Objective: This study was undertaken to characterize the role of CC chemokines and their receptors in rat adjuvant-induced arthritis (AIA), a model for rheumatoid arthritis (RA). Furthermore, we investigated the signaling pathways associated with CC receptors as well as the cell type distribution of the receptors.
Methods: Using TaqMan real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry, we defined chemokine and chemokine receptor messenger RNA (mRNA) expression, CC chemokine receptor (CCR) protein activation during the disease course, CCR-associated signaling pathways, and immunopositive CCR5, phosphorylated signal transducer and activator of transcription 1 (p-STAT-1), and p-STAT-3 cells in rat AIA versus control joints.
Results: We showed significant up-regulation of CCR1, CCR2, CCR5, and macrophage inflammatory protein 1beta/CCL4 mRNA in AIA on post-adjuvant injection day 18, coincident with peak inflammation. Additionally, increases in tyrosine phosphorylation of CCR1 (days 14, 18, 21, and 24), CCR2 (days 14 and 18), and CCR5 (days 14, 18, and 21) were detected in AIA rats compared with control (nonarthritic) rats. JAK-1, STAT-1, and STAT-3 were associated with CCR1 and were highly tyrosine phosphorylated on days 14 and 18. Moreover, CCR2 was associated with JAK-2, STAT-1, and STAT-3 on day 18. The association of STAT-1 and STAT-3 with CCR5 on days 18 and 21 correlated with JAK-1 phosphorylation and binding on day 18. However, the activation of JNK was not associated with CCR5 activation in rat AIA. Immunohistochemical analysis demonstrated that the expression of CCR5, p-STAT-1, and p-STAT-3 was detected on synovial lining cells, macrophages, and endothelial cells in arthritic rat ankles on post-adjuvant injection day 18. While the majority of the CCR5 and p-STAT-1 immunostaining was on synovial lining cells and macrophages, p-STAT-3 was predominantly expressed on endothelial cells.
Conclusion: CCR1, CCR2, and CCR5 mRNA expression and tyrosine phosphorylation increased with peak inflammation in the AIA model. CCR1, CCR2, and CCR5 tyrosine phosphorylation are associated with the JAK/STAT-1/STAT-3 pathway at different stages of rat AIA, as well as with macrophage and endothelial cell infiltration. However, their signaling activation overlaps with peak inflammation. Up-regulation and activation of CCRs may play a role in macrophage and endothelial cell infiltration in rat AIA joints in addition to activating the associated signaling pathways. The downstream intermediate signaling proteins associated with CC receptors may be used as potential tools to control inflammation in RA.