Background/Purpose: Aminopeptidase N (CD13, EC 3.4.11.2) is a metalloproteinase expressed on the surface of fibroblast like synoviocytes (FLS) that is also found in soluble form in serum and synovial fluid.  We have shown that CD13 can be released from FLS in culture, and that CD13 is higher in amount and activity in Rheumatoid arthritis (RA) synovial fluids compared to osteoarthritis (OA).  Recombinant human CD13 (rhCD13) can act as a chemokine for cytokine activated T cells (Tck), and it has been previously suggested but not proven that CD13 may contribute to the T cell chemotactic activity of synovial fluid.  The goal of this study was to determine potential functions of CD13 in the RA joint by examining its effects on migration of T cells and FLS.

Methods: Tck were generated using IL-6, TNFα, and IL-2. Tck chemotaxis was measured using an under agarose system.  Synovial fluids were immunodepleted with 591.1D7.34 (an anti-CD13 antibody developed in our laboratory) bound to agarose beads.  FLS cell growth and migration were measured using an Incucyte (Essen Biosciences) imaging system.  Growth was measured by confluence and migration by relative wound density.  FLS were grown in 20% CMRL and switched to 10% media for assays.

Results: Pertussis toxin treatment of the Tcks prior to chemotaxis significantly reduced positive chemotaxis toward 200ng/ml rhCD13 either with a fibronectin coating (p=0.047) or without (p=0.00096), and also inhibited migration toward SDF/CXCL12 and TARC/CCL17 positive controls.  Immunodepletion of synovial fluids significantly reduced chemotaxis across an uncoated surface compared to mock depleted fluids, Chemotactic Index (CI=Ln # of cells toward the chemokine – Ln # of cells toward media control) was 0.56±0.21 versus -0.045±0.21 p=0.047 pre- versus post-immunodepletion.  Immunodepletion of CD13 reduced CI across a fibronectin coating, from 1.12±0.43 to 0.31±0.50, however it was not statistically significant.  Addition of 200ng/ml rhCD13 to the depleted synovial fluids partially restored the depleted activity.  Treatment of FLS with anti-CD13 mAbs 1D7 or WM15 (50ng/ml) or the chemical inhibitors, bestatin (100µM) or actinonin (10µM) significantly decreased FLS confluence at time points from 24-120hours.  CD13 inhibitors were also able to significantly decrease FLS migration in a wound healing assay.  Actinonin (10µM) and 1D7 (25ng/ml) both slowed migration so that wound healing was significantly lower at 48hr, while WM15 (25ng/ml) showed a significant difference by 24hr.  Bestatin, however, had no significant effect on FLS migration.  Addition of rhCD13 was not able to further increase cell growth or wound healing beyond FLS alone.

Conclusion: Together this data demonstrates multiple pathways by which CD13 may contribute to RA pathogenicity.  CD13 significantly contributes to the chemotactic ability of synovial fluids for a population of T cells similar to those found in the RA joint. This process is mediated through a G-protein coupled receptor.  Increased levels of CD13 in the RA joint may also contribute to synovial tissue hyperplasia by increasing FLS proliferation and migration.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/functions-of-aminopeptidase-ncd13-in-vitro-on-rheumatoid-arthritis-synovial-cells/