Cannabinoids have been shown to possess anti-inflammatory properties, suppressing the production of several key inflammatory mediators including IL-8 (Ihenetu et al., 2003). However, despite the well documented immunosuppressive and historical use of these compounds as anti-inflammatory agents, the mechanisms underlying their anti-inflammatory actions are poorly understood. Experiments were therefore carried out to identify the molecular mechanism(s) through which cannabinoids modulate inflammation. These studies have focused mainly on NF-κB and have examined the effects of the cannabinoid receptor agonist Win55212-2 on the activation of this ubiquitous transcription factor in parallel with the regulation of IL-8 release induced by TNF-α in the human HT-29 colon epithelial cell line.

Release of IL-8 was initiated by stimulating confluent monolayers of HT-29 cells with TNF-α (100 ng ml^-1) for 24h. When present, Win55212-2 (10^-10-10^-5 M) was added to cultures 30 min prior to stimulation with TNF-α. IL-8 release was determined by ELISA using a biotinylated anti-human IL-8 antibody (0.5 µg ml^-1) and a streptavidin linked peroxidase conjugate (0.5 µg ml^-1). Changes in expression of IκB-α in lysates obtained from cells treated with TNF-α (100 ng ml^-1) in the absence and presence of Win5512-2 (10^-10-10^-5 M) were determined by western blotting using an anti-IκB-α antibody. Modulation of TNF-α-induced NFκB-DNA binding by Win5512-2 (10^-10-10^-5M) was monitored by electromobility shift assays in nuclear extracts from cells treated with these agents.

Incubation of cells with TNF-α at a concentration of 100 ng ml^-1 resulted in a time-dependent increase in IL-8 release which was enhanced from a basal value of 890 ± 91 pg ml^-1 to 19181 ±182 pg ml^-1 at 24 h (n=6). This increase was inhibited in a concentration dependent manner by Win55212-2 which, when added prior to TNF-α, reduced IL-8 releases by approximately 50% at 10^-6 M. In contrast, the addition of Win55212-2 simultaneously with or after the addition of TNF-α did not produce any significant decrease in the release of IL-8. Pretreatment of cells with Win55212-2 reversed the degradation of IκB-α and subsequent activation of NF-κB induced by TNF-α. These effects occurred at concentrations which overlapped with those that caused inhibitions of IL-8 release, and at 10^-5 M Win55212-2 virtually abolishing both TNF-α induced IκB degradation and NF-κB-DNA binding.

In conclusion, the data obtained from these studies show that IL-8 release in HT-29 cells can be inhibited by Win55212-2 and further suggests that this action may be mediated by Win55212-2 negatively regulating the signaling cascade that leads to the activation of NF-κB.