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1017/S000711450769936X Published online by Cambridge University Press

British Journal of Nutrition (2007), 98, 237–252 doi: 10.1017/S000711450769936X
q The Authors 2007

Review Article

Amino acids and immune function

Peng Li1, Yu-Long Yin1,2, Defa Li3, Sung Woo Kim1,4 and Guoyao Wu1,2,4*
1
Faculty of Nutrition and Department of Animal Science, Texas A&M University, College Station, TX 77843, USA
2
Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, Hunan, China 41012
3
National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China 100094
4
Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX 79409, USA
(Received 20 October 2006 – Revised 9 January 2007 – Accepted 22 January 2007)

A deficiency of dietary protein or amino acids has long been known to impair immune function and increase the susceptibility of animals and
humans to infectious disease. However, only in the past 15 years have the underlying cellular and molecular mechanisms begun to unfold. Protein
malnutrition reduces concentrations of most amino acids in plasma. Findings from recent studies indicate an important role for amino acids in
immune responses by regulating: (1) the activation of T lymphocytes, B lymphocytes, natural killer cells and macrophages; (2) cellular redox
state, gene expression and lymphocyte proliferation; and (3) the production of antibodies, cytokines and other cytotoxic substances. Increasing evi-
dence shows that dietary supplementation of specific amino acids to animals and humans with malnutrition and infectious disease enhances the
immune status, thereby reducing morbidity and mortality. Arginine, glutamine and cysteine precursors are the best prototypes. Because of a nega-
tive impact of imbalance and antagonism among amino acids on nutrient intake and utilisation, care should be exercised in developing effective
strategies of enteral or parenteral provision for maximum health benefits. Such measures should be based on knowledge about the biochemistry and
physiology of amino acids, their roles in immune responses, nutritional and pathological states of individuals and expected treatment outcomes.
New knowledge about the metabolism of amino acids in leucocytes is critical for the development of effective means to prevent and treat immu-
nodeficient diseases. These nutrients hold great promise in improving health and preventing infectious diseases in animals and humans.

Diet: Immunity: Lymphocyte: Macrophage: Protein: Health

Malnutrition and infection are major obstacles to survival, in malnourished subjects (McMurray et al. 1981), this is
health, growth and reproduction of animals and humans not feasible for patients who cannot tolerate enteral feeding. Con-
worldwide (Field et al. 2002; Calder & Yaqoob, 2004). This sequently, defining the roles of individual amino acids in immune
global concern has led to the development of nutritional responses can aid in developing effective strategies to improve
immunology as a new scientific discipline that integrates nutri- health and prevent infectious diseases. However, only in the
tion and immunology research methodologies to define a role past 15 years have the underlying cellular and molecular mechan-
for nutrients in the metabolism and function of cells of the isms begun to unfold (e.g. Wu & Brosnan, 1992; Yaqoob &
immune system at molecular, cellular, tissue and whole- Calder, 1997; Newsholme et al. 2003; Field, 2005; Calder,
body levels. Recent studies indicate that dietary protein 2006). The major objective of this article is to provide an
deficiency, which reduces concentrations of most amino insight into the specific roles of amino acids in immune function.
acids in plasma (Wu et al. 1999) and compromises the
immune system, remains a significant nutritional problem in
developing countries and also occurs in subpopulations (e.g. An overview of the immune system and assessments
the elderly or hospitalised patients) of developed nations of immune function
(Woodward, 1998; Dasgupta et al. 2005). Thus, there is grow-
ing interest in the role of amino acids in the immune function The immune system
of mammals, birds, fish and other species (Roch, 1999; Calder, Because leucocytes are important targets for the actions of
2006; Grimble, 2006; Kim et al. 2007). amino acids, here we briefly review the pertinent literature
Although dietary supplementation with high-quality protein related to the defence against infectious diseases. The
may be effective in improving protein nutritional status immune system protects the host from various pathogens and

Abbreviations: BCAA, branched-chain amino acids; GSH, glutathione; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NK, natural
killer; NO, nitric oxide; TNF, tumour necrosis factor; TPN, total parenteral nutrition; UCA, urocanic acid.
* Corresponding author: Dr Guoyao Wu, fax þ1 979 845 6057, email [email protected]
 https://doi.org/10.1017/S000711450769936X Published online by Cambridge University Press
238 P. Li et al.

consists of the innate (natural, non-specific) and the acquired are generally cleared by cytotoxic T lymphocytes after the
(adaptive, specific) systems (Calder, 1995). These two systems fragments of pathogens and the major histocompatibility com-
are highly inter-related through cytokines and signalling mol- plex are presented on the host cell surface and recognised by
ecules (Table 1). The innate immune system consists of physi- the T cells.
cal barriers (e.g. skin, the endothelial cell layer in the Innate and acquired immune systems are regulated by a highly
respiratory tract, and the gastrointestinal tract), mononuclear interactive network of chemical communications (Fig. 1), which
phagocytes (e.g. monocytes and macrophages), dendritic includes the synthesis of the antigen-presenting machinery,
cells, polymorphonuclear granulocytes (e.g. neutrophils, immunoglobulins and cytokines (Calder, 2006). Both immune
eosinophils and basophils), mast cells, natural killer (NK) systems are highly dependent upon an adequate availability of
cells, platelets and humoral factors including collectins, comp- amino acids for the synthesis of these proteins and polypeptides,
lements, lysozymes, C-reactive proteins and interferons. as well as other molecules with enormous biological importance
Recently, neutrophil extracellular traps, comprising DNA and (Kim et al. 2007). These substances include nitric oxide (NO),
proteins as major structural components, were discovered as a superoxide, hydrogen peroxide, histamine, glutathione and
mechanism against bacterial infection (Buchanan et al. 2006). anthranilic acid (Table 2). Individual amino acids affect
The innate immune system can rapidly respond to invading immune responses either directly or indirectly through their
microbes, but its major disadvantages include non-specificity metabolites. While the immune system is vital to health, it can
and a lack of memory effect. When infection cannot be fully be dysfunctional under certain conditions, resulting in the
cleared by the innate immunity over a short period, the adaptive development of autoimmune and hypersensitivity diseases,
immune system is activated to destroy infectious agents. such as insulin-dependent diabetes mellitus, rheumatoid arthritis
The adaptive (acquired) immune system consists of T lym- and asthma (Wu, 1995; Field, 2005).
phocytes, B lymphocytes and humoral factors (Calder, 2006).
The bone marrow is primarily responsible for haematopoiesis
Assessments of immune function
and lymphopoiesis, while the thymus is required for T-cell
development. The spleen, lymph nodes and the mucosa- It is likely that nutrients influence several or all aspects of the
associated lymphoid tissues in the gastrointestinal, respiratory immune system. Thus, there are multiple, complex methods
and reproductive tracts, and other organs are secondary lym- for assessing immune function in individuals, depending on
phoid tissues. Because each lymphocyte carries surface recep- experimental conditions, the availability of analytical facilities
tors for a single antigen, the acquired immune response is and the investigator’s interest (Field, 1996; Calder & Yaqoob,
highly specific. This immune system becomes effective over 1999). The classic functional measurements in vivo include:
days after initial stimulation and possesses immunological (1) the delayed-type hypersensitivity response measured by
memory. B lymphocytes are unique in their ability to produce skin testing; (2) serum antibody titres or the humoral immu-
and release specific antibodies in the humoral immunity. The nity in response to primary or secondary (booster) immunis-
antibodies can neutralise microorganisms (including viruses) ation; (3) blood levels of different lymphocyte subsets as
or toxins by binding to them; activate complement proteins well as serum concentrations of cytokines and other immune
in plasma for the destruction of bacteria by phagocytes; immo- mediators; (4) the weights of lymphoid organs; and (5) mor-
bolise bacteria; and opsonise various pathogens. When patho- bidity and recovery from infectious disease. The in vitro
gens escape the humoral immunity, they are targeted by the assays of immune function often consist of: (1) the metabo-
cell-mediated immunity that involves the production of cyto- lism of immunocytes; (2) lymphocyte blastogenesis (cell pro-
kines (e.g. interferon-g (IFNg)) and other cytotoxic proteins liferation) in response to mitogens; (3) cell morphology and
by T lymphocytes. Antibodies are highly effective against apoptosis; (4) the phagocytosis of particles by monocytes
extracellular pathogens. In contrast, host cells infected with and macrophages; and (5) the production of antibodies, cyto-
intracellular pathogens (e.g. viruses and certain bacteria) kines and low-molecular weight cytotoxic substances.

