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© 2007 The American Society for Nutrition J. Nutr. 137:106-111, January 2007

Nutritional Immunology

Arginine Does Not Exacerbate Markers of Inflammation in Cocultures of Human Enterocytes and Leukocytes1,2

Alexandr Parlesak3,*, Isabelle Negrier4, Nathalie Neveux4,5, Christiane Bode3 and Luc Cynober4,5

3 Department of Physiology of Nutrition, Hohenheim University, D-70593 Stuttgart, Germany; 4 Faculty of Pharmacy, Laboratory of Biological Nutrition, University René Descartes Paris 5, F-75270 Paris Cedex 06, France; and 5 Laboratory of Clinical Chemistry, F-75181 Paris Cedex 04, France

* To whom correspondence should be addressed. E-mail: parlesak{at}uni-hohenheim.de.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Enteral arginine supplementation in the critically ill has become a matter of controversy. In this study, we investigated effects of the addition of 0.4 and 1.2 mmol/L arginine in a coculture model on markers of inflammation, enterocyte layer integrity, and amino acid transport. In this model, a monolayer of intestinal epithelial cells (Caco-2) separated compartments with nonpathogenic Escherichia coli and mononuclear leukocytes. Activation of enterocytes and leukocytes was assessed by the measurement of nitric oxide, TNF-{alpha}, IL-6, IL-8, IL-10, and IFN-{gamma}. Further outcomes were the transepithelial flux of 22 amino acids, their catabolism, and the integrity of the enterocyte layer assessed as permeability of fluorescein dextran (Mr 4400). Bacterial stimulation of intestinal epithelial cells enhanced the basolateral concentration of nitric oxide and all cytokines measured. Supplementation with arginine did not affect epithelial integrity, production of any of the cytokines investigated, or the amount of nitric oxide. The amino acid used primarily by nonstimulated intestinal epithelial cells cocultured with leukocytes was glutamine. Activation of IEC with bacteria significantly enhanced the catabolism of serine, asparagine, and lysine, and reduced glutamine catabolism. Addition of arginine increased ornithine formation and moderately reduced transepithelial transport of methionine and other amino acids. Hence, arginine supplementation does not interfere with inflammation-associated cross-talk between human enterocytes and leukocytes. Because it also does not seem to affect the integrity of enterocyte layers, a detrimental role of arginine during septic-like conditions seems unlikely.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Arginine, which is not an essential amino acid (AA)6 for humans under normal conditions, can become essential in stress situations, especially when the intestine is involved in systemic inflammation (1). Therefore, the addition of arginine to enteral feeding solutions improves immune responses, especially in the critically ill (2). Besides its ability to serve as a substrate for polyamine synthesis (3), a key pathway of immunoregulation by arginine is its conversion into nitric oxide (and citrulline). Nitric oxide (NO) is an important signaling molecule regulating the severity of inflammation. It can be synthesized from the guanidine group of arginine by 2 types of nitric oxide synthases, which are either constitutive (cNOS) or inducible (iNOS). The activation of the latter enzyme by cytokines and LPS can lead to the production of large amounts of NO for extended periods of time (4). With respect to exacerbation of inflammation and damage of the intestinal tissue, NO plays an ambiguous role. On the one hand, suppression of basal NO levels results in hallmark features of intestinal inflammation such as increased neutrophil infiltration, mast cell degranulation, and increased microvascular permeability (5–7).

On the other hand, time-delayed suppression of NO production after being challenged with endotoxin has protective effects in rats (8). Also, inhibition of NO synthesis in human macrophages in vitro results in a reduced production of inflammatory cytokines such as TNF-{alpha} (9). In stages of acute inflammation or sepsis, the availability of arginine has been proposed to become the limiting factor for the production of NO (10).

In the face of these discrepant results, our working hypothesis was that arginine supplementation does not enhance bacteria-induced production of both proinflammatory cytokines and nitric oxide by intestinal epithelial cells (IEC) cocultured with human leukocytes and that it does not initiate a loss of epithelial integrity. Moreover, we sought to understand how bacterial challenge and arginine supplementation affect the metabolism and the transepithelial transport of 22 amino acids.

