Background/Purpose: This study was conducted to determine the expression pattern and function of CCL28 and its corresponding receptor CCR10 in the pathogenesis of rheumatoid arthritis (RA).

Methods: Expression of CCL28 and CCR10 was examined in RA compared to osteoarthritis (OA) and normal (NL) synovial tissue (ST) and/or fluid (SF) by histological studies and real-time RT-PCR. Next, the factors modulating CCL28 and CCR10 expression were identified in RA peripheral blood (PB) in vitro differentiated macrophages and endothelial cells by real-time RT-PCR. Based on the elevated expression of CCR10 on RA blood vessels, significance of CCL28 expressed in RA SF was evaluated on human endothelial progenitor cell (EPC) migration and tube formation. Finally, the mechanism by which CCL28 mediates angiogenesis was determined in knockdown CCR10 endothelial cells by Western blot analysis, endothelial chemotaxis and tube formation.

Results: We demonstrate that expression of CCL28 and CCR10 are markedly higher in RA and OA ST lining macrophages and sublining endothelial cells compared to NL ST. Consistently, comparable levels of CCL28 were expressed in RA and OA SF which were 4 to 33 fold greater than those detected in RA and NL serum. Since both CCL28 and CCR10 are mainly expressed in RA myeloid and endothelial cells, we asked whether their expression levels are regulated in a similar manner in these cells. Interestingly we found that expression of CCL28 and CCR10 was very responsive to stimulation in RA PB in vitro differentiated macrophages and was similarly enhanced by TNF-a (8-18 folds), IL-1b (6-32 folds) or IL-6 (12-18 folds) treatment. In contrast, concentrations of CCL28 and CCR10 were differentially regulated in endothelial cells. We show that while in endothelial cells, IL-1b (5 fold) or IL-17 (3 fold) were responsible for increasing CCL28 mRNA levels, CCR10 (11 fold) expression levels were modulated by TNF-a stimulation. We uncovered that CCL28 can strongly attract endothelial cells starting at 0.1 ng/ml, indicating that CCL28 (up to 3300 pg/ml expressed in RA SF) can contribute to migration of endothelial cells at a physiologically relevant concentration. We further document that ligation of SF CCL28 to endothelial CCR10 is involved in RA angiogenesis, as neutralization of CCL28 in RA SF or blockade of CCR10 on human EPCs significantly reduce RA SF induced endothelial migration and tube formation. To determine the mechanism by which CCL28 promotes RA angiogenesis, endothelial cells stimulated with CCL28 were examined for activation of MAPK and AKT pathways. We found that while ERK was phosphorylated by CCL28 stimulation in endothelial cells, JNK, p38 and AKT pathways were unaffected by this process. We further show that knockdown of endothelial CCR10 significantly reduces CCL28 mediated ERK phosphorylation as well as endothelial migration and tube formation compared to the control siRNA cells.

Conclusion: We found that CCL28 and CCR10 are coexpressed in RA ST blood vessels and as a result, ligation of RA SF CCL28 to endothelial CCR10 facilitates endothelial cell migration and tube formation through activation of ERK pathway. This study identifies for the first time the presence and function of CCL28 as a novel mediator of RA angiogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterizing-the-expression-and-function-of-ccl28-and-its-corresponding-receptor-ccr10-in-the-pathogenesis-of-ra/