Immunoregulatory Effects of L-Arginine and Therapeutical Implications

M.A. Potenza*, C. Nacci and D. Mitolo-Chieppa

Department of Pharmacology and Human Physiology, Section of Pharmacology, Medical School, University of Bari, Italy

*Address correspondence to this author at the Department of Pharmacology and Human Physiology, Section of Pharmacology, Medical School, Policlinico, PIAZZA G. Cesare, 70124 Bari, Italy, Phone: +39 080 5478425; Fax: +39 080 5478449; e-mail: potenza@farmacol.uniba.it

Abstract: Arginine, initially classified as a non-essential amino acid, participates to multiple biological processes including release of several hormones, collagen synthesis during wound healing, antitumor and antibacterial activities and non-specific immunity. Nitric oxide synthase and arginase competes for L-arginine as a substrate and this event appears to play a key role in the regulation of the inflammatory process. In this framework recent studies have identified complex patterns of interactions among these enzymes.

This review will emphasizes some effects of L-arginine on immune cell functions, including triggering
of L-arginine-nitric oxide and arginase pathways, its biological properties and therapeutical applications.

INTRODUCTION

L-Arginine-Mediated Immune Responses

     Arginine, has been classified as a semiessential aminoacid because of its nutritional requirement for the optimal growth of some species, but not humans. This aminoacid can be obtained from exogenous sources via blood circulation, from intracellular protein degradation, or by the endogenous synthesis of arginine. Major sites of arginine synthesis in ureotelic animals are the liver, where arginine, generated in the urea cycle, is rapidly converted to urea and ornithine by arginase, and the kidney, where arginine is synthesized from citrulline and released into blood circulation [1].

     Over the past two decades, studies have shown that arginine plays an important role in many physiological, biological and immunological processes beyond its role as a protein-incorporated aminoacid. Early animal experiments have delineated some the effects that arginine exerts on the immune system. In normal rodents the administration of 1% arginine HCl to their normal diet (1.8% L-Arg content) increased thymic weight secondary to increased numbers of total thymic T lymphocyte [2]. This thymotropic effect was functionally correlated with enhancement of cell-mediated immunity and T-lymphocyte responses to mitogenic stimulation [3]. In the athymic mouse, supplemental arginine increased the number of T cells and augmented delayed-type hypersensitivity responses, indicating that it can exert its effects on peripheral lymphocytes and not just those within the thymus. Other studies performed in healty human volunteers and in severely ill intensive care patients indicated that the mitogenic response of peripheral blood lymphocytes is similarly increased by arginine when given at doses of 30g/day [4,5]. Following injury, arginine could reduce or abrogate the thymolytic and immunosuppressive effects of trauma and enhance rejection of allogeneic skin graft [6, 7]. Dietary supplementation with the semiessential amminoacid arginine enhanced T-cell-mediated immune function and stimulated wound healing and reparative collagen synthesis in healthy animals and human beings [8].

     Intravenous infusion of a mixture of arginine and lysine increased autonomic nervous signals to the thymus while simultaneously decreasing sympathetic conduction to the spleen. Vagal stimulation of the thymus resulted in T cell release from this gland, whereas decreased splenic efferent activity facilitated splenic natural killer (NK) cell activity [9].

     At cellular level, arginine is metabolized by different enzymes to various end products that are involved in immunomodulation.

 

    

In addition to its importance as a structural semiessential amino acid and its role in a variety of physiological functions, it has been postulated that L-arginine (L-Arg) is a potent modulator of macrophage functions as these phagocytes are able to metabolize L-Arg via two major pathways as shown in “Fig. (1)”: a) the arginase pathway, by which the guanidino nitrogen is incorporated into urea, with the other product being L-ornithine; and b) the nitric oxide synthase pathway, which results in oxidation of the guanidino nitrogen, in production of nitric oxide (NO) and yielding nitrite, nitrate, and citrulline as stable end products [10]. NO is highly lipophilic and therefore rapidly traverses cell membranes making it an effective intra – and inter- cellular messenger. It has a very short life (3-9 seconds) and must be produced in large quantities or over long periods to have prolonged biological effects [11]. When produced by macrophages it can rapidly enter microrganisms and tumor cells and exert cytostatic and cytotoxic effects by increasing cyclic-GMP synthesis and inhibiting host mitochondrial electron transport and DNA replication [12, 13] . However, macrophages also play a central role in the regulation of specific and nonspecific immunity [14] directly or by cytokine secretion [i.e. interleukin-1 (IL-1) and tumor necrosis factor (TNF-a)] and are actually involved in immunological functions such as phagocytosis, tumoricidal and antibacterial activities. On the bases of the above concepts, one can infer that alterations in macrophage functions could have cascading effects on other immune cells.