As an experimental approach, a coculture model of human leukocytes and an IEC line (Caco-2) was used for investigating the effects of arginine supplementation on the cross-talk between human enterocytes and leukocytes after a bacterial challenge (11,12). Furthermore, the effect of bacterial stimulation and arginine supplementation on the flux of AA and their catabolism was measured.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Human epithelial intestinal cells (Caco-2). Cells (1 x 106) of the IEC line Caco-2 (DSM 169; DSMZ) were seeded on cell culture inserts, which were embedded in tissue culture plates (both from Becton Dickinson), and cultured for 15 to 17 d in DMEM high glucose (PAA) supplemented with nonessential AA (PAA) and 17% endotoxin-free fetal calf serum (Biochrom). The bottom of the inserts (4.3 cm2) consisted of a membrane with pores (0.4 µm) enabling polarized secretion of cytokines by enterocytes into the basal compartment. Only Caco-2 monolayers on inserts having a transepithelial electrical resistance (Millicell-ERS, Millipore) higher than 450 {Omega} x cm2 (11,12) were used for experiments.

    Preparation of mononuclear leukocytes/bacteria. Mononuclear cell fraction from peripheral venous blood (peripheral blood mononuclear cells (PBMC): ~74% lymphocytes, ~15% monocytes, ~9% natural killer cells, <2% neutrophils) of 9 healthy men aged 43.8 ± 12.5 y (Blood Transfusion Medicine, Katharinenhospital Stuttgart) were isolated by density gradient centrifugation (550 x g; 30 min, ambient temperature) as described earlier (12). The nonpathogenic strain Escherichia coli K12 was cultured for 2 breeding periods for 12 and 24 h (37°C) in Luria broth [1% Trypton (Oxoid); 0.5% yeast extract (Difco); 0.5% NaCl] and washed before application.

    Cocultivation of enterocytes, leukocytes, and bacteria. PBMC (4 x 106 in 2 mL completed DMEM with gentamicin 20 mg/L) (Gibco) were added to the basolateral compartment beneath the insert bottoms with the differentiated and confluent enterocyte layer. The apical compartment (2 mL) contained either completed DMEM with gentamicin (120 mg/L: for growth arrest of E. coli) only (negative control) or with E. coli added (2 x 1010 CFU/L; positive control). Arginine concentration in completed DMEM was 340 µmol/L. In further experiments, either 400 µmol/L (Arg1) or 1.2 mmol/L (Arg2) arginine were added to the apical compartment.

    Catabolism, production, and net flux of AA and glucose catabolism/transport. Changes in the concentration of AA after 72 h were measured in both the basolateral and the apical compartment by an ion-exchange HPLC method as described earlier (13) using an AminoTac JLC-500/V apparatus (JEOL). Glucose concentration (0 h and 72 h) in the apical and basal compartment was measured enzymatically with a commercially available kit (GlucoQuant, Roche Diagnostics). The catabolism (formation) of AA and glucose was calculated as the difference between the amounts after and before incubation (72 h).

    Integrity of the Caco-2 cell layer. To control the confluence of the Caco-2-monolayer and to assess changes of its integrity by the different treatments, the permeability of fluorescein isothiocyanate (FITC)-dextran (Mr 4400; Sigma) was measured after 12, 24, and 72 h. Results are expressed as a relative percentage of transmigrating FITC-dextran.

    Release of cytokines and NO formation. To assess changes in the production of cytokines, the basolateral concentrations of TNF-{alpha} (12 h), IL-8 (24 h), IL-6 (24 h), IL-10 (24 h), and IFN-{gamma} (72 h) were measured as previously described (12) with OptEIA ELISA sets (BD Pharmingen). The formation of NO in the basolateral medium was measured by nitrite derivatization with 2,3-diaminonaphatalene (14). The reaction product [1-(H)-naphtotriazole] was quantified in a microtiter plate fluorescence reader (Fluorstar, BMG; excitation: 355 nm; emission 460 nm).

