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. 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) 1The abbreviations used are: PMN, polymorphonuclear neutrophils; FITC, fluorescein isothiocyanate; fMLP, formyl-methionyl-leucyl-phenylalanine; GA, glutaminase; GST, glutathione S-transferase; IL, interleukin; LGA, liver-type GA; MFI, mean fluorescence intensity; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PVDF, polyvinylidene fluoride; ROS, reactive oxygen species; TBS, Tris-buffered saline. 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 (O2–), hydrogen peroxide (H2O2), and hydroxyl radicals (OH–). The generation of ROS requires the cytosolic NADPH oxidase proteins to form a complex with several of the membrane-bound parts of the oxidases. Once assembled, direct stimulation of cells results in the release of O2·¯ 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 (1Cassatella M.A. Immunol. Today. 1995; 16: 21-26Google Scholar, 2Eggleton P. Anticancer Res. 1999; 19: 3711-3715Google Scholar). Recent work has shown that glutamine supplementation in vitro enhances both phagocytosis and ROS in isolated PMN (3Furukawa S. Saito H. Inoue T. Matsuda T. Fukatsu K. Han I. Ikeda S. Hidemura A. Nutrition. 2000; 16: 323-329Google Scholar) and, in vivo, suppresses IL-8 production by PMN (4Castell L. Sports Med. 2003; 33: 323-345Google Scholar). 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 (5Ogle C.K. Ogle J.D. Mao J.X. Simon J. Noel J.G. Li B.G. Alexander J.W. J. Parenter. Enteral Nutr. 1994; 18: 128-133Google Scholar) or after surgery (6Furukawa S. Saito H. Fukatsu K. Hashiguchi Y. Inaba T. Lin M.T. Inoue T. Han I. Matsuda T. Muto T. Nutrition. 1997; 13: 863-869Google Scholar) 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 (7Ardawi M.S. Newsholme E.A. Essays Biochem. 1985; 21: 1-44Google Scholar, 8Newsholme P. Newsholme E.A. Biochem. J. 1989; 261: 211-218Google Scholar). 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 evidence suggests that glutamine is conditionally essential when it becomes rapidly depleted in the blood in stressful situations. Plasma glutamine concentrations are substantially decreased by clinical trauma such as in major surgery by 37% (9Powell H. Castell L.M. Parry-Billings M. Desborough J.P. Hall G.M. Newsholme E.A. Clin. Physiol. 1994; 14: 569-580Google Scholar) or after prolonged, exhaustive exercise by 20–25% (10Castell L.M. Poortmans J.R. Newsholme E.A. Eur. J. Appl. Physiol. Occup. Physiol. 1996; 73: 488-490Google Scholar). The provision of glutamine or glutamine precursors to endurance athletes has resulted in a decreased self-reported incidence of illness, particularly for upper respiratory tract infections (URTI), in four studies (see Castell, 2003; Ref. 4Castell L. Sports Med. 2003; 33: 323-345Google Scholar). Increasing evidence, in vitro and in vivo, suggests that PMN may benefit from exogenous glutamine, which repletes the decrease in the blood concentration observed after stress. The energy substrate for PMN has traditionally been thought to be glucose. However, it is possible that PMN could use glutamine, particularly in cases such as severe infection in which glucose is restricted. Evidence for a direct effect of glutamine on neutrophil function has been demonstrated in rats, where it was shown that isolated PMN utilized glutamine at a rate of 12.8 nmol min–1 mg–1 protein (11Curi T.C. De Melo M.P. De Azevedo R.B. Zorn T.M. Curi R. Am. J. Physiol. 1997; 273: C1124-C1129Google Scholar) in the absence of glucose. The same workers further confirmed the presence of phosphate-dependent glutaminase (GA) in rat PMN using Western blots and immunocytochemistry methods. To date, the presence of GA in human PMN has not been demonstrated, despite several attempts to do so. Strenuous exercise stimulates leukocytosis and neutrophilia (12McCarthy D.A. Dale M.M. Sports Med. 1988; 6: 333-363Google Scholar, 13Woods J.A. Davis J.M. Smith J.A. Nieman D.C. Med. Sci. Sports Exerc. 1999; 31: 57-66Google Scholar, 14Larrabee R.C. J. Med. Res. 1902; 2: 76-82Google Scholar) and the release of immature PMN from bone marrow (15Hansen J.B. Wilsgard L. Osterud B. Eur. J. Appl. Physiol. Occup. Physiol. 1991; 62: 157-161Google Scholar, 16Suzuki K. Yamada M. Kurakake S. Okamura N. Yamaya K. Liu Q. Kudoh S. Kowatari K. Nakaji S. Sugawara K. Eur. J. Appl. Physiol. 2000; 81: 281-287Google Scholar). However, there are conflicting reports upon the effects of exercise on ROS release by PMN (17Suzuki K. Sato H. Kikuchi T. Abe T. Nakaji S. Sugawara K. Totsuka M. Sato K. Yamaya K. J. Appl. Physiol. 1996; 81: 1213-1222Google Scholar). Significant increases have been observed in the plasma concentration of IL-8 (∼2-fold) immediately after and 1.5 h after a marathon (18Nieman D.C. Henson D.A. Smith L.L. Utter A.C. Vinci D.M. Davis J.M. Kaminsky D.E. Shute M. J. Appl. Physiol. 