• View in gallery
    Figure 1—

    Mean ± SD phagocytic capacity (A) and OBA (B) in PMNs isolated from blood samples obtained from 15 healthy Beagles (n = 5/group) undergoing MPSS treatment that received an IV infusion of saline (0.9% NaCl) solution alone (control; white bars), admixture of saline solution with 8.5% amino acids solution (2.3 g/kg/d; gray bars), or admixture of saline solution with 8.5% amino acids solution (1.8 g/kg/d) supplemented with 20% Ala-Gln solution (0.5 g/kg/d; black bars). *Mean values at 2 hours after MPSS injections ceased to differ significantly (P < 0.05) between the treatment groups. †Mean values of the control and generic amino acids groups differ significantly (P < 0.05) at 2 hours after injection relative to values before injection. MFI = Mean fluorescence intensity.

  • View in gallery
    Figure 2—

    Mean ± SD total degree of cellular F-actin expression in the same dogs as in Figure 1. *Mean values at 2 hours after MPSS injection differ significantly (P < 0.05) between the control and Ala-Gln groups. †Mean values of each group differ significantly (P < 0.05) at 2 hours after MPSS injection relative to values immediately before injection. See Figure 1 for remainder of key.

  • View in gallery
    Figure 3—

    Representative fluorescence images of PMNs isolated from blood samples obtained from one of the control dogs (top row) and one of the Ala-Gln–treated dogs (bottom row), represented in Figure 1, at the points of PMN contact with microspheres. Filamentous polymeric actin fluorescence (green) was merged with nuclear fluorescence (propidium iodide counterstain; red). Bar = 8 μm.

  • 1.

    Segal AW. How neutrophils kill microbes. Annu Rev Immunol 2005; 23:197223.

  • 2.

    Nauseef WM. How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 2007; 219:88102.

  • 3.

    English D. The remarkable neutrophil! Developing a blueprint for integrated cellular signaling. In: Gabrilovich DI, ed. The neutrophils: new outlook for old cells. 2nd ed. London: Imperial College Press, 2005:134.

    • Search Google Scholar
    • Export Citation
  • 4.

    Appelberg R. Neutrophils and intracellular pathogens: beyond phagocytosis and killing. Trends Microbiol 2007; 15:8792.

  • 5.

    Lee WLHarrison REGrinstein S. Phagocytosis by neutrophils. Microbes Infect 2003; 5:12991306.

  • 6.

    Torres MCoates TD. Function of the cytoskeleton in human neutrophils and methods for evaluation. J Immunol Methods 1999; 232:89109.

  • 7.

    May RCMachesky LM. Phagocytosis and the actin cytoskeleton. J Cell Sci 2001; 114:10611077.

  • 8.

    Kang JHYang MP. Effect of a short-term infusion with soybean oil-based lipid emulsion on phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes. J Vet Intern Med 2008; 22:11661173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Dahlgren CKarlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999; 232:314.

  • 10.

    Fialkow LWang YDowney GP. Reactive oxygen and nitrogen species as signaling molecules regulating neutrophil function. Free Radic Biol Med 2007; 42:153164.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Brown SAHall ED. Role of oxygen-derived free radicals in the pathogenesis of shock and trauma, with focus on central nervous system injuries. J Am Vet Med Assoc 1992; 200:18491859.

    • Search Google Scholar
    • Export Citation
  • 12.

    Bracken MBHolford TR. Effects of timing of methylprednisolone or naloxone administration on recovery of segmental and long-tract neurological function in NASCIS 2. J Neurosurg 1993; 79:500507.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Kim YTCaldwell JMBellamkonda RV. Nanoparticle-mediated local delivery of methylprednisolone after spinal cord injury. Biomaterials 2009; 30:25822590.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Braughler JMHall EDMeans ED, et al. Evaluation of an intensive methylprednisolone sodium succinate dosing regimen in experimental spinal cord injury. J Neurosurg 1987; 67:102105.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Hurlbert RJ. Methylprednisolone for acute spinal cord injury: an inappropriate standard of care. J Neurosurg 2000; 93(suppl 1):17.

  • 16.

    Olby N. Current concepts in the management of acute spinal cord injury. J Vet Intern Med 1999; 13:399407.

  • 17.

    Sayer FTKronvall ENilsson OG. Methylprednisolone treatment in acute spinal cord injury: the myth challenged through a structured analysis of published literature. Spine J 2006; 6:335343.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Gerndt SJRodriguez JLPawlik JW, et al. Consequences of high-dose steroid therapy for acute spinal cord injury. J Trauma 1997; 42:279284.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Kang JHYang MP. In vitro evaluation of the effect of trans-10, cis-12 conjugated linoleic acid on phagocytosis by canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to methylprednisolone sodium succinate. Am J Vet Res 2008; 69:494500.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Shimamura SKanayama KShimada T, et al. Evaluation of the function of polymorphonuclear neutrophilic leukocytes in healthy dogs given a high dose of methylprednisolone sodium succinate. Am J Vet Res 2010; 71:541546.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Chan DL. The role of nutrients in modulating disease. J Small Anim Pract 2008; 49:266271.

  • 22.

    Saker KE. Nutrition and immune function. Vet Clin North Am Small Anim Pract 2006; 36:11991224.

  • 23.

    Roth E. Nonnutritive effects of glutamine. J Nutr 2008; 138:2025S2031S.

  • 24.

    Novak FHeyland DKAvenell A, et al. Glutamine supplementation in serious illness: a systematic review of the evidence. Crit Care Med 2002; 30:20222029.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Furukawa SSaito HFukatsu K, et al. Glutamine-enhanced bacterial killing by neutrophils from postoperative patients. Nutrition 1997; 13:863869.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Furukawa SSaito HInoue T, et al. Supplemental glutamine augments phagocytosis and reactive oxygen intermediate production by neutrophils and monocytes from postoperative patients in vitro. Nutrition 2000; 16:323329.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Mauldin GEReynolds AJMauldin GN, et al. Nitrogen balance in clinically normal dogs receiving parenteral nutrition solutions. Am J Vet Res 2001; 62:912920.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    DiBartola SPBateman S. Introduction to fluid therapy. In: DiBartola SP, ed. Fluid, electrolyte, and acid-base disorders in small animal practice. 3rd ed. St Louis: Saunders Elsevier, 2006:325344.

    • Search Google Scholar
    • Export Citation
  • 29.

    Thomas WB. Disorders of the spinal cord. In: Morgan RVBright RMSwartout MS, eds. Handbook of small animal practice. 4th ed. Philadelphia: Saunders, 2003:256275.

    • Search Google Scholar
    • Export Citation
  • 30.

