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Biochemical and Molecular Action of Nutrients

Dietary Glutamine Enhances Murine T-Lymphocyte Responsiveness¹

Institute of Human Nutrition, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom

²To whom correspondence and reprint requests should be addressed.

	ABSTRACT

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To examine the effects of dietary glutamine on lymphocyte function, male mice aged 6 wk were fed for 2 wk one of three isonitrogenous, isocaloric diets, which varied in glutamine concentration. The control diet included 200 g casein/kg, providing 19.6 g glutamine/kg; the glutamine-enriched diet provided 54.8 g glutamine/kg partly at the expense of casein; and the alanine + glycine-enriched diet provided 13.3 g glutamine/kg. The plasma concentrations of a number of amino acids varied because of the diet fed. The plasma glycine concentration was greater in mice fed the alanine + glycine-enriched diet (380 ± 22 µmol/L) than in mice fed the control (177 ± 17 µmol/L) or the glutamine-enriched (115 ± 18 µmol/L) diets. The plasma glutamine concentration was greater in mice fed the glutamine-enriched diet (945 ± 117 µmol/L) than in those fed the diet enriched with alanine + glycine (561 ± 127 µmol/L), but was not different from that in mice fed the control diet (791 ± 35 µmol/L). There was a significant linear relationship between the amount of glutamine in the diet and plasma glutamine concentration (r = 0.655, P = 0.015). Plasma alanine concentration was unaffected by diet. The reason for the lack of effect of increasing the amount of alanine in the diet upon its concentration in the circulation may relate to its use by the liver. Thymidine incorporation (56 ± 18 kBq/well versus <10 kBq/well), expression of the -subunit of the interleukin-2 receptor (62 versus 30% receptor positive cells) and interleukin-2 production [189 ± 28 versus 106 ± 5 (control) or 61 ± 13 (alanine + glycine enriched) ng/L] were greater for concanavalin A-stimulated spleen lymphocytes from mice fed the glutamine-enriched diet compared to those from mice fed the other two diets. Thus, increasing the amount of glutamine in the murine diet enhances the ability of T lymphocytes to respond to mitogenic stimulation. Taken together, these observations suggest that increasing the oral availability of glutamine could promote the T-cell driven, cell-mediated immune response.

KEY WORDS: • lymphocyte • glutamine • cytokine • interleukin-2 • mice

	INTRODUCTION

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Glutamine is used at a high rate by lymphocytes, even in the resting state (Ardawi 1988 , Ardawi and Newsholme 1983 , Brand 1985 , Brand et al. 1989 , Calder 1995 for a review, O'Rourke and Rider 1989 ); the rate of glutamine use is increased if lymphocytes are activated, for example, following stimulation with mitogens (Ardawi 1988 , Ardawi and Newsholme 1983 , Brand 1985 , Brand et al. 1989 , O'Rourke and Rider 1989 ). The high rate of glutamine use and its increase upon activation suggest that glutamine plays an important role in these cells. Indeed, glutamine is required for lymphocyte proliferation: the in vitro proliferative response of rat (Ardawi and Newsholme 1983 , Szondy and Newsholme 1989 ), mouse (Griffiths and Keast 1990 , Yaqoob and Calder 1997 ) and human lymphocytes (Parry-Billings et al. 1990 ) to the T-cell mitogens concanavalin A (Con A)⁴ and phytohemagglutinin depends upon the glutamine concentration (for a review, Calder 1994 ). It was also demonstrated that the availability of glutamine enhances the production of the cytokine interleukin(IL)-2 by cultured rodent lymphocytes (Calder and Newsholme 1992 , Yaqoob and Calder 1997 ) and of IL-2, IL-10 and interferon- (IFN-) by cultured human lymphocytes (Rohde et al. 1996b , Yaqoob and Calder 1998 ).