Table 1. Innate and adaptive immunity

Innate/non-specific Adaptive/acquired

Anatomical components Skin, respiratory tract, gastrointestinal tract Bone marrow, thymus, mucosal-associated lymphoid
tissue, spleen, lymph node
Cells Neutrophils, monocytes, macrophages, dendritic B lymphocytes, T lymphocytes
cells, mast cells, eosinophils, basophils, natural killer cells
Protein Interferons, complements, collectin and lysozymes Immunoglobulins
Receptors Pattern recognition receptors, encoded in a germline Antigen-specific receptors, rearranged during
development (somatic recombination)
Distribution of receptors Non-clonal Clonal: clones of cells have distinct specificities and
express different receptors
Specificity Non-specific nature of activity Discriminate and specific molecular entities
Rapidity No induction time Take days to activate
Immunological memory Functioning is not increased as a result of Increased functioning as a result of previous exposure
previous exposure
Stage Early stage of defence Later stage of defence
Self-discrimination Distinguish self from non- self components, Distinguish self from non- self components, but is
but indiscriminate tissue damage can occur imperfect (autoimmunity)
 https://doi.org/10.1017/S000711450769936X Published online by Cambridge University Press
Amino acids and immunity 239

Fig. 1. Interactions among immunocytes through production of regulatory molecules. Abbreviations: ab T, ab T cell; Abs, antibodies; Ags, antigens; AbPC, anti-
body-producing cells; AgPC, antigen-presenting cells; B, B lymphocytes; CD8, cytotoxic T cells carrying CD8 maker; DC, dendritic cell; EC, endothelial cells; ESP,
eisinophil; GM-CSF, granulocyte/macrophage colony-stimulating factor; IFNg, interferon g; IL, interleukin; LPS, lipopolysaccharide; Mf, macrophage; M-CSF,
macrophage colony-stimulating factor; Mast, mast cells; MC, monocyte; NETs, neutrophil excellular traps; NK, natural killer cells; NO, nitric oxide; NTP, neutrophil;
PMA, phorbol myristate acetate; SCF, stem cell factor; Th0 CD4, T cells carrying CD4 marker; Th1, T helper cell 1; Th2, T helper cell 2; TNFa, tumour necrosis
factor a.

Roles of amino acids in immune function and proline (Wu, 1998). Plasma concentrations of both
arginine and citrulline decrease markedly in subjects with pro-
Alanine
tein malnutrition, fasting, trauma, burn injury, inflammation,
Alanine is a major substrate for the hepatic synthesis of glu- sepsis and liver transplantation (Bansal & Ochoa, 2003).
cose, a significant energy substrate for leucocytes (Newsholme Under these conditions, arginine must be provided from the
& Newsholme, 1989), thereby influencing immune function. diet to support nitrogen balance and the health of animals
There is evidence that supplementation with 2 mM -alanine to and humans (Flynn et al. 2002).
the culture medium prevented apoptosis, enhanced cell Due to membrane depolarisation coupled to transport of
growth and augmented antibody production in B-lymphocyte the positively charged amino acid, arginine is a potent
hybridoma (Duval et al. 1991; Franek & Sramkova, 1996). secretagogue for insulin, growth hormone, prolactin and
This supplemental concentration of alanine is approximately insulin-like growth factor-I (Newsholme et al. 2005).
2– 4 times that in the plasma of animals and represents 8 % These hormones can mediate an NO-independent effect of
of that in ovine allantoic fluid on day 60 of gestation (Kwon arginine on immune function. In particular, insulin and
et al. 2003). The underlying mechanism is not known, but growth hormone regulate the metabolism of glucose and
may involve an alanine-mediated inhibition of protein degra- amino acids in major tissues, including skeletal muscle, adi-
dation in immunocytes, as reported for hepatocytes through pose tissue, liver and heart (Meijer & Dubbelhuis, 2004),
cellular signalling pathways (Meijer & Dubbelhuis, 2004). thereby influencing the availability of these nutrients for leu-
At present, little information is available regarding an effect cocytes. Growth hormone can also increase the production
of dietary supplementation with alanine on the immune of T-lymphocytes in the thymus, the number of haemato-
response in any animal species. However, in patients with poietic progenitor cells in the bone marrow, the response
total parenteral nutrition (TPN), inclusion of alanine could of T cells to cytokines and the antigen-presenting capability
be highly beneficial for supporting gluconeogensis and leuco- of dendritic cells (Calder & Yaqoob, 2004). Interestingly,
cyte metabolism (Kudsk, 2006). prolactin enhances the release of cytokines by Th1 lympho-
cytes and expression of the antigen-presenting major histo-
compatibility complex class II molecules (Dorshkind &
Arginine, citrulline and ornithine
Horseman, 2000). In addition, insulin-like growth factor-I
Arginine is synthesised from citrulline as an immediate pre- promotes the maturation of lymphocytes in the bone
cursor in virtually all cell types (Wu & Morris, 1998). The marrow, ameliorates ageing-related thymic involution and
small intestine of most mammals, except for cats and ferrets, increases lymphocyte number and activity (Dorshkind &
is capable of synthesising citrulline from glutamine, glutamate Horseman, 2000).
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240 P. Li et al.

Table 2. Roles of amino acids in immune responses

Amino acid Products Major functions

Amino acids Proteins Humoral and cellular immune factors and enzymes
Alanine Directly Inhibition of apoptosis; stimulation of lymphocyte proliferation; and enhancement of
Ab production probably through cellular signalling mechanism
Arginine NO Signalling molecule; killing of pathogens; regulation of cytokine production; and mediator
of autoimmune diseases
BCAA Directly Regulation of protein synthesis and activation of cytokine and Ab production through
cellular mTOR signalling
Glutamine A major fuel for cells of the immune system; regulation of T-lymphocyte proliferation,
protein synthesis, as well as cytokine and Ab production; activation of macrophage
function; inhibition of apoptosis
Cysteine Taurine Antioxidant; regulation of cellular redox state
Glutamate GABA Neurotransmitter; inhibition of T-cell response and inflammation
Glutamine Glu and Asp Neurotransmitters; components of the malate shuttle; cell metabolism
Glycine Directly Calcium influx through a glycine-gated channel in the cell membrane
Serine One-carbon unit metabolism; ceramide and phosphatidylserine formation
Haem Haemoproteins (e.g. haemoglobin, myoglobin, catalase, and cytochrome c); production
of carbon monoxide (CO, a signalling molecule)
Histidine Histamine Allergic reaction; vasodilator; and central acetylcholine secretion
Urocanic acid Modulation of the immune response in skin
Leucine HMB Regulation of immune responses
Lysine Directly Regulation of NO synthesis; antiviral activity
Methionine Homocysteine Oxidant; inhibitor of NO synthesis
Betaine Methylation of homocysteine to methionine; one-carbon unit metabolism
Choline Synthesis of betaine, acetylcholine and phosphatidylcholine
Cysteine Glutathione synthesis and production of H2S (signalling molecule)
DCSAM Methylation of proteins and DNA; polyamine synthesis; gene expression
Phenylalanine Directly Regulation of tetrahydrobiopterin (a cofactor for NO synthesis) synthesis
Tyrosine Synthesis of neutrotransmitters that regulate neuronal function and cell metabolism
Proline H2O2 Killing pathogens; intestinal integrity; a signalling molecule; immunity
P5C Cellular redox state; DNA synthesis; lymphocyte proliferation; ornithine and polyamine
formation; gene expression
Serine Glycine Antioxidant; one-carbon unit metabolism; neurotransmitter
Directly Inhibition of apoptosis; stimulation of lymphocyte proliferation; and enhancement of Ab
production probably through cellular signalling mechanism
Taurine TauCl Anti-inflammation
Threonine Directly Synthesis of the mucin protein that is required for maintaining intestinal immune function;
inhibition of apoptosis; stimulation of lymphocyte proliferation; and enhancement
of Ab production
Tryptophan Serotonin Neurotransmitter; inhibition of the production of inflammatory cytokines and superoxide
NAS Inhibitor of tetrahydrobiopterin synthesis; antioxidant; inhibition of the production of
inflammatory cytokines and superoxide
Melatonin Antioxidant; inhibition of the production of inflammatory cytokines and superoxide
ANS Inhibiting production of proinflammatory T-helper-1 cytokines; preventing autoimmune
neuroinflammation; enhancing immunity
Tyrosine Dopamine Neurotransmitter; regulation of immune response
EPN and NEPN Neurotransmitters; cell metabolism
Melanin Antioxidant; inhibition of the production of inflammatory cytokines and superoxide
Arg and Met Polyamines Gene expression; DNA and protein synthesis; ion channel function; apoptosis; signal
transduction; antioxidants; cell function; lymphocyte proliferation and differentiation
Arg, Met and Gly Creatine Antioxidant; antiviral; antitumour
Arg, Pro and Gln Ornithine Glutamate, glutamine and polyamine synthesis; mitochondrial integrity
Cys, Glu and Gly Glutathione Free radical scavenger; antioxidant; cell metabolism (e.g. formation of leukotrienes,
mercapturate, glutathionylspermidine, glutathione – NO adduct and
glutathionylproteins; signal transduction; gene expression; apoptosis; cellular redox
state; immune response
Gln, Asp and Gly Nucleic acids Coding for genetic information; gene expression; cell cycle and function; protein and uric
acid synthesis; lymphocyte proliferation
Uric acid An antioxidant
Gln, Glu and Pro Citrulline Antioxidant; arginine synthesis
Gln and Trp NAD(P) Coenzymes for oxidoreductases; substrate of poly(ADP-ribose) polymerase
Lys, Met and Ser Carnitine Transport of long-chain fatty acids into mitochondria for oxidation; storage of energy as
acetylcarnitine

