Abstract
Objective
Interleukin (IL)-10 has been demonstrated to have anti-inflammatory and anti-tumour activity. Because aberrant angiogenesis is a significant pathogenic component of tumor growth and chronic inflammation, we investigated the effect of IL-10 on the production of vascular endothelial growth factor (VEGF) by the synovial fibroblasts derived from the patients with rheumatoid arthritis (RA).
Methods
Fibroblast-like synoviocytes (FLS) were cultured with transforming growth factor (TGF-β) alone or with IL-10. The level of VEGF was measured by RT-PCR and enzyme-linked immunosorbent assay (using the 24, 48 and 72 h culture supernatants). The FLSs were cultured with TGF-b for 48 hr in the presence of PD98059 (an ERK inhibitor), curcumin and SP600125 (a JNK and Ap-1 inhibitor, respectively). The level of VEGF in the supernatants was measured by ELISA. Cell viability was assessed using MTT assay. The expressions of VEGF, ERK, AP-1 and IL-10 in the synovial tissue were quantified by immunohistochemistry.
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Fig. 1.
IL-10 inhibits the VEGF mRNA in the TGF-β stimulated RA synovial fibroblasts. The FLSs were cultured with TGF-β (10 ng/mL) alone or with the indicated concentrations of IL-10 (1∼50 ng/mL) for 12 h. The mRNA level of VEGF was measured by RT-PCR. The data is expressed as the mean±s.d. of three independent experiments (∗p<0·05, ∗∗p<0.01 versus the that were well treated with TGF-β alone).
![jkra-16-213f1.tif](/upload/SynapseXML/0010jkra/thumb/jkra-16-213f1.gif)
Fig. 2.
IL-10 inhibits the TGF-β induced VEGF production in synovial fibroblasts. The FLSs were cultured for 24, 48 and 72 h with TGF-β (10 ng/mL) in the presence of increasing doses of IL-10 (1∼50 ng/mL). The level of VEGF in the supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was assessed using MTT assay. The data is expressed as the mean±s.d. of three independent experiments (∗p<0.05, ∗∗p<0.01 versus the that were well treated with TGF-β alone).
![jkra-16-213f2.tif](/upload/SynapseXML/0010jkra/thumb/jkra-16-213f2.gif)
Fig. 3.
IL-10 inhibits the TGF-β-induced phospho ERK and phospho C-Jun expressions in synovial fibroblasts. The FLSs were cultured for 48 h with medium alone or with 10 ng/mL of TGF-β in the presence of PD98059 (an ERK inhibitor), curcumin and SP600125 (a JNK and Ap-1 inhibitor, respectively). The level of VEGF in the supernatants was measured by ELISA. The levels of phosphor ERK and phosphor C-Jun in the cell lysates were measured by western blotting. This data shows the dose-dependent inhibition of the phospho ERK and phospho C-Jun expressions by IL-10 (10 ng/mL, 50 ng/mL). The data is expressed as the mean±s.d. of three independent experiments (∗∗p<0.01 versus the that were well treated with TGF-β alone).
![jkra-16-213f3.tif](/upload/SynapseXML/0010jkra/thumb/jkra-16-213f3.gif)
Fig. 4.
VEGF was highly expresses by RA patients, as compared to the OA patients, in the synovial tissue and synovial fluid mononuclear cells. Immunostaining was performed using the specific antibodies against VEGF, ERK, AP-1 and IL-10 in the synovial tissue samples from the patients with RA and OA. VEGF, ERK, AP-1 and IL-10 stained a brown color. All the tissues were counterstained with hematoxylin (original magnification ×400). The levels of mRNA VEGF and IL-10 in the synovial fluid were measured by RT-PCR. The data is expressed as the mean±s.d. of three independent experiments (∗∗p<0.01 versus OA SFMC).
![jkra-16-213f4.tif](/upload/SynapseXML/0010jkra/thumb/jkra-16-213f4.gif)