Plasma glutamine concentrations are lowered in a variety of stressful conditions, such as following burns (Parry-Billings et al. 1990 , Stinnett et al. 1982 ), during sepsis (Askanazi et al. 1980 , Milewski et al. 1982 , Roth et al. 1982 ) and post surgery (Askanazi et al. 1978 , Jensen et al. 1996 , Lund et al. 1986 , Parry-Billings et al. 1992a , Powell et al. 1994 ), and following endurance exercise (Castell et al. 1997 , Parry-Billings et al. 1992b , Rohde et al. 1996a ), athletic training (Hack et al. 1997 ) and overtraining (Parry-Billings et al. 1992b ). These situations are associated with an increased susceptibility to infections, and it has been suggested that this may be partly because of a diminished supply of glutamine to immunocompetent cells, such as lymphocytes (Newsholme and Calder 1997 , Newsholme et al. 1989 ). As a result, there is great interest in the provision of glutamine to subjects in stressful situations (see Calder 1994 ). Despite this interest, very little is known about the effect of dietary glutamine upon cells of the immune system; indeed, it is not clear whether glutamine supplied orally will influence immune function. Therefore, in the current study we investigated the effects upon spleen lymphocyte proliferation, expression of the IL-2 receptor and production of a range of cytokines when the amount of glutamine in the diets of mice is increased.

	METHODS

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Animals and diets.

Male C57BL6 mice aged 5 wk and weighing ~20 g were purchased from Harlan-Olac, Bicester, Oxfordshire, UK. They were housed individually in plastic cages in controlled environmental conditions [21.5 ± 0.5°C, 45 ± 2% humidity; 14 h light/10 h dark cycle (lights off 2000 h; lights on 0600 h)]. After acclimatization for 1 wk, during which they were a fed standard, non-purified diet (SDS Rat and Mouse No. 1; Special Diet Services, Witham, Essex, UK), the mice were randomly allocated to receive one of three diets, which were provided in powdered form by Oxford Nutrition, Witney, Oxfordshire, UK. To measure lymphocyte function, 5 or 6 mice were fed each diet, while to measure plasma amino acid concentrations, 10 or 12 mice were fed each diet. The diets were a control diet (Control) in which all amino acids were provided in casein, a glutamine-enriched diet (+Gln), and a second control diet (+AlaGly), which was enriched with alanine and glycine to provide a physiologically neutral amino acid control diet (Table 1 ). The diets contained equal amounts of carbohydrate, fat, fiber, vitamins and minerals (Table 1) . The amount of casein was decreased in the +Gln and +AlaGly diets to ensure that all diets were isonitrogenous (Table 1) . The control diet contained 19.6 g glutamine/kg, the +Gln contained 54.8 g glutamine/kg and the +AlaGly diet contained 13.3 g glutamine per kg (Table 2 ). All diets were isocaloric (15.6 kJ/g). The mice were fed the diets for 2 wk and were killed in the fed state between 0800 and 0930 by an overdose of CO₂. All procedures involving animals were approved under the Home Office Animals (Scientific Procedures) Act of 1986.

Histopaque (a mixture of Ficoll and sodium metrizoate with a density of 1.077 kg/L), glutamine, arginine, fetal calf serum (FCS), streptomycin, penicillin, bovine serum albumin, formaldehyde, Con A and sodium azide were purchased from Sigma Chemical, Poole, Dorset, UK; the FCS was dialyzed for 48 h against several changes of PBS prior to use. Components for the enzymatic glutamine assay (NADH, 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 potassium phosphate buffer/L, pH 6.6, prior to use. Arginine- and glutamine-free minimal essential medium (MEM) was purchased from ICN Flow, Thame, Oxfordshire, UK. [6-³H]Thymidine was purchased from Amersham International, Amersham, Buckinghamshire, UK. Fluorescein-isothiocyanate—labeled rat monoclonal antibodies to murine CD4 (clone YTS 177.9; immunoglobulin G2a) and CD8 (clone KT15; immunoglobulin G2a) and a phycoerythrin-labeled rat monoclonal antibody to the -chain of the IL-2 receptor (CD25) (clone PC61.5.3; immunoglobulin G1) were from Serotec, Kidlington, Oxfordshire, UK. Cytokine ELISA kits were purchased from BioSource International, Camarillo, CA.

