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.
Poster presentation. Abstract: It has been shown that the innate immune system plays an important role in the pathogenesis of diabetes. However, the effects of diabetes on the innate immunity have not been well understood. CD14, a receptor for lipopolysaccharide (LPS) and one of the pattern recognition receptors (PRRs), plays an essential role in the innate immune responses. Although clinical studies have shown that CD14 expression in monocytes from diabetic patients is higher than that from nondiabetic individuals, the underlying mechanism has not been defined. In the present study, we determined the effect of elevated concentration of glucose on CD14 expression by U937 mononuclear cells. Real-time PCR showed that high glucose augmented LPS-stimulated CD14 expression by 15-fold as compared with normal glucose. Immunoassay showed a marked enhancement of both membrane-associated and soluble CD14 levels by high glucose. Further investigations revealed that high glucose augmented LPS-stimulated CD14 expression by enhancing NF?B and AP-1 activities. Finally, studies showed that augmentation of LPS-stimulated MMP-1 expression by high glucose was inhibited by anti-CD14 antibodies, suggesting a crucial role of CD14 in high glucose-enhanced
gene expression. Taken together, this study has demonstrated a robust augmentation by high glucose of LPS-stimulated CD14 expression, suggesting that hyperglycemia may enhance the innate immunity.