Dietary glutamine enhances cytokine production by murine macrophages

Dietary glutamine enhances cytokine production by murine macrophages

BASIC NUTRITIONAL INVESTIGATION Nutrition Vol. 15, Nos. 11/12, 1999 Dietary Glutamine Enhances Cytokine Production by Murine Macrophages SHARON M. W...

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BASIC NUTRITIONAL INVESTIGATION

Nutrition Vol. 15, Nos. 11/12, 1999

Dietary Glutamine Enhances Cytokine Production by Murine Macrophages SHARON M. WELLS, BSC, SAMANTHA KEW, BSC, PARVEEN YAQOOB, MA, DPHIL, FIONA A. WALLACE, BSC, MMEDSCI, AND PHILIP C. CALDER, PHD, DPHIL From the Institute of Human Nutrition, University of Southampton, Southampton, UK ABSTRACT

To examine the effects of dietary glutamine on cytokine production by macrophages, mice were fed for 2 wk on a control diet that included 200.0 g casein/kg providing 19.6 g glutamine/kg or a glutamine-enriched diet that provided 54.8 g glutamine/kg partly at the expense of casein. There were no differences in weight gain between animals fed the two diets. The plasma concentrations of a number of amino acids differed according to the diet fed; this variation largely reflected the variation in the levels of the different amino acids in the diets. Plasma glutamine concentration was not significantly affected by dietary glutamine level. The production of three cytokines, tumor necrosis factor-␣, interleukin-1␤, and interleukin-6, was greater for lipopolysaccharide-stimulated macrophages from mice fed the glutamine-enriched diet. Thus, increasing the amount of glutamine in the murine diet enhances the ability of macrophages to respond to stimulation, at least in terms of cytokine production. These observations suggest that increasing the availability of glutamine orally could promote immune responses involving macrophagederived cytokines. Nutrition 1999;15:881– 884. ©Elsevier Science Inc. 1999 Key words: macrophage, glutamine, cytokine, interleukin, tumor necrosis factor

INTRODUCTION

Mononuclear phagocytes (i.e., monocytes and macrophages) play a key part in both natural and acquired immunity.1 They have been shown to use the amino acid glutamine at a high rate2,3 (for a review, see Calder4) and this has been taken as suggestive that glutamine plays an important role in these cells.5 In accordance with this idea, the level of cell surface expression of certain molecules involved in phagocytosis and in intercellular interactions by human monocytes is influenced by the concentration of glutamine in which the cells are cultured, and this is associated with altered function (i.e., phagocytosis of immunoglobulin G or complement opsonized particles, antigen presentation).6,7 Likewise, phagocytosis by cultured murine macrophages is dependent on glutamine availability.8,9 Cytokines are soluble proteins produced by leukocytes and other cells that act as chemical communicators between cells. Cytokines include the interleukins (ILs) and tumor necrosis factors (TNFs). These cytokines act to control the activity of different types of leukocytes and thus to regulate and optimize the immune and inflammatory responses. Macrophages are an important source of such immunoregulatory cytokines. Given the key role of cytokines in regulating the function of cells of the immune system, there has been some interest in the role of glutamine in modulating cytokine production by various cell types.9 –14 However, studies of the role of glutamine in modulating production of cytokines by

macrophages or their precursors, monocytes, are few.9,12–14 Maximal IL-1 and TNF-␣ production by cultured murine macrophages9,14 and maximal production of IL-6 and IL-8 by cultured human monocytes12,14 require a sufficient supply of glutamine. Plasma glutamine concentrations are lowered under a variety of “stress” conditions such as after burns,8,15 during sepsis16 –18 and after surgery,19 –23 and after endurance exercise,24 –26 athletic training,27 and over-training.24 These situations are associated with an increased susceptibility to infections, and it has been suggested that this may be due in part to a diminished supply of glutamine to immunocompetent cells such as macrophages.5,28 As a result, there is great interest in the provision of glutamine to subjects in stress situations.29 Despite this interest, it is not yet clear whether glutamine supplied orally will influence immune function. Therefore, in the current study we investigated the effect of increasing the amount of glutamine in the diet of mice on the production of immunoregulatory cytokines by macrophages. To our knowledge, this is the first study to investigate the effect of dietary glutamine on macrophage function. MATERIALS AND METHODS

Animals and Diets Adult male C57Bl6 mice were purchased from Harlan-Olac (Bicester, Oxfordshire, UK). They were housed individually and

This work was supported by Oxford Nutrition Ltd. Correspondence to: Philip C. Calder, PhD, Institute of Human Nutrition, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.

