Chou Wen K, Yu Ting C, Chen Shuen E, Peh Ho C, Liu Wen B, Chen Ming T, Nagahata Hajime, Chang Chai J
Department of Animal Science, National Chung Hsing University, Taichung 402, Taiwan ROC.
J Dairy Res. 2009 Nov;76(4):459-68. doi: 10.1017/S0022029909990069. Epub 2009 Jul 29.
Interactions between inflammatory cytokines and plasminogen (Pg) activation system on immune cells are yet to be established. In previous studies we reported a somatic cell-associated elevation of proteolytic activity in mammary secretion of drying-off goats and cows. The purposes of the present study were to examine the role of TNF-alpha in polymorphonuclear neutrophil (PMN)-associated Pg activation, and the significance of this activation pathway for overall plasmin (Pm) activity in mammary secretion of drying-off cows. Results of experiments in vitro showed that the spontaneous Pg activation observed on fresh preparations of bovine blood PMN was completely blocked by anti bovine TNF-alpha antibody, and was further up-regulated by exogenous bovine TNF-alpha. Monitoring the parameters of mammary secretion of drying-off cows revealed that both somatic cell counts and differential PMN ratio was significantly elevated at weeks 1, 2 and 3 of milk stasis. Nevertheless, specific activity of soluble Pm in mammary secretion increased and the level of 17-kDa TNF-alpha decreased immediately following milk stasis. Iimmunoblotting revealed that although both 26-kDa pro-TNF-alpha and 17-kDa TNF-alpha were consistently present in somatic cells of mammary secretion collected at weeks 0, 1, 2 and 3 of milk stasis, only 26-kDa pro-TNF-alpha was present in somatic cells of milk during lactation. In-vitro assay indicated that cell-free mammary secretion of drying-off cows exerted no Pg activation bioactivity towards bovine blood PMN. Altogether, the current study suggests the existence of an active TNF-alpha-Pg-Pm autocrine/paracrine loop on the massively infiltrated PMN inside udders of drying-off cows, which involves extensive binding and internalization of 17-kDa TNF-alpha on PMN and consequently activation of Pg, resulting in high Pm activity and low 17-kDa TNF-alpha level in mammary secretion. These coordinated mechanisms may play a role in the defence of drying-off mammary gland.
炎症细胞因子与免疫细胞上纤溶酶原（Pg）激活系统之间的相互作用尚未明确。在之前的研究中，我们报道了干奶期山羊和奶牛乳腺分泌物中与体细胞相关的蛋白水解活性升高。本研究的目的是检测肿瘤坏死因子-α（TNF-α）在多形核中性粒细胞（PMN）相关的Pg激活中的作用，以及该激活途径对干奶牛乳腺分泌物中总纤溶酶（Pm）活性的意义。体外实验结果表明，抗牛TNF-α抗体可完全阻断新鲜制备的牛血PMN上观察到的自发Pg激活，而外源性牛TNF-α可进一步上调该激活。监测干奶牛乳腺分泌物参数发现，在乳汁淤积的第1、2和3周，体细胞计数和PMN差异比例均显著升高。然而，乳汁淤积后，乳腺分泌物中可溶性Pm的比活性增加，17-kDa TNF-α水平立即下降。免疫印迹显示，尽管在乳汁淤积第0、1、2和3周收集的乳腺分泌体细胞中始终存在26-kDa前体TNF-α和17-kDa TNF-α，但泌乳期乳汁中的体细胞中仅存在26-kDa前体TNF-α。体外实验表明，干奶牛的无细胞乳腺分泌物对牛血PMN没有Pg激活生物活性。总之，当前研究表明，在干奶牛乳房内大量浸润的PMN上存在一个活跃的TNF-α-Pg-Pm自分泌/旁分泌环，这涉及17-kDa TNF-α在PMN上的广泛结合和内化，从而激活Pg，导致乳腺分泌物中Pm活性高而17-kDa TNF-α水平低。这些协调机制可能在干奶期乳腺的防御中发挥作用。
