Background/Purpose:   RANTES/CCL5 (RANTES) is a C-C chemokine that binds to its receptor (CCR5) and initiates inflammatory processes in rheumatoid arthritis (RA) by facilitating leukocyte infiltration. Syndecans (also known as heparin sulfate proteoglycans, HSPGs) modulate chemokine interaction with the endothelium thereby facilitating leukocyte infiltration. However, the role of RANTES/CCL5 beyond its chemotactic activity and the role of syndecans in RANTES/CCL5 induced matrix metalloproteinase (MMP)-1 and MMP-13 expression is not yet studied. The present study was carried out to determine the role of syndecans on the RANTES/CCL5 induced MMP-1 and MMP-13 expression in human rheumatoid arthritis synovial fibroblasts (RASFs).

Methods:   Human RASFs and normal SFs (NLSFs) were isolated from de-identified RA synovial tissues and healthy synovial tissues, respectively, under the IRB approved protocol. Basal cell lysates were prepared to study the expression of syndecan-1, -2, -3, and -4) was studied using qRT-PCR and Western immunoblotting methods. RASFs were treated with RANTES/CCL5 (20-100 ng/ml), IL-1β (10 ng/ml), or TNF-α (20 ng/ml) for 24 h alone or in presence of a known HSPG inhibitor, Heparinase III (0.5 U/ml). Effect of RANTES/CCL5 on MMP-1 and MMP-13 expression was evaluated using qRT-PCR and ELISA methods. Conditioned media was collected and concentrated for to determine MMP-1 and MMP-13 expression using Western immunoblotting. p<0.05 was considered significant.

Results:   Our results showed that RANTES/CCL5 significantly induced MMP-1 and MMP-13 mRNA and protein expression (p<0.05). Since syndecans influence inflammatory processes by interacting with chemokines, we compared the basal expression levels of syndecans in NLSFs and RASFs. Our qRT-PCR results showed that the expression of syndecan-2 (~320%) and syndecan-4 (~80%) was significantly higher in RASFs when compared to NLSFs (p<0.05). No significant changes were observed in syndecan-1 and syndecan-3 expression in RASFs compared to NLSFs. Furthermore, Western immunoblotting results confirmed that RASFs showed an increase in expression of syndecan-2 (~300 %) and syndecan-4 (~290 %) with (p<0.05) with no significant change in syndecan-1 and syndecan-3 expression compared to NLSFs. In addition, further stimulation of RASFs with RANTES/CCL5, IL-1β, or TNF-α for 24 h resulted in a selective induction in syndecan-2 and syndecan-4 expression (p<0.01) compared to the non-stimulated control. RANTES/CCL5, IL-1β, or TNF-α stimulation did not alter syndecan-1 and syndecan-3 expression in RASFs. To study whether RANTES/CCL5 induced MMP-1 and MMP-13 expression is modulated by syndecans in RASFs, we evaluated the effect of Heparinase III (HSPG inhibitor) on the ability of RANTES/CCL5 to induce MMP-1 and MMP-13 expression in RASFs. Analysis of the conditioned media showed that pretreatment with Heparinase III significantly reduced the ability of RANTES/CCL5 to induce MMP-1 and MMP-13 protein expression in RASFs (p<0.05).

Conclusion:   Human RASFs express higher levels of syndecan-2 and syndecan-4 compared to NLSFs. In general, syndecans may also contribute to the process of tissue destruction in RA by mediating RANTES/CCL5-induced MMP-1 and MMP-13 expression in RASFs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/syndecans-mediate-rantesccl5-induced-mmp-1-and-mmp-13-expression-in-rheumatoid-arthritis-synovial-fibroblasts/