Session Type: Abstract Submissions (ACR)
Background/Purpose: We previously showed that citrullinated epithelial-derived neutrophil-activating peptide 78/CXCL5 (citENA-78/CXCL5) was significantly higher in rheumatoid arthritis (RA) synovial fluids (SFs) compared to osteoarthritis (OA) and other inflammatory rheumatic diseases (OD) SFs, and its concentration correlates with RA disease activity. Furthermore, we have shown that citrullination of ENA-78/CXCL5 results in conversion from a non-monocyte recruiting to a monocyte recruiting chemokine. We now examine the cellular receptors utilized by citENA-78/CXCL5 to induce monocyte migration in vitro.
Methods: Citrullination of ENA-78/CXCL5 was verified by mass spectrometry and Western blot analysis with anti-modified citrulline antibody (AMC). We initially performed polymorphonuclear neutrophil(PMN) chemotaxis assays to citENA-78/CXCL5 using a 48-well Boyden chamber system to determine if citrullination altered recruitment activity of ENA-78/CXCL5 for PMNs. Next, we performed monocyte chemotaxis assays to determine whether citENA-78/CXCL5 induced monocyte chemotaxis. Finally, we investigated whether cell recruitment could be inhibited by pertussis toxin (PTX), which is a G protein-coupled receptor antagonist, or with anti-CXCR1 and/or anti-CXCR2 blocking antibodies. For in vivo analysis, C57BL/6 mice were injected intra-articularly (i.a.) with non-cit or citENA-78/CXCL5 to induce inflammation. Immunofluorescence (IF) staining was performed on injected joint tissues using anti-F4/80 antibody, which is specific for monocytes/macrophages, to determine the number of recruited monocytes/macrophages to mouse joints.
Results: citENA-78/CXCL5 was recognized by AMC, while non-citENA-78/CXCL5 was not. Chemotaxis assays showed that citENA-78/CXCL5 induced less PMN migration compared to non-citENA-78/CXCL5, indicating that the binding affinity for PMN chemokine receptors was altered by citrullination of ENA-78/CXCL5. Monocyte chemotaxis assays showed that citrullinated ENA-78/CXCL5 significantly enhanced monocyte migration, while non-citENA-78/CXCL5 did not. PTX completely inhibited citENA-78/CXCL5-induced monocyte chemotaxis, indicating that citENA-78/CXCL5 is binding to a G-protein coupled receptor. Anti-CXCR1 and 2 antibodies both partially reduced the monocyte chemotaxis, and a combination of anti-CXCR1 and 2 antibodies completely inhibited monocyte chemotaxis. Lastly, IF staining showed that citENA-78/CXCL5 recruited more F4/80 positive monocytes/macrophages into mouse knee joints compared to non-citENA-78/CXCL5.
Conclusion: Our results show that citENA-78 induces monocyte migration via CXCR1 and CXCR2, and indicate that citrullination may increase the affinity of ENA-78/CXCL5 for CXCR1. Our data also shows that citENA-78/CXCL5 is a potent recruitment factor for monocytes, but a poor recruitment factor for PMNs. citENA-78/CXCL5 also induced more macrophage accumulation in synovium when injected into mouse knee joints compared to non-citENA-78/CXCL5. These results show that citrullination of ENA-78/CXCL5 can alter its receptor affinity and cellular recruitment properties.
A. E. Koch,
Eli Lilly and Company: I am currently employed by Eli Lilly with an adjunct appointment at University of Michigan. My work at Lilly is not relevant to the content of the abstract.,
R. A. Ohara,
P. L. Campbell,
M. A. Amin,
D. A. Fox,
J. H. Ruth,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrullination-of-ena-78cxcl5-changes-its-receptor-affinity-from-cxcr2-to-cxcr1-and-induces-monocyte-migration/