l -Arginine modulates CD3ζ expression and T cell function in activated human T lymphocytes

ellular Immunology Cellular Immunology 232 (2004) 21–31 www.elsevier.com/locate/ycimm

L-Arginine

modulates CD3
expression and T cell function in activated human T lymphocytes

Arnold H. Zeaa,b,¤, Paulo C. Rodrigueza,h, Kirk S. Culottac, Claudia P. Hernandeza, Joanna DeSalvod, Juan B. Ochoae, Hae-Joon Parkf, Jovanny Zabaletag, Augusto C. Ochoaa,h b

a Stanley S. Scott Cancer Center, LSUHSC, New Orleans, LA, USA Microbiology Immunology and Parasitology, LSUHSC, New Orleans, LA, USA c UT M.D. Anderson Cancer Center-PDC, Houston, TX, USA d Tulane University, New Orleans, LA, USA e Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA f MOGAM Biotechnology Research Institute, Yogin-City, Republic of Korea g Department of Pathology, LSUHSC, New Orleans, USA h Department of Pediatrics, LSUHSC, New Orleans, USA

Received 1 September 2004; accepted 10 January 2005 Available online 23 February 2005

Abstract Engagement of the T cell receptor (TCR) by antigen or anti-CD3 antibody results in a cycle of internalization and re-expression of the CD3
. Following internalization, CD3
is degraded and replaced by newly synthesized CD3
on the cell surface. Here, we provide evidence that availability of the amino acid L-arginine modulates the cycle of internalization and re-expression of CD3
and cause T cell dysfunction. T cells stimulated and cultured in presence of L-arginine, undergo the normal cycle of internalization and re-expression of CD3
. In contrast, T cells stimulated and cultured in absence of L-arginine, present a sustained down-regulation of CD3
preventing the normal expression of the TCR, exhibit a decreased proliferation, and a signiWcantly diminished production of IFN, IL5, and IL10, but not IL2. The replenishment of L-arginine recovers the expression of CD3
. The decreased expression of CD3
is not caused by a decreased CD3
mRNA, an increased CD3
degradation or T cell apoptosis.  2005 Elsevier Inc. All rights reserved. Keywords: CD3
; L-Arginine; Lymphocytes; T cell function; T cell receptor; IL-2

1. Introduction L-Arginine is a semi-essential amino acid important in diVerent biological and metabolic systems including the immune system. In macrophages, L-arginine is metabolized by nitric oxide synthase (NOS)1 to pro-

¤

Corresponding author: Fax: +1 504 599 0864. E-mail address: [email protected] (A.H. Zea). 1 Abbreviations used: C-RPMI, conventional RPMI; Arg-free-RPMI, RPMI media without L-arginine; CD3
, CD3 zeta chain; NO, nitric oxide; NOS, nitric oxide synthase; IL2R, interleukin 2 receptor. 0008-8749/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2005.01.004

duce nitric oxide (NO) [1,2] or by arginase to produce urea and ornithine, the latter being a precursor of polyamines [3–5]. NO is one of the principal cytolytic mechanisms in macrophages while polyamines are essential for cell proliferation [6–8]. Various conditions including liver transplantation and trauma, can lead to the depletion of L-arginine in vivo, resulting in a profound decrease in T cell function [9,10]. L-Arginine supplementation in these patients results in the recovery of normal T cell responses [11,12], suggesting that L-arginine may play an important role in regulating T cell function.

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A.H. Zea et al. / Cellular Immunology 232 (2004) 21–31

The T cell receptor
chain (CD3
) is the principal signal transduction element of the TCR and is the ratelimiting step in its assembly and membrane expression [13,14]. The decreased expression of CD3
and a decreased in vitro response to antigens or mitogens has been demonstrated in patients with cancer [15–19], chronic infectious diseases [20,21] and autoimmunity [22]. The mechanisms leading to the decreased expression of CD3
and T cell dysfunction are poorly understood. We recently demonstrated that the depletion of L-arginine from the tissue culture medium blocked proliferation of the Jurkat T cell line and induced the loss of CD3
due mostly to a decreased CD3
mRNA stability [23,24]. However, the eVect of L-arginine depletion on normal human T cells was signiWcantly diVerent. The data presented here demonstrates that L-arginine depletion does not aVect the expression of CD3
in resting T cells. Instead, it appears to impair the cycle of internalization and re-expression of CD3
after antigen stimulation. The absence of L-arginine also blocked cytokine production and cell proliferation. However, this eVect was not caused by T cell apoptosis a decreased expression in the CD3
mRNA or to increased CD3
degradation. Instead, preliminary data suggest that the absence of L-arginine prevents the synthesis of new CD3
. The changes in signal transduction and T cell function were reversible by the replenishment of L arginine.