Nutrition 15:881– 884, 1999 ©Elsevier Science Inc. 1999 Printed in the USA. All rights reserved.

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GLUTAMINE AND MACROPHAGE CYTOKINES TABLE I. COMPOSITION OF THE DIETS USED

Component (g/kg) Casein Protein N Extra glutamine Total N Carbohydrate Fat Fiber Ash Water

Control

⫹Glutamine

200.0 31.3 — 31.3 655.0 35.0 60.0 30.0 20.0

151.0 23.6 40.0 31.3 664.0 35.0 60.0 30.0 20.0

allowed free access to water and to one of two diets provided in powdered form by Oxford Nutrition Ltd. (Witney, Oxfordshire, UK). These diets were a control diet in which all amino acids were provided in casein and a glutamine-enriched diet (Table I). The amount of casein was decreased in the glutamine-enriched diet to ensure that both diets were isonitrogeneous (Table I). The diets contained equal amounts of carbohydrate, fat, and fiber (Table I). Table II shows the amino acid composition of the diets: the control diet contained 19.6 g glutamine/kg, and the glutamine-enriched diet contained 54.8 g glutamine/kg. The diets were isocaloric (3730.0 kcal/kg). The mice were fed on the diets for 2 wk and were killed by an overdose of CO2; 4 d before being killed, the mice were injected intraperitoneally with 1.0 mL Brewer’s thioglycollate broth to elicit macrophage migration to the peritoneal cavity. All procedures involving animals were approved under the Animals (Scientific Procedures) Act 1986 by the Home Office. Materials Glutamine, arginine, fetal calf serum (FCS), streptomycin, penicillin, and Eschericia coli lipopolysaccharide (LPS) were TABLE II. AMINO ACID COMPOSITION OF THE EXPERIMENTAL DIETS Amino acid (g/kg diet) Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Histidine Alanine Arginine Aspartic acid Cysteine Glutamic acid Glutamine Glycine Proline Serine Tyrosine

Control 9.02 17.64 15.1 5.69 10.0 8.43 2.35 11.18 5.69 5.88 7.25 13.53 0.78 21.37 19.61 3.53 20.39 11.37 10.78

⫹Glutamine 6.81 13.47 11.4 4.29 7.55 6.37 1.78 8.44 4.29 4.44 5.48 10.21 0.59 16.14 54.8 2.66 15.4 8.59 8.14

purchased from Sigma Chemical Co. (Poole, Dorset, UK); the FCS was dialyzed for 48 h against several changes of phosphatebuffered saline prior to use. Components for the enzymatic glutamine assay (reduced nicotinamide adenine dinucleotide, glutamate dehydrogenase, asparaginase, and ␣-ketoglutarate) were purchased from Boehringer (Mannheim, Germany); the asparaginase was dialyzed for 48 h against several changes of 0.1 mol/L potassium phosphate buffer, pH 6.6, before use. Arginine- and glutamine-free minimal essential medium (MEM) was purchased from ICN Flow (Thame, Oxfordshire, UK). Cytokine enzymelinked immunosorbent assay (ELISA) kits were purchased from BioSource International (Camarillo, CA, USA). Plasma Amino Acid Concentrations Immediately after death, blood from the mice was collected into heparin by cardiac puncture, and the spleen was removed. Plasma was prepared by centrifugation of the blood. Plasma amino acid concentrations were determined with high performance liquid chromatography (HPLC) by Dr. A. C. Willis (MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, UK). Plasma glutamine concentrations were also measured enzymatically according to the method of Parry-Billings et al.30 Macrophage Preparation Immediately after death, the peritoneal cavity was lavaged with 5.0 mL phosphate-buffered saline, and the cell suspension was filtered through lens tissue to remove debris. The cells were collected by centrifugation (1000.0 g, 5 min), washed, and then resuspended in MEM supplemented with 0.9 mmol/L glutamine, 0.04 mmol/L arginine, 100.0 mL/L dialyzed FCS, and antibiotics (streptomycin and penicillin). Typically, this cell preparation comprises ⬎85% macrophages; the majority of the remaining cells are lymphocytes. Measurement of Cytokine Production Macrophages were cultured at 37°C in an air:CO2 (19:1) atmosphere in 24-well culture plates at a density of 1 ⫻ 106 cells/well, and a total culture volume of 2.0 mL in MEM supplemented with 0.9 mmol/L glutamine, 0.04 mmol/L arginine, 100.0 mL/L dialyzed FCS, antibiotics (streptomycin and penicillin), and 5.0 ␮g/mL LPS. After 24 h, the culture medium was removed and frozen (⫺70°C) for later analysis of cytokines (TNF-␣, IL-1␤, IL-6) by ELISA. All measurements were made according to the instructions given by the manufacturers of the ELISA kits. Data Presentation and Statistical Analysis All data are mean ⫾ SEM from the indicated number of animals fed on each diet. Data were analyzed statistically with Student’s t test; analysis was performed with SPSS version 6.0 (SPSS Inc., Chicago, IL, USA), and a value of P ⬍ 0.05 was considered statistically significant. RESULTS

