MEASUREMENT OF NITRIC
OXIDE PRODUCTION BY CHRONIC GRANULOMATOUS DISEASE PATIENTS
Tsuji S, Taniuchi S,
Hasui M, Yamamoto A, Kobayashi Y
Department of
Pediatrics, Kansai Medical University, Osaka, Japan
Objective: Nitric oxide
synthase(NOS) in human neutrophils is not well characterized. We have
evaluated the direct measurement of NO production in the patients with
chronic granulomatous disease.
Methods: Venous blood was
obtained from healthy volunteers and from patients with chronic
granulomatous disease (CGD). One ml of whole blood was incubated with
lipopolysaccharide (LPS), methylisothiourea (EIT) and 7-nitroindazole
(7-NI). Then the mixture was incubated for the indicated periods of time,
to which diaminofluorescein-2 (DAF-2) was added together with LPS, EIT and
7-NI 120 min before termination of the incubation period A mixture of whole
blood, DCFH-DA and PMA was incubated. At the end of the incubation period,
the sample was prepared for flow cytometry analysis.
Results: Intracellular NO
production increased time-dependently when stimulated by LPS, and its
inhibition by EIT. All PMNs from the patients with CGD failed to generate
hydrogen peroxide, but NO production of CGD PMNs was significantly
increased when compared with that of control PMNs (p<0.05).
Conclusion: A significantly higher
NO production by CGD PMNs than control cells clearly indicates that DAF-2
does not measure hydrogen peroxide, and this observation may be ascribed to
the failure of CGD PMNs to produce hydrogen peroxide and peroxynitrite.
Therefore an overproduction of NO by PMNs of CGD patients may be explained
by the overproduction of iNOS stimulated by several inflammatory cytokines.