2. Materials and methods 2.1. T cell preparations The peripheral blood mononuclear cells (PBMCs) from normal donors to be used for in vitro experiments were purchased from the Blood Center of New Orleans. The use of samples from normal donors is currently approved under the LSU-IRB protocols 4761 and 4867 that are currently active. The PBMCs were separated over Ficoll–Paque (Amersham Biosciences, Uppsala, Sweden) and passed through T cell enrichment columns (R&D Systems, Minneapolis, MN). After a 10 min incubation the columns were washed. The resulting enriched T cells were counted and tested for surface markers by Xow cytometry. The resulting T cell preparation contained >95% CD3+ (Clone HIT3a) cells, <3% CD16+ (Clone 3G8) cells, and <1% each of B (CD19+) (Clone HIB19) cells and monocytes (CD14+) (Clone ME52). 2.2. Tissue culture media and cell culture Tissue culture dishes (100 mm) (Corning, Corning, NY) were coated overnight at 4 °C with PBS containing 10 g/ml of anti-CD3 (OKT-3 Ortho Pharmaceutical, Raritan, NJ) and then carefully washed once with cold phosphate-buVered saline (PBS). Ten million enriched

T cells were then added to each dish, resuspended in 10 ml of conventional RPMI-1640 (C-RPMI) which contains 1140M L-arginine (Cambrex, Walkersville, MD) or in RPMI-1640 without L-arginine (Arg-freeRPMI) (Gibco-Invitrogen, Carlsbad, CA) or RPMI-1640 media without L-glutamine or L-glycine. Anti-CD28 (Becton–Dickinson, San Jose, CA) at 100 ng/ml was added to each dish. Then the T cells were cultured for the diVerent time periods at 37 °C in 5% CO2. As a negative control, T cells were plated under the same media culture conditions but without OKT-3 and CD28. All cultures were supplemented with 10% fetal calf serum (FCS) (Hyclone, Logan, UT), 25 mM Hepes buVer (Invitrogen, Carlsbad, CA), and 4 mM L-glutamine (Cambrex). 2.3. Flow cytometry To measure CD3
expression, puriWed T cells were incubated for 15 min at 4 °C with anti-CD3-FITC (Clone UCHT1) or the isotype control (Beckman-Coulter, Miami, FL) at 1 g of antibody/106 cells. Cells were washed and re-suspended in PBS containing digitonin at 500 g/ml (Wako, BioProducts, Richmond, VA) and 2.5 g of anti-CD3
-PE (Clone2H2D9) antibody (Beckman-Coulter, Miami, FL). Cells were incubated for 8 min at 4 °C, washed, and re-suspended in PBS for analysis. For surface markers, 3 £ 105 T cells were plated on a 96-well/U-bottom plates (Corning, Corning, NY) and incubated with 1 g of isotype control, CD3 (Clone HIT3a), CD4 (Clone RPA-T4), CD8 (Clone HIT8a), CD14 (Clone M5E2), CD45RO (Clone UCHL1), CD45RA (Clone HI100), CD69 (Clone FN50) or CD25 (Clone M-A251) antibodies (Becton–Dickinson, San Jose, CA) for 15 min at 4 °C, followed by two washes with PBS containing 2% bovine serum albumin (BSA) (Sigma, St. Louis, MO), then Wxed in 1% paraformaldehyde. For apoptosis, T cells were stained with Annexin V FITC apoptosis detection kit (Oncogene, San Diego, CA). Fluorescence analysis was done using a CoulterEPICS Xow cytometer (Beckman-Coulter, Miami, FL). 2.4. Western blots and protein tyrosine phosphorylation For Western blot, 10 £ 106 stimulated T cells were harvested after culture at diVerent time points in the presence or absence of L-arginine and lysed in Triton X-100 buVer with protease inhibitors as described before [20]. Lysates were electrophoresed in 14% Tris–glycine gels (Invitrogen, Carlsbad, CA), transfer to PVDF membranes (Invitrogen), immunoblotted with the diVerent antibodies and detected by horseradish peroxidase conjugated antibodies and ECL (Amersham Biosciences, Little Chalfont, England). Gels were autoradiographed on X-OMAT AR Wlms (Eastman Kodak, Rochester, NY).

