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-
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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,
