Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Functional Expression of CD13/Aminopeptidase N by RA Synovial Cells In Vitro and In Vivo.
Morgan, Rachel, Endres, Judith, A. Fox, David
Aminopeptidase N (CD13, EC 126.96.36.199) is a metalloproteinase expressed on and secreted by myeloid cells and fibroblasts. Previously published data has documented increased CD13-related enzymatic activity in RA synovium compared to OA, and has proposed a role for CD13 as a T cell chemoattractant. The mechanisms whereby T cells become localized to the synovial lining and contribute to synovial inflammation and joint destruction remain incompletely understood. While CD13 has been identified on synovial fibroblasts (FLS), ELISA assay of CD13 in synovial fluid, serum or culture supernantants has not yet been reported. The purpose of this study was to critically examine CD13 expression and function in RA synovium.
Novel monoclonal antibodies (mAb) were developed from splenocytes of mice immunized with an RA FLS line that had been treated with IL-17. Hybridoma supernatants were screened by flow cytometry to detect cell surface structures upregulated by IL-17 but not TNF. This approach yielded one mAb, termed 591.1D7.34, which was used to immunoaffinity purify a protein that was identified as CD13 by amino acid sequencing. 1D7 and another anti-CD13 mAb, WM15, were then used to create a sandwich ELISA for CD13. CD13 enzymatic activity was measured in parallel by cleavage of L-Leucine 7-amido-4-methyl coumarin hydrochloride (L-leu-AMC) to release the fluorescent molecule AMC.
We detected substantial amounts of CD13 in synovial fluids and synovial fibroblast cell lysates, sera and culture supernatants by ELISA, with a significant (p<0.0001) increase in CD13 in RA synovial fluids (1191±121.6 ng/ml) when compared to OA (646.1±45.64 ng/ml). Similar to previously published data we also found a significant difference in CD13-type enzymatic activity between RA and OA synovial fluids (3402±239.6 nmoles of substrate cleaved per hour per ml of synovial fluid versus 2250±93.18 nM/hr, p<0.0001). This activity was depleted by 1D7 immunoprecipitation. Recombinant CD13 was chemotactic for T cells.
CD13 is released by synovial fibroblasts into the synovial fluid and its expression is upregulated on FLS by IL-17. These findings suggest a mechanism by which IL-17 may enhance synovial inflammation in RA independent of TNFa. We have shown that the CD13 protein is present in significant amounts and accounts for most of the L-leu-AMC cleavage activity in synovial fluids. These data support the concept of CD13 as a multifunctional pro-inflammatory mediator that can serve as a T cell chemoattractant in the RA joint.
To cite this abstract, please use the following information:
Morgan, Rachel, Endres, Judith, A. Fox, David; Functional Expression of CD13/Aminopeptidase N by RA Synovial Cells In Vitro and In Vivo. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1520