Burn injury induces the expression of cystine/glutamate transporter (xc−) in mouse T cells
Introduction
Antigenic stimulation of T lymphocytes induces a series of activation processes including tyrosine phosphorylation in various proteins, increase of Ca2+, induction of various genes such as IL-2 and IL-2Rα-chain. T cell activation also stimulates reactive oxygen species (ROS) production [1], [2]. It has been reported that mitogenically induced lymphocyte proliferation is affected or regulated by various thiol compounds [3]. Glutathione (GSH) is the major scavenger for ROS in T cells. A direct relationship exists between GSH availability and the proliferative response of T lymphocytes because when cells are cultured with the specific GSH inhibitor l-buthionine-S,R-sulfoximine (BSO), proliferation in response to mitogens is inhibited [4], [5]. l-Cysteine and GSH enhance the proliferation of rabbit peripheral lymphocytes stimulated by mitogens [6]. GSH is a tripeptide and cysteine can be rate limiting in GSH synthesis. The addition of cysteine to fresh resting lymphocytes only partially prevented the rapid decrease of GSH levels. However, in lipopolysaccharide (LPS)-activated splenocytes the addition of cysteine enhanced cellular GSH levels in a dose-dependent manner [7].
Previous studies in lymphocytes identified cysteine as a rate limiting step for cell growth and proliferation after antigen receptor activation [8]. An equilibrium exists between cysteine, in the reduced form, and cystine, in the oxidized form. Cystine is predominant in the extracellular environment, while cysteine levels are elevated in the reduced intracellular environment [9], [10]. The entry of cystine into cells is regulated by the expression of system xc− transporter which exchanges one glutamate for one cystine molecule. System xc− is formed by two proteins, CD98 and xCT [11]. Even though CD98 is expressed in mature T cells and upregulated following activation, T lymphocytes express low levels of xCT and are not able to take up cystine from the surrounding environment and convert it to be cysteine [12]. Furthermore, lymphocytes have been reported to lack cystathionase, an enzyme essential for the production of cysteine from methionine, and thus cysteine is essential for lymphocytes [13]. During a specific immune response, T cells interact with antigen-presenting cells (APC). It has been shown that this interaction increased both cystines uptake and cysteine production by macrophages or dendritic cells [14]. Unlike lymphocytes, APC expresses high levels of xCT which is the main regulator of the system xc− activity and its expression is induced by electrophilic agents like diethyl maleate [15], oxygen [16], hydrogen peroxide [17], bacterial LPS [18] and amino acid deprivation [19]. Unless APC makes cysteine available, antigen-stimulated T cells will be unable to increase protein synthesis, proliferate or secrete immunoregulatory cytokines [8], [14], [20].
It is well known that the addition of 10–50 μM of 2-mercaptoethanol (2-ME) to cell media culture supports the growth of primary T lymphocytes cultures [21], [22]. The action of 2-ME on mouse spleen lymphocytes was related to the membrane transport of cysteine [7], [23]. In vitro culture of T cells with 2-ME produced profound effects such as preventing the loss of GSH, enhancing their proliferation, preventing their apoptosis and functionally replacing antigen-presenting cells [24], [25], [26], [27], [28].
Burn injuries are associated with oxidative stress, GSH depletion and immunosuppression. Macrophage hyperactivity has been related to T cell dysfunction post-burn injury [29]. This condition leads to increased susceptibility to bacterial infections and sepsis which is associated with high lethality in post-burn patients. A recent study has demonstrated the induction of cystine/glutamate transporter in the spleen by LPS in a murine model of endotoxemia [30]. Only a few reports have shown a link between the intracellular cysteine equilibrium and the immune system following thermal injury. Antioxidant therapy has been introduced to early post-burn injury protocols to prevent oxidative stress damage [31]. N-Acetyl-l-cysteine given to burned rats has demonstrated a cytoprotective role in intestinal mucosal barrier and preventive effects against burn-induced bacterial translocation [32]. Furthermore, it was shown in a rat model of burn injury that antioxidant therapy given for 7 days following the trauma significantly improved cell-mediated immunity [33].
Sulfasalazine (SASP) is a drug commonly used in the treatment of inflammatory bowel disease, a T cell mediated autoimmune disease. This drug has recently been found to specifically inhibit the xc− cystine transporter [34]. It is believed that SASP is effective in inflammatory bowel disease because it inhibits the growth and proliferation of autoreactive T cells that initiate the inflammatory response [35]. Collectively these data suggested that intracellular cysteine levels regulate T cell activity. We hypothesize that thermal injury affected T cell proliferation by modifying its dependency for low molecular weight thiols such as 2-ME or cysteine delivered by APC. Surprisingly, our results indicate that in vitro proliferation of burn-injured T cells is independent of 2-ME supplementation in cell culture media and that these cells express xCT. In addition, the xc− cystine/glutamate transporter was found to be active in burn-injured T cells and cystine import was inhibited by SASP. Moreover, SASP significantly reduced burn-injured T cell proliferation. These results show for the first time the expression of system xc− in lymphocytes and suggests that intracellular cysteine content is important in regulating burn-injured T cell response.
Section snippets
Animals
All experiments were performed on 6-week-old male C57BL/6 mice (Charles River Laboratories, St-Constant, Quebec, Canada). The mice were acclimatized for a period of 2 weeks prior to the initiation of any procedures and were housed in a central animal facility under strictly controlled temperature, relative humidity and a 12-h light/dark cycle. They were kept in cages, each containing five mice. Standard chow (Richmond Standard Lab Diet; Lab Diet, Richmond, IN, USA) and water was provided ad
2-ME is dispensable for in vitro burn-injured T cell proliferation
Accessory cells control T lymphocyte proliferation by maintaining a cysteine-rich extracellular environment and macrophage hyperactivity post-thermal injury was related to T cell dysfunction [29]. As previously reported, intracellular thiol equilibrium is critical for T cell proliferation [3]. In order to evaluate T cell dependency for low molecular weight thiols and to avoid any effect related to APC, we used purified T cells. Phenotyping analysis revealed that for both groups more than 98% of
Discussion
T cell immunosuppression following a thermal injury is widely documented in studies using in vitro cell culture systems and is characterized by decreased proliferation, IL-2 production, increased apoptosis and anergy [40], [41], [42]. Since normal T cells have weak membrane transport activity for the disulfide cystine, all of these studies have used cell culture media supplemented with 2-ME [43] to facilitate the entry of cysteine into T cells and their proliferation [44]. However,
Acknowledgements
The present study was supported by a grant from the Fondation des Pompiers du Québec pour les Grands-Brûlés. Michele D’Elia and Julie Patenaude were supported by a research award from the FRSQ-Fondation de la Recherche en Santé du Québec.
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