ANS, anthranilic acid; BCAA, branched-chain amino acids (isoleucine, leucine and valine); DCSAM, decarboxylated S-adenosylmethionine; EPN, epinephrine; GABA, g-amino-
butyrate; HMB, b-hydroxy-b-methylbutyrate; mTOR, the mammalian target of rapamycin; NAS, N-acetylserotonin; NEPN, norepinephrine; P5C, pyrroline-5-carboxylate,
TauCl, taurine chloramine.
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Amino acids and immunity 241

Early in vitro studies have identified that 0·2 mM -arginine tumour-bearing or septic rats increased thymic weight, the
(close to its post-absorptive level in plasma) is required for number of thymic lymphocytes, T-lymphocyte proliferation,
the maximal proliferation of rodent and human T lymphocytes the cytotoxicity of specific cells (T lymphocytes, macrophages
in response to mitogens and for the killing of tumour cells by and NK cells), interleukin (IL)-2 production, IL-2 receptor
activated macrophages (Hibbs et al. 1987). There is also evi- expression on T lymphocytes and the delayed-type hypersen-
dence that high concentrations of arginine (e.g. 2 mM ) increase sitivity response (Calder & Yaqoob, 2004). In addition, sup-
the cytotoxicity of monocytes and NK cells in vitro (Abumrad plementing 1 or 2 % arginine to the diet for rats with trauma
& Barbul, 2004). Note that an extracellular concentration of injury ameliorated thymic involution and body weight loss,
arginine . 2 mM occurs in certain physiological fluids. For while improving wound healing and survival rate (Field et al.
example, the arginine concentration is as high as 4–6 mM in 2002). In animals with burn injury, lethal bacterial peritonitis
porcine allantoic fluid during early pregnancy (Wu et al. or intestinal damage, arginine supplementation (1 % of the
2006). Intensive research over the past decade has established diet) reduced bacterial translocation, increased the bactericidal
that NO synthesis by inducible NO synthase (iNOS) in macro- activity of host phagocytes and prolonged host survival
phages and neutrophils is an essential mechanism against (Abumrad & Barbul, 2004). Further, dietary supplementation
viruses, bacteria, fungi, malignant cells, intracellular protozoa, with 0·83 % arginine enhanced the immune status of pregnant
and parasites in mammals, birds, terrestrial animals, low ver- sows and neonatal pigs, thereby reducing morbidity and mor-
tebrates (e.g. freshwater and marine fishes) and invertebrates tality in response to infectious pathogens (Kim et al. 2007).
(e.g. shrimp) (Bronte & Zanovello, 2005). Expression of Like arginine, ornithine a-ketoglutarate supplementation
iNOS is induced in leucocytes in response to IFNg and lipo- (1 g/kg body weight per day) modulated immune function in
polysaccharide (LPS), and NO is now recognised to play an various rat models of catabolic conditions, including burns,
important role in both innate and acquired immunity sepsis, tumour bearing and glucocorticoid-induced stress
(Bogdan et al. 2000). Therefore, NO production by iNOS is (Cynober, 2004).
of most relevance to the immune response. Because arginase Clinical studies have shown that enteral or parenteral pro-
and iNOS compete for arginine as a common substrate, mod- vision of arginine (e.g. 8– 20 g/d; corresponding to 1·5 –3·6
ulating arginase expression and activity plays a critical role in times the arginine intake by an average adult) improves
NO generation by leucocytes (Kepka-Lenhart et al. 2000). immune functions and clinical outcomes in patients with
Interestingly, some bacteria, such as Helicobacter pylori, burn injury, cancer, HIV infection, major traumas and gas-
develop a strategy of survival from NO killing through consti- trointestinal surgical operations (Field et al. 2002; Suchner
tutive expression of arginase, which consumes arginine and et al. 2002). The benefits are indicated by enhanced T-cell
thus reduces its availability for NO production by iNOS function, increased antibody production, accelerated wound
(Gobert et al. 2001). An exciting new development is that healing mediated by immune cells or a reduction in infec-
physiological levels of arginine (e.g. 150 mM ) modulates tion, ventilator days, intensive care unit stay and hospital
expression of the T-cell receptor z chain (CD3z) that is stay. The most important outcome of this research is the
required for T-cell receptor integrity (Rodriguez et al. 2003). current availability of arginine-supplemented ‘immunonutri-
Further, the addition to culture medium of citrulline at tion’ (Kudsk, 2006). However, these formulas have sup-
0·1 mM (close to its plasma level) or 1 mM (10 % of its concen- plemental L -arginine, n-3 fatty acids and nucleotides,
tration in ovine allantoic fluid during early pregnancy (Kwon making it unclear whether their benefit derives entirely
et al. 2003)) promoted the synthesis of CD3j by prolonging from arginine. Nevertheless, recent studies with surgical
the half-life of its mRNA (Bansal et al. 2004). This result pro- patients have shown that these formulas may save hospital
vides a basis for the use of citrulline to increase arginine avail- costs by decreasing the length of stay and reducing infection
ability under immunodeficient conditions associated with rates (Senkal et al. 1997; Braga et al. 1999). In contrast,
elevated arginase activity in blood. benefits of arginine supplementation to critically ill patients
A large body of evidence from animal studies indicates that with severe systemic inflammatory response syndrome,
adequate provision of arginine is required for lymphocyte sepsis or multiple organ failure are less clear (Suchner
development and that dietary arginine supplementation en et al. 2002). This is probably owing to the complex
hances immune function in various models of immunological nature of arginine metabolism and NO production in vivo
challenges (Field et al. 2000; Calder & Yaqoob, 2004). In par- (Wu & Morris, 1998). Further, it is important to recognise
ticular, a deficiency of arginine in mice (its plasma concen- that NO is an oxidant and inhibitor of enzymes that contain
tration ,0·1 mM ) resulting from overexpression of arginase an iron–sulphur centre and that high levels of NO rapidly
in the small intestine impaired the development of progeni- react with H2O2 to form peroxynitrite (ONOO2) (Fang
tor-B to precursor-B lymphocytes in the bone marrow and et al. 2002). NO and peroxynitrite oxidise biomolecules
decreased the number of B lymphocytes in secondary lym- (e.g. proteins, amino acids, lipids and DNA), which leads
phoid organs (De Jonge et al. 2002). Importantly, these defects to cell injury and death. Thus, large amounts of NO pro-
were reversed by subcutaneous administration with arginine duced by iNOS can exert a deleterious effect on mammalian
(5 mmol/kg body weight twice daily). Also, inadequate cells and mediate the pathogenesis of many diseases, includ-
intake of dietary arginine (e.g. 0·3 % arginine in the diet) ing the autoimmune destruction of pancreatic b-cells in
impairs NO synthesis by both constitutive NOS and iNOS in type I diabetes mellitus, arthritis, glomerulonephritis, inflam-
young rats (Wu et al. 1999) and reduces the immune response matory bowel disease and neurological disorders (Flynn et al.
in growing chickens (Konashi et al. 2000). Further, dietary 2002). Under these conditions, arginine supplementation
supplementation with 1 or 2 % arginine (,1 or 2 times the could ‘fuel the fire’ to worsen clinical outcomes (Wu
arginine content of the regular diet) to healthy rodents and et al. 2000).
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242 P. Li et al.