Effect of L-Arginine on Phagocytosis

     Under normal steady-state conditions the population of macrophages is constant, which means that in the liver, spleen, lung and peritoneal cavity macrophage populations are maintained mainly by the influx of monocytes that differentiate into macrophages. On the other hand, during an inflammatory reaction there is an increase in the number of macrophages due to the influx of bone marrow-derived monocytes at the site of the lesion and by production of macrophages in the inflammatory exudate by locally dividing cells [15].

     It was observed that sites of inflammation with prominent macrophage infiltration, such as wounds and certain tumors, are deficient in free arginine. In this regard Albina et al. (1988) [16] reported that low concentrations of L-Arg in culture media (< 0.1 mM) enhanced activation-associated functions in rat resident peritoneal macrophages, including citotoxicity against tumor cells, superoxide production, and phagocytosis. In contrast, when L-Arg is added to the culture media in concentrations ranging from the plasmatic one (about 0.1 mM) to 1.2 mM (the concentration in RPMI 1640), a suppression of superoxide production, cytotoxicity, phagocytosis and protein synthesis were observed in resident peritoneal macrophages [17]. Moreover, the same supplemental L-Arg concentrations were able to induce an increase of cytotoxicity in Corynebacterium parvum-elicited macrophages [18].

     In particular, a decrease in arginine availability may contribute to the activation of unprimed macrophages migrating at inflammatory sites. The reduction in free L-Arg observed in inflammatory milieu is due to the activity of macrophage-derived arginase, rather than to L-Arginine/NO pathway, since ornithine, the product of arginase activity, accumulates within extracellular space rather than citrulline, the product of L-Arg/NO pathway [16,19]. These results provide evidence that the NO-pathway may not be preferentially expressed in sites of inflammation during maximal macrophage infiltrations because the remarkably low extracellular L-Arg concentrations become rate limiting for its expression [20].

     Many other mammalian cells, as well as neutrophils (PMN) use L-Arg as the substrate for production of reactive nitrogen species, such as NO [21-24].

     However, PMN produce NO from L-Arg in lower quantities than macrophages [25] and this production often stimulated by the same activators, proceeds by separate and independent pathways [23, 26, 27]. Compared to macrophages, little is known about the relevance of the nitric oxide synthase (NOS) pathway to immune competence of PMN. Kaplan et al. (1989) [25] observed that PMN treated with the NOS inhibitor (L-NMA) showed marked inhibition of formylated polypeptide-stimulated chemotaxis which could be reversed by addition of L-Arg or dibutyril cyclic guanosine monophosphate, while L-NMA did not inhibit microbial killing. However, since the chemotaxis was impaired by L-NMA, while bacterial killing remained unaffected this would imply that these two functions are modulated differently by NOS products.

     Other findings obtained in vitro on bacterial phagocytosis in human PMN, showed that incubation with supplemental L-Arg concentrations until 380 mM induced a concentration-dependent increase of phagocytosis, but with higher supplemental L-Arg concentrations this enhancement of phagocytosis was progressively lost. Further addition of NOS inhibitors abolished L-Arg-induced increase of bacterial phagocytosis. These results suggest that enhancement of phagocytosis by L-Arg is mediated through the NOS pathway and that there is an optimal level of NO above which it interferes with PMN metabolic processes [3].

