Southern Arizona Veterans Affairs Health Care Systems, Tucson, AZ 85723, USA.
BMC Microbiol. 2012 Jan 9;12:2. doi: 10.1186/1471-2180-12-2.
A common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs) in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET), which is formed by the combination of two proteins produced by the organism, edema factor (EF), which is an adenyl cyclase, and protective antigen (PA). Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse.
Pretreatment of human microvascular endothelial cell(EC)s of the lung (HMVEC-L) with ET decreased interleukin (IL)-8-driven transendothelial migration (TEM) of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-α or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA) activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP.
We conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.
吸入性炭疽病患者的一个常见特征是，尽管循环中性粒细胞数量丰富，但受感染组织中却缺乏多形核白细胞（PMN）。炭疽的主要毒力决定因素是水肿毒素（ET），它由该生物体产生的两种蛋白质组合而成，即水肿因子（EF），它是一种腺苷酸环化酶，和保护性抗原（PA）。由于环腺苷酸（cAMP）是腺苷酸环化酶的产物，已知它可以增强内皮屏障完整性，因此我们想知道 ET 是否可以通过关闭PMN 穿过的细胞旁途径来减少PMN 渗出到组织中。
ET 预处理人肺微血管内皮细胞（HMVEC-L）可使白细胞介素（IL）-8 驱动的PMN 跨内皮迁移（TEM）减少近 60%，最大减少量。这种效应需要 EF 和 PA 的存在。相反，ET 不会减少无 EC 的系统中的PMN 趋化性。与培养基对照相比，亚汇合 HMVEC-L 的预处理使 14 C-白蛋白的跨内皮通量减少了约 50%。将 ET 与肿瘤坏死因子-α或细菌脂多糖（均为 100ng/ml）共同给药，可减弱两种药物单独作用引起的跨内皮 14 C-白蛋白通量的增加。ET 对 TEM 的抑制作用与蛋白激酶 A（PKA）活性的增加平行，但不能通过用 H-89 或 KT-5720 抑制 PKA 来阻断。最后，我们无法用 forskolin 或 3-异丁基-1-甲基黄嘌呤复制 ET 的作用，这两种药物已知可增加 cAMP。
我们得出结论，ET 独立于 cAMP/PKA 活性，可降低 IL-8 驱动的 HMVEC-L 单层 PMN 的 TEM。