Asparagine 2003). Finally, glutamate and aspartate mediate the transfer

of reducing equivalents across the mitochondrial membrane
An early study suggested that asparagine availability could
and thus regulate glycolysis and the cellular redox state in
modulate lymphocyte blastogenesis as a possible means to
cells via the malate/aspartate shuttle (Newsholme et al. 1999).
treat childhood acute lymphoblastic leukaemia (Ohno &
GABA administration (0·6 mg/d) inhibited the development
Hersh, 1970). Depletion of asparagine by exogenous asparagi-
of the proinflammatory T-cell response and retarded the adop-
nase had been considered to be immunosuppressive until Kaf-
tive transfer of type I diabetes in NOD/SCID mice (Tian et al.
kewitz & Bendich (1983) reported that this effect might also
2004). Interestingly, Lin et al. (1999) reported that supplemen-
be attributable to glutamine depletion by a glutaminase
tation with 4 and 8 % glutamate to a glutamine- and gluta-
activity present in asparaginase. However, subsequent
mate-free diet enhanced the delayed-type hypersensitivity
research has provided the following lines of evidence that
and lymphoproliferation responses in rats recovering from
asparagine plays a significant role in immune function. First,
methotrexate treatment. Notably, the beneficial effect of diet-
the expression of asparagine synthetase was markedly
ary glutamate was dose dependent and more pronounced
enhanced in lymphocytes and macrophages in response to
after a longer period of supplementation (Lin et al. 1999).
mitogens and other stimuli (Suzuki et al. 2002). Secondly,
These results suggest that dietary glutamate is necessary for
an increase in intracellular provision of asparagine increased
maintaining an optimal immune status under conditions of
the expression and activity of ornithine decarboxylase for
immunosuppression.
polyamine synthesis in thymocytes (Brand, 1987) and of
iNOS in activated macrophages (Suzuki et al. 2002). Thirdly,
asparagine (2 mM ) prevented apoptosis and increased cell Branched-chain amino acids (BCAA)
growth in lymphocytes (Duval et al. 1991). Thus, asparagine
is beneficial for mounting a successful immune response in Lymphocytes express BCAA transaminase and branched-chain
normal subjects but can also contribute to abnormal lympho- 2-oxoacid dehydrogenase for BCAA degradation (Schafer &
blastic growth in patients with leukaemia. At present, little Schauder, 1988). The transport and utilisation of BCAA by lym-
is known about an effect of dietary supplementation with phocytes are dramatically increased in response to mitogens,
asparagine on immune function in animals or humans. with their uptake being highest during the S phase of the cell
cycle (Koch et al. 1990). Importantly, BCAA provide the
a-amino group for the endogenous synthesis of glutamine pri-
Aspartate and glutamate marily in skeletal muscle (Fig. 2), which has been considered
as part of the immune system (Newsholme and Calder, 1997).
Aspartate and glutamate play versatile roles in the metabolism
and function of leucocytes (Newsholme et al. 2003). As a sub-
strate for the synthesis of purine and pyrimidine nucleotides,
aspartate is crucial for the proliferation of lymphocytes
(Newsholme & Calder, 1997). Moreover, aspartate is required
for the recycling of the citrulline produced by iNOS into argi-
nine in activated macrophages (Wu & Brosnan, 1992). This
helps maintain an adequate intracellular concentration of argi-
nine for sustaining a high rate of NO production in response to
immunological challenges. As noted above, through aspara-
gine synthesis, aspartate contributes to the modulation of
immune function. In addition, glutamate regulates iNOS
expression in certain tissues (e.g. brain), thereby indirectly
modulating immunocompetence of animals (Wu & Meininger,
2002). Moreover, glutamate is a substrate for the synthesis of
g-aminobutyrate (GABA), which is present in both lympho-
cytes (Tian et al. 2004) and macrophages (Stuckey et al.
2005). Interestingly, T cells express GABA receptors, which
mediate an inhibitory effect of GABA on their proliferation
(Tian et al. 2004). Further, as an immediate precursor for glu-
tathione synthesis, glutamate plays an important role in the
removal of oxidants and regulation of the immune response
(Wu et al. 2004a). Importantly, dietary aspartate and gluta-
mate, along with glutamine, are the major fuels for enterocytes Fig. 2. Inter-organ metabolism of branched-chain amino acids, glutamine
and arginine, and their role in immune function. Skeletal muscle takes up
(Wu, 1998). Together, these amino acids help maintain intes-
BCAA from the arterial blood, synthesises both alanine and glutamine from
tinal barrier integrity and prevent the translocation of intestinal BCAA and a-ketoglutarate, and releases these two amino acids into the cir-
microorganisms to the systemic circulation (Van der Hulst culation. The small intestine utilises glutamine to synthesise citrulline, which
et al. 1993). Besides their role in leucocyte metabolism as is converted into arginine in kidneys, cells of the immune system and other
energy substrates, both aspartate and glutamate are excitatory cell types. The liver is the primary organ for the synthesis of glutathione from
glutamate, glycine and cysteine, and of glucose from alanine for use by
neurotransmitters in central and peripheral nervous systems, extrahepatic cells (including leukocytes) and tissues. Abbreviations: Arg, argi-
acting on ionotropic and metabotropic receptors, which play nine; Asp, aspartate; Cit, citrulline; BCAA, branched-chain amino acids;
a role in modulating the immune systems (Newsholme et al. BCKA, branched-chain a-ketoacids; Gluc, glucose; GSH, glutathione.
 https://doi.org/10.1017/S000711450769936X Published online by Cambridge University Press
Amino acids and immunity 243

