Lipopeptide Pam3Cys4 Synergizes N-Formyl-Met-Leu-Phe (Fmlp)-Induced Calcium Transients in Mouse Neutrophils.

N-Formyl-Met-Leu-Phe (fMLP), a mimic of N-formyl oligopeptides that are released from bacteria, is a potent leukocyte chemotactic factor. It induces intracellular calcium ([Ca2+](i)) transient that is important for various neutrophil biological functions, e. g., adhesion, ROS, and cytokine productions. Toll-like receptors (TLRs), an essential part of host innate immunity, regulate neutrophil activities, but their role in [Ca2+](i) signaling is less clear. In the present study, we examined the effect of several TLR ligands, including Pam3Cys4 (TLR1/2), lipopolysaccharide (LPS, TLR4), and lipoteichoic acid (LTA, TLR2/6), on calcium signaling and on the fMLP-induced [Ca2+](i) transients in mouse neutrophils loaded with Fura2/AM. We found that unlike fMLP, the three TLR ligands tested did not elicit any detectable Ca2+ flux. However, Pam3Cys4, but not LPS or LTA, markedly synergized the fMLP-induced [Ca2+](i)transients, and had no effect on the host component keratinocyte-derived cytokine (KC)-or C5a-induced calcium flux. The effect of Pam3Cys4 on the fMLP-induced [Ca2+](i) transients is by enhancing extracellular Ca2+ influx, not intracellular Ca 2thorn release. Surprisingly, deletion of TLR2 or MyD88 in neutrophils had no impact on the Pam3Cys4's effect, suggesting a TLR2-MyD88-independent mechanism. Finally, using the pan PKC activator and inhibitor, we demonstrated that PKC negatively regulated fMLP-induced [Ca2+](i) transients and that inhibition of PKC did not prohibit Pam3Cys4's synergistic effect on the fMLP-induced calcium influx. In conclusion, the present study identified a novel synergistic effect of Pam3Cys4 on fMLP-induced [Ca2+](i) transients, a process important for many neutrophil biological functions.