Peripheral blood mononuclear cells from patients with rheumatoid arthritis spontaneously secrete vascular endothelial growth factor (VEGF): specific up-regulation by tumour necrosis factor-alpha (TNF-alpha) in synovial fluid

Abstract

This study was designed to investigate VEGF production from peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) compared with healthy controls and to identify the predominant cellular source in PBMC isolated from RA patients. The regulation of PBMC VEGF production by cytokines and synovial fluid (SF) was studied. PBMC were isolated from RA patients and healthy controls and stimulated with lipopolysaccharide (LPS), IL-1beta, IL-4, IL-6, IL-8, IL-10, TNF-alpha and transforming growth factor-beta (TGF-beta) isoforms for varying time points up to 72 h at 37 degrees C/5% CO2. The effect of SF on VEGF secretion by PBMC was also studied. Supernatant VEGF levels were measured using a flt-1 receptor capture ELISA. RA patients had significantly higher spontaneous production of VEGF compared with controls, and monocytes were identified as the predominant cellular source. RA PBMC VEGF production was up-regulated by TGF-beta isoforms and TNF-alpha and down-regulated by IL-4 and IL-10, with no effect observed with IL-1beta, IL-6 and IL-8. Antibody blocking experiments confirmed that TNF-alpha and not TGF-beta isoforms in SF increased VEGF secretion by RA PBMC. These results emphasize the importance of monocytes as a source of VEGF in the pathophysiology of RA. Several cytokines known to be present in SF can modulate the level of VEGF secretion, but the predominant effect of SF in VEGF up-regulation is shown to be dependent on TNF-alpha.

Fig. 1

Levels of VEGF secretion by unstimulated and stimulated (10 U/ml lipopolysaccharide (LPS)) peripheral blood mononuclear cells (PBMC) isolated from 21 rheumatoid arthritis (RA) patients (▪) and 21 healthy controls (□) after 24 h and 72 h in culture. Spontaneous secretion of VEGF by RA patients was significantly higher after 24 h and 72 h compared with controls. *P < 0.02 (unpaired t-test).

Fig. 2

Levels of VEGF secretion from peripheral blood mononuclear cells (PBMC) isolated from 10 rheumatoid arthritis (RA) patients in response to transforming growth factor-beta 1 (TGF-β1) (a) and TNF-α (b) at 100, 10, 1, 0.1 and 0.01 ng/ml after 24 h in culture. *P < 0.05 compared with control (one-way anova with Bonferroni correction).

Fig. 3

Levels of VEGF secretion from peripheral blood mononuclear cells (PBMC) isolated from 10 rheumatoid arthritis (RA) patients in response to IL-4 (a) and IL-10 (b) at 100, 10, 1, 0.1 and 0.01 ng/ml after 24 h in culture. *P < 0.05 compared with control (one-way anova with Bonferroni correction).

Figure 4

The effect of 10% synovial fluid (SF) from rheumatoid arthritis (RA), primary inflammatory (PI), osteoarthritis (OA) and non-inflammatory (NI) groups on VEGF secretion by normal peripheral blood mononuclear cells (PBMC) (a). The results are expressed as a percentage of VEGF secretion by cells alone and the VEGF already present in the SF was corrected for by subtracting 10% of the neat SF VEGF level. No significant differences were observed between groups. The effect of 10% RA SF (n = 10) alone, or in combination with 20 μg/ml of anti-TNF-α, anti-transforming growth factor-beta 1,2,3 (TGF-β1,2,3) or mouse immunoglobulin as control on VEGF secretion by normal PBMC after 24 h in culture (b). *P < 0.001 compared with cells with SF alone (one-way anova with Bonferroni correction).