    Moritz AFickenscher YMeyer K, et al. Canine and feline hematology reference values for the ADVIA 120 hematology system. Vet Clin Pathol 2004; 33:3238.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Richardson MPAyliffe MJHelbert M, et al. A simple flow cytometry assay using dihydrorhodamine for the measurement of the neutrophil respiratory burst in whole blood: comparison with the quantitative nitrobluetetrazolium test. J Immunol Methods 1998; 219:187193.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Ziegler TRBye RLPersinger RL, et al. Effects of glutamine supplementation on circulating lymphocytes after bone marrow transplantation: a pilot study. Am J Med Sci 1998; 315:410.

    • Search Google Scholar
    • Export Citation
  • 33.

    Mondello SItaliano DGiacobbe MS, et al. Glutamine-supplemented total parenteral nutrition improves immunological status in anorectic patients. Nutrition 2010; 26:677681.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Cetinbas FYelken BGulbas Z. Role of glutamine administration on cellular immunity after total parenteral nutrition enriched with glutamine in patients with systemic inflammatory response syndrome. J Crit Care 2010; 25:661:e1e6.

    • Search Google Scholar
    • Export Citation
  • 35.

    Newsholme PCuri RPithon-Curi TC, et al. Glutamine metabolism by lymphocytes, macrophages, and neutrophils: its importance in health and disease. J Nutr Biochem 1999; 10:316324.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Li PYin YLLi D, et al. Amino acids and immune function. Br J Nutr 2007; 98:237252.

  • 37.

    Yao ZDuBois DCAlmon RR, et al. Pharmacokinetic/pharmacodynamic modeling of corticosterone suppression and lymphocytopenia by methylprednisolone in rats. J Pharm Sci 2008; 97:28202832.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Feduska NJTurcotte JGGikas PW, et al. Reversal of renal allograft rejection with intravenous methylprednisolone “pulse” therapy. J Surg Res 1972; 12:208215.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Gold RButtgereit FToyka KV. Mechanism of action of glucocorticoid hormones: possible implications for therapy of neuroimmunological disorders. J Neuroimmunol 2001; 117:18.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40.

    Buttgereit FScheffold A. Rapid glucocorticoid effects on immune cells. Steroids 2002; 67:529534.

  • 41.

    Liu LWang YXZhou J, et al. Rapid non-genomic inhibitory effects of glucocorticoids on human neutrophil degranulation. Inflamm Res 2005; 54:3741.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Clements MKSiemsen DWSwain SD, et al. Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses. J Leukoc Biol 2003; 73:344355.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Pithon-Curi TCDe Melo MPCuri R. Glucose and glutamine utilization by rat lymphocytes, monocytes, neutrophils in culture: a comparative study. Cell Biochem Funct 2004; 22:321326.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Castell LVance CAbbott R, et al. Granule localization of glutamine in human neutrophils and the consequence of glutamine utilization for neutrophil activity. J Biol Chem 2004; 279:1330513310.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Garcia Cde Oliveira MCVerlengia R, et al. Effect of dexamethasone on neutrophil metabolism. Cell Biochem Funct 2003; 21:105111.

  • 46.

    Kong SEHall JCCooper D, et al. Glutamine-enriched parenteral nutrition regulates the activity and expression of intestinal glutaminase. Biochim BiophysActa 2000; 1475:6775.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47.

    Holten ATGundersen V. Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase. J Neurochem 2008; 104:10321042.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Røshol HSkrede KKAErø CE, et al. Dexamethasone and methylprednisolone affect rat peritoneal phagocyte chemiluminescence after administration in vivo. Eur J Pharmacol 1995; 286:917.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49.

    Yoshida TTanaka MSotomatsu A, et al. Effect of methylprednisolone-pulse therapy on superoxide production of neutrophils. Neurol Res 1999; 21:509512.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50.

    Mouithys-Mickalad ADeby-Dupont GMathy-Hartert M, et al. Effect of glucocorticoids on the respiratory burst of Chlamydia-primed THP-1 cells. Biochem Biophys Res Commun 2004; 318:941948.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51.

    Pithon-Curi TCLevada ACLopes LR, et al. Glutamine plays a role in superoxide production and the expression of p47phox, p22phox and gp91phox in rat neutrophils. Clin Sci (Lond) 2002; 103:403408.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52.

    Wernerman J. Clinical use of glutamine supplementation. J Nutr 2008; 138:2040S2044S.

Advertisement

Effect of parenteral l-alanyl-l-glutamine administration on phagocytic responses of polymorphonuclear neutrophilic leukocytes in dogs undergoing high-dose methylprednisolone sodium succinate treatment

Ji-Houn KangLaboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361–763, Republic of Korea.

Search for other papers by Ji-Houn Kang in
Current site
Google Scholar
PubMed
Close
 DVM, PhD
,
Sung-Soo KimLaboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361–763, Republic of Korea.

Search for other papers by Sung-Soo Kim in
Current site
Google Scholar
PubMed
Close
 DVM, MS
, and
Mhan-Pyo YangLaboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361–763, Republic of Korea.

Search for other papers by Mhan-Pyo Yang in
Current site
Google Scholar
PubMed
Close
 DVM, PhD

Abstract

Objective—To determine whether parenteral l-alanyl-l-glutamine (Ala-Gln) administration modulated phagocytic responses of polymorphonuclear neutrophilic leukocytes (PMNs) from dogs undergoing high-dose methylprednisolone sodium succinate (MPSS) treatment.

Animals—15 healthy Beagles.

Procedures—Dogs were randomly assigned to 3 treatment groups (n = 5/group): 38-hour IV infusion of saline (0.9% NaCl) solution (control group), saline solution with 8.5% amino acids (2.3 g/kg/d), or saline solution with 8.5% amino acids (1.8 g/kg/d) and 20% l-alanyl-l-glutamine (Ala-Gln; 0.5 g/kg/d). High-dose MPSS treatment was initiated at the same time that IV infusions began, such that a total dose of 85 mg of MPSS/kg was administered through multiple IV injections over a 26-hour period. The infusions were maintained until 12 hours after the last MPSS injection. Blood samples collected before MPSS injections began and 2, 12, and 24 hours after injections ceased were used to evaluate PMN function.

Results—MPSS injections resulted in an increase in the total number of circulating leukocytes and increases in neutrophil and monocyte counts but did not affect lymphocyte, eosinophil, or basophil counts. Lymphocyte counts in the Ala-Gln group were higher than in the control group 12 hours after MPSS injections finished. Relative to preinfusion values, phagocytic capacity, oxidative burst activity, and filamentous actin polymerization of PMNs were suppressed in all dogs except those that received Ala-Gln.

Conclusions and Clinical Relevance—Parenteral Ala-Gln administration in dogs resulted in an increase in PMN phagocytic responses that were suppressed by high-dose MPSS treatment.

