chemotactic migration via PTX-sensitive G-pro- tein(s) (Figure 2F and data not shown). Formyl peptide receptors are representative chemoattrac- tant receptors in human neutrophils (Ye et al., 2009). Here, we attempted to determine whether or not the three peptides acted through FPR1 and related receptors. For this purpose, we used FPR1 antagonist (CsH) (de Paulis et al., 1996) and FPR2 antagonist (WRW4) (Bae et al., 2004). As shown in Figures 6A and 6C, GMMWAI- and MMHWFM-in- duced Ca2+ increases were totally inhibited by CsH 4 but not by WRW . However, MMHWAM-induced Ca2+ increase was completely blocked by WRW4 but not by CsH (Figure 6B). These results suggest that GMMWAI and MMHWFM stimulated Ca2+ increases via FPR1 but not FPR2. On the other hand, MMHWAM stimulated a Ca2+ increase via FPR2 but not FPR1. We also used vector, FPR1-, or FPR2-expressing RBL-2H3 cells as previously reported (Lee et al., 2008). As shown in Figure 6E, stimulation of FPR1-expressing RBL-2H3 cells with the two novel peptides (GMMWAI and MMHWFM) elicited a dramatic increase in intracellular Ca2+. However, the two peptides did not induce an intracellular Ca2+ increase in vector- or FPR2- expressing RBL-2H3 cells (Figures 6D and 6F). These results strongly indicate that the two pep- tides (GMMWAI and MMHWFM) stimulated FPR1 but not FPR2, resulting in an increase in Ca2+. For MMHWAM, Ca2+ increase was observed in FPR2- expressing RBL-2H3 cells but not in FPR1-ex- pressing RBL-2H3 cells (Figure 6E). The result indicates that MMHWAM acted through FPR2, increasing intracellular Ca2+. Discussion Since neutrophils perform important roles in early defense against invading pathogens and other harmful agents (Borregaard, 2010; Kumar and Sharma, 2010), the identification of agonists that enhance neutrophil function is of paramount importance. Here, we screened hexapeptide com- binatorial libraries containing more than 47 million different peptide sequences, and we identified three novel hexapeptides (GMMWAI, MMHWAM, and MMHWFM) that stimulate intracellular Ca2+ increase in human neutrophils. GMMWAI and MMHWFM were shown to have selectivity on FPR1 but not on FPR2. In the case of MMHWAM, the peptide showed selectivity on FPR2 but not on FPR1. Specific ligands are recognized by specific receptors, and this specificity results from a com- bination of subtle conformational and amino acid Novel neutrophil-activating peptides 135 sequence differences. Previously some synthetic hexapeptides, WKYMVm, MFYLPM, HFYLPM, and HFYLPm, which act at two fMLF receptor subtypes have been described (Seo et al., 1997; Bae et al., 2001). In our previous reports, we demonstrated that the Y in the 3rd position of the hexapeptide and the M in the 6th position are important for stimula- tion of FPR family proteins (Bae et al., 2001). Here, we found that other peptides having different amino acids at the 3rd and 6th positions, such as H/M (for 3rd position) and I (for 6th position), showed stimulating activity on FPR family proteins. These results suggest that FPR1 has a broad spectrum of ligand recognition capacity. On the receptor speci- ficity of MMHWAM and MMHWFM, we demon- strated that MMHWFM and MMHWAM acted on FPR1 and FPR2, respectively (Figures 6E and 6F). The result indicates that the 5th amino acid is important for the recognition of the peptides by FPR1 or FPR2. Since GMMWAI, MMHWAM, and MMHWFM were identified by screening artificially synthesized peptides, we looked for sequence similarities between the novel peptides and known proteins by searching databases (SWISS-PROT and TrEMBL), and found that several bacterial proteins such as aminoacyltransferase femB contained the MMHWFM sequence. At this time, we cannot attach any significance to this sequence homology between our novel peptides and bacterial proteins. However, since FPRs recognize certain patterns derived from pathogens, the information that our peptides carry certain bacterial sequences may provide us useful insights into understanding the pattern involved in the recognition of pathogens. Even though various chemokines and chemo- attractants have been identified, only a few short peptides acting on human leukocytes have been known until now. The previously reported peptides WKYMVm (Seo et al., 1997) and fMLF (Spilberg et al., 1981) have been useful tools for studying neutrophil activation. Here, we demonstrated that the novel peptides GMMWAI, MMHWAM, and MMHWFM stimulate human neutrophils by acting on FPR1 or FPR2 (Figure 6). Therefore, these three peptides can also be used as agonists to study neutrophil activation and signaling. Methods Materials Peripheral blood mononuclear cell (PBMC) separation me- dium (Histopaque-1077) and cytochrome c were pur- chased from Sigma (St. Louis, MO). Fura-2 pentaacetoxy- methylester (fura-2/AM) was purchased from Molecular