Macrophage killing of Leishmania amazonensis amastigotes requires both nitric oxide and superoxide (original) (raw)
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Leishmania amazonensis and macrophage interactions: immune factors necessary to kill the parasite
List of Tables vi List of Figures vii CHAPTER 1 General Introduction V 2.4.7 Glutathione 3. Host immune response to Leishmania 3.1 CD4 + T cells and L. major infection 3.2 CD4 + T cells and L. amazonensis 3.3 B cells 4. General conclusion References CHAPTER 2 Macrophage killing of Leishmania amazonensis amastigotes requires both nitric oxide and superoxide Abstract Introduction Material and methods Results Discussion References CHAPTER 3 Leishmania major specific CD4 + T cells and B cells limit L. amazonensis amastigote survival within in vitro infected macrophages through IgG mediated superoxide production Abstract Introduction Material and methods Results Discussion References CHAPTER 4 Leishmania infection modulates the expression of Fey receptors Abstract CHAPTER 2 Macrophage killing of Leishmania amazonensis amastigotes requires both nitric oxide and superoxide
Leishmania (L.) amazonensis-induced inhibition of nitric oxide synthesis in host macrophages
Microbes and Infection, 2002
Inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production was demonstrated in J774-G8 macrophages infected with Leishmania (L.) amazonensis promastigotes. The downmodulation of NO production observed in infected and LPS-stimulated J774-G8 cells correlated with a reduction in inducible nitric oxide synthase (iNOS) activity. Reduction in iNOS activity was not paralleled by decreased iNOS mRNA expression, suggesting that the parasite affects post-transcriptional events of NO synthesis. Supplementation with L-arginine or tetrahydrobiopterin did not increase NO production, suggesting that inhibition is not due to an insufficiency of substrate or co-factor. Treatment with anti-IL-10, anti-IL-4 or anti-TGF-neutralizing antibodies also failed to increase NO production, indicating that these cytokines are not involved in the observed parasite-induced inhibition of NO synthesis. However, treatment of the cultures with IFN-γ resulted in a marked increase in NO production by infected LPS-stimulated cells. These results show that although L.(L.) amazonensis infection inhibits iNOS activity and NO production by J774-G8 cells, activation by IFN-γ is capable of overriding the suppression of NO synthesis.
Role of superoxide dismutase in survival of Leishmania within the macrophage
Biochemical Journal, 2003
Intracellular parasitic protozoans of the genus Leishmania depend for their survival on the elaboration of enzymic and other mechanisms for evading toxic free-radical damage inflicted by their phagocytic macrophage host. One such mechanism may involve superoxide dismutase (SOD), which detoxifies reactive superoxide radicals produced by activated macrophages, but the role of this enzyme in parasite survival has not yet been demonstrated. We have cloned a SOD gene from L. tropica and generated SOD-deficient parasites by expressing the corresponding antisense RNA from an episomal vector. Such parasites have enhanced sensitivity to menadione and hydrogen peroxide in axenic culture, and a markedly reduced survival in mouse macrophages. These results indicate that SOD is a major determinant of intracellular survival of Leishmania.
Parasite Immunology, 2012
CBA mouse macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. It has been established that some Leishmania species are destroyed by reactive oxygen species (ROS). However, other species of Leishmania exhibit resistance to ROS or even down-modulate ROS production. We hypothesized that L. amazonensis-infected macrophages reduce ROS production soon after parasite-cell interaction. Employing a highly sensitive analysis technique based on chemiluminescence, the production of superoxide (O ÁÀ 2) and hydrogen peroxide (H 2 O 2) by L. majoror L. amazonensis-infected CBA macrophages were measured. L. major induces macrophages to release levels of O ÁÀ 2 3AE5 times higher than in uninfected cells. This O ÁÀ 2 production is partially dependent on NADPH oxidase (NOX) type 2. The level of accumulated H 2 O 2 is 20 times higher in L. major-than in L. amazonensis-infected cells. Furthermore, macrophages stimulated with L. amazonensis release amounts of ROS similar to uninfected cells. These findings support previous studies showing that CBA macrophages are effective in controlling L. major infection by a mechanism dependent on both O ÁÀ 2 production and H 2 O 2 generation. Furthermore, these data reinforce the notion that L. amazonensis survive inside CBA macrophages by reducing ROS production during the phagocytic process.
