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 lactate induces insulin resistance. However, the underlying mechanismshave not been well understood. Based on our observation that lactate augments lipopolysccharide (LPS)-stimulated inflammatory gene expression, we proposed that lactate may enhance Toll-like receptor 4 (TLR4) signaling in macrophages, which has been shown to play an important role in insulin resistance in adipocytes. In this study, we demonstrated that lactate stimulated MD-2, a co-receptor for TLR4 signaling activation, NF?B transcriptional activity, and the expression of inflammatory genes by human U937 macrophages. Similar enhancement of the inflammatory gene expression by lactate was also observed in human monocyte-derived macrophages. The essential role of MD-2 in lactate-augmented TLR4 signaling was confirmed by observation that the suppression of MD-2 expression by small interference RNA (siRNA) led to significant inhibition of inflammatory gene expression. To further elucidate how lactate treatment enhances TLR4 activation, we showed that the augmentation of inflammatory gene expression by lactate was abrogated by antioxidant treatment, suggesting a critical role of reactive oxygen species (ROS) in the enhancement of TLR4 activation by lactate. Finally, we showed that ?-cyano-4-hydroxycinnamatic acid (?-CHCA), a classic inhibitor for monocarboxylate transporters (MCTs), blocked lactate-augmented inflammatory gene expression and nuclear NF?B activity. Taken together, this study has documented that lactate boosts TLR4 activation and NF?B-dependent inflammatory gene expression via monocarboxylate transporters and MD-2 upregulation.
Poster presentation. Abstract: Matrix metalloproteinase (MMP) plays a crucial role in vascular diseases and inflammatory mediator lipopolysaccharide (LPS) is a powerful stimulator of MMP expression. Thus, researchers have long sought to discover agents that are potent in inhibiting LPS-stimulated proinflammatory gene expression and useful to treat vascular diseases. In this study, we demonstrated that simvastatin, a 3-hydroxyl-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, effectively inhibited LPS-stimulated MMP-1 as well as MMP-8 and MMP-9 expression by U937 mononuclear cells. Furthermore, results showed that geranylgeranyl transferase inhibitor (GGTI) inhibited LPS-stimulated MMP-1 expression and addition of isoprenoid intermediate geranylgeranyl pyrophosphate (GGPP) diminished the inhibitory effect of simvastatin on LPS-stimulated MMP-1 expression. Results also showed that simvastatin inhibited the membrane association of Ras and Rac and the inhibition was abolished by addition of GGPP. In addition, inhibition of Ras and Rac isoprenylation by Ras and Rac inhibitors led to inhibition of MMP-1 expression. These results indicate that Ras and Rac protein isoprenylation is targeted by simvastatin in the inhibition of LPS-stimulated MMP-1 expression. Moreover, we showed that simvastatin inhibited LPS-stimulated nuclear transcription factor AP-1, but not NFkB activity, and the inhibition was reversed by addition of GGPP. Simvastatin also inhibited LPS-stimulated extracellular signal-regulated kinase (ERK), but not p38 mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK). Finally, we showed that the inhibitory effect of simvastatin on LPS-stimulated ERK activation was significantly reduced by addition of GGPP. Taken together, this study showed that simvastatin suppresses LPS-induced MMP-1 expression in U937 mononuclear cells by targeting Ras and Rac protein isoprenylation-mediated ERK activation.