Weight Gain The diets appeared palatable and were readily consumed by the mice. Because of their powdered nature, there was some wastage and soiling of the food, and so it was not possible to measure food intake accurately. All mice gained weight, and there were no differences in weight gain over the 2-wk period between mice fed the two diets (Table III). Plasma Amino Acid Concentrations The plasma concentrations of a number of amino acids were different between mice fed the two diets (Table IV); this variation largely reflected the variation in the levels of the amino acids in

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TABLE III.

TABLE V.

BODY WEIGHT AND WEIGHT GAIN OF MICE FED THE DIFFERENT DIETS*

CYTOKINE PRODUCTION BY MACROPHAGES FROM MICE FED THE DIFFERENT DIETS*

Initial body weight (g) Final body weight (g) Body weight gain (g)

Control

⫹Glutamine

23.2 ⫾ 0.5 24.7 ⫾ 0.6 1.5 ⫾ 0.4

22.6 ⫾ 0.9 23.6 ⫾ 1.4 1.0 ⫾ 0.5

* Data are mean ⫾ SEM from five mice fed on each diet.

the diets. Plasma glutamine concentration, as measured by HPLC, was not affected by dietary glutamine level (Table IV). Plasma glutamine concentrations were also measured by an enzymatic procedure; concentrations obtained were 791.2 ⫾ 34.7 mmol/L (control diet) and 944.7 ⫾ 116.8 mmol/L (glutamine-enriched diet). These values were not significantly different from one another (P ⬎ 0.05). Cytokine Production The production of TNF-␣ was 3.7-fold greater for LPSstimulated macrophages from mice fed the glutamine-enriched diet than for those from mice fed the control diet (Table V). LPS-stimulated IL-1␤ production was 1.7-fold higher for cells from the glutamine-fed mice, although this concentration was not significantly different from that observed with cells from the control mice (Table V). LPS-stimulated IL-6 production was significantly increased with glutamine enrichment of the diet (Table V). DISCUSSION

This study investigated the effect of increasing the amount of glutamine in the diet from 19.6 to 54.8 g/kg on the production of TABLE IV. PLASMA AMINO ACID CONCENTRATIONS IN MICE FED THE DIFFERENT DIETS* Amino acid (␮mol/L) Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Histidine Alanine Arginine Aspartic acid Asparagine Cysteine Glutamic acid Glutamine Glycine Proline Serine Tyrosine

Control

⫹Glutamine

179.3 ⫾ 13.6 282.3 ⫾ 29.4 315.3 ⫾ 38.7 115.9 ⫾ 11.8 127.6 ⫾ 18.9 160.2 ⫾ 27.6 438.5 ⫾ 43.6 38.3 ⫾ 6.7 662.3 ⫾ 67.9 36.3 ⫾ 6.1 15.4 ⫾ 2.8 62.9 ⫾ 8.4 16.0 ⫾ 2.1 83.9 ⫾ 13.5 739.8 ⫾ 98.1 177.7 ⫾ 17.2 195.4 ⫾ 30.6 121.6 ⫾ 17.1 229.6 ⫾ 59.5

133.1 ⫾ 14.3† 193.6 ⫾ 14.1† 210.8 ⫾ 11.4† 79.4 ⫾ 5.6† 101.8 ⫾ 11.9 97.4 ⫾ 11.1 296.5 ⫾ 23.0† 23.9 ⫾ 6.0 524.1 ⫾ 60.9 41.5 ⫾ 10.8 10.9 ⫾ 3.0 62.8 ⫾ 18.9 15.2 ⫾ 2.9 75.9 ⫾ 12.8 551.3 ⫾ 70.8 114.6 ⫾ 18.0† 156.7 ⫾ 30.4 84.2 ⫾ 13.3 170.0 ⫾ 33.3

* Data are mean ⫾ SEM for 10 –12 mice fed on each diet. † Significantly different from control (Student’s t test).