Abstract

Objective—To determine whether parenteral l-alanyl-l-glutamine (Ala-Gln) administration modulated phagocytic responses of polymorphonuclear neutrophilic leukocytes (PMNs) from dogs undergoing high-dose methylprednisolone sodium succinate (MPSS) treatment.

Animals—15 healthy Beagles.

Procedures—Dogs were randomly assigned to 3 treatment groups (n = 5/group): 38-hour IV infusion of saline (0.9% NaCl) solution (control group), saline solution with 8.5% amino acids (2.3 g/kg/d), or saline solution with 8.5% amino acids (1.8 g/kg/d) and 20% l-alanyl-l-glutamine (Ala-Gln; 0.5 g/kg/d). High-dose MPSS treatment was initiated at the same time that IV infusions began, such that a total dose of 85 mg of MPSS/kg was administered through multiple IV injections over a 26-hour period. The infusions were maintained until 12 hours after the last MPSS injection. Blood samples collected before MPSS injections began and 2, 12, and 24 hours after injections ceased were used to evaluate PMN function.

Results—MPSS injections resulted in an increase in the total number of circulating leukocytes and increases in neutrophil and monocyte counts but did not affect lymphocyte, eosinophil, or basophil counts. Lymphocyte counts in the Ala-Gln group were higher than in the control group 12 hours after MPSS injections finished. Relative to preinfusion values, phagocytic capacity, oxidative burst activity, and filamentous actin polymerization of PMNs were suppressed in all dogs except those that received Ala-Gln.

Conclusions and Clinical Relevance—Parenteral Ala-Gln administration in dogs resulted in an increase in PMN phagocytic responses that were suppressed by high-dose MPSS treatment.

Neutrophils are key players in the immune response.1–3 Morbidity and mortality rates increase dramatically in humans with infections when these patients have low neutrophil counts or neutrophil defects.2 Neutrophils have the capacity to phagocytose and thereby eliminate pathogens and cell debris.3 Various neutrophil receptors recognize chemoattractant molecules, which stimulate neutrophil migration to sites of infection.4 Contact with invading foreign particles activates phagocytic receptors, which then mediate ingestion of particles.5 Phagocytic responses involve the polymerization and rearrangement of cellular actin filaments.6–8 Once ingested, microbes are killed by microbicidal enzymes and the oxidative burst caused by ROS formation.9,10

Methylprednisolone sodium succinate is a synthetic glucocorticoid that at high doses has free radical–scavenging properties.11 In human medicine, the second National Acute Spinal Cord Injury Study12 showed that high-dose treatment with MPSS has neuroprotective effects.13 Evidence from an experiment14 involving cats suggests that MPSS treatment is useful in minimizing damage after spinal cord injury. High-dose and timely MPSS administration are important for the drug to provide a neuroprotective effect. However, because of reports of immunosuppressive complications in humans15 and dogs16 and a paucity of evidence that MPSS treatment limits neural damage, high-dose MPSS treatment remains controversial.

In humans with spinal cord injuries, MPSS treatment is associated with an increase in the risk of infections17; for example, the incidence of pneumonia is higher in methylprednisolone-treated patients than in other patients.18 Experiments involving dogs have recently shown that high-dose MPSS treatment can temporarily suppress the innate immune functions of neutrophilic leukocytes.19,20

Balanced nutrition is important for maintaining health and managing various diseases in animals.21 Cells involved in immunity require balanced and adequate nutrition to function optimally.22 Glutamine is the preferred fuel source for these cells,23 and it acts as a potent immune stimulator in vitro and in vivo.23 Intravenous administration of Gln reduces rates of infectious complications in critically ill people.24 It also enhances in vitro bacterial killing in neutrophils obtained from people who have undergone surgery.25 Neutrophils respond to Gln added to cell culture medium by increasing the activity of phagocytes and the rate of ROS production.26 However, to our knowledge, little is known about the effect of Gln infusion on in vivo phagocytic responses of PMNs in dogs. The purpose of the study reported here was to investigate the effect of IV infusion with Ala-Gln solution (as a Gln-containing dipeptide solution) on the phagocytic responses of PMNs in dogs and determine whether treatment with this solution would restore phagocytic capacity, OBA, and F-actin expression in PMNs in dogs treated with MPSS.

Materials and Methods

Animals—The subjects of this study were fifteen 3-year-old Beagles (mean ± SD body weight, 8.34 ± 0.54 kg). Nine dogs were neutered males, and 6 were spayed females. All were healthy, as judged through physical examination, indirect measurement of systolic arterial blood pressure, parasitological examination of fecal specimens, heartworm antigen test, CBC, serum biochemical analysis, urinalysis, ACTH response test, abdominal ultrasonography, and thoracic radiography. Each dog was housed separately in a cage with a cycle of 12 hours of light and 12 hours of dark. Dogs were fed a commercial dieta and provided with tap water. All experimental procedures were approved by the ethics committee of the Chungbuk National University.

Experimental design—The 15 dogs were randomly assigned to 3 treatment groups (5 dogs/treatment): IV infusion of saline (0.9% NaCl) solution (control group), an admixture of saline solution with 8.5% amino acids solutionb (2.3 g of amino acids/kg/d; generic amino acids group), or an admixture of saline solution with 8.5% amino acids (1.8 g/kg/d) and 20% Ala-Gln solutionc (0.5 g/kg/d; Ala-Gln group; Appendix). The amino acid requirements (g/d) of the dogs were calculated via the following formula: 2.3 × body weight in kilograms.27 Maintenance fluid requirements were estimated to be 60 mL/kg/d.28 Therefore, dogs in the generic amino acids and Ala-Gln groups also received the appropriate volume of saline solution to achieve this total fluid intake, resulting in different total infusion volumes among the groups. The 8.5% amino acids and Ala-Gln solutions were aseptically compounded in a parenteral nutrition bag immediately before administration.

Food was withheld from all dogs for a 24-hour period. Afterward, high-dose MPSS treatment, as has been suggested for dogs with acute spinal cord injury,29 was initiated in all dogs in accordance with protocols described elsewhere.19,29 Briefly, dogs received an initial bolus dose of MPSSd (30 mg/kg, IV) over 5 minutes through an over-the-needle catheter inserted into a cephalic vein, followed by a second bolus dose (15 mg/kg, IV) 2 hours later. Additional doses of MPSS (10 mg/kg, IV) were injected 8, 14, 20, and 26 hours after the first dose to achieve a total dose of 85 mg/kg over a 26-hour period. At the same point MPSS injections began, IV infusions of the assigned treatment were initiated through an over-the-needle catheter inserted into a jugular vein. Infusions continued until 12 hours after MPSS injections were completed.

