The immunomodulatory effects of nociceptin – development and validation of a biosensor assay

Opioids have long been associated with increased susceptibility to infectious diseases, and the role of the classical opioids in immunomodulation is a subject of some debate. A fourth opioid-like receptor, sharing significant sequence homology, and Gi mechanism, to the classical MOP, DOP and KOP opioid receptors has emerging evidence for its expression throughout the immune system. Furthermore, observational evidence suggests that the concentration of nociceptin, the endogenous agonist for this receptor increases in the human plasma, synovial fluid and sputum sampled in the context of inflammatory conditions. Administration of exogenous nociceptin worsens mortality in an animal model of sepsis, further suggestive that this non-classical opioid may have an immunomodulatory role in-vivo.

Despite limited observational evidence, localising the source of the increased nociceptin concentration has been challenging, because of its low concentration and problems with existing use of ELISA and RIA tests. Chimeric G-proteins facilitate non-classical coupling between intracellular pathways, allowing the use of new markers as a readout of receptor activation, and therefore local detection of agonist ligands and their concentration.

This work validated the design of a chimeric G-protein based biosensor test to detect nociceptin release from a single cell. In principle, the biosensor facilitated nociceptin receptor coupling via Gq to calcium flux for a non-invasive and non-radioactive readout of receptor activation. The validated test was used to investigate nociceptin release from immunocytes (and subtypes) from healthy individuals and patients admitted to the intensive care unit with sepsis. These data were correlated with additional PCR and immunohistochemistry tests to determine expression of nociceptin, its receptor and their precursors.

In general, mixed granulocytes were found to release nociceptin. In evaluating immunocyte subsets, both nociceptin and its receptor are found in eosinophils and neutrophils from individuals with sepsis, and when incubated in an environment mimicking sepsis, suggesting upregulation of this system in sepsis.