Analysis of apoptosis in alveolar macrophages induced by periodontopathic bacteria and development of a method of protection from pneumonia

牙周病细菌伴生放线杆菌感染体外培养的小鼠巨噬细胞诱导细胞凋亡。在这项研究中，我们研究了caspase在伴放线放线杆菌感染的巨噬细胞的凋亡死亡中的作用。半胱氨酸天冬氨酸酶的两种多肽抑制剂benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl酮(Z-VAD-FMK)和benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe)-fluoromethyl酮(Z-DEVDFMK)可抑制伴放线放线菌感染的小鼠巨噬细胞株J774.1的细胞死亡。在细胞凋亡过程中，J774.1细胞培养上清液中检测到IL-1β(IL-1β)。IL-1β的分泌被Caspase-1抑制剂Z-VAD-FMK阻断，表明Caspase-1不仅参与诱导放线菌感染的J774.1细胞的凋亡，而且还参与IL-1β的分泌。免疫印迹分析显示伴生放线杆菌感染J774.1细胞后，视网膜母细胞瘤蛋白(Rb)发生裂解，提示伴生放线杆菌感染激活了caspase-3。伴放线放线杆菌感染J774.1细胞的胞浆在体外诱导Rb蛋白降解，这种作用可被caspase-3抑制剂Z-DEVD-FMK抑制。此外，伴随放线放线杆菌感染J774.1细胞12~24 h后，caspase-3样活性显著增强，而caspase-3抑制剂Z-DEVD-FMK可抑制这一作用。综上所述，这些结果提示caspase-1和caspase-3参与了伴生放线杆菌感染的巨噬细胞的凋亡诱导过程。

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A.actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone(Z-VAD-FMK)and benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe)-fluoromethyl katone(Z-DEVD-FMK), inhibited apoptotic cell death of mouse macrophage cell line J774.1 infected with A.actinomycetemcomitans. During the process of apoptosis, interleukin-1β(IL-1β) was detected in the culture supernatants of J774.1 cells. IL-1β secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1β secretion from A.actinomycetemcomintas-infected J774.1 cells. Immunoblot analysis revealed that the infection of A.actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein(Rb), suggesting that caspase-3 was activated by A.actinomycetemcomitans-infection. The cytosol from A.actinomycetemcomitans-infected J774.1 cells induced Rb proteollysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK.Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK.These findings indings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A.actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A.actinomycetemcomitans-infected macrophages.