Tyrosine phosphatase SHP-1 in oxidative stress and development of allergic airway inflammation

Abstract

Oxidative stress has been implicated in allergic responses. SHP-1 is a target of oxidants and has been reported as a negative regulator in a mouse model of asthma. We investigated the effect of oxidative stress on the development of allergic airway inflammation in heterozygous viable motheaten (mev/+) mice deficient of SHP-1. Wild-type (WT) and mev/+ mice were compared in this study. Human alveolar epithelial cells (A549) transfected with mutant SHP-1 gene were used to evaluate the role of SHP-1 in lung epithelial cells. Hydrogen peroxide (H(2)O(2)) and Paraquat were used in vitro and in vivo, respectively. We also investigated whether mev/+ mice can break immune tolerance when exposed to aeroallergen intranasally. Compared with WT mice, bronchoalveolar lavage (BAL) cells and splenocytes from mev/+ mice showed a different response to oxidant stress. This includes a significant enhancement of intracellular reactive oxygen species and STAT6 phosphorylation in vitro and increased CCL20, decreased IL-10, and increased number of dendritic cells in BAL fluid in vivo. Mutant SHP-1-transfected epithelial cells secreted higher levels of CCL20 and RANTES after exposure to oxidative stress. Furthermore, break of immune tolerance, as development of allergic airway inflammation, was observed in mev/+ mice after allergen exposure, which was suppressed by antioxidant N-acetylcystein. These data suggest that SHP-1 plays an important role in regulating oxidative stress. Thus, increased intracellular oxidative stress and lack of SHP-1 in the presence of T helper cell type 2-prone cellular activation may lead to the development of allergic airway inflammation.

Figure 1.

SHP-1 protein phosphatase activity in lung tissues. SHP-1 protein was immunoprecipitated from lung homogenates of wild-type (WT), heterozygous viable motheaten (mev/+), and viable motheaten (mev/mev) mice and the phosphatase activity was determined using substrate pNPP in a colorimetric assay. The data are expressed as percent of SHP-1 phosphatase activity relative to that of WT mice. Shown is a representative of two experiments with similar results (WT, n = 3; mev/+, n = 4; mev/mev, n = 4; *P < 0.05 and **P < 0.01).

Figure 2.

In vitro cellular responses to oxidative stress. (A) Ratio of mean fluorescent intensity (MFI) for intracellular reactive oxygen species in H₂O₂-treated (100 μM) bronchoalveolar lavage (BAL) cells over basal conditions. Data were collected from 5,000 events (n = 3, *P < 0.05). The data are the mean ± SEM. (B) Nrf2 in nucleus and cytoplasm of splenocytes from WT and mev/+ mice 1 hour after H₂O₂ treatment. (C) Total and phosphorylated STAT6 and I-κBα in the splenocytes from WT and mev/+ mice. Data are representative of three experiments.

Figure 2.

In vitro cellular responses to oxidative stress. (A) Ratio of mean fluorescent intensity (MFI) for intracellular reactive oxygen species in H₂O₂-treated (100 μM) bronchoalveolar lavage (BAL) cells over basal conditions. Data were collected from 5,000 events (n = 3, *P < 0.05). The data are the mean ± SEM. (B) Nrf2 in nucleus and cytoplasm of splenocytes from WT and mev/+ mice 1 hour after H₂O₂ treatment. (C) Total and phosphorylated STAT6 and I-κBα in the splenocytes from WT and mev/+ mice. Data are representative of three experiments.

Figure 2.

In vitro cellular responses to oxidative stress. (A) Ratio of mean fluorescent intensity (MFI) for intracellular reactive oxygen species in H₂O₂-treated (100 μM) bronchoalveolar lavage (BAL) cells over basal conditions. Data were collected from 5,000 events (n = 3, *P < 0.05). The data are the mean ± SEM. (B) Nrf2 in nucleus and cytoplasm of splenocytes from WT and mev/+ mice 1 hour after H₂O₂ treatment. (C) Total and phosphorylated STAT6 and I-κBα in the splenocytes from WT and mev/+ mice. Data are representative of three experiments.

