Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins

doi:10.1016/j.imlet.2003.11.030

Immunology Letters

Volume 92, Issues 1–2, 29 March 2004, Pages 143-147

https://doi.org/10.1016/j.imlet.2003.11.030 Get rights and content

Abstract

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4⁺-T cells derived from MIF −/− mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

Introduction

Thioredoxin (TRX) is a 12 kDa protein with redox-active dithiol in the conserved active site; -Cys³²-Gly-Pro-Cys³⁵- [1]. Human TRX was originally cloned as a soluble factor named adult T-cell leukemia-derived factor (ADF) produced by human T cell leukemia virus type-I transformed ATL2 cells [2]. TRX superfamily in mammalian possessing the redox-active site; -Cys-Xa.a.-Xa.a.-Cys- is composed of 17 proteins; TRX, TRX-2 [3], spTRX [4], TMX [5] or etc. TRX is induced by a variety of oxidative stresses including viral infection and anti-cancer agents. It was recently reported that antigen-presenting cells secrete TRX for the activation of T lymphocytes [6]. Although the releasing mechanism is still unclear, the secretion of TRX is associated with the cellular resistance to cisplatin- or ethanol-induced oxidative stress [7], [8]. Intracellular TRX plays crucial roles in the scavenging of reactive oxygen species with peroxiredoxin, the regulation of redox-sensitive transcription factors including AP-1 and NF-κB or the negative regulator of apoptosis signal-regulating kinase (ASK) 1, suggesting that TRX functions for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine- and stress-induced apoptosis [9]. As other TRX family proteins for regulation of apoptosis, proinflammatory cytokines or immune response, macrophage migration inhibiting factor (MIF) and glycosylation inhibitory factor (GIF) are addressed [10]. Although MIF and GIF share an identical structure gene, the generation of immunosuppressive activity of GIF is required for cysteinylation at Cys⁶⁰ in the redox-active site; -Cys⁵⁷-Ala-Leu-Cys⁶⁰ [11]. MIF is a pivotal mediator of innate immunity and sustains macrophage proinflammatory function by inhibiting p53 [12], [13]. It is recently reported that MIF is a modulator of pro-oxidative stress-induced apoptosis of HL-60 [14]. An interest has herein arisen how TRX and MIF/GIF regulate oxidative stress-induced apoptosis of T lymphocytes. We reveal that both oxidative stress and overexpression of intracellular TRX suppress MIF expression in T cell line and TRX expression is accelerated in CD4⁺ T cells derived from MIF-deficient mice.

Section snippets

Reagents and cells

Jurkat (human T lymphocyte cell line) cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO₂ at 37 °C. CD4⁺ T cells were purified from spleens of MIF −/− mice [15] by using magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany).

Plasmid DNA

The DNA fragments for encoding TRX gene were amplified by PCR method using the pcDNA3.1 encoding human TRX cDNA as template [2] and were inserted to p3xFLAG-CMV-10

Hydrogen peroxide-induced apoptosis is inhibited by overexpression of intracellular TRX

In this report, we tried to elucidate the cross-talk among TRX superfamily proteins in oxidative stress-induced apoptosis of T cells. To assess the functions of TRX superfamily proteins, Jurkat T cells transfected with human TRX-wild-type gene (TRX-transfectant) or control vector (mock-transfectant) was prepared, respectively. As a preliminary experiment to test whether hydrogen peroxide (H₂O₂) induces apoptosis of TRX-tranfectant or mock-transfectant, both caspase-3 activity and sub G1

Discussion

In the present study, we have shown that the expression level of both TRX and MIF/GIF in TRX superfamily is regulated each other in the course of oxidative stress-induced apoptosis on T lymphocytes. We have already reported that human TRX has been shown to be a scavenger of reactive oxygen species (ROS), and recombinant human TRX performs a protective activity against hydrogen peroxide-induced cytotoxicity [18]. In fact, overexpression of human TRX in Jurkat T cell-transfectant resulted in the

Acknowledgements

We thank H. Nakamura, H. Masutani, Y.C. Kim and A. Teratani for excellent technical assistance and discussion, and both Y. Kanekiyo and Maina Ueshima for secretarial help.

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Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins

Abstract

Introduction

Section snippets

Reagents and cells

Plasmid DNA

Hydrogen peroxide-induced apoptosis is inhibited by overexpression of intracellular TRX

Discussion

Acknowledgements

Annu. Rev. Biochem.

EMBO J.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Proc. Natl. Acad. Sci. U.S.A.

Antioxid. Redox Signal.

Biochem. Biophys. Res. Commun.

EMBO J.

J. Biol. Chem.

Proc. Natl. Acad. Sci. U.S.A.