Related Experiment Video
Updated: Jul 29, 2026
Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics
Published on: June 7, 2018
Inhibition by manoalide of fMLP-stimulated elastase release from human neutrophils
G W De Vries1, L D Amdahl, K D Kramer
1Department of Biological Sciences, Allergan, Inc., Irvine, CA 92713.
Abstract:
Incubation of human polymorphonuclear leukocytes (PMNLs) with the chemotactic factor N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) resulted in a concentration-dependent release of the neutral protease elastase. This response was inhibited by pretreatment of the PMNLs with manoalide (IC50 approximately 0.08 microM). To understand the mechanism of this inhibition, we examined the effect of manoalide on the signal-transduction pathway believed to mediate fMLP stimulation. We observed in fura-2 loaded cells that pretreatment with manoalide blocked fMLP-induced increases in cytosolic free-calcium (IC50 approximately 0.15 microM). However, manoalide had no effect on inositol 1,4,5-trisphosphate (IP3) production at concentrations which completely inhibited the Ca2+ signal. Furthermore, manoalide was approximately 50-fold less potent as an inhibitor of phospholipase C activity in membrane preparations of PMNLs than as an inhibitor of calcium mobilization in whole cells. These data indicate that manoalide can block stimulation of human PMNLs through inhibition of Ca2+ mobilization, but that this occurs at a site beyond phospholipase C activation and inositol phosphate turnover.
Insights
Manoalide inhibits human white blood cell (PMNL) activation by blocking calcium (Ca2+) mobilization. This occurs downstream of phospholipase C and inositol phosphate signaling, offering a novel therapeutic target.
Area of Science:
- Immunology
- Cell Signaling
- Pharmacology
Background:
- Human polymorphonuclear leukocytes (PMNLs) release elastase upon stimulation with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP).
- This fMLP-induced response is mediated by intracellular signaling pathways, including calcium mobilization.
Purpose of the Study:
- To elucidate the mechanism by which manoalide inhibits fMLP-induced PMNL activation.
- To investigate the effect of manoalide on the signal-transduction pathway of fMLP stimulation.
Main Methods:
- Human PMNLs were incubated with fMLP and varying concentrations of manoalide.
- Calcium (Ca2+) mobilization was measured in fura-2 loaded cells.
- Inositol 1,4,5-trisphosphate (IP3) production and phospholipase C activity were assessed.
Main Results:
- Manoalide inhibited fMLP-induced elastase release and Ca2+ mobilization in a dose-dependent manner.
- Manoalide did not affect IP3 production or phospholipase C activity at effective concentrations.
- The IC50 for manoalide inhibition of Ca2+ mobilization was significantly lower than its effect on phospholipase C.
Conclusions:
- Manoalide inhibits human PMNL stimulation by blocking Ca2+ mobilization.
- The inhibitory site of manoalide is downstream of phospholipase C activation and IP3 turnover.
- Manoalide represents a potential therapeutic agent targeting PMNL activation pathways.
