SF

We hypothesized that neutrophils are an important source of the VEGF found in the SF of RA patients. To test this hypothesis, neutrophils were freshly isolated from the PB of RA patients and healthy controls and from the SF of RA patients, and cell-associated VEGF was assayed by ELISA. As shown in Fig. 1a, SF neutrophils from RA patients contained greater amounts of VEGF (mean 103·1 pg/5 × 106 cells) than either normal or rheumatoid PB neutrophils (8·8 pg/5 × 106 and 16·8 pg/5 × 106 cells, respectively). In paired samples, levels of neutrophil-associated VEGF correlated significantly with free VEGF in SF (rs = 0·501, P = 0·022) (Fig. 1b), but there was no obvious correlation between PB and SF neutrophil-associated VEGF (data not shown). In addition, higher levels of VEGF were detected in the SF of RA patients (697·1 pg/ml) than in that of patients with OA (6·5 pg/ml), which is in agreement with previous reports (Fig. 1c) [6,7].

Fig. 1.

Fig. 1

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Concentrations of vascular endothelial growth factor (VEGF) in lysates of freshly isolated neutrophils or in synovial fluids. (a) Neutrophils were obtained from peripheral blood (PB) of RA patients (n = 12) and healthy controls (n = 15) and from the synovial fluid (SF) of RA patients (n = 19), after which cell-associated VEGF (pg/5 × 106 cells) was assayed by ELISA. (b) Correlation between neutrophil-associated and free VEGF in SF. Each point represents an individual RA patient. (c) SF was obtained from RA patients (n = 10) or osteoarthritis (OA) (n = 8) patients and assayed by ELISA. Data are expressed as means ± s.e.m. *P < 0·01 versus rheumatoid or normal PB (a), or OA (c).

The cellular source of VEGF protein was then confirmed immunohistochemically. Although rheumatoid SF neutrophils were not stained by preimmune IgG (Fig. 2A), when labelled with an anti-VEGF antibody they exhibited significant VEGF antigenicity (Fig. 2B; arrows). In contrast, no positive staining for VEGF was observed among PB neutrophils obtained from either healthy controls or RA patients (data not shown).

Fig. 2.

Fig. 2

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Representative photomicrograph showing the immunohistochemical localization of antigenic vascular endothelial growth factor (VEGF) in freshly isolated rheumatoid synovial fluid (SF) neutrophils. (A) Cells stained with control IgG. (B) Cells stained by anti-VEGF antibody, demonstrating the presence of neutrophil-associated VEGF antigen (arrows).

Because the levels of VEGF protein were significantly elevated in rheumatoid SF neutrophils (Fig. 1a), we compared the steady-state expression of VEGF mRNA in SF and PB neutrophils using RT-PCR. Several alternative splice variants of VEGF have been described [3], and in fact the primers used in the present experiments were capable of yielding four spliced forms 435 bp, 384 bp, 312 bp and 180 bp in length [19]. We found that VEGF transcripts, especially the 180-bp form, corresponding to VEGF121, were strongly expressed in freshly isolated SF neutrophils (Fig. 3), whereas there was little or no transcription of VEGF in PB neutrophils from either healthy controls or RA patients.

Fig. 3.

Fig. 3

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Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of vascular endothelial growth factor (VEGF) mRNA in freshly isolated neutrophils obtained from rheumatoid synovial fluid (SF) and peripheral blood (PB) or from normal PB. (a) Representative neutrophil-derived VEGF mRNA. GAPDH primers were used as an internal control. Lane 1, Normal PB neutrophils; lane 2, RA PB neutrophils; lane 3, RA SF neutrophils. Lane M contains molecular weight markers (100-bp ladder). (b) VEGF mRNA expression was quantified and normalized to GAPDH as the VEGF/GAPDH ratio. Data are expressed as the means ± s.e.m. of three independent experiments.

The effects of TNF-α were then analysed to assess the extent to which neutrophil activation by other cytokines affects VEGF secretion. We found that even in the absence of TNF-α, rheumatoid SF neutrophils secreted 219·7 ± 68·2 pg of VEGF/ml over the course of 24 h, which was significantly higher than was secreted by neutrophils obtained from healthy or rheumatoid PB over the same period (68·5 ± 26·2 pg/ml and 75·7 ± 17·9 pg/ml, respectively). Exposing the cells to TNF-α for 24 h increased VEGF secretion from all three neutrophil populations; nonetheless, secretion from rheumatoid SF neutrophils remained significantly higher (464·6 ± 49·8 pg/ml) than from normal (144·7 ± 76·3 pg/ml) or rheumatoid PB neutrophils (112·7 ± 29·1 pg/ml).

