TLR-9 stimulation leads to the activation of plasminogen/plasmin and MMP-9 with the activation of NF-κB signaling in macrophages. (A) Gene expression of uPA, uPAR, and MMP9 in RAW264.7 cells stimulated with CpG for 3 hours was determined by quantitative PCR (qPCR) (n = 3 per group). (B) RAW264.7 cells pretreated with PBS or YO-2 were stimulated with CpG for 3 hours. Culture supernatants were assayed for plasmin (n = 3 per group). (C) RAW264.7 cells pretreated with PBS or YO-2 were stimulated with CpG for 8 hours. TNF-α levels were determined using ELISA (n = 3 per group). (D-F) RAW264.7 cells were stimulated with plasmin, CpG, or plasmin/CpG for 3 hours. The gene expression of (D) TNF-α, (E) CCL2, and (F) MMP-9 in cultured cells was determined by qPCR (n = 3 per group). (G) Western blot of phosphorylated-NF-κB p65 in RAW264.7 cells stimulated with plasmin, CpG, or plasmin/CpG for 30 minutes (n = 3 per group). (H) Proposed mechanism by which plasmin enhances the cytokine storm in MAS. Data represent mean ± SEM. *P < .05, **P < .01, ***P < .001, using 1-way ANOVA with the Tukey posttest or the unpaired, 2-tailed Student t test for significance. mRNA, messenger RNA; mTNF, membrane TNF-α; sTNF, soluble TNF-α.