Suppressive Role of L-Arginine on Rat Splenic Macrophage Phagocytosis

     It is well known that L-Arg is a potent modulator of macrophage phagocytosis. Previous findings demonstrated that elevated concentrations of L-Arg, such as the concentration in culture medium, were able to inhibit rat peritonal macrophages phagocytosis [20]. Thus, we have evaluated the regulatory role of L-Arg on phagocytic activity of rat resident splenic macrophages (SpM_) treated with or without LPS to further investigate the pathway used by these cells for metabolizing L-Arg .

     Male Wistar rats (220-250 g) were used as sources of splenic macrophages. Spleens were excised aseptically and pressed through wire sieves to make single cell preparations. To obtain the mononuclear cell fraction, we applied washed cells on Ficoll Hypaque density gradient and centrifuged them for 30 min at room temperature. Cell suspensions recovered at the interface were removed, washed twice with Hank’s balanced salt solution (HBSS) and then resuspended in RPMI 1640 without L-Arg. In order to obtain enriched SpM_ suspensions, 5x10⁶ mononuclear cells resuspended in 2 ml of complete medium, were cultured in 60-mm plastic petri dishes and incubated at 37°C to allow macrophage adesion [27]. To control the L-Arg level in the medium using a Select-Amine kit (GIBCO), we formulated RPMI 1640 lacking in L-Arg, since it usually contains approximately 1.15 mM L-Arg. After gentle agitation, the non-adherent cells were discarded and the adherent cells were enriched about 85% of SpM_, as determined by Wright staining and about 99% viable as assessed by tripan blue staining.

 

    

1x10⁶/ml monolayers of male Wistar rat SpM_ were incubated for 4h in the absence or in the presence of 10mg/ml LPS (from E.coli 0.55:B5) in culture medium supplemented or not with different concentrations of L-Arg (80, 500, 1000 mM). After stimulation, 3x10⁶/ml Candida albicans were added to SpM_ and incubated in 5% CO2 and 95% air at 37°C for 1h. Then, SpM_ were stained with acridine orange, examined immediately with fluorescence microscope and the number of cells that engulfed 1, 2, 3 C. albicans respectively, were counted.

     Rat SpM_ exhibited a significant decrease of phagocytic activity which was inversely correlated to the L-Arg concentrations in culture medium. In rat resident SpM_ incubated with LPS, a similar effect was observed because phagocytosis was significantly reduced compared to basal levels when different concentrations of L-Arg were added to the culture medium “Fig. (2)”.

     Control and LPS-stimulated SpM_ were incubated for 4h with L-arginase (50 units/ml) or indomethacin (10mM) in culture medium lacking in L-Arg or containing different L-Arg concentrations (80, 500 or 1000 mM). Then, C.albicans was added to SpM_ and after1h incubation the phagocytic activity was assayed.

     In control SpM_, the addition of L-arginase to culture media was able to antagonize L-Arg effects produced at 500 and 1000 mM, as the basal levels of phagocytosis were fully restored by this enzyme “Fig. (3A)”. On the contrary, no effect was observed when arginase was added to the culture media of SpM_ incubated with LPS “Fig.(3B)”.

 

    

The effect of indomethacin, a cyclo-oxygenase inhibitor, on resident SpM_ phagocytic ability is shown in “Fig. (4A)”. This agent is able to restore the phagocytic activity reduced by 500 and 1000 mM concentrations of L-Arg to basal level. Moreover, LPS-stimulated SpM_, treated with L-Arg 80 mM (a concentration corresponding to the plasmatic one) exhibited a significant increase of phagocytosis after indomethacin was added to the culture medium. However, this inhibitor was able to fully reverse the suppressive effects on phagocytic activity induced by higher concentrations of L-Arg “Fig. (4B)”.

B: Effect of supplemental concentrations of L-Arginine (L-Arg) on phagocytosis of C. albicans exerted by rat resident splenic macrophages (SpM_) stimulated with (10 mg/ml) LPS incubated in absence (black bars) or in presence (hatched bars) of arginase (50U/ml). Each bar represents the mean ± SD of values from 8 separate experiments. Student’s paired or unpaired t test was used, as appropriate, to determine the significance of differences between means. ^#P< 0.001 vs L-Arg 0mM.

