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Oxidized {alpha}1-antitrypsin stimulates the release of monocyte chemotactic protein-1 from lung epithelial cells: potential role in emphysema.

Li Z, Alam S, Wang J, Sandstrom CS, Janciauskiene S, Mahadeva R - Am. J. Physiol. Lung Cell Mol. Physiol. (2009)

Bottom Line: Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion.The effect of Ox-AT was dependent on NF-kappaB and activator protein-1 (AP-1)/JNK.They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the antielastase lung protection, but also converts AT into a proinflammatory stimulus.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medicine, Univ. of Cambridge, Addenbrookes Hospital, United Kingdom.

ABSTRACT
alpha(1)-Antitrypsin (AT) is a major elastase inhibitor within the lung. Oxidation of critical methionine residues in AT generates oxidized AT (Ox-AT), which has a greatly diminished ability to inhibit neutrophil elastase. This process may contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD) by creating a functional deficiency of AT permitting lung destruction. We show here that Ox-AT promotes release of human monocyte chemoattractant protein-1 (MCP-1) and IL-8 from human lung type epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells. Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion. These findings were supported by the fact that instillation of Ox-AT into murine lungs resulted in an increase in JE (mouse MCP-1) and increased macrophage numbers in the bronchoalveolar lavage fluid. The effect of Ox-AT was dependent on NF-kappaB and activator protein-1 (AP-1)/JNK. These findings have important implications. They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the antielastase lung protection, but also converts AT into a proinflammatory stimulus. Ox-AT generated in the airway interacts directly with epithelial cells to release chemokines IL-8 and MCP-1, which in turn attracts macrophages and neutrophils into the airways. The release of oxidants by these inflammatory cells could oxidize AT, perpetuating the cycle and potentially contributing to the pathogenesis of COPD. Furthermore, these data demonstrate that molecules such as oxidants, antiproteinases, and chemokines, rather than act independently, are likely to interact to cause emphysema.

