Human lymphocytes express cannabinoid (CB) receptors and endogenous cannabinoids are thought to play a role as immune modulators. The aim of this study was to examine the long term changes in CB2 receptor protein expression in activated T lymphocytes and to determine whether the receptors expressed were biochemically functional. Human peripheral blood lymphocytes (PBLs) derived from whole blood of normal, healthy donors were activated by Staphylococcal enterotoxin B (SEB) and then clonally expanded for up to 14 days with IL-2. Aliquots of PBLs were collected each day, whole cell lysates prepared and CB2 receptor levels determined by Western blotting. CB2 receptor protein levels increased after 72 hours of activation and were maintained up to 7 days after initial PBL isolation. Levels were then downregulated and remained weak for the rest of the culture period. The CB2 receptor protein could not be detected in resting T cells purified from PBLs. Aliquots of 5 day old PBLs were stimulated with cannabinoids and whole cell lysates subsequently analysed for changes in ERK phosphorylation by Western blotting to establish whether the receptors couple to signal transduction pathways. Although unstimulated cells express some phosphorylated ERK, both anandamide ( 100nM) and 2-arachidonoyl glycerol (2-AG; 5 µM) can induce an increase in levels above basal. Thus, our results demonstrate that CB2 receptor protein expression is differentially increased upon T cell activation and that the upregulated receptor is biochemically functional.

A leukemic T cell line, Jurkat, was used as a model to examine the effects of 2-AG on T cell chemotaxis as demonstrated in a transwell migration assay system. The chemotactic index, where basal migration is indicated by a value of 1, was used to analyse the data obtained. Jurkats did not migrate towards 2-AG (10nM-10 µM), eliciting changes in chemotactic index no bigger than +/- 0.32 compared to the basal. As previously published (Curnock et al, 2003), Jurkats chemotax to the CXCR4 ligand stromal cell-derived factor (SDF)-1 (0.1-10nM). Subsequent experiments were designed to determine whether 2-AG could modulate this effect. Pretreating cells with 2-AG (1 µM) alone had no significant effect on the migration to SDF-1. MAFP is an inhibitor of one of the enzymes involved in anandamide and 2-AG breakdown. Interestingly, when cells were pretreated with both MAFP (1 µM) and 2-AG (1 µM) the number of cells migrating towards SDF-1 increased. An increase in chemotactic index from 4.25 ( ±0.26, n=3) with 2-AG alone to 14.18 ( ±0.58, n=3) with MAFP and 2-AG was elicited (p<0.05). Thus it appears that although 2-AG does not itself induce chemotaxis of Jurkats, it may modulate the migration of cells to SDF-1 when it is present in high enough concentrations.

Curnock et al (2003) J. Immunol., 170: 4021-4030