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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 92, Num. s2, 1997, pp. 233-235
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 92 (Suppl.II), 1997, pp.
233-235
Nitric Oxide Mediates the Microbicidal Activity of Eosinophils
Sandra HP Oliveira, Simone G Fonseca, Pedro RT Romao, Sergio H Ferreira,
Fernando Q Cunha^+
Departamentos de Farmacologia e Microbiologia, Parasitologia e Imunologia,
Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Av.
Bandeirantes 900, 14049-900 Ribeirao Preto, SP, Brazil
^+Corresponding author. Fax: +55-16-633.2301. E-mail: fdqcunha@fmrp.usp.br
Received 3 September 1997; Accepted 30 September 1997
Code Number:OC97194
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There are several experimental evidences that nitric oxide (NO) is
involved in the microbicidal activity of macrophages against a number of
intracellular pathogens including Leishmania major, Trypanozoma cruzi,
Toxoplasma gondii. It is also well known that eosinophils (EO) have
microbicidal activity against many parasites such as Schistosoma
mansoni, Trichinella spiralis, T. cruzi and L. amazonensis. The
purpose of this study was to investigate if NO is involved in the
microbicidal activity of EO against L. major. Eosinophils harvested
from peritoneal cavity of rats released spontaneously after 24 and 48 hr a
small amount of nitrite. This release was enhanced by the treatment of
cells with IFN-gamma (200 IU/ml). This release was blocked by addition of the
NO synthase inhibitor, L-NIO (100 mM) into the culture. To determinate the
leishmanicidal activity of eosinophils the parasites were incubated with
activated eosinophils with IFN-gamma and the ability of surviving parasites to
incorporate [^3H]thymidine was evaluated. IFN-gamma-activated eosinophils were
able to kill L. major and to release high levels of nitrite. The
ability to destroy L. major and the release of NO were completely
blocked by L-NIO. These results indicate that activated eosinophils release
NO which is involved in the microbicidal activity of these cells against
L. major.
Key words: nitric oxide - eosinophils - microbicidal activity -
Leishmania major
Nitric Oxide Mediates the Microbicidal Activity of Eosinophils (Full
Text)
The microbicidal activity of eosinophils has been associated with
degranulation and release of granule-protein such as major basic protein
(MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP) and
eosinophil-derived neurotoxin (EDN). These substances are cytotoxic to
cells and to several parasites in vitro (Gleich et al. 1992), among
them, Trichinella spiralis (Wassom & Gleich 1979), Onchocerca
volvulus (Greene et al. 1981), Toxocara canis (Badley et al.
1987), Fasciola hepatica (Duffus et al. 1980), Necator
americanus (Desakorn et al. 1987), Nippo-strongylus brasiliensis
(Kojima et al. 1985), Schistosoma mansoni (Capron et al. 1979,
Butterworth et al. 1979), Trypanosoma cruzi (Villalta & Kierszenbaum
1984), Plasmodium falciparum (Waters et al. 1987), Leishmania
mexicana amazonensis (Pimenta et al. 1987) and L. donovani
(Pearson et al. 1987). In addition to the granule proteins, oxygen-free
radicals, including the toxic singlet oxygen, seem to contribute to the
microbicidal activity of eosinophils (Pincus et al. 1984).
Nitric oxide (NO) or nitrogen-derived metabolites have been identified as
major effector molecules involved in the macrophage microbicidal activity
against most intracellular pathogens, including L. major (Liew &
Millott 1990), Toxoplasma gondii (Adams et al. 1990), T.
musculi (Vincendeau & Dalouede 1991), T. cruzi (Gazzinelli et
al. 1992), P. berghei (Mellouk et al. 1991), Mycobacterium
leprae (Adams et al. 1991), M. avium (Denis 1991), Candida
albicans (Cenci et al. 1993) and the virus Ectromelia, vaccinia and
herpes simplex-1 (Gunasegaran et al. 1993). Recently, it was demonstrated
that the microbicidal activity of neutrophils against C. albicans is
also mediated by NO (Fierro et al. 1996).
Since at present there is no experimental demonstration of the contribution
of nitric oxide to the microbicidal activity of eosinophils, the aim of
this study was to investigate whether eosinophils produce nitric oxide and
whether NO is involved in the microbicidal activity of these cells against
L. major.
To investigate if eosinophils are able to produce nitric oxide, these cells
were harvested from peritoneal cavities of rats and incubated in vitro
with IFN-gamma. Eosinophils released spontaneously a small amount of
nitrite when incubated in vitro. Levels of nitrite in the medium
were already present after 12 hr of incubation, which increased
continuously within 48 hr. The stimulation of eosinophils with mIFN-gamma
enhanced the release of the nitrite after 24 and 48 hr of incubation. A
similar effect of IFN-gamma has been described in macrophages (Liew & Millott,
1990, Cunha et al. 1993). Nitrite production by the stimulated eosinophils
was abolished by the NO synthase inhibitor L-NIO^ (McCall et al. 1992),
indicating that the nitrite measured by the Griess method resulted from the
oxidation of L-arginine by NO synthase (Fig. 1).
Fig. 1: level of
nitrite in the culture supernatants of
eosinophils incubated with RPMI, mIFN-gamma (200 IU/ml) in the presence or
absence of L-NIO (100 mM). The nitrite concentration was determined at 12
hr , 24 hr and 48 hr after eosinophils stimulation by the Griess methods.
Data are reported as means +/- SEM of assays performed in triplicate and
are representative of two different experiments. * p < 0.05 compared to
RPMI (ANOVA followed by Bonferroni t-test).
An important protective role for reactive nitrogen intermediates has been
established in macrophage killing of intracellular protozoa (Liew & Millott
1990, Adams et al. 1990, Vincendeau & Dalouede 1991, Gazzinelli et al.
1992), bacteria (Adams et al. 1991, Denis 1991), fungus (Cenci et al. 1993)
and virus (Gunasegaran et al. 1993). A similar role has also been
demonstrated in neutrophil killing of C. albicans (Fierro et al.
1996) and Staphilococcus aureus (Malawista et al. 1992). Since there
are data showing that eosinophils are able to kill Leishmania
(Pimenta et al. 1987, Pearson et al. 1987), we investigated whether NO
mediates the killing of this intracellular parasite by stimulated
eosinophils. IFN-gamma-activated eosinophils were able to kill L. major
and to release high levels of nitrite. The ability to destroy L.
major and the release of NO were completely blocked by L-NIO,
suggesting that NO mediate the leishmanicidal activity of eosinophils.
Leishmania survival inside the unstimulated eosinophils was not
affect by L-NIO treatment (Fig. 2). These results, together with the
findings that eosinophils are present in Leishmania lesions
(Katakura et al. 1993)^ , reinforce the importance to investigate the role
of eosinophils in the evolution of leishmaniasis.
Figure 2: killing of
Leishmania major by
eosinophils incubated with RPMI, or stimulated with mIFN-gamma (200 IU/ml) in
the presence or absence of L-NIO (100 mM) (panel a). Panel b: level of
nitrite in the supernatant of eosinophils incubated with L. major
and stimulated with IFN-gamma (200 IU/ml). Leishmanicidal activity is reported
as the ability of residual parasites to incorporate [^3H]thymidine. Data
are presented as means +/- SEM of assays performed in triplicate and are
representative of three different experiments. * p < 0.05 compared to RPMI
(ANOVA followed by Bonferroni t-test).
Thus, the data obtained in the present study indicate that activated
eosinophils release NO which may be involved in the microbicidal activity
of these cells against L. major.
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Copyright 1997 Fundacao Oswaldo Cruz - Fiocruz
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