Background 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.
Objectives To explore the expression of CCL18 in RA patients and its effect on RA fibroblast-like synoviocytes (FLS).
Methods CCL18 in serum and synovial fluid was measured by ELISA. Synovial tissue was obtained by closed-needle biopsy for H&E and immunohistochemistry staining, and FLS culture in vitro. 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.
Results (1) Serum CCL18 of 51 active RA patients was 112 ng/mL (IQR, 85–132), which was significantly higher than healthy controls (n=11, 64 ng/mL (IQR, 36–70), p<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 (p<0.001) and synovial fluid CCL18 of osteoarthritis patients (n=7, 33 ng/mL (IQR, 25–229), p=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 p=0.001, Figure 1B∼D). Double-immunocytofluorescent staining showed vimentin-positive RA-FLS in vitro 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).
Conclusions 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.
Acknowledgement 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).
Disclosure of Interest None declared