Poster presentation. Abstract: Matrix metalloproteinases (MMPs) play a crucial role in the destabilization of atherosclerotic plaques that may lead to plaque rupture-triggered acute coronary syndrome. Although it is known that interleukin 6 (IL-6) is associated with atherosclerosis, and diabetes increases acute coronary events, the regulation of MMP expression in mononuclear cells by IL-6 and the impact of high glucose on the regulation have not been well documented. In the present study, we found that IL-6 stimulated MMP-1 expression in a concentration- and time-dependent manner in U937 mononuclear cells and high glucose further increased IL-6-stimulated MMP-1 expression by 2-fold. By comparing the stimulatory effect of IL-6 with that of lipopolysaccharide (LPS) on MMP-1 expression, we found that IL-6 was 3-fold more effective than LPS at 1 and 10 ng/ml, suggesting that IL-6 is a potent stimulator on MMP-1 expression by U937 cells. Furthermore, results showed that high glucose, IL-6 and LPS stimulated MMP-1 expression by 1.4-, 3.9-, 6.1-fold, respectively, after 24 h treatment, but the combination of high glucose, IL-6 and LPS led to a 53.3-fold increase, revealing a remarkable synergistic effect between high glucose, IL-6, and LPS on MMP-1 expression. In the studies to elucidate how high glucose and IL-6 act synergistically for MMP-1 upregulation, we found that while either high glucose or IL-6 increased c-Jun expression by 2.5-fold, the combination of high glucose and IL-6 stimulated c-Jun expression by 5-fold. In contrast, high glucose did not enhance IL-6-stimulated c-Fos expression. These results suggest that high glucose and IL-6 exert synergy on MMP-1 expression by upregulating c-Jun, an immediate early gene and a major subunit for transcription factor AP-1 that is known to play a key role in MMP-1 transcription. Our further studies showed that the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways, but not Janus kinases /signal transducer and activator of transcription (JAK/STAT)3 cascade, were involved in high glucose and IL-6-stimulated MMP-1 expression. Taken together, this study showed that high glucose and IL-6 synergistically stimulate MMP-1 expression in U937 mononuclear cells through c-Jun and ERK and JNK cascades.
Poster presentation. Abstract: Although it is known that macrophage content in visceral adipose tissue is significantly increased in obese humans and mice, the crosstalk between macrophages and adipocytes and its impact on the expression of genes involved in inflammation that contributes to insulin resistance have not been well elucidated. In this study, we incubated human monocytes, macrophages, or U937 mononuclear cells with adipocytes in a transwell system and studied how cell interaction regulated the expression of osteopontin, a matrix glycoprotein and proinflammatory cytokine that mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance. Results showed that coculture of human monocytes, macrophages or U937 cells with adipocytes led to a 3- to 5-fold increase in osteopontin production when compared to that released by independent cultures of monocytes, macrophages or U937 cells and adipocytes. Moreover, LPS or palmitic acid, which activates Toll-like receptor (TLR)4, and high glucose augmented the coculture-stimulated osteopontin secretion. Real-time PCR studies showed that coculture of U937 cells and adipocytes led to increased osteopontin mRNA in U937 cells, but not adipocytes, suggesting that adipocyte-derived soluble factors may stimulate osteopontin expression in U937 cells. This notion was supported by the observation that osteopontin expression by U937 cells was increased by 2-fold by treatment with adipocyte-conditioned medium. In our studies to explore the mechanism involved in the coculture-stimulated osteopontin expression, we found that the neutralizing antibodies against IL-6 and IL-1b, but not those against IL-1a and TNFa, effectively blocked coculture-augmented osteopontin expression, indicating that IL-6 and IL-1b may stimulate osteopontin expression in the coculture. In support of this finding, experiments showed that IL-6 and IL-1b stimulated osteopontin expression. In conclusion, this study demonstrated that the crosstalk between adipocytes and U937 cells stimulated osteopontin expression via IL-6 and IL-1b and that TLR4 activation and high glucose further enhanced the adipocyte/U937 cell interaction-stimulated osteopontin 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.
Poster presentation. Abstract: Objectives: Epidemiological studies have established that patients with diabetes have increased prevalence and severity of periodontal disease. However, the periodontal expression of inflammatory cytokines and matrix metalloproteinases (MMPs) in diabetic patients has not been well characterized. The objective of this study is to determine the difference in the periodontal expression of MMP-1, MMP-8, interleukin-6 (IL-6), tumor necrosis factor (TNF) alpha and IL-1 beta between diabetic and nondiabetic patients.
