Increased 15-HPETE production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells.

Selenium (Se) is an integral component of glutathione peroxidase and is able to detoxify peroxides that can affect arachidonic acid (AA) metabolism, thereby influencing eicosanoid biosynthesis. This study investigated the effects of oxidant stress, a consequence of Se deficiency, on eicosanoid formation and important key enzyme expression in bovine aortic endothelial cells (BAEC). Bovine aortic endothelial cells cultured in Se-deficient media and stimulated with tumor necrosis factor alpha or H2O2 produced significantly less prostacyclin (PGI(2)) and more 15-hydroxyeicosatetraenoic acid, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and thromboxane than Se-supplemented BAEC. Additionally, reverse transcription polymerase chain reaction and immunoblotting determined that the mRNA and protein levels of the eicosanoid forming enzymes cyclooxygenase-1 (COX1), cyclooxygenase-2 (COX2), and PGI synthase were not significantly changed. The addition of 15-HPETE to Se-supplemented BAEC inhibited the production of PGI(2) suggesting that the accumulation of lipid hydroperoxides during Se-deficiency may be the underlying factor in the altered eicosanoid production during Se deficiency. Furthermore, inhibition of COX and addition of PGH(2) to Se-deficient or Se-supplemented BAEC still resulted in lower PGI(2) formation by Se-deficient cells. Together, these results suggest that Se deficiency modifies eicosanoid production by affecting the activity of key enzymes, particularly PGI synthase, rather than their transcription or translation.

Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium.

Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor.

Lipoxin A4 (LXA4) triggers selective responses with human neutrophils that are pertussis toxin sensitive and binds to high affinity receptors (Kd = 0.5 +/- 0.3 nM) that are modulated by stable analogues of guanosine 5'-triphosphate (GTP). Here, we characterized [11,12-(3)]LXA4 specific binding with neutrophil granule and plasma membranes, which each display high affinity binding sites (Kd = 0.7 +/- 0.1 nM) that were regulated by GTP gamma S. Since functional LXA4 receptors are inducible in HL-60 cells, we tested orphan cDNAs encoding 7-transmembrane region receptors cloned from these cells for their ability to bind and signal with LXA4. Chinese hamster ovary (CHO) cells transfected with the orphan receptor cDNA (pINF114) displayed specific 3H-LXA4 high affinity binding (1.7 nM). When displacement of LXA4 binding with pINF114-transfected CHO cells was tested with other eicosanoids, including LXB4, leukotriene D4 (LTD4), LTB4, or prostaglandin E2, only LTD4 competed with LXA4, giving a Ki of 80 nM. In transfected CHO cells, LXA4 also stimulated GTPase activity and provoked the release of esterified arachidonate, which proved to be pertussis toxin sensitive. These results indicate that pINF114 cDNA encodes a 7-transmembrane region-containing protein that displays high affinity for 3H-LXA4 and transmits LXA4-induced signals. Together, they suggest that the encoded protein is a candidate for a LXA4 receptor in myeloid cells.

Selenium deficiency alters the lipoxygenase pathway and mitogenic response in bovine lymphocytes.

We investigated the effect of altered selenium (Se) nutrition on arachidonic acid oxidation in immune cells. Experiments were conducted with peripheral blood lymphocytes obtained from dairy cattle fed diets either supplemented with or deficient in Se. The results indicate that the concanavalin A-stimulated lymphocyte proliferation was significantly lower in Se-deficient cows. When stimulated by calcium ionophore A23187, the lymphocytes derived from Se-deficient cows produced significantly less 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) than those obtained from Se-supplemented cows. When included in cell cultures from animals fed +Se diets, 5-HETE and LTB4 elicited a partial reversal of the inhibition of lymphocyte proliferation by either hydrocortisone or nordihydroguaiaretic acid. Based on this information, we postulate that dietary Se status, which in turn determines tissue Se concentration, plays an important role in the regulation of arachidonate metabolism by way of the 5-lipoxygenase pathway. This may be one of the biochemical mechanisms underlying the inhibition of lymphocyte proliferation and the decrease in resistance to infectious diseases observed in Se-deficient animals.

Increased 12-HETE production in bovine lymphocytes during selenium deficiency.

When peripheral blood lymphocytes were incubated with arachidonic acid in the presence of Ca++ ionophore (A23187), the cells from the selenium-deficient dairy cows produced significantly greater quantities of 12-hydroxyeicosatetraenoic acid (12-HETE) than the cells from the selenium-supplemented animals. The major product of reaction was verified as 12-HETE by cochromatography with a 12-HETE standard on an HPLC and structural analysis by GC-MS. Additionally, concanavalin A-stimulated lymphocyte proliferation was significantly decreased in cells from the Se-deficient cows. Furthermore, 12-HETE generated by the A23187-stimulated lymphocytes inhibited lymphocyte proliferation when added to Se-supplemented cell cultures. These observations suggest that self-regulation of lymphocyte proliferation might be mediated by 12-HETE production, especially during an altered nutritional state such as Se deficiency.

Dietary selenium effects on milk eicosanoid concentration in dairy cows during coliform mastitis.

The effect of selenium deficiency on the product profile of arachidonic acid oxidation by enzymatic pathways in Holstein cows with experimentally-induced coliform mastitis was investigated. The animals were fed dairy rations containing 0.05 mg Se/kg dry matter, with the supplemented group receiving additional Se to increase the dietary concentration to approximately 0.35 mg Se/kg dry matter. Cows were inoculated intracisternally with 30 colony-forming-units of Escherichia coli at 14-16 weeks of lactation. Eicosanoids and bacteria numbers were recorded at various intervals of time for 60 h postinoculation. Milk from cows fed the Se-depleted diet had significantly higher (p less than 0.05) concentrations of TXB2 between 24 and 48 h and 6-keto-PGF1 alpha between 24 and 60 h postinoculation. Milk PGE2 concentration was significantly higher in the Se-deficient group at 24 h, whereas LTB4 was higher between 36 and 60 h postinoculation in the Se-deficient cows (p less than 0.05). Milk bacteria numbers were significantly higher between 16 and 24 h postinoculation in the Se-deficient group and three of the four cows in this group required euthanasia, whereas all four cows in the Se-supplemented group recovered without therapeutic intervention. These data indicate marked effects of dietary Se on milk eicosanoid concentrations in response to an E. coli infection. The changes in eicosanoid concentrations may be associated with the altered pathogenesis and outcome of mastitis in a Se-deficient state.