Novel neutrophil-activating peptides 133 Figure 3. Cell specificity of the peptides. Prepared human primary monocytes, neutrophils, NIH3T3, 3Y1, 3T3L1, and PC12 cells were loaded with fura-2 and stimulated with three peptides at a concentration of 10 μM. The peak level of the Ca2+ increase was recorded. Data are presented are means ± S.E. from at least three independent experiments. Figure 4. Chemotactic effects of the peptides. Assays were performed using a modified Boyden chamber assay. Isolated human neutrophils (1 × 106 cells/ml of serum-free RPMI 1640) were added to the upper wells of a 96-well chemotaxis chamber, and migration across the polycarbonate membrane with 3-μm pore size was assessed after 1.5 h of incubation at 37oC (A-C). Human neutrophils were incubated with or without 1 μg/ml of PTX for 4 h. Then, the cells were used for chemotaxis assay using 5 μM GMMWAI, MMHWAM, or MMHWFM (D-F). The numbers of migrated cells were determined by counting them in high power field (400X). The data are presented as mean ± S.E. of three independent experiments, each performed in duplicate. *P # ＜ 0.05, compared with the value obtained from the vehicle control; P ＜ 0.05, significantly different from the -PTX control. (rat adrenal pheochromocytoma) cells showed no response to the three peptides in terms of Ca2+ increase (Figure 3). From these results, we suggest that the effects of the three peptides were specific to human leukocytes. Chemotactic effects of the novel peptides on human neutrophils We demonstrated that the novel peptides sti- mulated Ca2+ increase in human neutrophils in a PTX-sensitive manner. Since peptides-induced and chemoattractant-induced neutrophil activation are similar to each other, we investigated whether or not the peptides exhibit chemotactic activity on human neutrophils. GMMWAI, MMHWAM, and MMHWFM induced migration of human neutrophils within a 0.1-5 μM concentration, showing concen- tration response curves (Figures 4A-4C). This implies that the three novel peptides induced chemotaxis in human neutrophils. Various extracellular stimuli, including many chemoattractants, activate neutrophils via PTX- sensitive G-protein(s) (Badolato et al., 1995; Bae et al., 1999). Here, we also examined the involve- ment of PTX-sensitive G-protein(s) on peptide-in-