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Related in: MedlinePlus

A: effect of Ox-AT on induction of NF-κB activity in lung tissue. There was a significant increase in NF-κB activity following intratracheal Ox-AT compared with PBS (4 h, P = 0.028; 24 h, P = 0.029; 72 h, P = 0.037). Intratracheal N-AT did not have any significant effect on NF-κB activity. The positive control was Jurkat cells with high endogenous NF-κB activity (P = 0.032 compared with PBS). Data are means (SE) from 3 separate experiments. *P ≤ 0.05. B: effect of Ox-AT on induction of AP-1 activity in lung tissue. There was a significant increase in AP-1 activity following intratracheal Ox-AT compared with PBS (4 h, P = 0.019; 24 h, P = 0.033; 72 h, P = 0.049). Intratracheal N-AT did not have any significant effect on AP-1 activity. The positive control was K562 cells with high endogenous AP-1 activity (P = 0.022 compared with PBS). Data are means (SE) from 3 separate experiments. *P ≤ 0.05. C: Western blot analysis of homogenized lung tissue for phosphorylated JNK. The p54 JNK isoform was expressed highly in lung homogenates from Ox-AT-treated mice compared with lung homogenates from N-AT-treated mice. Densitometric analysis of each band using ImageJ software demonstrated the level of p54 phospho-JNK was significantly higher and peaking at 24 h in lung homogenates from Ox-AT-treated mice compared with lung homogenates from N-AT-treated mice (4 h, P = 0.031; 24 h, P = 0.012; 72 h, P = 0.023). Lane 1, positive control A431 cell lysate; lane 2, 4 h N-AT; lane 3, 24 h N-AT; lane 4, 72 h N-AT; lane 5, 2 h Ox-AT; lane 6, 24 h Ox-AT; lane 7, 72 h Ox-AT. Gel is representative of 3 independent experiments. Data are means (SE) of 3 independent experiments. *P ≤ 0.05. D: Western blot analysis of homogenized lung tissues for total JNK. Western blot analysis for total JNK shows total (phosphorylated and nonphosphorylated) JNK ranging in size between 46 and 54 kDa. Densitometric analysis of both bands from each sample (expressed cumulatively) using ImageJ software demonstrated total JNK was expressed in comparable levels in lung homogenates from both Ox-AT- and N-AT-treated mice. Densitometric analysis of bands showed there was no significant difference in total JNK in lung homogenates from Ox-AT- and N-AT-treated mice at 4, 24, or 72 h. Lane 1, positive control A431 cell lysate; lane 2, 4 h N-AT; lane 3, 24 h N-AT; lane 4, 72 h N-AT; lane 5, 4 h Ox-AT; lane 6, 24 h Ox-AT; lane 7, 72 h Ox-AT. Gel is representative of 3 independent experiments. Data are means (SE) of 3 independent experiments.
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f9: A: effect of Ox-AT on induction of NF-κB activity in lung tissue. There was a significant increase in NF-κB activity following intratracheal Ox-AT compared with PBS (4 h, P = 0.028; 24 h, P = 0.029; 72 h, P = 0.037). Intratracheal N-AT did not have any significant effect on NF-κB activity. The positive control was Jurkat cells with high endogenous NF-κB activity (P = 0.032 compared with PBS). Data are means (SE) from 3 separate experiments. *P ≤ 0.05. B: effect of Ox-AT on induction of AP-1 activity in lung tissue. There was a significant increase in AP-1 activity following intratracheal Ox-AT compared with PBS (4 h, P = 0.019; 24 h, P = 0.033; 72 h, P = 0.049). Intratracheal N-AT did not have any significant effect on AP-1 activity. The positive control was K562 cells with high endogenous AP-1 activity (P = 0.022 compared with PBS). Data are means (SE) from 3 separate experiments. *P ≤ 0.05. C: Western blot analysis of homogenized lung tissue for phosphorylated JNK. The p54 JNK isoform was expressed highly in lung homogenates from Ox-AT-treated mice compared with lung homogenates from N-AT-treated mice. Densitometric analysis of each band using ImageJ software demonstrated the level of p54 phospho-JNK was significantly higher and peaking at 24 h in lung homogenates from Ox-AT-treated mice compared with lung homogenates from N-AT-treated mice (4 h, P = 0.031; 24 h, P = 0.012; 72 h, P = 0.023). Lane 1, positive control A431 cell lysate; lane 2, 4 h N-AT; lane 3, 24 h N-AT; lane 4, 72 h N-AT; lane 5, 2 h Ox-AT; lane 6, 24 h Ox-AT; lane 7, 72 h Ox-AT. Gel is representative of 3 independent experiments. Data are means (SE) of 3 independent experiments. *P ≤ 0.05. D: Western blot analysis of homogenized lung tissues for total JNK. Western blot analysis for total JNK shows total (phosphorylated and nonphosphorylated) JNK ranging in size between 46 and 54 kDa. Densitometric analysis of both bands from each sample (expressed cumulatively) using ImageJ software demonstrated total JNK was expressed in comparable levels in lung homogenates from both Ox-AT- and N-AT-treated mice. Densitometric analysis of bands showed there was no significant difference in total JNK in lung homogenates from Ox-AT- and N-AT-treated mice at 4, 24, or 72 h. Lane 1, positive control A431 cell lysate; lane 2, 4 h N-AT; lane 3, 24 h N-AT; lane 4, 72 h N-AT; lane 5, 4 h Ox-AT; lane 6, 24 h Ox-AT; lane 7, 72 h Ox-AT. Gel is representative of 3 independent experiments. Data are means (SE) of 3 independent experiments.

Mentions: Following intratracheal instillation of Ox-AT, NF-κB was found to be significantly increased in lung homogenates in a time-dependent manner compared with PBS (4 h, P = 0.028; 24 h, P = 0.029; and 72 h, P = 0.037). Intratracheal N-AT had no significant effect on the activity of NF-κB in lung homogenates (Fig. 9A).