Also, leucine is an activator of the mTOR signalling pathway Glutamine

that regulates protein synthesis and degradation in cells
(Meijer & Dubbelhuis, 2004). This may explain why reducing Glutamine is an abundant amino acid in plasma, skeletal
extracellular concentrations of each BCAA below 0·2 mM muscle, fetal fluids and milk (Wu & Knabe, 1994; Newsholme
(close to the plasma level), as occurs in patients with protein mal- & Calder, 1997; Self et al. 2004). As a major energy substrate
nutrition, impairs lymphocyte proliferation (Skaper et al. 1976). for cells of the immune system (Wu et al. 1991; Newsholme
There is a paucity of data on an effect of BCAA on the pro- et al. 1999), glutamine plays an important role in their func-
duction of cytokines and antibodies by lymphocytes in vitro tion and homeostasis. Interestingly, glutamine is extensively
(Calder, 2006). Because the carbon skeletons of BCAA are catabolised via the glutaminolysis pathway to yield primarily
not synthesised in leucocytes, a lack of leucine, isoleucine glutamate and, to a lesser extent, aspartate, alanine, lactate,
or valine in the culture medium results in the complete pyruvate and CO2 in cells of the immune system, including
absence of protein synthesis or proliferation of lymphocytes thymocytes, lymph node lymphocytes, blood lymphocytes,
in response to mitogens (Waithe et al. 1975). However, alter- intraepithelial lymphocytes, neutrophils and macrophages
ing medium concentrations of BCAA between 0·2 and 1 mM (Field et al. 1994; Wu, 1996; Newsholme et al. 1999). The
(, 1 and 5 times plasma levels) had no effect on lymphocyte action of the NADPþ-dependent malate dehydrogenase,
proliferation (Skaper et al. 1976). This finding suggests that which is present in lymphocytes, macrophages, monocytes
normal levels of BCAA in plasma do not limit T-cell and neutrophils, converts malate and NADPþ to pyruvate
responses to mitogens. and NADPH (Newsholme, 2001). Notably, NADPH is
A number of animal studies indicate that an inadequate required by NOS and NADPH oxidase for the production of
intake of BCAA results in immune impairment. In particular, NO and superoxide anion, respectively (Fang et al. 2002).
Jose & Good (1973) reported that dietary restriction of leucine As a major source of glutamate, glutamine regulates the syn-
and valine (25 and 50 % of the control dietary intake) caused thesis of glutathione, a tripeptide crucial for defending cells
approximately 80 –90 % decreases in lymphocyte-mediated from oxidative stress (Wu et al. 2004b). As an essential pre-
tumour cell lysis. Interestingly, leucine appears to exert a cursor for the synthesis of purine and pyrimidine nucleotides,
greater effect on immune function than isoleucine and valine glutamine is required for proliferation of lymphocytes (Ardawi
(Konashi et al. 2000), which may be explained in part by & Newsholme, 1983; Wu et al. 1992). Increasing extracellular
their differential actions on the mTOR signalling (Meijer & concentrations of glutamine from 0·01 to 0·5 mM (a physio-
Dubbelhuis, 2004). Also, mice fed a BCAA-deficient diet logical level in plasma) dose-dependently increases lympho-
for 3 weeks exhibited enhancement of susceptibility to Sal cyte proliferation (Wu et al. 1992). There is also evidence
monella typhimurium, impairment in antibody production, that glutamine is required for NO synthesis in macrophages
reductions in serum concentrations of transferrin and comp- and monocytes via arginine synthesis. Indeed, arginine derived
lement C3, and increased numbers of bacteria in liver and from glutamine appears to be essential for macrophage
spleen (Petro & Bhattacharjee, 1981). In tetrachloride-induced activity (Murphy & Newsholme, 1998). Mitogens, changes
cirrhotic rats fed a 14 % casein diet, supplementing 10 % in cell volume (an early event in the activation of lymphocytes
BCAA increased the number of liver-associated lymphocytes and macrophages in response to immunological stimulation),
(especially CD8-positive cells) and NK cells, as well as inflammatory cytokines and an acid –base balance are major
lectin-dependent cellular cytotoxicity, compared with the con- regulators of glutamine metabolism in leucocytes (Wu &
trol rats consuming a 24 % casein diet (Tsukishiro et al. 2000). Flynn, 1995a, b; Newsholme et al. 2003).
Consistent with the animal studies, supplementing 35 % Several lines of evidence from in vitro studies show that
BCAA (or 0·7 g/kg body weight per day) to TPN solution glutamine affects various components of the immune
increased blood lymphocyte counts in patients recovering response. First, glutamine is necessary for the proliferation
from surgery and decreased mortality in septic patients of lymphocytes in response to stimulation by T-cell mitogens
(Freund et al. 1978; Cerra et al. 1984). Further, dietary sup- and activation of protein kinase C (Parry-Billing et al. 1990;
plementation with a 6 g BCAA mixture (60 % leucine, 20 % Wu et al. 1992; Wu, 1996). Secondly, addition of 2 mM -gluta-
isoleucine and 20 % valine) to athletes increased IFNg pro- mine to the culture medium prevented apoptosis, stimulated
duction, decreased IL-4 release, prevented an exercise-associ- cell growth and promoted antibody production in lymphocytes
ated decrease in tumour necrosis factor-a (TNFa) and IL-1 (Franek & Sramkova, 1996). Thirdly, maximal NO production
synthesis by mononuclear cells and stimulated lymphocyte by activated macrophages occurs in the presence of an extra-
proliferation (Bassit et al. 2002). Because BCAA share the cellular concentration of 1 mM -glutamine (Wu & Meininger,
same transporter on the cell membrane, an imbalance in 2002). Fourthly, at or near physiological levels in plasma, glu-
their dietary composition can result in immunity impairment, tamine (0·5 –2 mM ) modulates the production of cytokines by
especially when animals are fed a low-protein diet (Aschken- monocytes and macrophages (Spittler et al. 1997; Yaqoob &
asy, 1979). Calder, 1998). Indeed, a sufficient supply of extracellular glu-
b-Hydroxy-b-methyl-butyrate (HMB), a leucine metabolite, tamine (e.g. 2 mM ) is required for the maximal production of
may play a role in immune function. In vitro studies have IL-1 and TNFa by murine macrophages, and of IL-6 and
shown that HMB (0·1 –2 mM ) increased the proliferation, pha- IL-8 by human monocytes (Field et al. 2002). Fifthly, the
gocytosis and expression of Fc receptors in a chicken macro- maximum phagocytosis of murine macrophage depends on
phage cell line (Peterson et al. 1999). Dietary supplementation adequate provision of extracellular glutamine (0·6 mM )
with 0·1 % HMB improved immune function and reduced (Parry-Billing et al. 1990; Newsholme et al. 2003). Similarly,
mortality in several animal models of infection, including glutamine (0·5 –2 mM ) influences the expression of various
chickens, fish and pigs (Nissen & Abumrad, 1997). genes related to (1) intercellular interactions; (2) the
 https://doi.org/10.1017/S000711450769936X Published online by Cambridge University Press
244 P. Li et al.