A.H. Zea et al. / Cellular Immunology 232 (2004) 21–31

For protein tyrosine phosphorylation, 5 £ 106 stimulated T cells were cultured in C-RPMI or in Arg-freeRPMI for 12, 24, and 48 h harvested and washed with cold PBS. As a negative control unstimulated T cells were cultured in C-RPMI for the same time periods. To trigger protein phosphorylation the cells were re-suspended in 200 l of media, incubated with 1 g of OKT-3 (Ortho Pharmaceutical) on ice for 10 min, followed by cross-linking with 1.25 g of goat anti-mouse (GAM) (Kirkegaard and Perry Labs., Gaithersburg, MD) for 2 min at 37 °C. The reaction was stopped by washing the cells in cold PBS containing 400 M sodium orthovanadate and 1 mM EDTA (Sigma, St. Louis, MO). The cells were then lysed and electrophoresed in 10% polyacrylamide gels, transferred to PVDF membranes and immunoblotted with anti-phosphotyrosine 4G10-antibody (Upstate Biotechnology, Lake Placid, NY). 2.5. Calcium Xux One million of stimulated T cells cultured in C-RPMI or in Arg-free-RPMI for 12 and 24 h were harvested and loaded with 2 mM Flou-3 AM (Molecular Probes, Eugene, OR) and 0.02% Pluronic 127F (Molecular Probes) for 30 min at 37 °C. The cells were then washed once and incubated in calcium buVer (HBSS, 1 mM CaCl2, 1 mM MgCl2) for 20 min at room temperature in the dark. Calcium Xux was measured at 488 nm as a function of time (up to 450 s) in response to 1 g of OKT-3 as a positive control or PBS as a negative control 30 s after initial monitoring using a Coulter-EPICS Xow cytometer (Beckman-Coulter, Miami, FL). The Xow cytometry data were analyzed using WinMDI 2.8 software (TSRI, La Jolla, CA). 2.6. Cytokine production Supernatants from the T cells cultures were collected at 48, 72, and 96 h and tested for IL2, IFN, IL5, and IL10 production by ELISA. BrieXy, 96-well plates (Immulon IV, Dynatech, Burlington, MA) were coated with the respective capture monoclonal antibody (Biosource, Camarillo, CA) and the culture supernatants incubated for 30 min. The reaction was detected by biotin–streptavidin conjugated with horseradish peroxidase (BD-Pharmingen, San Diego, CA) using 3,3⬘,5,5⬘-tetramethylbenzidine (Roche, Indianapolis, IN) as a substrate. The reaction was stopped with 0.8 M sulfuric acid and the absorbances were read at 450 nm. The minimum level of cytokines detectable by the assay was 30 pg/ml. 2.7. Proliferation assay Unstimulated or stimulated cells were plated at 1 £ 105 per well and cultured in RPMI with or without arginine for 24, 48, and 72 h. [3H]Thymidine (0.5 Ci)

23

(Perkin-Elmer Life Sciences, Boston, MA) was added to each well and allowed to incubate for an additional 18 h at 37 °C. Each condition was tested in triplicate. Cells were lysed by freezing and thawing, harvested onto a UniWlter-96 GF/B (Packard, Meriden, CT) and counted using a TOPCOUNT Microplate Scintillation Counter (Packard, Meriden, CT). 2.8. Lysosome and proteasome inhibition To inhibit lysosome function 2 £ 106 T cells cultured in the presence or absence of L-arginine for 24 h, were treated with 1 M bafylomycin A1 (Calbiochem, San Diego, CA). Similar treatment was done at 48 and 72 h in culture. To inhibit proteasome activity 2.5 M lactacystin (Calbiochem) was added to the cultures at the same time points. DMSO was used as a control vehicle in all the cases. The CD3
expression was tested by Xow cytometry 24 and 48 h after the addition of the inhibitors. 2.9. Isolation of RNA for Northern blots and ribonuclease protection assay Total RNA was extracted from 107 T cells by lysis with TRIzol (Invitrogen, Carlsbad, CA). Ten micrograms of total RNA was electrophoresed under denaturing conditions, blotted onto nytran membranes (Schleicher & Schuell, Keene, NH), and cross-linked by UV irradiation. Membranes were prehybridized at 42 °C in ULTRAhyb buVer (Ambion, Austin, TX) and hybridized overnight with 1 £ 106 cpm/ml of 32Plabeled speciWc probes. Membranes were washed three times, and auto-radiographed at ¡70 °C using Kodak BIOMAX-MR Wlms (Eastman Kodak, Rochester, NY). The murine cDNA glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (Clontech, Palo Alto, CA) and the human CD3
(a kind gift from Dr. Allan Weissman, National Institutes of Health, Bethesda, MD) were labeled by random priming using a RediPrime Kit (Amersham Biosciences) and [-32P]dCTP 3,000 Ci/mmol (Perkin-Elmer Life Sciences, Boston, MA). All signal intensities were normalized to GAPDH. For the ribonuclease protection assay (RPA), 5 g of RNA was mixed with the templates (BD-Pharmingen, San Diego, CA) and incubated Wrst at 90 °C allowing the temperature to decrease slowly to 56 °C. The samples were treated with RNAse followed by proteinase K. After extraction with phenol–chlorophorm, the sample was precipitated in 100% ethanol for 30 min at ¡70 °C and recovered by centrifugation at 12,000 rpm, resuspended in loading buVer and separated on a polyacrylamide gel containing 8 M Urea. Gels were dried and exposed to BIO-MAX Wlms (Eastman Kodak, Rochester, NY).