Plasma amino acid concentrations.

At killing, blood was collected into heparin by cardiac puncture, and the spleen was removed. Plasma was prepared by centrifugation (6000 x g, 10 min) of the blood within 60 min of collection and was then stored at -20°C. Plasma amino acid concentrations were determined within 5 d by Dr A. C. Willis, MRC Immunohistochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, UK, by using HPLC. Plasma glutamine concentrations were measured enzymatically according to Parry-Billings et al. (1989) .

Lymphocyte preparation.

The spleen was teased apart, and the cell suspension was filtered through lens tissue to remove debris. The cells were collected by centrifugation (1000 x g, 5 min), and then the lymphocytes were purified by centrifugation (1200 x g, 20 min) on Histopaque. The lymphocytes were washed with MEM and finally resuspended in MEM supplemented with 0.9 mmol glutamine/L, 0.04 mmol arginine/L, 100 mL dialyzed FCS/L and antibiotics (streptomycin and penicillin) (for cell culture) or PBS supplemented with 1 g bovine serum albumin/L and 10 mmol sodium azide/L (modified PBS) (for flow cytometry).

Flow cytometry. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ lymphocytes in fresh cell preparations. Approximately 10⁶ cells resuspended in 100 of µL modified PBS were incubated for 20 min at 4°C with 10 µL of a fluorescein isothiocyanate-labeled monoclonal antibody to CD4 (50 mg/L) or CD8 (25 mg/L). After this, cells were washed twice with modified PBS and then suspended in 20 mL formaldehyde in PBS/L and examined for fluorescence by using a Becton Dickinson FACScan fluorescence-activated cell sorter (San Jose, CA). Fluorescence data were collected on 10⁴ viable cells (determined according to forward angle light scatter). Figure 1 shows typical flow cytometry profiles for one of the samples from this study.

Flow cytometry was also used to determine the proportions of cultured lymphocytes expressing CD25 and co-expressing CD4 and CD25 or CD8 and CD25. After culture of lymphocytes for 24 h (see below), the cells were washed and ~10⁶ cells resuspended in 100 µL of modified PBS. The cells were incubated for 20 min at 4°C with 10 µL of a fluorescein isothiocyanate-labeled monoclonal antibody to CD4 or CD8. Cells were then washed with modified PBS and incubated for 20 min at 4°C with 10 µL of a phycoerythrin-labeled monoclonal antibody to CD25 (100 mg/L). They were then washed twice with modified PBS and then suspended in 20 mL formaldehyde in PBS/L and examined for fluorescence by using a Becton Dickinson FACScan fluorescence-activated cell sorter. Fluorescence data were collected on 10⁴ viable cells (determined according to forward angle light scatter).

View larger version (10K): [in this window] [in a new window]   Figure 1. Flow cytometry profiles for murine spleen lymphocytes. Freshly prepared spleen lymphocytes were incubated in the absence of (A) monoclonal antibody or in the presence of monoclonal antibodies to (B) CD4 or (C) CD8 as described in Materials and Methods. They were then analyzed by flow cytometry as described in Materials and Methods. The bar labeled M2 indicates the position of cells staining positively.

Spleen lymphocytes were cultured at 37°C in an air/CO₂ (19:1) atmosphere in 96-well, micro-titer culture plates at a density of 5 x 10⁵ cells/well and a total culture volume of 200 µL in MEM supplemented with 0.9 mmol glutamine/L, 0.04 mmol arginine/L, 100 mL dialyzed FCS/L, antibiotics (streptomycin and penicillin) and, for stimulated cells, 5 mg Con A/L; Con A was omitted from cultures of unstimulated lymphocytes. After 48 h of culture, [6-³H]thymidine was added to each well (7.4 kBq/well), and the cells were incubated for a further 18 h. The cells were then harvested onto glass fiber filters and washed and dried by using a Skatron Cell Harvester (Skatron, Lier, Norway). Radioactive thymidine incorporation was determined by liquid scintillation counting. The stimulation index was calculated as

Measurement of cytokine production.