production of cytokines by T lymphocytes; (3) phagocytosis of (Morlion et al. 1998). Similar results have also been reported
immunoglobulin G or complement-opsonised particles; (4) for patients receiving bone marrow transplants (McBurney
antigen presentation; and (5) opsonisation of human mono- et al. 1994). Consistent with the favourable clinical outcomes,
cytes (Spittler et al. 1997; Yaqoob & Calder, 1997; Wells patients receiving glutamine in TPN (5 g/100 ml) exhibited
et al. 1999; Newsholme et al. 2003). Sixthly, glutamine higher total counts of blood lymphocytes (including CD4 and
(0·1 –30 mM ) in the culture medium enhances the bactericidal CD8 T lymphocytes) when compared with patients receiving
function of neutrophils isolated from burn patients in a dose- glutamine-free TPN (Ziegler et al. 1998). In addition, oral
dependent manner (Ogle et al. 1994). Note that an extracellu- administration of glutamine (27 mg/kg body weight) increased
lar concentration of glutamine . 20 mM occurs in certain concentrations of plasma growth hormone in humans (Wel-
physiological fluids. For example, glutamine concentration is bourne, 1995), which in turn beneficially modulate the
as high as 25 mM in ovine allantoic fluid during early preg- immune system (Newsholme et al. 2005). Thus, a reduced
nancy (Kwon et al. 2003). Finally, glutamine (2 mM ) affects availability of glutamine may impair immune function, thereby
the lytic potential of cultured lymphokine-activated killer increasing the susceptibility of humans to infectious diseases.
cells (Juretic et al. 1994) and is required for the activation Because these published clinical studies involved a relatively
of NK cells that are capable of spontaneous cytolytic activity small number of subjects, the efficacy of glutamine should
against a variety of tumour cells (Liang et al. 1989). In be verified in a larger, multicentre study.
addition, the activated NK cells participate in other functions
through the production of cytokines (Fig. 1).
Glycine
Animal studies show that enteral or parenteral provision of
glutamine enhances the immunity of the host. For example, diet- Glycine participates in the synthesis of many physiologically
ary supplementation with 4 % glutamine maintained intramus- important molecules, including purine nucleotides, glutathione
cular glutamine concentrations and normalised lymphocyte and haem (Kim et al. 2007). In addition, glycine itself is a
function in early-weaned pigs infected with endotoxin potent antioxidant, scavenging free radicals (Fang et al.
(Yoo et al. 1997). Also, supplementation with 3·5 % glutamine 2002). Thus, glycine is essential for the proliferation and anti-
to a casein-based diet resulted in an increased ability of oxidative defence of leucocytes. There is also molecular and
macrophages to produce TNFa, IL-1b and IL-6 (Wells et al. pharmacological evidence for a glycine-gated chloride channel
1999), and lymphocyte responsiveness to mitogens (Kew et al. in leucocytes (Froh et al. 2002). The activation of this channel
1999), providing the first evidence that glutamine supplemen- suppresses the agonist-induced opening of L-type voltage-
tation can enhance both macrophage and lymphocyte activities dependent calcium channels and, thus, attenuates intracellular
in vivo. Note that this supplemental glutamine corresponds to Ca2þ concentrations. As a result, glycine plays a role in regu-
1·8 times the glutamine content of the control diet. Further, lating the production of cytokines by leucocytes and immune
dietary provision of 2 % glutamine was essential for the function (Zhong et al. 2003). This notion is supported by in
maintenance of gut-associated lymphoid tissues and for the syn- vitro studies showing that an increase in extracellular glycine
thesis of secretary immunoglobulin A by the small intestine, concentration (0·1–1 mM ) within the physiological range acti-
thereby preventing TNFa-induced bacterial translocation from vated a glycine-gated chloride channel and hyperpolarised
the lumen of the gut into the circulation (Alverdy, 1990). More- the plasma membrane in a variety of cells types, including
over, dietary supplementation with 2 or 4 % glutamine increased macrophages, monocytes, lymphocytes and neutrophils (Froh
the survival of mice to bacterial challenges (Adjei et al. 1994), et al. 2002). In macrophages stimulated by LPS, glycine
improved tumour-directed NK cell cytotoxic activity in rats (0·1– 1 mM ) reduced the influx of Ca2þ and an increase in
(Shewchuk et al. 1997) and reduced the growth of implanted its intracellular concentration, thereby blunting the production
tumours in rats (Shewchuk et al. 1997). Likewise, parenteral of superoxide, IL-1 and TNFa (Wheeler & Thurman, 1999).
provision of glutamine (2 g/100 ml) attenuated the adverse Glycine did not affect IL-2 production in T-cells in response
effects of TPN on the gut and respiratory tract immunity in to stimulation by immobilised anti-CD3 antibody, but inhib-
rats (Li et al. 1997), and increased the survival of rats to endo- ited cell proliferation in a dose-dependent manner (0·1 –
toxin and bacterial challenges (Ardawi, 1991; Inoue et al. 1993). 1 mM ) by attenuating an increase in intracellular Ca2þ levels
Findings from a majority of human clinical trials indicate (Stachlewitz et al. 2000). Further, addition of 2 mM -glycine
that glutamine supplementation in the form of free glutamine to the culture medium prevented apoptosis and enhanced anti-
or alanyl-glutamine dipeptide (8 –30 g of oral glutamine per body production in B lymphocytes (Duval et al. 1991).
day or 0·3 g of alanyl-glutamine/kg body weight per day in There is also in vivo evidence that glycine reduces inflam-
TPN) is beneficial for the immune system in patients with matory reactions and morbidity in pathogen-infected animals.
burn injury and gastrointestinal surgical operations, as well A deficiency of dietary glycine impaired immune responses in
as in critically ill patients (Van der Hulst et al. 1993; Melis chickens treated with LPS, which was alleviated by its dietary
et al. 2004). These effects are indicated by increases in supplementation (Konashi et al. 2000). Additionally, dietary
lymphocyte number and function, as well as reductions in supplementation with 5 % glycine to rats infected with a
infectious complications, hospital stay, morbidity and mor- lethal dose of LPS reduced plasma levels of TNFa and
tality. Notably, a randomised, double-blind study with 28 improved survival rate (Ikejima et al. 1996). Similarly,
patients undergoing major abdominal surgery demonstrated supplementation with 1 % glycine to a liquid milk diet reduced
that pre-operative glutamine supplementation via TPN inflammation and attenuated a rise in body temperature
improved nitrogen balance, increased lymphocyte function, of calves infected with a low dose of endotoxin (Simon,
augmented leukotriene production by neutrophils, reduced 1999). Interestingly, glycine protected animals against pepti-
the incidence of infection and shortened hospital stay doglycan polysaccharide-induced arthritis; chemically and
 https://doi.org/10.1017/S000711450769936X Published online by Cambridge University Press
Amino acids and immunity 245

stress-induced gastrointestinal mucosal injury; the ischaemia/ apoptosis, increased cell growth and promoted antibody pro-
reperfusion injury of a variety of organs; and shock caused duction in lymphocytes (Duval et al. 1991). Surprisingly,
by haemorrhage, endotoxin and sepsis (Zhong et al. 2003). only a few studies have examined a role for dietary histidine
In particular, dietary supplementation with 5 % glycine pre- in immune function of animals. Nonetheless, a deficiency of
vented experimental colitis in rat models induced by an intra- dietary histidine can decrease plasma concentrations of pro-
colonic injection of 2,4,6-trinitrobenzene sulphonic acid or teins, including histidine-rich glycoprotein (Jones et al.
oral administration of dextran sulphate sodium (Tsune et al. 2005), which in turn impairs the immune response. Further,
2003). In both models of gut inflammation, dietary sup- an inadequate intake of dietary histidine reduced the
plementation with 5 % glycine abolished increases in colonic immune response in chickens, which could be reversed by
expression of IL-1b and TNFa, cytokine-induced neutrophil its dietary supplementation (Konashi et al. 2000). Moreover,
chemoattractant and macrophage inflammatory protein, feeding either a high (64 g/kg diet) or low (0·4 g/kg diet)
thereby ameliorating diarrhoea and body weight loss (Tsune level of dietary histidine to rats resulted in an increase of
et al. 2003). Collectively, these findings indicate that glycine skin trans-UCA levels above those in the control group and
is a novel anti-inflammatory, immunomodulatory and cytopro- attenuated the UV-induced immunosuppression (De Fabo
tective nutrient. Clinical trials are warranted to determine the et al. 1997). These findings suggest that dietary histidine sup-
efficacy of glycine supplementation on improving immune plementation may provide a potentially novel means to boost
function in humans. immune function, particularly in the skin.

