Advanced Search

Journal Navigation

Journal Home

Subscriptions

Archive

Contact Us

Table of Contents

Click here to sign up for SAGE Journal Email Alerts today!
Journal of Parenteral and Enteral Nutrition
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Saved Citations
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Request Reprints
Right arrow Add to My Marked Citations
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Right arrow Citing Articles via Scopus
Google Scholar
Right arrow Articles by Bansal, V.
Right arrow Articles by Ochoa, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bansal, V.
Right arrow Articles by Ochoa, J. B.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*(L)-ARGININE
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Original Communications

Citrulline Can Preserve Proliferation and Prevent the Loss of CD3 {zeta} Chain Under Conditions of Low Arginine

Vishal Bansal, MD§, Paulo Rodriguez, PhD*, Guoyao Wu, PhD{ddagger}, Duane C. Eichler, PhD{dagger}, Jovanny Zabaleta, PhD*, Faramarz Taheri, PhD* and Juan B. Ochoa, MD§

From the * Louisiana State University Health Sciences Center, New Orleans, Louisiana;{dagger} University of South Florida, Tampa, Florida;{ddagger} Texas A&M University, College Station, Texas; and § University of Pittsburgh Department of Surgery, Pittsburgh, Pennsylvania

Correspondence: Juan B. Ochoa, MD, FACS, Associate Professor of Surgery and Critical Care, UPMC Presbyterian Hospital, 200 Lothrop Street, Room F1267, Pittsburgh, PA 15213. Electronic mail may be sent to ochoajb{at}msx.upmc.edu.

Background: Arginine depletion by the enzyme Arginase I, decreases expression of the TCR {zeta} chain preventing T-cell activation and causing T-cell dysfunction. We hypothesized that citrulline could substitute for arginine under conditions of increased arginase expression. Thus, the goal was to establish a possible mechanism of how citrulline could overcome arginine depletion caused by arginase. Methods: Jurkat cells were cultured, with or without arginase, in media containing different amino-acid constituents: complete RPMI containing arginine (C-RPMI) (arginine), Arginine-Free-RPMI (Arg-Free RPMI) and Citrulline-containing RPMI (Cit RPMI). Incorporation of citrulline was measured via uptake of 3H-citrulline, whereas proliferation was measured via 3H-thymidine incorporation. {zeta} Chain was analyzed by 2-color flow cytometry. Argininosuccinate synthase (AS) and argininosuccinate lyase expression was detected using Northern blots, RT-PCR, and Western blots. Results: Jurkat cells exhibited a significant decrease in proliferation and {zeta} chain expression when cultured in the presence of arginase or in the absence of arginine. With citrulline, {zeta} chain expression and proliferation were maintained in the absence of arginine or in the presence of the enzyme arginase. Jurkat cells, cultured in the absence of arginine, were associated with a 5-fold increase in citrulline uptake. The absence of arginine was also associated with increased expression of AS. Conclusions: T cells exhibit the molecular capability of increasing citrulline membrane transport and up-regulating AS expression, thus exhibiting the necessary mechanisms for converting citrulline into arginine and escaping the ill effects of arginine depletion. Therefore, citrulline has the potential to be a substitute for supplemental arginine in diseases associated with arginase-mediated T cell dysfunction.

Arginine is an amino acid critical for immune function, including T lymphocyte proliferation and the production of nitric oxide.1,2 Arginine deficiency develops in adults subjected to stress, such as trauma and surgery, making it a conditionally essential amino acid.3,4 There are several mechanisms contributing to the development of arginine deficiency in vivo, including a decreased dietary intake, inefficient endogenous arginine generation, and an increase in use.5 Central to the increase in arginine use appears to be the induction of Arginase I expression in cells of the myeloid lineage (macrophages, monocytes), an enzyme that converts arginine to ornithine and urea.6 Increased Arginase I expression has been reported under several circumstances but is most prominent after surgery (both in humans and animal models), trauma, cancer, and in chronic infections. Arginase I, induced by T helper 2 cytokines, cyclic AMP analogs, and prostaglandin E2, is thought to be an integral part of the anti-inflammatory response after immune activation.7–12 By depleting arginine, arginase modulates the production of nitric oxide and may control T lymphocyte function.

Arginine is essential for normal T cell function. We have recently shown that arginine modulates proliferation partly through a rapid decrease in the expression of the T cell receptor {zeta} chain (CD3{zeta}), which results in the disruption of T cell receptor integrity.13,14 Decreased {zeta} chain expression, along with the lack of T cell proliferation, is observed in vivo under conditions of increased Arginase I expression and is characteristic in patients with cancer, chronic infections, and after surgery.15–17