    Statistics. All values are given as means ± SEM. Data were analyzed by 1-way ANOVA and Tukey's post hoc test when more than 2 means were compared. Prerequisites for the applicability of ANOVA (normality of distribution, homogeneity of variances) were checked with tests of Kolmogorov-Smirnov and Bartlett, respectively. If these prerequisites were not met, the statistical evaluation was performed with the logarithmically transformed values. Student's t test for matched pairs and Student's t test for independent samples were used for the calculation of P if 2 means were compared. Differences were considered as significant at P < 0.05. Correlations between AA and cytokine concentrations were calculated with the nonparametric Spearman test.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Catabolism, formation, and net flux of amino acids. In experiments where intestinal epithelial cells (IEC) were incubated with leukocytes on the basolateral side only (without bacteria), the total amount per well (sum of amounts in the basolateral and apical medium) of Gln, Cys, Lys, Ile, Leu, Arg, Thr, Val, Met, and Tyr decreased significantly after 72 h. In contrast, the amount of Ala, Glu, and Pro increased significantly (Fig. 1). Arginine, Glu, Lys, Cys, and Orn were the AA transported most actively through the layer of IEC into the basolateral compartment (Table 1).


Figure 1
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  		Figure 1  Catabolism (negative values) and production (positive values) of AA by leukocyte/enterocyte cocultures without bacterial challenge over 72 h. Each column represents the change in total AA (sum of basolateral and apical compartments) compared with the initial medium (0 h). Amounts are sums of the basolateral and the apical compartment. Amino acids with negligible changes (<0.1 µmol/well: Tau, Asp, Ser, Cit, and His) were omitted. Values are means ± SEM, n = 9. Asterisks indicate different from 0 h, *P < 0.05, **P < 0.01, ***P < 0.001.

 

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  		TABLE 1 Concentrations of AA in the apical and basolateral compartments of the coculture experiments with and without bacterial stimulation of enterocytes after 72 h of incubation1

 
The apical addition of E. coli resulted in a significant increase in the catabolism of Ser, Asn, and Lys and, to a lesser extent, also in that of Arg, Orn, Thr, and Glu (Fig. 2). In contrast, the amount of Gln and Gly increased after 72 h, when bacteria were added to the apical compartment. The net flux (= difference of AA concentration between the basolateral and the apical compartment after 72 h) of Ser, Ala, Leu, and Pro through the IEC layer increased significantly after the addition of E. coli (Table 1).


Figure 2
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  		Figure 2  Effect of addition of E. coli to the apical surface of IEC on total amount of AA per well (apical and basolateral compartment). Each column represents the change in total AA (sum of basolateral and apical compartments) compared with incubations without bacterial stimulation (72 h). If bacterial stimulation led to AA loss, values are negative. If surplus AA are formed in addition to those present in the medium, columns are in the positive range. Only AA of which the concentration changed significantly in the presence of bacteria are given in the figure. Values are means ± SEM, n = 9. Asterisks indicate different from experiments without bacteria, *P < 0.05, **P < 0.01, ***P < 0.001.

 
Supplementation of the apical compartment with Arg1 (400 µmol/L) in the presence of bacteria increased the concentration of Arg and Orn only in the basolateral compartment (Table 2). Elevation of the concentration of arginine by 1200 µmol/L (Arg2) resulted in a pronounced increase of arginine in both the apical and the basolateral medium and in an impaired translocation of Met, Leu, Tyr, Phe, Orn, and Lys into the basolateral compartment (Table 2).


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  		TABLE 2 Amino acid concentrations in both compartments of the coculture experiments with bacterial stimulation with and without arginine supplementation12

 
    Changes in glucose concentration. After 72 h of coincubation with PBMC, the apical addition of bacteria decreased glucose concentration in both the apical (from 21.1 ± 4.0 to 17.6 ± 2.8 mmol/L, P = 0.021) and basolateral (from 18.3 ± 4.1 to 14.1 ± 2.9 mmol/L, P = 0.008) compartments. The difference in concentrations of glucose between the apical and the basolateral compartments was significant only in the presence of bacteria (P = 0.038).

    Release of cytokines. All cytokines measured increased significantly when E. coli K12 was added to the apical compartment of the coculture model. The mean concentrations of TNF-{alpha} and IL-10 increased ~150-fold, that of IL-6 ~210-fold, that of IL-8 ~8-fold, and that of IFN-{gamma} ~25-fold (Table 3). The addition of arginine to the apical medium did not affect any of the cytokine concentrations measured (Table 3).


