Presentation: IL-17 Induces MCP-1 Production Through PI3K and ERK Pathways in Peripheral Blood Differentiated Macrophages (2007)

139 IL-17 Induces MCP-1 Production Through PI3K and ERK Pathways in Peripheral Blood Differentiated Macrophages

Introduction: Macrophages, derived from circulating monocytes are the key producers of chemotactic cytokines in the rheumatoid arthritis (RA) synovium. IL-17 is a proinflammatory cytokine that is produced by RA synovial tissue (ST) and high levels have been detected in RA synovial fluid (SF). IL-17 promotes its proinflammatory effect by synergizing with TNF-α and IL-1β in producing macrophage inflammatory protein (MIP)-3alpha/CCL20, IL-6 and IL-8 from RA ST fibroblasts.
Purpose: Since macrophages are a major source of chemokines in RA, these studies were performed to examine the effect and mechanism by which IL-17 induces CC chemokines in macrophages.
Methods: Macrophages differentiated in vitro from peripheral blood monocytes were transfected with siRNA, dominant negative adenovirus or specific controls prior to treatment with IL-17. Results were obtained using real-time RT-PCR, ELISA and Western blot analysis.
Results: Our results demonstrate that IL-17 increases macrophage MCP-1/CCL2 mRNA at all time points examined (8 h time frame) with highest effect after 2h (30 fold increase) and 4h (20 fold increase) stimulation compared to baseline. Additionally, IL-17 increases MCP-1 protein levels by 2.5 fold compared to control (p<0.05). To examine the IL-17 activated signaling pathways, macrophages were stimulated with IL-17 from 0 to 120 min. Western blot results, employing phospho-specific antibodies, demonstrate that IL-17 activates p38 and AKT as early as 15 min, while the ERK pathway was activated at 60 min. To determine which signaling pathways are responsible for IL-17 induced MCP-1 production, macrophages were treated with chemical inhibitors (1h prior to treatment) or siRNA (48h prior to stimulation) for ERK, p38 and PI3K prior to IL-17 stimulation. Inhibition of ERK (50%-60% decrease) and PI3K (70%-90% decrease) pathways reduced IL-17-induced MCP-1 secretion. However, inhibition of p38 (1.5 fold) increased IL-17-induced MCP-1 production in macrophages. Next we asked whether PI3K signaling regulates IL-17-induced MAPK activity. For this purpose cells were pretreated with either chemical inhibitor, dominant negative AKT expressing adenoviral vector or appropriate controls prior to IL-17 stimulation. Results obtained demonstrated that inhibition of AKT activation suppressed IL-17-induced ERK phosphorylation however it had no effect on IL-17-mediated p38 activation suggesting that IL-17 modulates ERK signaling via PI3K.
Conclusions: PI3K and its downstream target ERK, regulates IL-17 mediated MCP-1 production in macrophages and thereby indirectly induces cell recruitment into the RA joint. Thus targeting IL-17 may be important for reduction of inflammation and cell trafficking in patients with RA by suppressing monocyte chemotaxis.

 S. Shahrara, None; B. Shi, None; R. Pope, None.