Histidine Lysine
Plasma contains a high level of a histidine-rich glycoprotein, Compelling evidence shows that a dietary deficiency of lysine
which has a multidomain structure, interacts with many limits the synthesis of proteins (including cytokines) and the
ligands and regulates a number of biological processes, includ- proliferation of lymphocytes, and impairs immune responses
ing cell adhesion and migration, complement activation, in chickens, resulting in increases in morbidity and mortality
immune complex clearance and phagocytosis of apoptotic in response to infection (Kidd et al. 1997; Konashi et al.
cells (Jones et al. 2005). Notably, an immunologically signifi- 2000). There are also findings that an inadequate intake of
cant pathway for histidine utilisation is initiated by histidine dietary lysine reduced antibody responses and cell-mediated
decarboxylase to produce histamine, a major mediator of immunity in chickens (Chen et al. 2003). By sharing the
inflammatory reactions (Tanaka & Ichikawa, 2006). It was same transport systems with arginine, the availability of diet-
previously thought that only mast cells and basophils could ary or extracellular lysine can modulate the entry of arginine
release histamine from storage during degranulation in into leucocytes and thus NO synthesis by iNOS (Wu &
response to various stimuli. However, it is now clear that Meininger, 2002). Indeed, increasing extracellular lysine
many tissues and cell types express this enzyme to synthesise concentrations (0·3 –2 mM ) reduced the intracellular arginine
histamine. These cells include haematopoietic progenitors, con- centration and NO synthesis in activated macrophages
macrophages, platelets, dendritic cells and T lymphocytes in a dose-dependent manner (Closs et al. 2000). The knowl-
(Dy & Schneider, 2004). Histamine regulates various physio- edge about an antagonism between lysine and arginine has
logical and immunological functions by activating various his- been exploited to treat effectively dermal infections caused by
tamine receptors on target cells. The overwhelming evidence the herpes simplex virus (Griffith et al. 1978). Topical application
that many cell types (e.g. medullary and peripheral haemato- of lysine (a therapeutic dosage of 0.8–1 g daily during an overt
poietic cells, eosinophils, basophils, mast cells, T lymphocytes infection) inhibited the replication of the virus, shortened the
and dendritic cells) express a histamine 4 receptor (H4R) course and duration of the disease, and improved clinical out-
suggests a possible role for these leucocytes in inflammation, comes (Griffith et al. 1978). The underlying mechanism involves
haematopoiesis and immunity (Tanaka & Ichikawa, 2006). In a decrease in the transport of arginine into the virus and an inhi-
addition, histamine mediates platelet aggregation and pro- bition of arginase activity by lysine, resulting in a depletion of
motes Th2 cell activity by both reducing IL-12 and enhancing polyamines for the growth of the virus (Griffith et al. 1981).
IL-10 production (Dy & Schneider, 2004). The successful use of lysine to treat infections by the herpes sim-
In addition to histamine synthesis, histidine can be deami- plex virus illustrates the power of basic research on amino acid
nated by the catalytic enzyme histidine-ammonia lyase to metabolism to solve a significant problem in medicine.
form urocanic acid (UCA). This substance is a unique photo-
receptor, and its conversion from cis-UCA to trans-UCS con-
Phenylalanine and tyrosine
trols the initiation of the immune suppressive action of solar
ultraviolet-B (De Fabo & Noonan, 1983). Further, cis-UCA Emerging evidence shows that an important function of
decreases the following capacities of murine spleenocytes: phenylalanine is to upregulate expression and activity of
(1) serving as antigen-presenting cells; (2) proliferating in GTP cyclohydrolase I, which is the first and rate-controlling
response to mitogens; (3) suppressing the production of IL-2 enzyme for the synthesis of tetrahydrobiopterin, an essential
and IFNg; or (4) increasing the production of IL-10 (Holan cofactor for NOS (Shi et al. 2004). Thus, phenylalanine can
et al. 1998). These effects resulted in a reduced T-lymphocyte regulates NO synthesis by leucocytes. Consequently, an ade-
activity and an increased risk of UV carcinogenesis (De Fabo quate intake of dietary phenylalanine is required to maintain
et al. 1997). a sufficient provision of tetrahydrobiopterin for the production
A limited number of in vitro studies show that supplemen- of NO by iNOS in activated macrophages and other leucocytes
tation of 2 mM -histidine to the culture medium prevented (Wu & Meininger, 2002).
 https://doi.org/10.1017/S000711450769936X Published online by Cambridge University Press
246 P. Li et al.

Tyrosine, a product of phenylalanine degradation, is the Serine

immediate precursor for the synthesis of catecholamine hor-
There are multiple pathways for serine utilisation, which
mones (epinephrine and norepinephrine), thyroid hormones
include one-carbon unit metabolism; the hepatic and renal
(triiodothyronine and thyroxine), as well as dopamine and
synthesis of glucose, particularly in ruminants; and the syn-
melanin (Kim et al. 2007). Norepinephrine is a key messenger
thesis of glycine, ceramide and phosphatidylserine as struc-
released from the sympathetic nervous system to act on the
tural components and signalling molecules of cells,
immune system (Kin & Sanders, 2006). Interestingly, both
including T and B lymphocytes (Jones et al. 1999; Kim
Th1 cells and B cells express b2-adrenergic receptors. The
et al. 2007). Indeed, phosphatidylserine has been implicated
binding of epinephrine and norepinephrine to the receptors
in the regulation of IL-2 production and T-lymphocyte acti-
triggers the generation of cAMP from ATP and the subsequent
vation in response to an immunological challenge (Pelassy
activation of protein kinase A, which stimulates the differen-
et al. 1991). Because glucose is a major fuel for lymphocytes
tiation and proliferation of Th1 cells and B cells (Kin & San-
and macrophages (Newsholme et al. 1999), an adequate avail-
ders, 2006). In addition, thyroid hormones regulate many
ability of serine is necessary for the function of these cells,
important physiological processes, including gene expression,
particularly during late pregnancy in ruminants (Wu et al.
and the metabolism and differentiation of leucocytes (Dorsh-
2006). Interestingly, addition to the culture medium of
kind & Horseman, 2000). Moreover, dopamine and melanin
2 mM -serine (,7 times its plasma levels and 10 % of its con-
reduce the synthesis of proinflammatory cytokines (including
centration in ovine allantoic fluid during late gestation (Kwon
TNFa, IL-1b, IL-6 and IL-10) by monocytes and macro-
et al. 2003)) prevented apoptosis, stimulated cell growth and
phages, induce the production of anti-inflammatory mediators
increased antibody production in lymphocytes (Duval et al.
by leucocytes, and regulate lymphocyte proliferation, platelet
1991; Franek & Sramkova, 1996). Further, there is evidence
aggregation and the phagocytic activity of neutrophils (Basu
that inadequate intake of dietary serine reduced the immune
& Dasgupta, 2000; Mohagheghpour et al. 2000). These bio-
response in chickens, which could be reversed by its dietary
chemical bases explain the finding that a deficiency of dietary
supplementation (Konashi et al. 2000).
phenylalanine plus tyrosine impaired the immune response in
chickens, which could be reversed by their supplementation to
the diet (Konashi et al. 2000).
Sulphur-containing amino acids
A sufficient intake of dietary methionine and cysteine is
important for the synthesis of proteins of the immune system
Proline
(Grimble, 2006). Through the generation of decarboxylated
Proline is catabolised via proline oxidase in a variety of organs, S-adenosylmethionine, methionine is a donor of the methyl
including the small intestine, liver, kidney, lymphoid organs and group that participates in the methylation of DNA and proteins,
placenta, to generate pyrroline-5-carboxylate (P5C) and H2O2 the synthesis of spermidine and spermine, and regulation of
(Wu, 1997; Wu et al. 2005). Interestingly, P5C can be reduced gene expression (Wu et al. 2006). Because polyamines are
to proline by the widespread NADPH-dependent P5C reductase. important for the proliferation and differentiation of lympho-
This proline–P5C cycle functions to regulate the cellular redox cytes (Flynn et al. 2002), methionine may play a role beyond
state and the proliferation of cells, including lymphocytes a protein constituent. In addition, methionine is a substrate
(Phang, 1985). This may provide a cellular mechanism respon- for the synthesis of choline and thus phosphatidylcholine and
sible for a role for proline in protecting lymphocytes from apop- acetylcholine that are essential for nerve function and leuco-
tosis, stimulating cell growth and promoting antibody cyte metabolism (Kim et al. 2007).
production (Duval et al. 1991). Moreover, proline constitutes Cysteine is the precursor of glutathione (GSH) and H2S (a
one-third of amino acids in collagen, and, thus, is crucial for signalling molecule) in animal cells, and its metabolism is
wound healing and injury recovery mediated by cells of the markedly altered in response to infection (Malmezat et al.
immune system (Abumrad & Barbul, 2004). 2000). Glutathione synthesis is influenced by dietary intakes
An intriguing new discovery is that proline oxidase may of sulphur amino acids (Wu et al. 2004b). Thus, there is a
play an important role in immunity (Ha et al. 2005). Notably, positive correlation between the transulphuration pathway
a lack of proline catabolism due to a deficiency of intestinal activity and glutathione concentrations in the liver, spleen
proline oxidase impairs immune function in the gut (Ha et al. and muscle (Malmezat et al. 2000). Glutathione scavenges
2005). H2O2, a major product of proline oxidation, is a signal- free radicals and other reactive oxygen species (e.g. hydroxyl
ling molecule (Shi et al. 2004) and a cytotoxic agent for radical, lipid peroxyl radical, peroxynitrite and H2O2) and con-
pathogenic bacteria (Kim et al. 2007). Therefore, a high jugates with various electrophils and xenobiotics for their
activity of proline oxidase in the porcine placenta (Wu et al. detoxification (Fang et al. 2002). There is evidence that the
2005) and the small intestine of piglets (Wu, 1997) may intracellular concentrations of GSH play an important role in
play a crucial role in protecting these organs from infections regulating cellular signalling pathways (including the nuclear
during the critical periods of fetal and neonatal development. transcription factor kB pathway) in response to immunological
Further, proline oxidase is present in milk and may aid in pro- challenges (Fratelli et al. 2005). In addition, GSH concen-
tecting the neonatal intestine from bacterial and viral chal- trations in antigen-processing cells modulate immune
lenges (Sun et al. 2002). This may explain, in part, why responses, including T-helper cell function and antibody pro-
neonates fed a diet of non-mother’s milk have a high risk of duction (Peterson et al. 1998). Thus, a deficiency of extra-
intestinal dysfunction in comparison with those nursed by cellular cysteine or intracellular GSH decreases the number
their mothers (Wu et al. 1996; Field, 2005). of CD4 cells, reduces the production of IFNg, impairs the
 https://doi.org/10.1017/S000711450769936X Published online by Cambridge University Press
Amino acids and immunity 247