The acute-phase response, as measured by CRP concentrations, is commonly used in clinical practice to monitor RA disease activity. Elevated levels of CRP are generally indicative of disease progression and lack of improvement under therapy. We therefore wondered whether there would be any correlation between RA disease activity, as indicated by serum CRP levels, and neutrophil-associated or free VEGF in SF. Figure 4 shows that, indeed, serum CRP levels significantly correlated with both cell-associated (rs = 0·509, P = 0·01) and free VEGF (rs = 0·504, P = 0·02) in SF. On the other hand, there was no correlation between levels of SF VEGF and serum CRP in OA patients (rs = 0·004, P = 0·879) (data not shown), nor was there any correlation between levels of cell-associated or free VEGF and the use of NSAIDs, oral or intra-articular glucocorticoids, or slow-acting anti-rheumatic drugs (data not shown).

Fig. 4.

Fig. 4

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Correlation between neutrophil-associated (a) or free vascular endothelial growth factor (VEGF) (b) in synovial fluid (SF) and serum C-reactive protein (CRP) concentrations. Each point represents an individual RA patient.

Correlation between integrin expression by SF neutrophils and levels of neutrophil-associated VEGF

Expression of cell surface molecules, such as integrins CD11b and CD18, is known to be up-regulated in neutrophils in rheumatoid SF, reflecting their activated state [20–23]. Interestingly, we found that the expression levels of both CD11b and CD18 were well correlated with the expression of VEGF in SF neutrophils (CD11b: rs = 0·574, P = 0·011; CD18: rs = 0·585, P = 0·010) (Fig. 5).

Fig. 5.

Fig. 5

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Correlation between levels of cell-associated vascular endothelial growth factor (VEGF) and expression of integrins CD11b (a) or CD18 (b) in freshly isolated synovial fluid (SF) neutrophils. Cell-associated VEGF and cell surface integrins were assayed by ELISA and flow cytometry, respectively. Each point represents an individual RA patient. MFI, Mean fluorescence intensity.

DISCUSSION

VEGF has been detected in rheumatoid SF and synovial tissues and may have an important role in angiogenesis and endothelial migration during development of synovitis in RA [2,6,7,24,25]. These observations led us to hypothesize that the presence of VEGF in rheumatoid SF may be dependent, at least in part, on SF neutrophils, which are the dominant cell type in the SF of RA patients.

In the present study, we demonstrated that freshly isolated rheumatoid SF neutrophils contain significant amounts of cell-associated VEGF, which correlate with levels of free VEGF in SF, integrin expression and RA disease activity (Figs 1, 2, 4 and 5); that they exhibit increased steady-state transcription of VEGF mRNA, especially the VEGF121 form (Fig. 3); and that in both the absence and presence of TNF-α, they secrete greater amounts of VEGF than either healthy or rheumatoid PB neutrophils. Taken together, these data strongly support the notion that once activated by exposure to cytokines within rheumatoid joints, SF neutrophils are an important source of VEGF. In this way, activated SF neutrophils may contribute to the neovascularization seen during RA synovitis by acting directly on endothelial cells through secretion of various cytokines, including VEGF, and may act indirectly through recruitment of monocytes: VEGF induces not only endothelial cell migration, but also monocyte activation and migration [26, 27].

Previous studies have shown that treating RA patients with methotrexate or methylprednisolone pulse therapy significantly reduces numbers of SF neutrophils, their expression of CD11b and CD18 and the concentration of IL-1β in SF, and is associated with impressive clinical improvement [28, 29]. Our findings shed new light on the possible role played by SF neutrophils in the perpetuation of inflammation in RA, and may serve as the basis for a new therapeutic approach, targeting activated neutrophils and neutrophil-derived VEGF in the synovial space.

Acknowledgments

This study was supported, in part, by Uehara Memorial Foundation, the High-Technology Research Centre Project (Ministry of Education, Science, Sport and Culture of Japan), Research Promotion of Emerging and Reemerging Infectious Diseases (Ministry of Health and Welfare of Japan), and Sasakawa Memorial Foundation.

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