2-acetylaminofluorene (AAF) have been established as being inhibitory on the lymphoproliferative response induced by lipopolysaccharide (LPS). We investigated the effects of AAF on protein kinase C (PKC) activation, an enzyme required for LPS-induced B-cell proliferation. PKC activity was studied in murine splenocytes by phosphorylation of PKC-specific substrate. The role of PKC as a positive signal in LPS-induced B-cell proliferation was confirmed by the observation that LPS increased the translocation of PKC to membrane and total PKC activity. In untreated splenocytes, approximately 70% of the total PKC activity was localized in the cytosol and 30% in the membrane fraction. Unexpectedly short-term treatment of AAF (10 min) to splenocytes culture significantly increased the membrane PKC activity while having little effect on total PKC activity. But, after treatment with 50 μM AAF for 18 hr, PKC activity in the cytosolic fraction decreased by 50% from control level, and splenocytes also lost 30% of total PKC activity. These results were confirmed by the observation that AAF also reduced the phosphorylation of the endogenous PKC-specific 80 kDa substrates. And, AAF was required in culture for ＞18 hr for maximum suppression of the LPS-induced B-cell proliferation, indicating that a sufficient time for depletion of PKC was need. Furthermore, as determined by the electrophoretic mobility shift assay (EMSA), AAF was shown to inhibit the binding activity of the transcription factor complex, NF-kB whose LPS-mediated induction in murine splenocytes is dependent on PKC activation. Also, a chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the NF-κB element was transfected into pre-B cell line, 70Z/3 or mature B-cell line, S194 and CH12, and assessed induction of CAT activity. AAF treatment of the transfected 70Z/3 cell lines but not S194 and CH12 cell lines, resulted in a significant inhibition of CAT activity induced by LPS. These results suggest that, although PKC may be not sole target of AAF action, AAF partially mediates its effect by a mechanism that is linked to the depletion of PKC.
Anandamide (arachidonylethanolamide), isolated from porcine brain, and 2-arachidonyl-glycerol (2-Ara-Gl), identified in canine gut, are two recently identified putative endogenous cannabinoid receptor ligands. Both ligands have been reported to possess binding affinity for cannabinoid receptor subtypes, CB1 (for brain) and CB2 (for peripheral tissue). The objective of the present studies was to investigate the immunomodulatory effects of both of these ligands in B6C3F1 mouse splenocytes. 2-Ara-Gl produced a marked and dose-related inhibition of the mixed lymphocyte response, anti-CD3 mAb-induced T-cell proliferation and LPS-induced B-cell proliferation while having no inhibitory effect on phorbol-12-myristate-13-acetate/ionomycin-induced cell proliferation. Interestingly, the inhibitory effects by 2-Ara-Gl on proliferation were at least partially dependent on cell density. At high cell density, 2-Ara-Gl enhanced lymphoproliferation while exhibiting marked inhibitory activity at low cell density. Similarly, in vitro primary IgM antibody forming cell responses which are dependent on high cell density were also found to be enhanced by 2-Ara-Gl. Conversely, anandamide exhibited no inhibitory effects on cell proliferative responses to stimulation by either anti-CD3 mAb, LPS or phorbol-12-myristate-13-acetate/ionomycin treatment. Anandamide also showed no effect on the in vitro sRBC AFC response.
Cannabinoid compounds inhibited the forskolin-stimulated adenylate cyclase through its receptor. Therefore, to elucidate whether the effects of 2-Ara-Gl was mediated through cannabinoid receptor, cannabinoid receptor expression was characterized in lymphoid preparations. Northern analysis of purified poly(A) RNA showed very low expression of cannabinoid receptor, CB1 (brain-type), in mouse spleen. Conversely, RNA transcripts for receptor subtype, CB2 (peripheral), were readily expressed in mouse and rat spleen and at low levels in mouse thymus. Northern analysis of human T-cell-lines, Jurkat E6-1 and HPB-ALL revealed mRNA transcripts for CB2 but not CB1. These results provide further evidence that CB2 is the predominant cannabinoid receptor in lymphoid cells. And 2-Ara-Gl markedly inhibited forskolin-stimulated cAMP accumulation in splenocytes. Additionally, anandamide showed negligible inhibitory effects at extremely high concentrations on forskolin-stimulated adenylate cyclase activity and no effect on basal adenylate cyclase activity in splenocytes. Taken together, these results demonstrated biological activity in mouse splenocytes for 2-Ara-Gl, putative endogenous cannabinoid receptor ligand.