Resumen de Stimulatory Effects of Glucose and Porphyromonas gingivalis Lipopolysaccharide on the Secretion of Inflammatory Mediators From Human Macrophages
Background: Hyperglycemia is widely considered to be the causal link between diabetes mellitus (DM) and diabetic complications. The purpose of this study is to determine the effects of high glucose in the presence of lipopolysaccharide (LPS) purified from the periodontal pathogen Porphyromonas gingivalis on human macrophages.
Methods: Macrophages (U937) were treated with various concentrations of P. gingivalis�LPS under normal (5.5 mM) or high (25 mM) glucose conditions. Mitochondrial dehydrogenase activity was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. The levels of inflammatory mediators secreted were determined using the enzyme-linked immunosorbent assay and the competitive enzyme immunoassay. The intracellular calcium chelator was used to examine whether the intracellular calcium was involved. Statistical differences were assessed using a one-way analysis of variance and Tukey multiple-comparison intervals with a = 0.05.
Results: High glucose condition enhanced the mitochondrial dehydrogenase activity in macrophages. P. gingivalis�LPS induced the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-a, and prostaglandin E2 (PGE2) in a dose-dependent manner both in normal and high glucose conditions. The stimulatory effects by P. gingivalis�LPS were more evident when cells were cultured under high glucose conditions. Changes of intracellular calcium concentration were involved not only in high glucose�induced mitochondrial dehydrogenase activity but also in P. gingivalis�LPS-induced production of IL-6, TNF-a, or PGE2, especially under the high glucose conditions.
Conclusions: High glucose appeared to enhance the inflammatory response induced by the periodontal pathogen. The information generated may help to delineate the possible mechanisms by which hyperglycemia compromises the periodontal health of patients with DM.
Diabetes mellitus (DM) is an endocrine disease affecting glucose tolerance and the metabolism of carbohydrates and lipids.1 Patients with DM suffer many complications as a result of hyperglycemia and inflammation.2 The complications include vascular disease, renal disease, poor wound healing, greater risk of infection, and an increased susceptibility to periodontitis.1-3 Periodontal disease is an inflammatory disease caused in part by Gram-negative bacteria. DM is a major risk factor for the development of periodontal disease.4 Patients with either type 1 or type 2 DM experience exacerbated periodontitis compared with individuals without DM.5-7 Increased levels of proinflammatory cytokines and bone-related factors in the gingival crevicular fluid (GCF) have been observed in patients with type 2 DM and chronic periodontitis (CP) compared with patients without DM.8 Treatment of periodontitis leading to improved periodontal status has been associated with significant improvements of glycemic control.7 Uncontrolled glucose regulation leading to hyperglycemia is widely considered as the causal link between DM and diabetic complications. The possible molecular mechanisms implicated in hyperglycemia-induced tissue damage include activation of protein kinase C (PKC) isoforms, increased hexosamine pathway flux, increased advanced glycation end product formation, and increased polyol pathway flux.9 Cytokine production may be affected by hyperglycemia.7,10,11 Moreover, overproduction of superoxide induced by hyperglycemia may be the causal link between high glucose and the pathways responsible for hyperglycemic damage.9 Glucose levels have an effect on bone-marrow-derived macrophage gene expression, including toll-like receptor (TLR) 2 and TLR4 and the cytokine tumor necrosis factor (TNF)-a.12 Hyperglycemia may alter lipopolysaccharide (LPS)-stimulated macrophage responses by augmenting the secretion of the inflammatory mediator prostaglandin E2 (PGE2).13 The innate immune responsiveness to LPS derived from Porphyromonas gingivalis may be affected in DM, suggesting that proper control of blood sugar levels may have an increased immunologic benefit when challenged with a periodontal infection.14 Interleukin-6 (IL-6), a cytokine with pleiotropic activities,15 plays an important role in many inflammatory and immunomodulatory processes. For instance, IL-6 supports differentiation of B lymphocytes, plays a role in T-cell differentiation, and inhibits myeloid-cell growth but induces differentiation of these cells into macrophages.15,16 IL-6 also accelerates bone resorption.17,18 The level of IL-6 in GCF has been reported to be a good indicator of periodontal disease severity.19 The expression levels of IL-6 in fibroblasts of the inflammatory human gingival tissues are higher than those in the healthy gingival tissues.20 Higher expression of periodontal IL-6 protein has been observed in patients with both DM and periodontal disease when compared with patients with periodontal disease alone or without both diseases.21 Elevated levels of PGE2 have also been demonstrated in the GCF of patients with periodontal infections and are associated with periodontal breakdown.22,23 PGE2 regulates immune response in multiple levels and is important in periodontal inflammation because of its ability to enhance osteoclastic bone resorption and increase vascular permeability.24 Macrophages may respond to microbial components and activate the host defense mechanisms through the production of inflammatory mediators. Alteration of macrophage functions may be involved in the pathogenesis of diabetic periodontitis.25 It is valuable to evaluate the effects of high glucose in the presence of virulence factor of periodontal pathogens on macrophages to understand the pathogenesis of diabetic periodontal disease. The purpose of this study is to determine the possible effects of high glucose and purified LPS from the periodontal pathogen P. gingivalis on the mitochondrial activity and the secretion of several inflammatory mediators from macrophages. The possible involvement of intracellular calcium was also evaluated.
