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Effects of wortmannin on human neutrophil respiratory burst and phagocytosis
P Santoro1, C Cacciapuoti, A Palumbo
1Department of Pediatrics, Università degli Studi di Napoli Federico II, Italy.
Abstract:
Modulation of neutrophil response to naturally occurring stimuli is important to avoid host tissue injure. Both soluble and particulate stimuli may induce superoxide anion generation in human polymorphonuclear leukocytes. Recently wortannin has been shown to inhibit the N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced activation of respiratory burst via phosphatidylinositol 3-kinase. However no data are available about the effect of the inhibitor on the respiratory burst induced by a particulate stimulus. In this paper we studied the effect of wortmannin on E. coli induced respiratory burst and phagocytosis by flow cytometry, which allows the quantitation of both H2O2 production and ingested bacteria in whole blood samples without the need of purification and concomitant manipulation of the cells. The effects of worthmannin on fMLP-induced chemotaxis was also examined by the under agarose method. Neither the E. coli nor the fMLP-induced responses were blocked by wortmannin, suggesting that PI 3-kinase activity is not required to activate these neutrophil functions. Since it is known that the respiratory burst elicited by fMLP is blocked by wortmannin, our results suggest that the generation of oxygen radicals is controlled via different signal transduction pathways, depending on the agonist used.
Insights
Wortmannin does not inhibit neutrophil responses to E. coli, suggesting different signaling pathways for respiratory burst activation by particulate versus soluble stimuli. Phosphatidylinositol 3-kinase is not essential for these neutrophil functions.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neutrophil responses to stimuli are crucial for host defense but must be modulated to prevent tissue damage.
- Soluble and particulate stimuli can trigger superoxide anion generation in human polymorphonuclear leukocytes.
- Wortannin, a phosphatidylinositol 3-kinase inhibitor, previously showed inhibition of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst.
Purpose of the Study:
- To investigate the effect of wortmannin on the respiratory burst and phagocytosis induced by the particulate stimulus E. coli.
- To examine the impact of wortmannin on fMLP-induced neutrophil chemotaxis.
- To determine if phosphatidylinositol 3-kinase (PI 3-kinase) activity is essential for neutrophil activation by particulate stimuli.
Main Methods:
- Flow cytometry was used to quantify hydrogen peroxide (H2O2) production and ingested bacteria in whole blood samples.
- The under agarose method was employed to assess fMLP-induced chemotaxis.
- Wortmannin was used as an inhibitor of PI 3-kinase.
Main Results:
- Wortmannin did not block the respiratory burst or phagocytosis induced by E. coli.
- Wortmannin also failed to inhibit fMLP-induced chemotaxis.
- These findings indicate that PI 3-kinase activity is not required for these specific neutrophil functions.
Conclusions:
- Neutrophil activation pathways differ depending on the stimulus used.
- The generation of oxygen radicals by neutrophils may be controlled by distinct signal transduction pathways for soluble versus particulate agonists.
- PI 3-kinase is not universally required for all neutrophil functions, including responses to particulate stimuli.