Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune disease with chronic inflammation characterized by synovial cells proliferation in response to inflammatory stimuli, leading to formation of very aggressive tissue called the rheumatoid pannus. Expansion of pannus induces bone erosion and cartilage thinning, leading to loss of joint function. Neovascularization is required to maintain the chronic inflammatory state by transporting inflammatory cells to the site of synovitis as well as by supplying nutrients and oxygen to the pannus [1]. A number of angiogenic factors are involved in the neovascularization and include acidic and basic fibroblast growth factors, platelet-derived endothelial cell growth factor, transforming growth factor(TGF)-α, TGF-β, angiogenin, and vascular endothelial growth factor (VEGF) [2, 3].

VEGF is a heparin-binding, dimeric glycoprotein that induces endothelial cell proliferation, angiogenesis, and capillary permeability [4, 5]. It has been documented that VEGF may be involved in the pathogenesis of RA. Significantly greater quantities of VEGF are found in the synovial fluid of RA patients than in osteoarthritis or other forms of arthritis [6, 7], and VEGF protein and messenger RNA (mRNA) were expressed by subsynovial macrophages and synovial lining cells in RA joints [6, 8]. Hypoxia is an important stimulus for VEGF release in rheumatoid synovial cells [9–11]. In addition, prostaglandin (PG), IL-1 [12], IL-6 [13], TGF-β [10, 14], TNF-α [15] and CD40 ligand [16] have been describe to induce VEGF.

IL-17 is s T cell-derived cytokine produced by activated T cells, predominantly activated CD4+, CD45RO+ memory T cells [17, 18]. This cytokine stimulate stromal cells to secrete various cytokines and growth factors associated with inflammation [17–20]. Increased IL-17 expression was detected in several diseases, such as systemic sclerosis [21], nephritic syndrome [22], systemic lupus erythematosus [23], and RA [24, 25]. IL-17 is spontaneously produced by rheumatoid synovium [24], and high levels have been detected in the synovial fluid of patients with RA [24, 25]. IL-17 can stimulate the production of IL-1β and TNF-α from macrophages [20] and triggers human synoviocytes to produce IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and prostaglandin E2[18, 26] suggesting that IL-17 could be an important mediator in the pathogenesis of arthritis. Furthermore, IL-17 may be involved in tissue destruction. IL-17 has biologic activities similar to those of IL-1β, and additive/synergic effects with IL-1β and TNF-α have been reported [27]. IL-17 suppresses matrix synthesis by murine articular chondrocytes through nitric oxide (NO) production [28, 29], and promotes bone erosion through loss RANKL/OPG balance in collagen-induced arthritis (CIA) [30].

There is paucity of data regarding the effect of IL-17 on VEGF production in RA synoviocytes. In this study, we investigate the effect of IL-17 on the production and mRNA expression of VEGF in rheumatoid FLS.

Materials and methods

Isolation and culture of synoviocytes

Fibroblast-like synoviocytes (FLS) were isolated by enzymatic digestion of synovial tissues, obtained from RA patients undergoing total joint replacement surgery, as described previously [16]. Tissues were minced into 2- to 3-mm pieces and treated for 4 h with 4 mg/ml of collagenase (type I; Worthington Biochemical, Freehold, NJ) in Dulbecco’s modified Eagle’s medium (DMEM) at 37 ° C in 5% CO2. Dissociated cells were then centrifuged at ×500g, resuspended in DMEM supplemented with 10 % fetal calf serum (FCS, Life Technologies, Grand Island, NY), 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml), and plated in 75-cm2 flasks. Cultures were kept at 37 ° C in 5% CO2, and the medium was replaced every 3 days. When cells approached confluence, they were passed by diluting 1:3 with fresh medium and were recultured until used.

VEGF production by cytokines

A homogeneous population of FLS from passage 4 to 8 was used for each experiment. FLS were seeded in 24-well plates at 6×104 cells/well in 1 ml DMEM supplemented with 5% FCS, and incubated at 37° C for 24 h. Cultures were washed with serum-free DMEM, and then incubated in serum-free DMEM supplements with insulin-transferrin-selenium A (ITSA, Life Technologies) for 48 h until the medium was replaced with fresh DMEM/ITSA. Subsequently, various concentrations of IL-17, IL-1β, TNF-α and TGF-β (5–400 pg/ml) were added to the each well, and cultures were incubated for 24 h. IL-17(20 pg/ml), IL-1β (20 pg/ml), TNF-α (20 pg/ml) and TGF-β (20 pg/ml), IL-17 with TNF-α, IL-17 with IL-β, and IL-17 with TGF-β were added to each well, and cultures were incubated for 12, 24, 48, and 72 h. Cell-free media were collected and stored at −20 ° C until used.