Spleen lymphocytes were cultured at 37°C in an air/CO₂ (19:1) atmosphere in 24-well culture plates at a density of 5 x 10⁶ cells/well and a total culture volume of 2 mL in MEM supplemented with 0.9 mmol glutamine/L, 0.04 mmol arginine/L, 100 mL dialyzed FCS/L, antibiotics (streptomycin and penicillin) and, for stimulated cells, 5 mg Con A/L; Con A was omitted from cultures of unstimulated lymphocytes. After 24 h, the culture medium was removed and frozen (-70°C) for later analysis of cytokines (IL-2, IL-4, IFN-, IL-10) by ELISA. All measurements were made according to the instructions given by the manufacturers of the ELISA kits. Lower limits of detection were 13 ng/L (IL-2), 1 ng/L (IFN-), 5 ng/L (IL-4) and 13 ng/L (IL-10).

Data presentation and statistical analysis.

All data are mean ± SEM from the indicated number of animals fed each diet. Data were checked for normality using the Kolmogorov-Smirnov test. Data for animal and tissue weights and for plasma amino acid concentrations were analyzed by one-way ANOVA and, where a significant effect of diet was found, differences between groups were determined by the post hoc least significant difference test by using Bonferroni's correction; where variances were unequal, data were log transformed prior to analysis. Data from flow cytometry and for lymphocyte proliferation and cytokine production were analyzed by Kruskal-Wallis one-way ANOVA, and where a significant effect of diet was found, differences between groups were determined by the Mann-Whitney U-test. The effect of stimulation with Con A on lymphocyte responses was determined by the Wilcoxon matched pairs test. In all cases a value for P < 0.05 was taken to indicate a significant difference. The relationships between the levels of glycine and glutamine in the diet and plasma glycine and glutamine concentrations were determined by calculating Spearman's linear correlation coefficient. All statistical analyses were performed using SPSS version 6.0 for Windows (SPSS, Chicago, IL).

	RESULTS

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Weight gain.

All 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 accurately measure food intake. All mice (n = 12 per group) gained weight (~0.5 g/wk), and there were no differences in weight gain over the 2-wk period among mice fed the different diets (P = 0.381; data not shown). There were no differences in the weights of the liver, thymus, lungs, spleen, kidneys or epididymal fat pads among mice fed the different diets (data not shown).

Plasma amino acid concentrations.

The plasma concentrations of six essential amino acids were significantly affected by diet (Table 3 ); this variation largely reflected the variation in the levels of these amino acids in the diets. Plasma alanine concentration was not affected by the level of alanine in the diet, whereas plasma glycine concentration was significantly greater in mice fed the +AlaGly diet than in those fed the other two diets (Table 3) . There was a significant linear correlation (r = 0.821, P < 0.001) between the level of glycine in the diet and plasma glycine concentration. Plasma glutamine concentration, measured enzymatically, was significantly higher in the mice fed the +Gln diet than in those fed the +AlaGly diet (Table 3) . There was a significant linear correlation (r = 0.655, P = 0.015) between the level of glutamine in the diet and plasma glutamine concentration.

The proportion of CD4⁺ and CD8⁺ cells was greater in the spleens of mice fed the +Gln diet than in the spleens of the other two groups (Table 4 ).

Thymidine incorporation and the concentrations of IL-2, IL-4 and IL-10 in the absence of Con A stimulation did not differ among the dietary groups (Table 5) ; IFN- was not detected in the medium of unstimulated lymphocytes. Stimulation of lymphocytes with Con A significantly increased thymidine incorporation (P = 0.0005), and production of the cytokines IL-2 (P = 0.0007), IFN- (P = 0.003) and IL-4 (P = 0.002) (Table 5) ; production of IL-10 was unaffected by Con A stimulation of the cells (P = 0.224) (Table 5) .

