Seitz Daniel H, Perl Mario, Mangold Stefanie, Neddermann Anne, Braumüller Sonja T, Zhou Shaoixa, Bachem Max G, Huber-Lang Markus S, Knöferl Markus W
Department of Trauma, Hand, Plastic and Reconstructive Surgery, University of Ulm, Ulm, Germany.
Shock. 2008 Nov;30(5):537-44. doi: 10.1097/SHK.0b013e31816a394b.
Alveolar type 2 (AT-2) cell apoptosis is an important mechanism during lung inflammation, lung injury, and regeneration. Blunt chest trauma has been shown to activate inflammatory cells such as alveolar macrophages (AMs) or neutrophils (polymorphonuclear granulocytes [PMNs]), resulting in an inflammatory response. The present study was performed to determine the capacity of different components/cells of the alveolar compartment (AMs, PMNs, or bronchoalveolar lavage [BAL] fluids) to induce apoptosis in AT-2 cells following blunt chest trauma. To study this, male Sprague-Dawley rats were subjected to either sham procedure or blunt chest trauma induced by a single blast wave. Various time points after injury (6 h to 7 d), the lungs were analyzed by immunohistochemistry, for AT-2 cells, or with antibodies directed against caspase 3, caspase 8, Fas, Fas ligand (FasL), BAX, and BCL-2. Bronchoalveolar lavage concentrations of TNF-alpha, IL-1beta, and soluble FasL were determined by enzyme-linked immunosorbent assay. Furthermore, cultures of AT-2 cells isolated from healthy rats were incubated with supernatants of AMs, PMNs, or BAL fluids obtained from either trauma or sham-operated animals in the presence or absence of oxidative stress. Annexin V staining or TUNEL (terminal deoxynucleotidyl transferase) assay was used to detect apoptotic AT-2 cells. Histological evaluation revealed that the total number of AT-2 cells was significantly reduced at 48 h following trauma. Fas, FasL, active caspase 8, and active caspase 3 were markedly up-regulated in AT-2 cells after chest trauma. BAX and BCL-2 did not show any significant changes between sham and trauma. IL-1beta, but not TNF-alpha, levels were markedly increased at 24 h after the injury, and soluble FasL concentrations were significantly enhanced at 6, 12, 24, and 48 h after the insult. Apoptosis of AT-2 cells incubated with supernatants from cultured AMs, isolated at 48 h following chest trauma was markedly increased when compared with shams. In contrast, no apoptosis was induced in AT-2 cells incubated with supernatants of activated PMNs or BAL fluids of traumatized animals. In summary, blunt chest trauma induced apoptosis in AT-2 cells, possibly involving the extrinsic death receptor pathway. Furthermore, mediators released by AMs appeared to be involved in the induction of AT-2 cell apoptosis.
肺泡Ⅱ型（AT-2）细胞凋亡是肺部炎症、肺损伤和再生过程中的重要机制。钝性胸部创伤已被证明可激活炎症细胞，如肺泡巨噬细胞（AMs）或中性粒细胞（多形核粒细胞[PMNs]），从而引发炎症反应。本研究旨在确定肺泡腔室的不同成分/细胞（AMs、PMNs或支气管肺泡灌洗[BAL]液）在钝性胸部创伤后诱导AT-2细胞凋亡的能力。为了研究这一点，将雄性Sprague-Dawley大鼠进行假手术或单次冲击波诱导的钝性胸部创伤。在损伤后的不同时间点（6小时至7天），通过免疫组织化学分析肺组织中的AT-2细胞，或使用针对半胱天冬酶3、半胱天冬酶8、Fas、Fas配体（FasL）、BAX和BCL-2的抗体进行分析。通过酶联免疫吸附测定法测定支气管肺泡灌洗中肿瘤坏死因子-α、白细胞介素-1β和可溶性FasL的浓度。此外，将从健康大鼠分离的AT-2细胞培养物与从创伤或假手术动物获得的AMs、PMNs或BAL液的上清液在有或没有氧化应激的情况下孵育。使用膜联蛋白V染色或TUNEL（末端脱氧核苷酸转移酶）测定法检测凋亡的AT-2细胞。组织学评估显示，创伤后48小时AT-2细胞总数显著减少。胸部创伤后，AT-2细胞中Fas、FasL、活性半胱天冬酶8和活性半胱天冬酶3明显上调。假手术组和创伤组之间BAX和BCL-2没有显示出任何显著变化。损伤后24小时白细胞介素-1β水平显著升高，但肿瘤坏死因子-α水平没有升高，损伤后6、12、24和48小时可溶性FasL浓度显著升高。与假手术组相比，用胸部创伤后48小时分离的培养AMs的上清液孵育的AT-2细胞凋亡明显增加。相反，用活化的PMNs上清液或创伤动物的BAL液孵育的AT-2细胞未诱导凋亡。总之，钝性胸部创伤诱导AT-2细胞凋亡，可能涉及外源性死亡受体途径。此外，AMs释放的介质似乎参与了AT-2细胞凋亡的诱导。
