Shahrara Shiva, Amin Mohammad A, Woods James M, Haines G Kenneth, Koch Alisa E
Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Arthritis Rheum. 2003 Dec;48(12):3568-83. doi: 10.1002/art.11344.
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.
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.
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.
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.
本研究旨在明确CC趋化因子及其受体在大鼠佐剂性关节炎（AIA）（类风湿关节炎（RA）的一种模型）中的作用。此外，我们还研究了与CC受体相关的信号通路以及受体的细胞类型分布。
我们运用TaqMan实时逆转录-聚合酶链反应、蛋白质免疫印迹分析和免疫组织化学，确定了趋化因子和趋化因子受体信使核糖核酸（mRNA）的表达、疾病过程中CC趋化因子受体（CCR）蛋白的激活、CCR相关信号通路，以及大鼠AIA关节与对照关节中免疫阳性的CCR5、磷酸化信号转导及转录激活因子1（p-STAT-1）和p-STAT-3细胞。
我们发现，在佐剂注射后第18天，AIA中CCR1、CCR2、CCR5和巨噬细胞炎性蛋白1β/CCL4 mRNA显著上调，与炎症高峰一致。此外，与对照（非关节炎）大鼠相比，在AIA大鼠中检测到CCR1（第14、18、21和24天）、CCR2（第14和18天）和CCR5（第14、18和21天）的酪氨酸磷酸化增加。JAK-1、STAT-1和STAT-3与CCR1相关，并在第14和18天高度酪氨酸磷酸化。此外，在第18天，CCR2与JAK-2、STAT-1和STAT-3相关。第18和21天，STAT-1和STAT-3与CCR5的关联与第18天JAK-1的磷酸化和结合相关。然而，在大鼠AIA中，JNK的激活与CCR5的激活无关。免疫组织化学分析表明，在佐剂注射后第18天，在关节炎大鼠踝关节的滑膜衬里细胞、巨噬细胞和内皮细胞上检测到CCR5、p-STAT-1和p-STAT-3的表达。虽然大多数CCR5和p-STAT-1免疫染色位于滑膜衬里细胞和巨噬细胞上，但p-STAT-3主要在内皮细胞上表达。
在AIA模型中，随着炎症高峰的出现，CCR1、CCR2和CCR5 mRNA表达及酪氨酸磷酸化增加。在大鼠AIA的不同阶段，CCR1、CCR2和CCR5酪氨酸磷酸化与JAK/STAT-1/STAT-3通路相关，也与巨噬细胞和内皮细胞浸润相关。然而，它们的信号激活与炎症高峰重叠。CCR的上调和激活可能在大鼠AIA关节的巨噬细胞和内皮细胞浸润中发挥作用，此外还激活相关信号通路。与CC受体相关的下游中间信号蛋白可能用作控制RA炎症的潜在工具。
