134 Exp. Mol. Med. Vol. 44(2), 130-137, 2012 duced neutrophil chemotaxis. Preincubation of human neutrophils with PTX (1 μg/ml) for 4 h caused complete inhibition of peptides-induced neutrophil chemotaxis (Figure 4D-F). The results indicate that GMMWAI, MMHWAM, and MMHWFM stimulated human neutrophil chemotaxis via PTX- sensitive G-protein(s). MMHWAM and MMHWFM but not GMMWAI stimulate superoxide anion production from human neutrophils In this study, we found that the three novel pep- tides stimulated not only intracellular Ca2+ increase but also chemotactic migration via PTX-sensitive G-protein(s). Neutrophils perform an important role in the innate immune response by producing a large amount of superoxide anion after stimulation with extracellular stimuli (Dahlgren and Karlsson, 1999). Superoxide anion is a well-known method of killing invading pathogens employed by human neutrophils (Rossi, 1986; Segal and Abo, 1993; Dahlgren and Karlsson, 1999). Here, we also tested the effects of the three novel peptides on the generation of superoxide anion from human neutrophils. Stimulation of human neutrophils with various concentrations of GMMWAI failed to induce superoxide anion production (Figure 5A). However, the other two novel peptides (MMHWAM and MMHWFM) strongly increased superoxide anion production from human neutrophils (Figures 5B and 5C). Novel peptides stimulate formyl peptide receptor (FPR)1 or FPR2 The three peptides showed similar effects on human neutrophils, in terms of Ca2+ increase and Figure 5. Effects of peptides on superoxide anion production in human neutrophils. Human neutrophils were stimulated with various concentrations of GMMWAI, MMHWAM, or MMHWFM, and the amount of generated superoxide was measured using cytochrome c reduction assay. The data are pre- sented as mean ± S.E. of three independent experiments, each performed in duplicate. *P ＜ 0.01 versus vehicle treatment. Figure 6. Role of FPR1 or FPR2 in novel peptide-induced Ca2+ increase. Isolated human neutrophils were in- cubated in the presence or absence of 10 μM CsH or WRW4 prior to Ca2+ measurement using 5 μM GMMWAI (A), 5 μM MMHWAM (B), or 5 μM MMHWFM (C). Vector- (D), FPR1- (E), or FPR2- (F) expressing RBL-2H3 cells (1 × 106 cells/ml of serum-free RPMI 1640 medium) were stimulated with 5 μM GMMWAI, 5 μM MMHWAM, or 5 μM MMHWFM. The results represent one of two in- dependent experiments.