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Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

Furtado JL, Oliveira GA, Pontes AS, Setúbal Sda S, Xavier CV, Lacouth-Silva F, Lima BF, Zaqueo KD, Kayano AM, Calderon LA, Stábeli RG, Soares AM, Zuliani JP - Biomed Res Int (2014)

Bottom Line: The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis.After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min.Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunofarmacologia Aplicada à Saúde, Fundação Oswaldo Cruz, FIOCRUZ-Rondônia, Rua da Beira, 7671 Br364, Km 3.5, 76812-245 Porto Velho, RO, Brazil.

ABSTRACT
In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

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Effect of BaTX-I, BaTX-II, and BaPLA2 on superoxide production by J774A.1 macrophages. 2 × 105 macrophages were incubated with RPMI, 0.1% NBT, 10 uL of PMA (500 ng/mL) (positive control) or toxins (6 µg/mL) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 for the formation of formazan crystals resulting from the reduction of NBT by superoxide. The crystals were solubilized and the absorbance of the supernatant was determined at 620 nm in a spectrophotometer. Data were expressed as O.D. Values represent the mean ± S.E.M. from 3 independent experiments. *P ≤ 0.05 compared with control (ANOVA).
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fig6: Effect of BaTX-I, BaTX-II, and BaPLA2 on superoxide production by J774A.1 macrophages. 2 × 105 macrophages were incubated with RPMI, 0.1% NBT, 10 uL of PMA (500 ng/mL) (positive control) or toxins (6 µg/mL) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 for the formation of formazan crystals resulting from the reduction of NBT by superoxide. The crystals were solubilized and the absorbance of the supernatant was determined at 620 nm in a spectrophotometer. Data were expressed as O.D. Values represent the mean ± S.E.M. from 3 independent experiments. *P ≤ 0.05 compared with control (ANOVA).

Mentions: To investigate the ability of BaTX-I, BaTX-II, and BaPLA2 to induce the production of the superoxide anion by J774A.1 macrophages, the cells were incubated with a noncytotoxic concentration of BaTX-I, BaTX-II, or BaPLA2, or with RPMI (control), in the presence of NBT. As shown in Figure 6, J774A.1 macrophages incubated with RPMI showed an average superoxide anion production of 37 ± 2.8%. Incubation of macrophages with BaTX-I, BaTX-II, and BaPLA2 at a concentration of 6 μg/mL, induced significant production of O2− by J774A.1 macrophages.


Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

Furtado JL, Oliveira GA, Pontes AS, Setúbal Sda S, Xavier CV, Lacouth-Silva F, Lima BF, Zaqueo KD, Kayano AM, Calderon LA, Stábeli RG, Soares AM, Zuliani JP - Biomed Res Int (2014)

Effect of BaTX-I, BaTX-II, and BaPLA2 on superoxide production by J774A.1 macrophages. 2 × 105 macrophages were incubated with RPMI, 0.1% NBT, 10 uL of PMA (500 ng/mL) (positive control) or toxins (6 µg/mL) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 for the formation of formazan crystals resulting from the reduction of NBT by superoxide. The crystals were solubilized and the absorbance of the supernatant was determined at 620 nm in a spectrophotometer. Data were expressed as O.D. Values represent the mean ± S.E.M. from 3 independent experiments. *P ≤ 0.05 compared with control (ANOVA).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921937&req=5

fig6: Effect of BaTX-I, BaTX-II, and BaPLA2 on superoxide production by J774A.1 macrophages. 2 × 105 macrophages were incubated with RPMI, 0.1% NBT, 10 uL of PMA (500 ng/mL) (positive control) or toxins (6 µg/mL) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 for the formation of formazan crystals resulting from the reduction of NBT by superoxide. The crystals were solubilized and the absorbance of the supernatant was determined at 620 nm in a spectrophotometer. Data were expressed as O.D. Values represent the mean ± S.E.M. from 3 independent experiments. *P ≤ 0.05 compared with control (ANOVA).
Mentions: To investigate the ability of BaTX-I, BaTX-II, and BaPLA2 to induce the production of the superoxide anion by J774A.1 macrophages, the cells were incubated with a noncytotoxic concentration of BaTX-I, BaTX-II, or BaPLA2, or with RPMI (control), in the presence of NBT. As shown in Figure 6, J774A.1 macrophages incubated with RPMI showed an average superoxide anion production of 37 ± 2.8%. Incubation of macrophages with BaTX-I, BaTX-II, and BaPLA2 at a concentration of 6 μg/mL, induced significant production of O2− by J774A.1 macrophages.

Bottom Line: The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis.After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min.Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunofarmacologia Aplicada à Saúde, Fundação Oswaldo Cruz, FIOCRUZ-Rondônia, Rua da Beira, 7671 Br364, Km 3.5, 76812-245 Porto Velho, RO, Brazil.

ABSTRACT
In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

Show MeSH
Related in: MedlinePlus