[3 Enzyme Linked Immunospot Assay for Detection of Thioredoxin and Thioredoxin Reductase Secretion from Cells

By Bita Sahaf, Anita Sôderberg, Christina Ekerfelt, Staffan Paulie, and Anders Rosen

Oxidative stress response was determined in this study by enzyme-linked immunospot (ELISpot) assays for thioredoxin (Trx) and Trx reductase (TrxR). On exposure to oxidative stress, cells can launch a variety of defense mechanisms, including release of antioxidant proteins. The Trx system, consisting of Trx, TrxR, and NADPH, constitutes one of these cellular defense systems for maintenance of a healthy reduction-oxidation (redox) balance. Trx and TrxR are rapidly upregulated and released from monocytes, lymphocytes, and other normal and neoplastic cells on exposure. Secreted Trx and TrxR have proved to be eminent indicators of oxidative stress. Trx is a small, 12-kDa protein released through a leaderless pathway, whereas TrxR, which is a 116-kDa selenoprotein and required for regeneration of Trx, is secreted through the Golgi pathway. In this chapter we present a detailed laboratory bench protocol for enumeration of single cells secreting redox-active Trx and TrxR after oxidative stress exposure. Physiological stimuli (such as interferon y, lipopolysaccharide, interleukin 1, and CD23 ligation; and phorbol 12-myristate 13-acetate and ionophore) as well as UV light and hydrogen peroxide were used to generate oxidative stress, and some are presented in detail. The protocol includes a description of cell isolation, preparation, handling, and development of ELISpot plates, troubleshooting notes, presentation of results, statistical evaluation, and comments on alternative sources of materials and manufacturer Web addresses. We conclude that the ELISpot assay is a useful method for detection of single cells secreting the redox-active proteins Trx and TrxR after oxidative stress exposure.

Introduction

Cell growth and proliferation, programmed cell death, and cytoprotection are cellular events that involve signal transduction pathways, which depend on the generation of oxidants and their scavenging by antioxidants. For maintenance of a healthy reduction-oxidation (redox) balance, the thioredoxin (Trx) system, consisting of Trx, Trx reductase (TrxR), and NADPH, has been shown to play a pivotal role.1-2

1 B. Sahaf, Thioredoxin System in Normal and Transformed Human Cells. Medical Dissertation no.

642. Linkoping University, Linkoping, Sweden, 2000.

2 H. Nakamura, K. Nakamura, and J. Yodoi, Annu. Rev. Immunol. 15, 351 (1997).

Thioredoxin constitutes a family of small redox-active proteins with multiple functions, including cytoprotective antioxidant,3'4 cytokine,5 and chemokine6 activities. Trx is secreted by monocytes, lymphocytes, and other normal and neoplastic cells7 through a leaderless pathway.8 Mammalian cells have at least three different types of Trx: (1) cytoplasmic,9 (2) mitochondrial,10 and (3) plasma membrane bound.11 The cytoplasmic form has 104 amino acids and a molecular mass of 12 kDa, so-called full-length Trx. It can be cleaved into a 10-kDa form consisting of 80 to 84 amino acids, termed truncated Trx. Both forms of Trx can be secreted from cells.12

Thioredoxin reductase is required for redox regeneration of Trx. TrxR is a 116-kDa flavoprotein consisting of two homodimers of Mr 58,000. It was found to have a catalytically active COOH-terminal elongation containing the amino acid selenocysteine.13 Expression of constitutive Trx and TrxR has been observed in several cell types of the mammalian body, including keratinocytes of the skin, placental cells, hepatocytes, secretory cells and leukocytes.14'15 Physiological stimuli, including UV light, hydrogen peroxide, and mitogens, can induce the expression of Trx and TrxR, suggesting an important role in protection against oxidative stress.2'16 We found that TrxR was upregulated and released from human monocytes after mitogenic stimulation that generated oxidative stress.16

Dysregulation and overexpression of Trx and TrxR have also been found in a number of human primary cancers, such as malignant melanoma,11'17 adult T cell leukemia, lung, colon, cervical, and liver carcinoma.18 Previously, the lack

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of reliable techniques to assess redox changes in intact biological samples has been a challenge to investigators. Technical advances have allowed for significant development. We present a detailed protocol of an immunological technique called the enzyme-linked immunospot (ELISpot) assay. Single cells secreting redox-active proteins Trx and TrxR after the induction of a signal pathway involving oxidative stress can be enumerated. The basic principle of observing the secretion of immunoreactive substances from single cells was first explored in the hemolytic plaque assay by Niels Jerne and co-workers in 1963 (Jerne plaque assay).19 He was awarded the Nobel prize in 1984.20 Later, assays were developed for detection of single immunoglobulin (Ig)-secreting cells, using erythrocytes coated with anti-immunoglobulins or staphylococcal protein A.21'22 The instability of the erythrocytes and reagents in the plaque assay was, however, a drawback. The assays for detection of antibody (Ab)-secreting cells were considerably improved when solid-phase material such as polystyrene plastic surfaces,23"25 nitrocellulose,26,27 or polyvinylidene difluoride (PVDF)28 membranes was used for antigen coupling. Enzyme-linked anti-Ig conjugates and chromogenic substrates were used for visualization of secretory cells. Sedgwick and Holt named the technique "spot" ELISA23 and Czerkinsky et al. coined the ELISpot24 assay. The technique has been successfully adopted for enumeration of cytokine-secreting cells,25'26,29 or cells releasing redox-active proteins after an oxidative burst.12,16

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