2001; 91: 109-114Google Scholar) and in cell IL-8 production after a rowing ergo test (19Castell L.M. The Role of Some Amino Acids in Exercise, Fatigue, and Immunosuppression. University of Oxford, Oxford1996Google Scholar). This suggests enhanced post-exercise activation of PMN. Significant decreases in post-exercise neutrophilia (∼1.75-fold) and cell IL-8 production (∼3.5-fold) have been observed after a marathon in runners taking glutamine compared with a placebo (20Castell L.M. Newsholme E.A. Can. J. Physiol. Pharmacol. 1998; 76: 524-532Google Scholar). Since then, in three further studies in our laboratory, a reduction in IL-8 associated with glutamine feeding after exercise has been observed (see Castell, 2003; Ref. 4Castell L. Sports Med. 2003; 33: 323-345Google Scholar). Clinical studies (21de Beaux A.C. O'Riordain M.G. Ross J.A. Jodozi L. Carter D.C. Fearon K.C. Nutrition. 1998; 14: 261-265Google Scholar, 22O'Riordain M.G. De Beaux A. Fearon K.C. Nutrition. 1996; 12: S82-S84Google Scholar) established that IL-8 production was reduced in both surgical patients and patients with acute pancreatitis who received glutamine-enriched parenteral nutrition. IL-8 is a potent chemoattractant that perpetuates the inflammatory response and attracts PMN to the site of tissue damage. The present study was set up to establish whether glutamine supplementation affects human neutrophil respiratory burst via in vitro incubation of blood with/without glutamine and to determine whether human PMN utilizes glutamine via the presence of GA, using both immunochemical techniques and confocal microscopy. Subjects and Blood Samples—Ethical permission for the studies was obtained from the Central Oxford Research Ethics Committee (COREC). Resting blood samples were taken from healthy controls recruited from colleagues. Neutrophil function was measured with a novel chemiluminescence assay as described below. Isolation of PMN—Isolated whole cells and subcellular neutrophil fractions were assayed for the presence of glutaminase. Neutrophils were isolated as described previously (23Eggleton P. Wang L. Penhallow J. Crawford N. Brown K.A. Ann. Rheum. Dis. 1995; 54: 916-923Google Scholar). Briefly, 5 ml of anticoagulated (potassium EDTA, 1.5 mg/ml) blood was layered on top of 4 ml of PolymorphPrep™ (Nycomed Pharma AS) in a 15-ml conical tube that was then spun at 1000 × g for 45 min. The layer of PMN was removed using a Pasteur pipette and placed with an equal volume of hypertonic sodium chloride solution (0.45% w/v) in another 15-ml tube to reduce viscosity. Following another spin, the supernatant was aspirated and the pellet resuspended in lysis buffer (isotonic NH4Cl) and spun again. The pellet was resuspended in 0.5 ml of phosphate-buffered saline without calcium or magnesium (PBS2–), and the cells were counted and checked for viability by the exclusion of 0.02% (v/v) trypan blue and stored at 4 °C until required. Subcellular Fractionation of PMN—Subcellular fractionation of human PMN by nitrogen cavitation is an established technique for elucidating PMN structural composition (24Kjeldsen L. Sengelov H. Borregaard N. J. Immunol. Methods. 1999; 232: 131-143Google Scholar). Briefly, freshly isolated, unstimulated PMN (2 × 108 per condition) were resuspended in 5 ml of nitrogen cavitation buffer (0.34 m sucrose, 10 mm Hepes, 1 mm EDTA, 0.1 mm MgCl2, and 1 mm Na2ATP, pH 7.4), followed by lysis by nitrogen cavitation (15 min, 400 p.s.i.) (4 °C). The lysates were centrifuged (1000 × g), and supernatants were then subjected to isopycnic sucrose density gradient fractionation on linear 20–55% sucrose gradients in a Beckman SW-28 swinging bucket rotor (100,000 × g; 3 h, 4 °C) exactly as described previously. 1.5-ml fractions were collected from each gradient and analyzed for marker proteins, plasma membrane (alkaline phosphatase), primary granules (myeloperoxidase), and secondary granules (lactoferrin) as described previously (25Wirthmueller U. Dewald B. Thelen M. Schafer M.K. K. J. P. J. Immunol. 1997; Scholar). Blood Neutrophil with and without a novel chemiluminescent is to the burst in real time by This technique the which is isolated from the the when activated by radicals and reactive oxygen species such as released by activated blood was in with or without glutamine concentration 2 and with of and for 3 h at °C with the blood was assayed for PMN activity using the chemiluminescence assay and with the 1 as below. were further in 2 ml of blood buffer saline mm an of and assay buffer was added to each with of to the of and of whole The cells were stimulated by the addition of of concentration 1 or concentration and the response was measured until the with and with using a of of the human liver-type GA from cells A. De I. J.A. J. Biochem. J. 2000; Scholar) was and in the site of This for the of the of human LGA, was in as a protein and were as described previously A. De I. J.A. J. Biochem. J. 2000; Scholar) using a The protein was used for of and antibodies were as described J.A. S. I. J. 1995; 6: Scholar). and Western of of neutrophil membrane by nitrogen cavitation as described previously (25Wirthmueller U. Dewald B. Thelen M. Schafer M.K. K. J. P. J. Immunol. 