Dietary arginine supplementation, using supra-physiologic doses 10 times the normal daily intake, is used empirically to "enhance" the immune system. With these diets, a significant decrease in infection rates is observed in high-risk surgical populations.18,19 However, successful supplementation of these high arginine concentrations may be difficult and may also be associated with troublesome side effects, including diarrhea and abdominal distention. Thus, there is a need for newer dietary strategies that exhibit equal efficacy and have the potential for fewer side effects. Therefore, the main objective of this paper is to demonstrate the feasibility of overcoming T cell dysfunction caused by arginine deficiency using the amino acid citrulline. Arginine can maintain {zeta} chain expression in T cell lines and proliferation in mouse T lymphocytes.14,20 Citrulline can be converted to arginine through an enzymatic pathway involving 2 enzymes: argininosuccinate synthase (AS) and argininosuccinate lyase (AL).21 We hypothesized that a decrease in arginine, caused by arginase, would lead to increased expression of AS, hence an increase in citrulline incorporation, and ultimately the endogenous generation of arginine from citrulline. To test this hypothesis, Jurkat cells, a CD4+ T cell line, were cultured in the presence or absence of arginine or citrulline. Cellular proliferation and the expression of CD3{zeta} chain were measured at various time points in cell cultures either without arginine or with bovine Arginase I. As expected, T lymphocyte proliferation and CD3{zeta} chain expression were significantly decreased in the absence of arginine or by the addition of Arginase I to the culture media. The absence of arginine resulted in an increased expression of AS mRNA within 24 hours. The addition of citrulline, in the absence of arginine, restored Jurkat cell proliferation and maintained {zeta} chain expression. Thus, citrulline could be used as a substitute for an amino acid supplement regimen in arginine deficient states caused by arginase.


   MATERIALS AND METHODS
Top
MATERIALS AND METHODS
RESULTS
 DISCUSSION
 
Tissue Culture Media
Regular complete RPMI-1640 (C-RPMI) contains 1140 µM L-arginine. RPMI-1640 without L-arginine (Arg-free-RPMI) was obtained from GIBCO BRL/Life Technologies (Grand Island, NY). Tissue culture medium with citrulline or ornithine was made by adding citrulline or ornithine (Sigma, Steinbeim, Germany) to Arg-free-RPMI. The Cit-RPMI and Orn-RPMI contain 1000 µmol/L citrulline or ornithine, respectively, and no arginine. This concentration was chosen to be equivalent to the arginine concentration in C-RPMI. All tissue culture media was supplemented with 10% fetal calf serum (FCS; Hyclone, Roadlogan, UT), 25 mmol/L HEPES (GIBCO BRL/Life Technologies), 4 mmol/L L-glutamine (Bio-Whittaker, Walkers-ville, MD) and 100 U and 100 µg/mL of penicillin/streptomycin (GIBCO BRL/Life Technologies).

Cell Cultures
Jurkat cells (clone E6–1) were obtained from ATCC (Manassas, VA) and maintained in RPMI-1640 containing 10% FCS. Cells were cultured 3 times a week at 0.5 x 106 cells/mL. Cells used for the experiments were always taken 48 hours after passage. Cell cultures were started at 0.5 x 106 cells/mL and samples were obtained at different time points. Jurkat cells were cultured in C-RPMI, Cit-RPMI, Orn-RPMI, Arg-free-RPMI, or Arg-free-RPMI containing different concentrations of L-citrulline (0 to 1000 µmol/L) for various periods of time. Cells were harvested at different time points (according to the experiment design) and tested for proliferation and expression of the TCR proteins by flow cytometry.

Antibodies and Reagents
Conjugated antibodies for flow cytometry including PE-labeled anti-CD3{zeta}, FITC-labeled anti-CD3{epsilon}, and the isotype control antibodies were purchased from Beckman-Coulter (Miami, FL).

Proliferation Assay
3H-thymidine incorporation was performed in 96-well plates by incubating 1 x 106 cells per well in different tissue culture media plus 0.5 µCi of 3H-thy-midine per well. Each condition was tested in triplicate. After 24 hours of incubation at 37°C, cells were then harvested onto a Unifilter-96 GF/B (Packard, Meriden, CT) and washed 3 times with distilled water. 3H-Thymidine incorporation was measured using a TOPCOUNT microplate Scintillation Counter (Packard), and average counts of triplicate wells were calculated for each condition.

Flow Cytometry
Jurkat cells were stained with 1 µg FITC-labeled anti-CD3 or an isotype control antibody per 106 cells in 200 µL PBS for 15 minutes at room temperature and washed with PBS. Cells were then resuspended in 200 µL of digitonin solution (500 µg/mL in PBS) to which 2.5 µg of PE-labeled anti-CD3{zeta} or an isotype control antibody was added. Cells were incubated for 8 minutes at room temperature, washed once with and resuspended in PBS, and analyzed immediately.