Blood samples were collected via jugular venipuncture with a 21-gauge needle into bottles containing K2-EDTA or heparin immediately before the initial dose of MPSS was injected. Samples were also collected 2, 12, and 24 hours after MPSS injection ceased, corresponding to 28, 38, and 50 hours after treatment began, respectively.

CBCs—Blood samples from K2-EDTA bottles and an automated CBC analyzere were used to determine the total number of leukocytes as well as neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts. Results were compared with established reference intervals.30

PMN isolation—The PMNs were isolated via density-gradient centrifugation immediately after blood sample collection as described elsewhere.8 Briefly, heparinized blood samples were overlaid in a 1:1 ratio on a polysaccharide solutionf that had been adjusted to a specific gravity of 1.076 to 1.078. After centrifugation at 400 × g for 45 minutes at 20°C, the PMNs were harvested from the upper layer of sedimented erythrocytes. For purification of the PMNs, erythrocytes were allowed to sediment for 60 minutes in a PBS solution containing 1.5% dextrang (molecular weight, 200 kDa). Floating cells were gently collected and pelleted by centrifugation at 400 × g for 5 minutes at 4°C. Residual erythrocytes were lysed by treatment with 0.83% NH4Cl in a tri(hydroxymethyl) aminomethane buffer solution (pH, 7.2) for 5 minutes. Purity of PMNs in the final cell suspension was verified to be > 96% by cytocentrifugation and Wright-Giemsa staining analysis. Polymorphonuclear neutrophilic leukocytes were resuspended in RPMI 1640 mediumh supplemented with 2mM l-glutamine, 0.02 mg of gentamicin/mL, and 10% heat-inactivated Gln fetal bovine serum.i

Simultaneous measurement of phagocytic capacity and OBA—Phagocytic capacity and OBA were evaluated simultaneously as described elsewhere.8,19 Briefly, isolated PMNs were placed in 24-well plates at a density of 1 × 106 cells/mL and incubated for 2 hours at 37°C in a humidified 5% CO2 atmosphere. For the final hour of culture, 20 μL of a suspension of carboxylate-modified polystyrene fluorescent microspheresj (size, 1.0 μm) that had been adjusted to 1 × 109 beads/mL was added to each well. Fifteen minutes before the end of the culture period, 1μM dihydrorhodamine 123h was added.

The ROS-mediated conversion of the nonfluorescent dihydrorhodamine 123 into fluorescent rhodamine 123 was used to measure OBA,31 and phagocytic capacity was determined by estimating the number of PMNs containing phagocytosed fluorescent microspheres by flow cytometric analysis. The cultured cells were gently harvested, centrifuged at 400 × g for 3 minutes at 4°C, and washed 3 times with PBS solution containing 3mM EDTA. The viability of the PMNs was found to be > 97% on the basis of their ability to exclude trypan blue dye. The cells were then resuspended in fixation bufferk in accordance with the manufacturer's instructions. All steps after beginning the cell culture were conducted in the dark.

Cultured cells were analyzed with a multipurpose flow cytometerl and analysis software,m with an argon laser set at 488 nm. Samples of 10,000 cells were assayed in triplicate. The FL1 channel was set to 505 to 545 nm to detect the green fluorescent rhodamine 123, and the FL3 channel was set to 630 to 660 nm to detect the red fluorescent microspheres. The cells were gated on the basis of forward and side light-scattering characteristics, and cell viability was confirmed via cytometric evaluation of the ability of the cells to exclude propidium iodide. Phagocytic capacity and OBA were recorded as percentages and mean fluorescence intensities (arbitrary units), respectively.

Measurement of cellular F-actin expression—To evaluate the effect of Ala-Gln supplementation on actin polymerization in the PMNs at the points of contact with microspheres during phagocytosis, the total degree of cellular F-actin expression was measured as described elsewhere.6,8 The isolated PMNs were placed in 24-well plates at a density of 1 × 106 cells/mL and incubated for 80 minutes at 37°C in a humidified 5% CO2 atmosphere. For the final 20 minutes of culture, 20 μL of a suspension of carboxylate-modified polystyrene fluorescent microspheresj (size, 1.0 μm; 1 × 109 beads/mL) was added. The cultured cells were then gently harvested, centrifuged at 400 × g for 3 minutes at 4°C, washed 3 times with PBS solution, and fixed with fixation bufferk at 4°C in accordance with the manufacturer's instructions. The fixed cells were washed 3 times with PBS solution and then stained in the dark for 15 minutes at 37°C with 165nM fluorescein isothiocyanate–labeled phalloidink and 100 μg of lysophophatidylcholine/mL.h

Stained cells were washed and analyzed within 30 minutes by use of a multipurpose flow cytometerl and analysis software,m with the argon laser set at 488 nm. Samples of 10,000 cells each were assayed in triplicate. The FL1 channel was set to 505 to 530 nm to detect the green fluorescein isothiocyanate molecule. The degree of F-actin expression was recorded as mean fluorescence intensity (arbitrary units). To assess changes in the cell shape and F-actin distribution, the fluorescence images of PMNs were visualized by confocal laser scanning microscopyn and analyzed with appropriate software.o Cells were counterstained with propidium iodidee nuclear stain to show the cell shape clearly.

Statistical analysis—All analyses were performed with statistical software.p Differences among the 3 treatment groups or within each treatment group at the 4 blood collection time points were evaluated via a 1-way ANOVA or a repeated-measures ANOVA, respectively. Post hoc tests were performed with the Tukey test. Two-group comparisons were performed by means of a paired t test. Normality of data distribution was evaluated with the Kolmogorov-Smirnov test. A value of P < 0.05 was considered significant. Results are reported as mean ± SD.

Results

Animals—Dogs that received an infusion of saline solution (mean ± SD body weight, 8.3 ± 0.6 kg), saline solution and 8.5% generic amino acids (8.4 ± 0.5 kg), and saline solution and 8.5% generic amino acids supplemented with 20% Ala-Gln (8.4 ± 0.6 kg) were similar with respect to age. For dogs in the generic amino acids group, the mean total dose of amino acids infused over 38 hours was 30.4 ± 1.9 g. For dogs in the Ala-Gln group, this dose was 30.5 ± 2.3 g (23.9 ± 1.8 g of generic amino acids and 6.6 ± 0.5 g of Ala-Gln). The total volume of saline solution infused in each group was as follows: control group, 786.6 ± 55.6 mL; generic amino acids group, 436.0 ± 26.8 mL (also received 358.2 ± 22.0 mL of 8.5% amino acids); Ala-Gln group, 482.0 ± 36.2 mL (also received 281.0 ± 21.1 mL of 8.5% amino acids and 33.2 ± 2.5 of 20% Ala-Gln).