     In another set of experiments, in order to evaluate the possible involvement of L-Arg/NO pathway, we have tested the effect of N^w-Nitro-L-Arginine methyl esther (L-NAME 1 mM), an NO-synthase inhibitor, on phagocytic activity of LPS-stimulated SpM_ in culture medium supplemented with L-Arg 500 mM in presence or absence of indomethacin. L-NAME significantly reversed L-Arg inhibitory activity of phagocytosis, even though it was not able to restore it to basal values. Only when indomethacin was further added to the culture medium, the SpM_ phagocytic activity was completely re-established to basal values “Fig. (5)”.

 

    

 Our results suggest that rat resident SpM_ express a reduced phagocytosis which is inversely correlated to culture media L-Arg concentrations. The suppressive effect elicited by L-Arg could be due either to high, not physiological concentrations, of L-Arg in the culture media or to prostaglandin release from macrophages. In fact, the effects of L-Arg concentrations above 80 mM are antagonized by arginase. Moreover, indomethacin was able to restore to basal values SpM_ phagocytic activity in response to C. albicans as this antinflammatory agent was able to inhibit prostaglandin release.

     In rat resident LPS-stimulated SpM_ a reduced phagoctic activity compared to control SpM_ was evident and increases with increasing concentrations of L-Arg in culture media.

     Based on the reported experiments, two mechanisms could be involved in this effect. A first mechanism should involve the activation of LPS-induced L-Arg/NO pathway, that is able to generate NO starting from substrate L-Arg addedd to the culture media. In fact, the NOS expressed in macrophages [the inducible NOS (i-NOS)] is independent of Ca²⁺ and can be induced by certain cytokines, such as TNF-a, IL-1-b, and IFN-g, and by microbes or microbial membrane components such as LPS and lipid A [28, 29]. On the induction of iNOS, NO is produced continuously at high rate in the presence of adequate L-Arg supply. Because arginase and NOS both use L-Arg as a substrate, we hypothesize that in rat resident LPS-stimulated SpM_ arginase could not restore phagocytosis to basal values since its substrate is lacking through competition with NO production. This hypothesis is sustained by the increased phagocytosis activity in LPS-stimulated SpM_ after addition of L-NAME to the culture media.

 

   

  A second mechanism could involve the release of cyclo-oxygenase products. In fact, both C. albicans and LPS are able to stimulate the activation of inducible cyclo-oxygenase synthase (COX), whose effect is the release of prostaglandins [30, 31]. Moreover, both agents are able to stimulate NO and free radicals production from M_, which in turn are involved in the release of cyclooxygenase products “Fig. (6)”. In fact, indomethacin is able to restore phagocytosis to basal values as it can inhibit prostaglandin release. The two mechanisms seem to be synergic as the simultaneous addition of NO-synthase and cyclooxygenase inhibitors resulted in an increased phagocytic activity.

 

 

Effect of L-Arginine on Tumoricidal Activity and Antibacterial Function Elicited by Activated Macrophages

     L-Arg is one of the crucial components in the regulation of the antibacterial and antitumoral functions of macrophages under in vitro culture conditions and probably in vivo.

     In cytotoxic macrophages, among several enzymes NOS and arginase seem to play the most significant role in the metabolism of L-Arg.

     NO, an important regulator and mediator in many physiological and pathophysiological events, is produced by the oxidation of one of the guanidino nitrogens of L-Arg by a family of NOS isoforms. Although some NOS isoforms are constitutively expressed and Ca²⁺ dependent, the NOS expressed in M_ [inducible NOS (iNOS)] is independent of Ca²⁺ and can be induced by certain cytokines and by LPS, producing NO in the presence of L-Arg supply.

     This L-Arg-dependent production of NO has been implicated in mediating the cytotoxic actions of the activated macrophages against a variety of pathogens, including yeasts, helminths, protozoa, mycobacteria [32-35] and against various cellular targets, including tumor cells [36].

