The endocannabinoid anandamide, binds to cannabinoid CB₁ and CB₂ receptors and has both analgesic and anti-inflammatory actions (Rice et al, 2002). The non-psychoactive cannabis constituent, cannabidiol has low affinity for cannabinoid receptors yet it is an oral anti-arthritic therapeutic in murine collagen-induced arthritis (Malfait et al, 2000). Neutrophils play a critical role in the development of inflammation. The aim of this study was to investigate the effect of cannabinoids on the migration of human peripheral polymorphonuclear neutrophils (PMNs).

Peripheral PMNs were isolated from normal whole blood by centrifugation over Polymorphprep^TM . The isolated cells were resuspended at a concentration of 1x10⁶ cellsml^-1 in PBS containing CaCl₂ and MgCl₂. in vitro cell migration assays were performed using a modified 48-well Boyden Chamber. PMNs were pre-incubated with vehicle (0.01% DMSO) or test compound for 30mins at 37ºC before loading into the upper wells. The lower wells contained the corresponding concentration of test compound and N-formyl-methione-leucine-phenylalanine (fMLP). After incubation for a further 30 minutes in a 5% CO₂ atmosphere at 37° C, the migrated adherent cells on the underside of the 3 µm pore filter were stained using a Diff-Quik stain set. Each well was counted in ten non-overlapping fields (x40) using a light microscope.

Anandamide significantly (one-way ANOVA) inhibited neutrophil migration induced by fMLP (1 µM). The fMLP- induced migration was -3.87 ± 14.54% (P<0.001), 56.5 ± 10.2% (P<0.01), 44.2 ± 7.4 % (P<0.001), 56.4 ± 10.2% (P<0.01), and 75.2 ± 10.1% of vehicle control in the presence of 100nM, 10nM, 1nM, 0.1nM and 0.01nM (n = 8) anandamide respectively. Anandamide was considerably more potent than predicated for activation of CB₁ or CB₂ receptors, the pEC₅₀ value being 8.67 ± 0.35. Whilst the effect of anandamide was attenuated by the CB₁ receptor antagonist, SR141716A (1µM), the apparent K_B value of 700nM is considerably higher than that expected for antagonism of the CB₁ receptor (~2nM). The CB₂ receptor antagonist SR144528, did not attenuate anandamide-mediated inhibition of fMLP. Neither the potent non-selective CB₁/CB₂ receptor agonist CP55940 nor the endocannabinoid 2-arachidonoyl glycerol (0.01nM - 1µM) altered fMLP-induced migration of neutrophils: in the presence of these compounds the fMLP-induced migration was not significantly different from vehicle control (P>0.05, one-way ANOVA). In contrast, the plant-derived cannabinoid, cannabidiol was a highly potent inhibitor of PMN migration. The fMLP-induced migration was 18.09 ± 2.9% (P<0.001), 58.1 ± 4.5 % (P<0.05), 21.0 ± 3.7 % (P<0.001), 49.9 ± 2.4 (P<0.05) and 110 ± 13.9% (P>0.05) of control with 100nM, 10nM, 1nM, 0.1nM and 0.01nM (one-way ANOVA; n = 6) cannabidiol respectively.

These data demonstrate that anandamide and cannabidiol potently inhibit migration of PMNs and raise the possibility that they act via a novel SR141716A-sensititve non-CB₁, non-CB₂ receptor mechanism.

Malfait, A.M., Gallily, R., Sumariwalla, P.F., Malik, A.S., Andreakos, E., Mechoulam, R., & Feldmann. M. (2000) Proc Natl Acad Sci U S A.  97, 9561.
Rice, A.S.C., Farquhar-Smith, W.P. & Nagy, I. (2002). Prostaglandins Leukotrienes and Essential Fatty Acids, 66, 243.