Background/Purpose: CD6 is an important marker and regulator of T cells and interacts with its known ligand, CD166. Our group has previously shown that interferon gamma (IFNγ)-inducible antigen recognized by monoclonal antibody 3A11 is a new CD6 ligand distinct from CD166. And, it has recently been discovered that CD318 is the new CD6 ligand recognized by 3A11. CD318 is a cell surface protein widely expressed on epithelial tissues and tumor cells as an important regulator of cell adhesion through phosphorylation of its intracellular tyrosine domain. We currently examined CD318 expression on synovial tissues (STs) and soluble CD318 concentrations in synovial fluids (SFs) of rheumatoid arthritis (RA) patients. In addition, we investigated its potential roles in recruitment and retention of T cells in the RA joints. We hypothesize that CD6 on T cells interacts with CD318 on synovial fibroblasts and may lead to activation of either or both cellular subsets to induce proinflammatory responses in RA.

Methods: To determine the localization of CD318 in the RA joints, normal (NL), RA, and osteoarthritis (OA) ST sections were stained via both chromogenic and fluorescent immunohistochemistry using 3A11. To quantify the CD318 levels, STs were homogenized and measured for CD318 using ELISA. Additionally, to examine whether CD318 is shed in a soluble form, NL and RA peripheral blood sera and RA, OA, and juvenile idiopathic arthritis (JIA) SFs were measured for CD318 using ELISA. To confirm the expression of CD318 in vitro, synovial fibroblasts were treated with or without IFNγ and stained for CD318 for flow cytometric analysis. To investigate the role of CD318 in the interaction between synovial fibroblasts and T cells, adhesion assays were performed on synovial fibroblasts stimulated with or without IFNγ in the presence or absence of neutralizing antibodies to CD318 and/or CD166. To further characterize the function of CD318, T cell migration assay was performed in a modified 48-well Boyden chamber system.

Results: Immunohistochemistry revealed robust staining in the RA STs, specifically on the synovial fibroblasts and endothelial cells, greater in RA than in NL and OA STs. ELISA showed elevated levels of CD318 in the RA ST homogenates compared to that of NL and OA ST homogenates. ELISA for SFs also showed that soluble CD318 is selectively and significantly elevated in the SF from patients with RA and JIA. Flow cytometry showed upregulation of CD318 on synovial fibroblast lines upon IFNγ treatment. In adhesion assays, without IFNγ treatment, only CD166 was functionally involved, consistent with low expression of CD318 on these cells. In contrast, with IFNγ treatment, both CD318 and CD166 were functionally involved, and adhesion was substantially diminished only by antibodies to both CD166 and CD318. T cell migration assay confirmed CD318 as a chemoattractant at concentrations corresponding to the levels seen in RA sera and SFs.

Conclusion: CD318 is highly elevated in the RA joints and involved in T cell adhesion to synovial fibroblasts. Soluble CD318 is a T cell chemoattractant and its levels are similarly elevated in the SFs of RA and JIA patients. CD318 is a potential new biomarker or target for the diagnosis and treatment of RA and other inflammatory arthritides.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/synovial-fibroblast-cd318-expression-mediates-t-cell-adhesion-and-migration-in-rheumatoid-arthritis/