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A.H. Zea et al. / Cellular Immunology 232 (2004) 21–31

2.10. Radiolabeling and pulse-chase experiments Ten million T cells were cultured for 24, 48, 72, and 96 h in C-RPMI or Arg-free-RPMI. At the diVerent time points cells were harvested and resuspended in 2 ml of methionine-free C-RPMI or methionine-free-Arg-freeRPMI (Gibco, Invitrogen). After 1 h starvation the cells were labeled for 6 h with 1 mCi of 35S-methionine (Perkin-Elmer Life Sciences) in RPMI or Arg-freeRPMI media with 5% dialyzed FCS. For the chase the cells were cultured in excess of unlabeled methionine (15 mg/ml) for 2, 4, and 6 h. Cells were washed twice in cold PBS and lysed in 1% digitonin and 0.12% Triton X100 (BioRad Laboratories, Hercules, CA) buVer plus protease inhibitors. Cell lysates were incubated with protein G–Sepharose beads (Amersham–Pharmacia Biotech., Uppsala, Sweden) coated with 20 g of OKT-3 (Ortho-Pharmaceuticals). The immunoprecipitates were subjected to one-dimensional non-reducing SDS–polyacrylamide gel electrophoresis (PAGE) or to two-dimensional non-reducing (NR), reducing (R) SDS/PAGE. Gels were dried and exposed to Kodak BIOMAX MR (Eastman Kodak). 2.11. Statistical analysis Comparison between the groups were calculated by Student’s t test using the graph-pad statistical program (Graph-Pad, San Diego, CA).

3. Results 3.1. Depletion of L-arginine blocks the normal TCR cycling and CD3
re-expression in activated T cells T cells stimulated by cross-linked anti-CD3 plus antiCD28 and cultured in conventional RPMI (C-RPMI), which contains 1140 M L-arginine, showed the normal cycle of down-regulation of the TCR CD3 (Fig. 1A) and CD3
(Fig. 1B) by 24 h, followed by a gradual reexpression by 48–96 h. In contrast, T cells stimulated and cultured in RPMI without L-arginine (Arg-free-RPMI) showed an initial down-regulation of CD3
at 24 h, but failed to recover the expression of CD3
and consequently of the TCR, even after 96 h in culture. Control unstimulated T cells did not show any changes in the expression of the CD3 or CD3
when cultured in Argfree-RPMI or C-RPMI. The decrease and recovery of CD3
was similar in CD4+and CD8+ T cell subpopulations as well as in CD45RA+ and CD45RO+ naïve and memory T cells (data not shown). Western blots done at 72 h conWrmed the prolonged decrease of CD3
and CD3 in T cells cultured in Arg-free-RPMI (Fig. 1C). The eVect of L-arginine on CD3
expression was amino acid speciWc since the depletion of other amino

Fig. 1. The absence of L-arginine in tissue culture media induces a sustained decrease in CD3
and CD3. Expression of CD3 (A) and CD3
(B) was measured in T cells stimulated with cross linked antiCD3 plus anti-CD28 and cultured in C-RPMI (䊐) or Arg-free-RPMI (䉫), or cultured without stimulation in C-RPMI (䊊) or Arg-freeRPMI (䉮). Mean Xuorescence intensity (MFI) § SEM from four diVerent experiments is shown. (C) T cells stimulated and cultured in the presence (+) or absence (¡) of L-arginine, were tested at 24, 48, and 72 h for the expression of CD3
, CD3, and GAPDH by Western blot.