     Takema et al. [37] confirmed that the tumoricidal activity of macrophages activated by IFN-g plus LPS is dependent on L-Arg. In fact, concentrations ranging from 0.1 to 0.15 mM are necessary for this function. This range is almost equal to that for the microbiostatic activity of mouse M_ reported by Granger et al. [38], and for the cytotoxicity of rat M_ elicited by Corinebacterium parvum [20].

     Activated M_ inflict on tumor cells and fungi a form of oxidative injury associated with iron loss and involving inhibition of iron-sulfur enzymes [39]. A methodical analysis led Hibbs and colleagues [39] to conclude that L-Arg was a necessary and sufficient metabolite in the extracellular medium to sustain this form of macrophage-mediated cytotoxicity. Dissecting the L-Arg molecule, they discovered that L-Arg analogs, substituted on one or both of the guanidino nitrogens, blocked cytotoxicity in a stereospecific manner [18]. Nitrite or nitrate themselves could not replicate the cytotoxicity of the nitrite-producing M_ [18, 36] except at acidic pH [36], a condition in which nitrite can generate NO.

     On the other hand, there are studies demonstrating that low L-Arg levels can enhance oxygen radical generation and cytotoxicity in M_ [16, 17]. In fact, Xia and Zweier (1997) [40] demonstrated that iNOS-activation in M_ produces important amounts of O^2-, and this is triggered by L-Arg depletion [40]. The production of O^2- by macrophages is critical for host defense. The cytotoxic nature of O^2- not only contributes to the killing of invading microbes but also causes tissue damage in inflammation [41]. Thus, while L-Arg is required for NO generation from iNOS, partial L-Arg depletion is required to trigger O^2- and ONOO^- generation. Since L-Arg concentration is very low in inflammatory sites during M_ infiltration and would healing [16, 17], under these circumstances iNOS-mediated O^2- and ONOO^- could be particularly important in the cytotoxic actions of M_. Although iNOS-mediated formation of O^2- and ONOO^- may initially enhance M_ immune function, an overproduction of these oxidants could cause autotoxicity triggering cell death. Thus, by modulating cytosolic L-Arg it is possible to provide a therapeutical approach able to influence M_ immune functions in inflammatory disease [40].

     Besides iNOS, arginase, represents the other major enzyme that catalyze the conversion of L-Arg to ornithine and urea. There are two distinct isoforms of mammalian arginase, which are encoded by separate genes.They are quite similar with regard to enzymatic properties, but differ with regard to subcellular localization, tissue distribution, regulation of expression and immunological reactivity [42, 43]. Type I arginase, a cytosolic enzyme, is hyghly expressed in liver as a component of the urea cycle, and to a limited extent in a few other tissues. In contrast, type II arginase, a mitochondrial enzyme, is xpressed at lower levels in kidney, brain, small intestine, mammary gland and macrophages, but there is little or no expression in liver. Rat aortic endothelial cells and murine macrophages express both type I and type II arginases [44, 45] and many investigations indicate that both type of arginase have a high activity in activated macrophages [46-50]. Sufficient quantities of arginase can limit the availability of arginine for NO synthesis by intact cells. For example, in wounds [51, 52] and macrophage cultures [53, 54], the extracellular fluid becomes almost completely depleted of L-Arg, whereas ornithine increases, indicative of high arginase activity.

     More recently, inhibition of arginase in LPS-treated rodent macrophages was shown to result in enhanced conversion of arginine into citrulline indicating that arginase and iNOS can compete for arginine [55, 56]. Because arginase and iNOS both use L-Arg as a substrate and NO production from activated macrophages is dependent on extracellular supply of L-Arg [47], it is possible that the extracellular level of L-Arg might affect the modulatory role of arginase for NO production. This may be explained by the fact that the source of L-Arg for both arginase and NOS in macrophages is mainly exogenous [47] and that the relatively high L-Arg affinity for NOS versus arginase apparently reduces the L-Arg available to arginase and, thus, subsequently reduces the contribution of arginase to NO production. The modulation of NO production by arginase can be further supported by in vivo studies showing that NO production in LPS-treated rats was significantly reduced when plasma L-Arg was depleted by administration of arginase to animals [57, 58].