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  		TABLE 3 Induction of cytokine production in the basal compartment of the coculture model after addition of 2 x 107 E. coli K12 to the apical compartment and its modulation by apical arginine supplementation1

 
    Correlation between concentrations of AA and cytokine production. The concentrations of Arg, Tau, Ser, Asp, Asn, Orn, and Lys were inversely correlated with concentrations of most cytokines measured in experiments where bacterial stimulation of Caco-2 cells occurred (Supplemental Table 1). Other AA were less affected. In particular, Thr, Cit, Cys, Tyr, and histidine did not correlate significantly with any of the measured cytokine concentrations (not shown).

    Formation of NO. In the basolateral compartment of the coculture model, the concentration of nitrite was higher (P = 0.004) after 72 h when E. coli K12 was present in the apical compartment (8.25 ± 0.87 µmol/L) in comparison to experiments without bacteria (4.72 ± 0.70 µmol/L). Bacteria incubated for 72 h in completed medium (with gentamicin) without other cells did not produce measurable amounts of nitrite. Apical supplementation with arginine (+400 µmol/L: 8.61 ± 1.63 µmol/L; +1200 µmol/L: 8.54 ± 0.98 µmol/L) did not change the nitrite concentration significantly (72 h).

    Integrity of the Caco-2 cell monolayer. In general, the integrity of the Caco-2 cell monolayer (assessed as the permeability of FITC-dextran Mr 4000) was maintained over 72 h (negative control: 0.96 ± 0.50%), even in the presence of bacteria (positive control: 1.61 ± 0.26%). The concentration of FITC-dextran was below detection limit in the basolateral medium after 12 h and 24 h. After 72 h, arginine supplementation did not affect the permeability of the probe in experiments with bacteria in the apical compartment (+400 µmol/L: 0.90 ± 0.18%; +1200 µmol/L: 1.80 ± 0.12%).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
In the model used, Caco-2 cells form a confluent, differentiated, and polarized cell layer with tight junctions, desmosomes, and microvilli (15), thereby separating an apical (luminal) compartment from a basolateral one containing mononuclear human leukocytes (11). This model offers the unique opportunity to meet 2 conditions present in the intestine: the separation of bacteria from leukocytes by a confluent monolayer of IEC, and the enabling of the interaction between enterocytes and leukocytes. The activation of IEC and mononuclear leukocytes was repeatedly shown to underlie a cross-talk (11,12,16). At least at the level of mRNA induction of IL-8 in IEC, no differences were noted in the efficacy of cocultured mononuclear cells from the lamina propria (LPMC) or PBMC (16). However, differences in cytokine profiles may occur when LPMC are applied in the model. Further experiments are needed to better define the effects of arginine on the stimulation of LPMC, especially with respect to the release of cytokines and their importance for immunoregulation in the intestine (e.g., transforming growth factor-ß). The latter cytokine was not measured in the current study because in a previous study (12) we showed that its production occurs too late to affect the early increase of primary proinflammatory cytokines such as TNF-{alpha} or IL-8.

    Catabolism, conversion and transepithelial transport of AA and glucose. In critically ill patients, the gut is a key organ in the development of sepsis-derived multi-organ failure. Arginine supplementation in the critically ill has been shown to decrease morbidity and mortality (2,17). In contrast, the finding of some deleterious effects of arginine supplementation (18) led to the recommendation to avoid arginine supplementation in septic ICU patients (19), a suggestion that was questioned shortly after its publication (20).

In the current model, in which the AA metabolism of leukocytes and enterocytes is not under the influence of other organs such as the liver, either the catabolism of Ser after bacterial challenge was enhanced dramatically or its synthesis (e.g., from alanine) became impaired. Serine was hitherto barely considered as important for immune functions in the intestine (21). It plays an outstanding role in the sufficient availability of transferable methyl groups, being closely involved in the regeneration of methionine from homocysteine via 5-methylenetetrahydrofolate (22). Hence, the catabolism of serine may indicate an enhanced need for transferable methyl groups in the intestine during inflammation. This surprising result should stimulate additional studies that examine the metabolism of serine during an intestinal inflammatory response and makes it worthwhile to consider serine supplementation under sepsis-like circumstances.

As both the growth and the protein synthesis of the bacteria were blocked during incubation with gentamicin (23), the changes in AA composition can be presumed to be due only to an altered metabolism by PBMC and/or Caco-2 cells resulting from the bacterial challenge of these cells. The decreased glutamine catabolism by leukocytes/IEC after bacterial stimulation of IEC, especially, can be explained by a decreased activity of glutaminase, the reduced activity of which was also shown in sepsis (24).