Northern Blot
Jurkat cells (1.0 x 106) were used for RNA extraction. Total RNA was extracted by lysis with TRIzol (Invitrogen, Carlsbad, CA) and purified according to the manufacturer's specifications. Northern blot analysis was performed using 10 µg of total RNA from each sample. The RNA was electrophoresed under denaturing conditions, blotted to nytran membranes (Schleicher & Schuell Inc, Keene, NH) and cross-linked by UV irradiation. Membranes were prehybridized at 42°C in ULTRAhyb buffer (Ambion, Austin, TX) and hybridized overnight with 1 x 106 cpm/mL of 32P-labeled probe. Human AS and AL probes were labeled by random priming using a RediPrime Kit (Amersham, Arlington Heights, IL) and [32P]dCTP (3000 Ci/mmol; NEN Life Science Products, Boston, MA). Membranes were washed 3 times, once at 65°C for 30 minutes, using a buffer containing 2 x SSC and 0.1% sodium-dodecyl sulfate (SDS) and twice at 65°C for 30 minutes in 0.1 x SSC and 0.1% SDS. Membranes were subjected to autoradiography at –70°C using Kodak Biomax-MR (Eastman Kodak Company, Rochester, NY) films and intensifying screens. A densitometer, alphaImager 2000 (Alpha Innotech Corporation, San Leandro, CA) was used to analyze the band intensities.

RT-PCR Analysis of mRNA Expression
RNA expression for AS and AL was measured by sequential reverse transcription and cDNA amplification (RT-PCR). β-Actin (a house keeping gene) was used as an internal control. Total RNA was isolated from 10 x 106 Jurkat cells cultured in different conditions using Trizol reagent according to the manufacturer's directions. RNA samples were quantitated spectrophotometrically at 260/280 nm and stored at –70°C until used. Residual genomic DNA was eliminated from RNA samples using DNase I protocol (Life Technologies, Grand Island, NY). Then cDNA was synthesized by using SuperScriptII reagent and Random primers (Life Technologies) as instructed by the manufacturer. Gene-specific oligonucleotide primers based on sequences published in GenBank human DNA database included β-actin synthesized by IDT (Coralville, IA) and AS and AL synthesized by Life Technologies. Reverse transcription for cDNA synthesis was carried out in 20-µL volumes; 2 µL of the product was used for AS or β-actin and 4 µL for AL amplification. Amplification was performed under optimal conditions in a PTC-200 Peltier Thermal Cycler (MJ Research, Water-town, MA). Either 5 or 10 µL of PCR products were electrophoresed on 1% agarose gel and visualized with ethidium bromide staining. For semiquantitative analysis, the DNA PCR products were labeled using 0.2 µCi of Easytide [32P]dCTP 3000 Ci/mmol (NEN Life Science Products) per reaction. The reactions were carried out in 50-µL volumes, and either 5 or 10 µL of the PCR products were electrophoresed on 5% polyacrylamide gel prepared by mixing 2% bis-acrylamide, acrylamide, 10xTBE, double-distilled water, 10% ammonium persulfate, and TEMED. Densitometry analysis was performed using the AlphaImager 2000 to 3.3 software.

Western Blot
Cell lysate preparation and immunoblotting. Lysates from Jurkat cells (10 x 106) cultured in different conditions were prepared by placing cells for 7 minutes on ice in a Super Lysis Buffer complete with trypsin, apoprotein, leupopeptin, and PMSF (Gibco [BRL/Life Technologies, Grand Island, NY]). Cells were subsequently centrifuged for 15 minutes at 12,000 RPM, and supernatants were extracted as the cells' respective protein. Protein concentrations were determined by BCA Assay (Gibco). Equal amounts (10 µg) of protein were resolved on 4% to 15% polyacrylamide gels (Bio-Rad) and blotted onto Immobilon-P PVDF membranes (Invitrogen, Carlsbad, CA). Western blotting was performed as previously described.22 Briefly, membranes were blocked for 1 hour in 5% non-fat dry milk (NFDM) in TBS-T and subsequently washed. Membranes were incubated with primary antibody (1:2500 anti-AS; BD Transduction Labs), 1:7500 anti-Actin (Sigma) in 5% NFDM for 1 hour. After washing, membranes were incubated with secondary antibody in 5% NFDM for 1 hour. The signal was visualized by chemiluminescence using ECL reagent (Amersham Biosciences) and exposed to film. Band intensities were quantitated using ImageQuant software (Molecular Dynamics).

HPLC Analysis
HPLC-ECD was performed as previously reported using an ESA-Coul Array Model 540 (ESA Inc, Chelmsford, MA) with an 80 x 3.2 column with 120A pore size.23 Briefly, supernatants were deproteinized by methanol. After centrifugation at 6000 x g for 10 minutes at 4°C, the supernatant was derivatized with 0.2-M OPA/BME (o-phtaldialdehyde containing b-mer-captoethanol). Fifty microliters of the sample was injected to the column. The retention time for L-arginine was 10.2 minutes. Standards of L-arginine were prepared in methanol.

Statistics were done using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA). Data were analyzed using analysis of variance and a 2-tailed t test when appropriate. Significance was established at p < .05. All experiments were repeated at least 3 times.


