2019SCOTTSWMMD.pdf (5.44 MB)
N/OFQ and the Granulocyte
thesisposted on 2019-06-12, 15:50 authored by Simon Scott
Sepsis is a life-threatening syndrome associated with the deaths of 44,000 people in the UK per year. Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) have demonstrated associations with sepsis and cells of the immune system. This thesis describes studies examining the expression of mRNA transcripts for the NOP receptor and the N/OFQ precursor ppNOC. It further describes the development of a novel live-imaging assay for the study of N/OFQ release from single human granulocytes in vitro. mRNA experiments demonstrated that ppNOC is predominately expressed in eosinophils. Decreased expression of mRNA for NOP in isolated human PMNs was seen following 20 hours of exposure to 0, 1, 2.5 and 5 μg ml-1 LPS in vitro. NOP expression in PMNs after whole blood exposure to 5 hours of LPS at 0, 0.01, 0.1 and 1 μg ml-1 for 5 hours was decreased. The decrease in expression between 0 and 1 μg ml-1 for whole blood treated samples was ~200-fold; for isolated PMN treated samples it was 3-fold. There was an apparent rightward shift in the concentration-response curve. Other factors in whole blood are likely involved in modulation of NOP expression in PMNs to LPS. Determination of PMN release of N/OFQ was achieved through development of a novel bioassay. Chimeric hNOP CHOαi/q were loaded with Fluo-4 to detect N/OFQ-associated [Ca2+]i increases by fluorescence. Confocal microscopy was used to co-image CHO cells and live human PMNs, also loaded with Fluo-4. Fluorescence of PMNs was observed in fMLP-activated PMNs. Subsequent fluorescence was observed in individual CHO cells indicating release of N/OFQ by stimulated PMNs. Exposure of CHO cells alone to fMLP-stimulated PMN supernatant was tested in the presence of a NOP antagonist (TRAP-101) and / or purinergic type 2 receptor antagonist (PPADS). Fewer CHO cells fluoresced to supernatant in the presence of TRAP-101 compared with PPADS, suggesting fMLP-stimulated PMN supernatant contains N/OFQ. These data demonstrate for the first time the visualised real-time release of N/OFQ from human immune cells. PMNs both produce and respond to N/OFQ. Further work will determine the cell types involved and their relevance to sepsis.
Supervisor(s)Thompson, Jonathan P; Lambert, David G
Date of award2019-04-26
Author affiliationDepartment of Cardiovascular Sciences
Awarding institutionUniversity of Leicester