1997; were in buffer pH and blue with for 5 at and then to of a was in Tris-buffered saline at a of for 2 were The was with and was as and the proteins were by membrane for at a of of the nonspecific with the proteins were with a of glutaminase as described below. The was then in mm pH and mm and then for 1 h in an in the was to an substrate for 1 to and then the membrane was to for 5 min. and of GA on the surface of human PMN was using both flow cytometry and a and an work PMNs were with glutaminase for min, and, after three in the cells were with for at 4 °C and then three in In the flow cytometry studies, the PMNs were stimulated with or 0.1 or in phosphate-buffered saline with calcium and magnesium for at °C with test The cells were then in (v/v) and analyzed on a or spun at in a and were the using an and with The cells were on the with a confocal were taken from the top to the of each cell using a The appropriate and for were were collected for each were compared using with of in the of blood from healthy stimulated with concentration and concentration 1 were with the highly real time chemiluminescence to ROS release in the presence and absence of glutamine. of samples an increased response to when with glutamine compared with glutamine. In the there were who did not to the addition of glutamine. However, the response was not whether or not glutamine was In a four of four samples an increased response to Glutamine stimulated the burst from cells without the of response However, cells increased both the of response and the burst in the presence of glutamine. of on the of Human The that glutamine a effect on burst from to whether human PMN, as the of the enzyme GA on cell PMNs were isolated as described and for the presence of GA by flow shown in cells appeared as of cells and with to surface of PMN surface GA mean fluorescence of the cells were highly for GA for an of of the cells with to of surface GA and the of highly GA of cells In effect on surface GA levels in the and the surface of PMN in 0.5 with that the GA was on or the cell surface and could be into the cell surface at the surface of the of glutaminase at the cell surface of human confocal of isolated neutrophils were taken from the top of the cell 0.5 the surface and that the glutaminase is the of the which appears to of neutrophils of number of human including and have been to produce GA, to GA has not been in human PMN. To the of the GA from the PMNs stimulated the cells with and for min. The in which 5 × cells been were collected and to of were on a with GA, and and with glutaminase shown in Western revealed that the supernatants from cells that been with In supernatants from cells and cells GA as by The presence of GA in the cell that it be from the Thus, it was to neutrophil which been previously by nitrogen cavitation into four primary granules secondary granules and plasma and cell supernatant The protein of four fractions are shown in and the of protein fractions that a protein was in the secondary granules not in of the subcellular The that lymphocytes and macrophages use of glutamine has been for a number of (7Ardawi M.S. Newsholme E.A. Essays Biochem. 1985; 21: 1-44Google Scholar, 8Newsholme P. Newsholme E.A. Biochem. J. 1989; 261: 211-218Google Scholar). recent observations have evidence that glutamine is utilized by rat PMN T.C. Curi R. Biochem. Scholar). M. J. Physiol. 1998; Scholar) observed of glutamine by human neutrophils stimulated in vitro with D.A. Newsholme P. Clin. Sci. glutamine by human neutrophils The ability of glutamine to the function of PMN has been in patients (5Ogle C.K. Ogle J.D. Mao J.X. Simon J. Noel J.G. Li B.G. Alexander J.W. J. Parenter. Enteral Nutr. 1994; 18: 128-133Google Scholar) and (3Furukawa S. Saito H. Inoue T. Matsuda T. Fukatsu K. Han I. Ikeda S. Hidemura A. Nutrition. 