Poster presentation. Abstract:We have reported that high glucose and IFN? synergistically stimulate matrix metalloproteinase (MMP)-1, a proteinase involved in diabetic complications, by U937 mononuclear cells. We also reported that pre-exposure to high glucose and IFN? markedly increased lipopolysaccharide (LPS)-stimulated MMP-1 expression. To understand how high glucose and IFN? augment MMP-1 expression, we analyzed their effect on the expression of IFN? receptor, Toll-like receptor (TLR)4, CD14, and MD-2, a protein associated with TLR4 and conferring LPS responsiveness. Results showed that high glucose and IFN? had no effect on the expression of IFN? receptor, TLR4, and CD14, but stimulated MD-2. IFN? alone stimulated MD-2 expression by 3.4-fold (0.49 vs 0.15, MD-2/GAPDH mRNA) and high glucose alone had no significant stimulation. Interestingly, the combination of high glucose and IFN? further increased MD-2 expression by 1.7-fold as compared to the combination of normal glucose and IFN? (0.85 vs 0.49, p<0.01). These data indicate that MD-2 expression is specifically upregulated by high glucose and IFN?. More interestingly, we found that neutralizing antibody to MD-2 blocked high glucose and IFN?-stimulated MMP-1 expression. The antibody at 2.5 ?g/ml had 70% blocking while control antibody did not have significant effect. Since it is known that transcription factor AP-1 plays a crucial role in MMP-1 expression, we further determined the effect of high glucose and IFN? on the expression of c-Jun and c-Fos, two major subunits for AP-1. Results showed that high glucose and IFN? increased c-Jun expression by 1.8- and 2.1-fold, respectively, but high glucose plus IFN? increased c-Jun expression by 5.2-fold. Similarly, high glucose and IFN?increased c-Fos expression by 2.3- and 1.4-fold, respectively, but high glucose plus IFN? increased c-Fos expression by 3.6-fold. In conclusion, this study demonstrated that high glucose and IFN? stimulate MMP-1 expression in U937 cells by upregulating MD-2 and AP-1 expression.