Methods: Periodontal tissue specimens were collected from 9 non-diabetic patients without periodontal disease (group 1), 11 non-diabetic patients with periodontal disease (group 2) and 7 diabetic patients with periodontal disease (group 3). The expression of MMP-1, MMP-8, IL-6, TNF alpha and IL-1 beta was quantified using real-time PCR.
Results: The nonparametric Kruskal-Wallis test showed that the difference in IL-6 expression among the groups was statistically significant (p = 0.04). Furthermore, the generalized Kruskal-Wallis nonparametric linear-by-linear association test showed a statistically significant trend of increase in the expression of IL-6 from group 1 to group 2 to group 3 (p=0.02) and a suggestion of such a trend in MMP-1 (p=0.05). No increase in MMP-8 expression was observed in patients in group 3 as compared to patients in groups 1 and 2. Although the average expression levels of MMP-1, IL-1 beta, and TNF alpha were increased from group 1 to group 3, the differences were not statistically significant.
Conclusion: A trend with increased IL-6 expression in periodontal tissues was observed across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases.
Poster presentation. Abstract: Purpose. Sphingosine kinases (SKs) phosphorylate sphingosine to create sphingosine 1 phosphate (S1P), which is involved in cell survival, proliferation, motility, and inflammation via intracellular signaling through release of Ca2+ stores, activation of NF??B, and extracellular interactions with G protein-coupled receptors (S1P receptors 1-5). Inhibitors of SK1 have been used successfully to treat experimental murine inflammatory bowel disease, diabetic retinopathy, and rheumatoid arthritis. SK1 is expressed in mesangial cells and plays a role in mesangial cell migration. The role of SKs in promoting inflammation in lupus is unknown. We hypothesized that ABC294640 (ABC), an orally available SK inhibitor, would reduce markers of glomerular pathology in the MRL/MpJTnfrsf6lpr/ J (MRL/lpr) murine model of lupus. Methods. Eleven MRL/lpr mice were treated with either polyethylene glycol (PEG, n=5) or ABC in PEG (50 mg/kg/day, n=6) from 10 (before onset of disease) to 20 weeks of age (significant albuminuria present). Urine was collected every other week and analyzed for albumin. Moribund mice were sacrificed, and all measures were carried forward. At 20 weeks, kidney and total weights were determined. Renal, skin, and joint tissue were harvested for histopathological scoring, while renal tissue was stained for IgG/C3 deposition and examined by electron microscopy. Serum anti-double stranded DNA antibody levels were determined at 20 weeks. Urine thromboxane levels were determined at 16 weeks. Spleen cell subsets were determine by flow cytometry. Differences between groups were determined by t-test or Wilcoxon rank sum analysis. Results were reported as averages standard error. Results. Survival was greater in ABC-treated mice than controls (100 vs. 40% at 20 weeks). By 18 and 20 weeks, proteinuria was lower in ABC-treated mice than control mice (106 vs. 3818 and 191 vs. 6021). Kidney/total body weights were significantly lower in ABC-treated mice than controls (1.50.1 vs. 2.20.1 mg/g, p=0.001). There was a trend towards lower renal thromboxane production in treated mice. Renal and cutaneous pathologic scores were lower
in treated mice. Neither anti-double stranded DNA antibody production nor basement membrane and mesangial IgG/C3 staining were different between groups. Spleen marginal zone B cells were greater in treated mice. Conclusions. ABC294640 treatment improves survival, possibly due to reduced renal failure
and vasculitis. ABC294640 treatment reduces often clinically refractory necrotic/crescentic glomerular lesions associated with reduced renal function in humans, making SK an attractive target for investigation as adjunctive therapy ABC294640 treatment reduces podocyte flattening associated with nephrotic
range proteinuria in humans, making SK a possible target for investigation as adjunctive therapy in membranous nephritis with nephrotic range proteinuria. Improvements in outcomes are independent of changes in production and deposition of nephritic autoantibodies. We hypothesize that inhibition of SK
may reduce homing of inflammatory cells and activation of resident and inflammatory cells to produce inflammatory mediators such as thromboxane.