acids such as L-glutamine, L-glycine (Fig. 2A) or L-leucine and L-lysine (data not shown), did not alter the cycle of internalization and re-expression of CD3
after antigen stimulation. Furthermore, only the replenishment of L-arginine at doses of 150 M or higher, but not other amino acids, resulted in the rapid re-expression of CD3
(Fig. 2B). This phenomenon occurred even when L-arginine was added to the cells as late as 72 h in culture (data not shown). Arginine depletion did not alter the expression of other cell membrane receptors such as CD4 and CD8 (data not shown). Similarly it did not prevent the upregulation of the early T cell activation marker CD69 or the later IL2R chain (CD25) marker as seen in Table 1. However, although the expression of CD25 was gradually upregulated in a time dependent

A.H. Zea et al. / Cellular Immunology 232 (2004) 21–31

25

Fig. 2. (A) Decreased expression of CD3
is amino acid speciWc. T cells were stimulated and cultured in the absence of L-arginine (䉫), L-glutamine (䉭), L-glycine (䉮) or in C-RPMI (䊐). CD3
expression was measured by Xow cytometry. (B) Addition of L-arginine to the culture media induces the re-expression of CD3
. T cells were stimulated and cultured in Arg-free-RPMI (䉫) for 24 h. L-Arginine (150 M) was added to the media (䉬) and the expression of CD3
was measured by Xow cytometry. Table 1 CD25 and CD29 activation markers are up-regulated after stimulation of T cells cultured in the absence of L-arginine Time (h)

0 24 48 72

CD25 § SD (%)

CD69 § SD (%)

C-RPMI

Arg-free-RPMI

C-RPMI

Arg-free-RPMI

9.23 § 1.7 30.3 § 1.7 61.1 § 5.7 75.9 § 7.8

9.23 § 1.7 27.5 § 2.1 39.8 § 4.0 55.5 § 12.1

10.8 § 1.6 89.5 § 1.3 93.7 § 1.5 85.6 § 1.5

10.8 § 1.6 91.0 § 1.6 94.4 § 1.6 93.8 § 1.9

T cells stimulated with anti-CD3 plus CD28 were cultured in presence or absence of L-arginine for 24, 48, and 72 h. The expression of CD25 and CD69 were measured by Xow cytometry. The expression values are expressed as a percentage (%) § SD from four diVerent experiments.

manner, did not achieve the magnitude (after 48 h) of the cells cultured in C-RPMI (p < 0.05). 3.2. L-Arginine starvation markedly reduces cell proliferation and cytokine production T cells stimulated with anti-CD3 + anti-CD28 and cultured in Arg-free-RPMI also had a signiWcantly lower proliferation (p < 0.001) as compared to cells cultured in C-RPMI (Fig. 3). In addition, as shown in Fig. 4A, T lymphocytes cultured in the absence of L-arginine had a signiWcantly decreased production of IFN, IL5, and IL10 (p < 0.001). The decreased production of these cytokines was not caused by a delay in their kinetics since cytokine production after 72–96 h was still decreased in the absence of L-arginine. Interestingly IL2 production did not appear to be impaired by L-arginine depletion. The decreased cytokine production could in part be explained by changes in mRNA expression as shown by a ribonuclease protection assay (RPA). The mRNA expression for IFN, IL5, and IL10 in T cells stimulated and cultured in Arg-free-RPMI

Fig. 3. T cell proliferation is signiWcantly decreased (p < 0.001) in T cells cultured in Arg-free-RPMI after stimulation with cross linked anti-CD3 and anti-CD28 when compared to cells stimulated and cultured in C-RPMI. T cells were pulsed with [3H]thymidine after 18 h in culture. Data from three experiments are presented as a mean cpm § SEM.

were decreased as compared to those cultured in C-RPMI, while IL2 mRNA expression was similar in the presence or absence of L-arginine (Fig. 4B). 3.3. Calcium Xux and tyrosine phosphorylation patterns in T cells stimulated and cultured in absence of L-arginine The markedly low proliferation and the decreased production of cytokines in T cells cultured in the absence of L-arginine could also be explained by an impairment of the early signaling events such as Ca2+ Xux and tyrosine phosphorylation. However, the absence of L-arginine did not alter Ca2+ Xux in stimulated T cells after 12 and 24 h in culture, since Ca2+ Xux was similar to those observed in the T cells cultured in presence of L-arginine (Fig. 5A). No diVerences in the pattern of protein phosphorylation was observed in stimulated T cells during the Wrst

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A.H. Zea et al. / Cellular Immunology 232 (2004) 21–31

Fig. 4. (A) Production of IFN, IL5, and IL10 but not IL2 is signiWcantly decreased (*p < 0.001) in T cells stimulated and cultured in Arg-free-RPMI when compared to T cells stimulated and cultured in C-RPMI. These signiWcant diVerences were observed only after 48 h in culture. No cytokine production was observed in the control (unstimulated) T cells. Data from Wve experiments is presented as mean picograms § SEM. (B) Ribonuclease protection assay (RPA) shows the mRNA expression for the diVerent cytokine RNA in T cells stimulated and cultured for 24, 48, and 72 h in the presence or absence of L-arginine.