2000; 16: 323-329Google Scholar). The of the production of enhanced reactive oxygen by glutamine is Recent that glutamine the of some of the proteins that form the NADPH oxidase in turn, the production of the superoxide T.C. A.C. Curi R. Clin. Sci. Scholar). In glutamine appears to the of the cytosolic protein as as the and NADPH and glutamine by GA in PMN a decrease in superoxide the presence and subcellular of glutamine appears to be important for superoxide production by PMN. In the present the results obtained the that glutamine burst by human PMN in the presence of phorbol and These were using a real time in vitro assay of The supplementation of whole blood with 2 mm glutamine not to a production of superoxide increased the rate of GA activity has been in rat PMN T.C. Curi R. Biochem. and studies have GA in human tumor cells Res. Scholar, A. J.D. Biochem. J. 2003; where GA is an essential for cell In vitro from burst in culture with or without glutamine used to whole blood samples from marathon a role for glutamine and glutaminase in neutrophil during However, during with the extracellular at the cytosolic and membrane and the NADPH oxidase to form the complex upon glutamine a role in O2·¯ to GA in a The present results that GA is present on the surface of cells and is released into the medium upon PMA, not fMLP, In the present was obtained on the of GA by confocal microscopy. 0.5 PMN, it was observed that GA was into the cell PMNs are and the GA appeared to be in at or the plasma In an or evidence of the presence of GA in human PMN was using three and of techniques was to the to determine in the subcellular of glutamine in human PMN, techniques were cavitation and density gradient a and of granules granules and plasma membrane as as a GA was in the secondary granules not in the plasma membrane in PMN, GA is from the secondary granules and to the cell Following not the GA is removed from the membrane fractions and be in the extracellular culture It as granules with the plasma membrane at the of the NADPH oxidase and GA be in to the plasma membrane in response to This into with their a number of have of glutaminase in human tumor cell at the mRNA and protein A. J.D. Biochem. J. 2003; Scholar) and in cells L. A. J.A. J.A. J. J. Scholar). In the present of human PMN with PMA, a resulted in the release of glutaminase from the cell surface into cell culture However, three were by the This is with the results of that revealed of of GA L. J. N. B. Am. J. Med. Scholar). secondary granules were by Western a glutaminase of was observed and compared with that for rat neutrophil GA, which is (11Curi T.C. De Melo M.P. De Azevedo R.B. Zorn T.M. Curi R. Am. J. Physiol. 1997; 273: C1124-C1129Google Scholar). a in glutaminase in human neutrophils on the cell surface stimulation with PMA, there was in after stimulation with and are that protein kinase by via with surface and activation and The phorbol PMA, on the the cell membrane and a and of protein kinase of the release of and inflammatory mediators into the extracellular of stimulated In the of glutaminase appears to be in the secondary granules of and on the cell where it is for glutamine The of the observed be to the of activation in the that results in and with the plasma in activity and have been previously Y. Scholar). PKC, which may the release of glutaminase by as compared with J. Pharmacol. 1998; Scholar). studies be to the of is an in the plasma concentration of neutrophil after exercise B. U. J. Sports Med. 1989; Scholar). of PMN are by bone marrow on a in healthy J.D. 1988; Scholar). It is possible that to as the PMN the In glutamine an important role in cells in a of PMN of the glutathione J.A. for which glutamine is a The that in vivo glutamine feeding has cell production of IL-8 in several in vitro studies is IL-8 acts as a potent chemoattractant for PMNs and to the blood and into or IL-8 NADPH oxidase and in turn, the release of ROS from PMN from recent studies that glutamine has a effect on IL-8 production in cell during activation by inflammatory mediators (4Castell L. Sports Med. 2003; 33: 323-345Google Scholar, Beaux A.C. O'Riordain M.G. Ross J.A. Jodozi L. Carter D.C. Fearon K.C. Nutrition. 1998; 14: 261-265Google Scholar, 22O'Riordain M.G. De Beaux A. Fearon K.C. Nutrition. 1996; 12: S82-S84Google Scholar, M. R. P. P. Clin. Nutr. 2003; Scholar, Y. Li N. K. J. 2003; Scholar, M.T. Saito H. S. R. J. Parenter. Enteral Nutr. 2001; Scholar). The of neutrophil chemoattractant be of the effect of glutamine in the blood upon or immature PMN. of the are recruited during the leukocytosis that in response to exercise. However, glutamine supplementation has been shown to reduce the number of PMNs to normal levels compared with a placebo h after a (20Castell L.M. Newsholme E.A. Can. J. Physiol. Pharmacol. 1998; 76: 524-532Google Scholar). The of regulation of IL-8 production by blood is not It could in cell by such as M. R. P. P. Clin. Nutr. 2003; Scholar). In conclusion, the present study suggests that glutamine supplementation the respiratory burst of human PMN to inflammatory in the novel finding has been made that there is a of GA, the major enzyme for glutamine in the granules of human PMN. This is to to the of immune function of PMN in clinical and stress. are to of for and with the assay and to for the of the