Human neutrophil stimulation by influenza virus: relationship of cytoplasmic pH changes to cell activation

Area of Science:

• Immunology
• Cellular Biology
• Virology

Background:

• Influenza A virus (IAV) is known to stimulate human neutrophils.
• Previous work demonstrated IAV activates neutrophils through phospholipase C.
• Cytoplasmic pH changes accompany IAV-induced neutrophil activation.

Purpose of the Study:

• To investigate the mechanisms underlying cytoplasmic acidification and alkalinization in IAV-stimulated human neutrophils.
• To determine the roles of Na(+)-H+ antiporter, cytosolic Ca2+ (Cai2+), and protein kinase C (PKC) in these pH responses.
• To elucidate the relationship between pH changes and other neutrophil activation markers like the respiratory burst.

Main Methods:

• Utilized the fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor cytoplasmic pH (pHi).
• Employed pertussis toxin (PT) to assess PT-sensitive pathways.
• Used the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) to investigate PKC involvement.
• Dissociated responses by analyzing specific inhibitors and ion flux independence.

Main Results:

• IAV stimulation leads to both cytoplasmic acidification and alkalinization in human neutrophils.
• These pH responses are not inhibited by pertussis toxin.
• Alkalinization is largely mediated by the Na(+)-H+ antiporter and likely by PKC, independent of Cai2+.
• Acidification is independent of Cai2+ and PKC, and can be dissociated from alkalinization.
• Both pHi responses are independent of the respiratory burst.

Conclusions:

• Cytoplasmic alkalinization and acidification represent two independently mediated responses in activated neutrophils.
• Alkalinization is ultimately linked to phospholipase activation and involves PKC.
• Acidification results from other, yet uncharacterized, neutrophil activities.
• These findings offer insights into the complex signaling networks governing neutrophil activation by IAV.