Effect on numbers of circulating leukocytes—Compared with values before assigned treatments began, MPSS injections resulted in a significant increase in the total number of circulating leukocytes and neutrophils and monocytes counts in all treatment groups (Table 1). Lymphocyte, eosinophil, and basophil counts did not change significantly from values in any treatment group. No significant differences were evident in the total leukocyte, neutrophil, monocyte, eosinophil, and basophil counts among the 3 groups immediately before MPSS injection or at 2, 12, and 24 hours after injections ceased (P values ranged from 0.158 to 0.993). However, in the Ala-Gln group, lymphocyte numbers at 12 hours after MPSS injection were significantly (P = 0.031) higher than those of the control group.

Table 1—

Mean ± SD leukocyte counts (× 109 cells/L) for blood samples collected from healthy Beagles undergoing MPSS treatment that concurrently received an IV infusion of saline solution (n = 5; control group), an admixture of saline solution with 8.5% generic amino acids solution (2.3 g/kg/d; 5), or an admixture of saline solution with 8.5% generic amino acids (1.8 g/kg/d) and 20% Ala-Gln solutions (0.5 g/kg/d; 5) at various points before MPSS injections began and after they ceased.

Treatment, by time pointTotal leukocytesNeutrophilsMonocytesLymphocytesEosinophilsBasophils
Control
 Before injection8.96 ± 1.545.25 ± 1.250.70 ± 0.182.58 ± 0.290.42 ± 0.280.02 ± 0.02
 2 hours after injection22.19 ± 2.24a18.10 ± 2.03a1.55 ± 0.24a2.30 ± 0.610.23 ± 0.080.02 ± 0.02
 12 hours after injection20.28 ± 4.62a15.85 ± 5.08a1.91 ± 0.50a2.23 ± 0.370.28 ± 0.140.01 ± 0.01
 24 hours after injection20.07 ± 5.17a14.32 ± 3.06a1.54 ± 0.33a2.38 ± 0.460.28 ± 0.140.02 ± 0.01
 P value< 0.001< 0.001< 0.0010.3670.4320.899
8.5% generic amino acids
 Before injection9.00 ± 1.195.28 ± 1.150.73 ± 0.142.53 ± 0.290.45 ± 0.330.01 ± 0.01
 2 hours after injection22.04 ± 2.64a17.95 ± 3.02a1.45 ± 0.25a2.38 ± 0.610.24 ± 0.090.02 ± 0.02
 12 hours after injection19.92 ± 4.32a15.55 ± 4.67a1.79 ± 0.33a2.27 ± 0.540.31 ± 0.280.01 ± 0.01
 24 hours after injection18.85 ± 3.33a14.45 ± 3.25a1.85 ± 0.68a2.30 ± 0.520.23 ± 0.110.01 ± 0.01
 P value< 0.001< 0.0010.0010.5730.5040.694
Ala-Gln (n = 5)
 Before injection8.98 ± 1.035.34 ± 1.030.70 ± 0.142.54 ± 0.330.40 ± 0.260.01 ± 0.01
 2 hours after injection22.43 ± 1.25a17.98 ± 1.50a1.42 ± 0.34a2.74 ± 0.420.27 ± 0.160.01 ± 0.01
 12 hours after injection20.04 ± 3.22a15.16 ± 3.50a1.76 ± 0.34a2.85 ± 0.38b0.26 ± 0.120.01 ± 0.01
 24 hours after injection18.97 ± 3.63a14.46 ± 3.38a1.66 ± 0.52a2.58 ± 0.340.26 ± 0.260.01 ± 0.01
   P value< 0.001< 0.0010.0020.3980.7350.426

Value is significantly (P < 0.05) different from the value for the same treatment group immediately before MPSS injection.

Value is significantly (P < 0.05) differentfrom the value at 12 hours after MPSS injection for the control group.

Phagocytic capacity and OBA—Values for the phagocytic capacity of PMNs in the control (P = 0.001) and generic amino acids (P = 0.001) groups were significantly lower 2 hours after MPSS injections finished, compared with values in the same groups immediately before MPSS injection (Figure 1). Values for the OBA of PMNs from the control (P < 0.001) and generic amino acids (P < 0.001) groups 2 hours after MPSS injections ended were significantly lower than those in the same groups immediately before MPSS injection. In the Ala-Gln group, MPSS administration had no significant effect on PMN phagocytic capacity (P = 0.269) or OBA (P = 0.080) values, although the mean values were lower than immediately before MPSS injection. Values for phagocytic capacity (P = 0.007) and OBA (P = 0.002) of PMNs in the same group were significantly higher at 2 hours after MPSS injections ceased than they were in the control and generic amino acids groups. At 12 hours after MPSS injections ceased, phagocytic capacity and OBA of PMNs in the control and generic amino acids groups were at preinjection values.

Figure 1—
Figure 1—

Mean ± SD phagocytic capacity (A) and OBA (B) in PMNs isolated from blood samples obtained from 15 healthy Beagles (n = 5/group) undergoing MPSS treatment that received an IV infusion of saline (0.9% NaCl) solution alone (control; white bars), admixture of saline solution with 8.5% amino acids solution (2.3 g/kg/d; gray bars), or admixture of saline solution with 8.5% amino acids solution (1.8 g/kg/d) supplemented with 20% Ala-Gln solution (0.5 g/kg/d; black bars). *Mean values at 2 hours after MPSS injections ceased to differ significantly (P < 0.05) between the treatment groups. †Mean values of the control and generic amino acids groups differ significantly (P < 0.05) at 2 hours after injection relative to values before injection. MFI = Mean fluorescence intensity.

Citation: American Journal of Veterinary Research 73, 9; 10.2460/ajvr.73.9.1410

F-actin polymerization—At 2 hours after MPSS injections ceased, the total degree of F-actin expression was significantly lower in the control (P < 0.001) and generic amino acids (P < 0.001) groups than it was immediately before injection (Figure 2). Although MPSS injections did not significantly affect PMN phagocytic capacity and OBA in PMNs from dogs in the Ala-Gln group, at 2 hours after injection, F-actin expression was significantly (P = 0.008) lower than preinjection values. However, compared with the degree of expression in the control group at 2 hours after injection, there was a significant (P = 0.022) increase in F-actin expression in PMNs from the Ala-Gln group. Confocal fluorescence imaging analysis confirmed that, relative to PMNs obtained from the control group at 2 hours after MPSS injection, PMNs from the Ala-Gln group had a marked increase in actin polymerization at the microsphere contact points at the same time point (Figure 3).

