SAT0048 Overexpression of CCL18 Promotes Migration and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis
Authors: Y.-Q. Mo, J. Jing, J.-D. Ma, C. Deng, L.-F. Chen, D.-H. Zheng, L. Dai
Journal: Annals of the Rheumatic Diseases (2016)

<h3>Background</h3> CC chemokine ligand 18 (CCL18) has been reported overexpressed in numerous inflammatory disorders since 1997. However, its functional receptor, phosphatidylinositol transfer membrane-associated phosphatidylinositol transfer protein 3 (PITPNM3), has not been discovered until 2011 by our colleagues. Multiple functions of CCL18 besides its chemotactic activity were then identified, including promoting cytokine production, migration/invasion of cancer cells, type I collagen production by human fibroblasts of lung or skin. The effect on the pathogenesis of rheumatoid arthritis (RA) remains unclear. <h3>Objectives</h3> To explore the expression of CCL18 in RA patients and its effect on RA fibroblast-like synoviocytes (FLS). <h3>Methods</h3> CCL18 in serum and synovial fluid was measured by ELISA. Synovial tissue was obtained by closed-needle biopsy for H&amp;E and immunohistochemistry staining, and FLS culture <i>in vitro</i>. Expression of PITPNM3 on cell membrane was detected by western blot. Cell proliferation was identified by cell counting kit (CCK-8) and migration ability was analyzed by transwell assay. Cytokines (IL-1β, IL-6, IL-17A, TNF-α, GM-CSF), chemokines (CCL2, CCL3, CCL5, CCL8) and MMP3 in culture supernatant were measured by Cytometric Bead Array (CBA) or ELISA. <h3>Results</h3> (1) Serum CCL18 of 51 active RA patients was 112 ng/mL (IQR, 85–132), which was significantly higher than healthy controls (<i>n</i>=11, 64 ng/mL (IQR, 36–70), <i>p</i>&lt;0.001, Figure 1A). Eighteen RA patients had synovial fluid and CCL18 in synovial fluid was 1229 ng/mL (IQR, 934–1540) which was significantly higher than corresponding serum level (<i>p</i>&lt;0.001) and synovial fluid CCL18 of osteoarthritis patients (<i>n</i>=7, 33 ng/mL (IQR, 25–229), <i>p</i>=0.001). (2) Immunohistochemistry staining showed CCL18-positive cells in lining and sublining area of RA synovium (n=48), which were significantly greater than osteoarthritis synovium (both <i>p</i>=0.001, Figure 1B∼D). Double-immunocytofluorescent staining showed vimentin-positive RA-FLS <i>in vitro</i> expressed PITPNM3 (Figure 2A) and western blot of membrane protein showed the expression of PITPNM3 protein on RA-FLS. (3) RA-FLS was incubated with different concentrations of CCL18 (0, 100, 250, 500 ng/mL) and CCK-8 proliferation assay showed no significant impact throughout 9 days (Figure 2B). Transwell assay showed that the migration ability of RA-FLS was significantly enhanced after stimulation of CCL18 (500 ng/mL) for 48 hours (Figure 2C). IL-6, CCL2 and MMP3 in culture supernatant were significantly increased after stimulation of CCL18 (500 ng/mL) for 48 hours (Figure 2D∼F). <h3>Conclusions</h3> Our preliminary results show overexpression of CCL18 in synovial tissue and fluid of RA patients can stimulate migration and activation of RA-FLS probably through the interaction between CCL18 and PITPNM3. <h3>Acknowledgement</h3> This work was supported by National Natural Science Foundation of China (81471597), Specialized Research Fund for the Doctoral Program of Higher Education (20130171110075) and Guangdong Natural Science Foundation (2014A030313074). <h3>Disclosure of Interest</h3> None declared