proliferation of lymphocytes in response to mitogens and of 2 mM- threonine to the culture medium prevented apoptosis,
diminishes cytotoxic T-cell activity (Obled et al. 2004). stimulated cell growth and promoted antibody production in
Further, GSH depletion is associated with many diseases, lymphocytes (Duval et al. 1991). Animal feeding studies indi-
such as malaria, tuberculosis, cancer, AIDS and rheumatoid cate that changes in components of the immune system are
arthritis, and the requirement for sulphur amino acids sensitive to dietary threonine intake (Li et al. 1999). Indeed,
increases during trauma, sepsis and injury (Obled et al. serum antibody titres increased with increasing dietary
2004; Grimble, 2006). intake of threonine in chickens infected with the Newcastle
Dietary supplementation with methionine or cysteine is ben- disease virus (Bhargava et al. 1971). Also, dietary supplemen-
eficial for the immune system under various catabolic con- tation with threonine increased serum levels of IgG in sows
ditions. For example, increasing total methionine levels from (Cuaron et al. 1984). Further, increasing dietary threonine
0·35 to 1·2 % in the diet for chickens infected with the Newcastle intake increased antibody production, serum IgG levels and
disease virus markedly enhanced the following key aspects of jejunal mucosal concentrations of IgG and IgA, while decreas-
the immune responses: T-cell proliferation in response to mito- ing jejunal mucosal concentrations of IL-6 in young pigs chal-
gen stimulation (Tsiagbe et al. 1987a), plasma levels of immu- lenged with Escherichia coli (X. Wang et al. 2006). These
noglobulin G (Tsiagbe et al. 1987a), leucocyte migration and findings provide support for a role of dietary threonine in mod-
antibody titre (Swain & Johri, 2000). Dietary cysteine provided ulating immune function in livestock and perhaps humans.
similar effects to methionine with regard to the immune
responses of chickens (Tsiagbe et al. 1987b). However, high
Tryptophan
supplemental levels of methionine or cysteine (. 1·45 % in the
diet) were detrimental to the growth and immune responses of The products of tryptophan catabolism include serotonin,
chickens (Tsiagbe et al. 1987a, b), probably due to the excess N-acetylserotonin, melatonin and anthranilic acid (Kim et al.
production of highly toxic substances (e.g. homocysteine and 2007). Tryptophan catabolism is increased to generate anthra-
sulphuric acid) (Wu & Meininger, 2002). nilic acid through the indoleamine 2,3-dioxygenase (IDO)
Because cysteine is toxic at a high concentration, N-acetyl pathway during inflammation or stimulation by LPS or certain
cysteine (NAC) and L -2-oxothiazolidine-4-carboxylate (OTC, cytokines (Platten et al. 2005). Serotonin, melatonin and
an analogue of 5-oxoproline) are commonly used to deliver N-acetylserotonin can enhance host immunity by inhibiting
cysteine via intravenous infusion or drinking water to increase the production of superoxide, scavenging free radicals and
endogenous glutathione synthesis in cells (Wu et al. 2004b). attenuating the production of TNFa (Perianayagam et al.
Findings from clinical studies show that daily NAC provision 2005). In addition, N-acetylserotonin is an inhibitor of sepiap-
to septic patients (a bolus dose of 150 mg/kg body weight, fol- terin reductase, an enzyme for the synthesis of tetrahydrobiop-
lowed by a constant infusion of 50 mg/kg body weight over terin (Shi et al. 2004). By modulating inducible NO synthesis,
4 h) increased blood GSH content, decreased plasma IL-8 this tryptophan metabolite can affect both innate and acquired
levels and soluble TNF receptors, improved respiratory func- immunity systems. Excitingly, anthranilic acid was recently
tion, and reduced the number of days in the intensive care unit found to inhibit the production of proinflammatory Th1 cyto-
(Spapen et al. 1998). Further, oral administration of NAC kines and prevent autoimmune neuroinflammation (Platten
(0·6 g/d every second day) was also effective in raising GSH con- et al. 2005). Because there is a progressive decline in trypto-
centrations in blood and CD4 T cells, increasing NK cell activity phan concentrations in plasma of animals with inflammation,
and lymphocyte proliferation in response to mitogens, and its catabolism plays a critical role in the functions of both
prolonging the survival time of HIV patients (Breitkreutz et al. macrophages and lymphocytes (Melchior et al. 2004).
2000). Similarly, OTC supplementation to cirrhotic patients Early work indicated that tryptophan starvation resulting
(0·2 g/kg body weight per day) increased GSH content in mono- from IFNg treatment was associated with the antiproliferative
cytes and reduced the production of inflammatory cytokines, effect of this cytokine on intracellular parasites (Taylor &
including IL-1, IL-8 and TNFa (Obled et al. 2004). Feng, 1991) and tumours (Ozaki et al. 1988). Interestingly,
Taurine is the most abundant free amino acid in lympho- progressively increasing concentrations of IFNg were required
cytes and a potent antioxidant (Fang et al. 2002). Further, for its growth inhibition in the presence of elevated tryptophan
the reaction of taurine with hypochlorous acid, which is a concentrations (Pfefferkorn, 1984). Subsequently, Munn et al.
microbicidal agent produced by activated monocytes and neu- (1998) demonstrated that a pharmacological inhibition of IDO
trophils, yields taurine chloramine (Wright et al. 1986). This suppressed T-cell activity and induced fetal allograft rejection
long-lived oxidant reduces the production of proinflammatory in mice. More recently, N-(3,4,-dimethoxycinnamoyl) anthra-
cytokines (e.g. IL-1, IL-6 and TNFa) and prostaglandin E2 nilic acid, an orally active synthetic derivative of the trypto-
(Weiss et al. 1982; Chore˛ży et al. 2002), and increases hista- phan metabolite anthranilic acid, was found to protect
mine release from neutrophils of carrageenin-induced rats paralysed mice from experimental autoimmune encephalo-
(Wojtecka-Lukasik et al. 2004). Thus, dietary supplementation myelitis (Platten et al. 2005). Available evidence suggests
with taurine (1 % in drinking water) can reduce lung inflam- that tryptophan catabolism plays a role in immune responses
mation induced by bleomycin (Schuller-Levis et al. 2003). by producing a local immunosuppressive environment that is
able to control T-cell homeostasis and self-tolerance during
inflammation (Platten et al. 2005).
Threonine
A deficiency of dietary tryptophan impaired the immune
Threonine is a major component of intestinal mucin and response in chickens (Konashi et al. 2000). Conversely, oral
plasma g-globulin in animals (Kim et al. 2007). Through administration of 300 mg of tryptophan to rats enhanced
protein synthesis and cellular signalling mechanisms, addition phagocytosis by macrophages and the innate immune response
 https://doi.org/10.1017/S000711450769936X Published online by Cambridge University Press
248 P. Li et al.

(Esteban et al. 2004). Dietary supplementation with 0·22 % C. Jobgen and Frances Mutscher for assistance in manuscript
L -tryptophan also increased resistance to bacterial and para- preparation as well as anonymous reviewers for constructive
sitic infections in rats fed a 20 % zein diet (Watson & Petro, comments.
1984). At present, a potential use of crystalline tryptophan
for animal health management is not fully developed. How-
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