Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Aminopeptidase N/CD13 is a metalloproteinase expressed on the surface of fibroblast like synoviocytes (FLS). It is found in soluble form in serum and rheumatoid arthritis (RA) synovial fluid (SF). We have shown that CD13 induces T cell migration and FLS proliferation. In this study, we determine the role of CD13 in monocyte (MN) migration, signaling mechanisms involved and its role in acute inflammatory arthritis.
Methods: To examine the functional significance of CD13, we performed an in vitro migration assay using normal human MNs and U937 cells. We examined the signaling mechanisms involved in CD13-induced MN migration using signaling inhibitors and pertussis toxin (PT). To assess whether its enzymatic activity contributes to CD13-induced phosphorylation of signaling molecules and MN migration, we generated enzymatically inactive (mutant CD13) and active (WT CD13). MN chemotaxis was performed with CD13-depleted RA SF supplemented with mutant or WT CD13. To examine the role of CD13 in RA, MN migration was performed with CD13- and sham-depleted RA SF. To determine the crosstalk between CD13-induced signaling intermediates, RA FLS, MN, and U937 cells were treated with or without signaling inhibitors or PT before CD13 stimulation. To test the arthritogenicity of CD13 in vivo, CD13 or PBS was injected into mouse knees. Joint circumference was measured at 0 and 24h. Pro-inflammatory mediators in knee joint homogenates were measured by ELISA.
Results: We found that CD13 is a potent chemoattractant for MNs and U937 cells. Chemotaxis was significantly decreased by inhibitors of Erk1/2, Src, NFκB, and Jnk, and was strongly inhibited by PT, a G-protein coupled receptor (GPCR) inhibitor, indicating the role of these molecules in CD13-mediated MN migration. CD13-depleted RA SF induced significantly less MN migration than sham-depleted, while addition of mutant or WT CD13 to CD13-depleted RA SF restored MN migration similar to the sham-depleted. CD13 and WT and mutant CD13 had similar effects in activating signaling molecule phosphorylation, indicating that enzymatic activity had no effect on these CD13 functions. CD13 induced Erk1/2, Src, NFkB and Jnk phosphorylation in MNs and U937 cells. In RA FLS, phosphorylation of Erk1/2, Src, and NFκB showed similar results, but there was no phosphorylation of Jnk. We found that the GPCR inhibitor, PT, decreased CD13-stimulated phospho-Erk1/2 in MNs, U937, and RA FLS, suggesting that GPCRs are upstream in CD13-mediated signaling pathways. In an acute model of inflammation, there was a significant increase in the knee circumference in mice injected with CD13 compared to PBS, indicating that CD13 contributes to acute inflammatory arthritis. MCP-1/CCL2 and IL-1β were significantly higher in CD13-injected knee joint homogenates, while TNFα and IL-6 were not.
Conclusion: CD13 is strongly chemotactic for MNs and U937 cells. CD13 contributes to MN migration independent of its chemotactic activity. CD13 induces MN migration through Erk1/2, Src, NFkB, Jnk, and GPCR. CD13 plays an important role in RA SF mediated MN migration. CD13 contributes to acute inflammatory arthritis by increasing MCP-1/CCL2 and IL-1β. CD13 may be a potential therapeutic target in RA.
To cite this abstract in AMA style:Du Y, Stinson WA, Campbell P, Morgan R, Lepore N, Cealey E, Hervoso J, Cui H, Fox D, Amin MA. Aminopeptidase N/CD13 Induces Monocyte Migration in Vitro and In Vivo and Signals through GPCR, Erk1/2, Jnk, Src, and NFκB [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/aminopeptidase-ncd13-induces-monocyte-migration-in-vitro-and-in-vivo-and-signals-through-gpcr-erk12-jnk-src-and-nf%ce%bab/. Accessed October 23, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/aminopeptidase-ncd13-induces-monocyte-migration-in-vitro-and-in-vivo-and-signals-through-gpcr-erk12-jnk-src-and-nf%ce%bab/