   RESULTS
Top
MATERIALS AND METHODS
 RESULTS
DISCUSSION
 
Arginase Causes a Decrease in T Cell Proliferation and a Loss of CD3 {zeta} Chain Comparable to Withholding Arginine From the Culture Media
Arginase metabolizes arginine to ornithine and urea-depleting arginine from the culture media. The effects of arginase on T cell proliferation should be comparable to culturing cells in the absence of arginine. To test this hypothesis, Jurkat cells (1 x 106 cells/mL) were cultured for 24 hours in the presence of conventional complete RPMI (C-RPMI), containing 1.1 mmol/L arginine, or arginine free media (Arg-free-RPMI). Cellular proliferation was measured using 3H-thymidine incorporation. As expected, cellular proliferation decreased in cells grown in Arg-free-RPMI (38,808 ± 3454 CPM) when compared with cells grown in C-RPMI (116,221 ± 6555 CPM) (p < .0001). A similar decrease in cell proliferation was observed with addition of 10 U/mL of bovine arginase to culture media (116,221 ± 6555 CPM to 26,255 ± 6555 CPM) (p < .0001) (Fig. 1).


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 1. Jurkat cells grown in citrulline rich media maintain proliferation even with the addition of arginase. Jurkat proliferation was measured as counts per minute (CPM) of 3H thymidine uptake in C-RPMI (116,221 ± 6555 and 26,255 ± 6555 CPM), Arg-free-RPMI (38,808 ± 3454 and 14,476 ± 2061) and Cit-RPMI (91,862 ± 8131 and 90,554 ± 4886 CPM) with and without the addition of Arginase I, respectively. Arginase decreased proliferation in C-RPMI (p < .0001), whereas cells cultured in Cit-RPMI maintained normal proliferation (p = NS).

 

We have previously demonstrated that the absence of arginine in culture media decreases {zeta} chain expression, an integral and essential component of the T cell receptor. Alterations in CD3{zeta} chain expression have also been reported in vivo under circumstances of increased arginase expression and arginine depletion. Thus, the evaluation {zeta} chain expression serves as a sensitive biomarker of arginine deficiency. Jurkat cells were grown in the presence of arginine (C-RPMI) or in Arg-free-RPMI with or without arginase (10 U/mL). Jurkat cells were harvested after 24 hours and {zeta} chain expression was measured by flow cytometry. The absence of arginine was associated with a significant decrease in {zeta} chain expression as measured by mean gated fluorescence (MGF) from 9.22 ± 1.1 MGF to 4.4 ± 0.5 MGF. Arginase decreased {zeta} chain expression to levels comparable to that of cells grown in the absence of arginine (4.2 ± 0.4 MGF) (p < .0006) (Fig. 2A).


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 2. A, Flow cytometry showing Jurkat TCR {zeta} chain expression as mean gated fluorescence (MGF). Cells in grown in C-RPMI had a significant decrease in {zeta} chain expression as measured from 9.22 ± 1.1 MGF to 4.4 ± 0.5 MGF when compared with cells grown in Arg-free RPMI. The addition of arginase to C-RPMI showed a drop in {zeta} chain expression similar to that of {zeta} chain expression in Arg-free RPMI (4.2 ± 0.4 MGF) (p < .0006). {zeta} Chain expression in Cit-RPMI was 8.73 ± 0.7 MGF. The addition of arginase had a minimal effect on {zeta} chain expression (8.6 ± 0.5 MGF) when compared with Cit-RPMI alone (p = NS). Thus, citrulline effectively conserved the biomarkers of arginine deficiency in T cells. B, Histogram of TCR {zeta} chain expression. TCR {zeta} chain expression does not change in citrulline containing media even with the presence of arginase.

 

Citrulline Maintains Jurkat Cell Proliferation in the Presence of Arginase
To determine whether citrulline could substitute for arginine we compared cell proliferation and {zeta} chain expression results in cells grown in the absence of arginine but in the presence of 1 mmol/L citrulline (Cit-RPMI). A titration curve using citrulline demonstrated that significant increases in proliferation were obtained with citrulline levels as low as 200 µmol/L (58,980 ± 1907 CPM), but the same effect was observed with 1 mmol/L citrulline (60,780 ± 4407 CPM) comparable to C-RPMI (59,330 ± 1885 CPM) (p = NS). Because C-RPMI contains 1.1 mmol/L of arginine, we used similar concentrations of citrulline (1 mmol/L) for the rest of the experiments.

To determine whether citrulline could overcome the effects of arginase, Jurkat cells were grown in Cit-RPMI in the presence or absence of arginase and compared with the above Jurkat cultures in C-RPMI and Arg-free-RPMI with or without arginase. Cellular proliferation increased to 91,862 ± 8131 CPM in cells grown in Cit-RPMI when compared with cells grown in Arg-free-RPMI (38,808 ± 3454 CPM) or arginase containing C-RPMI (26,255 ± 6555 CPM) (p < .0001) (Fig. 1). Cellular proliferation was maintained (90,554 ± 4886 CPM) despite the addition of 10U/mL of bovine arginase and was not significantly different in comparison to Jurkats cultures in Cit-RPMI alone (p = NS).