Cytokine TNF-␣ (pg/mL) IL-1␤ (pg/mL) IL-6 (pg/mL)

Control

⫹Glutamine

73.0 ⫾ 31.9 47.9 ⫾ 9.9 6380.0 ⫾ 598.0

275.3 ⫾ 44.4† 81.4 ⫾ 15.5 9907.0 ⫾ 703.0†

* Data are mean ⫾ SEM for five or six mice fed on each diet. † Significantly different from control (Student’s t test). IL-1␤, interleukin 1␤; IL-6, interleukin-6; TNF-␣, tumor necrosis factor-␣.

immunoregulatory cytokines by peritoneal macrophages. These levels of dietary glutamine were selected on the basis of other animal studies that have shown beneficial immunologic effects of dietary glutamine. For example, Shewchuk et al.31 found enhanced spleen lymphocyte responsiveness and decreased tumor growth in rats fed diets containing 257.0 g casein plus 20.0 g glutamine/kg compared with those fed 257.0 g casein/kg; although the precise glutamine contents of these diets were not given, it can be estimated from the information provided that they contained approximately 45.0 –55.0 g and 20.0 –30.0 g glutamine/kg, respectively. In the current study, the increase in dietary glutamine supply resulted in increased ability of macrophages to produce TNF-␣, IL-1␤, and IL-6. This is the first study to show that glutamine supplied orally can enhance macrophage activity. These findings are supported by in vitro observations of increasing TNF-␣, IL-1, and IL-6 production by cultured macrophages and monocytes with increasing glutamine concentration in the cell culture medium.9,12–14 Interestingly, the increase in cytokine production observed in the current study occurred in the absence of a significant change in plasma glutamine concentration. The lack of effect of dietary glutamine level on plasma glutamine concentration agrees with findings of a recent study31 in which the plasma glutamine concentration did not differ between rats fed diets containing 257.0 g casein/kg or 257.0 g casein plus 20.0 g glutamine/kg. The lack of effect of dietary glutamine level on plasma glutamine concentration perhaps reflects use of glutamine at the gut level or a homeostatic mechanism to maintain its plasma concentration (e.g., decreasing release from muscle when dietary glutamine is available). Whatever the mechanism for the maintenance of plasma glutamine concentration, this study shows that macrophage function can be enhanced by dietary glutamine without a concomitant change in plasma glutamine concentration. The cytokines measured in this study play important roles in immunity and inflammation. TNF-␣ regulates the acute phase response, is antiparasitic, induces the production of other immunoregulatory cytokines, induces expression of major histocompatibility class I and II antigens and thus increases antigen presentation capacity, induces expression of adhesion molecules, and activates T cells, B cells, natural killer cells, and neutrophils. TNF-␣ is also able to directly destroy some tumor cells. IL-1 enhances the activity of T cells, B cells, natural killer cells, and neutrophils, promotes chemotaxis of monocytes, neutrophils, and granulocytes to areas of immune activity, and increases acute phase protein synthesis. IL-6 is a key regulator of hepatic acute phase protein synthesis and also activates T and B cells. Thus, between them these, three cytokines are important regulators of host defense against bacteria, viruses, parasites, and tumor cells. Therefore, the observed increased production of cytokines with increasing availability of glutamine in the diet would suggest an

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enhanced ability of the host to combat a variety of infections and also the growth of tumors. There is evidence that increasing the amount of glutamine in the diet increases survival of mice to bacterial challenge32,33 and reduces the growth of an implanted tumor in rats.31 Similarly, parenteral glutamine provision has been shown to increase the survival of rats after a bacterial challenge.34 –36 The findings of the current study suggest that at least part of the effect of glutamine in these studies may have been due to enhanced macrophage activity toward bacteria and tumors, respectively, and/or to the enhancing effect of macrophagederived cytokines on the cell-mediated immune response to bacteria and tumors, respectively. Furthermore, the increased survival of critically ill patients who received glutamine parenterally compared with those who did not37 may have been due, at least in part, to enhanced macrophage-mediated immunity, particularly when it is considered that more than 75% of the patients in the group

receiving glutamine were suffering from severe sepsis, pneumonia, or burns. In support of the immune-enhancing effect of glutamine are studies showing decreased clinical infection and microbial colonization in patients with bone marrow transplant who received glutamine-containing parenteral nutrition38 and a decreased rate of sepsis in very-low birthweight infants who received glutamine-supplemented formula.39 In summary, this study has shown that the production of cytokines by macrophages is enhanced by feeding mice a diet enriched in glutamine. This effect suggests that oral glutamine could enhance a variety of immune responses toward bacteria, viruses, parasites, and tumors. Further studies of the effectiveness of oral glutamine in in vivo models of such challenges to host defense are required. Furthermore, the dependence of the effect of glutamine on its level in the diet should be investigated.

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