Enzyme-linked immunosorbent assay (ELISA) of VEGF

VEGF in culture supernatants was measured by sandwitch ELISA, as previously described [16]. Ninety-six-well microtiter plates were coated with 100μl/well of 0.4μg/ml goat anti-human VEGF165 (R&D systems, Minneapolis, MN) buffered with 50 mM of sodium carbonate (pH 9.6). After overnight incubation at 4 ° C, the plates were blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 h at room temperature. The human recombinant VEGF 165 (R&D Systems) or test samples were added to the wells and incubated for 2 h at room temperature. Plates were incubated with 0.2 μg/ml biotinylated goat anti-human VEGD165 (R&D Systems) for 2 h at room temperature. Peroxidase-labeled extravidin (Sigma), diluted 1:1000, was added to react with the plates at room temperature for 1 h. A color reaction was induced by the addition of substrate solution (TMB/H2O2) and was stopped 30 min later by the addition of 1 M phosphoric acid. An automated microplate reader was used to measure the OD at a wave-length of 450 nm. Between each of these steps, the plates were washed four times with PBS containing 0.05% Tween 20. Human recombinant VEGF 165 diluted in culture medium was used as a calibration standard, ranging from 10 pg/ml to 2,000 pg/ml. A standard curve was drawn by plotting OD versus the log of the recombinant VEGF 165 concentration.

Quantitation of VEGF mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR)

Total cellular RNA was extracted from synovial cells as described previously [31] and was treated with amplification-grade DNase I (GIBCO BRL, Life Techonologies). RNA was then subjected to reverse transcription (RT) using the SuperScript Preamplification System for First Strand cDNA Synthesis (GIBCO BRL, Life Technologies). Serial dilutions of the cDNA template were prepared and PCR was carried out using a Lightcycler System (Roche Molecular Biochemicals, Palo Alto, CA). After each elongation phase, the fluorescence of SYBR Green I (Roche Molecular Biochemicals), which binds double-stranded DNA, was measured. Reactions (20 μl) were performed in microcapillary tubes using 5 μl of diluted cDNA with SYBR Green I, master mis, upstream and downstream primers, and MgCl2. Sequences of primer pairs were as follows: VEGF:upstream, 5′-TTCATGGATGTCTATCAGCG-3′, downstream, 5′-GCTCATCTCTCCTATGTGCT-3′, β-actin : upstream, 5′-AACACCCCAGCCATGTACG-3′, downstream, 5′-ATGTCACGCACGATTTCCC-3′. Reactions containing water or cDNA synthesized without reverse transcriptase, as template, resulted in no PCR products. LightCycler quantification software v3 was used to compare amplification in experimental samples during the log-linear phase to the standard curve from the dilution series of acute tissue. All experimental samples were normalized to β-actin expression levels for that tissue. The expression levels of each gene were plotted relative to the levels in normal tissue.

Data

Data were taken from at least three repeated experiments performed with each sample obtained from the three RA patients. Statistical analysis was performed using the SPSS statistical software package (version 10.0 for Windows). Comparisons among groups were performed by Mann–Whitney test. A 5% significance levels was accepted as showing statistical significance.

Results

IL-17 dose-dependently increased the production of VEGF in FLS

Stimulation of FLS by IL-17, IL-1β, TNF-α and TGF-β with 5, 10, 20, 50, 100, 200, 400 pg/ml increased VEGF production dose-dependently (Fig. 1).

Fig. 1
figure 1

Vascular endothelial growth factor (VEGF) production was dose-dependently increased by IL-17, IL-1β, tumor necrosis factor- α (TNF-α) and transforming growth factor-β (TGF-β). Human rheumatoid arthritis synoviocytes were stimulated with IL-17, IL-1β, TNF-α and TGF-β (5–400 pg/ml) and cultured for 24 h. Data were taken from at least three repeated experiments performed with each sample obtained from the three RA patients. The amount of VEGF in the supernatant was determined by sandwitch ELISA. Data were expressed as log scale of mean concentration (C : concentration of with IL-17, IL-1β, TNF-α and TGF-β, V : concentration of VEGF)

