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Mite and cockroach proteases activate p44/p42 MAP kinases in human lung epithelial cells.

Kuderer NM, San-Juan-Vergara HG, Kong X, Esch R, Lockey RF, Mohapatra SS - Clin Mol Allergy (2003)

Bottom Line: RESULTS: Each of these antigens induced a significant increase in IL-8 levels compared to the medium control.PD98059, a MEK1 inhibitor, significantly decreased phosphorylation of p44/p42 MAPKs and IL-8 production.Exposure of A549 cells with antigens, which had been preincubated with different protease inhibitors, also resulted in a reduction of both MAPK phosphorylation and IL-8 production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Joy McCann Culverhouse Airway Disease Center, University of South Florida and James A Haley VA Hospital, Tampa, FL, USA. smohapat@hsc.usf.edu

ABSTRACT
BACKGROUND: The mechanisms underlying epithelial cell activation by indoor inhaled antigens are poorly understood. METHODS: In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 epithelial cells upon exposure to antigens of house dust mite (HDMA), German cockroach (GCA), and American cockroach (ACA). RESULTS: Each of these antigens induced a significant increase in IL-8 levels compared to the medium control. Exposure of A549 cells to these antigens induced the phosphorylation of p44/42 MAPKs within 5 minutes, which reached a peak at 25 minutes later and reached baseline levels at 1 hour after exposure. PD98059, a MEK1 inhibitor, significantly decreased phosphorylation of p44/p42 MAPKs and IL-8 production. Exposure of A549 cells with antigens, which had been preincubated with different protease inhibitors, also resulted in a reduction of both MAPK phosphorylation and IL-8 production. CONCLUSION: Thus, proteolytic antigens present in HDMA, GCA and ACA activate the p44/42 MAPKs airway epithelial cells, which lead to elevated IL-8 production and initiation of the inflammatory cascade.

No MeSH data available.


Related in: MedlinePlus

Exposure to allergenic extracts induces both IL-8-transcription and IL-8 secretion. (A) Time-dependent IL-8 transcription after exposure to allergenic extracts. IL-8 mRNA expression was evaluated by RT-PCR and PCR. Total RNA of A549 cells exposed to Der f-HDMA (25 μg/ml) and GCA (12.5 μg/ml) was isolated at different time points. PCR-amplified IL-8 cDNA bands were quantified by densitometry and standardized with β-actin levels of intensity. Experiments were performed twice. Representative experiments are shown. (B) HDM, GCA and ACA induce IL-8 release in A549 cells in a concentration-dependent fashion. Following 24 hours of exposure to indicated concentrations of HDMA, GCA, and ACA. ELISA assessed IL-8 concentration in cell culture supernatants. The experiment was performed twice and mean values and standard deviations are shown, *, p < 0.05; * *, p < 0.005. (C) Allergens differ in their ability to induce detachment of A549 cells from monolayers. A549 cells were exposed to increasing concentrations (10–500 μg/ml) of Der f-HDMA, GCA or ACA. Cell detachment was assessed by methylene blue assay. For the assay, A549 cells were grown in 96-well plates (2.5 × 103 cells/well) and treated with various concentrations of allergen extracts. Following the 6-hour exposure period, the cells were fixed with formol saline, stained for 30 minutes with methylene blue and excess dye removed by several washings. The intracellular methylene blue was eluted with ethanol and the concentration of dye in each well quantified by spectrophotometry at a wavelength of 650 nm. The experiment was performed three times and a representative experiment is shown, *, p < 0.05.
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Figure 4: Exposure to allergenic extracts induces both IL-8-transcription and IL-8 secretion. (A) Time-dependent IL-8 transcription after exposure to allergenic extracts. IL-8 mRNA expression was evaluated by RT-PCR and PCR. Total RNA of A549 cells exposed to Der f-HDMA (25 μg/ml) and GCA (12.5 μg/ml) was isolated at different time points. PCR-amplified IL-8 cDNA bands were quantified by densitometry and standardized with β-actin levels of intensity. Experiments were performed twice. Representative experiments are shown. (B) HDM, GCA and ACA induce IL-8 release in A549 cells in a concentration-dependent fashion. Following 24 hours of exposure to indicated concentrations of HDMA, GCA, and ACA. ELISA assessed IL-8 concentration in cell culture supernatants. The experiment was performed twice and mean values and standard deviations are shown, *, p < 0.05; * *, p < 0.005. (C) Allergens differ in their ability to induce detachment of A549 cells from monolayers. A549 cells were exposed to increasing concentrations (10–500 μg/ml) of Der f-HDMA, GCA or ACA. Cell detachment was assessed by methylene blue assay. For the assay, A549 cells were grown in 96-well plates (2.5 × 103 cells/well) and treated with various concentrations of allergen extracts. Following the 6-hour exposure period, the cells were fixed with formol saline, stained for 30 minutes with methylene blue and excess dye removed by several washings. The intracellular methylene blue was eluted with ethanol and the concentration of dye in each well quantified by spectrophotometry at a wavelength of 650 nm. The experiment was performed three times and a representative experiment is shown, *, p < 0.05.

Mentions: In naive A549 cells, IL-8 mRNA transcripts were undetectable (Figure 4A). Antigens of house dust mite (HDMA), German cockroach (GCA) and American cockroach (ACA) induced a clear increase in IL-8 mRNA levels over 24 hours. In contrast, control β-actin mRNA levels did not change due to these stimuli.


