Granule Localization of Glutaminase in Human Neutrophils and the Consequence of Glutamine Utilization for Neutrophil Activity

Abstract

The provision of glutamine in vivo has been observed to reduce to normal levels the neutrophilia observed after exhaustive exercise and to decrease the neutrophil chemoattractant, interleukin-8. Thus, the role for glutamine in the regulation of inflammatory mediators of human neutrophil activation was investigated. The study sought to establish whether glutamine supplementation in vitro affects neutrophil function at rest and whether glutaminase, the major enzyme that metabolizes glutamine, is present in human polymorphonuclear neutrophils (PMN). During in vitro studies, the addition of 2 mM glutamine increased the respiratory burst of human PMN stimulated with both phorbol myristate acetate (PMA) and formyl-methionyl-leucyl-phenylalanine. These observations were made using a highly sensitive, real time chemiluminescent probe, Pholasin ® . Glutamine alone did not stimulate the release of reactive oxygen species. In a novel finding using glutaminase-specific antibodies in combination with flow cytometry and confocal microscopy, glutaminase was shown to be present on the surface of human PMN. Subcellular fractionation revealed that the enzyme was enriched in the secondary granules and could be released into cell culture medium upon stimulation with PMA. In conclusion, human PMN appeared to utilize glutamine and possess the appropriate glutaminase enzyme for metabolizing glutamine. This may depress some pro-inflammatory factors that occur during prolonged, exhaustive exercise. Human polymorphonuclear neutrophils (PMNs)1 are a major class of the nonspecific immune response against infections. This is due, in part, to their ability to produce toxic forms of reactive oxygen species (ROS), including superoxide anions (O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH Ϫ ).The generation of ROS requires the cytosolic NADPH oxidase proteins to form a complex with several of the membranebound parts of the oxidases. Once assembled, direct stimulation of cells results in the release of O 2 . into the extracellular compartment or into phagosomes. In turn, activated PMN synthesize and release a number of pro-inflammatory cytokines, including tumor necrosis factor (TNF), IL-1, IL-6, and IL-8 (1, 2). Recent work has shown that glutamine supplementation in vitro enhances both phagocytosis and ROS in isolated PMN (3) and, in vivo, suppresses IL-8 production by PMN (4). This has important consequences, as glutamine appears to exert a regulatory influence on inflammatory processes by PMN responding to inflammatory and infectious stimuli. For example, PMNs taken from patients with burns (5) or after surgery (6) have been shown to have improved bactericidal activity in vitro when glutamine is added to the culture medium for incubation. Glutamine is an important substrate for some key cells of the immune system, such as macrophages and lymphocytes (7,8). It acts as a nitrogen donor for purine and pyrimidine nucleotide synthesis for new DNA synthesis and for mRNA repair. Although classified as non-essential, recent eviden...

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