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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 lipid body formation by J774A.1 macrophages. 2 × 105 macrophages were incubated with toxins (6 µg/mL) or RPMI (control) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 before the addition of opsonized zymosan particles. The number of lipid bodies stained with osmium tetroxide within macrophages was determined using phase-contrast microscopy. Values represent the mean ± S.E.M. from 3 independent experiments. *P ≤ 0.05 compared with control and #P ≤ 0.05 compared with control submitted to phagocytosis (ANOVA).
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fig7: Effect of BaTX-I, BaTX-II, and BaPLA2 on lipid body formation by J774A.1 macrophages. 2 × 105 macrophages were incubated with toxins (6 µg/mL) or RPMI (control) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 before the addition of opsonized zymosan particles. The number of lipid bodies stained with osmium tetroxide within macrophages was determined using phase-contrast microscopy. Values represent the mean ± S.E.M. from 3 independent experiments. *P ≤ 0.05 compared with control and #P ≤ 0.05 compared with control submitted to phagocytosis (ANOVA).

Mentions: To determine whether these enzymes induce the formation of lipid bodies in J774A.1 macrophages, the cells were incubated with noncytotoxic BaTX-I, BaTX-II, or BaPLA2, or with RPMI (control) for 1 h. After that, the cells were submitted to phagocytosis and lipid bodies were counted. As shown in Figure 7, the incubation of macrophages with the toxins increased the number of lipid bodies compared to the control. In the cells submitted to phagocytosis, the amount of lipid bodies was significantly higher than cells not submitted to phagocytosis.


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 lipid body formation by J774A.1 macrophages. 2 × 105 macrophages were incubated with toxins (6 µg/mL) or RPMI (control) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 before the addition of opsonized zymosan particles. The number of lipid bodies stained with osmium tetroxide within macrophages was determined using phase-contrast microscopy. Values represent the mean ± S.E.M. from 3 independent experiments. *P ≤ 0.05 compared with control and #P ≤ 0.05 compared with control submitted to phagocytosis (ANOVA).
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Effect of BaTX-I, BaTX-II, and BaPLA2 on lipid body formation by J774A.1 macrophages. 2 × 105 macrophages were incubated with toxins (6 µg/mL) or RPMI (control) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 before the addition of opsonized zymosan particles. The number of lipid bodies stained with osmium tetroxide within macrophages was determined using phase-contrast microscopy. Values represent the mean ± S.E.M. from 3 independent experiments. *P ≤ 0.05 compared with control and #P ≤ 0.05 compared with control submitted to phagocytosis (ANOVA).
Mentions: To determine whether these enzymes induce the formation of lipid bodies in J774A.1 macrophages, the cells were incubated with noncytotoxic BaTX-I, BaTX-II, or BaPLA2, or with RPMI (control) for 1 h. After that, the cells were submitted to phagocytosis and lipid bodies were counted. As shown in Figure 7, the incubation of macrophages with the toxins increased the number of lipid bodies compared to the control. In the cells submitted to phagocytosis, the amount of lipid bodies was significantly higher than cells not submitted to phagocytosis.

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