{zeta} Chain expression in cells grown in Cit-RPMI was also increased to 8.6 ± 0.5 MGF when compared with Arg-free-RPMI (4.4 ± 0.5 MGF) or arginase containing C-RPMI (4.2 ± 0.4 MGF) (p < .001). When 10 U/mL of bovine arginase was added, there was a minimal decrease in {zeta} chain expression (8.73 ± 0.7 MGF) when compared with Cit-RPMI alone. Thus, citrulline effectively restored normal T cell function and normalized biomarkers of arginine deficiency suggesting that this amino acid overcame arginine deficiency caused by arginase (Fig. 2).

Arginine Depletion and Not the Products of Arginase Metabolism Cause a Decrease in Jurkat Cell Proliferation and {zeta} Chain Expression
Arginase generates ornithine, which in turn may be converted to polyamines when ornithine decarboxylase is present. Some reports suggest that ornithine may substitute for arginine under certain circumstances, whereas others suggest that ornithine, by competing with arginine for the same cationic amino acid transporters, can actually inhibit the effects of arginine. Polyamines, in turn, are important molecules for normal cellular proliferation and thus open the possibility that arginase, under certain circumstances, may play a dual role. Thus, the goal of these experiments was to determine the potential effects of the products of arginase metabolism of arginine on Jurkat cells. To determine whether ornithine or polyamines could alter the effects of arginine, Jurkat cells (1 x 106cells/mL) were cultured in C-RPMI in the presence or absence of ornithine (Orn-RPMI) or the polyamine putrescine (Put-RPMI). Cellular proliferation was measured as above. 3H thymidine incorporation was 122,160 ± 4561 CPM in Put-RPMI media and 124,160 ± 4020 CPM in Orn-RPMI media compared with 118,783 ± 3301 CPM in C-RPMI (p = NS). Thus, we conclude that products of arginase seem neither to promote nor inhibit T cell function under the circumstances tested. The effects of arginase can only be explained by the depletion of arginine. We proceeded to measure the concentration of arginine in the culture media of these cells. C-RPMI has an arginine concentration of 1140 µmol/L. After 24 hours, the arginine concentration drops to 920 ± 82 µmol/L. Arginine levels in cells cultured in Arg-free-RPMI were measured to be 0 µmol/L after 24 hours, as were cells grown in C-RPMI with 10 U/mL of bovine arginase. Therefore, arginase successfully depletes the extracellular concentration of arginine.

Citrulline Uptake Is Significantly Increased in the Absence of Arginine
Citrulline transport across the cell membrane and incorporation into the cell is necessary if it is to be a substrate for AS. To determine this, we measured incorporation of 3H citrulline in Jurkat cells grown for 24 hours in the presence or absence of arginine (C-RPMI contains 1.1 mmol/L arginine). A significant increase in 3H citrulline incorporation (1391 ± 60.9 to 5685 ± 273.0 CPM) (p < .0001) was observed when cells were grown in C-RPMI compared with Arg-free RPMI, respectively (Fig. 3). Thus, there is increased transport of citrulline when arginine is depleted from the culture media.


Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		FIG. 3. Citrulline uptake is increased in the absence of arginine. Jurkat cells were grown in either C-RPMI or Arg-free-RPMI in the presence of radioactive 3H citrulline. A significant increase in 3H citrulline incorporation, 1391 ± 60.9 to 5685 ± 273.0 CPM (p < .0001), was noted in Jurkat cells cultured in Arg-free-RPMI (p < .0001).

 

Arginine Deprivation in Jurkat Cells Stimulates the Expression of AS mRNAs and Protein Expression
Citrulline is converted to arginine through 2 enzymes: AS, which converts citrulline and aspartate to citrullyl-AMP, and AL, which produces arginine (Fig. 4). These 2 enzymes are poorly expressed in lymphocytes. Thus, for citrulline to be converted to arginine, induction and expression of these enzymes should occur. To investigate the effect of arginine deprivation and substitution with citrulline and the expression of the AS and AL genes, Jurkat cells were cultured in C-RPMI, Arg-free-RPMI, or Cit-RPMI. Total RNA was extracted after 24 hours in culture. AS and AL mRNA and protein expression were measured by Northern blot, RT-PCR, and Western blot. An increase in AS mRNA was observed in Jurkat cells cultured in the absence of arginine, followed by an increase in protein expression (Figs. 5 and 6). RT-PCR densitometric evaluation of mRNA abundance confirmed the increase in AS (data not shown). The increase in AS was not caused by the presence of citrulline because the addition of this amino acid at 1000 µmol/L to C-RPMI did not result in any changes in the level of AS mRNA expression. Changes in AL mRNA could not be consistently detected under the different conditions reported, suggesting that regulation of the expression of this enzyme might not be under nutrient control. These results suggest that in the absence of arginine, Jurkat cells increase the level of AS gene expression as a substitute pathway to provide arginine. In the presence of citrulline, the high level of mRNA expression will last for several months (we tested them after 4 months) as long as cells are cultured in the absence of arginine (data not shown).


