The role of NF-kB in human lung mast cells and peripheral blood eosinophils
The role of NF-kB in human lung mast cells and peripheral blood eosinophils
The hypothesis of this work is that upon allergen challenge, preformed TNF-α is released from mast cell granules, which in an autocrine manner activates the transcription nuclear factor kappa B (NF-κB), leading to increased transcription of the cytokines TNF-α, granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 8 (IL-8). In addition, TNF-α augments NF-κB activation within surrounding inflammatory cells, including peripheral blood eosinophils.
Using a monoclonal antibody specific for activated NF-κB, in conjunction with a novel immunocytochemical technique, activated NF-κB was localised to human purified lung mast cells and peripheral blood eosinophils. Activation of mast cells with stem cell factor in combination with anti-IgE for 2 hours or TNF-α for 0.25 hours induced 38% ± 5.44 and 77.4% ± 2.16 of mast cells to be immunoreactive for activated NF-κB, respectively. The autocrine effects of endogenous TNF-α were demonstrated by its modulation by a blocking monoclonal antibody for TNF-α. The use of inhibitors ascertained the involvement of tyrosine kinase, sphingosine and ceramide pathways in both anti-IgE and TNF-α induced activation of NF-κB, suggesting common elements in their signalling pathways. Activation of atopic peripheral blood eosinophils, with house dust mite and grass pollen allergens for 4 hours induced 23.6% ± 3.71 and 20.8% ± 3.47 of eosinophils to be immunoreactive for activated NF-κB, respectively. In both mast cells and eosinophils a decline in the percentage of cells immunoreactive for inhibitory kappa B alpha (IκB-α) were observed in association with the activation of NF-κB. In addition, inhibitors of NF-κB activation were demonstrated to reduce the percentage of cells immunoreactive for TNF-α, GM-CSF and IL-8 to varying extents, suggesting that NF-κB was directly involved in the transcription of these cytokines. Gel shift and supershift assays were performed to confirm the activation of NF-κB, and cytokine production was measured by ELISA.
University of Southampton
1998
Coward, William Ralph
(1998)
The role of NF-kB in human lung mast cells and peripheral blood eosinophils.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The hypothesis of this work is that upon allergen challenge, preformed TNF-α is released from mast cell granules, which in an autocrine manner activates the transcription nuclear factor kappa B (NF-κB), leading to increased transcription of the cytokines TNF-α, granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 8 (IL-8). In addition, TNF-α augments NF-κB activation within surrounding inflammatory cells, including peripheral blood eosinophils.
Using a monoclonal antibody specific for activated NF-κB, in conjunction with a novel immunocytochemical technique, activated NF-κB was localised to human purified lung mast cells and peripheral blood eosinophils. Activation of mast cells with stem cell factor in combination with anti-IgE for 2 hours or TNF-α for 0.25 hours induced 38% ± 5.44 and 77.4% ± 2.16 of mast cells to be immunoreactive for activated NF-κB, respectively. The autocrine effects of endogenous TNF-α were demonstrated by its modulation by a blocking monoclonal antibody for TNF-α. The use of inhibitors ascertained the involvement of tyrosine kinase, sphingosine and ceramide pathways in both anti-IgE and TNF-α induced activation of NF-κB, suggesting common elements in their signalling pathways. Activation of atopic peripheral blood eosinophils, with house dust mite and grass pollen allergens for 4 hours induced 23.6% ± 3.71 and 20.8% ± 3.47 of eosinophils to be immunoreactive for activated NF-κB, respectively. In both mast cells and eosinophils a decline in the percentage of cells immunoreactive for inhibitory kappa B alpha (IκB-α) were observed in association with the activation of NF-κB. In addition, inhibitors of NF-κB activation were demonstrated to reduce the percentage of cells immunoreactive for TNF-α, GM-CSF and IL-8 to varying extents, suggesting that NF-κB was directly involved in the transcription of these cytokines. Gel shift and supershift assays were performed to confirm the activation of NF-κB, and cytokine production was measured by ELISA.
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Published date: 1998
Identifiers
Local EPrints ID: 463642
URI: http://eprints.soton.ac.uk/id/eprint/463642
PURE UUID: 4c30cd46-0346-4cdf-9249-b4ec85bb2b00
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Date deposited: 04 Jul 2022 20:54
Last modified: 04 Jul 2022 20:54
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Author:
William Ralph Coward
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