Figure 2—
Figure 2—

Mean ± SD total degree of cellular F-actin expression in the same dogs as in Figure 1. *Mean values at 2 hours after MPSS injection differ significantly (P < 0.05) between the control and Ala-Gln groups. †Mean values of each group differ significantly (P < 0.05) at 2 hours after MPSS injection relative to values immediately before injection. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 73, 9; 10.2460/ajvr.73.9.1410

Figure 3—
Figure 3—

Representative fluorescence images of PMNs isolated from blood samples obtained from one of the control dogs (top row) and one of the Ala-Gln–treated dogs (bottom row), represented in Figure 1, at the points of PMN contact with microspheres. Filamentous polymeric actin fluorescence (green) was merged with nuclear fluorescence (propidium iodide counterstain; red). Bar = 8 μm.

Citation: American Journal of Veterinary Research 73, 9; 10.2460/ajvr.73.9.1410

Discussion

The purpose of the present study was to determine whether suppressive effects of high-dose MPSS treatment on neutrophil function in dogs could be modulated by Ala-Gln supplementation. As expected, high-dose MPSS treatment triggered an increase in total numbers of circulating leukocytes in each of the 3 treatment groups and corresponded with neutrophilia and monocytosis. However, a 38-hour infusion with an amino acid solution containing Ala-Gln had no effect on total leukocyte, neutrophil, monocyte, eosinophil, and basophil counts. Notably, Ala-Gln infusion resulted in an increase in circulating lymphocyte numbers at 12 hours after high-dose MPSS treatment ceased; this point was immediately after Ala-Gln infusion had finished.

Human studies have revealed similar findings. Parenteral nutrition supplemented with l-Gln increases the number of circulating lymphocytes in humans after allogeneic bone marrow transplantation.32 Anorectic patients receiving total parenteral nutrition with dipeptide glycyl-Gln supplementation have a significant increase in total lymphocyte count.33 Supplementation of total parenteral nutrition with Ala-Gln also resulted in an increase in total lymphocyte count in human patients with systemic inflammatory response syndrome, although this increase is not significant.34 Furthermore, Gln has been shown to be essential for the proliferation and differentiation of lymphocytes.35,36

In humans,32–34 the increase in circulating lymphocytes continues for a longer period than it did in the dogs in the present study. This difference was possibly related to MPSS treatment, given that glucocorticoids trigger lymphopenia.37 We observed a significant increase in the lymphocyte count only at 12 hours after high-dose MPSS treatment ceased, even though the duration of the Ala-Gln infusions was shorter than that of human studies33 (38 hours and 20 days, respectively). It is possible that if the duration of Ala-Gln infusion had been longer and without high-dose MPSS treatment, the increase in circulating lymphocyte count would have persisted for a longer period, as has been observed in humans.32–34

High-dose MPSS treatment resulted in suppression of phagocytic responses of canine neutrophilic leukocytes in 2 independent studies19,20; however, the duration of the inhibitory action of MPSS on PMN functions differed. In one of those studies,20 we found MPSS had suppressive effects only for a short period after injections ceased, and these suppressive effects were not detectable 24 hours later. We postulated that the loss of suppressive effects on canine PMN phagocytic functions after 24 hours was related to the short circulating half-life of MPSS (approx 3 hours) when administered IV.38 However, it is also possible that the difference in the duration of action between our previous study and the other study19 may have been attributable to the use of different foreign particles for the assessment of phagocytosis.

The effects of glucocorticoids on the immune system can be provoked both by genomic events regulated through the modulation of nuclear transcription and by a rapid, nongenomic mechanism.39–41 Nongenomic effects of glucocorticoids can be classified into specific and nonspecific effects.39,41 Specific nongenomic effects are mediated by steroid-selective membrane receptors, whereas nonspecific nongenomic effects appear to result from direct interactions with biological membranes only at high glucocorticoid dosages.39–41 In the present study, the suppressive effects of MPSS on the phagocytic capacity and OBA of canine PMNs in response to unopsonized foreign particles were possibly due to the rapid, nonspecific, nongenomic mechanism of action.

Infusion of the study dogs with an amino acid solution containing Ala-Gln overwhelmed the suppressive effect of high-dose MPSS treatment on the phagocytic capacity and OBA of the PMNs. Moreover, Ala-Gln supplementation enhanced the total degree of cellular F-actin expression relative to the effects of the other treatments, even though the inhibitory effect of MPSS treatment was evident in dogs that received Ala-Gln. Actin polymerization plays an essential role in various neutrophil functions, including chemotaxis, phagocytosis, and ROS production.6,7,42 However, this does not explain how Ala-Gln administration directly impacts the immunosuppressive effect of glucocorticoids.

Neutrophils use Gln as a fuel source for carrying out their functions.36,43 For this, these cells need the appropriate glutaminase enzyme for metabolizing Gln and Gln synthase.44 Administration of dexamethasone, a synthetic glucocorticoid, reportedly results in a decrease in glutaminase activity but not Gln oxidation in neutrophils.45 Glutamine modulates glutaminase activity by promoting the accumulation of glutaminase mRNA in various cells.46,47 This observation suggests that the action of Ala-Gln on the suppressive effect of MPSS could be related to the modulation of glutaminase activity.

Nicotinamide adenine dinucleotide phosphate oxidase generates the superoxide anions that elicit production of several ROS.2,5,10 Methylprednisolone sodium succinate decreases superoxide anion generation in mouse peritoneal macrophages48 and human neutrophils41,49 and downregulates the expression of p22phox, which is a protein subunit of NADPH-oxidase.50 Conversely, Gln enhances superoxide anion production in rat51 and human neutrophils,26,44 partly through an increase in the mRNA expression of NADPH-oxidase components such as p22phox and p47phox.51 These findings provide a plausible explanation for the effect of Ala-Gln on OBA in canine PMNs, which could be associated with modulation of expression of NADPH-oxidase components. However, although the immunomodulating effect of Gln has been studied extensively, the mechanism of action of Ala-Gln on the immunosuppressive effect of MPSS is not yet clear. Additional studies are required to investigate the change in Gln metabolism caused by Ala-Gln supplementation in dogs undergoing high-dose MPSS treatment.

The present study had some limitations. No serial plasma amino acid measurements were performed during the experimental period. Enteral or parenteral Gln supplementation leads to an increase in plasma Gln concentrations; therefore, if plasma amino acid measurements were performed, this would help clarify whether the stimulatory effect of Ala-Gln on the phagocytic response corresponds to the restoration of plasma Gln concentration. Although adult dogs generally require dietary protein at a rate of 2 to 3 g/100 kcal/d, we provided a relatively higher concentration of amino acids to dogs in the generic amino acids and Ala-Gln groups, as suggested elsewhere.27 This is relevant because an increase in protein intake may be necessary for patients with higher than usual protein requirements and because the metabolic demands on Gln can increase in the critically ill.21–24 Consequently, if lower concentrations of amino acids had been provided than were used in the present study, then a different interpretation of the Ala-Gln effect might have resulted.

