Apoptosis of inflammatory cells is a critical event in the resolution of
inflammation, as failure to undergo this form of cell death leads to increased tissue
damage and exacerbation of the inflammatory response. Prolonged survival of the
neutrophilic granulocyte has been implicated in the pathogenesis of many chronic
inflammatory conditions. Many factors are able to influence the rate of apoptosis in
neutrophils. Among these is the signalling molecule, nitric oxide (NO), which
possesses both anti-apoptotic and pro-apoptotic properties in many cell types,
depending on the concentration and flux of NO, and also the source from which NO
Characterisation of the NO-related species generated by four 'NO-donating'
compounds, spermine diazeniumdiolate (SPER/NO), diethylamine diazeniumdiolate
(DEA/NO), 1,2,3,4-oxatriazolium, 5-amino-3- (3,4-dichlorophenyl)-chloride (GEA
3162) and 3-morpholinosydnonime (SIN-1) was carried out. It was demonstrated
that the diazeniumdiolates, SPER/NO and DEA/NO both spontaneously liberated
free NO in aqueous solution at physiological temperature and pH. In contrast, both
GEA 3162 and SIN-1 produced NO and superoxide anion (O2") concomitantly, that
rapidly react to form the powerful oxidant species, peroxynitrite (ONOO"). These
two compounds should therefore be considered ONOO" donors rather than NO
Apoptosis studies demonstrated the biphasic pro- and anti-apoptotic effects of
pure NO donors, SPER/NO and DEA/NO, and the pro-apoptotic effects of ONOO"
donors, GEA 3162 and SIN-1, in human neutrophils. Low concentrations of the pure
NO donors delayed the rate of neutrophil apoptosis, while high concentrations of all
compounds tested accelerated cell death. Time course analyses of four independent
events of apoptosis revealed that morphological and biochemical parameters of
neutrophil apoptosis may proceed independently of internucleosomal DNA
fragmentation, which has long been considered a key hallmark of apoptosis and is
frequently used as the sole criterion for assessment of this form of cell death.
Treatment with high concentrations of ONOO" donors and, to a lesser extent, with
the longer-lasting pure NO donor, SPER/NO, induced morphological and cell surface
(CD 16 shedding and phosphatidylserine exposure) changes characteristic of
neutrophil apoptosis, but paradoxically inhibited internucleosomal DNA
fragmentation, as measured by propidium iodide intercalation and gel
electrophoresis. In contrast, treatment with the short-lasting NO donor, DEA/NO,
produced no such inhibition. An oxidation reaction was shown to be responsible for
the suppression of the DNA fragmentation pathway, as the reducing agent,
dithiothreitol, restored DNA fragmentation back to control levels. However, GEA
3162-mediated inhibition of DNA fragmentation did not occur upstream or at the
level of degradation of inhibitor of caspase-activated DNase (ICAD/DFF45), as
Western blotting showed breakdown of this protein was enhanced rather than
inhibited in GEA 3162-treated neutrophils.
Therefore, NO can exert either pro- or anti-apoptotic effects on human
neutrophil apoptosis, depending on its concentration and flux, whereas only proapoptotic effects are achieved with ONOO" donors. Cell death promoted by ONOO"
or SPER/NO is independent of an increase in internucleosomal DNA fragmentation;
this process is inhibited via an oxidative mechanism, but not through inhibition of
ICAD/DFF45 breakdown or upstream mechanisms. Thus, NO and ONOO" are able
to modulate the rate of neutrophil apoptosis, which may have implications for
chronic inflammatory conditions.