Abstract
Abstract: :
Purpose: In ocular infectious diseases prominent expression of class II MHC molecules has been detected in microglia and perivascular cells. We have shown that cannabinoids affect MHC class II protein expression at the transcriptional level through the action on CIITA (Class II Transactivator). Promoter IV of the mouse CIITA gene primarily controls IFN–γ inducible expression. The INF–γ protein binds to its receptor, leading to STAT–1 activation, which is necessary for the expression of IRF–1. STAT–1 also binds cooperatively with USF–1 to a composite GAS/E box motif present in promoter IV of the CIITA gene. Synergistic activation of promoter IV by STAT–1, USF–1, IRF–1 and IRF–2 leads to expression of CIITA, which then activates transcription of Class II MHC. This study was undertaken to determine whether the modulation of CIITA by cannabinoids is dependent on STAT–1. Methods: EOC 20 microglial cells were maintained according to manufacturer’s (ATCC) protocol. Confluent cells received the following treatment 1) media alone, 2) media with 100U/ml IFN–γ, 3) media with 50 nM CP55, 940, 4) media with INF–γ and CP55, 940. 5) media with 150nM SR141716A, 6) media with INF–γ, CP55, 940 and SR14176A. Protein extracts for gel–electrophoresis were made from the cells treated as above according to standard techniques and Western blotted with monoclonal antibodies to either STAT 1, STAT–1 that recognizes the phosphorylated form, IRF–1 and IRF–2. Results: Our western blot data indicate that 50 nM of CP55, 940 effects the phosphorylation of STAT–1 and downregulates the expression of IRF–1. No changes in the immunoreactivity of STAT–1 and IRF–2 were observed between cells exposed to cannabinoids and untreated cells. The effect is receptor mediated since the receptor antagonist, SR141716A (150nM) abolishes the inhibitory effect of CP55, 940. Conclusions: Our data suggest that cannabinoids effect the phosphorylation of STAT–1 and down regulate the expression of IRF–1, therefore cannabinoids may reduce cAMP–dependent kinase activity which effects activation, phosphorylation and nuclear import of STAT–1 as well as expression of IRF–1. Other possibilities may exist as to how cannabinoids influence CIITA expression. A detailed understanding of the molecular mechanisms whereby cannabinoids modulate CIITA expression may lead to therapeutic insights into ocular infectious disease.
Keywords: antigen presentation/processing • microglia • gene/expression