Mite and cockroach proteases activate p44/p42 MAP kinases in human lung epithelial cells.

Kuderer NM, San-Juan-Vergara HG, Kong X, Esch R, Lockey RF, Mohapatra SS - Clin Mol Allergy (2003)

Exposure to allergenic extracts induces both IL-8-transcription and IL-8 secretion. (A) Time-dependent IL-8 transcription after exposure to allergenic extracts. IL-8 mRNA expression was evaluated by RT-PCR and PCR. Total RNA of A549 cells exposed to Der f-HDMA (25 μg/ml) and GCA (12.5 μg/ml) was isolated at different time points. PCR-amplified IL-8 cDNA bands were quantified by densitometry and standardized with β-actin levels of intensity. Experiments were performed twice. Representative experiments are shown. (B) HDM, GCA and ACA induce IL-8 release in A549 cells in a concentration-dependent fashion. Following 24 hours of exposure to indicated concentrations of HDMA, GCA, and ACA. ELISA assessed IL-8 concentration in cell culture supernatants. The experiment was performed twice and mean values and standard deviations are shown, *, p < 0.05; * *, p < 0.005. (C) Allergens differ in their ability to induce detachment of A549 cells from monolayers. A549 cells were exposed to increasing concentrations (10–500 μg/ml) of Der f-HDMA, GCA or ACA. Cell detachment was assessed by methylene blue assay. For the assay, A549 cells were grown in 96-well plates (2.5 × 103 cells/well) and treated with various concentrations of allergen extracts. Following the 6-hour exposure period, the cells were fixed with formol saline, stained for 30 minutes with methylene blue and excess dye removed by several washings. The intracellular methylene blue was eluted with ethanol and the concentration of dye in each well quantified by spectrophotometry at a wavelength of 650 nm. The experiment was performed three times and a representative experiment is shown, *, p < 0.05.
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Related In: Results  -  Collection

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Figure 4: Exposure to allergenic extracts induces both IL-8-transcription and IL-8 secretion. (A) Time-dependent IL-8 transcription after exposure to allergenic extracts. IL-8 mRNA expression was evaluated by RT-PCR and PCR. Total RNA of A549 cells exposed to Der f-HDMA (25 μg/ml) and GCA (12.5 μg/ml) was isolated at different time points. PCR-amplified IL-8 cDNA bands were quantified by densitometry and standardized with β-actin levels of intensity. Experiments were performed twice. Representative experiments are shown. (B) HDM, GCA and ACA induce IL-8 release in A549 cells in a concentration-dependent fashion. Following 24 hours of exposure to indicated concentrations of HDMA, GCA, and ACA. ELISA assessed IL-8 concentration in cell culture supernatants. The experiment was performed twice and mean values and standard deviations are shown, *, p < 0.05; * *, p < 0.005. (C) Allergens differ in their ability to induce detachment of A549 cells from monolayers. A549 cells were exposed to increasing concentrations (10–500 μg/ml) of Der f-HDMA, GCA or ACA. Cell detachment was assessed by methylene blue assay. For the assay, A549 cells were grown in 96-well plates (2.5 × 103 cells/well) and treated with various concentrations of allergen extracts. Following the 6-hour exposure period, the cells were fixed with formol saline, stained for 30 minutes with methylene blue and excess dye removed by several washings. The intracellular methylene blue was eluted with ethanol and the concentration of dye in each well quantified by spectrophotometry at a wavelength of 650 nm. The experiment was performed three times and a representative experiment is shown, *, p < 0.05.
Mentions: In naive A549 cells, IL-8 mRNA transcripts were undetectable (Figure 4A). Antigens of house dust mite (HDMA), German cockroach (GCA) and American cockroach (ACA) induced a clear increase in IL-8 mRNA levels over 24 hours. In contrast, control β-actin mRNA levels did not change due to these stimuli.

Bottom Line: RESULTS: Each of these antigens induced a significant increase in IL-8 levels compared to the medium control.PD98059, a MEK1 inhibitor, significantly decreased phosphorylation of p44/p42 MAPKs and IL-8 production.Exposure of A549 cells with antigens, which had been preincubated with different protease inhibitors, also resulted in a reduction of both MAPK phosphorylation and IL-8 production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Joy McCann Culverhouse Airway Disease Center, University of South Florida and James A Haley VA Hospital, Tampa, FL, USA. smohapat@hsc.usf.edu

ABSTRACT
BACKGROUND: The mechanisms underlying epithelial cell activation by indoor inhaled antigens are poorly understood. METHODS: In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 epithelial cells upon exposure to antigens of house dust mite (HDMA), German cockroach (GCA), and American cockroach (ACA). RESULTS: Each of these antigens induced a significant increase in IL-8 levels compared to the medium control. Exposure of A549 cells to these antigens induced the phosphorylation of p44/42 MAPKs within 5 minutes, which reached a peak at 25 minutes later and reached baseline levels at 1 hour after exposure. PD98059, a MEK1 inhibitor, significantly decreased phosphorylation of p44/p42 MAPKs and IL-8 production. Exposure of A549 cells with antigens, which had been preincubated with different protease inhibitors, also resulted in a reduction of both MAPK phosphorylation and IL-8 production. CONCLUSION: Thus, proteolytic antigens present in HDMA, GCA and ACA activate the p44/42 MAPKs airway epithelial cells, which lead to elevated IL-8 production and initiation of the inflammatory cascade.

No MeSH data available.


Related in: MedlinePlus