Abstract
OBJECTIVE: To investigate MRP8/MRP14-induced expression of inflammatory cytokines in mouse peritoneal macrophages and explore the mechanism. METHODS: Tumor necrosis factor-α (TNF-α), interferon-γ inducible protein 10 (IP-10), interleukin-2 (IL-2), IL-6, IL-5 and interferon-γ (IFN-γ) proteins in the culture supernatants of mouse peritoneal macrophages treated with recombinant MRP8/MRP14 were quantified using Luminex xMAP system. TNF-α, IP-10 and IL-6 levels were detected in the culture supernatants of the peritoneal macrophages after treatment with different domains of MRP14. Western blotting was used to detect the phosphorylation of p38 MAPK, JNK and ERK in the cells after MRP8/MRP14 treatment. The effects of p38 MAPK, JNK, ERK inhibitors, TLR4 or RAGE receptor antagonists on MRP8/MRP14-induced expressions of TNF-α, IP-10 and IL-6 were tested. RESULTS: MRP8/MRP14 significantly increased TNF-α, IP-10 and IL-6 levels in mouse peritoneal macrophages by 98.2, 378.6 and 6.3 folds (P<0.01), respectively, but did not obviously affect IL-2, IL-5 and IFN-α levels. MRP14 protein and its calcium binding motifs such as EF hand-1, EF hand-2, EF hand-1+2, but not CT terminal domain, all induced TNF-α, IP-10 and IL-6 expressions (P<0.01). Phosphorylation of p38 MAPK, JNK and ERK were detected by Western blotting in the cells at 1 h after MRP8/MRP14 stimulation and sustained to 2 h. Compared with MRP8/MRP14, SB203580 (p38 MAPK inhibitor) significantly inhibited TNF-α, IP-10 and IL-6 expression (P<0.05), and SP600125 (JNK inhibitor) inhibited the expression of TNF-α and IP-10 (P<0.05) but not IL-6, PD98059 and U0126 (ERK and MEK1/2 inhibitor) reduced IL-6 expression (P<0.05). TNF-α, IP-10 and IL-6 levels were inhibited by TAK242 (P<0.05); IL-6 level in the cells was also partially inhibited by RAGE neutralizing antibody (P<0.05). CONCLUSION: MRP8/MRP14 can induce the expression of TNF-α, IP-10 and IL-6 in mouse peritoneal macrophages. MRP14 protein, which contain calcium binding motifs, has the biological activity of inducing cytokine expression. TNF-α and IP-10 expressions are related with TLR4 and its downstream p38 MAPKs and JNK; IL-6 is regulated by both TLR4 and RAGE and their downstream p38 MAPKs and ERK.