Regulation of eicosanoid enzyme expression in inflammatory leukocytes in asthma
Regulation of eicosanoid enzyme expression in inflammatory leukocytes in asthma
Peripheral blood eosinophils from asthmatic patients generate more cys-LT in vitro than normal eosinophils, and this can be mimicked by priming with eosinophilopoietic cytokines such as IL-5. Using immunocytochemistry, immunofluorescence and SDS-PAGE/Western blotting, the expression of 5-LO and FLAP was examined in blood eosinophils from normal and asthmatic subjects, and following in vitro culture with IL-5 and the glucocorticoid dexamethasone. IL-5 significantly increased the proportion of eosinophils immunostaining for FLAP (65±4%) when compared to control (34±4%, n=14, p<0.0001), enhanced protein levels of FLAP 51±14% (n=5, p=0.03), and increased ionophore-stimulated synthesis of cys-LT 4-fold from 5.7 to 20.8 ng/106 cells, as measured by ELISA (n=10, p<0.02). The IL-5 induced FLAP expression was abolished by the protein synthesis inhibitor cycloheximide and the transcription inhibitor actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (n=8, p<0.0001), but this did not alter cys-LT synthesis. In contrast, neither IL-5 nor dexamethasone altered the expression of 5-LO, although IL-5 significantly increased 5-LO immunofluorescence localising to the nucleus. A higher proportion of eosinophils isolated from atopic asthmatics immunostained for FLAP when compared to those of normal subjects, and IL-5 significantly increased 5-LO immunofluorescence localising to the nucleus. A higher proportion of eosinophils isolated from atopic asthmatics immunostained for FLAP when compared to those of normal subjects, and IL-5 induced increases in FLAP immunoreactivity were smaller than observed in normals (p<0.05). IL-5 up-regulates the expression of FLAP and translocates 5-LO to the nucleus in normal blood eosinophils in vitro, leading to an enhanced capacity for cys-LT synthesis, mimicking in vivo changes in the 5-LO pathway in eosinophils of allergic asthmatics.
These studies indicate that the activity of the cys-LT pathway can be modulated in vitro and in vivo by genetic factors, by the cytokine microenvironment and by drugs. Understanding how these influences interact may throw light on mechanisms central to allergic and non-allergic inflammation in asthma, and help to characterise asthma phenotypes and asthmatic individuals most likely to benefit from anti-LT therapy.
University of Southampton
1998
Cowburn, Andrew Stephen
(1998)
Regulation of eicosanoid enzyme expression in inflammatory leukocytes in asthma.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Peripheral blood eosinophils from asthmatic patients generate more cys-LT in vitro than normal eosinophils, and this can be mimicked by priming with eosinophilopoietic cytokines such as IL-5. Using immunocytochemistry, immunofluorescence and SDS-PAGE/Western blotting, the expression of 5-LO and FLAP was examined in blood eosinophils from normal and asthmatic subjects, and following in vitro culture with IL-5 and the glucocorticoid dexamethasone. IL-5 significantly increased the proportion of eosinophils immunostaining for FLAP (65±4%) when compared to control (34±4%, n=14, p<0.0001), enhanced protein levels of FLAP 51±14% (n=5, p=0.03), and increased ionophore-stimulated synthesis of cys-LT 4-fold from 5.7 to 20.8 ng/106 cells, as measured by ELISA (n=10, p<0.02). The IL-5 induced FLAP expression was abolished by the protein synthesis inhibitor cycloheximide and the transcription inhibitor actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (n=8, p<0.0001), but this did not alter cys-LT synthesis. In contrast, neither IL-5 nor dexamethasone altered the expression of 5-LO, although IL-5 significantly increased 5-LO immunofluorescence localising to the nucleus. A higher proportion of eosinophils isolated from atopic asthmatics immunostained for FLAP when compared to those of normal subjects, and IL-5 significantly increased 5-LO immunofluorescence localising to the nucleus. A higher proportion of eosinophils isolated from atopic asthmatics immunostained for FLAP when compared to those of normal subjects, and IL-5 induced increases in FLAP immunoreactivity were smaller than observed in normals (p<0.05). IL-5 up-regulates the expression of FLAP and translocates 5-LO to the nucleus in normal blood eosinophils in vitro, leading to an enhanced capacity for cys-LT synthesis, mimicking in vivo changes in the 5-LO pathway in eosinophils of allergic asthmatics.
These studies indicate that the activity of the cys-LT pathway can be modulated in vitro and in vivo by genetic factors, by the cytokine microenvironment and by drugs. Understanding how these influences interact may throw light on mechanisms central to allergic and non-allergic inflammation in asthma, and help to characterise asthma phenotypes and asthmatic individuals most likely to benefit from anti-LT therapy.
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Published date: 1998
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Local EPrints ID: 463543
URI: http://eprints.soton.ac.uk/id/eprint/463543
PURE UUID: 273b9a37-8e20-420a-9b9b-ca72ccdc76f8
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Date deposited: 04 Jul 2022 20:53
Last modified: 04 Jul 2022 20:53
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Author:
Andrew Stephen Cowburn
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