Department of Medicine A, University Medicine, Ernst-Moritz-Arndt-University, Greifswald, Friedrich-Loeffler-Str. 23a, 17475 Greifswald, Germany.
Gut. 2013 Mar;62(3):430-9. doi: 10.1136/gutjnl-2011-300771. Epub 2012 Apr 5.
Acute pancreatitis has long been considered a disorder of pancreatic self-digestion, in which intracellular activation of digestive proteases induces tissue injury. Chemokines, released from damaged pancreatic cells then attract inflammatory cells, whose systemic action ultimately determines the disease severity. In the present work the opposite mechanism is investigated; that is, whether and how inflammatory cells can activate intracellular proteases.
Using mice either deficient for the CD18-α subunit of the membrane attack complex-1 (MAC-1) complex or tumour necrosis factor (TNF)α, as well as after depletion of leucocyte subpopulations, pancreatitis was induced by 7-hourly caerulein injections (50 μg/kg, intraperitoneally). Pancreatic acini were coincubated in vitro from wild-type and cathepsin-B-deficient animals with phorbol-12-myristate-13-acetate (PMA)-activated neutrophils and macrophages, caerulein or TNFα, and activities of trypsin, cathepsin-B and caspase-3 were measured, as well as necrosis using fluorogenic substrates. TNFα was inhibited with monospecific antibodies.
Deletion of CD18 prevented transmigration of leucocytes into the pancreas during pancreatitis, greatly reduced disease severity and abolished digestive protease activation. Depletion of neutrophils and macrophages equally reduced premature trypsinogen activation and disease severity. In vitro activated neutrophils and macrophages directly induced premature protease activation and cell death in pancreatic acini and stimulation of acini with TNFα induced caspase-3 activation and necrosis via a cathepsin-B and calcium-dependent mechanism. Neutralising antibodies against TNFα and genetic deletion of TNFα prevented leucocyte-induced trypsin activity and necrosis in isolated acini.
The soluble inflammatory cell mediator TNFα directly induces premature protease activation and necrosis in pancreatic acinar cells. This activation depends on calcium and cathepsin-B activity. The findings from the present work further suggest that targeting TNFα, for which pharmaceutical agents are readily available, could be an effective treatment strategy that directly addresses the cellular causes of pancreatitis.
急性胰腺炎长期以来一直被认为是一种胰腺自我消化的紊乱，其中细胞内消化蛋白酶的激活导致组织损伤。趋化因子从受损的胰腺细胞释放出来，然后吸引炎症细胞，其全身作用最终决定疾病的严重程度。在目前的工作中，研究了相反的机制；也就是说，炎症细胞是否以及如何激活细胞内蛋白酶。
使用缺乏膜攻击复合物-1 (MAC-1) 复合物的 CD18-α 亚基或肿瘤坏死因子 (TNF)α 的小鼠，以及白细胞亚群耗竭后，通过 7 小时间隔的 caerulein 注射（50 μg/kg，腹腔内）诱导胰腺炎。从野生型和组织蛋白酶-B 缺陷动物中提取的胰腺腺泡与佛波醇-12-肉豆蔻酸-13-乙酸酯 (PMA) 激活的中性粒细胞和巨噬细胞、caerulein 或 TNFα 共孵育，测量胰蛋白酶、组织蛋白酶-B 和半胱天冬酶-3 的活性，以及使用荧光底物测量坏死。用单特异性抗体抑制 TNFα。
CD18 的缺失阻止了白细胞在胰腺炎期间向胰腺的迁移，大大降低了疾病的严重程度，并消除了消化蛋白酶的激活。中性粒细胞和巨噬细胞的耗竭同样减少了过早的胰蛋白酶原激活和疾病的严重程度。体外激活的中性粒细胞和巨噬细胞直接诱导胰腺腺泡中的过早蛋白酶激活和细胞死亡，并用 TNFα 刺激腺泡通过组织蛋白酶-B 和钙依赖性机制诱导 caspase-3 激活和坏死。针对 TNFα 的中和抗体和 TNFα 的基因缺失可防止白细胞诱导的胰蛋白酶活性和分离腺泡中的坏死。
可溶性炎症细胞介质 TNFα 直接诱导胰腺腺泡细胞中的过早蛋白酶激活和坏死。这种激活依赖于钙和组织蛋白酶-B 活性。目前工作的结果进一步表明，针对 TNFα 的靶向治疗可能是一种有效的治疗策略，直接针对胰腺炎的细胞原因。
