BBSRC Funded Biosensor Development

Biosensor based approach to measure release of Nociceptin/Orphanin FQ from live single immune cells and consequences for immune cell function.

The aim of this BBSRC-funded project is to determine the role the NOP-N/OFQ system plays in regulated immune cells. We have preliminary data using reverse-transcription polymerase chain reactions (rtPCR) looking at RNA levels, which cells process into protein, as well as initial studies looking at mixed groups of immune cells to determine whether N/OFQ is present in this cells (figure 1). We have detected the presence of N/OFQ using a confocal microscope to measure the increase in calcium in cells expressing the NOP receptor which has been modified to interact with calcium stores in the cell (Gq coupled receptor) immune cells are responsible for the innate and adaptive protection of the human body from invading pathogens. In a healthy system, these cells will identify an invading pathogen, destroy it (the inflammatory response) and 'switch off' allowing the body to recover and function as normal. In certain cases, immune cells activate either in the presence of a pathogen or of their own accord and do not switch off, leading to numerous health issues (e.g. asthma or sepsis). When immune cells are activated, they release a number of cytokines (small proteins) and peptides which modulate the environment the immune cells have been drawn to, as well as modulating the immune cells themselves. We believe one such peptide realised by certain types of immune cells is nociception/orphanin FQ (N/OFQ).

PCR distribution of NoP and ppNoc

Figure 1: A: PCR distribution of NOP and ppNoc in different immune cell populations separated using FACS. B: ERK1/2 phosphorylation in EOL-1 (eosinophil-like cancerous cell line) cells in response to N/OFQ (1μM) stimulation for naive and SB-612,111 (NOP antagonist) (1μM) treated (for 72h) cells. The substantial reduction in basal in the treated cells suggests that EOL-1 cells release N/OFQ, activates NOP and elevates basal phosphorylation. An N/OFQ response is ‘unmasked’ in SB-612,111 treated cells.

N/OFQ is an opioid peptide which binds to, and activates, a receptor protein on the surface of cells, called the Nociceptin Opioid Peptide (NOP) receptor. The NOP receptor is part of the opioid receptor family, with the most well-known receptor in this family being the Mu (µ) receptor which is activated by morphine to relieve pain. The NOP receptor is found throughout the body and has been implicated in a wide range of functions. We believe the NOP receptor is found on immune cells and, when activated, can regulate immune cell function and migration. Furthermore, we also believe that specific members of the immune cell family that contain N/OFQ, release this peptide to inhibit movement of immune cells away from the site of infection. This leads to a build-up of active immune cells, which can then lead to a prolonged immune response.

CHO biosensor to PMN

Figure 2: Response of CHO-biosensor to PMN degranulation. In (A) (1-4) the PMN is stimulated with fMLP leading to degranulation. There is subsequent increased fluorescence of the CHO biosensor (A, 5-7) suggesting stimulation by products of PMN degranulation. All cells return to baseline fluorescence (A, 7-8). This response is displayed graphically (B). The effect of antagonists on CHO-biosensors when exposed to the supernatant from fMLP-stimulated PMNs is displayed in panel C; in the presence of a NOP antagonist the CHO-biosensor response is inhibited, but not when solely in the presence of the non-specific purinergic receptor antagonist PPADS. This suggests the supernatant contains N/OFQ.

This initial work has been based on mixed groups of immune cells, otherwise known as polymorphonuclear cells (PMNs). Future work will see us separate these mixed cells into their individual cells types (eosinophils, neutrophils, basophils, etc) to determine which cells contain N/OFQ and/or the NOP receptor. We will confirm our findings by using both western blot and immunofluorescence assays (specific antibodies for N/OFQ will attach to the peptide and allow us to detect it in the immune cell of interest).

The project is led by Professor David Lambert, a peptide pharmacologist who has been working predominantly on opioids for over 25 years with that last 19 years (since its discovery) on Nociceptin/Orphanin FQ (N/OFQ). We have been heavily involved with collaborators in Italy (Dr Girolamo Caló and Dr Remo Guerrini of the University of Ferrara) looking at very basic questions such as structure-activity relationships for peptide-receptor interaction through to modulation of the release of glutamate in the rat brain. Doctor Jonathon Willets is a co-supervisor on the project and is a molecular pharmacologist whose primary interests are in GPCR regulation and how this controls smooth muscle tone. Over the last 15 years we have pioneered the use of molecular probes and confocal imaging techniques to monitor intracellular signalling pathways in real time. These powerful techniques have enabled the examination of the regulation of endogenous GPCR signalling in a number of cell lines, and complex primary cells including hippocampal neurones.

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