Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Aminopeptidase N/CD13 Localization and Chemotaxis in Rheumatoid Arthritis In Vivo and In Vitro.
Morgan1, Rachel, Behbahani-Nejad1, Nilofar, Endres1, Judith, Fox2, David A.
University of Michigan, Ann Arbor, MI
Univ of Michigan Med Ctr, Ann Arbor, MI
Background/Purpose:
Aminopeptidase N (CD13, EC 3.4.11.2) is a metalloproteinase expressed on the cell surface of fibroblast like synoviocytes (FLS) that has also been found in soluble form in serum and synovial fluid. We have shown that CD13 is higher in amount and activity in RA synovial fluids compared to OA. It has been suggested but not proven that CD13 can act as a chemokine for T cells in RA. The goal of this study was to determine whether FLS contribute to the CD13 in synovial fluid, and if CD13 could play a role in bringing T cells to the RA joint.
Methods:
FLS were cultured in serum free media (Peprogrow) either alone, with cytokines, or with protease inhibitors. Cytokine activated T cells (Tck) were generated using IL-6, TNFa, and IL-2. The antibody 591.1D7.34, developed by our laboratory and another anti-CD13 mAb, WM15, were used to create a novel 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. T cell chemotaxis was measured using an under agarose system. MMP14 or control siRNA was transfected into FLS and the FLS were grown up in 20% serum media and switched to serum free conditions for 6 hours prior to harvesting. GFP siRNA was used to determine success of transfection. Exosomes were isolated by centrifugation at 110,000 xg from synovial fluid, RA FLS culture supernatant or serum. Following centrifugation over an Optiprep density gradient, exosomes were obtained at 1.110–1.163 gm/ml density.
Results:
Recombinant human CD13 was strongly chemotactic for cytokine activated T cells (Tck) over a range of concentrations with peak effect at 200ng/ml CD13 (3.85 chemotactic index, p=0.0031). Soluble CD13 was found in supernatants of FLS by ELISA (45.72±10.28 ng/ml) and enzymatic assay (497.96±167.27 nmoles of substrate cleaved per hour per ml). Among various protease inhibitors added to FLS cultures only GM6001 (a metalloprotease inhibitor) prevented release of CD13 into culture supernatants. Knockdown of MMP14 decreased FLS shedding of CD13 protein into the supernatant, as measured both by ELISA and enzymatic assay. We also found CD13 on exosomes in serum (19.16±0.64ng/ml), synovial fluid (66.55±4.68 ng/ml), and FLS culture supernatants (44.28±2.54ng/ml). In addition, the proinflammatory cytokines IFNg, TNFa, and IL-17 all upregulated expression of CD13 mRNA by FLS, but with distinct kinetics of protein upregulation and compartmentalization induced by each cytokine.
Conclusion:
CD13 is shed from the cell surface of FLS into culture supernatants and is found in synovial fluid. This process is mediated by one or more metalloproteases, including MMP14. CD13 is upregulated by proinflammatory cytokines that are commonly found in the RA joint. The combination of upregulation and shedding from FLS contributes to the high levels of CD13 in RA synovial fluid. CD13 induces chemotaxis of Tck, T cells that are similar to those found in the RA joint, at concentrations of CD13 detected in vivo in humans. Together this data demonstrates that CD13 could play an important role as a T cell chemoattractant, in a positive feedback loop that contributes to RA synovitis.
To cite this abstract, please use the following information:
Morgan, Rachel, Behbahani-Nejad, Nilofar, Endres, Judith, Fox, David A.; Aminopeptidase N/CD13 Localization and Chemotaxis in Rheumatoid Arthritis In Vivo and In Vitro. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :2572
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