Alarcón P, Conejeros I, Carretta M D, Concha C, Jara E, Tadich N, Hidalgo M A, Burgos R A
Laboratory of Inflammation Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Science, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile.
Vet Immunol Immunopathol. 2011 Nov 15;144(1-2):68-78. doi: 10.1016/j.vetimm.2011.07.012. Epub 2011 Jul 23.
D-lactic acidosis occurs in ruminants, such as cattle, with acute ruminal acidosis caused by ingestion of excessive amounts of highly fermentable carbohydrates. Affected animals show clinical signs similar to those of septic shock, as well as acute laminitis and liver abscesses. It has been proposed that the inflammatory response and susceptibility to infection could both be caused by the inhibition of phagocytic mechanisms. To determine the effects of d-lactic acid on bovine neutrophil functions, we pretreated cells with different concentrations of D-lactic acid and measured intracellular pH using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and calcium flux using FLUO-3 AM-loaded neutrophils. Reactive oxygen species (ROS) production was measured using a luminol chemiluminescence assay, and MMP-9/gelatinase-B granule release was measured by zymography. CD11b and CD62L/l-selectin expression, changes in cell shape, superoxide anion production, phagocytosis of Escherichia coli-Texas red bioparticles, and apoptosis were all measured using flow cytometry. Our results demonstrated that D-lactic acid reduced ROS production, CD11b upregulation and MMP-9 release in bovine neutrophils treated with 100 nM platelet-activating factor (PAF). D-lactic acid induced MMP-9 release and, at higher concentrations, upregulated CD11b expression, decrease L-selectin expression, and induces late apoptosis. We concluded that D-lactic acid can interfere with neutrophil functions induced by PAF, leading to reduced innate immune responses during bacterial infections. Moreover, the increase of MMP-9 release and CD11b expression induced by 10mM D-lactic acid could promote an nonspecific neutrophil-dependent inflammatory reaction in cattle with acute ruminal acidosis.
D-乳酸性酸中毒发生于反刍动物，如牛，由摄入过量高度可发酵碳水化合物引起急性瘤胃酸中毒所致。患病动物表现出与感染性休克相似的临床症状，以及急性蹄叶炎和肝脓肿。有人提出，炎症反应和对感染的易感性可能均由吞噬机制受抑制所致。为确定D-乳酸对牛中性粒细胞功能的影响，我们用不同浓度的D-乳酸预处理细胞，使用2',7'-双-(2-羧乙基)-5-(和-6)-羧基荧光素乙酰氧基甲酯（BCECF-AM）测量细胞内pH，并使用负载FLUO-3 AM的中性粒细胞测量钙通量。使用鲁米诺化学发光测定法测量活性氧（ROS）生成，通过酶谱法测量MMP-9/明胶酶-B颗粒释放。使用流式细胞术测量CD11b和CD62L/L-选择素表达、细胞形状变化、超氧阴离子生成、大肠杆菌-德克萨斯红生物颗粒的吞噬作用以及细胞凋亡。我们的结果表明，D-乳酸降低了用100 nM血小板活化因子（PAF）处理的牛中性粒细胞中的ROS生成、CD11b上调和MMP-9释放。D-乳酸诱导MMP-9释放，且在较高浓度时上调CD11b表达、降低L-选择素表达并诱导晚期细胞凋亡。我们得出结论，D-乳酸可干扰PAF诱导的中性粒细胞功能，导致细菌感染期间固有免疫反应降低。此外，10 mM D-乳酸诱导的MMP-9释放增加和CD11b表达上调可促进急性瘤胃酸中毒牛的非特异性中性粒细胞依赖性炎症反应。