Oxidized {alpha}1-antitrypsin stimulates the release of monocyte chemotactic protein-1 from lung epithelial cells: potential role in emphysema.

Li Z, Alam S, Wang J, Sandstrom CS, Janciauskiene S, Mahadeva R - Am. J. Physiol. Lung Cell Mol. Physiol. (2009)

A: effect of Ox-AT on induction of NF-κB activity in lung tissue. There was a significant increase in NF-κB activity following intratracheal Ox-AT compared with PBS (4 h, P = 0.028; 24 h, P = 0.029; 72 h, P = 0.037). Intratracheal N-AT did not have any significant effect on NF-κB activity. The positive control was Jurkat cells with high endogenous NF-κB activity (P = 0.032 compared with PBS). Data are means (SE) from 3 separate experiments. *P ≤ 0.05. B: effect of Ox-AT on induction of AP-1 activity in lung tissue. There was a significant increase in AP-1 activity following intratracheal Ox-AT compared with PBS (4 h, P = 0.019; 24 h, P = 0.033; 72 h, P = 0.049). Intratracheal N-AT did not have any significant effect on AP-1 activity. The positive control was K562 cells with high endogenous AP-1 activity (P = 0.022 compared with PBS). Data are means (SE) from 3 separate experiments. *P ≤ 0.05. C: Western blot analysis of homogenized lung tissue for phosphorylated JNK. The p54 JNK isoform was expressed highly in lung homogenates from Ox-AT-treated mice compared with lung homogenates from N-AT-treated mice. Densitometric analysis of each band using ImageJ software demonstrated the level of p54 phospho-JNK was significantly higher and peaking at 24 h in lung homogenates from Ox-AT-treated mice compared with lung homogenates from N-AT-treated mice (4 h, P = 0.031; 24 h, P = 0.012; 72 h, P = 0.023). Lane 1, positive control A431 cell lysate; lane 2, 4 h N-AT; lane 3, 24 h N-AT; lane 4, 72 h N-AT; lane 5, 2 h Ox-AT; lane 6, 24 h Ox-AT; lane 7, 72 h Ox-AT. Gel is representative of 3 independent experiments. Data are means (SE) of 3 independent experiments. *P ≤ 0.05. D: Western blot analysis of homogenized lung tissues for total JNK. Western blot analysis for total JNK shows total (phosphorylated and nonphosphorylated) JNK ranging in size between 46 and 54 kDa. Densitometric analysis of both bands from each sample (expressed cumulatively) using ImageJ software demonstrated total JNK was expressed in comparable levels in lung homogenates from both Ox-AT- and N-AT-treated mice. Densitometric analysis of bands showed there was no significant difference in total JNK in lung homogenates from Ox-AT- and N-AT-treated mice at 4, 24, or 72 h. Lane 1, positive control A431 cell lysate; lane 2, 4 h N-AT; lane 3, 24 h N-AT; lane 4, 72 h N-AT; lane 5, 4 h Ox-AT; lane 6, 24 h Ox-AT; lane 7, 72 h Ox-AT. Gel is representative of 3 independent experiments. Data are means (SE) of 3 independent experiments.
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Related In: Results  -  Collection

