Kido Jun-ichi, Hayashi Noriko, Kataoka Masatoshi, Nagata Toshihiko
Department of Periodontology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia.
J Periodontol. 2005 Mar;76(3):437-42. doi: 10.1902/jop.2005.76.3.437.
Calprotectin is a major cytosolic protein of monocytes and granulocytes. It is increased in inflammatory tissues and is detected at high levels in the gingival crevicular fluid (GCF) of periodontitis patients. We previously reported that lipopolysaccharide of Porphyromonas gingivalis (P-LPS) and cytokines induced the release of calprotectin from monocytes isolated from human peripheral blood. The mechanisms of calprotectin expression and presence of its regulation factors in periodontal disease are unknown. On the other hand, P-LPS and cytokines are significant etiologic factors in the initiation and progression of periodontal diseases. In this study, we investigated the expression and production of calprotectin from human monocytes by examining the effects of lipopolysaccharide of P-LPS, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta).
Monocytes were isolated from the peripheral blood of healthy donors and cultured in the presence or absence of P-LPS, TNF-alpha, or IL-1beta. The expressions of calprotectin mRNAs (MRP8 and MRP14) were detected by Northern blotting. The contents of calprotectin in the cells and medium fractions were determined by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha, a transcription factor of MRP14, was assessed by electrophoretic mobility shift DNA-binding assay (EMSA).
P-LPS, TNF-alpha, and IL-1beta induced MRP8/14 mRNAs and calprotectin production in monocytes. These factors also induced DNA CEBPalpha binding activity in monocytes. P-LPS increased MRP14 mRNA expression in monocytes to the maximum level, about two times the control level after 24 hours treatment, but did not enhance the basal level of MRP8. When the effects of TNF-alpha and IL-1beta on those mRNAs were investigated, both MRP8 and MRP14 significantly increased to about 2- and 2.5-fold the control level, respectively. Increases of MRP8/14 mRNA expression were followed by their protein production at about 2-fold the basal amount. DNA binding activity of C/EBPalpha was increased in P-LPS, TNF-alpha, and IL-1beta-treated monocytes.
These results demonstrate that P-LPS, TNF-alpha, and IL-1beta induce calprotectin production from human monocytes and that this production is associated with the activation of DNA C/EBPalpha binding complex.
钙卫蛋白是单核细胞和粒细胞的一种主要胞质蛋白。它在炎症组织中含量增加，在牙周炎患者的龈沟液（GCF）中可检测到高水平。我们之前报道牙龈卟啉单胞菌的脂多糖（P-LPS）和细胞因子可诱导从人外周血分离的单核细胞释放钙卫蛋白。牙周病中钙卫蛋白表达的机制及其调节因子的存在尚不清楚。另一方面，P-LPS和细胞因子是牙周病发生和发展的重要病因。在本研究中，我们通过检测P-LPS的脂多糖、肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的作用，研究了人单核细胞中钙卫蛋白的表达和产生。
从健康供体的外周血中分离单核细胞，并在有或无P-LPS、TNF-α或IL-1β的情况下进行培养。通过Northern印迹检测钙卫蛋白mRNA（MRP8和MRP14）的表达。通过酶联免疫吸附测定（ELISA）测定细胞和培养基组分中钙卫蛋白的含量。通过电泳迁移率变动DNA结合测定（EMSA）评估MRP14的转录因子C/EBPα的DNA结合活性。
P-LPS、TNF-α和IL-1β诱导单核细胞中MRP8/14 mRNA和钙卫蛋白的产生。这些因子还诱导单核细胞中的DNA CEBPα结合活性。P-LPS将单核细胞中MRP14 mRNA表达增加到最高水平，处理24小时后约为对照水平的两倍，但未提高MRP8的基础水平。当研究TNF-α和IL-1β对这些mRNA的影响时，MRP8和MRP14均显著增加至对照水平的约2倍和2.5倍。MRP8/14 mRNA表达增加后，其蛋白质产生量约为基础量的2倍。在P-LPS、TNF-α和IL-1β处理的单核细胞中，C/EBPα的DNA结合活性增加。
这些结果表明，P-LPS、TNF-α和IL-1β诱导人单核细胞产生钙卫蛋白，且这种产生与DNA C/EBPα结合复合物的激活有关。