The Journal of Immunology
Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin...
Oxidative Responses of Human and Murine Macrophages During Phagocytosis ofLeishmania chagasi
The Journal of Immunology, 2001
Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2− during phagocytosis of opsonized promastigotes. Addition of the O2− scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2− enhances macrophage leishmanicidal activity. The importance of NO· produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO· in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor NG-monomethyl-l-arginine inhibited IFN-γ-mediated intracellular killing by both murine and human macrophage...
Parasites & vectors, 2017
Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease caused by protozoa of the genus Leishmania. In Morocco, CL is a public health problem mainly caused by Leishmania major and Leishmania tropica, which are responsible for zoonotic and anthroponotic CL, respectively. Macrophages are the primary cells infected by Leishmania parasites and their capacity to produce nitric oxide (NO) is of critical importance for parasite elimination. To our knowledge, the role of NO on autochthonous infections has never been investigated before. In this study, we evaluated in vitro the capacity of autochthonous primary dermotropic strains of L. major and L. tropica to modulate NO production by J774-macrophages and determine the sensitivity of both species to exogenous NO. The infectivity of the J774 cell line was analyzed by optical microscopy. NO production by macrophages was measured by the Griess method. The sensitivity to NO by the two strains was assessed by the MTT assay using NO dono...
Journal of Parasitology, 2000
Leishmaniasis is a parasitic disease that leads to chronic inflammation. Macrophages, depending on their activation state, are either hosts or killers of the parasites. Downregulation of nitric oxide (NO) synthesis by the parasite infecting the macrophages has been proposed to be an important evading mechanism based on in vitro studies. We confirmed inhibition of NO release by macrophages infected with Leishmania amazonensis in vitro. To examine the role of the parasite in regulating NO production in vivo, we monitored systemic NO levels elicited by challenging naive and L. amazonensis-infected BALB/c mice with lipopolysaccharide (LPS). Animals were challenged after 1, 2, 6, and 9 wk of infection. NO production was monitored by electron paramagnetic resonance spectroscopy as the levels of hemoglobin nitrosyl complexes (HbNO) present in the animal's blood. No significant differences in HbNO levels were observed between LPS-treated naive and inoculated mice at any time during infection. To control for increased macrophage numbers in infected mice, naive mice were injected with a macrophage cell line before LPS challenge; this treatment did not increase produced NO levels. The results argue against a major role for the parasite in downregulating NO production in vivo.
Journal of Biochemistry, 2000
Pretreatment of macrophages with PMA, an agonist of PKC, showed diverse effects on degradation and survival of two virulent strains of Leishmania donovani promastigotes. Treatment of macrophages with PMA for 45 min at 37°C generated significant amounts of superoxide anions and reduced the parasite burden of macrophages by up to 48 and 43% when AG83 and GE-1 strains were used for infection. Staurosporine, an inhibitor of PKC, inhibited PMA-dependent killing of the parasites, while tyrphostin AG 126, an inhibitor of protein tyrosine kinase, showed very little effect. Depletion of PKC by prolonged incubation with PMA drastically reduced the superoxide anion generation and increased the uptake and multiplication of the parasites. Finally, to understand the mechanism of higher uptake of the parasites by PKC-depleted macrophages, membrane microviscosity was measured by fluorescence depolarization. Membrane microviscosity was found to be approximately 40% lower in PKC-depleted macrophages than in normal macrophages, indicating the role of membrane fluidity in the infection process. Together, these data suggest PKC activation, superoxide generation, and membrane fluidity are essential factors in the efficient regulation of leishmanial infection.