Figure 3.

In vivo responses to oxidative stress. (A) Total cell counts of BAL cells from mev/+ and WT mice with or without Paraquat. (B) Percentage of CD11c+CD11b+ dendritic cells and CD11b+CD11c− macrophages in the lung after exposure to Paraquat. (C) CCL20 in BAL fluids. Dotted line represents detection limit (n = 3–6, *P < 0.05 compared with other groups). The data are the mean ± SEM.

Figure 3.

In vivo responses to oxidative stress. (A) Total cell counts of BAL cells from mev/+ and WT mice with or without Paraquat. (B) Percentage of CD11c+CD11b+ dendritic cells and CD11b+CD11c− macrophages in the lung after exposure to Paraquat. (C) CCL20 in BAL fluids. Dotted line represents detection limit (n = 3–6, *P < 0.05 compared with other groups). The data are the mean ± SEM.

Figure 3.

In vivo responses to oxidative stress. (A) Total cell counts of BAL cells from mev/+ and WT mice with or without Paraquat. (B) Percentage of CD11c+CD11b+ dendritic cells and CD11b+CD11c− macrophages in the lung after exposure to Paraquat. (C) CCL20 in BAL fluids. Dotted line represents detection limit (n = 3–6, *P < 0.05 compared with other groups). The data are the mean ± SEM.

Figure 4.

Levels of (A) CCL20 and (B) RANTES produced by human lung epithelial cells transfected with plasmid vector containing WT or mutant human SHP-1 gene after H₂O₂ exposure. The data are the mean ± SEM of triplicate samples. *P < 0.05 compared with groups transfected with plasmid vectors containing WT SHP-1 gene in each pair of experiments.

Figure 4.

Levels of (A) CCL20 and (B) RANTES produced by human lung epithelial cells transfected with plasmid vector containing WT or mutant human SHP-1 gene after H₂O₂ exposure. The data are the mean ± SEM of triplicate samples. *P < 0.05 compared with groups transfected with plasmid vectors containing WT SHP-1 gene in each pair of experiments.

Figure 5.

Allergic inflammatory responses to ovalbumin (OVA) challenge. (A and B) Total and differential cell counts of BAL cells. (C) Lung histopathology of mev/+ mice and WT mice after intranasal OVA stimulation in the presence or absence of antioxidant NAC (hematoxylin and eosin, ×100). (D) Serum OVA-specific IgE. Dotted line represents detection limit. (n = 4–6, *P < 0.05 and **P < 0.05 compared with groups without NAC). The data are the mean ± SEM.

Figure 5.

Allergic inflammatory responses to ovalbumin (OVA) challenge. (A and B) Total and differential cell counts of BAL cells. (C) Lung histopathology of mev/+ mice and WT mice after intranasal OVA stimulation in the presence or absence of antioxidant NAC (hematoxylin and eosin, ×100). (D) Serum OVA-specific IgE. Dotted line represents detection limit. (n = 4–6, *P < 0.05 and **P < 0.05 compared with groups without NAC). The data are the mean ± SEM.

Figure 5.

Allergic inflammatory responses to ovalbumin (OVA) challenge. (A and B) Total and differential cell counts of BAL cells. (C) Lung histopathology of mev/+ mice and WT mice after intranasal OVA stimulation in the presence or absence of antioxidant NAC (hematoxylin and eosin, ×100). (D) Serum OVA-specific IgE. Dotted line represents detection limit. (n = 4–6, *P < 0.05 and **P < 0.05 compared with groups without NAC). The data are the mean ± SEM.

Figure 5.

Allergic inflammatory responses to ovalbumin (OVA) challenge. (A and B) Total and differential cell counts of BAL cells. (C) Lung histopathology of mev/+ mice and WT mice after intranasal OVA stimulation in the presence or absence of antioxidant NAC (hematoxylin and eosin, ×100). (D) Serum OVA-specific IgE. Dotted line represents detection limit. (n = 4–6, *P < 0.05 and **P < 0.05 compared with groups without NAC). The data are the mean ± SEM.