Fig. 5. Calcium Xux is not aVected by the absence of L-arginine, but tyrosine phosphorylation is decreased a later times. (A) Stimulated T cells cultured in C-RPMI (A+) or Arg-free-RPMI (A¡) for 12 and 24 h were harvested, washed, and loaded with Flou-3 and washed as described in Section 2. After 30 s (indicated by arrows) of Ca2+ monitoring, the cells were pulsed with PBS (negative control) or with 1 g OKT-3 (positive control). Then, Ca2+ mobilization was monitored for the rest of time span. Data are representative of at least three independent experiments. (B) Stimulated T cells cultured in the presence of absence of L-arginine were harvested after 12, 24, and 48 in culture. To trigger protein phosphorylation the cells were pulsed with 1 g OKT-3 cross-linked with 2.5 g GAM and the reaction stopped with EDTA/sodium–orthovanadate buVer. The cells were lysed, electrophoresed, and blotted with anti-phosphotyrosine monoclonal antibody 4G10. Unstimulated T cells cultured in the presence or absence of Larginine for the same time periods were used as negative controls.

A.H. Zea et al. / Cellular Immunology 232 (2004) 21–31

27

12 h of culture in C-RPMI (lane 2) or Arg-free-RPMI (lane 3). However, after 24 h in culture, the overall phosphorylation patterns were markedly decreased in the cells cultured in Arg-free-RPMI (lane 6) as compared to those seen in the T cells cultured in C-RPMI (lane 5). These diVerences in phosphorylation patterns were more prominent after 48 hours in culture (C-RPMI lane 9 vs. Arg-free-RPMI lane 8). At the diVerent time points, the unstimulated T cells used as control presented a quite similar decreased phosphorylation patterns as observed in stimulated T cells cultured in absence of L-arginine (lanes 1, 4, and 7). 3.4. The decreased expression of CD3
it is not caused by apoptosis of T cells, an increased protein degradation or a decreased mRNA expression The inability to re-express CD3
in T cells cultured in the absence of L-arginine could be caused by several mechanisms including an increased T cell apoptosis, an increase in the degradation of CD3
protein, a decrease in CD3
mRNA expression or a decrease in CD3
chain synthesis. Viability of T cells (by trypan blue exclusion) cultured in the absence of L-arginine was >92% over the 96 h of culture in these experiments. Furthermore, apoptosis, tested by annexin V expression was not increased in T cells cultured in the absence of L-arginine (Table 2). We then tested whether an increased degradation of CD3
could explain the inability to recover the expression of this TCR chain. Experiments blocking lysosome and proteasome function with baWlomycin and lactacystin respectively, failed to show the re-expression of CD3
in T cells cultured in the absence of L-arginine (Fig. 6A). We then tested whether the inhibition of CD3
could be caused by a decrease in CD3
mRNA. Northern blots showed no diVerences in CD3
mRNA expression at 1, 12, 24, 48, and 72 h in culture (Fig. 6B), and did not show a decrease in mRNA half life (data not shown). We then tested the possibility that the absence of L-arginine impaired the synthesis of CD3
or other components of the TCR. Fig. 7A shows pulse experiments of Table 2 The down-regulation of CD3
is not due to an increase in T cell apoptosis Time (h)

0 24 48 72

Percentage § SD C-RPMI

Arg-free-RPMI

1.36 § 0.6 3.12 § 1.2 2.04 § 0.5 1.68 § 1.0

1.08 § 0.6 1.80 § 1.6 1.14 § 0.6 1.58 § 0.9

Stimulated T cells were cultured in presence or absence of L-arginine for 24, 48, and 72 h. Annexin V-FITC apoptosis detection kit (Oncogene, San Diego, CA) was used to measure apoptosis at the diVerent time points of the experiments. The results are expressed as a percentage (%) § SD of four diVerent experiments.