Thymidine incorporation was greater into Con A-stimulated lymphocytes from mice fed the +Gln diet compared with those fed the other two diets (Table 5) . This was reflected in the significantly higher stimulation index in this group (Table 5) . IL-2 production by Con A-stimulated lymphocytes from mice fed the +Gln diet was higher than by those from mice fed the other diets and was lowest for cells from mice fed the +AlaGly diet (Table 5) . Compared with the control diet, feeding the +Gln diet did not affect IFN- production by Con A-stimulated cells (Table 5) . However, Con A-stimulated cells from mice fed the +AlaGly diet exhibited lower IFN- production than cells from mice fed the other two diets. (Table 5) ; this was significantly lower than for cells from mice fed either of the other two diets. There were no significant differences in IL-4 production among Con A-stimulated lymphocytes from mice fed the different diets (Table 5) . Although the concentration of IL-10 was not significantly affected by mitogenic stimulation, it was greater in the medium of Con A-stimulated cells from mice fed the Control or +Gln diets than in that from mice fed the +AlaGly diet (Table 5) .

	DISCUSSION

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Increasing the amount of glutamine in the mouse diet from 19.6 to 54.8 g/kg resulted in greater proliferation of lymphocytes, IL-2 production and expression of the IL-2 receptor, when the cells were stimulated by Con A. The effects of glutamine on IL-2 production and IL-2 receptor expression may account for the enhanced proliferative response because T lymphocyte proliferation is dependent upon the supply of IL-2 and upon expression of the IL-2 receptor (Smith 1988 ). The responsiveness of lymphocytes was examined by stimulation with a single concentration of the mitogen Con A, which was found from preliminary experiments to induce maximal thymidine incorporation. In using this single concentration in this study, it was assumed that the dietary manipulations did not affect the dose-response curve to Con A and that the values for thymidine incorporation reported represent maximal incorporation. There is some evidence from in vitro studies that this will be the case. Griffiths and Keast (1990) cultured murine spleen lymphocytes in the presence of different concentrations of glutamine and measured thymidine concentration in response to various concentrations of Con A and phytohemagglutinin. They reported that maximal thymidine incorporation occurred at the same concentration of Con A or phytohemagglutinin irrespective of glutamine concentration and that the shape of the mitogen dose-response curve was unaffected by glutamine availability.

To our knowledge, the current study is the first to show that increasing the amount of glutamine in the diet enhances the production of a cytokine and is the third to show increased lymphocyte proliferation in response to dietary glutamine enrichment. This latter effect was recently reported by Shewchuk et al. (1997) , who compared the proliferation of spleen lymphocytes taken from tumor-bearing rats fed diets containing 257 g casein/kg or 257 g casein plus 20 g glutamine/kg and stimulated with Con A; the precise glutamine concentrations of these diets were not given but it can be estimated from the information provided that they contained ~20–30 g and 45–55 g glutamine/kg for 257 g casein/kg or 257 g casein plus 20 g glutamine/kg, respectively. Proliferation was significantly higher when extra glutamine was included in the diet (Shewchuk et al. 1997 ). Likewise, Yoo et al. (1997) found that proliferation of blood lymphocytes from Eschericia coli-infected piglets was significantly higher if the piglets consumed a diet containing 40 g glutamine/kg compared with a diet that did not contain glutamine. These effects occurred in the absence of a difference in plasma glutamine concentration (Shewchuk et al. 1997 , Yoo et al. 1997 ). In the current study, plasma glutamine concentration was not significantly different in mice fed the control and glutamine-enriched diets, indicating that significant effects upon lymphocyte function can occur in the absence of a significant difference in plasma glutamine concentration. This may suggest that glutamine exerts effects principally at the level of the gut-associated immune system. The limited effect of dietary glutamine on plasma glutamine concentration perhaps reflects the use of glutamine in the gut or liver or a homeostatic mechanism to maintain its plasma concentration (e.g., decreasing release from muscle when dietary glutamine is available). The plasma concentration of alanine was unaffected by the level of alanine in the diet, which varied 10-fold. Alanine is readily metabolized by the liver, where it acts as a gluconeogenic substrate. The lack of effect of dietary alanine on plasma alanine concentration perhaps reflects its use by the liver or, as suggested for glutamine, a homeostatic mechanism to regulate its plasma concentration (e.g., decreasing release from muscle when dietary alanine is available). In contrast, plasma glycine concentration was greatly influenced by its level in the diet. A 10-fold variation in the amount of glycine in the diet resulted in a threefold difference in plasma glycine concentration. This perhaps suggests that mechanisms for the regulation of plasma glycine concentration are not as efficient as those for glutamine and alanine. One important metabolic fate of glycine is conversion to serine. Although the level of serine varied among the diets, plasma serine concentration was not affected by diet. This might reflect the conversion of excess dietary glycine to serine in the mice fed the +AlaGly diet.

