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Nitrite production by stimulated human polymorphonuclear leukocytes supplemented with azide and catalase
1Department of Medicine, University of Washington School of Medicine, Seattle 98195.
Abstract:
The formation of nitric oxide by human phagocytes as measured by nitrite production is controversial. We report here that nitrite production by phorbol myristate acetate (PMA)-stimulated human polymorphonuclear leukocytes (PMN) is considerably increased by the addition of azide and a further increase occurs when catalase also is added. Nitrite production by the PMN-PMA-azide-catalase system is unaffected by superoxide dismutase or monomethylarginine but is markedly reduced by the substitution of chronic granulomatous disease for normal neutrophils. The stimulated neutrophils could be replaced by the H2O2-generating enzyme system glucose-glucose oxidase. These findings suggest that nitrite production does not, in this instance, reflect nitric oxide synthase activity by human neutrophils but rather the catalase-catalyzed conversion of azide to nitrite in the presence of H2O2 generated by the stimulated PMN.
Insights
Nitrite production in human neutrophils stimulated with PMA does not indicate nitric oxide synthase activity. Instead, it results from catalase converting azide to nitrite using hydrogen peroxide.
Area of Science:
- Immunology
- Biochemistry
- Cell Biology
Background:
- The role of nitric oxide (NO) production in human phagocytes, particularly polymorphonuclear leukocytes (PMN), is debated.
- Nitrite production is often used as a surrogate marker for NO synthesis.
Purpose of the Study:
- To investigate the source of nitrite production in phorbol myristate acetate (PMA)-stimulated human PMN.
- To determine if nitrite production reflects nitric oxide synthase (NOS) activity in this context.
Main Methods:
- Human PMN were stimulated with PMA.
- Nitrite production was measured under various conditions, including the addition of azide, catalase, superoxide dismutase (SOD), and monomethylarginine (MMA).
- Experiments utilized neutrophils from patients with chronic granulomatous disease (CGD) and a glucose-glucose oxidase system to generate hydrogen peroxide (H2O2).
Main Results:
- Nitrite production by PMA-stimulated PMN increased significantly with the addition of azide and further with catalase.
- Superoxide dismutase and monomethylarginine did not affect nitrite production.
- Replacing normal neutrophils with those from CGD patients markedly reduced nitrite production.
- The H2O2-generating system of glucose-glucose oxidase could substitute for stimulated neutrophils.
Conclusions:
- Nitrite production in PMA-stimulated human PMN, under the tested conditions, does not originate from nitric oxide synthase activity.
- The findings suggest a catalase-dependent conversion of azide to nitrite, mediated by hydrogen peroxide generated by stimulated phagocytes.