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		FIG. 4. The metabolism of arginine. A, Arginine is metabolized to ornithine and urea by the enzyme Arginase I decreasing the concentration of extracellular arginine. B, In an arginine-deficient environment, citrulline is transported across the cell membrane, into the T cell, by a specific membrane transport protein. C, Now intracellular, citrulline is metabolized by argininosuccinate synthase (AS) to citrullyl-AMP, which is further metabolized by argininosuccinate lyase (AL) into D, arginine.

 

Figure 5
View larger version (30K):
[in this window]
[in a new window]

  		FIG. 5. Representative Northern blot demonstrates increased mRNA AS expression when Jurkat cells are grown in the absence of arginine (lanes 2, 4) or in the presence of arginase (lanes 5–7) or both (lane 8). Lane 1 represents C-RPMI and lane 3 represents C-RPMI with 1 mmol/L citrulline. No such changes can be reliably detected for AL. AS = argininosuccinate synthase; AL = argininosuccinate lyase.

 

Figure 6
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 6. This representative Western blot demonstrates the β-actin and the increase in expression of AS mRNA expression in Jurkat cells grown in the absence of arginine (lanes 2, 4), in the presence of arginase (lanes 5–7), or under both conditions (lane 8). The corresponding numbers are the ratio of AS/β-actin band densities. Lane 1 represents C-RPMI and lane 3 represents C-RPMI with 1 mmol/L citrulline.

 


   DISCUSSION
Top
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 
Arginine is necessary for many aspects of immune function, including T lymphocyte proliferation and the normal expression of the T cell receptor including the {zeta} chain. 5,14,20 Arginine is also necessary for the normal expression of key receptors such as CD3 and CD8, the development of memory, and expression of key elements of the signal transduction pathway of T cell activation such as NF-{kappa}B.20 In some disease states, endogenous arginine generation is insufficient to meet increased demands, and thus, arginine becomes "conditionally essential." The fact that arginine deficiency develops in disease states underscores the possibility that cells that are dependent on arginine, such as T lymphocytes, can be adversely affected by arginine deprivation.

Several investigators, including ourselves, have demonstrated that increased arginase I expression is an important cause of T cell dysfunction. In cancer, for example, increased Arginase I expression in myeloid cells appears to be the cause for decreased immune clearance of tumor cells.24–26 Similar events seem to happen in chronic infections such as Leishmaniasis.27 Several years ago, we described a large increase in Arginase I expression in peripheral mononuclear cells trauma patients and in patients undergoing surgical interventions.11,12

Arginase I expressed in myeloid cells depletes arginine from the pericellular media. In addition, in certain disease processes dietary intake and arginine generation by the kidney are decreased. Thus, for example, in trauma, there is a rapid and prolonged decrease in circulating arginine levels, which do not recover despite normal dietary arginine intake.

We suggest that arginine deficiency in trauma can be a cause of T cell dysfunction. In fact, abnormal T cell function after surgery or trauma mimics the molecular changes observed with arginine deficiency such as decreased T cell proliferation, {zeta} chain expression, and production of {gamma}-IFN.28–30 Thus, there is significant evidence linking arginine deficiency caused by Arginase I as a mechanism of immune suppression.

What is the treatment of immune dysfunction caused by Arginase I? Two decades ago, investigators reported that dietary arginine supplementation with supra-physiologic concentrations (10 times the normal dietary intake) "enhanced" the immune system after surgery or trauma.3 Though the mechanisms behind these beneficial effects were unknown, arginine supplementation was incorporated to other nutrients, such as {omega}-3 fatty acids, into so-called immune-enhancing diets, which have been tested extensively in surgical and critically ill populations.18,31 To date, the benefits of immune-enhancing diets in elective surgical populations are undisputed and should be ordered early (hopefully in the preoperative period) in patients undergoing major elective surgery such as a colon resection, pancreaticoduodenectomy, or esophageal surgery.19 Thus, we hypothesize that immune-enhancing diets overcome arginine deficiency in states of increased Arginase I expression. Achieving the high concentrations of arginine needed to see the beneficial effects using these current diets, however, is difficult and is a major cause in the lack of efficacy in clinical trials involving trauma patients.32 different In addition, side effects such as diarrhea and abdominal bloating are common with such large doses of arginine supplementation, prompting a less-than-ideal use of these diets.19 Therefore, new dietary strategies are needed. The fact that citrulline can promote T cell proliferation in arginine deficient media suggests that it is useful to explore this amino acid as a potential mechanism of overcoming the effects of Arginase I.

Mammalian cells, like unicellular organisms, have the capacity to detect alterations in the availability of specific nutrients and alter the expression of key genes necessary to adapt under new nutritional conditions. Up-regulation of AS expression has been described in several cell lines, including lymphoblasts grown in the presence of canavanine, a closely related toxic arginine metabolite.33–36 According to these observations, we tested whether Jurkat cells, a CD4+ T cell line, would up-regulate citrulline uptake and AS expression when cultured in the absence of arginine. The results reported in this article not only demonstrate that T cells are capable of regenerating arginine from citrulline but also that citrulline is a potentially useful substitute for arginine that can overcome Arginase I–mediated immune suppression. Thus, this work sets the stage for further investigations in vivo to determine the usefulness of citrulline supplementation under conditions of arginine deprivation.

