Abstract 1647: RAGE & HMBG1: Key Roles in Dendritic and T Cell Activation
The Receptor for Advanced Glycation Endproducts (RAGE) plays key roles in inflammatory mechanisms linked to cardiac allograft rejection and atherosclerosis. We previously reported that mechanisms underlying these observations link RAGE to both T cell and macrophage activation. We hypothesized that RAGE and its key inflammatory ligand, HMGB1, are regulated during dendritic cell (DC) and T cell activation. To test this premise, bone marrow derived DCs from C57BL/6 mice were matured in vitro for 24 hours in the presence of LPS (0.5 ug/ml); IgM antibodies to CD40 (CD40-IgM, 1ug/1e6 cells); ovalbumin-immunecomplexes (OVA/IC); or polyclonal antibodies to HMGB1 (HMGB1 Ab, 100 ug/ml). For antigen-specific T cell proliferation, DC from RAGE null (Rko) mice were incubated with OVA/IC. After 24 hours DC were washed and OVA-specific OTI CD8 T cells (MHC I restricted) or OTII CD4 T cells (MHCII restricted) were added for 6, 24, 48 or 72 hours. Polyclonal activation of T cells was performed with antibodies to CD3 (1ug/ml, plate bound) and CD28 (250 ng/ml, soluble). Total RNA was isolated and Real Time pcr performed with Taqman probes for RAGE, HMGB-1 and 18sRNA on a sequence-detection system, and results analyzed by 2−ΔΔct analysis. Normalized RAGE or HMGB1 transcripts relative to cells cultured under the same conditions without the specific treatment at the indicated time points are shown. Steady state mRNA levels in Rko cells were undetectable under all conditions. Only antigen-driven (OVA/IC) but not CD40- or LPS-mediated DC maturation leads to increased RAGE transcripts; a process blocked by addition of HMGB1 Abs. HMGB-1 mRNA levels for DC maturation in wild-type and Rko cells remained below a 2-fold change under the same conditions. Antigen-specific and polyclonal T cell activation result in increased RAGE and HMGB1 mRNA levels (see Table⇓). Thus, RAGE and HMGB1 are upregulated during cellular activation, consistent with a role of the RAGE axis in pro-inflammatory immune responses.