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IL-17 had additive effect with TNF-α, IL-1β and TGF-β on the production and expression of VEGF in FLS

VEGF production of FLS stimulated by PBS, IL-17 (20 pg/ml), IL-1β (20 pg/ml), TNF-α(20 pg/ml), TGF-β (20 pg/ml), IL-17 (20 pg/ml)+IL-1β (20 pg/ml), IL-17 (20 pg/ml)+TNF-α (20 pg/ml) and IL-17 (20 pg/ml)+TGF-β (20 pg/ml) was 190±21 pg/ml, 456±52, 547±35, 487±42, 423±46, 820±54, 680±43 and 676±54 pg/ml after 12 h culturing, 213±25, 497±38, 587±58, 587±47, 467±35, 880±57, 750±46, 840±78 pg/ml after 24 h culturing, 220±31, 492±32, 652±45, 610±66, 580±57, 1043±87, 1044±68 and 986±57 pg/ml after 48 h culturing, 232±37, 543±46, 663±35 and 578±35, 612±57, 1259±89, 1162±74 and 979±74 pg/ml after 72 h culturing (Table 1).

Table 1 IL-17 had additive effect with IL-1β, TNF-α and TGF-β on the production of vascular endothelial growth factor (VEGF)
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The mRNA VEGF/β-actin relative density of FLS untstimulated, stimulated by IL-17 (20 pg/ml), IL-1β (20 pg/ml), TNF-α (20 pg/ml), TGF-β (20 pg/ml), IL-17 (20 pg/ml)+IL-1β (20 pg/ml), IL-17 (20 pg/ml)+TNF-α(20 pg/ml) and IL-17 (20 pg/ml)+TGF-β (20 pg/ml) was 1, 2.2±0.6, 3±1, 1.8±0.3, 2±0.5, 3.4±0.8, 3.2±1 and 2.8±1.3 after 24 h culturing (Fig. 2).

Fig. 2
figure 2

IL-17 increased the vascular endothelial growth factor (VEGF) mRNA expression and had additive effect with TNF-α, IL-1β, and TGF-β on VEGF mRNA expression. Human rheumatoid arthritis synoviocytes were stimulated with IL-17, IL-1β, TNF-α, transforming growth factor-β (TGF-β) (20 pg/ml), IL-17+IL-1β, IL-17+TNF-α, and IL-17+TGF-β and cultured for 24 h. Data were taken from at least three repeated experiments performed with each sample obtained from the three RA patients. The VEGF mRNA expression was determined by real-time RT-PCR. Data were expressed as mean±SD *, **, *** ; P<0.05 by Mann–Whitney test

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Discussion

In this study, we demonstrated that IL-17 increased the production and mRNA expression of VEGF, and had additive effect with TNF-α, IL-1β and TGF-β on the production of VEGF in RA synoviocytes.

IL-17 may contribute to joint inflammation and destruction in RA. In vivo studies, IL-17 increased joint inflammation, cartilage destruction and bone erosion [30, 31] in CIA. It promotes bone erosion through loss of receptor activator NF-κB ligand/osteoprotegerin balance. Blocking of IL-17 reduced joint inflammation, cartilage destruction and bone erosion [32–34] and these results suggest blocking of IL-17 may provide a therapeutic strategy for RA.

Apart from inflammatory and destructive responses, IL-17 has been shown to promote angiogenesis in tumor models and correlates well with the numbers of blood vessels in human ovarian cancers [35]. IL-17 promotes tumorgenicity of human cervical tumors in nude mice and is associated with an increased level of IL-6 expression at the tumor sites [36]. Increased levels of IL-6 correlate well with the invasiveness of cervical tumors [37]. IL-17 promotes angiogenesis and tumor growth [38].

IL-17 had synergic effect with IL-1 on the production of IL-6, leukemia inhibitory factor (LIF) [39], and had synergic effects with TNF-α on the production of macrophage inhibitory protein −3α (MIP-3α) and IL-6 in rheumatoid arthritis synoviocytes [40]. In our study, IL-17 had no synergic but rather additive effect with TNF-α, IL-1β and TGF-β on the production and expression of VEGF in RA synoviocytes.

In summary, we have demonstrated that IL-17 increase the production and expression of VEGF and had additive effect with TNF-α, IL-1β and TGF-β on the production of VEGF in RA synoviocytes. Our data strongly suggest that IL-17 is implicated in the angiogenesis of RA synovium.