Kato Satsuki, Sugimura Norihiko, Nakashima Keisuke, Nishihara Tatsuji, Kowashi Yusuke
Department of Periodontology and Endodontology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan 2Department of Oral Microbiology, Kyusyu Dental College, Fukuoka 803-8580, Japan.
J Med Microbiol. 2005 Mar;54(Pt 3):293-298. doi: 10.1099/jmm.0.45693-0.
It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-alpha) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-alpha levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-alpha antibody. However, exogenous TNF-alpha could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-alpha antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-alpha may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans-induced apoptosis of human immune cells may be important in terms of initiation and progression of periodontal diseases.
此前有报道称，鼠巨噬细胞系J774.1和人口腔上皮细胞系KB会因伴放线放线杆菌感染而发生凋亡。最近的研究表明，凋亡调控是由几种不同蛋白激酶的多重磷酸化调节的，包括丝裂原活化蛋白激酶（MAPK）家族的主要亚型。MAPK家族在生长因子刺激下促进细胞存活和/或增殖，或在各种应激刺激下促进凋亡。本研究的主要目的是阐明人免疫细胞在伴放线放线杆菌感染后是否会发生凋亡，如果是，则确定MAPK家族的参与情况。将人单核细胞THP-1细胞在微量管中用伴放线放线杆菌感染。监测乳酸脱氢酶释放到培养上清液中的情况以及细胞中的DNA片段化。DNA片段化也通过琼脂糖凝胶电泳进行鉴定。伴放线放线杆菌感染后的细胞死亡是由凋亡引起的，表现为片段化DNA比例增加以及典型的凋亡指示性DNA片段梯形图谱。此外，伴放线放线杆菌感染后p38 MAPK活性和肿瘤坏死因子α（TNF-α）水平升高。相反，添加p38抑制剂或抗TNF-α抗体后，感染细胞中的细胞死亡和TNF-α水平降低。然而，外源性TNF-α不能诱导未感染的THP-1细胞凋亡。有趣的是，在抗TNF-α抗体存在下p38 MAPK活性降低。这些发现表明，伴放线放线杆菌感染诱导THP-1细胞凋亡，并且p38 MAPK活性直接参与凋亡。TNF-α可能通过增强p38 MAPK活性在凋亡中发挥间接作用。伴放线放线杆菌诱导的人免疫细胞凋亡在牙周疾病的发生和发展方面可能很重要。
