Department of Internal Medicine, Pulmonary, Critical Care, Sleep & Allergy Section, University of Nebraska Medical Center, 985300 Nebraska Medical Center, Omaha, NE 68198-5300, USA.
Am J Respir Cell Mol Biol. 2010 Jun;42(6):706-15. doi: 10.1165/rcmb.2009-0065OC. Epub 2009 Jul 27.
Dust samples collected from Nebraska swine confinement facilities (hog dust extract [HDE]) are known to elicit proinflammatory cytokine release from human bronchial epithelial (HBE) cells in vitro. This response involves the activation of two protein kinase C (PKC) isoforms: PKCalpha and PKCepsilon. Experiments were designed to investigate the relationship between the two isoenzymes and the degree to which each is responsible for cytokine release in HBE. Experiments also examined the contribution of TNF-alpha to IL-6 and IL-8 release. PKCalpha and PKCepsilon activities were inhibited using isoform-specific pharmacologic inhibitors and genetically modified dominant-negative (DN) expressing cell lines. Release of the proinflammatory cytokines IL-6, IL-8, and TNF-alpha was measured and PKC isoform activities assessed. We found that HDE stimulates PKCalpha activity by 1 hour, and within 6 hours the activity returns to baseline. PKCalpha-specific inhibitor or PKCalphaDN cells abolish this HDE-mediated effect. Both IL-6 and IL-8 release are likewise diminished under these conditions compared with normal HBE, and treatment with TNF-alpha-neutralizing antibody does not further inhibit cytokine release. In contrast, PKCepsilon activity was enhanced by 6 hours after HDE treatment. TNF-alpha blockade abrogated this effect. HDE-stimulated IL-6, but not IL-8 release in PKCepsilonDN cells. The concentration of TNF-alpha released by HDE-stimulated HBE is sufficient to have a potent cytokine-eliciting effect. A time course of TNF-alpha release suggests that TNF-alpha is produced after PKCalpha activation, but before PKCepsilon. These results suggest a temporal ordering of events responsible for the release of cytokines, which initiate and exacerbate inflammatory events in the airways of people exposed to agricultural dust.
从内布拉斯加州养猪场（猪尘提取物 [HDE]）收集的粉尘样本已知可在体外引起人支气管上皮细胞（HBE）的促炎细胞因子释放。这种反应涉及两种蛋白激酶 C（PKC）同工型的激活：PKCalpha 和 PKCepsilon。实验旨在研究两种同工型之间的关系，以及每种同工型在 HBE 细胞因子释放中的作用程度。实验还研究了 TNF-α对 IL-6 和 IL-8 释放的贡献。使用同工型特异性药理抑制剂和遗传修饰的显性负（DN）表达细胞系抑制 PKCalpha 和 PKCepsilon 活性。测量促炎细胞因子 IL-6、IL-8 和 TNF-α的释放，并评估 PKC 同工型活性。我们发现，HDE 在 1 小时内刺激 PKCalpha 活性，并且在 6 小时内活性恢复到基线。PKCalpha 特异性抑制剂或 PKCalphaDN 细胞消除了这种 HDE 介导的作用。与正常 HBE 相比，在这些条件下，IL-6 和 IL-8 的释放都减少，并且用 TNF-α中和抗体进一步抑制细胞因子释放。相比之下，HDE 处理 6 小时后 PKCepsilon 活性增强。TNF-α阻断消除了这种作用。HDE 刺激的 PKCepsilonDN 细胞中 IL-6 但不是 IL-8 释放。HDE 刺激的 HBE 释放的 TNF-α浓度足以产生强烈的细胞因子诱导作用。TNF-α释放的时间过程表明，TNF-α是在 PKCalpha 激活之后但在 PKCepsilon 之前产生的。这些结果表明，负责细胞因子释放的事件存在时间顺序，从而引发和加剧暴露于农业粉尘的人呼吸道中的炎症事件。
