The role of Cysteinyl-Leukotriene receptors in asthma

Hao, Lanping (2003) The role of Cysteinyl-Leukotriene receptors in asthma. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The main aim of this study was to investigate the genetic and immunological factors that may regulate the expression and activity of the CysLT₁ receptor. Such factors may contribute to variable tissue responsiveness to cys-LTs and to variable clinical responses to LTRA.

Detection of novel mutations in the CysLT₁R gene was performed by solid-phase chemical cleavage of mismatches (CCM). A polymorphism in the coding region was detected in 13 out of 30 asthmatic samples, and subsequent sequencing identified a nt927 T to C transition, which did not result in an amino acid substitution. Oligonucleotide ligation assays (OLA) were used to genotype two polymorphisms (T617G and G898A) previously reported by other workers in 60 asthmatic and 90 non-asthmatic individuals from the Southampton area, but neither polymorphism was found, indicating an allele frequency of less than 0.004. To validate OLA, two fragments containing the 617G and 898A alleles respectively were cloned into the pUC18 vector. OLA of these engineered pUC18 constructs confirmed the specificity and sensitivity of the assay. Together, the data suggest that CysLT₁R gene polymorphisms are either common and conservative (not affecting the amino acid sequence), or non-conservative and very rare in the population studied.

Eosinophils are key sources of cys-LTs and other mediators in asthma, and recruitment of eosinophils to the asthmatic lung depends at least in part on their responsiveness to cys-LTs, mediated by CysLT₁ and/or CysLT₂ receptors. The constitutive expression of these receptors was characterised in eosinophils and other leukocyte sub-types, and their modulation by immunopharmacological factors was investigated by flow cytometry, immunohistochemistry and Western blotting. Eosinophils transcribed mRNA for both CysLT₁R and CysLT₂R as confirmed by DNA sequencing of the RT-PCR products, but flow cytometry showed that only CysLT₁R protein, but not CysLT₂R, was expressed on the eosinophil surface, while both receptors were readily detectable on blood monocytes.