The effects of dietary glutamine observed here are in accordance with the in vitro effects of glutamine; this amino acid has been shown to enhance the proliferative response of rodent and human lymphocytes to T-cell mitogens (Ardawi and Newsholme 1983 , Griffiths and Keast 1990 , Parry-Billings et al. 1990 , Szondy and Newsholme 1989 , Yaqoob and Calder 1997 ), to increase mitogen-stimulated IL-2 production by rodent and human lymphocytes (Calder and Newsholme 1992 , Rohde et al. 1996b , Yaqoob and Calder 1997 and 1998 ) and to increase IL-2 receptor expression on Con A-stimulated mouse lymphocytes (Yaqoob and Calder 1997 ). Furthermore, parenteral administration of glutamine has been reported to increase mitogen-stimulated proliferation of blood lymphocytes taken from patients post surgery (O'Riordain et al. 1994 ). These effects are similar to those reported here for glutamine supplied orally to healthy mice.

Although glutamine has been regarded as a nonessential amino acid (Rose 1949 ), it has been known for many years that it is essential for the maintenance of viability and growth of isolated mammalian cells in culture (Eagle et al. 1956 ). There is now much evidence that the omission of glutamine from the medium of cultured lymphocytes results in their inability to divide when mitogenically stimulated and in decreased cytokine production and cytokine receptor expression (see Introduction for references). Thus, glutamine is essential for the function of lymphocytes cultured in isolation. There is also increasing evidence that glutamine is a conditionally essential amino acid in pathophysiological situations (discussed by Lacey and Wilmore 1990 ). Such conditions are characterized by decreased muscle stores of glutamine and by lowered plasma glutamine concentrations (see Introduction for references), suggesting that the demand for glutamine exceeds the supply. Thus, the most likely occasions for the application of glutamine are in such stressful situations (see Introduction). However, the current study investigated the effect of glutamine enrichment in the diet of healthy (i.e., unstressed) mice. It was found that, even in the healthy state, the addition of glutamine to the diet (concomitant with a reduction in the levels of other amino acids) improved measures of cell-mediated immunity. This suggests either a specific pharmacological effect of glutamine or that glutamine may be an essential amino acid, a notion that clearly requires more extensive investigation, including controlled dose-response studies.