Parenteral nutrition can be a cornerstone in the management of critically ill animals that are unable to receive oral or enteral nutrition and must provide a source of essential and nonessential amino acids as well as lipids and dextrose. In human23,52 and veterinary medicine,20,21 Gln is considered a conditionally essential amino acid during serious illness, injury, or other catabolic states because of the profound depletion of Gln in plasma and tissues that can occur. In addition to the important requirement for Gln to maintain the function of cells involved in the immune system,22,36 Gln has beneficial effects at the intestinal barrier and modulates the gut-associated immune system by decreasing the production of proinflammatory cytokines and increasing heat shock protein expression.23 Nevertheless, Gln is not yet a component of standard amino acid solutions in total nutrient admixtures because of its instability in solution; only stable Gln-containing dipeptides such as Ala-Gln as used in the present study and glycyl-Gln are available.52

In the present study, treatment with Ala-Gln solution arrested the suppressive effect of high-dose MPSS on phagocytic responses of PMNs and induced a transient increase in the number of circulating lymphocytes in healthy dogs. The results suggested that Ala-Gln supplementation may help alleviate the immunosuppressive complications caused by high-dose MPSS treatment. However, population-based studies are required to determine whether treatment with Ala-Gln decreases susceptibility to infection in patients receiving MPSS treatment and to clarify the mechanism by which Ala-Gln directly modulates MPSS-mediated downregulation of immune function.

ABBREVIATIONS

Ala-Gln

l-alanyl-l-glutamine

F-actin

Filamentous polymeric actin

Gln

Glutamine

MPSS

Methylprednisolone sodium succinate

NADPH

Nicotinamide adenine dinucleotide phosphate

OBA

Oxidative burst activity

PMN

Polymorphonuclear neutrophilic leukocyte

ROS

Reactive oxygen species

a.

ProPlan, Nestle Purina PetCare Korea Ltd, Seoul, Republic of Korea.

b.

Fravasol CJ Inj 8.5%, CJ Pharma, Seoul, Republic of Korea.

c.

Dipeptiven, Fresenius Kabi Korea, Seoul, Republic of Korea.

d.

Solu-Medrol (62.5 mg/mL), Pfizer Pharmaceuticals Korea, Seoul, Republic of Korea.

e.

CELL-DYN 3700, Abbott Diagnostics, Abbott Park, Ill.

f.

Histopaque-1077, Sigma Chemical Co, St Louis, Mo.

g.

Wako Pure Chemical Industries Ltd, Osaka, Japan.

h.

Sigma Chemical Co, St Louis, Mo.

i.

Invitrogen Co, Grand Island, NY.

j.

TransFluoSpheres, Molecular Probes Inc, Eugene, Ore.

k.

BD Cytofix, BD Biosciences, San Jose, Calif.

l.

FACSCalibur system, Becton Dickinson Immunocytometry Systems, San Jose, Calif.

m.

CELLQuest, version 3.3, Becton Dickinson Immunocytometry Systems, San Jose, Calif.

n.

Leica TCS SP2 AOBS Confocal System, Leica Microsystems GmBH, Wetzlar, Germany.

o.

Leica confocal software, Leica Microsystems GmBH, Wetzlar, Germany.

p.

GraphPad Prism 5, version 5.04, GraphPad Software Inc, La Jolla, Calif.

Appendix

Composition of solutions containing 8.5% amino acids and 20% Ala-Gln.

Component (g/L)8.5% amino acids20% Ala-Gln
Aminoacetic acid8.8
l-alanine17.682.0
l-Gln134.6
l-arginine9.78
l-histidine4.1
l-isoleucine5.1
l-leucine6.2
l-lysine4.9
l-methionine3.4
l-phenylalanine4.8
l-proline5.8
l-serine4.3
l-threonine3.6
l-tryptophan1.5
l-tyrosine0.3
l-valine4.9

— = Not applicable.

References

  • 1.

    Segal AW. How neutrophils kill microbes. Annu Rev Immunol 2005; 23:197223.

  • 2.

    Nauseef WM. How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 2007; 219:88102.

  • 3.

    English D. The remarkable neutrophil! Developing a blueprint for integrated cellular signaling. In: Gabrilovich DI, ed. The neutrophils: new outlook for old cells. 2nd ed. London: Imperial College Press, 2005:134.

    • Search Google Scholar
    • Export Citation
  • 4.

    Appelberg R. Neutrophils and intracellular pathogens: beyond phagocytosis and killing. Trends Microbiol 2007; 15:8792.

  • 5.

    Lee WLHarrison REGrinstein S. Phagocytosis by neutrophils. Microbes Infect 2003; 5:12991306.

  • 6.

    Torres MCoates TD. Function of the cytoskeleton in human neutrophils and methods for evaluation. J Immunol Methods 1999; 232:89109.

  • 7.

    May RCMachesky LM. Phagocytosis and the actin cytoskeleton. J Cell Sci 2001; 114:10611077.

  • 8.

    Kang JHYang MP. Effect of a short-term infusion with soybean oil-based lipid emulsion on phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes. J Vet Intern Med 2008; 22:11661173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Dahlgren CKarlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999; 232:314.

  • 10.

    Fialkow LWang YDowney GP. Reactive oxygen and nitrogen species as signaling molecules regulating neutrophil function. Free Radic Biol Med 2007; 42:153164.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Brown SAHall ED. Role of oxygen-derived free radicals in the pathogenesis of shock and trauma, with focus on central nervous system injuries. J Am Vet Med Assoc 1992; 200:18491859.

    • Search Google Scholar
    • Export Citation
  • 12.

    Bracken MBHolford TR. Effects of timing of methylprednisolone or naloxone administration on recovery of segmental and long-tract neurological function in NASCIS 2. J Neurosurg 1993; 79:500507.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Kim YTCaldwell JMBellamkonda RV. Nanoparticle-mediated local delivery of methylprednisolone after spinal cord injury. Biomaterials 2009; 30:25822590.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Braughler JMHall EDMeans ED, et al. Evaluation of an intensive methylprednisolone sodium succinate dosing regimen in experimental spinal cord injury. J Neurosurg 1987; 67:102105.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Hurlbert RJ. Methylprednisolone for acute spinal cord injury: an inappropriate standard of care. J Neurosurg 2000; 93(suppl 1):17.

  • 16.

    Olby N. Current concepts in the management of acute spinal cord injury. J Vet Intern Med 1999; 13:399407.

  • 17.

