Opioid receptor expression on neutrophils: effect of tumour necrosis factor alpha treatment
© BioMed Central Ltd. 2007
Published: 22 March 2007
Opioids and endogenous opioid peptides possess immunomodulating properties and are involved in the regulation of immunocyte activity. These effects are mediated by opioid receptors (OR) on peripheral white blood cells that can be detected and quantified by flow cytometry. We investigated OR expression on neutrophils using polyclonal antibodies against δ-opioid, κ-opioid and μ-opioid receptors (DOR, KOR, MOR) to assess spontaneous and TNFα-induced OR expression on neutrophils.
After approval by the local ethics committee and informed consent, 100 μl whole blood samples from 11 healthy volunteers (EDTA served as anticoagulant) was incubated with 10 μl TNF-α (100 ng/ml) for 3, 6, 12 and 24 hours. Samples were washed and incubated with 5 μl fluorescein-isothiocynate (FITC)-labelled polyclonal antibodies against human MOR, KOR and DOR. Rabbit IgG antibodies served as a negative control. After red cell lysis, flow cytometry was performed to quantify OR expression using live gating on neutrophils. The percentage of positive cells as well as mean fluorescent intensities (MFIs) were determined.
Stimulation with TNFα increased the percentage of DOR-expressing cells significantly from 2.1% to 12.8% positive cells after 6 hours. After 12 hours of stimulation 28.6%, and after 24 hours even 68.1%, of neutrophils expressed DOR. The MFI increased during TNFα stimulation from 47.6 ± 15.7 to 254.8 ± 110 after 6 hours, staying levelled at this height. After 6 hours of stimulation, the MFI for KOR reached a maximum, rising from 59.5 ± 19.4 to 513.9 ± 162. During TNFα stimulation, the percentage of positive cells increased from 5.4 to 65.0% after 24 hours (17.5% at 6 hours). Fourteen per cent of neutrophils expressed MOR after 6 hours (initially 5.4%) and up to 57.2% after 24 hours. The MOR MFI was measured at 43.4 ± 16.3 to 410 ± 263.6 6 hours after stimulation with TNFα.
Our results display that stimulation of whole blood with TNFα amplifies OR expression of all subtypes significantly on neutrophils. We suggest further studies to clarify the specific actions of opioids and their receptors in health and acute inflammation.