Zay K, Loo S, Xie C, Devine D V, Wright J, Churg A
Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5.
Am J Physiol. 1999 Feb;276(2):L269-79. doi: 10.1152/ajplung.1999.276.2.L269.
Mineral dusts produce emphysema, and administration of dust to rats results in the rapid appearance of desmosine and hydroxyproline in lavage fluid, confirming that dusts directly induce connective tissue breakdown. To examine the role of neutrophils and alpha1-antitrypsin (alpha1-AT) in this process, we instilled silica or coal into normal rats or rats that had been pretreated with antiserum against neutrophils. One day after dust exposure, lavage fluid neutrophils and desmosine and hydroxyproline levels were all elevated; treatment with antiserum against neutrophils reduced neutrophils by 75%, desmosine by 40-50%, and hydroxyproline by 25%. By 7 days, lavage fluid neutrophils and desmosine level had decreased, whereas macrophages and hydroxyproline level had increased. By ELISA analysis, lavage fluid alpha1-AT levels were increased four- to eightfold at both times. On Western blot, some of the alpha1-AT appeared as degraded fragments, and by HPLC analysis, 5-10% of the methionine residues were oxidized. At both times, lavage fluid exhibited considerably elevated serine elastase inhibitory capacity and also showed elevations in metalloelastase activity. We conclude that, in this model, connective tissue breakdown is initially driven largely by neutrophil-derived proteases and that markedly elevated levels of functional alpha1-AT do not prevent breakdown, thus providing in vivo support for the concept of quantum proteolysis proposed by Liou and Campbell (T. G. Liou and E. J. Campbell. Biochemistry 34: 16171-16177, 1995). Macrophage-derived proteases may be of increasing importance over time, especially in coal-treated animals.
矿物粉尘可导致肺气肿，给大鼠吸入粉尘后，灌洗液中迅速出现异锁链双氢脲酸和羟脯氨酸，这证实粉尘可直接诱发结缔组织破坏。为研究中性粒细胞和α1-抗胰蛋白酶（α1-AT）在此过程中的作用，我们将二氧化硅或煤滴注到正常大鼠或用抗中性粒细胞抗血清预处理过的大鼠体内。接触粉尘一天后，灌洗液中的中性粒细胞、异锁链双氢脲酸和羟脯氨酸水平均升高；用抗中性粒细胞抗血清处理可使中性粒细胞减少75%，异锁链双氢脲酸减少40%-50%，羟脯氨酸减少25%。到第7天，灌洗液中的中性粒细胞和异锁链双氢脲酸水平下降，而巨噬细胞和羟脯氨酸水平升高。通过酶联免疫吸附测定（ELISA）分析，两个时间点灌洗液中的α1-AT水平均升高了4至8倍。在蛋白质印迹法中，一些α1-AT表现为降解片段，通过高效液相色谱（HPLC）分析，5%-10%的甲硫氨酸残基被氧化。在两个时间点，灌洗液的丝氨酸弹性蛋白酶抑制能力均显著升高，金属弹性蛋白酶活性也升高。我们得出结论，在这个模型中，结缔组织破坏最初主要由中性粒细胞衍生的蛋白酶驱动，功能性α1-AT水平显著升高并不能阻止破坏，从而为Liou和Campbell提出的量子蛋白水解概念（T.G.Liou和E.J.Campbell。《生物化学》34:16171-16177，1995）提供了体内支持。随着时间的推移，巨噬细胞衍生的蛋白酶可能变得越来越重要，尤其是在接受煤处理的动物中。