    Sayer FTKronvall ENilsson OG. Methylprednisolone treatment in acute spinal cord injury: the myth challenged through a structured analysis of published literature. Spine J 2006; 6:335343.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Gerndt SJRodriguez JLPawlik JW, et al. Consequences of high-dose steroid therapy for acute spinal cord injury. J Trauma 1997; 42:279284.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Kang JHYang MP. In vitro evaluation of the effect of trans-10, cis-12 conjugated linoleic acid on phagocytosis by canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to methylprednisolone sodium succinate. Am J Vet Res 2008; 69:494500.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Shimamura SKanayama KShimada T, et al. Evaluation of the function of polymorphonuclear neutrophilic leukocytes in healthy dogs given a high dose of methylprednisolone sodium succinate. Am J Vet Res 2010; 71:541546.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Chan DL. The role of nutrients in modulating disease. J Small Anim Pract 2008; 49:266271.

  • 22.

    Saker KE. Nutrition and immune function. Vet Clin North Am Small Anim Pract 2006; 36:11991224.

  • 23.

    Roth E. Nonnutritive effects of glutamine. J Nutr 2008; 138:2025S2031S.

  • 24.

    Novak FHeyland DKAvenell A, et al. Glutamine supplementation in serious illness: a systematic review of the evidence. Crit Care Med 2002; 30:20222029.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Furukawa SSaito HFukatsu K, et al. Glutamine-enhanced bacterial killing by neutrophils from postoperative patients. Nutrition 1997; 13:863869.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Furukawa SSaito HInoue T, et al. Supplemental glutamine augments phagocytosis and reactive oxygen intermediate production by neutrophils and monocytes from postoperative patients in vitro. Nutrition 2000; 16:323329.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Mauldin GEReynolds AJMauldin GN, et al. Nitrogen balance in clinically normal dogs receiving parenteral nutrition solutions. Am J Vet Res 2001; 62:912920.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    DiBartola SPBateman S. Introduction to fluid therapy. In: DiBartola SP, ed. Fluid, electrolyte, and acid-base disorders in small animal practice. 3rd ed. St Louis: Saunders Elsevier, 2006:325344.

    • Search Google Scholar
    • Export Citation
  • 29.

    Thomas WB. Disorders of the spinal cord. In: Morgan RVBright RMSwartout MS, eds. Handbook of small animal practice. 4th ed. Philadelphia: Saunders, 2003:256275.

    • Search Google Scholar
    • Export Citation
  • 30.

    Moritz AFickenscher YMeyer K, et al. Canine and feline hematology reference values for the ADVIA 120 hematology system. Vet Clin Pathol 2004; 33:3238.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Richardson MPAyliffe MJHelbert M, et al. A simple flow cytometry assay using dihydrorhodamine for the measurement of the neutrophil respiratory burst in whole blood: comparison with the quantitative nitrobluetetrazolium test. J Immunol Methods 1998; 219:187193.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Ziegler TRBye RLPersinger RL, et al. Effects of glutamine supplementation on circulating lymphocytes after bone marrow transplantation: a pilot study. Am J Med Sci 1998; 315:410.

    • Search Google Scholar
    • Export Citation
  • 33.

    Mondello SItaliano DGiacobbe MS, et al. Glutamine-supplemented total parenteral nutrition improves immunological status in anorectic patients. Nutrition 2010; 26:677681.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Cetinbas FYelken BGulbas Z. Role of glutamine administration on cellular immunity after total parenteral nutrition enriched with glutamine in patients with systemic inflammatory response syndrome. J Crit Care 2010; 25:661:e1e6.

    • Search Google Scholar
    • Export Citation
  • 35.

    Newsholme PCuri RPithon-Curi TC, et al. Glutamine metabolism by lymphocytes, macrophages, and neutrophils: its importance in health and disease. J Nutr Biochem 1999; 10:316324.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Li PYin YLLi D, et al. Amino acids and immune function. Br J Nutr 2007; 98:237252.

  • 37.

    Yao ZDuBois DCAlmon RR, et al. Pharmacokinetic/pharmacodynamic modeling of corticosterone suppression and lymphocytopenia by methylprednisolone in rats. J Pharm Sci 2008; 97:28202832.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Feduska NJTurcotte JGGikas PW, et al. Reversal of renal allograft rejection with intravenous methylprednisolone “pulse” therapy. J Surg Res 1972; 12:208215.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Gold RButtgereit FToyka KV. Mechanism of action of glucocorticoid hormones: possible implications for therapy of neuroimmunological disorders. J Neuroimmunol 2001; 117:18.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40.

    Buttgereit FScheffold A. Rapid glucocorticoid effects on immune cells. Steroids 2002; 67:529534.

  • 41.

    Liu LWang YXZhou J, et al. Rapid non-genomic inhibitory effects of glucocorticoids on human neutrophil degranulation. Inflamm Res 2005; 54:3741.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Clements MKSiemsen DWSwain SD, et al. Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses. J Leukoc Biol 2003; 73:344355.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Pithon-Curi TCDe Melo MPCuri R. Glucose and glutamine utilization by rat lymphocytes, monocytes, neutrophils in culture: a comparative study. Cell Biochem Funct 2004; 22:321326.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Castell LVance CAbbott R, et al. Granule localization of glutamine in human neutrophils and the consequence of glutamine utilization for neutrophil activity. J Biol Chem 2004; 279:1330513310.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Garcia Cde Oliveira MCVerlengia R, et al. Effect of dexamethasone on neutrophil metabolism. Cell Biochem Funct 2003; 21:105111.

  • 46.

    Kong SEHall JCCooper D, et al. Glutamine-enriched parenteral nutrition regulates the activity and expression of intestinal glutaminase. Biochim BiophysActa 2000; 1475:6775.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47.

    Holten ATGundersen V. Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase. J Neurochem 2008; 104:10321042.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Røshol HSkrede KKAErø CE, et al. Dexamethasone and methylprednisolone affect rat peritoneal phagocyte chemiluminescence after administration in vivo. Eur J Pharmacol 1995; 286:917.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49.

    Yoshida TTanaka MSotomatsu A, et al. Effect of methylprednisolone-pulse therapy on superoxide production of neutrophils. Neurol Res 1999; 21:509512.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50.

    Mouithys-Mickalad ADeby-Dupont GMathy-Hartert M, et al. Effect of glucocorticoids on the respiratory burst of Chlamydia-primed THP-1 cells. Biochem Biophys Res Commun 2004; 318:941948.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51.

    Pithon-Curi TCLevada ACLopes LR, et al. Glutamine plays a role in superoxide production and the expression of p47phox, p22phox and gp91phox in rat neutrophils. Clin Sci (Lond) 2002; 103:403408.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52.

    Wernerman J. Clinical use of glutamine supplementation. J Nutr 2008; 138:2040S2044S.

Contributor Notes

Presented in abstract form at the American College of Veterinary Internal Medicine Forum and Canadian Veterinary Medical Association Convention, Montreal, June 2009.

Address correspondence to Dr. Yang (mpyang@chungbuk.ac.kr).