The influence of endotoxin on the expression of the ORL-1 receptor

Objective

The ORL-1 receptor (Orphan opioid receptor) has been discovered recently and is involved in pain perception and immune function [1,2,3]. The regulation of the ORL-1 receptor in patients with systemic inflammation has not been elucidated yet. This study investigates the influence of different doses of LPS on ORL-1 expression in peripheral blood cells ex vivo.

Methods

Human whole blood from healthy volunteers was cultured at 37°C and 5% CO₂ without LPS or LPS 0.1 ng, 10 ng and 100 ng/ml for 3, 6, 12 and 24 hours. Reverse transcriptase polymerase chain reaction (rt-PCR) using specific primers was performed and RNA contents was estimated by semiquantitative analysis employing a housekeeping gene as internal standard. Southern blot analysis and hybridisation to a specific DIG-labeled probe confirmed the identity of the ORL-1 transcripts.

Statistics

Mean ± SEM, repeated measures ANOVA.

Results

ORL-1 receptor was expressed constitutively in human peripheral blood cells. Mean baseline expression resulted in a ratio of ORL/GAPDH of 1.0 ± 0.07. Semiquantitative rt-PCR revealed a dose and time dependent down regulation of ORL-1 expression (P < 0.05). Incubation with LPS 0.1 ng/ml decreased the ratio ORL/GAPDH from 0.95 ± 0.06 at 3 hours to 0.19 ± 0.02 at 24 hours. In contrast, incubation with LPS 100 ng/ml already suppressed the ORL-1 message after 3 hours of incubation. Southern blot analysis and hybridisation proved the specificity of the amplified PCR products for ORL-1 transcripts.

Conclusions

Endotoxin decreased ORL-1 expression in human peripheral blood cells. The results suggest that the ORL-1 receptor is involved in immune response to infection.