Six of the AA measured were transported to a high degree through the IEC layer (Table 1), an observation that was also made previously for individual AA in Caco-2 cells (25). The transport of Ser, Asp, Tyr, Pro, and Met was more pronounced than that of other AA or occurred only in cases of bacterial stimulation of the IEC (Table 2). The transport of arginine was reported to be enhanced in cytokine-activated Caco-2 cells (25), an effect that might have taken place but that was not evident in the current study as, after 72 h, arginine was transported almost completely into the basolateral compartment in all experiments.

Arginine supplementation of the bacteria-stimulated IEC with PBMC affected the concentrations of the other AA only marginally, especially when a low arginine concentration (0.4 mmol/L) was added (Table 2). The amount of ornithine (Orn) increased significantly and dose-dependently after arginine addition, probably due to altered arginase (EC 3.5.3.1) activity (26). The impaired transport of 7 AA into the basolateral compartment (5 essential AA, Tyr, and Orn), after supplementation with 1.2 mmol/L arginine, may be the result of competitive mechanisms for AA transporters.

    Cytokine and NO production. Apical stimulation of Caco-2 cells by nonpathogenic bacteria (with the consequent activation of leukocytes) leads to an enhanced production of cytokines in the basolateral compartment of the coculture model (11,12) (Table 3). The concentration of AA, which were catabolized to a higher extent after bacterial stimulation (e.g., Ser, Asp, Asn, Orn, and Lys; Fig. 2), correlated inversely and closely to the production of cytokines (Supplemental Table 1), indicating that their catabolism was associated closely with the immune activation of leukocytes and IEC. Surprisingly, the concentrations of other AA were not affected by an enhanced cytokine production.

cNOS and iNOS are found in the gastrointestinal tract (5), but iNOS is expressed in large quantities only if stimulation with cytokines or bacterial toxins occurs (4,27). Differentiated Caco-2 cells also express cNOS and iNOS, the latter of which is expressed after stimulation with cytokines such as IFN-{gamma} and TNF-{alpha} (28,29). The induction of iNOS by the cytokines secreted into the basolateral compartment of the current coculture model may explain the increase in basolateral NO production. However, in this model, the source of NO cannot be identified, because both Caco-2 cells and monocytes are able to form NO, a situation that also occurs in the intestine.

The results of the current study suggest that the addition of arginine to enterocytes and leukocytes increases neither the release of proinflammatory cytokines nor the production of NO. Hence, in enterocyte/leukocyte cocultures, substrate availability for NOS is unlikely to be the regulating factor for NO production. This finding confirms the results of a study in which the enhancement of cytokine production by arginine supplementation was not found in biopsies from the human intestine (30), but stands in contradiction to the results of experiments with rodents (31). Hence, species differences may play a role in arginine-induced modulation of inflammation.

    Integrity of the enterocyte monolayer. Exposure of enterocyte layers to NO increases paracellular permeability in both animal models (32) and Caco-2 cell layers (33). Also, cytokines such as TNF-{alpha} and IFN-{gamma} can increase the permeability of the enterocyte layer (34) by mechanisms not necessarily dependent on NO (35). In the present study, arginine supplementation altered neither the permeability of FITC-dextran through the Caco-2 cell monolayer nor the release of NO, indicating, along with the other findings of the current study, that arginine supplementation does not impair the functionality of intestinal epithelial cells during inflammation.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge S. Scharr and M. Ostertag for their technical assistance as well as A. Bäuerlein for help in the measurement of intestinal integrity.


   FOOTNOTES
 
1 Supported by a fellowship grant from the Leonardo/Erasmus program to I. N. Back

2 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org. Back

6 Abbreviations used: AA, amino acid(s); cNOS, constitutive nitric oxide synthase; FITC, fluorescein isothiocyanate; IEC, intestinal epithelial cells; iNOS, inducible nitric oxide synthase; LPMC, lamina propria mononuclear cells; NO, nitric oxide; PBMC, peripheral blood mononuclear cells. Back

Manuscript received 28 July 2006. Initial review completed 29 August 2006. Revision accepted 8 November 2006.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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