Leukotriene E4 receptors in bronchial asthma

Abstract

Cysteinyl leukotriene (LTC₄, LTD₄ and LTE₄) are proinflammatory lipid mediators involved in the pathogenesis of asthma. LTE₄ the most stable of the cysteinyl leukotrienes elicits bronchoconstriction, and unlike LTC₄ and LTD₄ it enhances bronchial hyperresponsiveness. LTE₄ can also mediate other features of asthma, such as eosinophilia, increased mucus secretion and vascular permeability. None of these LTE₄ activities can be explained by current understanding of classical cysteinyl leukotriene receptors, CysLT₁ and CysLT₂, suggesting the presence of a novel receptor that preferentially responds to LTE₄. LTE₄ mediated pulmonary inflammation in a murine model was shown to remain intact in the absence of CysLT₁ and CysLT₂, and was abrogated by the knockout of the purinergic receptor, P2Y₁₂. This study aimed to elucidate whether LTE₄ directly signalled through the P2Y₁₂ receptor. Models of human CysLT₁, CysLT₂ and P2Y₁₂ overexpressed in HEK293T, CHO cells and human platelets were used to characterise responsiveness to cysteinyl leukotrienes. LTE₄ failed to induce any specific signalling responses in cells expressing P2Y₁₂ or in human platelets, showing that LTE₄ does not directly signal through P2Y₁₂. Previous reports have shown that LTE₄ is a potent agonist in human mast cells and so the aim was to identify the receptor responsible for LTE₄-induced responses using two human mast cell lines, LAD2 and LUVA. When intracellular signalling and gene expression were compared in response to stimulation, LUVA cells responded to LTC₄ and LTD₄ but lacked the potent responses to LTE₄ observed in LAD2 cells. Gene screening by microarrays was used to identify 4 target GPCRs which were cloned and their responsiveness to cysteinyl leukotrienes analysed in HEK293T overexpression model. CysLT₁ was identified as the only GPCR responding to cysteinyl leukotrienes and differently expressed in LAD2 cells. Stable knockdowns were created and analysed to reveal CysLT₁ was responsible for the augmented responses to LTE₄ observed in LAD2 cells.

Date of Award	1 Jan 2015
Original language	English
Awarding Institution	King's College London
Supervisor	Greg Woszczek (Supervisor), Christopher Corrigan (Supervisor) & James Pease (Supervisor)