The current study used a design where dietary protein was in part substituted with glutamine (+Gln diet) or alanine and glycine (+AlaGly diet). As such, the levels of all amino acids other than those under study were decreased in the +Gln and +AlaGly diets compared to the control diet. Thus, the results reported here may reflect in part a decrease in the availability of certain amino acids. However, the levels of all amino acids in each of the diets were sufficient for maintaining rodents (Reeves et al. 1993 ), and the adequacy of these diets is supported by the lack of differences in weight gain and tissue weights among mice fed the different diets. The combination of alanine plus glycine was selected as an inactive isonitrogenous control for the addition of free glutamine to the diet. However, cells from mice fed the +AlaGly diet behaved differently than those fed the control diet: they produced lower levels of IL-2, IFN- and IL-10 after Con A stimulation. This suggests either that the level of one or more amino acids in the +AlaGly diet was too low to maintain the production of certain cytokines or that high levels of alanine or glycine decrease the production of certain cytokines. There is some evidence for the latter suggestion: glycine reduces lipopolysaccharide (LPS)-induced tumor necrosis factor- (TNF-) production by cultured rat Kupffer cells (Ikejima et al. 1997 ); partly abolishes the rise in serum TNF- concentrations after LPS injection in rats, thus increasing survival (Ikejima et al. 1996 ); and reduces TNF- messenger RNA levels in ethanol-treated rats (Yin et al. 1998 ). These observations along with those of the current study suggest that glycine is not inactive with respect to cytokine production.

The cytokines measured in this study play important roles in immune responses. IL-2 and IFN- are Th1-type cytokines, which play a role in cell-mediated immunity. In contrast, IL-4 and IL-10 are Th2-type cytokines, which are involved in controlling antibody production by B cells. The data obtained here suggest that glutamine has an enhancing effect upon the Th1-type cytokines, in particular IL-2, and will enhance responsiveness to IL-2 by increasing the number of cells bearing the IL-2 receptor. IL-2 and IFN- activate helper T cells, cytotoxic T cells, natural killer cells, monocytes and macrophages, and IFN- has some antiviral activity. Thus, these two cytokines are important regulators of host defenses against bacteria, viruses and tumor cells. Therefore, the observed increased production of IL-2 and IFN- with increasing availability of glutamine in the diet would suggest an 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 the survival of mice subjected to bacterial challenge (Adjei et al. 1994 , Suzuki et al. 1993 ) and reduces the growth of an implanted tumor in rats (Shewchuk et al. 1997 ). Similarly, parenteral glutamine provision increases the survival of rats following a bacterial challenge (Ardawi 1991 , Inoue et al. 1993 , Naka et al. 1996 ). At least part of the effect of glutamine in these studies might have been due to enhanced lymphocyte-mediated responses towards bacteria and tumors. Furthermore, the increased survival of critically ill patients who received glutamine parenterally compared to those who did not (Griffiths et al. 1997 ) might have been at least partially caused by enhanced lymphocyte-mediated immunity, particularly considering that >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 that show decreased clinical infection and microbial colonization in bone marrow transplant patients who received glutamine-containing parenteral nutrition (Ziegler et al. 1992 ) and a decreased rate of sepsis in very low-birth weight infants who received glutamine-supplemented formula (Neu et al. 1997 ).

Although an increase in the proportion (and so presumably the number) of various T cell subsets and in the reactivity of T lymphocytes, as observed in the current study, might result in enhanced cell-mediated immunity towards pathogens, thus protecting against infections, it might also cause lymphocytes to be more reactive against self-antigens and so could contribute to autoimmune responses.

In summary, this study has shown that lymphocyte reactivity is higher after feeding mice a diet enriched in glutamine. This effect suggests that oral glutamine might enhance a variety of immune responses towards bacteria, viruses and tumors and so could be useful in various clinical situations. Further studies of oral glutamine in in vivo models of challenges to host defense are required before the real nature of its effectiveness can be determined. Furthermore, the dependence of the effect of glutamine upon its level in the diet should be investigated.

	FOOTNOTES

 
¹ This work was supported by Oxford Nutrition Ltd.

³ Present address: Hugh Sinclair Unit of Human Nutrition, Department of Food Science & Technology, University of Reading, Whiteknights, Reading, UK.

⁴ Abbreviations used: +AlaGly, alanine + glycine enriched diet; +Gln, glutamine-enriched diet; Con A, concanavalin A; FCS, fetal calf serum; IFN-, interferon-; IL, interleukin; LPS, lipopolysaccharide; MEM, minimal essential medium; TNF, tumor necrosis factor.

Manuscript received October 22, 1998. Initial review completed December 14, 1998. Revision accepted April 26, 1999.

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