Mochizuki S, Kobayashi M, Suzuki T, Oikawa A, Koseki T, Nishihara T, Hasegawa K
Department of Periodontology, Showa University Dental School, Tokyo, Japan.
J Periodontal Res. 2004 Oct;39(5):333-43. doi: 10.1111/j.1600-0765.2004.00749.x.
CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts.
Following treatment with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or gamma-interferon (IFN-gamma), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-gamma, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IkappaBalpha was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits.
IFN-gamma stimulated expression of mCD14, whereas -1beta and TNF-alpha did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-gamma. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IkappaBalpha and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-gamma augmented the following activation of MAP kinases and IkappaBalpha and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide.
These results suggest that the augmentation by IFN-gamma of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IkappaBalpha and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.
已证实CD14、Toll样受体4（TLR4）和髓样分化因子88（MyD88）可介导宿主细胞对脂多糖的反应性。我们在此研究了炎性细胞因子对人牙龈成纤维细胞中膜CD14（mCD14）、TLR4和MyD88表达的调节作用，以及对随后来自伴放线放线杆菌的脂多糖的反应性。
用白细胞介素-1β、肿瘤坏死因子-α（TNF-α）或γ-干扰素（IFN-γ）处理后，分别通过流式细胞术和蛋白质印迹法测定mCD14/TLR4和MyD88的表达。用IFN-γ预处理细胞后，先用抗CD14抗体MY4或抗TLR4抗体HTA125预孵育，随后用伴放线放线杆菌脂多糖处理。然后，通过蛋白质印迹法检测丝裂原活化蛋白（MAP）激酶和IκBα的磷酸化，并通过各自的酶联免疫吸附测定（ELISA）试剂盒测量白细胞介素-6和白细胞介素-8的产生。
IFN-γ刺激mCD14的表达，而白细胞介素-1β和TNF-α则无此作用。IFN-γ还增强了MyD88的表达，但未增强TLR4的表达。脂多糖激活了MAP激酶，如细胞外信号调节激酶（ERK）、c-Jun氨基末端激酶（JNK）和p38，以及IκBα，并刺激了白细胞介素-6和白细胞介素-8的产生。MY4或HTA125可显著抑制脂多糖刺激的白细胞介素-6和白细胞介素-8的产生。IFN-γ预处理增强了随后对脂多糖的MAP激酶和IκBα的激活以及白细胞介素-6和白细胞介素-8的产生。
这些结果表明，IFN-γ增强人牙龈成纤维细胞对伴放线放线杆菌脂多糖的反应性，如MAP激酶和IκBα的激活以及终末细胞因子的产生，可能部分是由CD14和MyD88表达的上调介导的。