In conclusion, our work provides a basic, molecular cornerstone of citrulline metabolism in an arginine-deficient environment. This serves as an important building block to eventually developing in vivo dietary trials using citrulline to overcome arginine deprivation caused by increased arginase. Citrulline should become a good candidate for the development of a second generation of immune-enhancing diets, hopefully being easier to deliver and causing fewer overall side effects.

The authors want to thank Dr Sidney Morris for his kind support and encouragement, and also the technical assistance of Brenda Flam who carried out the Western blot analysis.


  
Top
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 
The work presented here is partially funded by grant NIH KO8 GM 0646–02 (J.B.O.), and NCI-R21CA83198, NCI-R21CA83198, NCI-CA82689, and NCI-CA88885 (A.C.O.).

Received for publication March 17, 2004. Accepted for publication August 6, 2004.

  1. Hibbs JB Jr. Synthesis of nitric oxide from L-arginine: a recently discovered pathway induced by cytokines with antitumour and antimicrobial activity. Res Immunol.1991; 142:565 –569.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  2. Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry.1988; 27:8706 –8711.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  3. Barbul A, Lazarou SA, Efron DT, Wasserkrug HL, Efron G. Arginine enhances wound healing and lymphocyte immune responses in humans.Surgery. 1990;108:331 –337.[Web of Science][Medline] [Order article via Infotrieve]
  4. Barbul A. Arginine and immune function. Nutrition.1990; 6:53 –62.[Web of Science][Medline] [Order article via Infotrieve]
  5. Bansal V, Ochoa JB. Arginine availability, arginase, and the immune response. Curr Opin Clin Nutr Metab Care.2003; 6:223 –228.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  6. Morris SM Jr, Kepka-Lenhart D, Chen LC. Differential regulation of arginases and inducible nitric oxide synthase in murine macrophage cells.Am J Physiol. 1998;275:E740 –E747.[Web of Science][Medline] [Order article via Infotrieve]
  7. Bernard AC, Mistry SK, Morris Jr SM, et al. Alterations in arginine metabolic enzymes in trauma. Shock.2001; 15:215 –219.[Web of Science][Medline] [Order article via Infotrieve]
  8. Chang CI, Zoghi B, Liao JC, Kuo L. The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production. J Immunol.2000; 165:2134 –2141.[Abstract/Free Full Text]
  9. Hesse M, Modolell M, La Flamme AC, et al. Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism.J Immunol. 2001;167:6533 –6544.[Abstract/Free Full Text]
  10. Liu Y, Van Ginderachter JA, Brys L, De Baetselier P, Raes G, Geldhof AB. Nitric oxide-independent CTL suppression during tumor progression: association with arginase-producing (M2) myeloid cells. J Immunol. 2003;170:5064 –5074.[Abstract/Free Full Text]
  11. Ochoa JB, Bernard AC, O'Brien WE, et al. Arginase I expression and activity in human mononuclear cells after injury. Ann Surg.2001; 233:393 –399.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  12. Tsuei BJ, Bernard AC, Shane MD, et al. Surgery induces human mononuclear cell arginase I expression. J Trauma.2001; 51:497 –502.[Web of Science][Medline] [Order article via Infotrieve]
  13. Rodriguez PC, Zea AH, DeSalvo J, et al. L-Arginine consumption by macrophages modulates the expression of CD3 zeta chain in T lymphocytes.J Immunol. 2003;171:1232 –1239.[Abstract/Free Full Text]
  14. Taheri F, Ochoa JB, Faghiri Z, et al. L-Arginine regulates the expression of the T-cell receptor zeta chain (CD3zeta) in Jurkat cells.Clin Cancer Res. 2001;7:958s –965s.[Web of Science][Medline] [Order article via Infotrieve]
  15. Ichihara F, Kono K, Sekikawa T, Matsumoto Y. Surgical stress induces decreased expression of signal-transducing zeta molecules in T cells.Eur Surg Res. 1999;31:138 –146.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  16. Mizoguchi H, O'Shea JJ, Longo DL, Loeffler CM, McVicar DW, Ochoa AC. Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science.1992; 258:1795 –1798.[Abstract/Free Full Text]
  17. Zea AH, Ochoa MT, Ghosh P, et al. Changes in expression of signal transduction proteins in T lymphocytes of patients with leprosy. Infect Immun. 1998;66:499 –504.[Abstract/Free Full Text]
  18. Beale RJ, Bryg DJ, Bihari DJ. Immunonutrition in the critically ill: a systematic review of clinical outcome. Crit Care Med.1999; 27:2799 –2805.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  19. Braga M, Gianotti L, Radaelli G, et al. Perioperative immunonutrition in patients undergoing cancer surgery: results of a randomized double-blind phase 3 trial. Arch Surg.1999; 134:428 –433.[Abstract/Free Full Text]