Fig. 6. Decrease expression of CD3
in absence of L-arginine it is not due to protein degradation or to a decreased mRNA expression. (A) EVect of lysosome (baWlomycin) and proteasome (lactasystin) inhibitors on T cells stimulated and cultured in C-RPMI or Arg-free-RPMI for 24 h. CD3
expression was tested 24 and 48 h later by Xow cytometry. (B) Stimulated T cells were cultured in conventional RPMI or Arg-free-RPMI for 1, 12, 24, 48, and 72 h. Northern blot analysis was used to test for CD3
RNA expression using 10 g of total RNA. GAPDH was used as housekeeping gene.

T cells labeled with 35S-methionine after being cultured in C-RPMI or Arg-free-RPMI for 24, 48, 72, and 96 h. CD3
and other TCR chains were isolated by immunoprecipitation with anti-CD3 monoclonal antibody or an irrelevant control antibody. Immunoprecipitates were analyzed by one-dimensional SDS–PAGE under non reducing conditions or by two-dimensional SDS–PAGE under non-reducing (NR), reducing (R) conditions as seen in Fig. 7B. T cells stimulated and cultured in CRPMI normally synthesized CD3
and CD3. In contrast, T cells stimulated and cultured in the absence of L-arginine, showed an inability to synthesize new CD3
, although the synthesis of CD3 was normal. The , , and  chains of the TCR were similarly synthesized in C-RPMI and Arg-free-RPMI. Chase experiments done in T cells cultured in C-RPMI showed a long half life of the newly synthesized CD3
and CD3, respectively (data not shown). Therefore, initial data suggests that the depletion of L-arginine may speciWcally impair the process of CD3
synthesis.

4. Discussion L-Arginine is a semi-essential amino acid that plays an important role in the immune response. In macrophages, L-arginine can be metabolized by the inducible nitric oxide synthase (iNOS) to produce nitric oxide (NO), important in the cytotoxic mechanism of these cells [25], or by arginase I or arginase II to produce urea

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A.H. Zea et al. / Cellular Immunology 232 (2004) 21–31

Fig. 7. Decreased CD3
synthesis in T cells stimulated and cultured in Arg-free-RPMI. Stimulated T cells were cultured for 24, 48, 72, and 92 h in C-RPMI or Arg-free-RPMI, then pulsed with 35S-methionine for 6 h, lysed and immunoprecipitated with anti-CD3. (A) Analysis of the immunoprecipitates was done in one dimension under non-reducing conditions (NR) or (B) by two dimensional (NR), reducing (R) SDS–PAGE in a 14% gels at 72 h of culture. The position of the expected proteins is indicated by the arrows.

and L-ornithine the latter being the substrate for polyamines, important for cell proliferation [26]. An increased arginase activity has been reported in patients following liver transplantation [9,16] and in patients with severe trauma [27,28], resulting in the depletion of serum L-arginine levels and a decreased T cell proliferation. In vitro models have demonstrated that the addition of L-arginine to tissue culture medium increases the response of CD8+ T cells to antigen stimulation and increases the relative density of the TCR on the cell membrane [29]. Furthermore, the infusion of high doses of L-arginine in trauma patients reestablishes T cell proliferation and increases the number of CD4 T cells in circulation [30–32]. Therefore L-arginine availability appears to play an important role in T cell function however, the mechanisms remain poorly understood. We recently reported that L-arginine starvation induced a rapid decrease in both CD3
expression and proliferation of the Jurkat T cell line and murine T cells [23,24]. However, the eVect of L-arginine depletion on normal human T cells was not known. The work presented here shows that the absence of L-arginine in tissue culture media does not alter the expression of CD3
in resting T cells. However, when T cells were stimulated and cultured in absence of L-arginine there was a sustained down-regulation of CD3
which only recovered with the replenishment of this amino acid. The decreased expression of CD3
appears to be amino acid speciWc since the depletion of other amino acids such as L-glutamine or L-lysine did not decrease the expression of this protein. The sustained down-regulation of CD3
does

