Neutrophils (polymorphonuclear leukocytes) are short lived granulocytes that playa primordial role in host innate defense against invading pathogens. Freshly isolated neutrophils spontaneously undergo apoptosis when cultured, which is associated with oxidative stress. We found that there is a dramatic shift in the metabolism of the 5-lipoxygenase product 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) from its biologically inactive o-oxidation product in freshly isolated neutrophils to the potent granulocyte chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) in neutrophils cultured for 24 h. o-oxidation of the chemoattractant leukotriene B4 (LTB4) was also reduced in aging neutrophils incubated with arachidonic acid, resulting in higher levels of LTB4. The reduced o-oxidation activity appeared to be due to a decrease in active LTB4 20-hydroxylase. In contrast, the increased 5-oxo-ETE formation was not associated with an increase in the amount of active 5-hydroxyeicosanoid dehydrogenase, which is required for its formation, but rather with a dramatic increase in its cofactor NADP +. NAD+ levels also increased, but NADPH levels remained unchanged after 24 h. There was also evidence for increased oxidative stress (high GSSG/GSH) in aging neutrophils. The changes in 5-HETE metabolism and pyridine nucleotides in cultured neutrophils could be inhibited by neutrophil survival factors and antioxidants. These results suggest that in severe inflammation, aging neutrophils that have evaded rapid uptake by macrophages may produce increased amounts of the chemoattractants 5-oxo-ETE and LTB4, resulting in delayed resolution of inflammation. Similarly, we found that the NADPH oxidase activator PMA caused a very rapid and dramatic increase in NADP + levels in both freshly isolated and cultured neutrophils, accompanied by a rapid increase in 5-oxo-ETE synthesis and reduced o-oxidation activity. Surprisingly, this was not accompanied by a corresponding decline in NADPH levels, which instead initially increased, but rather by a precipitous reduction in NAD+, which mirrored the increase in NADP+. These results suggest that the phosphorylation of NAD+ by NAD kinase may be very important for providing both NADP+ for 5-oxo-ETE synthesis and NADPH for the respiratory burst.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.116087 |
| Date | January 2008 |
| Creators | Graham, François. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 003164025, proquestno: AAIMR67029, Theses scanned by UMI/ProQuest. |
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