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Show All Figures
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f9: A: effect of Ox-AT on induction of NF-κB activity in lung tissue. There was a significant increase in NF-κB activity following intratracheal Ox-AT compared with PBS (4 h, P = 0.028; 24 h, P = 0.029; 72 h, P = 0.037). Intratracheal N-AT did not have any significant effect on NF-κB activity. The positive control was Jurkat cells with high endogenous NF-κB activity (P = 0.032 compared with PBS). Data are means (SE) from 3 separate experiments. *P ≤ 0.05. B: effect of Ox-AT on induction of AP-1 activity in lung tissue. There was a significant increase in AP-1 activity following intratracheal Ox-AT compared with PBS (4 h, P = 0.019; 24 h, P = 0.033; 72 h, P = 0.049). Intratracheal N-AT did not have any significant effect on AP-1 activity. The positive control was K562 cells with high endogenous AP-1 activity (P = 0.022 compared with PBS). Data are means (SE) from 3 separate experiments. *P ≤ 0.05. C: Western blot analysis of homogenized lung tissue for phosphorylated JNK. The p54 JNK isoform was expressed highly in lung homogenates from Ox-AT-treated mice compared with lung homogenates from N-AT-treated mice. Densitometric analysis of each band using ImageJ software demonstrated the level of p54 phospho-JNK was significantly higher and peaking at 24 h in lung homogenates from Ox-AT-treated mice compared with lung homogenates from N-AT-treated mice (4 h, P = 0.031; 24 h, P = 0.012; 72 h, P = 0.023). Lane 1, positive control A431 cell lysate; lane 2, 4 h N-AT; lane 3, 24 h N-AT; lane 4, 72 h N-AT; lane 5, 2 h Ox-AT; lane 6, 24 h Ox-AT; lane 7, 72 h Ox-AT. Gel is representative of 3 independent experiments. Data are means (SE) of 3 independent experiments. *P ≤ 0.05. D: Western blot analysis of homogenized lung tissues for total JNK. Western blot analysis for total JNK shows total (phosphorylated and nonphosphorylated) JNK ranging in size between 46 and 54 kDa. Densitometric analysis of both bands from each sample (expressed cumulatively) using ImageJ software demonstrated total JNK was expressed in comparable levels in lung homogenates from both Ox-AT- and N-AT-treated mice. Densitometric analysis of bands showed there was no significant difference in total JNK in lung homogenates from Ox-AT- and N-AT-treated mice at 4, 24, or 72 h. Lane 1, positive control A431 cell lysate; lane 2, 4 h N-AT; lane 3, 24 h N-AT; lane 4, 72 h N-AT; lane 5, 4 h Ox-AT; lane 6, 24 h Ox-AT; lane 7, 72 h Ox-AT. Gel is representative of 3 independent experiments. Data are means (SE) of 3 independent experiments.
Mentions: Following intratracheal instillation of Ox-AT, NF-κB was found to be significantly increased in lung homogenates in a time-dependent manner compared with PBS (4 h, P = 0.028; 24 h, P = 0.029; and 72 h, P = 0.037). Intratracheal N-AT had no significant effect on the activity of NF-κB in lung homogenates (Fig. 9A).

Bottom Line: Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion.The effect of Ox-AT was dependent on NF-kappaB and activator protein-1 (AP-1)/JNK.They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the antielastase lung protection, but also converts AT into a proinflammatory stimulus.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medicine, Univ. of Cambridge, Addenbrookes Hospital, United Kingdom.

ABSTRACT
alpha(1)-Antitrypsin (AT) is a major elastase inhibitor within the lung. Oxidation of critical methionine residues in AT generates oxidized AT (Ox-AT), which has a greatly diminished ability to inhibit neutrophil elastase. This process may contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD) by creating a functional deficiency of AT permitting lung destruction. We show here that Ox-AT promotes release of human monocyte chemoattractant protein-1 (MCP-1) and IL-8 from human lung type epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells. Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion. These findings were supported by the fact that instillation of Ox-AT into murine lungs resulted in an increase in JE (mouse MCP-1) and increased macrophage numbers in the bronchoalveolar lavage fluid. The effect of Ox-AT was dependent on NF-kappaB and activator protein-1 (AP-1)/JNK. These findings have important implications. They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the antielastase lung protection, but also converts AT into a proinflammatory stimulus. Ox-AT generated in the airway interacts directly with epithelial cells to release chemokines IL-8 and MCP-1, which in turn attracts macrophages and neutrophils into the airways. The release of oxidants by these inflammatory cells could oxidize AT, perpetuating the cycle and potentially contributing to the pathogenesis of COPD. Furthermore, these data demonstrate that molecules such as oxidants, antiproteinases, and chemokines, rather than act independently, are likely to interact to cause emphysema.

Show MeSH
Related in: MedlinePlus