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Effect of increasing intravesicular pH on nitrite production and leishmanicidal activity of activated macrophages
Y Buchmüller-Rouiller1, S B Corradin, J Smith
1Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Abstract:
We examined the effect of bafilomycin A1 (BAF), an inhibitor of vacuolar-type H(+)-ATPases, on macrophages activation (measured as increased nitrite production and leishmanicidal activity) induced by interferon gamma alone or together with lipopolysaccharide or tumour necrosis factor alpha. BAF increased intravesicular pH and enhanced nitrite release by activated macrophages; however, the NO concentration necessary to kill parasites was higher in BAF-exposed than control macrophages, suggesting that microbicidal nitrogen derivatives were less active at alkaline pH. Antibody to tumour necrosis factor alpha inhibited BAF-induced nitrite production in interferon-activated cultures. To determine if enhanced NO synthesis was related to vesicular alkalinization, macrophages were incubated with the lysosomotropic bases NH4Cl and methylamine. These agents also increased intravesicular pH and nitrite production. Nitrite production was correlated with enhanced NO synthase activity in cytosolic extracts of the activated cells.
Insights
Bafilomycin A1 (BAF) enhances macrophage nitrite production but reduces parasite killing, indicating alkaline pH impairs microbicidal activity. This suggests vacuolar-type H(+)-ATPases are crucial for macrophage antimicrobial function.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophages are key immune cells involved in pathogen killing.
- Interferon gamma (IFN-γ) and other stimuli activate macrophages.
- Vacuolar-type H(+)-ATPases regulate intracellular pH and cellular functions.
Purpose of the Study:
- To investigate the impact of vacuolar-type H(+)-ATPase inhibition on macrophage activation.
- To determine how altered intracellular pH affects macrophage leishmanicidal activity.
- To explore the role of vesicular alkalinization in nitric oxide (NO) synthesis.
Main Methods:
- Macrophages were treated with bafilomycin A1 (BAF), an inhibitor of vacuolar-type H(+)-ATPases.
- Macrophage activation was induced by interferon gamma (IFN-γ) alone or with lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α).
- Intravesicular pH, nitrite production, and leishmanicidal activity were measured. NO synthase activity was assessed in cytosolic extracts.
Main Results:
- BAF treatment increased intravesicular pH and enhanced nitrite production in activated macrophages.
- Higher NO concentrations were required to kill parasites in BAF-exposed macrophages, suggesting reduced microbicidal activity at alkaline pH.
- Lysosomotropic bases (NH4Cl, methylamine) mimicked BAF's effects, increasing intravesicular pH and nitrite production.
- Nitrite production correlated with increased NO synthase activity.
Conclusions:
- Inhibition of vacuolar-type H(+)-ATPases enhances NO synthesis but impairs the microbicidal efficacy of activated macrophages.
- Alkaline intracellular pH reduces the effectiveness of NO-derived microbicidal agents.
- Vacuolar-type H(+)-ATPases play a critical role in maintaining the acidic environment necessary for optimal macrophage antimicrobial function.