not appear to be caused by a decrease in CD3
mRNA or a decrease in its half life, as has been shown previously in Jurkat T cells [24]. CD3
down-regulation can not be explained by an increase in apoptosis of T cells or an increase in proteosomal or lysosomal degradation of the protein. Instead, preliminary data from pulse chase experiments suggests a decreased synthesis of CD3
in the absence of L-arginine. How L-arginine depletion selectively decreases CD3
chain synthesis is still unclear. Possible explanations such as the number of arginine residues in
chain does not appear to be the case, since other proteins that are not altered by arginine depletion, such as the , , and  chains of the IL2 receptor, have similar numbers of arginine residues. Another possibility could be a selective decrease in the translation of CD3
, since a continuous supply of amino acids is a pre-requisite for maintenance of optimal rates of protein synthesis [33]. General amino acid starvation of mammalian cells results in a pronounced decrease in the overall rate of protein synthesis [34], associated with an increased phosphorylation of the -subunit of the initiation factor eIF2, which in turn would impair the activity of the guanine nucleotide exchange factor, eIF-2 [35,36]. However, our initial experiments have failed to show changes in the phosphorylation of any of the eIF-2 subunits (data not shown). Therefore, further research in the eVect of Larginine on the regulation of translation may help understand its role in the loss of CD3
. Although the depletion of L-arginine does not aVect the RNA expression of CD3
, it does cause a decreased RNA expression for certain cytokines including IL5,

A.H. Zea et al. / Cellular Immunology 232 (2004) 21–31

IL10, and IFN. The inability to up-regulate the genes for these cytokines could in part be explained by the decreased tyrosine phosphorylation observed in the absence of L-arginine after 24 h. Interestingly, IL2 production was not aVected by the depletion of L-arginine. One way to explain this is that enough intracellular arginine allowed the activation of CD69 and the IL2 receptor  chain-CD25 that are synthesized and expressed soon after activation of T cells. Similarly, early T cell functions such as Ca2+ Xux and tyrosine phosphorylation were not aVected in the Wrst 24 h of culture. However, phosphorylation patterns changed signiWcantly after 48 h in the absence of L-arginine. It is possible that certain cytokines genes such as IL2, which is expressed soon after activation, is not impaired. Instead, other cytokines genes such as IL4, IL5, and IL10 may require a longer sustained signaling which is impaired after 48 h of culture in the absence of L-arginine. It is also possible that the availability of amino acids regulates the transcription of cytokine genes [37–39]. Recent publications have shown that the depletion of the amino acid tryptophan by cells producing indoleamine 2,3-dioxygenase (IDO) can severely impair T cell gene expression and function [40]. It is also possible that other amino acids may also play an important role in the regulation of cytokine production. L-Glutamine enhances the production of IL2, IL10, and IFN in T lymphocytes [41,42] and a recent study demonstrated that adequate concentrations of glutamine increase a Th1 responses [43]. However, the eVect seen here is caused exclusively by the depletion of L-arginine since glutamine was present in all cultures. It is diYcult to determine with the results shown here whether or not there is a link between the absence of L-arginine and their eVect on cytokine production as an independent eVect on CD3
synthesis. More research targeting these questions need to be done. The Wndings described here could be important in explaining the diminished expression of CD3
and other T cell signal transduction proteins described in patients with cancer [16,17,44,45], chronic infections [20,21] and more recently in trauma [46]. The similarity in T cell signal transduction alterations in diseases with diVerent pathophysiology suggests the possibility of a common mechanism as the cause for such changes. Several mechanisms have been postulated to cause a decrease in CD3
chain expression in cancer, including Fas–FasL interactions [47,48], the production of H2O2 by macrophages and neutrophils [49,50] and more recently the depletion of L-arginine by macrophages [51]. L-Arginine levels are regulated in vivo by dietary intake and by three enzymatic pathways in macrophages namely iNOS, arginase I or II. Increased arginase activity, with the depletion of L-arginine and an impaired T cell function has been reported in clinical conditions such as severe trauma [27] or following liver transplantation [9,52]. Increased arginase production has been reported

29

in colon and gastric cancer [53,54] some leukemias [55] and in chronic infections such as leprosy [56]. Therefore, it is possible that the depletion of L-arginine in the microenvironment (at the tumor or sites of infection) or systemically (in metastatic disease) may decrease the expression of speciWc signal transduction proteins in T cells that are activated by antigen, resulting in an impaired T cell response. Despite the diVerent pathophysiology of these diseases the regulation of L-arginine availability could be a common mechanism responsible for immune dysfunction. Additional clinical research will test this possibility and determine its impact on the outcome of disease and the potential for the development of new therapies that may overcome them.

Acknowledgments This work was supported in part by Grants RO1CA82689, RO1-CA88885, and R21-CA83198 from the National Cancer Institute, National Institutes of Health(NIH-NCI), Bethesda MD. We thank Drs. Michael Hagensee, Diego Aviles, Alison Quayle, and Imtiaz Khan for their comments to this paper. We are grateful Mr. Kevin Zwezdaryk for his technical assistance as well as Ms. Sandra Lee and Ms. Nicole Barron for their assistance in preparing the manuscript.

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