  20. Ochoa JB, Strange J, Kearney P, Gellin G, Endean E, Fitzpatrick E. Effects of L-arginine on the proliferation of T lymphocyte subpopulations.JPEN J Parenter Enteral Nutr.2001; 25:23 –29.[Abstract/Free Full Text]
  21. Wu G, Morris SMJ. Arginine metabolism: nitric oxide and beyond.Biochem J. 1998;336:1 –17.[Web of Science][Medline] [Order article via Infotrieve]
  22. Flam BR, Hartmann PJ, Harrell-Booth M, Solomonson LP, Eichler DC. Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase. Nitric Oxide. 2001;5:187 –197.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  23. Tcherkas YV, Kartsova LA, Krasnova IN. Analysis of amino acids in human serum by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection. J Chromatogr A.2001; 913:303 –308.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  24. Bronte V, Serafini P, Apolloni E, Zanovello P. Tumor-induced immune dysfunctions caused by myeloid suppressor cells. J Immunother.2001; 24:431 –446.[Medline] [Order article via Infotrieve]
  25. Bronte V, Serafini P, Mazzoni A, Segal DM, Zanovello P. L-Arginine metabolism in myeloid cells controls T-lymphocyte functions. Trends Immunol. 2003;24:302 –306.[Web of Science][Medline] [Order article via Infotrieve]
  26. Hallemeesch MM, Soeters PB, Deutz NE. Tracer methodology in whole body and organ balance metabolic studies: plasma sampling is required: a study in post-absorptive rats using isotopically labeled arginine, phenylalanine, valine and leucine. Clin Nutr.2000; 19:157 –163.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  27. Iniesta V, Gomez-Nieto LC, Corraliza I. The inhibition of arginase by N(omega)-hydroxy-l-arginine controls the growth of Leishmania inside macrophages. J Exp Med.2001; 193:777 –784.[Abstract/Free Full Text]
  28. Miller-Graziano CL, De AK, Kodys K. Altered IL-10 levels in trauma patients' M phi and T lymphocytes. J Clin Immunol.1995; 15:93 –104.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  29. Miller-Graziano CL, Szabo G, Griffey K, Mehta B, Kodys K, Catalano D. Role of elevated monocyte transforming growth factor beta (TGF beta) production in posttrauma immunosuppression. J Clin Immunol.1991; 11:95 –102.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  30. Miller-Graziano CL, Fink M, Wu JY, Szabo G, Kodys K. Mechanisms of altered monocyte prostaglandin E2 production in severely injured patients.Arch Surg. 1988;123:293 –299.[Abstract/Free Full Text]
  31. Heyland DK, Novak F, Drover JW, Jain M, Su X, Suchner U. Should immunonutrition become routine in critically ill patients? a systematic review of the evidence. JAMA.2001; 286:944 –953.[Abstract/Free Full Text]
  32. McCowen KC, Bistrian BR. Immunonutrition: problematic or problem solving? Am J Clin Nutr.2003; 77:764 –770.[Abstract/Free Full Text]
  33. Jacoby LB. Canavanine-resistant variants of human lymphoblasts.Somat Cell Genet. 1978;4:221 –231.[CrossRef][Medline] [Order article via Infotrieve]
  34. Irr JD, Jacoby LB. Control of argininosuccinate synthetase by arginine in human lymphoblasts. Somat Cell Genet.1978; 4:111 –124.[CrossRef][Medline] [Order article via Infotrieve]
  35. Amos JA, Fleming BC, Gusella JF, Jacoby LB. Relative argininosuccinate synthetase mRNA levels and gene copy number in canavanine-resistant lymphoblasts. Biochim Biophys Acta.1984; 782:247 –253.[Medline] [Order article via Infotrieve]
  36. Boyce FM III, Freytag SO. Regulation of human argininosuccinate synthetase gene: induction by positive-acting nuclear mechanism in canavanine-resistant cell variants. Somat Cell Mol Genet.1989; 15:113 –121.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

Journal of Parenteral and Enteral Nutrition, Vol. 28, No. 6, 423-430 (2004)
DOI: 10.1177/0148607104028006423
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Nutr.Home page
P. J. Popovic, H. J. Zeh III, and J. B. Ochoa
Arginine and Immunity
J. Nutr., June 1, 2007; 137(6): 1681S - 1686S.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
P. C. Rodriguez, D. G. Quiceno, and A. C. Ochoa
L-arginine availability regulates T-lymphocyte cell-cycle progression
Blood, February 15, 2007; 109(4): 1568 - 1573.
[Abstract] [Full Text] [PDF]

Home page
 JPEN J Parenter Enteral NutrHome page
C. W. Van Way III
Of Glutamine and Arginine
JPEN J Parenter Enteral Nutr, May 1, 2005; 29(3): 218 - 219.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Saved Citations
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Request Reprints
Right arrow Add to My Marked Citations
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Right arrow Citing Articles via Scopus
Google Scholar
Right arrow Articles by Bansal, V.
Right arrow Articles by Ochoa, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bansal, V.
Right arrow Articles by Ochoa, J. B.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*(L)-ARGININE
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?