McClenahan David J
Department of Biology, University of Northern Iowa, Cedar Falls, IA, USA50614.
J Anim Sci. 2025 Jan 4;103. doi: 10.1093/jas/skaf274.
Movement of leukocytes from the circulatory system into a site of inflammation is a highly complex process. The migration of neutrophils into the lumen of the mammary gland during mastitis is no exception. There is information about the involvement of bacterial-produced products during this process, but less is known regarding the role of host products. Two bovine cell lines, a primary mammary gland endothelial cell line and an immortalized bovine mammary gland epithelial cell line (Mac-T), along with freshly isolated bovine neutrophils, were used to study this further. The cell lines were grown on inserts and in wells of tissue-culture plates. In the initial set of experiments, neutrophils were added to the inserts, and then their migration into the tissue-culture plate wells was monitored using a hemocytometer or a flow cytometer. Lipopolysaccharide was added to some of the wells to induce migration. This was then followed by a similar series of experiments that were initialized by the addition of inhibitors to interleukin-8 (IL-8), platelet-activating factor (PAF), tumor-necrosis factor-α (TNF-α), or lipoxygenase (LOX) prior to the addition of the neutrophils and their enumeration. In addition, integrin expression (CD11a/18) by the neutrophils was measured using flow cytometry. In our insert/tissue culture plate well system, neutrophils readily migrated towards the epithelial cells when they were separated from them either by the insert alone or the insert plus a layer of endothelial cells. The presence of LPS in the system allowed this migration to occur without the involvement of epithelial cells. The inhibition of PAF or TNF alone did not alter migration, while the inhibition of either IL-8 or LOS did significantly reduce the movement of neutrophils. Only the migrating neutrophils had upregulated levels of CD11a/18 on their surface. From a host perspective, it appears that products of the LOX enzyme system and IL-8 were the primary inducers of neutrophil migration, and that mammary gland epithelial cells were capable of driving this process on their own. Understanding the role of host-produced chemotactic agents that are involved in mammary gland inflammation may allow better regulation of this activity.
白细胞从循环系统向炎症部位的移动是一个高度复杂的过程。乳腺炎期间中性粒细胞向乳腺管腔的迁移也不例外。关于此过程中细菌产生的产物的参与情况已有相关信息，但对于宿主产物的作用了解较少。为了进一步研究，使用了两种牛细胞系，一种是原代乳腺内皮细胞系，另一种是永生化牛乳腺上皮细胞系（Mac-T），以及新鲜分离的牛中性粒细胞。这些细胞系在插入物和组织培养板的孔中生长。在最初的一组实验中，将中性粒细胞添加到插入物中，然后使用血细胞计数器或流式细胞仪监测它们向组织培养板孔中的迁移。向一些孔中添加脂多糖以诱导迁移。随后进行了一系列类似的实验，在添加中性粒细胞并计数之前，先添加白细胞介素-8（IL-8）、血小板活化因子（PAF）、肿瘤坏死因子-α（TNF-α）或脂氧合酶（LOX）的抑制剂来启动实验。此外，使用流式细胞术测量中性粒细胞的整合素表达（CD11a/18）。在我们的插入物/组织培养板孔系统中，当中性粒细胞仅通过插入物或插入物加一层内皮细胞与上皮细胞分离时，它们很容易向上皮细胞迁移。系统中脂多糖的存在使得这种迁移能够在没有上皮细胞参与的情况下发生。单独抑制PAF或TNF不会改变迁移，而抑制IL-8或LOS则会显著减少中性粒细胞的移动。只有迁移的中性粒细胞表面的CD11a/18水平上调。从宿主的角度来看，似乎脂氧合酶系统和IL-8的产物是中性粒细胞迁移的主要诱导物，并且乳腺上皮细胞能够自行驱动这一过程。了解参与乳腺炎症的宿主产生的趋化剂的作用可能有助于更好地调节这种活动。