Therapeutical implications

     The immunostimulatory effects of L-Arg in animal models and in cell culture have suggested that this aminoacid is a therapeutic agent in various immunological dysfunctions. L-Arg is involved in wound healing and repair and promotes collagen deposition at the wound site [59].

     In healthy humans, as in animals studies oral L-Arg supplementation has numerous effects on the immune system including increase in peripheral blood lymphocyte mitogenesis, increase of the T-helper to T-cytotoxic (c) cell ratio and in macrophage activity against microrganisms and tumor cells and decrease of Tc cells [6]. Furthermore, the delayed type hypersensitivity response is increased as it is the number of circulating NK and lymphokine-activated killer cells [6, 60, 61]. Therefore supplemental L-Arg may be necessary in patients undergoing major surgery after trauma and sepsis.

     Moreover, in many pathophysiological conditions, such inflammation and sepsis an increase of NO is evident. Since this production requires extracellular L-Arg, the manipulation of substrate availability for NOS could be an attractive target for therapeutic intervention. In fact, regulation of the L-Arg level by its transport [62] and administration of arginase has been shown to be beneficial in experimental models of septic shock [57, 58]. NO has important homeostatic roles, but increased production by immune cells may result in pathophysiological changes in conditions such as inflammatory disease [63]. In these conditions when increased production of NO is detrimental and stimulates the inflammatory process, the administration of NO-inhibitors may have therapeutical effects.

     It is even probably that alteration of arginase activity may alter the NO production. For example, a high arginase activity in the infiltrating macrophages was found to be associated with cancer development [64, 49]. Although the metabolism of L-Arg via NO is important for the macrophage antitumoral ability, an enhancement of arginase activity could compromise this tumoricidal activity. In fact, the M_ metabolism of L-Arg to ornithine and subsequently towards polyamines induced by arginase may provoke tumor cell proliferation [64, 65]. Therefore, L-Arg metabolism in M_ within a tumor could promote inhibition or growth of the tumor, depending whether the NOS or arginase pathway is prevailing.

Conclusions

     In view of the multiple actions of L-Arg and the different pathways that might be induced by its administration a single specific mechanism that would fully explain the effects of L-Arg on the immune system seems still unknown.

     For example Albina et al. [14] reported that in rat resident peritoneal M_ the phagocytosis was enhanced by low concentrations of L-Arg in inflammatory milieu and this was probably due to M_ derived arginase activity.

     These results are in agreement with our data obtained on rat resident control SpM_ phagocytosis. On the other hand, in rat resident LPS-stimulated SpM_, we have found that higher and not physiological concentrations of L-Arg produced a more pronounced decrease of phagocytic activity in comparison with control SpM_. This effect was probably due to NO-production induced by L-Arg/NO pathway. Our hypothesis was supported by the observation that after addition of L-NAME to the culture media this immune dysfunction was restored to basal values, implying that this disease may be, at last in part, dependent of the L-Arg/NO pathway.

     Furthermore, a second mechanism involving the release of COX products seems probable, since indomethacin inhibiting prostaglandin release was able to restore the phagocytic activity to basal values.

     In conclusion, we could hypothesize that the two mechanisms are synergic, as both LPS and C. albicans are able to stimulate NO and free radicals production from M_, which in turn exert a stimulatory action on COX activity, enhancing the production of prostaglandins [31].

     However, still much remains to be learned about the interactions between the two pathways of arginine utilization and removal (urea and ornithine production, on one hand, and nitric oxide formation on the other).

     Moreover, further investigations must be carried out to better understand how stress, including trauma and sepsis all alter ornithine-citrulline-arginine interrelations and how these conditions modulate the availability of endogenously synthesized substrate L-Arg.

ACKNOWLEDGEMENTS

     This work was supported by grants from MURST to Dr. Delia Mitolo-Chieppa and from Fondo Sociale Europeo (FSE) to Dr. Maria Assunta Potenza

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